Characterization and the comprehensive expression analysis of tobacco valine-glutamine genes in response to trichomes development and stress tolerance

Abstract

Valine-glutamine genes (VQ) acted as transcription regulators and played the important roles in plant growth and development, and stress tolerance through interacting with transcription factors and other co-regulators. In this study, sixty-one VQ genes containing the FxxxVQxxTG motif were identified and updated in the Nicotiana tobacum genome. Phylogenetic analysis indicated that NtVQ genes were divided into seven groups and genes of each group had highly conserved exon-intron structure. Expression patterns analysis firstly showed that NtVQ genes expressed individually in different tobacco tissues including mixed-trichome (mT), glandular-trichome (gT), and nonglandular-trichome (nT), and the expression levels were also distinguishing in response to methyl jasmonate (MeJA), salicylic acid (SA), gibberellic acid (GA), ethylene (ETH), high salinity and PEG stresses. Besides, only NtVQ17 of its gene family was verified to have acquired autoactivating activity. This work will not only lead a foundation on revealing the functions of NtVQ genes in tobacco trichomes but also provided references to VQ genes related stress tolerance research in more crops.

Introduction

Valine-glutamine genes (VQ) were kinds of transcription regulators and played the important roles in plant growth, development and responses to various environmental stimulus. VQ proteins were labeled with a conserved single short FxxxVQxxTG amino acid sequence motif (pfam 05678), which was essential between protein interaction with transcript factors like WRKY (Cheng et al. 2012), and dispensable in the interaction with other genes like MAPKs (Pecher et al. 2014). Multiple VQ gene families from plant species have been identified and studied. Taking monocotyledons for examples, there were forty VQ genes in Oryza sativa (Kim et al. 2013; Jiang et al. 2018), sixty-one in Zea mays (Song et al. 2016), and eighteen in Vitis vinifera (Wang et al. 2015); taking dicotyledons for another examples, there were thirty-four VQ genes in Arabidopsis thaliana (Cheng et al. 2012), twenty-six in Solanum lycopersicum (Ding et al. 2019), and eighty-nine in Gossypium hirsutum (Chen et al. 2020b).

VQ genes were involved in multiple regulatory aspects of plant growth and development. For instance, AtVQ8 mutants showed pale-green and stunted-growth, which was similar with the roles of AtVQ17/18/22 in overexpression Arabidopsis plants (Cheng et al. 2012). AtVQ14 (HAIKU1, IKU1) regulated endosperm growth and seeds size by interacting with AtWRKY10 (Wang et al. 2010). AtVQ20 participated in pollen development via inhibiting the expression of downstream MYBs genes (Lei et al. 2017). AtVQ29 acted as a repressor in the light-meditated inhibition of hypocotyl elongation during early seedling development (Cheng et al. 2012). Moreover, OsVQ13 could positively regulate grain size in rice (Uji et al. 2019). Soybean VQ genes overexpression plants showed altered leaf morphology and flowering time (Zhou et al. 2016).

VQ genes were also verified to manage responses to plant biotic and abiotic stress. In Arabidopsis, both AtVQ4 (MPK3/6-targeted VQ protein1, MVQ1) and AtVQ21 (Mitogen-activated Protein Kinnase4 Substrate1, MKS1) met with the quick response to WRKY mediated immune defense (Andreasson et al. 2005; Pecher et al. 2014). AtVQ5/20, AtVQ16 (Sigma Factor-Interacting Protein2, SIB2) and AtVQ23 (SIB1) were proved to be involved in the resistance to Botrytis cinerea (Lai et al. 2011; Cheng et al. 2012). AtVQ9 and AtVQ15 (Arabidopsis CaM-binding protein, AtCaMBP25) were demonstrated to regulate salinity and osmotic stresses, responsively (Hu et al. 2013b; Perruc et al. 2004). AtVQ22 (Jasmonate-associated VQ motif gene1, JAV1) negatively defined the transcriptional activity of WRKY28/51 responsive to injury (Hu et al. 2013a; Yan et al. 2018). Moreover, OsVQ13/14/32 overexpression plants increased resistance to rice bacterial blight (Uji et al. 2019; Li et al. 2021). BnVQ7 from Brassica napus, a MKS1 homologous gene, enhanced disease resistance to Leptosphaeria maculans (Zou et al. 2021). The function of soybean GmVQ58 were similar with AtVQ4 and AtVQ21 to participate in WRKY meditated immune defense responses (Li et al. 2020). These results indicated that most VQ genes were efficiently responsive to environmental conditions (Cheng et al. 2012).

As the epidermal outgrowths, trichomes have been divided into glandular- (gT) and nonglandular- (nT) type according to the secretory ability. Glandular-trichome were the site of biosynthesis and storage of large quantities of specialized metabolites (Chalvin et al. 2020), and played essential roles in the defense against biotic and abiotic factors such as pathogens attack and osmotic stress (Schuurink and Tissier 2020). However, little studies about VQ genes have focused on the trichomes development.

As one worldwide cultivated industrial crop, tobacco leaves were the main source of economic value surrounding with high density of trichomes. Tobacco glandular-trichome were related to various responses to salinity and heavy metal stresses (Yan et al. 2021; Zhang et al. 2021a, b), and the industrial quality of flue-cured leaves (Li et al. 2017). In this study, the information of tobacco NtVQ gene family were collected and updated for the performance of bioinformatics analysis. Then, the wild flue-cured tobacco K326 covered with mixed trichomes (mT) contained gT and nT, Tobacco Introduction 1112 (T.I.1112) characterized only by nT and T.I.1068 only shown gT were used to manifest the NtVQ genes comprehensive expression patterns, thus further indicating the importance of NtVQ in trichomes formation and development process. Meanwhile, several hormone treatments and abiotic stresses were conducted to evaluate NtVQ genes values in order to demonstrate the broad spectrum resistance functions. After the above analysis, the representative NtVQ genes were tested for autoactivation activity to facilitating a complete research system. This work would lay a solid theoretical foundation for exploring the crucial functions of VQ genes in more plants.

Materials and methods

Plant material and growth conditions

K326 was stored in the lab, T.I.1112 and T.I.1068 were provided from Oxford Tobacco Research Station (Oxford, North Carolina, USA). K326, T.I.1112 and T.I.1068 seedlings were grown at 25℃, 70–80% proportional humidity under 12 h / 12 h day / night cycles. For all experiments, four-week old seedlings were used. All experiments were repeated in triplicate.

Identification of tobacco VQ genes

The VQ proteins of Arabidopsis were obtained from TAIR database (http://www.arabidopsis.org/). A Hidden Markov Model (HMM) with VQ motif was extracted from Pfam database (http://pfam.sanger.ac.uk/) (Finn et al. 2010; Guo et al. 2014) to identify all putative NtVQ genes from tobacco genome database (https://solgenomics.net/) and NCBI (https://www.ncbi.nlm.nih.gov/). The identified VQ proteins were determined using the SMART server (http://smart.embl.de/), and annotated based on their phylogenetic relationships (Zhang et al. 2019, Zhang et al. 2021a, b).

Bioinformatics analysis of tobacco VQ genes

A phylogenetic tree of NtVQ proteins was constructed with MEGA 7.0 software using the neighbor-joining method (Kumar et al. 2016). The exon-intron structures were analyzed with their coding regions and full-length sequences and generated using the Gene Structure Display SERVER 2.0 (http://gsds.cbi.pku.edu.cn/). The NtVQ protein structures were determined using SMART server. NtVQ genes duplications were identified as previously described (Yan et al. 2017). The syntenic blocks were used to construct a synteny analysis map of the NtVQ genes from the Plant Genome Duplication Database (Tang et al. 2008). Diagrams were generated using Circos version 0.63 (http://circos.ca/) (Guo et al. 2014).

Acquisition of tobacco tissues and trichomes phenotyping

Roots, stem, leaves (with its epidermis) and flowers from K326 were collected for tissue-specific expression analysis. Trichomes from K326, T.I.1112 and T.I.1068 leaves were removed using freeze-thawing method with liquid nitrogen (Yan et al. 2021). After staining by 2% Rhodamine B for 30 min, 1 cm wide leaf filaments without veins were cut and observed using the depth-of-field digital microscope (VHX-2000; KEYENCE, Osaka, Japan).

Phytohormone treatments and abiotic stress

For exogenous phytohormone treatment, nine four-week old seedlings were sprayed with 150 µM methyl jasmonate (MeJA), 2.0 mM salicylic acid (SA), 150 µM gibberellic acid (GA), and 100 µM ethylene (ETH), respectively. Controls were cultured without any treatment. Samples were collected at 0, 1, 3, 6, and 12 h after phytohormone treatments and stored at -80℃. For abiotic stress, nine four-week old seedlings were separately treated with 300 mM NaCl and PEG-6000 (-0.5 MPa) solutions. Samples were collected at 0, 6, 24, 48, and 72 h after stress treatments and stored at -80℃.

Transcriptional activity assay

The full-length coding sequences (CDS) of NtVQ genes were cloned using the tobacco genome as the template and the relative primers in Table S1. CDS of NtVQ genes were fused into the pGBKT7 vector, and the empty pGBKT7 vector was used as the control. All constructions were transformed into the Y2H Gold yeast strain and selected on SD/-Trp/X (X: X-α-gal) and SD/-Trp/X/A medium (A: AbA), respectively (Yan et al. 2021).

RNA extraction and sqRT-PCR analysis

Tobacco RNA was extracted using the Total RNA Extraction Kit (R6827-01, Omega Bio-tek, USA). First-strand cDNAs were synthesized using a PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa Biotechnology, Dalian, China). NtVQ gene-specific primers were designed using Primer Premier 5.0 and listed in Table S1. NtL25 was used as the reference gene. The semi-quantitative RT-PCR reactions profiles and methods were described in previous studies (Yan et al. 2017, 2021). The data were analyzed with 2^−ΔΔCt method, quantified using the Gene Tools software, and visualized into heat maps with TBtools software (Guo et al. 2014; Chen et al. 2020a).

Data statistics

Data were presented using Microsoft Excel and Sigma Plot 10.0. One-way ANOVA analysis was performed using the SPSS Statistics 20.0 software (IBM China Company Ltd. Beijing, China) to assess significant differences.

Results

Identification and synteny analysis of VQ genes families

Sixty-one candidate NtVQ genes were identified in the tobacco genome sequence. The coding length of sixty-one NtVQ gene sequences ranged from 216 to 1404 bp (Table 1). The exon numbers of NtVQ genes ranged from one to nine, and 77.05% of these genes had one exon. The isoelectric points of thirty NtVQ proteins were alkaline and these of the remaining proteins were acidic, which indicated that NtVQ gene family had nearly harmonious relationship between alkaline and acidic amino acids.

As shown in Fig. 1a; Table 2, a total of nineteen NtVQ genes were clustered into twelve tandem duplication event regions on tobacco chromosome 2, 4, 5, 8, 10, 14, 15, 17, 20, 21, 22 and 24, indicating that less than half of the NtVQ genes were generated by tandem duplication. A synteny analysis from tobacco and Arabidopsis further showed five syntenic relations that contained four NtVQ genes and five AtVQ genes (Fig. 1b; Table 3).

Fig. 1

Chromosome distribution and synteny analysis of tobacco and Arabidopsis thaliana VQ genes (a) Chromosomes 1–24 were shown in different colors in a circular diagram. Colored curves denoted the details of syntenic regions between tobacco VQ genes. (b) The chromosomes of tobacco and A. thaliana were depicted as a circle. Colored curves denoted the details of syntenic regions between tobacco and A. thaliana VQ genes

Table 1 Tobacco VQ genes and accession numbers. CDS: coding sequence

Table 2 The details of syntenic regions of tobacco VQ genes

Table 3 The details of syntenic regions between tobacco and Arabidopsis thaliana VQ genes

Phylogenetic analysis of VQ gene families

According to the phylogenetic tree (Fig. 2), ninety-five VQ proteins derived from N. tobacum and A. thaliana were clustered into seven groups named from I to VII. Group I - VI contained VQ proteins from both tobacco and Arabidopsis, and group VII contained only tobacco VQ proteins. Group III (29.50%) contained the largest number of genes, followed by group I (16.39%), group IV (14.75%), group II (13.11%), group VII (13.11%), group V (6.56%) and group VI (6.56%). Moreover, a subset of the Arabidopsis VQ proteins phosphorylated by the MPK3a and MPK6 (named as MVQs) were uniformly clustered into the group I and II (Pecher et al. 2014), which predicated that eighteen tobacco VQ proteins from group I and II may be involved in the cellular process of protein phosphorylation. This comparison between VQ proteins clarified that genes in the same group may have similar functions.

Fig. 2

Phylogenetic analysis of VQ proteins among tobacco and Arabidopsis thaliana The full-length amino acid sequences of sixty-one tobacco VQ genes and thirty-four Arabidopsis VQ genes were aligned by using ClustalX and the phylogenetic tree was constructed using MEGA 7.0 by the neighbor-joining method with 1000 bootstrap. λ: N. tabacum, ϒ: A. thaliana

Structure and phylogenetic analysis of NtVQ gene family

A phylogenetic tree was constructed using sixty-one NtVQ protein sequences (Fig. 3a). The NtVQ genes could be divided into thirty-four categories (NtVQ1 to NtVQ34) marked with different colours. The analysis of protein domain organization showed that all NtVQ proteins delineated the VQ-motif using SMART database and NCBI, and protein structures were highly similar (Fig. 3b). Protein structure of NtVQ3 (Nitab4.5_0000443g0140.1) contained a coiled-coil region. Protein structures of NtVQ16 (Nitab4.5_0008558g0010.1 and Nitab4.5_0000786g0140.1) had two transmembrane helix regions, respectively. The analysis of gene structure showed that NtVQ genes in the same branch shared a similar exon-intron distribution, except NtVQ5, NtVQ8, NtVQ16 and NtVQ33 (Fig. 3c). Fourteen NtVQ genes contained introns in the genomic sequences, the number of introns varied significantly from one to eight, indicating that NtVQ genes usually varied in the exon-intron distribution profile and gene length of the tobacco genomic sequences.

Fig. 3

Genome wide organization of tobacco NtVQ genes a) Phylogenetic tree based on the protein sequences of sixty-one NtVQ genes. Phylogenetic tree was constructed using MEGA 7.0 by the neighbor-joining method with 1000 bootstrap. (b) Structures of NtVQ proteins: Conserved domains were showed in different colored boxes. (c) Exon-intron structure of NtVQ genes: black rectangles indicated coding sequence (CDS), blue indicated untranslated 5′- and 3′- regions, black lines indicated introns

Tissue expression pattern of NtVQ genes

To determine the potential functions of NtVQ genes in tobacco development, the expression profiles of thirty-four NtVQ genes were conducted in four tissues and three types of trichomes. Lots of trichomes were shown on the surface of stem and leaf, and tobacco root and flower were also covered with epidermis (Fig. 4a). As shown in Fig. 4b, the result of tissue expression profiles showed that NtVQ11 and NtVQ30 were barely expressed in different tissues including trichomes, NtVQ16 were barely expressed in tissues, and NtVQ19 were barely expressed in different type of trichomes. Seven genes (NtVQ3, NtVQ8, NtVQ17, NtVQ25, NtVQ27, NtVQ29 and NtVQ32) were highly expressed in root, and NtVQ28 was expressed only in root. Five genes (NtVQ9, NtVQ13, NtVQ18, NtVQ21 and NtVQ26) were highly expressed in stem. Eight genes (NtVQ2, NtVQ5, NtVQ10, NtVQ12, NtVQ20, NtVQ22, NtVQ24 and NtVQ33) were highly expressed in leaf. NtVQ15 was highly expressed in flower, NtVQ1 and NtVQ8 were barely expressed in flower. These results forecasted that NtVQ genes played essential roles in tobacco tissues growth and development. Besides, eleven genes (NtVQ8, NtVQ9, NtVQ12, NtVQ13, NtVQ16, NtVQ22, NtVQ24, NtVQ25, NtVQ26, NtVQ31 and NtVQ34) were highly expressed in mT. Four genes (NtVQ1, NtVQ2, NtVQ17 and NtVQ20) were highly expressed in gT. Five genes (NtVQ3, NtVQ4, NtVQ5, NtVQ28 and NtVQ33) were highly expressed in nT. The expression profiles in different type of trichomes firstly clarified the important functions of NtVQ genes in glandular- and nonglandular-trichome formation and development.

Fig. 4

Expression patterns of thirty-four NtVQ genes in different tissues and trichomes (a) Epidermal phenotypes of tobacco roots, stems, leaves, flowers, mixed-trichome (mT), glandular-trichome (gT) and nonglandular-trichome (nT). (b) Gene expression profiles in different tissues and trichomes

Expression patterns of NtVQ genes following phytohormone treatments and abiotic stresses

To elucidate the roles of NtVQ genes under different phytohormone, sqRT-PCR was conducted to achieve the relative expression values of each NtVQ gene (Fig. 5). The expression levels of thirteen genes (NtVQ3, NtVQ4, NtVQ6, NtVQ13, NtVQ15, NtVQ16, NtVQ20, NtVQ22, NtVQ28, NtVQ29, NtVQ31, NtVQ33 and NtVQ34) increased following MeJA treatment. The expression levels of twenty-two genes (NtVQ3, NtVQ4, NtVQ5, NtVQ7, NtVQ9, NtVQ10, NtVQ12, NtVQ14, NtVQ15, NtVQ16, NtVQ17, NtVQ18, NtVQ19, NtVQ21, NtVQ23, NtVQ24, NtVQ25, NtVQ29, NtVQ31, NtVQ32, NtVQ33 and NtVQ34) increased following SA treatment. The expression levels of eleven genes (NtVQ2, NtVQ7, NtVQ10, NtVQ16, NtVQ20, NtVQ23, NtVQ26, NtVQ27, NtVQ29, NtVQ33 and NtVQ34) increased following GA treatment. The expression levels of eight genes (NtVQ1, NtVQ4, NtVQ6, NtVQ7, NtVQ9, NtVQ14, NtVQ15 and NtVQ29) increased following ETH treatment. Among these genes, the expression levels of NtVQ29 was simultaneously up-regulated following the four phytohormones treatments. These results uncovered that all NtVQ genes were involved in the intricate signaling pathways and each gene had different regulatory characteristics.

Fig. 5

Expression profiles of thirty-four NtVQ genes under four phytohormones, NaCl and PEG stresses. The expression data from the semi-quantitative RT-PCR analysis were analyzed and visualized into heat maps using the TBtools software and MeV 4.8.1. The color scale represented relative expression levels with red and blue indicating increased or decreased transcript abundance, respectively

A systematic expression analysis of NtVQ genes was conducted following the abiotic stresses. The expression levels of eight genes (NtVQ5, NtVQ18, NtVQ20, NtVQ21, NtVQ24, NtVQ26, NtVQ28 and NtVQ34) increased following high salinity stress. The expression levels of six genes (NtVQ2, NtVQ7, NtVQ12, NtVQ16, NtVQ20 and NtVQ24) increased following PEG stress. NtVQ20 and NtVQ24 were simultaneously up-regulated following these two stresses. While the expression levels of fifteen genes (NtVQ1, NtVQ3, NtVQ4, NtVQ6, NtVQ8, NtVQ13, NtVQ15, NtVQ17, NtVQ19, NtVQ22, NtVQ23, NtVQ25, NtVQ29, NtVQ32 and NtVQ33) were simultaneously down-regulated. These results predicated that most NtVQ genes showed the negative regulatory responses to abiotic stress.

Transcriptional activity analysis of NtVQ genes

Several NtVQ genes have been chosen to investigate the potential transcriptional activity (Fig. 6). Compared with the control, pGBKT7-NtVQ4, pGBKT7-NtVQ28 and pGBKT7-NtVQ29 could only grow on SD/-Trp medium, while pGBKT7-NtVQ17 grew normally on both selective media and showed alpha-galactosidase activity, which speculated that most NtVQ genes might have no transcription activity.

Fig. 6

Transcriptional activity assay of several NtVQ genes. pGBKT7 was used as the control. The yeast colonies were cultivated and photographed after being cultured for 3 days at 30 °C

Discussion

VQ gene has been extensively identified in various plants including angiosperms, gymnosperms and mosses. Some VQ gene were known to be related to the plant development and stress tolerance (Jiang et al. 2018; León et al. 2021). According to the tobacco genome data, sixty-one NtVQ genes containing VQ-motif were identified and their protein lengths ranged from 71 to 467 amino acids in the current study (Table 1), which was similar with these in Arabidopsis but smaller than these in moss (Li et al. 2014; Jing and Lin 2015). Then NtVQ gene structures, gene expression patterns, protein characteristics and their primary functions have already been confirmed.

Gene duplication and phylogenetic analysis of NtVQ and AtVQ genes

Gene duplication event played a major role in plant genome rearrangement and expansion (Vision et al. 2000), and segregation duplication events were shown to provide references for the evolutionary relationship between VQ genes (Fig. 1), thereby enabling functional predictions (Panchy et al. 2016). For example, AtVQ11 (MVQ5, AT1G80450), AtVQ19 (MVQ4, AT3G15300) and AtVQ33 (MVQ3, AT5G53830) interacted with specific subgroups of WRKY transcription factors and their proteins stability were mediated by phosphorylation (Pecher et al. 2014). The phylogenetic analysis revealed that the VQ genes from tobacco and Arabidopsis were classified into seven groups and the orthologous genes were clad in the same group (Fig. 2). Pecher (2014) has also proved that AtVQ4, AtVQ6, AtVQ9, AtVQ13, AtVQ14, AtVQ31 and AtVQ32 were all targeted by MPK3 and MPK6, which speculated that tobacco NtVQ proteins from the group I and II might be involved in the phosphorylation process. These results indicated that the related VQ proteins in each group could be equipped with the similar functions.

Structural and conserved domain analysis of NtVQ genes

Cultivated tobacco N. tobacum was an allotetraploid plant, thereby one gene might have two homologous sequences from two ancestors of N. sylvestris and N. tomentosiformis (Renny-Byfield et al. 2011). In this study, thirty-four NtVQ genes were grouped referring to the phylogenetic tree (Fig. 3a). Structure analysis showed that NtVQ proteins in the same group contained the similar type of motifs (Fig. 3b), indicating that close proteins shared similar functions. NtVQ3 contained a coiled-coil region which was found in more than two hundred proteins and used to predicate regions of protein discontinuity and folding stability (Lupas et al. 1991). NtVQ16 had two transmembrane helix regions and marked as the integral membrane protein (Krogh et al. 2001). Interestingly, most VQ genes in higher plants did not have any intron (Jing and Lin 2015), for instance, 90% AtVQ genes (Cheng et al. 2012) and 76% PoVQ genes (Chu et al. 2016) had no introns. Consistently, introns of twenty NtVQ genes were lost (Fig. 3c), which meant that NtVQ genes might have experienced different selective pressures during evolution.

Analysis of NtVQ gene expression patterns in different tissues and trichomes

Tissue transcription patterns could exhibit genes involvement in functional or differential events. Thirty-four AtVQ genes were induced and differentially expressed in different tissues (Cheng et al. 2012). Herein, NtVQ11 and NtVQ30 were barely expressed in different tissues including trichomes. Consistently, the homologous gene AtVQ20 expressed strongly in the male gametophytic tissues, but barely in seedling, leaf, stem and root (Lei et al. 2017). AtVQ12 was mainly expressed in the root and leaf (Wang et al. 2015). AtVQ29 was mainly expressed in the root, leaf, hypocotyl and silique base (Jing and Lin 2015). Moreover, GmVQ58 was highest expressed in the cotton leaf and root (Li et al. 2020). Almost all CsVQ genes were more highly expressed in root, stem and leaf of tea plant, while only a few genes were more highly expressed in the flower (Guo et al. 2018). Some uncharacterized VQ proteins were also found in response to meditate plant growth. In our results, the tissues expression profiles showed that almost all NtVQ genes were responsive to organogenesis, thus pointing to the important regulatory roles of NtVQ genes in tobacco development.

Trichomes were one of the most important accommodative traits in plants (Ishida et al. 2008). Trichomes were associated with several important features that were involved in phytohormone responses, resistance to biotic and abiotic stress (Schuurink and Tissier 2020; Yan et al. 2021). gT and nT not only formed the physical barriers against UV radiation, waster loss and excess light (Schuurink and Tissier 2020), but also were crucial in the process of chemical secretion such as phenylpropanoids, alkaloids, sugars and some other metabolic compounds against insects and arthropods (Yang and Ye 2013; Maurya et al. 2019). However, the expression patterns and molecular regulatory mechanisms of NtVQ genes in trichomes had never been published. Here, eleven NtVQ genes highly expressed in mT from K326, four NtVQ genes highly expressed in gT from T.I.1068, and five NtVQ genes highly expressed in nT from T.I.1112, which made it clear that NtVQ genes had unique potentials and momentous values to participate in trichomes development responsive to the multiple external stimulus.

Gene expression patterns under environmental stresses

Plants had generated kinds of effective defense systems against biotic and abiotic stresses. Jasmonic acid (JA) and SA were two of the best-known signaling molecules that regulated plant development and triggered defense responses (Dubrovsky 2005; Jing and Lin 2015) pointed that VQ genes were involved in JA- and SA-meditated defense responses.

In this study, NtVQ4 was significantly and consistently up-regulated upon JA treatment (Fig. 5), and its highly homologous gene AtVQ22 (JAV1) transcript significantly increased and negatively regulated the transcriptional activity of WRKY28/51 involved in the JA-meditated defense response (Andreasson et al. 2005; Hu et al. 2013a; Yan et al. 2018). Moreover, OsVQ13 positively regulated JA-meditated grain size by activating the OsMPK6-OsWRKY45 signaling pathway in rice (Uji et al. 2019). The expression levels of MaVQ5 was up-regulated after MeJA treatment (Ye et al. 2016). Moreover, the roles of JA in promoting leaf senescence and epidermogenesis were affirmed in many plant species (Zhang et al. 2019; Wang et al. 2022). AtVQ18, AtVQ26 and ZmVQ52 could effectively regulate JA-mediated leaf senescence (Pan et al. 2018; Yu et al. 2019). Herein, a detailed expression analysis revealed that nineteen NtVQ genes were obviously involved in the formation and development of gT and nT (Fig. 4), and thirteen NtVQ genes were up-regulated after MeJA treatment (Fig. 5). These results proved that NtVQ genes emitted important response functions to JA signaling, which was similar with the result of around 32% AtVQ genes up-regulated in senescing leaves (Schmid et al. 2005).

NtVQ34 were highly expressed after SA treatment (Fig. 5), and its homologous gene AtVQ21 played a positive role in SA signaling (Andreasson et al. 2005). While NtVQ22 and NtVQ23 were not sensitive to SA, which was opposite to the functions of their highly homologous genes AtVQ16 (SIB2) and AtVQ23 (SIB1) strongly induced by SA treatment (Xie et al. 2010; Lai et al. 2011). These results verified that NtVQ genes interlaced to form a variety of complex regulatory mechanisms in tobacco. In addition, most NtVQ genes were up-regulated after SA treatment and conjectured to play the important roles in SA-meditated plant growth, development and resistance responses, which was consistent with the results that thirty-four AtVQ genes were responsive to SA treatment (Cheng et al. 2012), and sixteen VvVQ genes were induced by SA treatment (Wang et al. 2015).

Recent studies in Arabidopsis and other crop species highlighted the emerging key roles for GA and ETH in the regulation of nearly all aspects of plant organs growth and yield under abiotic stress (Yamaguchi 2008; Dubois et al. 2018). In this study, part of NtVQ genes were positively induced after these two phytohormone treatments. The VQ genes from other plant also showed the similar function, for example, PbrVQ9 was the top highly expressed gene after GA treatment (Cao et al. 2018), VvVQ2 was highly up-regulated in grapevine after ETH treatment (Wang et al. 2015).

Salt and drought were the main factors of reducing crop production. The expression levels of NtVQ16 and NtVQ17 decreased after NaCl treatment (Fig. 5), while their homologous gene AtVQ9 transcript increased (Perruc et al. 2004; Hu et al. 2013b). Some studies have indicated that some up-regulated VQ genes might have a negative effect on abiotic stress resistance. AtVQ9 expression was induced by salinity stress, but AtVQ9 overexpression increased plants hypersensitive to salinity stress (Hu et al. 2013b), which conjectured that the functional roles of NtVQ genes to abiotic stresses were characterized by inconsistency. Moreover, AtVQ15 (AtCaMBP25) was induced by various abiotic stresses, including dehydration and high salinity, and AtVQ15 overexpression plants displayed increasing sensitivity to both NaCl and mannitol (Perruc et al. 2004; Hu et al. 2013b). AtVQ24, a closed protein of AtVQ15, was clarified to have the negative role in osmotic stress (Hu et al. 2013b). And their homologous tobacco genes NtVQ9 and NtVQ10 showed the similar negative regulation. Besides, GhVQ18 and GhVQ84 were highly expressed under NaCl and PEG treatments (Chen et al. 2020b). Bamboo PeVQ28 overexpression in Arabidopsis showed increased resistance to salinity stress (Cheng et al. 2020). Here, eight and six NtVQ genes were up-regulated after NaCl and PEG treatments, respectively. While the expression profiles of most NtVQ genes decreased. Previous studies showed the consistent results that ten BrVQ genes (Zhang et al. 2015), eight PtVQ genes (Chu et al. 2016), eighteen VvVQ genes (Wang et al. 2015), and twenty-two OsVQ genes (Kim et al. 2013) were up-regulated after drought stress. And most VQ genes from poplar (Chu et al. 2016), maize (Song et al. 2016), and tea (Guo et al. 2018) were also responsive to drought or PEG and NaCl stress.

Most NtVQ genes lacked the transcriptional activity

Studies have proved that AtVQ1 and AtVQ10 did not have transcriptional activity (Jing and Lin 2015). Consistently, the homologous genes NtVQ28 and NtVQ29 were shown no self-activating activity (Fig. 6), and NtVQ4 also showed no self-activating activity in the Y2H assay. Acted as the homologous gene of AtVQ14 (IKU1/MVQ9), NtVQ17 displayed the transcriptional activity, which was same with the result of the most prominent interactions between MPK3, MPK6 and MVQ proteins from group I and II (Pecher et al. 2014). All these data predicted that most NtVQ genes acted as transcription regulators might have no transcriptional activity, and functions of NtVQ genes, especially NtVQ17, needed further verification and evaluation.

Conclusions

The transcriptions of VQ genes were modulated by multiple endogenous and environmental signals, consistent with their diverse roles in stress responses, plant growth and development. Study about NtVQ gene family provided a glimpse into the potential biological functions in tissues development, trichomes formation, and resistance to abiotic stress, indicating that members of NtVQ family played important roles in plant growth and responses to environmental conditions.