Role of L-ascorbate in alleviating abiotic stresses in crop plants

  • Jelli Venkatesh1 and

    Affiliated with

    • Se Won Park1Email author

      Affiliated with

      Botanical StudiesAn International Journal201455:38

      DOI: 10.1186/1999-3110-55-38

      Received: 8 October 2012

      Accepted: 16 December 2013

      Published: 9 April 2014

      Abstract

      L-ascorbic acid (vitamin C) is a major antioxidant in plants and plays a significant role in mitigation of excessive cellular reactive oxygen species activities caused by number of abiotic stresses. Plant ascorbate levels change differentially in response to varying environmental stress conditions, depending on the degree of stress and species sensitivity. Successful modulation of ascorbate biosynthesis through genetic manipulation of genes involved in biosynthesis, catabolism and recycling of ascorbate has been achieved. Recently, role of ascorbate in alleviating number of abiotic stresses has been highlighted in crop plants. In this article, we discuss the current understanding of ascorbate biosynthesis and its antioxidant role in order to increase our comprehension of how ascorbate helps plants to counteract or cope with various abiotic stresses.

      Keywords

      Abiotic stress Antioxidant L-ascorbate Reactive oxygen species Transgenics

      Review

      Introduction

      Adverse environmental factors such as excessive cold, heat, drought and salinity stresses result in a considerable yield loss of crop plants all over the world. These abiotic stresses elicit complex cellular responses in the plant system, resulting in the production of excessive reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyperoxyl (HO2 ·), superoxide (O2 -) and singlet oxygen (1O2) radicals. Excessive ROS generated in plant cells tends to interact with different macromolecules resulting in oxidation of proteins, membrane lipids and nucleic acids and causes cellular damage, ultimately affecting the growth and productivity of plants (Wang et al. 2003). To protect themselves from adverse conditions, plants have evolved a number of cellular defense mechanisms including antioxidants such as ascorbate, glutathione and tocopherols as well as ROS-detoxifying enzymes such as superoxide dismutases, peroxidases and catalases (Inzé and Van Montagu 1995; Noctor and Foyer 1998).

      Among the plant antioxidants, L-ascorbate is a major antioxidant playing a vital role in the mitigation of excessive ROS activity through enzymatic as well as non-enzymatic detoxification (Mittler 2002). It also acts as a cell signaling modulator in numerous cellular processes including cell division, cell expansion and cell wall growth (Liso et al. 1984; Conklin and Barth 2004; Wolucka et al. 2005; Zhang et al. 2007). It is a cofactor for the number of enzymes such as violaxanthin de-epoxidase (VDE, xanthophyll cycle), 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ethylene biosynthesis) and 2-oxoacid-dependent dioxygenases (ABA and GA biosynthesis) (Eskling et al. 1997; Davey et al. 2000; Smirnoff 2000). Plants with low ascorbate biosynthesis are rather sensitive to various environmental stress conditions affecting their growth and development (Müller-Moulé et al. 2004; Huang et al. 2005; Alhagdow et al. 2007; Gao and Zhang 2008). Recently, it has been reported that ascorbate plays a crucial role in protection against various environmental stresses such as, drought (Hemavathi et al. 2011; Fotopoulos et al. 2008), salinity (Kwon et al. 2003; Huang et al. 2005; Wang et al. 2005; Sun et al. 2010a; Zhang et al. 2011; Venkatesh et al. 2012), ozone (Zheng et al. 2000; Sanmartin et al. 2003; Feng et al. 2010), low/high temperatures (Kwon et al. 2003; Larkindale et al. 2005) and high light intensity (Müller-Moulé et al. 2004; Talla et al. 2011). These studies on mutant and/or transgenic plants (summarized in the Table 1) with altered endogenous ASA levels proved that ascorbate plays a significant role in plant growth and development as well as abiotic stress tolerance. In this article, an attempt has been made to illustrate the role of ascorbate in various abiotic stresses in crop plants by exploring transgenic technology.
      Table 1

      Role of ascorbate in plant growth and development and abiotic stress tolerance

      Enzyme/protein

      Target plant

      Gene

      Gene source

      Type of genetic manipulation

      Ascorbate content

      Phenotypic changes

      Reference

      GDP-mannose pyrophosphorylase

      Tobacco

      GMPase

      Tomato

      Overexpression

      2.0–4.0-fold increase

      Increased tolerance to temperature stress

      Wang et al. 2011

      Phosphomannose Isomerase

      Arabidopsis

      PMI1

      Arabidopsis

      RNAi

      0.47–0.65-fold decrease

      No phenotypic changes under normal growth conditions in both mutants

      Maruta et al. 2008

       

      PMI2

      T-DNA knockout

      No change

       

      Phosphomannomutase

      Tobacco

      NbPMM

      Tobacco

      VIGS

      Up to 3.0-fold decrease

      Qian et al. 2007

      Arabidopsis

      NbPMM

      Tobacco

      VVMEE

      0.2–0.5-fold increase

       

      AtPMM

      Arabidopsis

      Overexpression

      0.25–0.33-fold increase

      Increased tolerance to MV stress

      Tobacco

      PMM

      Acerola

      Overexpression

      2.0-fold increase

      Badejo et al. 2009

      VTC4/Myoinositol monophosphatase (IMP)

      Arabidopsis

      VTC4

      T-DNA knockout

      0.61–0.75-fold decrease

      22.4% –34% decreases in myoinositol content

      Torabinejad et al. 2009

           

      Slow seed germination under control conditions

           

      Slightly hypersensitive to ABA and NaCl during seed germination

      GDP-L-galactose phosphorylase

      Arabidopsis

      vtc5-1 and vtc5-2

      Arabidopsis

      T-DNA knockout

      0.2-fold decrease

      Plant growth retardation and bleaching of the cotyledons

      Dowdle et al. 2007

      L-Galactose dehydrogenase

      Tobacco (BY–2 cells)

      L–GalLDH

      Tobacco

      Overexpression

      1.5–2.0-fold increase

      Higher mitotic index in cells

      Tokunaga et al. 2005

           

      Reduced browning and cells death of cultures

           

      Increased tolerance to MV

      L-galactono-1,4-lactone dehydrogenase

      Tobacco (BY–2 cells)

      GLDH

      Tobacco

      Antisense downregulation

      0.30-fold decrease

      Adversely effected plant cell division, growth and structure of plant cell

      Tabata et al. 2001

      Tobacco

      RrGalLDH

      Rosa roxburghii

      Overexpression

      2.1-fold increase

      Enhanced tolerance to salt stress

      Liu et al. 2013a

      Monodehydroascorbate reductase

      Tobacco

      AtMDAR1

      Arabidopsis

      Overexpression

      Up to 2.2-fold increase

      Enhanced tolerance to ozone, salt and PEG stresses

      Eltayeb et al. 2007

      Tobacco

      Am-MDAR

      Avicennia marina

      Overexpression

      Up to 2.0-fold increase

      Increased tolerance to salt stress

      Kavitha et al. 2010

      Tobacco

      MDAR-OX

      Arabidopsis

      Overexpression

      Up to 1.1-fold increase

      No change in Aluminium tolerance

      Yin et al. 2010

      Dehydroascorbate reductase

      Tobacco

      DHAR-OX

      Arabidopsis

      Overexpression

      Up to 1.3-fold increase

      Increased tolerance to Al stress

      Yin et al. 2010

      Tobacco

      DHAR

      Arabidopsis

      Overexpression

      1.9–2.1-fold increase

      Enhanced tolerance to ozone, drought and salinity

      Eltayeb et al. 2006

      Tobacco

      DHAR

      Wheat

      Overexpression

      2.1-fold increase

      Increased ozone tolerance and NPR

      Chen and Gallie 2005

        

      Tobacco

      Antisense downregulation

      0. 29-fold decrease

      Substantially reduced stomatal area and low NPR

      Tobacco

      DHAR

      Human

      Overexpression

      No significant change

      Enhanced tolerance to low temperature and NaCl

      Kwon et al. 2003

      Ascorbate peroxidase

      Tobacco

      tAPx

      Tobacco

      Overexpression

      No change

      Increased tolerance to MV and chilling stresses under light conditions

      Yabuta et al. 2002

        

      Tobacco/ Spinach

      Antisense downregulation

      Plants failed to grow

      Arabidopsis

      HvAPX1

      Barley

      Overexpression

      Increased tolerance to salt stress

      Xu et al. 2008

      Arabidopsis

      OsAPXa and OsAPXb

      Rice

      Overexpression

      Increased tolerance to salt stress

      Lu et al. 2007

      Tobacco

      CAPOA1

      Pepper

      Overexpression

      Increased plant growth

      Sarowar et al. 2005

           

      Increased tolerance to MV stress

      Tobacco BY-2 cells

      cAPX

      Arabidopsis

      Antisense downregulation

      No change

      Increased tolerance against heat and salt stresses

      Ishikawa et al. 2005

      Tobacco

      StAPX

      Tomato

      Overexpression

      Improved seed germination

      Sun et al. 2010a

           

      Increased tolerance to salt and osmotic stresses

      Rice

      Apx1/ Apx2

      Rice

      RNAi (Apx1+ Apx2)

      Up to 1.5-fold decrease

      No change in plant growth and development

      Rosa et al. 2010

           

      Increased tolerance to aluminium

         

      RNAi (Apx1 or Apx2)

      Produced semi-dwarf phenotype

      Rice

      OsAPx-R

      Rice

      RNAi

      Delayed plant development

      Lazzarotto et al. 2011

      Rice

      OsAPXa

      Rice

      Overexpression

      Increased spikelet fertility under cold stress

      Sato et al. 2011

      Rice

      Osapx2

      Rice

      Overexpression

      Enhanced stress tolerance

      Zhang et al. 2013

           

      Sensitive to abiotic stresses

        

      T-DNA knockout

      Semi-dwarf seedlings, yellow-green leaves, leaf lesion-mimic and seed sterility

      Alfalfa

      Osapx2

      Rice

      Overexpression

      Increased salt resistance

      Guan et al. 2012

      Tomato

      cAPX

      Pea

      Overexpression

      Enhanced tolerance to UV-B and heat stresses

      Wang et al. 2006

      Tomato

      cAPX

      Pea

      Overexpression

      Enhanced tolerance to chilling and salt stresses

      Wang et al. 2005

      Tomato

      LetAPX

      Tomato

      Antisense downregulation

      No significant change

      Transgenic plants photosynthetically less efficient and sensitive to chilling stress

      Duan et al. 2012b

      Ascorbate oxidase

      Tobacco

      AAO

      Cucumber

      Overexpression

      No change

      Plants become susceptible to ozone

      Sanmartin et al. 2003

      Tobacco

      AAO

      Cucumber

      Overexpression

      No change

      Increased drought tolerance due to reduced stomatal conductance

      Fotopoulos et al. 2008

      Tobacco

      AAO

      Pumpkin

      Overexpression

      2.0-fold increase in apoplastic ASA

      Number of smaller flowers significantly increased 6% to 14% reduction of in seed weight

      Pignocchi et al. 2003

        

      Tobacco

      Antisense downregulation

      2.0-fold increase in apoplastic ASA

      No significant changes

      Tobacco

      AAO

      Tobacco

      Overexpression

      Severe inhibition of germination and seed yield under high salinity

      Yamamoto et al. 2005

      Tobacco

      AAO

      Tobacco

      Antisense downregulation

      Increased tolerance to salt stress

      Yamamoto et al. 2005

          

      Increased seed yield under salt stress

      Arabidopsis

      AAO

      T-DNA knockout

       

      Increased tolerance to salt stress

           

      Increased seed yield under salt stress

      Myoinositol oxygenase

      Rice

      OsMIOX

      Rice

      Overexpression

      No change

      Increased drought tolerance

      Duan et al. 2012a

      ASA mannose pathway regulator 1

      Arabidopsis

      AMR1

      T-DNA knockout

      2.0–3.0-fold increase

      Increased ozone tolerance

      Zhang et al. 2009

      APx-R, APX-related; CAPOA1, Capsicum annuum ascorbate peroxidase-like 1 gene; MV, methyl viologen; NPR, net photosynthetic rate; PEG, polyethylene glycol; RNAi, RNA interference; VIGS, Virus-induced gene silencing; VVMEE, Viral-vector-mediated ectopic- expression.

      Overview: ascorbic acid biosynthesis, transportation, recycling and degradation processes in plants

      In plants, the accumulation or steady level of ascorbate is maintained in homeostasis through the rate of synthesis, recycling and degradation, as well as intra- and inter-cellular transport (Horemans et al. 2000a; Pallanca and Smirnoff 2000; Green and Fry 2005).

      Biosynthesis

      Characterization of low ascorbate producing mutants (vtc) of Arabidopsis has helped us to better understand the essential role of enzymes involved in the biosynthesis of L-ascorbate (Conklin et al. 1996; Conklin et al. 2000; Huang et al. 2005; Conklin et al. 2006; Müller-Moulé 2008). Now it is well known that in higher plants, ascorbate biosynthesis occurs through well-characterized D-mannose/L-galactose pathway (Smirnoff-Wheeler pathway), where D-mannose is converted to L-galactose via GDP-sugar intermediates (Wheeler et al. 1998) (Figure 1). L-galactose is further oxidized to L-galactono-1,4-lactone, which is converted into ascorbate, by L-galactono-1,4-lactone dehydrogenase (L-GalLDH), located on the inner mitochondrial membrane (Siendones et al. 1999; Smirnoff 2001). All of the genes that are involved in this pathway have been well-characterized; these include genes encoding GDP-D-mannose pyrophosphorylase (Conklin et al. 1999), GDP-D-mannose-3’,5’-epimerase (Wolucka and Van Montagu 2003; Watanabe et al. 2006), GDP-L-galactose phosphorylase (L-galactose guanylyltransferase) (Dowdle et al. 2007; Linster and Clarke 2008), L-galactose-1-phosphate phosphatase (Laing et al. 2004a), L-galactose dehydrogenase (Gatzek et al. 2002; Laing et al. 2004b) and L-GalLDH (Imai et al. 1998; Siendones et al. 1999; do Nascimento et al. 2005; Tokunaga et al. 2005; Alhagdow et al. 2007).
      http://static-content.springer.com/image/art%3A10.1186%2F1999-3110-55-38/MediaObjects/40529_2012_90_Fig1_HTML.jpg
      Figure 1

      L-ascorbic acid biosynthesis pathways in plants (modified after Hemavathi et al. 2010 ): (1) Smirnoff-Wheeler pathway, (2) L-gulose pathway, (3) Myoinositol-based pathway, (4) D-galacturonic acid pathway.

      In addition to the Smirnoff-Wheeler pathway, three other potential pathways of ascorbate biosynthesis have been identified in plants. It was demonstrated that in addition to production of GDP-L-galactose, GDP-D-mannose-3’,5’-epimerase can also produce GDP-L-gulose (Davey et al. 1999; Wolucka and Van Montagu 2003). Moreover, exogenous L-gulose and L-gulono-1,4-lactone were shown to serve as direct precursors of ascorbate in Arabidopsis cell cultures (Davey et al. 1999). These observations led to a proposal for an alternative L-gulose pathway in which L-gulose and L-gulono-1,4-lactone are important intermediates (Wolucka and Van Montagu 2003). However, the intermediate steps in this pathway have not yet been characterized in plants. D-galacturonic acid pathway involves the conversion of D-galacturonic acid, a product of the degradation of cell wall pectins to L-ascorbate via L-galactono-1,4-lactone (Agius et al. 2003; Cruz-Rus et al. 2011; Badejo et al. 2012) (Figure 1). Following the cloning of Arabidopsis myoinositol oxygenase (MIOX) gene by Lorence et al. (2004), a myoinositol-based pathway (animal-like pathway) was proposed (Figure 1). MIOX converts myoinositol to D-glucuronate and plants can catalyze the conversion of D-glucuronate into L-gulonic acid. However, recently, Endres and Tenhaken (2009), proved that the MIOX is involved mainly in the modulation of the metabolite level of myoinositol and plays a negligible role in the plant ascorbate biosynthesis.

      Ascorbate transport

      Once the ascorbate is synthesized on the inner mitochondrial membrane, it is transported to different cellular compartments including the apoplast. Both the ascorbate and DHA transport is mainly mediated by facilitated diffusion or active transport systems (Ishikawa et al. 2006). In contrast to ascorbate, DHA tends to be more efficiently transported across plant membranes with a higher affinity and capacity (Horemans et al. 1998; Szarka et al. 2004). It was proposed that specific plasma membrane transporters transport ASA or DHA in plants (Horemans et al. 2000b). However, either the protein or the gene associated with this transport and the nature of the mechanisms driving these carrier proteins are still inconclusive. Several other putative ascorbate transporters are associated with the plant plasma membrane (reviewed in Horemans et al. 2000a); however, the specific mechanisms by which they transport ASA or DHA have not been well elucidated.

      Ascorbate biosynthesis occurs in almost all plant cells and tissues. However, its level is generally high in photosynthetic tissues, meristematic tissues, flowers, young fruits, root tips, and apices of stolons or tubers (Gest et al. 2013). In certain fruits, such as Ribes nigrum (by galactose pathway, Hancock et al. 2007) and strawberry (by D-galacturonic acid pathway, Agius et al. 2003), increased accumulation of ascorbate occurs by a combination of long-distance transport and in situ biosynthesis. High ascorbate demand in developing sink tissues is probably because it is critical for cell cycle and cell division/growth, which cannot be met entirely by sink tissue alone (Smirnoff 2000; Franceschi and Tarlyn 2002). Ascorbate accumulation in sink tissue is controlled to some extent by ascorbate biosynthesis in source tissues (Franceschi and Tarlyn 2002; Tedone et al. 2004). Franceschi and Tarlyn (2002), demonstrated that the long-distance transport of ASA in plants occurs via phloem, where L-ascorbate was found to be loaded into the phloem of source leaves and transported to sink tissues. In addition, ascorbate biosynthesis, which occurs in phloem tissue via the D-Man/L-Gal pathway could also contribute to ASA accumulation in plant storage organs (Hancock et al. 2003).

      In mammals, sodium-dependent ascorbate transporters (SVCT1 and SVCT2), which belong to the nucleobase-ascorbate transporter (NAT) family, have been identified and well characterized as an active ascorbate transport system (Daruwala et al. 1999; Tsukaguchi et al. 1999; Ishikawa et al. 2006). Although numerous NATs have been identified in plants (Li and Schultes 2002; Maurino et al. 2006), their role in ASA transportation has not been established. Further studies are required to determine the definitive role in plant ascorbate transportation.

      Ascorbate recycling

      ASA pool in cells is maintained through synthesis, recycling and transportation, and plays an important role in adaptation of plant to various stresses (Stevens et al. 2008). Ascorbate takes part in several enzymatic and non-enzymatic mechanisms for elimination of deleterious ROS (Asada and Takahashi 1987), and as a result, MDHA and DHA accumulates in the cells. The two enzymes involved in the oxidation of ascorbate are ascorbate oxidase (AAO) and ascorbate peroxidase (APX). AAO is an apoplastic enzyme that catalyzes the oxidation of ASA to MDHA using oxygen and is associated with cell wall metabolism and cell expansion (Smirnoff 1996). Ascorbate peroxidase (APX) is a class I peroxidase catalyzes the conversion of H2O2 into H2O, using ascorbate as a specific electron donor, thus resulting in the accumulation of MDHA as a by-product (Teixeira et al. 2004).

      The ASA pool size is dependent, on both the rate of synthesis and the rate of reduction of MDHA and DHA back to ascorbate. MDHA and DHA produced as a result of activities of APX and AAO, respectively, should be efficiently recycled to maintain the reduced pool of ASA. MDHA is reduced back to ASA by MDAR using NADH/NADPH as electron donors. In addition, plant PM cyt b 561 (plasma membrane b-type cytochrome c) is also associated with the recycling of ASA from MDHA on the cytoplasmic side of the plasma membrane (Trost et al. 2000; Asard et al. 2001; Pignocchi and Foyer 2003). DHA is reduced to ASA by dehydroascorbate reductase (DHAR) using reduced glutathione (GSH) as an electron donor or by the electron-transport chain (ETC.) electron carriers (Szarka et al. 2007). Thus, DHAR and MDAR are crucial components in the maintenance of the reduced pool of ASA and are of prime importance in oxidative stress tolerance (Eltayeb et al. 2006).

      Ascorbate degradation

      Although the pathway of ascorbate synthesis is distributed between the cytosol and the mitochondrion (Foyer 2004; Smirnoff et al. 2004), the ascorbate degradation pathway appears to reside in the apoplast (Green and Fry 2005). In most plants, ascorbate degradation can occur via dehydroascorbate, yielding oxalate (OxA) and L-threonate (ThrO). However, in some plants (Vitaceae eg. grape), ascorbate can also be degraded via L-idonate to L-threarate (L-tartrate) (Green and Fry 2005). A degradation pathway for ASA/DHA catabolism in plants has been reported recently (Simpson and Ortwerth 2000; Parsons and Fry 2012). Ascorbate degradation pathway involves enzymic and/or non-enzymic oxidation to dehydroascorbic acid (DHA), which may irreversibly hydrolyze to 2,3-diketogulonate (DKG). However, many of the enzymes involved in the degradation pathway of ASA are not well characterized in plants. Both DHA and DKG prone to further oxidation under the same physiological conditions as that of apoplast (Parsons and Fry 2012). DHA can be oxidized by H2O2 non-enzymatically to a monoanion (cyclic-oxalyl-threonate; cOxT) and a dianion (oxalyl-threonate [OxT] isomers, 3-OxT and 4-OxT) independently through formation of a reactive intermediate cyclic-2,3-O-oxalyl-L-threonolactone (Parsons et al. 2011). In the absence of H2O2, DKG is relatively stable, however slowly generates a range of products, such as 2-carboxy-l-xylonolactone, 2-carboxy-l-lyxonolactone and 2-carboxy-l-threo-pentonate (Parsons et al. 2011). In the presence of apoplastic plant esterases or prolonged non-enzymatic incubations, substantial hydrolysis of cOxT to OxT and then OxT to OxA and ThrO would take place (Parsons et al. 2011).

      Genetic modulation of plant ascorbate pathway has become feasible with advancements made in plant genomics and genetic engineering. Several possible strategies have been followed to increase ascorbate production in plants via genetic engineering of enzymes involved in the biosynthesis and recycling of ascorbate. Several transgenes, which are of plant and animal origins, have been successfully used for increasing biosynthesis of ascorbic acid. Mouse L-gulono-c-lactone oxidase (GLOase) gene in tobacco, lettuce and potato (Jain and Nessler 2000; Hemavathi et al. 2010), human dehydroascorbate (DHAR) gene in tobacco (Kwon et al. 2003), wheat DHAR gene in tobacco and maize (Chen et al. 2003; Naqvi et al. 2009), Arabidopsis MDAR gene (AtMDAR1) in tobacco, strawberry D-galacturonic acid reductase (GalUR) gene in Arabidopsis and potato (Agius et al. 2003; Hemavathi et al. 2009) and rice L-GalLDH gene in rice (Liu et al. 2011) have been successfully cloned and expressed (summarized in the Table 2).
      Table 2

      Transgenic approaches for overproduction of L-ascorbate in plants

      Enzyme

      Target plant

      Gene

      Gene source

      Type of genetic manipulation

      Ascorbate content

      Phenotypic change

      Reference

      GDP-l-galactose phosphorylases

      Tomato

      GGP/ VTC2

      Actinidia chinensis

      Overexpression

      3.0–6.0-fold increase in fruits

      Bulley et al. 2012

      Strawberry

        

      Overexpression

      2.0-fold increase in fruits

      Potato

       

      Potato/Arabidopsis

      Overexpression

      Up to 3.0-fold increase in tuber

      GDP-mannose pyrophosphorylase

      Potato

      GMPase

      Potato

      Antisense downregulation

      0.88–1.44-fold reduction in leaves

      Dark spots on leaf veins and stems

      Keller et al. 1999

          

      0.56-fold reduction in tubers

      Early senesce

      GDP-Mannose 3’,5’-epimerase

      Tomato

      SlGME1

      Tomato

      Overexpression

      Up to 1.42-fold increase in leaves

      Improved tolerance to various abiotic stresses such as cold, salt and MV

      Zhang et al. 2011

          

      Up to 1.60-fold increase in fruits

       
       

      SlGME2

       

      Overexpression

      Up to 1.37-fold increase in leaves

       
          

      Up to 1.24-fold increase in fruits

       

      L-galactose guanyltransferase

      Tobacco

      GalT

      Kiwifruit

      Transient expression (leaves)

      Up to 3.0-fold increase

      Laing et al. 2007

      L-Galactose dehydrogenase

      Tobacco

      L-GalDH

      Arabidopsis

      Overexpression

      No change

      Gatzek et al. 2002

      Arabidopsis

       

      Arabidopsis

      Antisense downregulation

      0.7-fold decrease

       

      L-galactono-1,4-lactone dehydrogenase

      Rice

      L-GalLDH

      Rice

      RNAi

      0.6–0.87-fold decrease

      Slow plant growth rate and poor seed set

      Liu et al. 2011

        

      Rice

      Overexpression

      Up to 1.48-fold increase

      Increased NPR and higher seed set

      Tomato

      SlGalLDH

      Tomato

      RNAi

      No change

      Slow plant growth rate

      Alhagdow et al. 2007

           

      Strong reduction in leaf and fruit size

      Rice

      L-GalLDH

      Rice

      RNAi

      0.3– 0.5-fold decrease

      Slow growth rate, reduced tiller number, decreased NPR and premature senescence

      Liu et al. 2013b

      L-gulono-c-lactone oxidase

      Arabidopsis

      GLOase

      Rat

      Overexpression

      Up to 2.0–3.0-fold increase

      Radzio et al. 2003

      Lettuce

      GLOase

      Rat

      Overexpression

      4.0–7.0-fold increase

      Jain and Nessler 2000

      Tobacco

        

      Overexpression

      Up to 7.0-fold increase

      Tomato

      GLOase

      Rat

      Overexpression

      1.5-fold increase in fruits

      Enhanced tolerance to MV, NaCl, and mannitol

      Lim et al. 2012

      Potato

      GLOase

      Rat

      Overexpression

      Up to 1.41-fold increase

      Enhanced tolerance to MV, NaCl, and mannitol

      Hemavathi et al. 2010

      D-galacturonic acid reductase

      Arabidopsis

      GalUR

      Strawberry

      Overexpression

      2.0–3.0-fold increase

      Agius et al. 2003

      Potato

      GalUR

      Strawberry

      Overexpression

      1.6–2.0-fold increase

      Enhanced tolerance to MV, NaCl, and mannitol

      Hemavathi et al. 2009

      Tomato (Hairy Roots)

      GalUR

      Strawberry

      Overexpression

      2.5-fold increase

      High growth rate

      Wevar Oller et al. 2009

      Monodehydroascorbate reductase

      Tomato

      LeMDAR

      Tomato

      Overexpression

      Up to 1.18-fold increase

      Enhanced tolerance to temperature (low/high) and MV stresses

      Li et al. 2010

           

      High NPR

         

      Antisense downregulation

      Up to 1.3-fold decrease

      Susceptible to various abiotic stresses

      Tomato

      MDAR

      Tomato

      Overexpression

      0.7-fold reduced in fruits

      Haroldsen et al. 2011

          

      No change in leaves

       

      Dehydroascorbate reductase

      Tomato

      DHAR

      Tomato

      Overexpression

      1.6-fold increase in fruits

      Haroldsen et al. 2011

          

      No change in leaves

       

      Maize (Kernels)

      DHAR

      Wheat

      Overexpression

      6.0-fold increase

      Naqvi et al. 2009

      Maize

      DHAR

      Wheat

      Overexpression

      Up to 1.8-fold (leaves) and 1.9–fold (kernels) increase

      Chen et al. 2003

      Tobacco

      DHAR

      Wheat

      Overexpression

      2.2–3.9-fold increase

      Chen et al. 2003

      Tobacco

      DHAR

      Rice

      Overexpression

      Up to 1.6-fold increase

      Enhanced tolerance to salt and cold stresses

      Le Martret et al. 2011

      Tobacco

      DHAR

      Human

      Overexpression (chloroplasts)

      1.1-fold increase

      Increased SOD and APX activities in conjunction via triple gene construct

      Lee et al. 2007

           

      Increased tolerance to MV and NaCl induced stress

      Potato

      DHAR

      Sesame

      Overexpression

      1.1–1.3-fold increase in tuber with patatin promoter

      Goo et al. 2008

         

      Overexpression

      1.5- and 1.6-fold increase in leaves and tuber respectively, with CaMV35S promoter

      1.5- and 1.6-fold increase in leaves and tuber respectively, with CaMV35S promoter

      Potato

      StDHAR1

      Potato

      Overexpression (Cytosol)

      Up to 0.69-fold increase in leaves

      Qin et al. 2011

          

      Up to 0.29-fold increase in tubers

       
          

      Up to 0.50-fold increase in leaves

       

      StDHAR2

       

      Overexpression (Chloroplast)

      No significant change in tubers

       

      Arabidopsis

      DHAR1

      Rice

      Overexpression

      > 1.4-fold increase

      Enhanced tolerance to salt stress

      Ushimaru et al. 2006

      Arabidopsis

      DHAR

      Arabidopsis

      Overexpression

      2.0–4.25-fold increase

      Enhanced tolerance to high–light and high–temperature stress

      Wang et al. 2010

      Myoinositol oxygenase

      Arabidopsis

      miox4

      Arabidopsis

      Overexpression

      2.0–3.0-fold increase

      Lorence et al. 2004

      MV, methyl viologen; NPR, net photosynthetic rate; RNAi, RNA interference.

      Role of ascorbate in photosynthesis as a photoprotectant

      A high concentration of ascorbate in chloroplasts would imply its central role in photosynthesis (Smirnoff 1996). Ascorbate plays a crucial roles in scavenging the deleterious ROS that are generated as by-products of photosynthesis and as a key component in excess photonic energy dissipation mechanisms, such as the water-water cycle (WWC) (Neubauer and Yamamoto 1992; Asada 1999) and the xanthophyll cycle (Müller-Moulé et al. 2002; Yabuta et al. 2007). WWC, which is also known as Mehler peroxidase reaction, is one of the most important detoxification systems functioning in intact chloroplasts (Asada 1994 1999,2006). It involves the photoreduction of O2 by PSI to a superoxide radical, followed by the dismutation of superoxide radical by superoxide dismutase (SOD) to hydrogen peroxide and oxygen (Müller-Moulé et al. 2002). The hydrogen peroxide is reduced to water by ascorbate, catalyzed by ascorbate peroxidase (APX), and the resulting by-product monodehydroascorbate (MDA) is directly reduced to ascorbate either by reduced ferredoxin of PSI (Miyake and Asada 1992; Miyake and Asada 1994; Asada 1999) or by NAD(P)H-dependent chloroplastic MDHA reductase using NADH or NADPH as electron donor (Sano et al. 2005). MDHA can spontaneously disproportionate to ascorbate and dehydroascorbate (DHA) (Asada 1999). DHA is unstable at the physiological pH and irreversibly degrade to 2,3 diketo-1-gulonic acid if not recycled back to ascorbate. To preserve the ascorbate pool, DHA should be rapidly reduced back to ascorbate. DHA is recycled back to ascorbate via the ascorbate-glutathione cycle by reduced glutathione (GSH), catalyzed by DHAR (Shimaoka et al. 2003). Finally, glutathione reductase (GR) converts glutathione disulfide (GSSG) back into GSH using NAD(P)H as a reducing agent (Figure 2). Recently, Huang et al. (2008), reported that thioredoxin h2 (Trx h2) having both DHA reductase (in the presence of glutathione) and MDA reductase (in the presence of NADH) activity may also involve in the regeneration of ascorbate from DHA and MDHA, respectively.
      http://static-content.springer.com/image/art%3A10.1186%2F1999-3110-55-38/MediaObjects/40529_2012_90_Fig2_HTML.jpg
      Figure 2

      Multiple functions of L-ascorbate in plants. During abiotic stress conditions, scavenging of ROS by APX increases MDA content in both apoplast and symplast. If the MDA is not rapidly reduced back to ascorbate by MDAR, spontaneously disproportionate into ascorbate and DHA. Cytoplasmic DHAR can reduce DHA back to ascorbate using GSH, and the resulting GSSG is regenerated back to GSH through the action of GR in a NADPH dependent reaction. Furthermore, during oxidative stress conditions, L-ascorbate acts as a cofactor for violaxanthin de-epoxidase for the formation of zeaxanthin and also involves in the regeneration of α-tocopherol from tocotrienoxyl radicals.

      Increased biosynthesis of ascorbate in high light exposed plants and enhanced photoinhibition and oxidative damage in ascorbate-deficient plants would imply its role in excess light energy dissipation (Smirnoff 2000; Müller-Moulé et al. 2004; Yabuta et al. 2007). It was previously reported that high light stress results in the induction of the cytosolic APX and protects the cytosol and other cellular compartments from high light induced oxidative stress (Mittler 2002; Mullineaux and Karpinski 2002). Several isoforms of APX have been found in many plant species including both monocots and dicots, and are localized to various subcellular compartments. In Arabidopsis, nine APX genes were described (Panchuk et al. 2002; Mittler et al. 2004; Narendra et al. 2006; Koussevitzky et al. 2008): two cytosolic, two microsomal, three chloroplastic, one mitochondrial, and one dual-targeted to mitochondria and chloroplasts (Chew et al. 2003). In tomato, APX gene family comprises of seven genes encoding three cytosolic, two peroxisomal, and two chloroplastic APXs (Najami et al. 2008). Whereas, in rice, the APX gene family consists of eight genes encoding two cytosolic, two peroxisomal, and three chloroplastic isoforms and one is targeted to the mitochondria (Teixeira et al. 2004,2006; Hong et al. 2007). Recently, Lazzarotto et al. (2011), characterized a new class of rice putative heme peroxidases, APX-R (APX-related), a dually localized protein, targeted to both chloroplasts and mitochondria, which is functionally associated with APX. APX genes have been partially characterized in some plant species such as spinach (Ishikawa et al. 1995,1996,1998), cowpea (D’Arcy-Lameta et al. 2006) and eggplant (Lin et al. 2007). The large functional diversity and subcellular localization of the APX genes suggest the degree of complementation and coordination of the antioxidant defences in different cellular compartments during development and abiotic stress (Teixeira et al. 2004,2006).

      APX is highly responsive to various abiotic stresses and plays an important role in the scavenging of ROS in plants. Mutant studies in Arabidopsis revealed that cytosolic APXs (APX1 and APX2) are critical for cellular H2O2 homeostasis and play an important role in growth, development and oxidative protection of chloroplasts under various abiotic stresses (Pnueli et al. 2003; Davletova et al. 2005; Koussevitzky et al. 2008; Zhang et al. 2013). In particular, Arabidopsis APX1 is important for plant growth and development (Pnueli et al. 2003), whereas APX2 is critical for drought tolerance (Rossel et al. 2006). Thylakoid-bound APXs (tAPXs) are crucial for photosynthesis and photoprotection under photo-oxidative stress in Arabidopsis (Kangasjarvi et al. 2008). In rice, expressions of OsAPX genes are modulated by various abiotic stresses and exogenous ABA as well as by biotic stresses (Agrawal et al. 2003; Teixeira et al. 2006; Hong et al. 2007; Rosa et al. 2010). The expressions of two cytosolic APX genes, OsAPX1 and OsAPX2, are developmentally regulated (Agrawal et al. 2003) and the suppression of either of these genes resulted in strong effects on plant growth and development and produced semi-dwarf rice phenotypes (Rosa et al. 2010). Zhang et al. (2013), reported similar results wherein, downregulation of OsAPX2 gene affected the growth and development of rice seedlings, resulting in semi-dwarf and lesion-mimic seedlings, yellow-green leaves, and seed sterility. In contrast, the overexpression of OsAPX2 gene increased APX enzyme activity and thus resulted in enhanced stress tolerance.

      Davletova et al. (2005), demonstrated the role of cytosolic APX1 in cross-compartment protection of thylakoid/stromal and mitochondrial APXs during light stress. Despite the protection of each individual cellular compartment by its own set of ROS-scavenging enzymes, APX1-deficient Arabidopsis plants exhibited the oxidation of chloroplastic, mitochondrial and membrane-bound proteins, suggesting the key role of cytosolic APX1 enzyme in the cross-compartment protection of adjacent compartments (Davletova et al. 2005). However, some early studies certainly suggest that thylakoid membrane-bound APX (tAPX) is a limiting factor of antioxidative systems under photo-oxidative stress in chloroplasts and that the enhanced activity of tAPX under stress is to maintain the redox status of ascorbate (Yabuta et al. 2002). Moreover, transgenic Arabidopsis plants overexpressing Suaeda salsa chloroplastic stromal APX (sAPX) and thylakoid-bound APX (tAPX) also showed an increased tolerance to high light oxidative stress by efficient detoxification of ROS (Pang et al. 2011).

      Ascorbate also plays a significant role in formation of zeaxanthin during photo-oxidative stress (Figure 2). The excess excitation energy from the incidence of high light is invariably dissipated as heat by zeaxanthin in the light harvesting complex of the photosynthetic apparatus (Demmig-Adams and Adams 1996). Zeaxanthin is regenerated (via Xanthophyll cycle) in the successive de-epoxidation of violaxanthin and antheroxanthin by the enzyme VDE, which is located in the thylakoid lumen, and requires ascorbate as a cofactor (Neubauer and Yamamoto 1993; Müller-Moulé et al. 2002). Müller-Moulé et al. (2003), demonstrated the role of ascorbate in regeneration of zeaxanthin in ascorbate-deficient mutant of Arabidopsis, vtc2. These plants are characterized with an increased degree of lipid peroxidation and photoinhibition, and the regeneration of zeaxanthin from violaxanthin was slower due to insufficient ascorbate content.

      Role of l-ascorbate in salinity and drought tolerance

      In the cell, ROS is continuously produced during normal functioning of the photosynthesis, respiration and photorespiration as well as in various enzyme-catalyzed redox reactions (Dat et al. 2000; Moller 2001). However, ROS activity increases several folds under stress conditions and can serve as a signal that activates defense responses by specific signal transduction pathway in which hydrogen peroxide acts as secondary messenger (Helena and de Carvalho 2008). However, an increased ROS activity for the prolonged period can cause oxidative stress in plants. If ROS is not efficiently scavenged and quenched, it can cause membrane lipid peroxidation, inactivation of cellular enzymes and degradation of nucleic acids, which may eventually lead to the death of plant cells.

      Plants with higher ascorbate content can effectively scavenge the excessive ROS generated during stress conditions, and confer increased tolerance to abiotic stresses. Increased salt stress sensitivity of the Arabidopsis vtc mutant is attributed to the low intrinsic ascorbate levels and impaired ascorbate-glutathione cycle, which resulted in an enhanced ROS activity and a significant decrease in the CO2 assimilatory capacity (Huang et al. 2005). Moreover, deficiency of ascorbate may limit the recycling of α-tocopheroxyl radicals to α-tocopherol, which may, in turn, increase the oxidation of thylakoid membrane lipids under drought conditions (Munné-Bosch and Alegre 2002). Several transgenic plants overproducing ascorbate showed an enhanced salt and drought tolerance with reduced membrane lipid peroxidation and chlorophyll content loss. These plants also exhibited higher survival rate and a significantly higher seed germination rate, fresh weight and root length (Wang et al. 2005; Sun et al. 2010a; Zhang et al. 2011). Transgenic potato plants expressing strawberry GalUR gene and rat GLOase gene with several-fold increased biosynthesis of ascorbate also exhibited a better survival under salinity and drought stresses conditions including a reduction in the level of lipid peroxidation (Hemavathi et al. 2009; Hemavathi et al. 2011; Upadhyaya et al. 2011).

      Regulation of plant ascorbate redox state by means of synthesis, degradation and transport plays an essential role in plant adaptation to the stress (Stevens et al. 2008; Yin et al. 2010). MDAR and DHAR are key enzymes involved in the regulation of the ascorbate redox state and are of vital importance in the oxidative stress tolerance. MDAR maintains higher redox state of ascorbate by recycling the oxidized MDHA. Several isoforms of MDAR have been found in different cellular compartments, such as chloroplasts (Miyake and Asada 1994; Sano et al. 2005), cytosol and mitochondria (De Leonardis et al. 1995; Jiménez et al. 1997; Mittova et al. 2003), peroxisomes (Mittova et al. 2003; Leterrier et al. 2005) and glyoxysomes (Bowditch and Donaldson 1990), to serve the specific physiological role in each cellular compartment. The level of MDAR expression increases in response to oxidative stress triggered by several stress conditions (Yoon et al. 2004; Leterrier et al. 2005; Kavitha et al. 2010). Transgenic tobacco plants overexpressing a salt-inducible chloroplastic MDAR from halophyte Avicennia marina survived better under conditions of salt stress compared with wild-type plants (Kavitha et al. 2010). Similarly, transgenic potato plants overexpressing the Arabidopsis DHAR gene in the cytosol exhibited enhanced DHAR activity with faster growth, even under drought and salt stress conditions (Eltayeb et al. 2011).

      High salt and drought tolerances were also observed in transgenic plants overexpressing APX gene. Heterologous expression of OsAPX2 gene improved salt tolerance in transgenic Arabidopsis and alfalfa (Lu et al. 2007; Guan et al. 2012). Increased APX activity was observed in roots of etiolated rice seedlings in response to NaCl stress and was correlated with upregulation of chloroplastic OsAPX8 expression; however, no effect on the expression of the rest of the rice APX isoforms was observed (Hong et al. 2007). In contrast, Teixeira et al. (2006), reported the enhanced expression of OsAPX2 and OsAPX7, and severe downregulation of OsAPX8 in rice seedlings under NaCl stress. This observed discrepancy in the above results seemed to be differ with cultivars, plant age, tissues, and growing conditions (Hong et al. 2007).

      It has been demonstrated that OsAPX gene expression and H2O2 production were increased in response to NaCl in roots of etiolated rice seedlings (Tsai et al. 2004,2005). However, OsAPX8 expression and APX activity induced by NaCl are not mediated through H2O2 in rice roots (Tsai et al. 2005; Hong et al. 2007). In rice roots, accumulation of ABA in response to NaCl was correlated with upregulation of OsAPX8 expression (Hong et al. 2007). Moreover, exogenous application of ABA also specifically enhanced the expression of OsAPX8. Similarly, application of ABA increased the expression of APX genes in pea, rice, and sweet potato (Mittler and Zilinskas 1992; Agrawal et al. 2003; Park et al. 2004). These findings indicate that NaCl induced expression of APX is mediated through an accumulation of the ABA.

      Transgenic plants overexpressing a heterologous cytosolic APX gene showed an enhanced tolerance to salt stress with lower ROS activity (Badawi et al. 2004; Wang et al. 2005; Lu et al. 2007; Faize et al. 2011). These transgenic plants exhibited lower electrolyte leakage and lipid peroxidation, higher water use efficiency, minimal leaf damage and better photosynthetic performance. Similar results were obtained in the transgenic tobacco overexpressing Solanum lycopersicum thylakoid-bound APX (tAPX) and showed a better performance in terms of photosynthetic efficiency, root lengths and fresh and dry weights of the plants with enhanced tolerance to salt and osmotic stresses (Sun et al. 2010a).

      Yamamoto et al. (2005), demonstrated that downregulation of apoplastic AAO (ascorbate oxidase) confers higher salt tolerance in tobacco and Arabidopsis plants. It was suggested that under salt stress conditions, suppressed expression of apoplastic AAO led to a relatively low level of hydrogen peroxide accumulation and a high redox state of symplastic and apoplastic ascorbate, which, in turn, increased the salt tolerance. Interestingly, transgenic tobacco plants with elevated levels of hydrogen peroxide by overexpression of a cell wall-localized cucumber AAO conferred increased drought tolerance due to reduced stomatal conductance (Fotopoulos et al. 2008).

      Control of the stomatal aperture is essential for the plant adaptation to changes in its ambient environment. Several mechanisms for the regulation of stomatal aperture have been proposed (Kim and Lee 2007; Araújo et al. 2011). It has been found that O2 - and other activated oxygen species are involved in the regulation of stomatal movement (Purohit et al. 1994). Zhang et al. (2001), demonstrated that hydrogen peroxide may function as an intermediate in ABA signalling in guard cells. During stress conditions ABA causes an increase in hydrogen peroxide production and induces stomatal closure. Stomatal closure induced by hydrogen peroxide was reversed by exogenous application of ascorbate because of hydrogen peroxide detoxification activity of ascorbate (Zhang et al. 2001). Earlier, Chen and Gallie (2004), demonstrated that transgenic plants with DHAR overexpression exhibited an increase in the ascorbate redox state and reduced levels of hydrogen peroxide in guard cells and leaves showed greater stomatal opening, increased transpiration rate and stomatal conductance even under normal growth conditions. Whereas, plants with suppression of DHAR activity showed an elevated level of hydrogen peroxide and conferred increased drought tolerance with a decreased ascorbate redox state.

      It has been known that the enzyme AAO, which catalyzes the oxidation of ASA to DHA exclusively located in the apoplast, plays an important role in the maintenance of the redox state of the apoplastic ascorbate levels (Pignocchi and Foyer 2003; Sanmartin et al. 2003; Pignocchi et al. 2006). However, the mechanism of regulation of AAO gene expression and stomatal moments is not clearly understood. It has been suggested that the signal perception of stomatal closure is altered by AAO overexpression (Pignocchi and Foyer 2003; Fotopoulos et al. 2008). Transgenic tobacco leaves overexpressing a cell wall-localized cucumber AAO contained elevated levels of hydrogen peroxide and ABA content, thereby resulting in reduced stomatal conductance and reduced rates of water loss (Fotopoulos et al. 2008). Based on these results, it is predictable that either the suppression of DHAR expression or the overexpression of AAO would result in the decrease in the ascorbate redox state and causes increased accumulation of hydrogen peroxide levels resulting in stomatal closure, lower transpiration thus providing drought tolerance. However, in both, suppression of DHAR expression or overexpression of AAO would result in greater accumulation of apolastic DHA levels which may play a key role in the regulation of stomatal aperture.

      Ascorbate as an ozone protectant

      An increasing concentration of ambient ozone was observed during recent decades in many industrial and rural regions of the world and poses a hazard for vegetation. The ozone exposure of plants causes extensive visible leaf damage and decreased rates of stomatal conductance and photosynthesis (Pell et al. 1997; Torsethaugen et al. 1997; Zheng et al. 2000; Sanmartin et al. 2003). Ozone entered through stomata reacts with apoplastic and symplastic components of the cell (Long and Naidu 2002; Castagna and Ranieri 2009; Cho et al. 2011) resulting in a greater accumulation of ROS, which causes an oxidative damage to the photosynthetic membranes and finally leads to the death of photosynthetic mesophyll cells (Godde and Buchhold 1992; Ciompi et al. 1997; Chen et al. 2005). It was suggested that ozone exposure directly affects guard cells by inhibiting the ion channels (K+1 channel) activity in the guard cell plasma membrane (Torsethaugen et al. 1999). Protection of crop plants from ozone damage could be accomplished by replacement of sensitive biotypes with more tolerant ones as well as by application of synthetic ozone protectants such as ethylene diurea, azoxystrobin, epoxiconazole and penconazole (Blum et al. 2011; Didyk and Blum 2011). However, application of synthetic ozone protectants will pollute the environment and may affect the crop production. Therefore, it is necessary to develop alternative ecofriendly strategies to minimize the ozone damage in plants by using plant-based natural antioxidants such as ascorbic acid.

      Apoplastic ascorbate is assumed to represent the first line of defence against potentially damaging pollutants (Plöchl et al. 2000; Barnes et al. 2002). Apoplastic ascorbate can protect plants from ozone-induced damage by directly reacting with ozone (Chameides 1989; Plöchl et al. 2000) and ROS (D’Haese et al. 2005) or by serving as a substrate in ROS-scavenging enzymatic reactions (Chen and Gallie 2005). Plant species that are resistant to ozone showed an increased apoplastic ascorbate levels (Lee 1991; Turcsányi et al. 2000; Zheng et al. 2000; Burkey et al. 2006; Feng et al. 2010). Moreover, exogenous application of plants with ascorbate prevented the foliar injury and alleviated the decline in photosynthesis rate caused by ozone stress (Maddison et al. 2002; Zheng et al. 2000). The lower levels of apoplastic ascorbate content greatly enhanced foliage injury upon chronic ozone exposure in tobacco (Sanmartin et al. 2003). Furthermore, Arabidopsis mutants (vtc1) with low foliar content of ascorbate exhibit hypersensitivity to ozone (Conklin and Barth 2004). Similarly, rice TOS17 insertional mutant (ND6172) for a GDP-D-mannose-3’,5’-epimerase gene, which is characterized with 20–30% lower ascorbate level than the wild type, showed a higher level of visible leaf damage upon ozone exposure (Frei et al. 2012).

      Maintenance of the apoplastic ascorbate redox state is crucial for ozone-induced oxidative stress tolerance of plants and is influenced by activities of enzymes such as AAO and APX. Altered expression of these enzymes was normally observed in plants exposed to ozone (Kubo et al. 1995; Sanmartin et al. 2003; Pignocchi et al. 2006). Transgenic tobacco plants with overexpressing AAO (Sanmartin et al. 2003) or downregulation of cytosolic APX (Orvar and Ellis 1997) resulted in the increased susceptibility of tobacco plants to ozone-induced damage. However, transgenic tobacco plants overproducing chloroplastic APX could not protect from ozone injury (Torsethaugen et al. 1997). The apoplastic ascorbate redox state also depends on the balance between oxidation of ascorbate to DHA in apoplast and reduction of MDA and DHA to ascorbate in cytoplasm. During the detoxification process, DHA produced in the apoplast diffuses into the cytoplasm and recycled back to ascorbate by cytDHAR (via ascorbate-glutathione cycle, Figure 2) on the plasma membrane. The regenerated ascorbate can be transported back into the apoplast for the detoxification of ozone (Luwe et al. 1993; Horemans et al. 2000a; Yoshida et al. 2006). Transgenic tobacco plants overexpressing MDAR gene conferred enhanced tolerance to ozone due to increased recycling of ascorbate from MDA (Eltayeb et al. 2007). Similarly, DHAR-overexpressing plants also showed an increased ozone tolerance with a higher level of photosynthetic activity despite exhibiting a larger stomatal area (Chen and Gallie 2005). In converse, plants with suppressed DHAR activity showed a substantially reduced stomatal area and lower level of photosynthetic activity. Yoshida et al. (2006), demonstrated that Arabidopsis mutant with completely lacking cytDHAR activity showed a significantly lower level of apoplastic ascorbate and was highly sensitive to ozone (Yoshida et al. 2006). Increased level of ascorbate through enhanced ascorbate recycling by DHAR overexpression offered greater protection against oxidative stress despite poor ability to respond to ozone through stomatal closure (Chen and Gallie 2005; Eltayeb et al. 2007).

      Role of ascorbate in temperature stress tolerance

      Temperature stress is one of the most important environmental factors affecting the crop yields and geographic distribution of plants. Temperature stresses such as heat, cold or freezing result in excessive ROS production and cause severe damage to cell membranes and proteins (O’Kane et al. 1996; Larkindale and Knight 2002; Suzuki and Mittler 2006; Hu et al. 2008; Yamashita et al. 2008) and also cause impairments in the chloroplast and mitochondrial metabolism (Salvucci and Crafts-Brandner 2004; Vacca et al. 2004; Barra et al. 2005; Nguyen et al. 2009; Barta et al. 2010; Tóth et al. 2011).

      Several studies demonstrated that ROS-mitigating mechanisms play an important role in protecting crops against extreme temperature stresses (Iba 2002; Yoshimura et al. 2004; Hu et al. 2008). For instance, overexpression of cytosolic APX in transgenic tomato enhances heat and chilling stress tolerance (Wang et al. 2005,2006). Similarly, transgenic potato plants overexpressing APX under the control of an oxidative stress inducible SWPA2 promoter showed increased tolerance to high temperature stress (Tang et al. 2006). In rice, overexpression of OsAPX1 enhanced tolerance to chilling stress at the booting stage (Sato et al. 2011). Increased temperature stress tolerance was also observed in transgenic tobacco plants overexpressing the thylakoid-bound APX gene from tomato. These transgenic tobacco lines, under stress condition, showed a higher APX activity and contained less hydrogen peroxide and malondialdehyde than wild-type plants (Sun et al. 2010b). Moreover, under chilling and heat stresses, the photochemical efficiency of PSII in the transgenic lines was distinctly higher than that of wild-type plants. Wang et al. (2011), reported the similar results in transgenic tobacco plants overproducing ascorbate through the expression of tomato GMPase and observed the reduced ROS activity in the transgenic plants under high or low temperature stress conditions.

      L-ascorbate may also act as an alternative electron donor of PSII; in those cases electron transfer is inhibited due to inactivation of oxygen evolving complex (OEC) (Mano et al. 2004; Guiss´e et al. 1995; Strasser 1997; Tóth et al. 2009; Gururani et al. 2012). Heat-induced inactivation of PSII was strongly influenced by the ascorbate content of leaves (Tóth et al. 2011). Tóth et al. (2011), experimentally proved the physiological role of ascorbate as alternative PSII electron donor in heat-stressed leaves with inactive OEC. This result suggests that the role of ascorbate as an alternative PSII electron donor is to decelerate the processes of photoinactivation and minimize the ROS activity in the photosynthetic thylakoid membranes, and thus minimize the damage to the entire photosynthetic apparatus.

      Conclusion

      In higher plants, ascorbate biosynthesis occurs through D-mannose/L-galactose pathway, which is a most important source of ascorbate. Ascorbate plays a major role in cellular ROS-scavenging activity. It also influences many stress responsive enzyme activities through synergic action with the other antioxidants such as glutathione and α-tocopherol and reduces the oxidative damage to cells. Recent studies suggest its role in photosynthesis as an alternative electron donor to PSII under abiotic stress conditions and play a major role in protection of photosynthetic apparatus in chloroplast by keeping the ROS activity under check.

      Several ascorbate biosynthetic pathway transgenes have been introduced into plants through genetic engineering to elevate the ascorbate level. These transgenic plants also provided better stress tolerance to various abiotic stresses such as high light, low/high temperature, ozone, salinity and drought. The role of ascorbate goes beyond that of simply an antioxidant given its apparent involvement in a complex signalling pathway that mediates responses to biotic and abiotic stresses as it is a cofactor for plant hormones such as ABA, GA and ethylene (Conklin and Barth 2004). However, role of ascorbate in signal transduction needs to be clarified further, particularly with respect to drought tolerance provided by altered stomatal movements.

      Abbreviations

      ABA: 

      Abscisic acid

      AGC: 

      The ascorbate-glutathione cycle

      AAO: 

      Ascorbate oxidase

      APX: 

      Ascorbate peroxidase

      DHA: 

      Dehydroascorbate

      DHAR: 

      Dehydroascorbate reductase

      GA: 

      Gibberellic acid

      GalUR: 

      D-galacturonic acid reductase

      GLOase: 

      L-gulono-c-lactone oxidase

      GR: 

      Glutathione reductase

      GSH: 

      Glutathione

      GSSG: 

      Glutathione disulfide

      MDAR: 

      Monodehydroascorbate reductase

      MDHA: 

      Monodehydroascorbate

      MIOX: 

      Myoinositol oxygenase

      L-GalLDH: 

      L-galactono-1,4-lactone dehydrogenase

      OEC: 

      Oxygen evolving complex

      PSI: 

      Photosystem I

      PSII: 

      Photosystem II

      SOD: 

      Superoxide dismutase

      ROS: 

      Reactive oxygen species

      VDE: 

      Violaxanthin de-epoxidase

      WWC: 

      The water-water cycle.

      Declarations

      Acknowledgements

      This paper resulted from the Konkuk University research support program.

      Authors’ Affiliations

      (1)
      Department of Molecular Biotechnology, Konkuk University

      References

      1. Agius F, Lamothe RG, Caballero JL, Blanco JM, Botella MA, Valpuesta V: Engineering increased vitamin C levels in plants by over-expression of a D-galacturonic acid reductase. Nat Biotechnol 2003, 21:177–181. 10.1038/nbt777PubMed
      2. Agrawal GK, Jwa NS, Iwahashi H, Rakwal R: Importance of ascorbate peroxidases OsAPX1 and OsAPX2 in the rice pathogen response pathways and growth and reproduction revealed by their transcriptional profiling. Gene 2003, 322:93–103.PubMed
      3. Alhagdow M, Mounet F, Gilbert L, Nunes-Nesi A, Garcia V, Just D, Petit J, Beauvoit B, Fernie AR, Rothan C, Baldet P: Silencing of the mitochondrial ascorbate synthesizing enzyme L-galactono-1,4-lactone dehydrogenase affects plant and fruit development in tomato. Plant Physiol 2007, 145:1408–1422. 10.1104/pp.107.106500PubMed CentralPubMed
      4. Araújo WL, Fernie AR, Nunes-Nesi A: Control of stomatal aperture: a renaissance of the old guard. Plant Signal Behav 2011, 6:1305–1311. 10.4161/psb.6.9.16425PubMed CentralPubMed
      5. Asada K: Mechanisms for Scavenging Reactive Molecules Generated in Chloroplasts Under Light Stress. In Photoinhibition of Photosynthesis. From Molecular Mechanisms to the Field. Edited by: Baker NR, Bowyer JR. Oxford: Bios Scientific Publishers; 1994:129–142.
      6. Asada K: The water–water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu Rev Plant Physiol Plant Mol Biol 1999, 50:601–639. 10.1146/annurev.arplant.50.1.601PubMed
      7. Asada K: Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiol 2006, 141:391–396. 10.1104/pp.106.082040PubMed CentralPubMed
      8. Asada K, Takahashi M: Production and Scavenging of Active Oxygen in Photosynthesis. In Photoinhibition. Edited by: Kyle DJ, Osmond CB, Arntzen CJ. Amsterdam: Elsevier; 1987:227–287.
      9. Asard H, Kapila J, Verelst W, Berczi A: Higher-plant plasma membrane cytochrome b561: a protein in search of a function. Protoplasma 2001, 217:77–93. 10.1007/BF01289417PubMed
      10. Badawi GH, Kawano N, Yamauchi Y, Shimada E, Sasaki R, Kubo A, Tanaka K: Over–expression of ascorbate peroxidase in tobacco chloroplasts enhances the tolerance to salt stress and water deficit. Physiol Plant 2004, 121:231–238. 10.1111/j.0031-9317.2004.00308.xPubMed
      11. Badejo AA, Eltelib HA, Fukunaga K, Fujikawa Y, Esaka M: Increase in ascorbate content of transgenic tobacco plants overexpressing the acerola ( Malpighia glabra ) phosphomannomutase gene. Plant Cell Physiol 2009, 50:423–428.PubMed
      12. Badejo AA, Wada K, Gao Y, Maruta T, Sawa Y, Shigeoka S, Ishikawa T: Translocation and the alternative D–galacturonate pathway contribute to increasing the ascorbate level in ripening tomato fruits together with the D-mannose/L-galactose pathway. J Exp Bot 2012, 63:229–239. 10.1093/jxb/err275PubMed CentralPubMed
      13. Barnes JD, Zheng Y, Lyons TM: Plant Resistance to Ozone: The Role of Ascorbate. In Air Pollution and Plant Biotechnology. Edited by: Omasa K, Saji H, Youssefian S, Kondo N. Tokyo: Springer–Verlag; 2002:235–254.
      14. Barra M, Haumann M, Dau H: Specific loss of the extrinsic 18 KDa protein from photosystem II upon heating to 47°C causes inactivation of oxygen evolution likely due to Ca release from the Mn–complex. Photosynth Res 2005, 84:231–237. 10.1007/s11120-004-7158-xPubMed
      15. Barta C, Dunkle AM, Wachter RM, Salvucci ME: Structural changes associated with the acute thermal instability of Rubisco activase. Arch Biochem Biophys 2010, 499:17–25.PubMed
      16. Blum O, Didyk N, Pavluchenko N, Godzik B: Assessment of protective effect of some modern agrochemicals against ozone-induced stress in sensitive clover and tobacco cultivars. J Toxicol 2011. doi:10.1155/2011/308598
      17. Bowditch MI, Donaldson RP: Ascorbate free–radical reduction by glyoxysomal membranes. Plant Physiol 1990, 94:531–537. 10.1104/pp.94.2.531PubMed CentralPubMed
      18. Bulley S, Wright M, Rommens C, Yan H, Rassam M, Lin-Wang K, Andre C, Brewster D, Karunairetnam S, Allan AC, Laing WA: Enhancing ascorbate in fruits and tubers through over-expression of the L-galactose pathway gene GDP-L-galactose phosphorylase. Plant Biotechnol J 2012, 10:390–397. 10.1111/j.1467-7652.2011.00668.xPubMed
      19. Burkey KO, Neufeld HS, Souza L, Chappelka AH, Davison AW: Seasonal profiles of leaf ascorbic acid content and redox state in ozone–sensitive wildflowers. Environ Pollut 2006, 143:427–434. 10.1016/j.envpol.2005.12.009PubMed
      20. Castagna A, Ranieri A: Detoxification and repair process of ozone injury: from O3 uptake to gene expression adjustment. Environ Pollut 2009, 157:1461–1469. 10.1016/j.envpol.2008.09.029PubMed
      21. Chameides WL: The chemistry of ozone deposition to plant leaves: role of ascorbic acid. Environ Sci Technol 1989, 23:595–600. 10.1021/es00063a013
      22. Chen Z, Gallie DR: The ascorbic acid redox state controls guard cell signaling and stomatal movement. Plant Cell 2004, 16:1143–1162. 10.1105/tpc.021584PubMed CentralPubMed
      23. Chen Z, Gallie DR: Increasing tolerance to ozone by elevating foliar ascorbic acid confers greater protection against ozone than increasing avoidance. Plant Physiol 2005, 138:1673–1689. 10.1104/pp.105.062000PubMed CentralPubMed
      24. Chen Z, Young TE, Ling J, Chang SC, Gallie DR: Increasing vitamin C content of plants through enhanced ascorbate recycling. Proc Natl Acad Sci U S A 2003, 100:3525–3530. 10.1073/pnas.0635176100PubMed CentralPubMed
      25. Chew O, Whelan J, Millar H: Molecular definition of the ascorbate-glutathione cycle in Arabidopsis mitochondria reveals dual targeting of antioxidant defenses in plants. J Biol Chem 2003, 278:46869–46877. 10.1074/jbc.M307525200PubMed
      26. Cho K, Tiwari S, Agrawal SB, Torres NL, Agrawal M, Sarkar A, Shibato J, Agrawal GK, Kubo A, Rakwal R: Tropospheric ozone and plants: absorption, responses and consequences. Rev Environ Contam Toxicol 2011, 212:61–111.PubMed
      27. Ciompi S, Castagna A, Ranieri A, Nali C, Lorenzini G, Soldatini GF: CO 2 assimilation, xanthophyll cycle pigments and PSII efficiency in pumpkin plants as affected by ozone fumigation. Physiol Plant 1997, 101:881–889. 10.1111/j.1399-3054.1997.tb01077.x
      28. Conklin PL, Barth C: Ascorbic acid, a familiar small molecule intertwined in the response of plants to ozone, pathogens, and the onset of senescence. Plant Cell Environ 2004, 27:959–970. 10.1111/j.1365-3040.2004.01203.x
      29. Conklin PL, Williams EH, Last RL: Environmental stress sensitivity of an ascorbic acid–deficient Arabidopsis mutant. Proc Natl Acad Sci U S A 1996, 93:9970–9974. 10.1073/pnas.93.18.9970PubMed CentralPubMed
      30. Conklin PL, Norris SN, Wheeler GL, Smirnoff N, Williams EH, Smirnoff N, Last RL: Genetic evidence for the role of GDP–mannose in plant ascorbic acid (vitamin C) biosynthesis. Proc Natl Acad Sci U S A 1999, 96:4198–4203. 10.1073/pnas.96.7.4198PubMed CentralPubMed
      31. Conklin PL, Saracco SA, Norris SR, Last RL: Identification of ascorbic acid–deficient Arabidopsis thaliana mutants. Genetics 2000, 154:847–856.PubMed CentralPubMed
      32. Conklin PL, Gatzek S, Wheeler GL, Dowdle J, Raymond MJ, Rolinski S, Isupov M, Littlechild JA, Smirnoff N: Arabidopsis thaliana vtc4 encodes L-galactose-1-P phosphatase, a plant ascorbic acid biosynthetic enzyme. J Biol Chem 2006, 281:15662–15670. 10.1074/jbc.M601409200PubMed
      33. Cruz–Rus E, Amaya I, Sánchez-Sevilla JF, Botella MA, Valpuesta V: Regulation of L–ascorbic acid content in strawberry fruits. J Exp Bot 2011, 62:4191–4201. 10.1093/jxb/err122PubMed CentralPubMed
      34. D’Arcy-Lameta A, Ferrari-Iliou R, Contour-Ansel D, Pham-Thi AT, Zuily-Fodil Y: Isolation and characterization of four ascorbate peroxidase cDNAs responsive to water deficit in cowpea leaves. Ann Bot 2006, 97:133–140.PubMed CentralPubMed
      35. D’Haese D, Vandermeiren K, Asard H, Horemans N: Other factors than apoplastic ascorbate contribute to the differential ozone tolerance of two clones of Trifolium repens L. Plant Cell Environ 2005, 28:623–632. 10.1111/j.1365-3040.2005.01308.x
      36. Daruwala R, Song J, Koh WS, Rumsey SC, Levine M: Cloning and functional characterization of the human sodium-dependent vitamin C transporters hSVCT1 and hSVCT2. FEBS Lett 1999, 460:480–484. 10.1016/S0014-5793(99)01393-9PubMed
      37. Dat J, Vandenabeele S, Vranová E, Van Montagu M, Inzé D, Van Breusegem F: Dual action of the active oxygen species during plant stress responses. Cell Mol Life Sci 2000, 57:779–795. 10.1007/s000180050041PubMed
      38. Davey MW, Gilot C, Persiau G, Østergaard J, Han Y, Bauw GC, Van Montagu MC: Ascorbate biosynthesis in Arabidopsis cell suspension culture. Plant Physiol 1999, 121:535–543. 10.1104/pp.121.2.535PubMed CentralPubMed
      39. Davey MW, Van Montagu M, Inze D, Sanmartin M, Kanellis A, Smirnoff N, Benzie IJJ, Strain JJ, Favell D, Fletcher J: Plant l–ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. J Sci Food Agric 2000, 80:825–860. Publisher Full Text 10.1002/(SICI)1097–0010(20000515)80:7<825::AID-JSFA598>3.0.CO;2–6
      40. Davletova S, Rizhsky L, Liang H, Shengqiang Z, Oliver DJ, Coutu J, Shulaev V, Schlauch K, Mittler R: Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis . Plant Cell 2005, 17:268–281. 10.1105/tpc.104.026971PubMed CentralPubMed
      41. De Leonardis S, De Lorenzo G, Borraccino G, Dipierro S: A specific ascorbate free radical reductase isozyme participates in the regeneration of ascorbate for scavenging toxic oxygen species in potato tuber mitochondria. Plant Physiol 1995, 109:847–851.PubMed CentralPubMed
      42. Demmig-Adams B, Adams WW III: Xanthophyll cycle and light stress in nature: uniform response to excess direct sunlight among higher plant species. Planta 1996, 198:460–470. 10.1007/BF00620064
      43. Didyk NP, Blum OB: Natural antioxidants of plant origin against ozone damage of sensitive crops. Acta Physiol Plant 2011, 33:25–34. 10.1007/s11738-010-0527-5
      44. Do Nascimento J, Higuchi B, Gómez M, Oshiro R, Lajolo F: L–Ascorbate biosynthesis in strawberries: L-galactono-1,4-lactone dehydrogenase expression during fruit development and ripening. Postharvest Biol Tech 2005, 38:34–42. 10.1016/j.postharvbio.2005.05.014
      45. Dowdle J, Ishikawa T, Gatzek S, Rolinski S, Smirnoff N: Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability. Plant J 2007, 52:673–689. 10.1111/j.1365-313X.2007.03266.xPubMed
      46. Duan J, Zhang M, Zhang H, Xiong H, Liu P, Ali J, Li J, Li Z: OsMIOX , a myoinositol oxygenase gene, improves drought tolerance through scavenging of reactive oxygen species in rice ( Oryza sativa L.). Plant Sci 2012, 196:143–151.PubMed
      47. Duan M, Ma NN, Li D, Deng YS, Kong FY, Lv W, Meng QW: Antisense-mediated suppression of tomato thylakoidal ascorbate peroxidase influences anti-oxidant network during chilling stress. Plant Physiol Biochem 2012, 58:37–45.PubMed
      48. Eltayeb AE, Kawano N, Badawi GH, Kaminaka H, Sanekata T, Morishima I, Shibahara T, Inanaga S, Tanaka K: Enhanced tolerance to ozone and drought stresses in transgenic tobacco overexpressing dehydroascorbate reductase in cytosol. Physiol Plant 2006, 127:57–65. 10.1111/j.1399-3054.2006.00624.x
      49. Eltayeb AE, Kawano N, Badawi GH, Kaminaka H, Sanekata T, Shibahara T, Inanaga S, Tanaka K: Overexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone, salt and polyethylene glycol stresses. Planta 2007, 225:1255–1264. 10.1007/s00425-006-0417-7PubMed
      50. Eltayeb AE, Yamamoto S, Habora MEE, Yin L, Tsujimoto H, Tanaka K: Transgenic potato overexpressing Arabidopsis cytosolic AtDHAR1 showed higher tolerance to herbicide, drought and salt stresses. Breed Sci 2011, 61:3–10. 10.1270/jsbbs.61.3
      51. Endres S, Tenhaken R: Myoinositol oxygenase controls the level of myoinositol in Arabidopsis , but does not increase ascorbic acid. Plant Physiol 2009, 149:1042–1049.PubMed CentralPubMed
      52. Eskling M, Arvidsson PO, Akerlund HE: The xanthophyll cycle, its regulation and components. Physiol Plant 1997, 100:806–816. 10.1111/j.1399-3054.1997.tb00007.x
      53. Faize M, Burgos L, Faize L, Piqueras A, Nicolas E, Barba-Espin G, Clemente–Moreno MJ, Alcobendas R, Artlip T, Hernandez JA: Involvement of cytosolic ascorbate peroxidase and Cu/Zn–superoxide dismutase for improved tolerance against drought stress. J Exp Bot 2011, 62:2599–2613. 10.1093/jxb/erq432PubMed
      54. Feng Z, Pang J, Nouchi I, Kobayashi K, Yamakawa T, Zhu J: Apoplastic ascorbate contributes to the differential ozone sensitivity in two varieties of winter wheat under fully open–air field conditions. Environ Pollut 2010, 158:3539–3545. 10.1016/j.envpol.2010.08.019PubMed
      55. Fotopoulos V, De Tullio MC, Barnes J, Kanellis AK: Altered stomatal dynamics in ascorbate oxidase over–expressing tobacco plants suggest a role for dehydroascorbate signalling. J Exp Bot 2008, 59:729–737. 10.1093/jxb/erm359PubMed
      56. Foyer CH: The Role of Ascorbic Acid in Defence Networks and Signalling in Plants. In Vitamin C. Edited by: Asard H, May JM, Smirnoff N. Oxford, UK: Functions and Biochemistry in Animals and Plants. Bios Scientific Publishers; 2004:65–82.
      57. Franceschi VR, Tarlyn NM: L-ascorbic acid is accumulated in source leaf phloem and transported to sink tissues in plants. Plant Physiol 2002, 130:649–656. 10.1104/pp.007062PubMed CentralPubMed
      58. Frei M, Wissuwa M, Pariasca-Tanaka J, Chen CP, Südekum KH, Kohno Y: Leaf ascorbic acid level- Is it important for ozone tolerance in rice? Plant Physiol. Biochem 2012, 59:63–70.
      59. Gao Q, Zhang L: Ultraviolet-B-induced oxidative stress and antioxidant defense system responses in ascorbate-deficient vtc1 mutants of Arabidopsis thaliana . J Plant Physiol 2008, 165:138–148. 10.1016/j.jplph.2007.04.002PubMed
      60. Gatzek S, Wheeler GL, Smirnoff N: Antisense suppression of L-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated L–galactose synthesis. Plant J 2002, 30:541–553. 10.1046/j.1365-313X.2002.01315.xPubMed
      61. Gest N, Gautier H, Stevens R: Ascorbate as seen through plant evolution: the rise of a successful molecule? J Exp Bot 2013, 64:33–53. 10.1093/jxb/ers297PubMed
      62. Godde D, Buchhold J: Effect of long-term fumigation with ozone on the turnover of the D1 reaction centre polypeptide of photosystem II in spruce ( Picea abies ). Physiol Plant 1992, 86:568–574. 10.1111/j.1399-3054.1992.tb02171.x
      63. Goo YM, Hyun JC, Kim TW, Lee CH, Ahn MJ, Bae SC, Cho KJ, Chun JA, Chung CH, Lee SW: Expressional characterization of dehydroascorbate reductase cDNA in transgenic potato plants. J Plant Biol 2008, 51:35–41. 10.1007/BF03030738
      64. Green MA, Fry SC: Vitamin C degradation in plant cells via enzymatic hydrolysis of 4-O-oxalyl-L-threonate. Nature 2005, 433:83–87. 10.1038/nature03172PubMed
      65. Guan Q, Takano T, Liu S: Genetic transformation and analysis of rice OsAPX2 gene in Medicago sativa . PLoS One 2012, 7:e41233. 10.1371/journal.pone.0041233PubMed CentralPubMed
      66. Guiss´e B, Srivastava A, Strasser RJ: The polyphasicrise of the chlorophyll a fluorescence (O-K-J-I-P) in heat stressed leaves. Arch Sci Gen`eve 1995, 48:147–160.
      67. Gururani MA, Upadhyaya CP, Strasser RJ, Woong YJ, Park SW: Physiological and biochemical responses of transgenic potato plants with altered expression of PSII manganese stabilizing protein. Plant Physiol Biochem 2012, 58:182–194.PubMed
      68. Hancock RD, McRae D, Haupt S, Viola R: Synthesis of L-ascorbic acid in the phloem. BMC Plant Biol 2003, 3:7. 10.1186/1471-2229-3-7PubMed CentralPubMed
      69. Hancock RD, Walker PG, Pont SDA, Marquis N, Vivera S, Gordon SL, Brennan RM, Viola R: L-ascorbic acid accumulation in fruit of Ribes nigrum occurs by in situ biosynthesis via the l-galactose pathway. Funct Plant Biol 2007, 34:1080–1091. 10.1071/FP07221
      70. Haroldsen VM, Chi-Ham CL, Kulkarni S, Lorence A, Bennett AB: Constitutively expressed DHAR and MDHAR influence fruit, but not foliar ascorbate levels in tomato. Plant Physiol Biochem 2011, 49:1244–1249. 10.1016/j.plaphy.2011.08.003PubMed CentralPubMed
      71. Helena M, de Carvalho C: Drought stress and reactive oxygen species. Production, scavenging and signaling. Plant Signal. Behav 2008, 3:156–165.
      72. Hemavathi CP, Upadhyaya KE, Young N, Akula HS, Kim JJ, Heung MH, Oh AC, Reddy SC, Chun DHK, Park SW: Over–expression of strawberry D–galacturonic acid reductase in potato leads to accumulation of vitamin C with enhanced abiotic stress tolerance. Plant Sci 2009, 177:659–667. 10.1016/j.plantsci.2009.08.004
      73. Hemavathi CP, Upadhyaya KE, Young N, Akula KE, Young SC, Chun DHK, Park SW: Enhanced ascorbic acid accumulation in transgenic potato confers tolerance to various abiotic stresses. Biotechnol Lett 2010, 32:321–330. 10.1007/s10529-009-0140-0PubMed
      74. Hemavathi CP, Upadhyaya N, Akula HS, Kim JH, Jeon MH, Oh SC, Chun DHK, Park SW: Biochemical analysis of enhanced tolerance in transgenic potato plants overexpressing d–galacturonicacidreductase gene in response to various abiotic stresses. Mol Breed 2011, 28:105–115. 10.1007/s11032-010-9465-6
      75. Hong CY, Hsu YT, Tsai YC, Kao CH: Expression of ascorbate peroxidase 8 in roots of rice ( Oryza sativa L.) seedlings in response to NaCl. J Exp Bot 2007, 58:3273–3283. 10.1093/jxb/erm174PubMed
      76. Horemans N, Asard H, Van Gestelen P, Caubergs RJ: Facilitated diffusion drives transport of oxidised ascorbate molecules into purified plasma membrane vesicles of Phaseolus vulgaris. Physiol Plant 1998, 104:783–789. 10.1034/j.1399-3054.1998.1040439.x
      77. Horemans N, Foyer CH, Asard H: Transport and action of ascorbate at the plasma membrane. Trends Plant Sci 2000, 5:263–267. 10.1016/S1360-1385(00)01649-6PubMed
      78. Horemans N, Foyer CH, Potters G, Asard H: Ascorbate function and associated transport systems in plants. Plant Physiol Biochem 2000, 38:531–540. 10.1016/S0981-9428(00)00782-8
      79. Hu WH, Song XS, Shi K, Xia XJ, Zhou YH, Yu JQ: Changes in electron transport, superoxide dismutase and ascorbate peroxidase isoenzymes in chloroplasts and mitochondria of cucumber leaves as influenced by chilling. Photosynthetica 2008, 46:581–588. 10.1007/s11099-008-0098-5
      80. Huang C, He W, Guo J, Chang X, Su P, Zhang L: Increased sensitivity to salt stress in an ascorbate–deficient Arabidopsis mutant. J Exp Bot 2005, 56:3041–3049. 10.1093/jxb/eri301PubMed
      81. Huang GJ, Chen HJ, Chang YS, Lu TL, Lin YH: Sweet potato storage root thioredoxin h2 with both dehydroascorbate reductase and monodehydroascorbate reductase activities. Bot Stud 2008, 49:1–7.
      82. Iba K: Acclimative response to temperature stress in higher plants: approaches of gene engineering for temperature tolerance. Annu Rev Plant Biol 2002, 53:225–245. 10.1146/annurev.arplant.53.100201.160729PubMed
      83. Imai T, Karita S, Shiratori G, Hattori M, Nunome T, Oba K, Hirai M: L-galactono-γ-lactone dehydrogenase from sweet potato: purification and cDNA sequence analysis. Plant Cell Physiol 1998, 39:1350–1358. 10.1093/oxfordjournals.pcp.a029341PubMed
      84. Inzé D, Van Montagu M: Oxidative stress in plants. Curr Opin Biotechnol 1995, 6:153–158. 10.1016/0958-1669(95)80024-7
      85. Ishikawa T, Sakai K, Takeda T, Shigeoka S: Cloning and expression of cDNA encoding a new type of ascorbate peroxidase from spinach. FEBS Lett 1995, 367:28–32. 10.1016/0014-5793(95)00539-LPubMed
      86. Ishikawa T, Sakai K, Yoshimura K, Takeda T, Shigeoka S: cDNAs encoding spinach stromal and thylakoid bound ascorbate peroxidase, differing in the presence or absence of their 3’-coding regions. FEBS Lett 1996, 384:289–293. 10.1016/0014-5793(96)00332-8PubMed
      87. Ishikawa T, Yoshimura K, Sakai K, Tamoi M, Takeda T, Shigeoka S: Molecular characterization and physiological role of a glyoxysome-bound ascorbate peroxidase from spinach. Plant Cell Physiol 1998, 39:23–34. 10.1093/oxfordjournals.pcp.a029285PubMed
      88. Ishikawa T, Morimoto Y, Madhusudhan R, Sawa Y, Shibata H, Yabuta Y, Nishizawa A, Shigeoka S: Acclimation to diverse environmental stresses caused by a suppression of cytosolic ascorbate peroxidase in tobacco BY-2 cells. Plant Cell Physiol 2005, 46:1264–1271. 10.1093/pcp/pci135PubMed
      89. Ishikawa T, Dowdle J, Smirnoff N: Progress in manipulating ascorbic acid biosynthesis and accumulation in plants. Physiol Plant 2006, 126:343–355. 10.1111/j.1399-3054.2006.00640.x
      90. Jain AK, Nessler CL: Metabolic engineering of an alternative pathway for ascorbic acid biosynthesis in plants. Mol Breed 2000, 6:73–78. 10.1023/A:1009680818138
      91. Jiménez A, Hernández JA, del Río LA, Sevilla F: Evidence for the presence of the ascorbate–glutathione cycle in mitochondria and peroxisomes of pea leaves. Plant Physiol 1997, 114:275–284.PubMed CentralPubMed
      92. Kangasjärvi S, Lepistö A, Hännikäinen K, Piippo M, Luomala EM, Aro EM, Rintamäki E: Diverse roles for chloroplast stromal and thylakoidbound ascorbate peroxidases in plant stress responses. Biochem J 2008, 412:275–285. 10.1042/BJ20080030PubMed
      93. Kavitha K, Georg S, Venkataraman G, Parida A: A salt-inducible chloroplastic monodehydroascorbate reductase from halophyte Avicennia marina confers salt stress tolerance on transgenic plants. Biochimie 2010, 92:1321–1329. 10.1016/j.biochi.2010.06.009PubMed
      94. Keller R, Renz FSA, Kossmann J: Antisense inhibition of the GDP-mannose pyrophosphorylase reduces the ascorbate content in transgenic plants leading to developmental changes during senescence. Plant J 1999, 19:131–141. 10.1046/j.1365-313X.1999.00507.xPubMed
      95. Kim DJ, Lee JS: Current theories for mechanism of stomatal opening: Influence of blue light, mesophyll cells, and sucrose. J Plant Biol 2007, 50:523–526. 10.1007/BF03030704
      96. Koussevitzky S, Suzuki N, Huntington S, Armijo L, Sha W, Cortes D, Shulaev V, Mittler R: Ascorbate peroxidase 1 plays a key role in the response of Arabidopsis thaliana to stress combination. J Biol Chem 2008, 283:34197–34203. 10.1074/jbc.M806337200PubMed CentralPubMed
      97. Kubo A, Saji H, Tanaka K, Kondo N: Expression of Arabidopsis cytosolic ascorbate peroxidase gene in response to ozone or sulfur dioxide. Plant Mol Biol 1995, 29:479–489. 10.1007/BF00020979PubMed
      98. Kwon SY, Choi SM, Ahn YO, Lee HS, Lee HB, Park YM, Kwak SS: Enhanced stress–tolerance of transgenic plants expressing a human dehydroascorbate reductase gene. J Plant Physiol 2003, 160:347–353. 10.1078/0176-1617-00926PubMed
      99. Laing WA, Bulley S, Wright M, Cooney J, Jensen D, Barraclough D, MacRae E: A highly specific l-galactose-1-phosphate phosphatase on the path to ascorbate biosynthesis. Proc Natl Acad Sci U S A 2004, 101:16976–16981. 10.1073/pnas.0407453101PubMed CentralPubMed
      100. Laing WA, Frearson N, Bulley S, MacRae E: Kiwifruit l–galactose dehydrogenase: molecular, biochemical and physiological aspects of the enzyme. Funct Plant Biol 2004, 31:1015–1025. 10.1071/FP04090
      101. Laing WA, Wright MA, Cooney J, Bulley SM: The missing step of the l-galactose pathway of ascorbate biosynthesis in plants, an l-galactose guanyltransferase, increases leaf ascorbate content. Proc Natl Acad Sci U S A 2007, 104:9534–9539. 10.1073/pnas.0701625104PubMed CentralPubMed
      102. Larkindale J, Knight MR: Protection against heat stress–induced oxidative damage in Arabidopsis involves calcium, abscisic acid, ethylene, and salicylic acid. Plant Physiol 2002, 128:682–695. 10.1104/pp.010320PubMed CentralPubMed
      103. Larkindale J, Hall JD, Knightand MR, Vierling E: Heat stress phenotypes of Arabidopsis mutants implicate multiple signaling pathways in the acquisition of thermotolerance. Plant Physiol 2005, 138:882–897. 10.1104/pp.105.062257PubMed CentralPubMed
      104. Lazzarotto F, Teixeira FK, Rosa SB, Dunand C, Fernandes CL, Fontenele Ade V, Silveira JA, Verli H, Margis R, Margis-Pinheiro M: Ascorbate peroxidase-related (APx-R) is a new heme-containing protein functionally associated with ascorbate peroxidase but evolutionarily divergent. New Phytol 2011, 191:234–250. 10.1111/j.1469-8137.2011.03659.xPubMed
      105. Le Martret B, Poage M, Shiel K, Nugent GD, Dix PJ: Tobacco chloroplast transformants expressing genes encoding dehydroascorbate reductase, glutathione reductase, and glutathione-S-transferase, exhibit altered anti-oxidant metabolism and improved abiotic stress tolerance. Plant Biotechnol J 2011, 9:661–673. 10.1111/j.1467-7652.2011.00611.xPubMed
      106. Lee EH: Plant resistance mechanisms to air pollutants: rhythms in ascorbic acid production during growth under ozone stress. Chronobiol Int 1991, 8:93–102. 10.3109/07420529109059161PubMed
      107. Lee YP, Kim SH, Bang JW, Lee HS, Kwak SS, Kwon SY: Enhanced tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplasts. Plant Cell Rep 2007, 26:591–598. 10.1007/s00299-006-0253-zPubMed
      108. Leterrier M, Corpas FJ, Barroso JB, Sandalio L, del Río LA: Peroxisomal monodehydroascorbate reductase genomic clone characterization and functional analysis under environmental stress conditions. Plant Physiol 2005, 138:2111–2123. 10.1104/pp.105.066225PubMed CentralPubMed
      109. Li Q, Schultes NP: Arabidopsis thaliana locus At5g62890, a nucleobase-ascorbate transporter family member, is preferentially expressed in carpel transmitting tract and tapetal cells. Plant Sci 2002, 163:233–240. 10.1016/S0168-9452(02)00086-9
      110. Li F, Wu QY, Sun YL, Wang LY, Yang XH, Meng QW: Overexpression of chloroplastic monodehydroascorbate reductase enhanced tolerance to temperature and methyl viologen-mediated oxidative stresses. Physiol Plant 2010, 139:421–434.PubMed
      111. Lim MY, Pulla RK, Park JM, Harn CH, Jeong BR: Over-expression of l-gulono-γ-lactone oxidase (GLOase) gene leads to ascorbate accumulation with enhanced abiotic stress tolerance in tomato. In Vitro Cell Dev Biol Plant 2012, 48:453–461. 10.1007/s11627-012-9461-0
      112. Lin KH, Lo HF, Lin CH, Chan MT: Cloning and expression analysis of ascorbate peroxidase gene from eggplant under flooding stress. Bot Stud 2007, 48:25–34.
      113. Linster CL, Clarke SG: L-ascorbate biosynthesis in higher plants: the role of VTC2. Trends Plant Sci 2008, 13:567–573. 10.1016/j.tplants.2008.08.005PubMed CentralPubMed
      114. Liso R, Calabrese G, Bitonti MB, Arrigoni O: Relationship between ascorbic acid and cell division. Exp Cell Res 1984, 150:314–320. 10.1016/0014-4827(84)90574-3PubMed
      115. Liu YH, Yu L, Wang RZ: Level of ascorbic acid in transgenic rice for l-galactono-1, 4-lactone dehydrogenase overexpressing or suppressed is associated with plant growth and seed set. Acta Physiol Plant 2011, 33:1353–1363. 10.1007/s11738-010-0669-5
      116. Liu W, An HM, Yang M: Overexpression of Rosa roxburghii l-galactono-1,4-lactone dehydrogenase in tobacco plant enhances ascorbate accumulation and abiotic stress tolerance. Acta Physiol Plant 2013. doi:10.1007/s11738–012–1204–7
      117. Liu YH, Yu L, Tong J, Ding J, Wang R, Lu Y, Xiao L: Tiller number is altered in the ascorbic acid-deficient rice suppressed for l-galactono-1,4-lactone dehydrogenase. J Plant Physiol 2013, 170:389–396. 10.1016/j.jplph.2012.11.019PubMed
      118. Long SP, Naidu SL: Effects of oxidants at the biochemical, cell and physiological levels, with particular reference to ozone. In Air Pollution and Plant Life. Edited by: Bell JNB, Treshow M. Chichester: John Wiley & Sons Ltd.; 2002:69–88.
      119. Lorence A, Chevone BI, Mendes P, Nessler CL: Myoinositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Plant Physiol 2004, 134:1200–1205. 10.1104/pp.103.033936PubMed CentralPubMed
      120. Lu Z, Liu D, Liu S: Two rice cytosolic ascorbate peroxidases differentially improve salt tolerance in transgenic Arabidopsis . Plant Cell Rep 2007, 26:1909–1917. 10.1007/s00299-007-0395-7PubMed
      121. Luwe MWF, Takahama U, Heber U: Role of ascorbate in detoxifiying ozone in the apoplast of spinach ( Spinacia oleracea L.) leaves. Plant Physiol 1993, 101:969–976.PubMed CentralPubMed
      122. Maddison J, Lyons T, Plöchl M, Barnes J: Hydroponically cultivated radish fed L-galactono-1,4-lactone exhibit increased tolerance to ozone. Planta 2002, 214:383–391. 10.1007/s004250100625PubMed
      123. Mano J, Hideg E, Asada K: Ascorbate in thylakoid lumen functions as an alternative electron donor to photosystem II and photosystem I. Arch Biochem Biophys 2004, 429:71–80.PubMed
      124. Maruta T, Yonemitsu M, Yabuta Y, Tamoi M, Ishikawa T, Shigeoka S: Arabidopsis phosphomannose isomerase 1, but not phosphomannose isomerase 2, is essential for ascorbic acid biosynthesis. J Biol Chem 2008, 283:28842–28851. 10.1074/jbc.M805538200PubMed CentralPubMed
      125. Maurino VG, Grube E, Zielinski J, Schild A, Fischer K, Flügge UI: Identification and expression analysis of twelve members of the nucleobase-ascorbate transporter (NAT) gene family in Arabidopsis thaliana . Plant Cell Physiol 2006, 47:1381–1393. 10.1093/pcp/pcl011PubMed
      126. Mittler R: Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 2002, 7:405–410. 10.1016/S1360-1385(02)02312-9PubMed
      127. Mittler R, Zilinskas BA: Molecular cloning and characterization of gene encoding pea cytosolic ascorbate peroxidase. J Biol Chem 1992, 267:21802–21807.PubMed
      128. Mittler R, Vanderauwera S, Gollery M, Van Breusegem F: Reactive oxygen gene network of plants. Trends Plant Sci 2004, 9:490–498. 10.1016/j.tplants.2004.08.009PubMed
      129. Mittova V, Tal M, Volokita M, Guy M: Upregulation of the leaf mitochondrial and peroxisomal antioxidative systems in response to salt-induced oxidative stress in the wild salt-tolerant tomato species Lycopersicon pennellii . Plant Cell Environ 2003, 6:845–856.
      130. Miyake C, Asada K: Thylakoid–bound ascorbate peroxidase in spinach chloroplasts and photoreduction of its primary oxidation product monodehydroascorbate radicals in thylakoids. Plant Cell Physiol 1992, 33:541–553.
      131. Miyake C, Asada K: Ferredoxin-dependent photoreduction of the monodehydroascorbate radical in spinach thylakoids. Plant Cell Physiol 1994, 35:539–549.
      132. Moller IM: Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive oxygen species. Annu Rev Plant Physiol Plant Mol Biol 2001, 52:561–591. 10.1146/annurev.arplant.52.1.561PubMed
      133. Müller-Moulé P: An expression analysis of the ascorbate biosynthesis enzyme VTC2. Plant Mol Biol 2008, 68:31–41. 10.1007/s11103-008-9350-4PubMed
      134. Müller-Moulé P, Conklin PL, Niyogi KK: Ascorbate deficiency can limit violaxanthin de-epoxidase activity in vivo. Plant Physiol 2002, 128:970–977. 10.1104/pp.010924PubMed CentralPubMed
      135. Müller-Moulé P, Havaux M, Niyogi KK: Zeaxanthin deficiency enhances the high light sensitivity of an ascorbate–deficient mutant of Arabidopsis . Plant Physiol 2003, 133:748–760. 10.1104/pp.103.026252PubMed CentralPubMed
      136. Müller-Moulé P, Golan T, Niyogi KK: Ascorbate-deficient mutants of Arabidopsis grow in high light despite chronic photooxidative stress. Plant Physiol 2004, 134:1163–1172. 10.1104/pp.103.032375PubMed CentralPubMed
      137. Mullineaux P, Karpinski S: Signal transduction in response to excess light: getting out of the chloroplast. Curr Opin Plant Biol 2002, 5:43–48. 10.1016/S1369-5266(01)00226-6PubMed
      138. Munné-Bosch S, Alegre L: Interplay between ascorbic acid and lipophilic antioxidant defences in chloroplasts of water–stressed Arabidopsis plants. FEBS Lett 2002, 524:145–148. 10.1016/S0014-5793(02)03041-7PubMed
      139. Najami N, Janda T, Barriah W, Kayam G, Tal M, Guy M, Volokita M: Ascorbate peroxidase gene family in tomato: its identiWcation and characterization. Mol Genet Genomics 2008, 279:171–182. 10.1007/s00438-007-0305-2PubMed
      140. Naqvi S, Zhu C, Farre G, Ramessar K, Bassie L, Breitenbach J, Perez Conesa D, Ros G, Sandmann G, Capell T, Christou P: Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways. Proc Natl Acad Sci U S A 2009, 106:7762–7767. 10.1073/pnas.0901412106PubMed CentralPubMed
      141. Narendra S, Venkataramani S, Shen GX, Wang J, Pasapula V, Lin Y, Kornyeyev D, Holaday AS, Zhang H: The Arabidopsis ascorbate peroxidase 3 is a peroxisomal membrane-bound antioxidant enzyme and is dispensable for Arabidopsis growth and development. J Exp Bot 2006, 57:3033–3042. 10.1093/jxb/erl060PubMed
      142. Neubauer C, Yamamoto HY: Mehler–peroxidase reaction mediates zeaxanthin formation and zeaxanthin–related fluorescence quenching in intact chloroplasts. Plant Physiol 1992, 99:1354–1361. 10.1104/pp.99.4.1354PubMed CentralPubMed
      143. Neubauer C, Yamamoto HY: The role of ascorbate in the related ascorbate peroxidase, violaxanthin de-epoxidase and non-photochemical fluorescence–quenching activities. In Photosynthetic Responses to the Environment. Edited by: Yamamoto HY, Smith CM. Maryland: American Society of Plant Physiologists Rockville; 1993:166–171.
      144. Nguyen HT, Leipner J, Stamp P, Guerra-Peraza O: Low temperature stress in maize ( Zea mays L.) induces genes involved in photosynthesis and signal transduction as studied by suppression subtractive hybridization. Plant Physiol. Biochem 2009, 47:116–122.
      145. Noctor G, Foyer CH: Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 1998, 49:249–279. 10.1146/annurev.arplant.49.1.249PubMed
      146. O’Kane D, Gill V, Boyd P, Burdon R: Chilling, oxidative stress and antioxidant responses in Arabidopsis thaliana callus. Planta 1996, 198:371–377. 10.1007/BF00620053PubMed
      147. Orvar BL, Ellis BE: Transgenic tobacco plants expressing antisense RNA for cytosolic ascorbate peroxidase show increased susceptibility to ozone injury. Plant J 1997, 11:1297–1305. 10.1046/j.1365-313X.1997.11061297.x
      148. Pallanca JE, Smirnoff N: The control of ascorbic acid synthesis and turnover in pea seedlings. J Exp Bot 2000, 51:699–674.
      149. Panchuk II, Volkov RA, Schöz F: Heat stress- and heat shock transcription factor-dependent expression and activity of ascorbate peroxidase in Arabidopsis . Plant Physiol 2002, 129:838–853. 10.1104/pp.001362PubMed CentralPubMed
      150. Pang CH, Li K, Wang B: Overexpression of SsCHLAPXs confers protection against oxidative stress induced by high light in transgenic Arabidopsis thaliana . Physiol Plant 2011, 143:355–366. 10.1111/j.1399-3054.2011.01515.xPubMed
      151. Park SY, Ryu SH, Jang LC, Kwon SY, Kim JG, Kwak SS: Molecular cloning of a cytosolic ascorbate peroxidase cDNA from cell cultures of sweet potato and its expression in response to stress. Mol Gen Genomics 2004, 271:339–346. 10.1007/s00438-004-0986-8
      152. Parsons HT, Fry SC: Oxidation of dehydroascorbic acid and 2,3-diketogulonate under plant apoplastic conditions. Phytochemistry 2012, 75:41–49.PubMed
      153. Parsons HT, Yasmin T, Fry SC: Alternative pathways of dehydroascorbic acid degradation in vitro and in plant cell cultures: novel insights into vitamin C catabolism. Biochem J 2011, 440:375–383. 10.1042/BJ20110939PubMed
      154. Pell EJ, Schlagnhaufer CD, Arteca RN: Ozone-induced oxidative stress: mechanisms of action and reaction. Physiol Plant 1997, 100:264–273. 10.1111/j.1399-3054.1997.tb04782.x
      155. Pignocchi C, Foyer CH: Apoplastic ascorbate metabolism and its role in the regulation of cell signaling. Curr Opin Plant Biol 2003, 6:379–389. 10.1016/S1369-5266(03)00069-4PubMed
      156. Pignocchi C, Fletcher JM, Wilkinson JE, Barnes JD, Foyer CH: The function of ascorbate oxidase in tobacco. Plant Physiol 2003, 132:1631–1641. 10.1104/pp.103.022798PubMed CentralPubMed
      157. Pignocchi C, Kiddle G, Hernández I, Foster SJ, Asensi A, Taybi T, Barnes J, Foyer CH: Ascorbate oxidase–dependent changes in the redox state of the apoplast modulate gene transcript accumulation leading to modified hormone signaling and orchestration of defense processes in tobacco. Plant Physiol 2006, 141:423–435. 10.1104/pp.106.078469PubMed CentralPubMed
      158. Plöchl M, Lyons T, Ollerenshaw J, Barnes J: Simulating ozone detoxification in the leaf apoplast through the direct reaction with ascorbate. Planta 2000, 210:454–467. 10.1007/PL00008153PubMed
      159. Pnueli L, Liang H, Rozenberg M, Mittler R: Growth suppression, altered stomatal responses, and augmented induction of heat shock proteins in cytosolic ascorbate peroxidase (APX1)-deficient Arabidopsis plants. Plant J 2003, 34:187–203. 10.1046/j.1365-313X.2003.01715.xPubMed
      160. Purohit S, Kumar GP, Laloraya M, Laloraya MM: Involvement of superoxide radical in signal transduction regulating stomatal movements. Biochem Biophys Res Commun 1994, 205:30–37. 10.1006/bbrc.1994.2625PubMed
      161. Qian W, Yu C, Qin H, Liu X, Zhang A, Johansen IE, Wang D: Molecular and functional analysis of phosphomannomutase (PMM) from higher plants and genetic evidence for the involvement of PMM in ascorbic acid biosynthesis in Arabidopsis and Nicotiana benthamiana . Plant J 2007, 49:399–413. 10.1111/j.1365-313X.2006.02967.xPubMed
      162. Qin A, Shi Q, Yu X: Ascorbic acid contents in transgenic potato plants overexpressing two dehydroascorbate reductase genes. Mol Biol Rep 2011, 38:1557–1566. 10.1007/s11033-010-0264-2PubMed
      163. Radzio JA, Lorence A, Chevone BI, Nessler CL: L-Gulono-1,4-lactone oxidase expression rescues vitamin C-deficient Arabidopsis (vtc) mutants. Plant Mol Biol 2003, 53:837–844.PubMed
      164. Rosa SB, Caverzan A, Teixeira FK, Lazzarotto F, Silveira JA, Ferreira-Silva SL, Abreu-Neto J, Margis R, Margis-Pinheiro M: Cytosolic APX knockdown indicates an ambiguous redox response in rice. Phytochemistry 2010, 71:548–558. 10.1016/j.phytochem.2010.01.003PubMed
      165. Rossel JB, Walter PB, Hendrickson L, Chow WS, Poole A, Mullineaux PM, Pogson BJ: A mutation affecting ascorbate peroxidase 2 gene expression reveals a link between responses to high light and drought tolerance. Plant Cell Environ 2006, 29:269–281. 10.1111/j.1365-3040.2005.01419.xPubMed
      166. Salvucci ME, Crafts-Brandner SJ: Relationship between the heat tolerance of photosynthesis and the thermal stability of Rubisco activase in plants from contrasting thermal environments. Plant Physiol 2004, 134:1460–1470. 10.1104/pp.103.038323PubMed CentralPubMed
      167. Sanmartin M, Drogoudi PA, Lyons T, Pateraki I, Barnes J, Kanellis AK: Over-expression of ascorbate oxidase in the apoplast of transgenic tobacco results in altered ascorbate and glutathione redox states and increased sensitivity to ozone. Planta 2003, 216:918–928.PubMed
      168. Sano S, Tao S, Endo Y, Inaba T, Hossain MA, Miyake C, Matsuo M, Aoki H, Asada K, Saito K: Purification and cDNA cloning of chloroplastic monodehydroascorbate reductase from spinach. Biosci Biotechnol Biochem 2005, 69:762–772. 10.1271/bbb.69.762PubMed
      169. Sarowar S, Kim EN, Kim YJ, Ok SH, Kim KD, Hwang BK, Shin JS: Overexpression of a pepper ascorbate peroxidase-like 1 gene in tobacco plants enhances tolerance to oxidative stress and pathogens. Plant Sci 2005, 169:55–63. 10.1016/j.plantsci.2005.02.025
      170. Sato Y, Masuta Y, Saito K, Murayama S, Ozawa K: Enhanced chilling tolerance at the booting stage in rice by transgenic overexpression of the ascorbate peroxidase gene. OsAPXa Plant Cell Rep 2011, 30:399–406. 10.1007/s00299-010-0985-7
      171. Shimaoka T, Miyake C, Yokota A: Mechanism of the reaction catalyzed by dehydroascorbate reductase from spinach chloroplasts. Eur J Biochem 2003, 270:921–928. 10.1046/j.1432-1033.2003.03452.xPubMed
      172. Siendones E, Gonzalez-Reyes JA, Santos-Ocana C, Navas P, Cordoba F: Biosynthesis of ascorbic acid in kidney bean. L-galactono-gamma-lactone dehydrogenase is an intrinsic protein located at the mitochondrial inner membrane. Plant Physiol 1999, 120:907–912. 10.1104/pp.120.3.907PubMed CentralPubMed
      173. Simpson GL, Ortwerth BJ: The non-oxidative degradation of ascorbic acid at physiological conditions. Biochim Biophys Acta 2000, 1501:12–24. 10.1016/S0925-4439(00)00009-0PubMed
      174. Smirnoff N: The function and metabolism of Ascorbic acid in plants. Ann Bot 1996, 78:661–669. 10.1006/anbo.1996.0175
      175. Smirnoff N: Ascorbate biosynthesis and function in photoprotection. Philos Trans R Soc Lond B Biol Sci 2000, 355:1455–1464. 10.1098/rstb.2000.0706PubMed CentralPubMed
      176. Smirnoff N: L–Ascorbic acid biosynthesis. Vitam Horm 2001, 61:241–266.PubMed
      177. Smirnoff N, Running JA, Gatzek S: Ascorbate Biosynthesis: A Diversity of Pathways. In Vitamin C. Edited by: Asard H, May JM, Smirnoff N. Functions and Biochemistry in Animals and Plants: Bios Scientific Publishers, Oxford, UK; 2004:7–29.
      178. Stevens R, Page D, Gouble B, Garchery C, Zamir D, Causse M: Tomato fruit ascorbic acid content is linked with monodehydroascorbate reductase activity and tolerance to chilling stress. Plant Cell Environ 2008, 31:1086–1096. 10.1111/j.1365-3040.2008.01824.xPubMed
      179. Strasser BJ: Donor side capacity of photosystem II probed by chlorophyll a fluorescence transients. Photosynth Res 1997, 52:147–155. 10.1023/A:1005896029778
      180. Sun WH, Duan M, Shu DF, Yang S, Meng QW: Over–expression of StAPX in tobacco improves seed germination and increases early seedling tolerance to salinity and osmotic stresses. Plant Cell Rep 2010, 29:917–926. 10.1007/s00299-010-0878-9PubMed
      181. Sun WH, Duan M, Li F, Shu DF, Yang S, Meng QW: Overexpression of tomato tAPX gene in tobacco improves tolerance to high or low temperature stress. Biol Plant 2010, 54:614–620. 10.1007/s10535-010-0111-2
      182. Suzuki N, Mittler R: Reactive oxygen species and temperature stresses: a delicate balance between signaling and destruction. Physiol Plant 2006, 126:45–51. 10.1111/j.0031-9317.2005.00582.x
      183. Szarka A, Horemans N, Banhegyi G, Asard H: Facilitated glucose and dehydroascorbate transport in plant mitochondria. Arch Biochem Biophys 2004, 428:73–80.PubMed
      184. Szarka A, Horemans N, Kovacs Z, Grof P, Mayer M, Banhegyi G: Dehydroascorbate reduction in plant mitochondria is coupled to the respiratory electron transfer chain. Physiol Plant 2007, 129:225–232. 10.1111/j.1399-3054.2006.00810.x
      185. Tabata K, Oba K, Suzuki K, Esaka M: Generation and properties of ascorbic acid-deficient transgenic tobacco cells expressing antisense RNA for l-galactono 1,4-lactone dehydrogenase. Plant J 2001, 27:139–148. 10.1046/j.1365-313x.2001.01074.xPubMed
      186. Talla S, Riazunnisa K, Padmavathi L, Sunil B, Rajsheel P, Raghavendra AS: Ascorbic acid is a key participant during the interactions between chloroplasts and mitochondria to optimize photosynthesis and protect against photoinhibition. J Biosci 2011, 36:163–173. 10.1007/s12038-011-9000-xPubMed
      187. Tang L, Kwon SY, Kim SH, Kim JS, Choi JS, Cho KY, Sung CK, Kwak SS, Lee HS: Enhanced tolerance of transgenic potato plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against oxidative stress and high temperature. Plant Cell Rep 2006, 25:1380–1386. 10.1007/s00299-006-0199-1PubMed
      188. Tedone L, Hancock RD, Alberino S, Haupt S, Viola R: Long-distance transport of l-ascorbic acid in potato. BMC Plant Biol 2004, 17:1–16.
      189. Teixeira FK, Menezes-Benavente L, Margis R, Margis-Pinheiro MJ: Analysis of the molecular evolutionary history of the ascorbate peroxidase gene family: inferences from the rice genome. J Mol Evol 2004, 59:761–770. 10.1007/s00239-004-2666-zPubMed
      190. Teixeira FK, Menezes-Benavente L, Galvao VC, Margis R, Margis- Pinheiro M: Rice ascorbate peroxidase gene family encodes functionally diverse isoforms localized in diVerent subcellular compartments. Planta 2006, 224:300–314. 10.1007/s00425-005-0214-8PubMed
      191. Tokunaga T, Miyahara K, Tabata K, Esaka M: Generation and properties of ascorbic acid–overproducing transgenic tobacco cells expressing sense RNA for L–galactono– 1,4–lactone dehydrogenase. Planta 2005, 220:854–863. 10.1007/s00425-004-1406-3PubMed
      192. Torabinejad J, Donahue JL, Gunesekera BN, Allen-Daniels MJ, Gillaspy GE: VTC4 is a bifunctional enzyme that affects myoinositol and ascorbate biosynthesis in plants. Plant Physiol 2009, 150:951–961. 10.1104/pp.108.135129PubMed CentralPubMed
      193. Torsethaugen C, Pitcher LH, Zilinskas BA, Pell EJ: Overproduction of Ascorbate peroxidase in the tobacco chloroplast does not provide protection against ozone. Plant Physiol 1997, 114:529–537.PubMed CentralPubMed
      194. Torsethaugen G, Pell EJ, Assmann SM: Ozone inhibits guard cell K1 channels implicated in stomatal opening. Proc Natl Acad Sci U S A 1999, 96:13577–13582. 10.1073/pnas.96.23.13577PubMed CentralPubMed
      195. Tóth SZ, Puthur JT, Nagy V, Garab G: Experimental evidence for ascorbate–dependent electron transport in leaves with inactive oxygen–evolving complexes. Plant Physiol 2009, 149:1568–1578. 10.1104/pp.108.132621PubMed CentralPubMed
      196. Tóth SZ, Nagy V, Puthur JT, Kovács L, Garab G: The physiological role of ascorbate as photosystem II electron donor: protection against photoinactivation in heat–stressed leaves. Plant Physiol 2011, 156:382–392. 10.1104/pp.110.171918PubMed CentralPubMed
      197. Trost P, Berczi A, Sparla F, Sponza G, Marzadori B, Asard H, Pupillo P: Purification of cytochrome b-561 from bean hypocotyls plasma membrane. Evidence for the presence of two heme centers. Biochim Biophys Acta 2000, 1468:1–5. 10.1016/S0005-2736(00)00283-2PubMed
      198. Tsai YC, Hong CY, Liu LF, Kao CH: Relative importance of Na + and Cl - in NaCl-induced antioxidant systems in roots of rice seedlings. Physiol Plant 2004, 122:86–94. 10.1111/j.1399-3054.2004.00387.x
      199. Tsai YC, Hong CY, Liu LF, Kao CH: Expression of ascorbate peroxidase and glutathione reductase in roots of rice seedlings in response to NaCl and H 2 O 2 . J Plant Physiol 2005, 162:291–299. 10.1016/j.jplph.2004.06.004PubMed
      200. Tsukaguchi H, Tokui T, Mackenzie B, Berger UV, Chen XZ, Wang YX, Brubaker RF, Hediger MA: A family of mammalian Na + -dependent L-ascorbic acid transporters. Nature 1999, 399:70–75. 10.1038/19986PubMed
      201. Turcsányi E, Lyons T, Plöchl M, Barnes J: Does ascorbate in the mesophyll cell walls form the first line of defence against ozone? Testing the concept using broad bean (Vicia faba L.). J Exp Bot 2000, 51:901–910. 10.1093/jexbot/51.346.901PubMed
      202. Upadhyaya CP, Venkatesh J, Gururani MA, Asnin L, Sharma K, Ajappala H, Park SW: Transgenic potato overproducing L–ascorbic acid resisted an increase in methylglyoxal under salinity stress via maintaining higher reduced glutathione level and glyoxalase enzyme activity. Biotechnol Lett 2011, 33:2297–2307. 10.1007/s10529-011-0684-7PubMed
      203. Ushimaru T, Nakagawa T, Fujioka Y, Daicho K, Naito M, Yamauchi Y, Nonaka H, Amako K, Yamawaki K, Murata N: Transgenic Arabidopsis plants expressing the rice dehydroascorbate reductase gene are resistant to salt stress. J Plant Physiol 2006, 163:1179–1184. 10.1016/j.jplph.2005.10.002PubMed
      204. Vacca RA, de Pinto MC, Valenti D, Passarella S, Marra E, De Gara L: Production of reactive oxygen species, alteration of cytosolic ascorbate peroxidase, and impairment of mitochondrial metabolism are early events in heat shock-induced programmed cell death in tobacco bright-yellow 2 cells. Plant Physiol 2004, 134:1100–1112. 10.1104/pp.103.035956PubMed CentralPubMed
      205. Venkatesh J, Upadhyaya CP, Yu JW, Hemavathi A, Kim DH, Strasser RJ, Park SW: Chlorophyll a fluorescence transient analysis of transgenic potato overexpressing D-galacturonic acid reductase gene for salinity stress tolerance. Hort Environ Biotechnol 2012, 53:320–328. 10.1007/s13580-012-0035-1
      206. Wang W, Vinocur B, Altman A: Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. Planta 2003, 218:1–14. 10.1007/s00425-003-1105-5PubMed
      207. Wang Y, Wisniewski M, Meilan R, Webb R, Fuchigami L: Overexpression of cytosolic ascorbate peroxidase in tomato ( Lycopersicon esculentum ) confers tolerance to chilling and salt stress. J Amer Soc Hort Sci 2005, 130:167–173.
      208. Wang Y, Wisniewski M, Meilan R, Cui M, Fuchigami L: Transgenic tomato ( Lycopersicon esculentum ) overexpressing cAPX exhibits enhanced tolerance to UV–B and heat stress. J Appl Hort 2006, 8:87–90.
      209. Wang Z, Xiao Y, Chen W, Tang K, Zhang L: Increased vitamin C content accompanied by an enhanced recycling pathway confers oxidative stress tolerance in Arabidopsis . J Integr Plant Biol 2010, 52:400–409. 10.1111/j.1744-7909.2010.00921.xPubMed
      210. Wang HS, Yu C, Zhu ZJ, Yu XC: Overexpression in tobacco of a tomato GMPase gene improves tolerance to both low and high temperature stress by enhancing antioxidation capacity. Plant Cell Rep 2011, 30:1029–1040. 10.1007/s00299-011-1009-yPubMed
      211. Watanabe K, Suzuki K, Kitamura S: Characterization of a GDP-D-mannose 3’,5’-epimerase from rice. Phytochemistry 2006, 67:338–346. 10.1016/j.phytochem.2005.12.003PubMed
      212. Wevar Oller AL, Agostini E, Milrad SR, Medina MI: In situ and de novo biosynthesis of vitamin C in wild type and transgenic tomato hairy roots: A precursor feeding study. Plant Sci 2009, 177:28–34. 10.1016/j.plantsci.2009.03.017
      213. Wheeler GL, Jones MA, Smirnoff N: The biosynthesis pathway of vitamin C in higher plants. Nature 1998, 393:365–369. 10.1038/30728PubMed
      214. Wolucka BA, Van Montagu M: GDP-mannose 3’,5’- epimerase forms GDP-L-gulose, a putative intermediate for the de novo biosynthesis of vitamin C in plants. J Biol Chem 2003, 278:47483–47490. 10.1074/jbc.M309135200PubMed
      215. Wolucka BA, Goossens A, Inzé D: Methyl jasmonate stimulates the de novo biosynthesis of vitamin C in plant cell suspensions. J Exp Bot 2005, 56:2527–2538. 10.1093/jxb/eri246PubMed
      216. Xu WF, Shi WM, Ueda A, Takabe T: Mechanisms of salt tolerance in transgenic Arabidopsis thaliana carrying a peroxisomal ascorbate peroxidase gene from barley. Pedosphere 2008, 18:486–495. 10.1016/S1002-0160(08)60039-9
      217. Yabuta Y, Motoki T, Yoshimura K, Takeda T, Ishikawa T, Shigeoka S: Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress. Plant J 2002, 32:915–925. 10.1046/j.1365-313X.2002.01476.xPubMed
      218. Yabuta Y, Mieda T, Rapolu M, Nakamura A, Motoki T, Takanori M, Yoshimura K, Ishikawa T, Shigeoka S: Light regulation of ascorbate biosynthesis is dependent on the photosynthetic electron transport chain but independent of sugars in Arabidopsis . J Exp Bot 2007, 58:2661–2671. 10.1093/jxb/erm124PubMed
      219. Yamamoto A, Bhuiyan MNH, Waditee R, Tanaka Y, Oba MEK, Jagendorf AT, Takabe T: Suppressed expression of the apoplastic ascorbate oxidase gene increases salt tolerance in tobacco and Arabidopsis plants. J Exp Bot 2005, 56:1785–1796. 10.1093/jxb/eri167PubMed
      220. Yamashita A, Nijo N, Pospísil P, Morita N, Takenaka D, Aminaka R, Yamamoto Y, Yamamoto Y: Quality control of photosystem II: reactive oxygen species are responsible for the damage to photosystem II under moderate heat stress. J Biol Chem 2008, 283:28380–28391. 10.1074/jbc.M710465200PubMed CentralPubMed
      221. Yin L, Wang S, Eltayeb AE, Uddin MI, Yamamoto Y, Tsuji W, Takeuchi Y, Tanaka K: Overexpression of dehydroascorbate reductase, but not monodehydroascorbate reductase, confers tolerance to aluminum stress in transgenic tobacco. Planta 2010, 231:609–621. 10.1007/s00425-009-1075-3PubMed
      222. Yoon HS, Lee H, Lee IA, Kim KY, Jo J: Molecular cloning of the monodehydroascorbate reductase gene from Brassica campestris and analysis of its mRNA level in response to oxidative stress. Biochim Biopys Acta 2004, 1658:181–186. 10.1016/j.bbabio.2004.05.013
      223. Yoshida S, Tamaoki M, Shikano T, Nakajima N, Ogawa D, Ioki M, Aono M, Kubo A, Kamada H, Inoue Y, Saji H: Cytosolic dehydroascorbate reductase is important for ozone tolerance in Arabidopsis thaliana . Plant Cell Physiol 2006, 47:304–308.PubMed
      224. Yoshimura K, Miyao K, Gaber A, Takeda T, Kanaboshi H, Miyasaka H, Shigeoka S: Enhancement of stress tolerance in transgenic tobacco plants overexpressing Chlamydomonas glutathione peroxidase in chloroplasts or cytosol. Plant J 2004, 37:21–33. 10.1046/j.1365-313X.2003.01930.xPubMed
      225. Zhang X, Zhang L, Dong F, Gao J, Galbraith DW, Song CP: Hydrogen peroxide is involved in abscisic acid–induced stomatal closure in Vicia faba . Plant Physiol 2001, 126:1438–1448. 10.1104/pp.126.4.1438PubMed CentralPubMed
      226. Zhang L, Wang Z, Xia Y, Kai G, Chen W, Tang K: Metabolic engineering of plant L–ascorbic acid biosynthesis: recent trends and applications. Crit Rev Biotechnol 2007, 27:173–182. 10.1080/07388550701503626PubMed
      227. Zhang W, Lorence A, Gruszewski HA, Chevone BI, Nessler CL: AMR1 , an Arabidopsis gene that coordinately and negatively regulates the mannose/l-galactose ascorbic acid biosynthetic pathway. Plant Physiol 2009, 150:942–950. 10.1104/pp.109.138453PubMed CentralPubMed
      228. Zhang C, Liu J, Zhang Y, Cai X, Gong P, Zhang J, Wang T, Li H, Ye Z: Overexpression of SlGMEs leads to ascorbate accumulation with enhanced oxidative stress, cold, and salt tolerance in tomato. Plant Cell Rep 2011, 30:389–398. 10.1007/s00299-010-0939-0PubMed
      229. Zhang Z, Zhang Q, Wu J, Zheng X, Zheng S, Sun X, Qiu Q, Lu T: Gene knockout study reveals that cytosolic ascorbate peroxidase 2 (OsAPX2) plays a critical role in growth and reproduction in rice under drought, salt and cold stresses. PLoS One 2013,8(2):e57472. doi: 10.1371/journal.pone.0057472 10.1371/journal.pone.0057472PubMed CentralPubMed
      230. Zheng Y, Lyons T, Ollerenshaw JH, Barnes JD: Ascorbate in the leaf apoplast is a factor mediating ozone resistance in Plantago major . Plant Physiol Biochem 2000, 38:403–411. 10.1016/S0981-9428(00)00755-5

      Copyright

      © Venkatesh and Park; licensee Springer. 2014

      This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​4.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.