Qc-SNARE AtBS14b localized at vesicular compartments
SNARE proteins are subdivided into three Q- and one R-SNARE groups. AtBS14a, a Qc-SNARE, was reported to be ubiquitously expressed in all the tissues except pollen and lateral roots (Lipka et al. [2007]). Further, AtBS14b has been analyzed its Golgi localization, but function of AtBS14b in plant growth and development is unknown. To test AtBS14b subcellular localization, ORFs of AtBS14b was cloned into pEarly-GW 104 in which yellow fluorescence protein was N-terminally fused to AtBS14b. YFP-AtBS14b fusion protein was subsequently transformed into N.benthamiana leaves by using Agrobacterium-mediated transformation. To understand specific localization compartment, tans-Golgi network maker SYP41 was C-terminally fused to mCherry protein. AtBS14b and SYP41 localization were co-localized at vesicular compartments and displayed punctuated patterns surrounding plasma membrane in tobacco leaves (Figure 1).
AtBS14b overexpression plants exhibit short petioles and hypocotyls
SNAREs are known function in membrane tethering and fusion and play key roles in plant development. To analyze AtBS14a and AtBS14b function, AtBS14a and AtBS14b overexpression (AtBS14a and AtBS14b ox) plants were generated in WS2 background. More than 15 AtBS14a ox and AtBS14b ox plants respectively, were successfully selected and further transferred into soil. Short petioles are one of the typical brassinosteroids (BRs) mutant phenotypes. BR mutants also develop short hypocotyls and stem growth (Oh et al. [2012]). AtBS14b ox plants developed relatively short petioles in the growth chamber compared to normal leaf length and width (Figure 2), but no obvious differences were observed from AtBS14a ox compared to WS2 plants (data not shown). To identify the relationship between BR hormone and AtBS14b, AtBS14b expression patterns were analyzed upon BR supplementation. One-week-old seedlings were treated with BR, and AtBS14b expression was analyzed by qRT-PCR. The results showed that AtBS14b was down-regulated by BR and the suppression requires BRI1 activity (Figure 3a). Hypocotyl growth of AtBS14b ox plants was further analyzed in the early seedling stage. One-week-old plants were grown in low, continuous light chamber, and hypocotyl lengths were measured. AtBS14b ox plants developed shorter hypocotyls than wild type (Figure 3b) while AtBS14a ox plants showed normal hypocotyl growth (Additional file 2: Figure S1a, b). Afterwards, expression levels of AtBS14b in overexpression plants were analyzed (Figure 3c). Further, BR-mediated gene expression showed that AtBS14a level was not altered by exogenously supplied BR (Additional file 2: Figure S1c). These results indicate that AtBS14b, but not AtBS14a, is negatively regulated by BR and inhibit hypocotyl and petiole elongation in plants in which AtBS14b is overexpressed.
AtBS14b ox plants are insensitive to BL
Brassinosteriods are essential hormones in plants, promoting stem and hypocotyl elongation. To test BR sensitivity of AtBS14b ox plants, plants were germinated on MS medium containing BL (0, 10, 100 and 200 nM), and grown for 7 days. Hypocotyl and primary root lengths were measured with ImageJ software. Results show that BR-mediated hypocotyl elongation was partially inhibited in AtBS14b ox plants (Figure 4a, b). Also, high concentration of BR was shown to inhibit root growth along with root coiling. Primary root growth patterns indicated that AtBS14b ox plants are insensitive to BR especially since drastic differences were observed in 10 nM BR containing medium (Figure 4c). In addition, effects of high concentration of BL (1 μM) on AtBS14b ox hypocotyl growth were tested. The data showed that high concentration of BL inhibited hypocotyl growth of wild-type plants, but it promoted hypocotyl length of AtBS14b ox plants (Additional file 3: Figure S2a, b). To test effects of other hormones on AtBS14b ox hypocotyl growth, gibberellic acid (GA)-dependent hypocotyl growth was analyzed. The data showed that GA changed hypocotyl length of both WS2 and AtBS14b ox plants, but no obvious differences were observed between WS2 and AtBS14b ox plants (Additional file 3: Figure S2c, d).
Since AtBS14b overexpression affected normal plant growth and normal BR response, we further tested AtBS14b knock-out mutants. Two independent T-DNA lines were isolated from SALK and named atbs14b-1 and atbs14b-2. T-DNA was inserted in the promoter and first exon in atbs14b-1 and atbs14-2, respectively (Additional file 4: Figure S3). AtBS14b expression level was analyzed, and no transcription was detected in two mutant plants (Additional file 4: Figure S3). As shown in Figure 3B, AtBS14b ox plants developed shorter hypocotyls than wild type, so hypocotyl growth of AtBS14b mutants was further analyzed. As shown in Additional file 4: Figure S3, two mutant lines did not show differences in hypocotyl elongation. In addition, BR dependent primary root growth was analyzed in BL containing media, but no any differences were observed (Additional file 5: Figure S4), suggesting that functional redundancy of SNAREs is believed to occur in plant genomes.
BR-mediated suppression of three biosynthesis enzyme expressions was inhibited in AtBS14b ox plants
AtBS14b ox plants, but not AtBS14b mutants, show BR dependent phenotypes. Therefore, expression levels of BR signaling genes were further analyzed. In one week old WS2, bri1-5, a weak allele of BRI1 mutant, and AtBS14b ox plants were treated with BL for 3 hours and subsequently total RNA was extracted from those lines. qRT-PCR results showed that BR6OX2, CPD and DWF4 were the three biosynthesis enzymes (Choe et al. [2001]; Nole-Wilson et al. [2010]; Ohnishi et al. [2012]) whose expressions were dramatically repressed after BR treatment in WS2, but the suppression was inhibited in bri1-5 indicating gene expression relies on BR signaling. Expression of BR6OX2 was slightly lower in AtBS14b ox plants than WS2, and BR-mediated repression of three genes was inhibited in AtBS14b ox plants. Further BR-induced SAUR15 expression was also analyzed. The results showed that BR-mediated induction of SAUR15 was inhibited in bri1-5 and slightly lower in AtBS14b ox plants compared to wild type (Figure 5). In contrast, BR-dependent expressions of BR6OX2, CPD and DWF4 were examined in atbs14b knock-out mutants. The data showed that no significant differences between wild-type and mutants were detected (Additional file 6: Figure S5). Since expression levels of BR biosynthetic genes (BR6OX2, CPD and DWF4) were altered in AtBS14b ox plants, BR receptor BRI1 levels was further monitored in 7-day-old AtBS14b ox and knock-out mutants. As shown in Additional file 7: Figure S6, BRI1 transcript was higher in AtBS14b ox plants than in WS2 while no differences was observed in knock-out mutants compared to wild-type plants. Taken together, BR-mediated expression patterns of BR6OX2, CPD and DWF4 were similar to bri1-5 and SAUR15 induction was slightly inhibited in AtBS14b ox plants, suggesting that AtBS14b might regulate BR signaling rather than BR biosynthesis genes.
AtBS14b interacts with MSBP1
As shown in Figure 1A, YFP-AtBS14b localized at vesicular compartments somehow associated to plasma membrane (Figure 1a), and AtBS14b overexpression inhibited BR signaling. SNAREs are well known as key players that function in membrane tethering and fusion. Therefore, one possibility of AtBS14b regulatory role might directly interact with membrane localized protein involved in BR signaling pathway. To test the possibility, BR receptor BRI1, its co-receptor BAK1 and steroid binding protein MSBP1 were chosen to test direct interaction with AtBS14b. Split GFP system was used to test their interaction in tobacco plants. ORFs of MSBP1, BAK1 and BRI1 were cloned into PXNGW vector in which N-half of YFP sequences were C-terminally fused to three proteins while ORF of AtBS14b was cloned into PCXGW vector in which C-half of CFP sequences were N-terminally fused to AtBS14b. The interaction was tested by co-expressing the two fusion constructs transiently in N. benthamiana and by observing YFP fluorescence using confocal microscopy. Fluorescence was observed for co-expression of AtBS14b and MSBP1, but not of BAK1 + AtBS14b or BRI1 + AtBS14b (Figure 6). AtBS14b and MSBP1 interaction was mainly observed at vesicular compartments (Figure 6). To confirm AtBS14b-MSBP1 interaction, we performed a mating-based split-ubiquitin assay. MSBP1, BAK1 and BRI1 were cloned into the Nub vector pXN25_GW and AtBS14b was cloned into the Cub vector pMETYC_GW. Yeast growth assay showed that AtBS14b interacts with MSBP1 but not BAK1 and BRI1. These results are consistent with split-GFP assay. These data support the hypothesis that AtBS14b directly interact with MSBP1 to modulate BR-mediated plant growth and development.