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RhNAC3, a stress-associated NAC transcription factor, has a role in dehydration tolerance through regulating osmotic stress-related genes in rose petals

Authors


Summary

Petal cell expansion depends on cell wall metabolism, changes in cell turgor pressure and restructuring of the cytoskeleton, and recovery ability of petal cell expansion is defined as an indicator of dehydration tolerance in flowers. We previously reported that RhNAC2, a development-related NAC domain transcription factor, confers dehydration tolerance through regulating cell wall-related genes in rose petals. Here, we identify RhNAC3, a novel rose SNAC gene, and its expression in petals induced by dehydration, wounding, exogenous ethylene and abscisic acid (ABA). Expression studies in Arabidopsis protoplasts and yeast show that RhNAC3 has transactivation activity along its full length and in the carboxyl-terminal domain. Silencing RhNAC3 in rose petals by virus-induced gene silencing (VIGS) significantly decreased the cell expansion of rose petals under rehydration conditions. In total, 24 of 27 osmotic stress-related genes were down-regulated in RhNAC3-silenced rose petals, while only 4 of 22 cell expansion-related genes were down-regulated. Overexpression of RhNAC3 in Arabidopsis gave improved drought tolerance, with lower water loss of leaves in transgenic plants. Arabidopsis ATH1 microarray analysis showed that RhNAC3 regulated the expression of stress-responsive genes in overexpressing lines, and further analysis revealed that most of the RhNAC3-up-regulated genes were involved in the response to osmotic stress. Comparative analysis revealed that different transcription regulation existed between RhNAC3 and RhNAC2. Taken together, these data indicate that RhNAC3, as a positive regulator, confers dehydration tolerance of rose petals mainly through regulating osmotic adjustment-associated genes.

Introduction

Plants are frequently challenged by various harmful environmental conditions, such as drought, high salt and low temperatures. Drought stress has an adverse effect on the growth of plants and the productivity of crops (Yamaguchi-Shinozaki and Shinozaki, 2006). To reduce the adverse effect of drought stress, plants have evolved multifaceted strategies, including morphological, physiological, biochemical and genetic adaptations (Bohnert et al., 2006; Broun, 2004). These processes are regulated by a wide range of genes, comprising a complex network of stress signalling pathways (Hirayama and Shinozaki, 2010). The genes involved in the complex network of stress signals can be classified into two groups (Shinozaki et al., 2003). The first group is functional proteins, such as chaperones and osmotin. The second group comprises regulatory proteins, including transcription factors (TFs), protein kinases, protein phosphatases and signalling components. Various TFs, implicated in the response to drought stress, play central roles in the regulation of expression of target genes via specific binding to cis-acting elements in their promoters. These TFs function as transcriptional activators or repressors and control downstream gene expression in stress signal transduction pathways resulting in stress-tolerant phenotypes in transgenic plants (Hussain et al., 2011).

Among the TFs, NAC (for NAM, ATAF1, ATAF2 and CUC2) constitutes one of the largest TF families in plant genomes (Olsen et al., 2005; Ooka et al., 2003). Arabidopsis, rice, tobacco and soybean contain approximately 117, 151, 152 and 152 NAC members, respectively (Fang et al., 2008; Le et al., 2011; Nuruzzaman et al., 2010). The NAC family can be classified into six major groups according to their phylogeny: stress-associated NAC (SNAC), turnip crinkle virus interacting protein (TIP), ANAC034, no apical meristem/cup-shaped cotyledon (NAM/CUC3), ONAC4 and secondary wall-associated NAC domain protein (SND) (Nakashima et al., 2012). They play diverse roles in a variety of environmental stimuli (Jeong et al., 2010) and developmental processes, such as embryonic and floral formation (Souer et al., 1996), lateral root development (Xie et al., 2000), leaf senescence (Zhang and Gan, 2012) and secondary wall formation (Wang et al., 2011).

Many stress-responsive NAC genes are in the SNAC group, including three subgroups: SNAC-A, SNAC-B and SNAC-C (Nuruzzaman et al., 2010). There is increasing evidence demonstrating that SNAC proteins respond to environmental cues. For instance, ATAF1 of Arabidopsis (Mauch-Mani and Flors, 2009; Wu et al., 2009), OsNAC5 of rice (Takasaki et al., 2010) and VvNAC of grape (Vitis vinifera) (Wang et al., 2012) are induced by abiotic stresses. In general, overexpression of SNAC genes in transgenic plants confers improved tolerance to abiotic stress. For example, overexpression of ANAC019, ANAC055 or ANAC072 in Arabidopsis (Tran et al., 2004) and SNAC1 or OsNAC6/SNAC2 in rice (Hu et al., 2006; Nakashima et al., 2007; Ohnishi et al., 2005) resulted in enhanced tolerance to drought. Many other SNAC proteins have been isolated and identified in other plant species, such as wheat (Triticum aestivum) (Xue et al., 2011) and soybean (Hao et al., 2011). However, little is known about SNAC group members in rose, especially in response to dehydration during petal expansion of flowers.

Rose is the most important ornamental plant worldwide. Cut roses for commercial production are usually harvested at an open bud stage and are extremely susceptible to dehydration damage during postharvest handling, resulting in abnormal flower opening (Dai et al., 2012). It is known that petal expansion depends on three important processes: cell wall metabolism, changes in cell turgor pressure and restructuring of the cytoskeleton (Zonia and Munnik, 2007). In our previous work, we obtained the expression profiles of dehydration-induced genes in rose petals and identified that a NAM/CUC3 subgroup gene, RhNAC2, improves dehydration tolerance trough regulating the cell wall-related genes in rose petals (Dai et al., 2012). However, it is still remained unclear how stress-responsive NAC TFs contribute to cell expansion in rose petals during dehydration.

In this study, we isolated a novel SNAC TF gene, RhNAC3. The expression of RhNAC3 was induced by dehydration, abscisic acid (ABA), ethylene and wounding treatments. Silencing RhNAC3 in rose petals by virus-induced gene silencing (VIGS) approach significantly decreased dehydration tolerance of petals, and overexpressing RhNAC3 in Arabidopsis led to enhanced resistance to drought stress. RhNAC3 mainly regulated the expression of the osmotic stress-related genes in both rose petal and Arabidopsis. The data indicate that RhNAC3 functions in dehydration tolerance of rose petals mainly through regulating osmotic stress-related genes at the transcriptional level.

Results

Isolation and sequence analysis of the RhNAC3 gene

Based on the previous microarray results, a dehydration-induced unigene, JK617768, encoding NAC family TF was obtained. This unigene was designated RhNAC3 because RhNAC1 and RhNAC2 were already designated for two other unigenes described previously (Dai et al., 2012; Pei et al., under review). To further characterize RhNAC3 function in dehydration tolerance of rose petals, the full-length cDNA was first cloned. The full-length RhNAC3 was 1662 bp in length with a 72 bp 5′-untranslated region (UTR), a 531 bp 3′-UTR and a 1059 bp open reading frame encoding a polypeptide of 352 amino acids. Two putative nuclear localization signal sequences were also identified at amino acid residues 75–86 and 112–128, respectively (Figure 1a). Analysis of the deduced amino acid sequence indicated that the protein shared homology with known NAC family proteins and contained five conserved subdomains (motif 1–5) at the N-terminal region. The transcription regulation domain at the C-terminal region contained the same two motifs (motif 6 and motif 7) as ANAC072, BnNAC485, GmNAC4 and GhNAC4 (Figure S1). Phylogenetic analysis showed that RhNAC3 shares a close relationship with the cotton GhNAC4 protein and belongs to the SNAC group (Figure 1b).

Figure 1.

Structure and Phylogenetic analysis of RhNAC3.

Expression profiles of RhNAC3 under dehydration conditions

To investigate the response of RhNAC3 in petals under dehydration, we determined the expression level of RhNAC3 using qRT-PCR assays. The RhNAC3 transcripts showed no obvious accumulation after the first 6 h of dehydration, then was induced accumulate 3.2-fold and 6.2-fold at 12 h and 24 h dehydration, respectively, compared with the control (Figure 2a). RhNAC3 expression was also investigated under other stress conditions. RhNAC3 was induced by 24 h ethylene or ABA with approximately fourfold and twofold mRNA increases, respectively, whereas 24 h brassinosteroid (BR), gibberellic acid (GA) and naphthalene acetic acid (NAA) did not noticeably alter the expression levels of the gene. Wounding in petals resulted in the accumulation of RhNAC3 transcripts by about fourfold after 24 h treatment (Figure 2b). The floral organ-specific expression of RhNAC3 in rose flower was also examined. RhNAC3 expression levels were higher in sepals and receptacles and relatively lower in stamens compared with petals and pistils (Figure S2).

Figure 2.

Expression profiles of RhNAC3 under various conditions.

Nuclear localization and transcriptional activation of RhNAC3

To provide further evidence for the potential role of RhNAC3 in transcriptional regulation, we examined subcellular localization of RhNAC3 in onion (Allium cepa) epidermal cells or Arabidopsis leaf protoplasts. The GFP fluorescence of RhNAC3-GFP was exclusively located in the nuclei of the cells, whereas the GFP control was distributed throughout the whole cell both in onion cells (Figure 3a) and in Arabidopsis leaf protoplasts (Figure S3). These results indicate that RhNAC3 is a nuclear-localized protein.

Figure 3.

Nuclear localization and transcriptional activation of RhNAC3.

A yeast one-hybrid assay was performed to examine the transactivation activity of RhNAC3. On selection medium, yeast transformants harbouring pGAL4, pBD-RhNAC3F and pBD-RhNAC3C constructs grew well, whereas the yeast transformants containing the pBD alone and pBD-RhNAC3N could not grow. Consistently, the relative β-galactosidase activities of the transformants with pBD-RhNAC3F and pBD-RhNAC3C were about 59-fold and 88-fold higher than that of the negative control, respectively. However, pBD-RhNAC3N did not show the ability to activate the reporter gene (Figure 3b). A GAL4 transient expression assay of RhNAC3 was also performed in Arabidopsis protoplasts. Compared with the GAL4-BD negative control, both the full-length and C-terminal region of RhNAC3 strongly activated the reporter gene, and the relative GUS activities of RhNAC3F and RhNAC3C were 1.8-fold and 4.3-fold higher than that of the negative control, respectively (Figure 3c). These results indicate that RhNAC3 functions as a transcriptional activator with a transactivation domain in the C-terminal.

Silencing RhNAC3 by VIGS reduced dehydration tolerance in rose petals

The role of RhNAC3 in dehydration tolerance in rose petals was investigated by silencing RhNAC3 using a VIGS approach. To specifically silence RhNAC3, the RhNAC3-specific 3′ UTR (389 bp) region was chosen to construct a tobacco rattle virus (TRV)-RhNAC3 vector. Entire rose petals or petal discs were infiltrated with Agrobacterium tumefaciens containing TRV-RhNAC3. The RhNAC3 gene was successfully silenced with a ~70% decrease in gene expression compared with TRV controls (Figure 4a). After 12 h of dehydration, the fresh weight of all of the petals was about 67% of its initial value, and petal width and length were about 86% and 85% of their initial values, respectively. During 24 h of rehydration, petal fresh weight, width and length in TRV controls returned to 78%, 94% and 93% of their initial values, but were still only 72%, 87% and 87% in the RhNAC3-silenced petals, respectively (Figure 4b, c). The cell expansion in petals was examined using measurement of abaxial subepidermis (AbsE) cell numbers in TRV control and RhNAC3-silenced petals. Silencing of RhNAC3 resulted in more AbsE cells per microscope visual area from 136 ± 2.1 in TRV petals to 151 ± 4.3 in RhNAC3-silenced ones, an increase of about 11% (Figure 4d).

Figure 4.

Silencing of RhNAC3 in rose petals and discs by virus-induced gene silencing (VIGS).

The effects of gene silencing and dehydration were also tested in petal discs. The area of expanded discs provided a convenient rehydration assay to demonstrate the cell expansion under dehydration and rehydration conditions. Discs were dehydrated for 12 h and then rehydrated for 24 h. After 6 h of rehydration, approximately 66% of the disc area in the TRV controls had recovered compared with only 51% in the RhNAC3-silenced discs. The difference between TRV control and RhNAC3-silenced discs was still significant at 12 h rehydration (~82% vs ~66%), but disappeared after 24 h (Figure 4e). These results are consistent with those obtained from intact petals and suggest that RhNAC3 is involved in the tolerance of rose petals to dehydration stress.

Altered expression of genes involved in the response to osmotic stress and cell wall expansion in RhNAC3-silenced petals

To elucidate the possible molecular mechanisms underlying dehydration tolerance and petal expansion, we investigated the expression levels of genes involved in the response to osmotic stress and cell wall expansion in RhNAC3-silenced petals. Based on the previous Gene Ontology (GO) term analysis (Dai et al., 2012), 27 genes were selected for further qRT-PCR analysis from the biological process of response to osmotic stress. The qRT-PCR analysis showed that there were 12 genes obviously down-regulated (fold change <0.5) and 12 genes slightly repressed (fold change 0.5–0.8) in RhNAC3-silenced petals. These genes included the key components involved in the metabolism of osmolytes, such as proline, glyceraldehyde-3-phosphate, UDP-glucuronosyl, S-adenosylmethionine and protective proteins (Table 1). We cloned and analysed the regulatory sequences of five strongly down-regulated genes, which belong to putative protein families of sucrose nonfermenting 1-related protein kinase (RU09156), protein phosphatase 2C (RU23063), delta1-pyrroline-5-carboxylate synthase 1 (RU20896), glutathione S-transferase (RU07111) and TGF-beta receptor interacting protein (RU01501), respectively. The results showed that core DNA sequences CGT[G/A] of NAC binding were universally present within the promoters (Table S6), and EMSA revealed that RhNAC3 could bind to their putative NAC recognition sites, respectively (Figure S4). Reduced transcript levels of these osmolyte-related genes may result in poor osmotic adjustment ability, thereby contributing to reduced dehydration tolerance in RhNAC3-silenced petals.

Table 1. Expression levels of genes involved in the biological process of response to osmotic stress in RhNAC2 and RhNAC3-silenced petals
IDaDescriptionTRV-RhNAC3TRV-RhNAC2
ECFbSDECFbSD
  1. SD, Standard Deviation; TRV, tobacco rattle virus; ECF, Expression Changed Fold.

  2. a

    According to Dai et al. (2012). The bold ones underlined indicate the representative genes in which regulatory sequences were isolated and used for EMSA assays in Figure S4.

  3. b

    The ECF of each gene was calculated from the average of three independent experiments expression compared with TRV control. The expression levels of TRV control was set to 1.0. Numbers in boldface indicate the relative expression level is less than 0.8 in comparison with TRV control.

Regulatory proteins
RU05027Light-regulated zinc finger protein 10.61 0.051.180.17
RU05149 myb domain protein 21 0.72 0.181.230.36
RU03368 Homeobox 1 0.70 0.071.620.28
RU00522 Homeobox 12 0.40 0.051.230.14
RU09959 Integrase-type DNA-binding superfamily protein 0.58 0.061.250.15
RU24322 B-box zinc finger family protein1.010.132.310.26
RU20838 RING/U-box superfamily protein 0.46 0.101.540.31
RU09156 Sucrose nonfermenting 1(SNF1)-related protein kinase 2.3 0.33 0.061.530.44
RU05715 SOS3-interacting protein 3 0.48 0.131.100.13
RU23970 Protein phosphatases 2C0.23 0.040.930.14
RU23063 Protein phosphatases 2C 0.43 0.100.90.12
Functional proteins
RU20896 Delta1-pyrroline-5-carboxylate synthase 1 0.29 0.08 0.26 0.06
RU03595 Delta1-pyrroline-5-carboxylate synthase 2 0.67 0.051.190.12
RU06037 Sucrose synthase 12.700.341.660.21
RU02197 Aldehyde Dehydrogenase 7B4 0.51 0.093.330.21
RU03740 S-Adenosylmethionine synthetase 1 0.38 0.071.140.32
RU06324 Glyceraldehyde-3-phosphate dehydrogenase C2 0.52 0.080.950.21
RU04349 Pyruvate dehydrogenase complex E1 alpha subunit 0.65 0.110.980.24
RU07111 Glutathione S-transferase PHI 9 0.27 0.08 0.18 0.04
RU25000 Peroxidase superfamily protein 0.31 0.06 0.69 0.20
RU02138 UDP-Glycosyltransferase superfamily protein 0.77 0.171.890.37
RU07831 CAP160 protein 0.43 0.07 0.41 0.06
RU06233 tubulin beta chain 2 0.71 0.100.920.26
RU26791 40s ribosomal protein SA 0.61 0.152.550.65
RU09813 Major facilitator superfamily protein2.010.461.850.36
RU05549 Major facilitator superfamily protein 0.56 0.071.380.20
RU01501 TGF-beta Receptor Interacting Protein 1 0.39 0.032.670.39

Because petal expansion was reduced in RhNAC3-silenced treatments, 22 genes closely related to cell wall expansion were also investigated. The results showed that four of 22 genes were down-regulated (fold change <0.8), that is, three xyloglucan endotransglycosylase-related genes and one expansion gene (Table S1). The results indicate that RhNAC3 may not directly regulate cell wall-related gene to affect petal expansion in rose flower.

Enhancement of drought tolerance in RhNAC3-overexpressing Arabidopsis

The role of the RhNAC3 gene in drought tolerance was also investigated in RhNAC3-overexpressing Arabidopsis. Three representative lines were selected for further experiments with differential expression levels of RhNAC3 under the control of a strong constitutive promoter (Figure 5a). There were no obvious morphological differences between the RhNAC3-expressing and control plants in terms of size of rosette leaves (Figure S5a) and inflorescent plant height (Figure S5b).

Figure 5.

Tolerance of RhNAC3 overexpressors to water deficit stress.

To test the performance of RhNAC3 transgenic lines under drought stress in soil, 2-week-old RhNAC3-expressing and control plants were grown under gradually reduced water availability. When the relative soil water content (SWC) was decreased to ~10%, the control plants exhibited more wilting, and the relative aerial biomass production was reduced to ~45% (Figure 5b, c and Figure S6). Under dehydration conditions, rosette leaves from transgenic plants lost water more slowly than did leaves from control plants. After a 5-h incubation, three independent transgenic lines retained approximately 50%, 55% and 56% of their fresh weights. However, control plants retained only approximately 46% of their fresh weights (Figure 5d). These results indicated that RhNAC3 improved drought tolerance in transgenic Arabidopsis via higher water retaining abilities.

Overall gene expression changes in RhNAC3-overexpressing plants

The gene expression profile of the RhNAC3-overexpressing lines was compared with that of the control plants under normal conditions using ATH1 microarray analysis. This analysis revealed that 219 genes were up-regulated (Table S2). These up-regulated genes corresponded to signal transduction (21 genes), transcription regulation (32 genes), stress response (19 genes), enzymes and metabolism (55 genes), cell expansion (4 genes) and others (88 genes). To verify the results of microarray analysis, we tested the expression levels of nine strongly up-regulated genes in RhNAC3-overexpressors. The results of qRT-PCR analysis supported the reliability of the microarray data (Figure S7; Table S2). We searched the Arabidopsis genome database to obtain the promoter regions of the up-regulated genes listed in Table S2. Most of them (216/219) have the NAC core motif, CGT[G/A], within their promoter regions. We further analysed the response to osmotic stress of RhNAC3-up-regulated genes according to the AtGenExpress global stress expression data set described by Kilian et al. (2007). In total, 154 of 219 RhNAC3-up-regulated genes were also found to be involved in the response to osmotic stress, such as detoxification (e.g. glutathione S-transferases), osmolyte biosynthesis (e.g. galactinol synthase) and antioxidants (e.g. alcohol dehydrogenase) (Table S2).

Different transcription regulation of RhNAC2 and RhNAC3 in rose petals and Arabidopsis

In comparison with the effects of RhNAC2 in rose petals and Arabidopsis (Dai et al., 2012), RhNAC3 showed similar phenotypes in tolerance to dehydration. As RhNAC2 and RhNAC3 belong to different subgroups of NAC TF family, we further compared the difference of their transcription regulation. We determined the expression levels of genes in response to osmotic stress and related to cell expansion in RhNAC2-silenced petals (Table 1, Table S1). The results showed that, different with RhNAC3-silenced petals, only 4 of 27 osmotic stress-related genes were reduced at least by ~20%, while 17 of 22 cell expansion-associated genes were repressed in RhNAC2-silenced petals. Furthermore, we compared the difference of up-regulated genes between RhNAC2- and RhNAC3-overexpressing Arabidopsis. We found that only three genes were commonly up-regulated according to the ATH1 microarray analysis (Figure S8). According to the AtGenExpress global stress expression data set described by Kilian et al. (2007), only 24 of 136 RhNAC2-up-regulated genes were found to be involved in response to osmotic stress (Figure S8 and Table S3). Taken together, different from RhNAC2, RhNAC3 affects rose petal expansion may be mainly through regulating the expression of the osmotic stress-related genes.

Discussion

The NAC superfamily is one of the largest TF families found only in plants, and they play diverse roles in plant development and in the response to environmental stimuli (Olsen et al., 2005; Puranik et al., 2012). Among them, many stress-responsive NAC genes belong to the SNAC group, being involved in abiotic and biotic stress signalling pathways in plants (Nuruzzaman et al., 2010). In the last decade, genes belonging to the SNAC group have been identified and functionally characterized in many plant species, such as ANAC72 in Arabidopsis (Fujita et al., 2004), OsNAC5 and OsNAC6 in rice (Jeong et al., 2013; Nakashima et al., 2007) and GmNAC20 in soybean (Hao et al., 2011). Increasing evidence has shown that the expression of SNAC group genes is a response to abiotic- and biotic-stresses (Nakashima et al., 2012; Olsen et al., 2005; Puranik et al., 2012). In this study, a new member of the SNAC TFs, RhNAC3, was identified in rose flowers (Figure 1, Figure S1). The transcript levels of RhNAC3 were significantly induced by dehydration, ABA, ethylene and wounding treatments (Figure 2a, b). In addition, it was also shown that RhNAC3 was localized in the nucleus and exhibited transactivation in yeast and Arabidopsis protoplasts with higher activities of C-terminal than the full-length protein (Figure 3). This may be owing to the conserved hydrophobic LVFY motif in RhNAC3′s N-terminal (motif 2 in Figure S1b), which was identified as transcription repression domain in GmNAC20 (Hao et al., 2010). These data indicate that RhNAC3 encodes a typical transcriptional factor and might be involved in both abiotic and biotic stress in rose.

It has also been demonstrated that overexpressing SNAC genes improved drought or dehydration tolerance in some plant species, such as ANAC019, ANAC055 and ANAC072 in Arabidopsis (Tran et al., 2004), SNAC1 in rice (Hu et al., 2006) and ZmSNAC1 in maize (Lu et al., 2012). Recently, similar functions of SNAC genes have been reported in other plant species, such as soybean (Hao et al., 2011) and millet (Eleusine coracana) (Ramegowda et al., 2012). This study indicated a similar role for RhNAC3 in the dehydration tolerance of rose petals and Arabidopsis plants (Figures 4 and 5). Using a VIGS approach, RhNAC3 was silenced in rose petals, and silenced samples resulted in a significant increase in sensitivity to dehydration (Figure 4). Meanwhile, overexpressing RhNAC3 in Arabidopsis conferred significant drought tolerance in both seedlings and detached leaves (Figure 5). These results indicate that the RhNAC3 gene improved the dehydration tolerance in rose petals, similar to its homolog genes in other plant species.

Plants have evolved a set of tolerance mechanisms ranging from perception of the stress signal to a series of metabolic, physiological and molecular responses (Shao et al., 2009; Umezawa et al., 2006). The regulatory mechanisms of SNAC genes for enhanced drought or dehydration tolerance in plants have been explored through microarray analysis (Puranik et al., 2012).

In rose flowers, the indicator of petal tolerance to dehydration is defined as the ability of the dehydrated flowers to open fully during rehydration (Dai et al., 2012). Flower opening depends mainly on petal expansion, which is a coordination process including cell wall metabolism, changes in cell turgor pressure and restructuring of the cytoskeleton (Zonia and Munnik, 2007). Osmotic adjustment plays an important role in the petal tolerance to dehydration. One of the important stress responses in plants is the synthesis or degradation of a variety of solutes, such as proline, glucose and sucrose (Merchant et al., 2006; Shao et al., 2009; Warren et al., 2007). These solutes could contribute to maintenance of homeostasis, detoxification of harmful elements and recovery of growth through osmotic adjustment under stress conditions (Xiong and Zhu, 2002). On the other hand, cell expansion requires an increase in osmotic pressure. Osmotic adjustment is shown to be a major internal factor that influences flower opening. It can help to maintain cell turgor and stabilize cell proteins, which is necessary for cell expansion. An increase in cell turgor facilitates water influx into vacuoles, which is required for petal cell expansion and rose flower opening (Yamada et al., 2009).

A previous study showed that a range of osmotic adjustment-related biological processes responded to dehydration in rose petals according to GO term analysis, including the polysaccharide catabolic process, the proline biosynthetic process and the glutamine family amino acid biosynthetic process (Dai et al., 2012). Here, the expression levels of genes involved in the biological process of osmotic stress (Go ID: 0006970) were tested in RhNAC3-silenced petals, and 24 of the 27 genes were down-regulated compared with TRV control in RhNAC3-silenced petals (Table 1). Those genes may take part in the osmotic adjustment in different signalling pathways in plants. HAB1 and HAB2, belonging to plant protein phosphatase 2C (PP2C) type genes, have prominent roles in ABA signalling and mediate the changes in turgor pressure of guard cells (Fuchs et al., 2013; Israelsson et al., 2006). P5CS1, a key rate-limiting enzyme for proline synthesis, can help in raising the osmotic pressure and maintaining both the turgor pressure and the driving gradient for water uptake under stress conditions (Majumder et al., 2010; Szekely et al., 2008). The results showed the RhNAC3 improved petal cell expansion under dehydration mainly through regulating the expression of osmotic stress-related genes to adjust cell turgor pressure.

The microarray analysis of RhNAC3-overexpressing transgenic plants in Arabidopsis revealed that a number of genes related to signal transduction, stress response and key components of enzymes and metabolism were up-regulated (Table S2). Similarly, improvement in drought tolerance by SNAC proteins in Arabidopsis, rice and wheat appears to be associated with enhanced expression levels in transgenic lines of ANAC019, ANAC055 or ANAC072 (Tran et al., 2004), SNAC2 (Hu et al., 2008), OsNAC9 (Redillas et al., 2012) and TaNAC69 (Xue et al., 2011). Such profiling data are preliminary findings in revealing the molecular basis of SNAC genes for improving tolerance to drought in plants. Interestingly, among the up-regulated genes, many of them have been documented with functions in the responses to osmotic stress or adaptations to abiotic stresses according to Kilian et al. (2007) (marked with ‘O’ in Table S2). For instance, many of the calmodulin and calmodulin-binding proteins are involved in the osmotic stress response and are related to cell expansion in trichome stalk expansion or cotton fibre elongation (Bouché et al., 2005; Ji et al., 2003; Preuss et al., 2003). Mitochondrial dicarboxylate carriers (AGI code: At4g24570) confer high-osmolarity adaptation (Izumitsu et al., 2009; Yoshimi et al., 2003). These results in Arabidopsis also support the view that RhNAC3 can activate osmotic-related gene expression and are consistent with the results in rose petals here.

This regulatory mechanism of RhNAC3 is quite different from the previous observation of RhNAC2, a member of the development-related NAC subgroups, which improved petal cell expansion mainly through regulating the expression of cell wall-related genes, such as RhEXPA4 under dehydration in rose flower (Dai et al., 2012). The further comparison also revealed that RhNAC3 mainly regulated the osmotic stress-related genes, whereas RhNAC2 mainly regulated cell expansion-related genes in rose petals (Table 1, Table S1) and Arabidopsis (Table S3). Although both RhNAC2 and RhNAC3 showed a similar phenotype in conferring tolerance to dehydration in rose petal and Arabidopsis (Dai et al., 2012), they belong to different subgroups in NAC TF family (Figure 1b) and show obviously different transcription regulation in dehydration tolerance.

The stress-responsive ANAC072 is the close homologue of RhNAC3 in Arabidopsis (Figure 1b). The functional overlap of its downstream genes was also analysed in transgenic Arabidopsis (Table S4). Among the 61 ANAC072-up-regulated genes, 31 genes were also involved in the response to osmotic stress with only three ones commonly regulated by RhNAC3 (Figure S8 and Table S4). This differences may be partially owing to the experimental methods used in ANAC072 (cDNA microarray) (Tran et al., 2004) and RhNAC3 (ATH1 Genome Array). This result may also imply that RhNAC3, a heterologous TF, might have changed the binding specificity and regulate the diverse osmotic genes comparing with its homologue in Arabidopsis.

In conclusion, a rose SNAC gene, RhNAC3, was identified and functionally characterized. Silencing RhNAC3 in rose petals reduced the dehydration tolerance of petals and overexpressing it in Arabidopsis enhanced tolerance to drought stress of plants. RhNAC3 functions mainly through regulating osmotic stress-related genes. All of the data presented suggest that RhNAC3 positively regulates the osmotic stress-related genes when encountering drought stress.

Experimental procedures

Plant materials and treatments

Cut roses (Rosa hybrida cv. Samantha) were harvested at opening stage 2 and placed immediately in water. The definitions of flower opening stages and pretreatment procedures have been described previously (Ma et al., 2006).

For gene expression profile analysis, rose flowers at stage 2 were used. For dehydration treatment, the flowers were placed horizontally on a bench for 1, 3, 6, 12 and 24 h, and control flowers remained in water throughout the experiments. For phytohormone treatments, flowers were placed in a vase with 100 μm ABA, 20 μm NAA (Dafny-Yelin et al., 2005), 100 μm GA or 10 μm BR for 24 h, respectively. For ethylene (Eth) treatment, the flowers were put in a sealed airtight chamber for 24 h, and 1 m NaOH was placed in the chamber to prevent the accumulation of CO2 (Ma et al., 2006). A mock sample was treated with DMSO only without any phytohormones added for 24 h. Wounding treatment was performed as described previously (Ma et al., 2005).

Cloning and sequence analysis of RhNAC3

The full-length sequence of RhNAC3 was amplified using the Rapid Amplification of cDNA Ends (RACE) method and nested PCR according to the manufacturer's recommendations (Clontech, Palo Alto, CA). All PCR products were subcloned into pGEM T-Easy Vector (Promega, Madison, WI) and transformed into Escherichia coli DH5a cells and sequenced. Primer sequences are listed in Table S5. Protein motif analysis was conducted by Multiple EM for Motif Elicitation (MEME) (Bailey et al., 2009). Phylogenic analyses were conducted using the clustalw program (Thompson et al., 1994), and a phylogenetic tree was constructed by the neighbour-joining method with 500 bootstrap replicates using mega5.0 software (Tamura et al., 2011).

Quantitative RT-PCR analysis

For quantitative RT-PCR analysis, 1 μg of DNase-treated RNA was used for the first-strand cDNA synthesis (Invitrogen, Carlsbad, CA); 2 μL cDNA was used as the template in a 20 μL qRT-PCR using Applied Biosystems StepOnePlus™ real-time PCR system with KAPA™ SYBR® FAST qPCR kits (Kapa Biosystems, Woburn, MA). The rose Ubiquitin1 gene (Ubi1, GenBank accession no. JK622648) and the Arabidopsis Actin2 gene (GenBank accession no. NM_112764) were used as the internal control in rose and Arabidopsis, respectively. Each qRT-PCR was run in three biological replicates. Primer sequences are listed in Table S5.

Subcellular localization of RhNAC3

The coding sequence of RhNAC3 was cloned into the modified pCAMBIA 1300 vector at Hind III and Pst I restriction sites. Both the fusion construct (RhNAC3-GFP) and the control vector (GFP) were transformed into onion epidermal cells. After incubation for 24 h in the dark at 23 °C, these cells were analysed with bright field and fluorescence using confocal microscopy (Nikon Inc., Melville, NY).

Transactivation assay of RhNAC3 in yeast and Arabidopsis protoplasts

For the transactivation assay in yeast (Saccharomyces cerevisiae), different portions of RhNAC3 (the full-length, N-terminal and C-terminal of RhNAC3 protein) were PCR-amplified and inserted into the vector of pBD (Stratagene, La Jolla, CA). Expression vectors (pBD-X), with pGAL4 (positive vector) or pBD (negative vector), were introduced into the yeast host strain YRG-2, respectively, according to the manufacturer's protocol (Stratagene). The transcriptional activation activities and quantification of transformants were performed as described in the yeast protocols handbook (PT3024-1; Clontech, Mountain View, CA). Three transformants from each transfection were selected randomly.

For the transactivation assay in Arabidopsis protoplasts, a GAL4 transient expression system was used (Miura et al., 2007). The ORF of RhNAC3 and truncated fragments (N-terminal and C-terminal of RhNAC3) were amplified using specific primers and inserted into the GAL4-BD vector (Hao et al., 2011). The reporter plasmid contains the GUS reporter gene. Both the effector constructs and reporter construct were cotransfected into Arabidopsis protoplasts (Yoo et al., 2007). GUS activities were assayed by the fluorometric method (Jefferson et al., 1987). Four biological replicates were conducted. The primers are listed in Table S5.

Silencing of RhNAC3 in rose petals and petal discs by VIGS

Virus-induced gene silencing (VIGS) RhNAC3 was performed according to the procedures described previously (Dai et al., 2012) with some modifications. A 389-bp fragment of RhNAC3 was used to generate the pTRV2-RhNAC3 construct. pTRV1, pTRV2 and pTRV2-RhNAC3 vectors were transformed into Agrobacterium tumefaciens GV3101, respectively. The transformed A.tumefaciens lines were grown at 28 °C in Luria-Bertani medium supplemented with 10 mm MES, 20 mm acetosyringone and 50 μg/mL kanamycin and 50 μg/mL gentamycin sulphate with shaking at 2.3 g for about 24 h. A. tumefaciens cells were harvested and suspended in the infiltration buffer (10 mm MgCl2, 200 mm acetosyringone, and 10 mm MES, pH 5.6) to a final OD600 around 1.8. A mixture of A. tumefaciens cultures containing pTRV1 and pTRV2-RhNAC3 in a ratio of 1:1 (v/v)(TRV-RhNAC3), as well as a mixture containing pTRV1 and pTRV2 in a 1:1 ratio (the negative control, TRV), was placed at room temperature for 4 h in dark before vacuum infiltration.

Rose flowers at stage 2 were used. One-centimetre-diameter discs were taken from the centre of the petals with a hole punch. For vacuum infiltration, rose petals were placed into the bacterial suspension solution and infiltrated under vacuum at 0.5 MPa for 15 s. After release of the vacuum, petals were washed in deionized water and kept in deionized water for 3 days at 8 °C, then at 23 °C for 1 days. Petals were dehydrated for 12 h and then placed in deionized water for rehydration for 24 h. The width, length and fresh weight of all petals were determined at intervals. The petals were sampled after 12 h dehydration to determine the VIGS efficiency using qRT-PCR. AbsE cell photography and cell counting were performed as described previously (Ma et al., 2008).

After the VIGS procedure, petal discs were dehydrated for 12 h and then rehydrated for 24 h. The discs were monitored using a digital camera (Nikon D200). The discs area was determined by photoshop cs6 software. The experiments with petal discs were repeated five times using at least 90 discs in each repetition. Student's t-test (**:P < 0.01, *:P < 0 .05) was used for statistical analysis.

Determination of drought tolerance in RhNAC3-overexpressing Arabidopsis

The ORF of RhNAC3 was cloned into pCAMBIA 1300 binary vector under the control of constitutive super promoter, which consists of three copies of the octopine synthase enhancer in front of the manopine synthase promoter (Yang et al., 2008). The recombinant plasmid was introduced into Arabidopsis (ecotype Columbia) by the Agrobacterium tumefaciens-mediated floral dip method (Clough and Bent, 1998). Transgenic plant seeds were screened on MS agar medium containing 35 mg/L hygromycin. T3 homozygous plants were used for further analysis.

Water deficit stress was imposed by withholding water from containers with 80 ± 0.5 g (dry weight) of soil media (peat: vermiculite = 1:1) for 2-week-old plants grown in a controlled environment (23 °C ± 1 °C, 30%–40% relative humidity, and 12 h light / 12 h dark). Plants were irrigated with water to saturation and weighted at the start of the water deficit stress treatment (initial weight) and then periodically throughout the treatment period. Relative SWC was calculated as (final fresh weight − dry weight) / (initial weight − dry weight) × 100. Aerial parts of plants were detached at different relative SWC, and the dry weights were recorded as biomass production. Relative biomass production was calculated related to the dry weights of aerial parts under normal irrigation. Twelve plants were used for each experiment, and all experiments were repeated three times.

Water loss assay of the aerial parts of 3-week-old seedlings was conducted on the laboratory bench (23–25 °C, 30%–40% relative humidity, and 100 μmol/m2/s light intensity) and weighed at designated time intervals. Student's t-test (**P < 0.01, *P < 0.05) was used for statistical analysis.

ATH1 microarray analysis

Three-week-old seedlings of RhNAC3-transgenic plants and vector plants were harvested under normal conditions, and total RNAs from the aerial portion were isolated by the TRIzol method (Invitrogen); 20 μg of total RNA was used for microarray analysis according to the manufacturer's instructions (Affymetrix; http://www.affymetrix.com). The raw microarray data were normalized (GeneChip Robust Multiarray Averaging) and analysed using the Genespring GX package (Agilent Technologies). The genes with higher than twofold changes were considered as up-regulated. These genes were converted to their corresponding probe ID and description using the AGI number to search the mips database (http://mips.gsf.de). Some unannotated genes were further analysed using the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/). The 1000 bp regulatory sequences of RhNAC3-up-regulated genes were searched and analysed by TAIR Loci Upstream Seq—1000 bp of ‘Sequence Bulk Download and Analysis at www.arabidopsis.org’.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 31071827; 31372096) and Beijing Nova Program (Grant No. 2009B51).

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