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Optimization of TaDREB3 gene expression in transgenic barley using cold-inducible promoters

Authors


  • Accession numbers: TdCor39 promoter (Acc. JQ991624)

Correspondence (fax +61 8 830 37 102;

email sergiy.lopato@acpfg.com.au)

Summary

Constitutive over-expression of the TaDREB3 gene in barley improved frost tolerance of transgenic plants at the vegetative stage of plant development, but leads to stunted phenotypes and 3- to 6-week delays in flowering compared to control plants. In this work, two cold-inducible promoters with contrasting properties, the WRKY71 gene promoter from rice and the Cor39 gene promoter from durum wheat, were applied to optimize expression of TaDREB3. The aim of the work was to increase plant frost tolerance and to decrease or prevent negative developmental phenotypes observed during constitutive expression of TaDREB3. The OsWRKY71 and TdCor39 promoters had low-to-moderate basal activity and were activated by cold treatment in leaves, stems and developing spikes of transgenic barley and rice. Expression of the TaDREB3 gene, driven by either of the tested promoters, led to a significant improvement in frost tolerance. The presence of the functional TaDREB3 protein in transgenic plants was confirmed by the detection of strong up-regulation of cold-responsive target genes. The OsWRKY71 promoter–driven TaDREB3 provides stronger activation of the same target genes than the TdCor39 promoter. Analysis of the development of transgenic plants in the absence of stress revealed small or no differences in plant characteristics and grain yield compared with wild-type plants. The WRKY71–TaDREB3 promoter–transgene combination appears to be a promising tool for the enhancement of cold and frost tolerance in crop plants but field evaluation will be needed to confirm that negative development phenotypes have been controlled.

Introduction

Among the various transcription factors (TFs) reported to be associated with a response to abiotic and biotic stress in plants, the most studied are the C-repeat (CRT) binding factors (CBFs) or drought-responsive element (DRE) binding (DREB) proteins. CBF/DREB TFs regulate transcription of drought/cold stress–related genes by binding to an (A/G)CCGAC core motif (Gao et al., 2002; Kizis and Pages, 2002; Maruyama et al., 2004; Sakuma et al., 2006; Stockinger et al., 1997; Xue, 2002). The involvement of CBF/DREB TFs in the regulation of plant responses to abiotic stresses has been shown for many different plant species (reviewed in Lata and Prasad, 2011; Sanghera et al., 2011). The utilization of CBF/DREB genes for engineering the ability of plants to better survive extreme environmental conditions has also been demonstrated (Agarwal et al., 2006; James et al., 2008; Lata and Prasad, 2011; Sanghera et al., 2011). Enhanced cold/frost tolerance was achieved in agriculturally important species where CBF/DREB genes were constitutively expressed at high levels (Ito et al., 2006; James et al., 2008; Kasuga et al., 2004; Lee et al., 2004; Morran et al., 2011; Pellegrineschi et al., 2004; Zhang et al., 2011). Unfortunately, most reports on constitutive over-expression of CBF/DREB genes show that the value of positive results was reduced by the development of negative, pleiotropic phenotypes, which included stunted growth, drastically delayed flowering and lower yields compared to control plants (Hsieh et al., 2002; Ito et al., 2006; Kasuga et al., 2004; Liu et al., 1998; Morran et al., 2011). Attempts to decrease the pleiotropic effects of TF over-expression by using stress-inducible promoters have been reported (James et al., 2008; Kasuga et al., 1999, 2004; Morran et al., 2011; Xue et al., 2011). Several stress-inducible promoters have been assessed in transgenic plants (Fu et al., 2007; James et al., 2008; Kasuga et al., 1999; Morran et al., 2011; Msanne et al., 2011; Nakashima et al., 2007; Ouellet et al., 1998; Pellegrineschi et al., 2004; Takumi et al., 2003; Xiao and Xue, 2001; Xu et al., 1996).

The most important characteristics associated with activation of stress-inducible genes are the strength and duration of expression. Stress-activated genes also have different constitutive, stress-independent levels of expression. According to the time required for activation after the application of stress, these genes could be divided into early and late responsible (Mahajan and Tuteja, 2005). Furthermore, expression of stress-responsive genes and therefore the activity of their promoters can either be dependent or independent of ABA signalling (Shinozaki and Yamaguchi-Shinozaki, 2000; Yamaguchi-Shinozaki et al., 2006). The Cor39 gene from wheat, like most other COR genes, belongs to late stress-responsive genes with its maximal expression reached several hours after plant exposure to cold. Cor39 was originally identified as a gene encoding a group II LEA protein that accumulates in root, leaf and crown tissues of wheat during cold acclimation (Guo et al., 1992). In the absence of stress, transcripts of Cor39 were present at relatively high levels in wheat seeds and accumulated in plants in response to exogenous application of ABA and water-deficit stress (Guo et al., 1992). A close homologue of the Cor39 gene in wheat, WCS120, is strongly induced during cold acclimation (Houde et al., 1992b). The antibodies raised against the WCS120 recognize a small family of proteins coordinately induced by cold (Houde et al., 1992a). Accumulation of these proteins was higher in a freezing-tolerant genotype compared to a more sensitive genotype. Analysis of different species (eight monocots and four dicots) indicated that this protein family is specific for freezing-tolerant cereals (Houde et al., 1992a). Immunohistochemical localization showed that WCS120-like proteins are highly expressed in the vascular transition zone. The cold-sensitive cells of this zone are situated near the regions where water tends to freeze first. The importance of this zone has been supported by the observation that regrowth after freezing stress is highly dependent on the viability of the tissues in the crown (Houde et al., 1995). Electron microscopy showed that WCS120-like proteins are present in the cytoplasm and in the nucleoplasm. It was suggested that they might be involved in a general protection mechanism of the plant cells (Houde et al., 1995). The 860-bp-long WCS120 promoter was isolated and shown by transient expression to be cold inducible in different freezing-tolerant and freezing-sensitive monocot and dicot species, suggesting that common TFs responsive to low temperature may be present in all plants (Ouellet et al., 1998).

The barley homologue of Cor39, HvDHN5, was initially identified as a major cold-inducible dehydrin in barley (Van Zee et al., 1995). Facultative and winter barley cultivars showed a higher level of DHN5 accumulation and greater frost tolerance than spring cultivars (Kosova et al., 2008).

OsWRKY71 (Gene Bank Acc. BK005074; JRC0189 in Rabbani et al., 2003) belongs to the group of early stress-response genes and has a low level of constitutive expression. It reached a maximum level of expression within 1 h of incubation at 4 °C, but in contrast to genes encoding many other TFs, its expression was prolonged; after 24 h of stress, the level of expression remained constant (Rabbani et al., 2003). OsWRKY71 was strongly induced by cold and moderately by drought and salt stresses in rice. It is not up-regulated upon treatment with endogenous ABA, suggesting it is regulated through the ABA-independent pathway for stress responses (Rabbani et al., 2003). The OsWRKY71 gene was also shown to be up-regulated by several defence signalling molecules, such as salicylic acid, methyl jasmonate, 1-aminocyclo-propane-1-carboxylic acid, as well as biotic elicitors, wounding and pathogen infection. This suggests that OsWRKY71 might also function in rice biotic stress responses (Chujo et al., 2008; Liu et al., 2007). In the absence of stress, OsWRKY71 is highly expressed in aleurone cells, where it blocks GA signalling by functionally interfering with OsGAMYB (Zhang et al., 2004). The barley orthologue of OsWRKY71, HvWRKY38, was shown to be early and transiently expressed during exposure to low non-freezing temperatures, in an ABA-independent manner. It also showed continuous induction during dehydration and freezing treatments (Maré et al., 2004).

In this study, two promoters of stress-inducible genes, OsWRKY71 and TdCor39, with contrasting properties, including time of activation, strength and dependence upon ABA, were used for the cold-inducible expression of the wheat DREB gene TaDREB3 in transgenic barley and rice plants with the aim of optimizing transgene expression for engineering cold stress tolerance. The work includes the detailed analysis of promoter activation under cold stress, the frost tolerance of transgenic barley plants transformed with promoter–TaDREB3 constructs and characterization of the transgenic plants. The results show that application of cold-inducible promoters in combination with the TaDREB3 gene has improved frost tolerance and moderated the negative pleiotropic phenotypes typically observed in plants with strong constitutive over-expression of DREB genes.

Results

Analysis of TdCor39 and OsWRKY71 gene expression and cloning of promoters

Expression of the Cor39 gene was analysed in different tissues of unstressed bread wheat plants. Low levels of expression were detected in embryos, leaves and coleoptiles, and higher levels were observed in roots and reproductive tissues. The highest level of expression was observed in endosperm (Figure 1a). The Cor39 gene was induced by ABA treatment in the green part of 10-day-old seedlings (Figure 1b). In leaves, the Cor39 gene was strongly activated by cold and moderately by drought (Figure 1c–e). Induction by cold was initially detected after 4 h of incubation at 4 °C and reached a maximum after several hours. After 48 h of cold treatment, the level of TdCor39 expression remained high (Figure 1e). In contrast, activation by wounding was more moderate than by cold, but was detected much earlier, 15 min after stress was applied. Expression of the TdCor39 gene reached its maximum 2 h after wounding, while 4 h after stress, it returned to a low level and remained at this level for at least 48 h (Figure 1f).

Figure 1.

Expression of the Cor39 gene in various wheat tissues and activation by different stresses demonstrated by Q-PCR. (a) Expression of the TaCor39 gene in different tissues of bread wheat in the absence of stress. (b) Expression of TaCor39 in the green part of 10-day-old seedlings treated by 200 μm ABA; control—growth media contained DMSO, but no ABA. (c) Expression of TaCor39 gene in leaves of 4-week-old seedlings of bread wheat subjected to drought stress and following rewatering. (d) Expression of TdCor39 gene in leaves of 4-week-old seedlings of durum wheat subjected to drought stress and following re-watering. The right parts of graphs in (c) and (d) show soil water potential; the arrows show the time of re-watering. (e) Expression of TdCor39 gene in leaves of 4-week-old seedlings of durum wheat subjected to cold stress at constant 4 °C. (f) Expression of TaCor39 in leaves of 4-week-old seedlings subjected to wounding. Y-axes in all cases show ‘Normalized transcript levels in arbitrary units’.

Activation of the OsWRKY71 gene by ABA, cold, salt and drought was demonstrated by Rabbani et al. (2003) and activation by wounding and pathogen infection was shown by Liu et al. (2007), and therefore, these experiments were not repeated in this study.

The TdCor39 and OsWRKY71 promoters were inserted upstream of the TaDREB3 coding region as described in Materials and Methods and used for barley transformation. Transgenic plants were selected using PCR and/or Northern blot hybridization analysis for transgene (TaDREB3) expression.

Comparison of developmental phenotypes of transgenic barley plants

Seventeen independent transgenic barley lines transformed with the pCor39–TaDREB3 construct and fourteen transformed with pWRKY71–TaDREB3 were selected by PCR using transgene-specific primers. Five independent transgenic lines (L5, L12, L18, L19 and L20) transformed with the pCor39–TaDREB3 construct and three lines (L2, L5 and L16) for the pWRKY71–TaDREB3 construct were taken for the further analysis. The selection was based on basal levels of transgene expression in T0 plants and the extent of promoter activation by cold and dehydration in T1 progeny (data not shown).

Six of the selected transgenic lines (three for each promoter) were used for the comparison of phenotypes and grain yields of transgenic and control plants. Seven T1 plants for each transgenic line and seven control plants (wild-type barley) were grown in the glasshouse, and transgene copy number and basal levels of transgene expression were examined for each plant (Figure 2). All T1 plants except L16-4 (WRKY71 promoter) and L12-6 (Cor39 promoter) contained a single copy of the transgene per haploid genome (Figure 2a). All plants of Line 12 and four plants of Line 18 had high basal levels of transgene expression (Figure 2b). Eight null segregants were detected by both Q-PCR (genomic DNA was used as template) and confirmed on the level of transgene expression by Northern blot hybridization (data not shown) and quantitative RT-PCR (Figure 2). They were combined in a second group of control plants for analysis in addition to the wild-type plants (Figure 3). Plant height and the length of leaves were measured at the end of the fourth week after germination. Tiller number was evaluated at the beginning of flowering. Spike number, spike length, number of spikelets per spike, grain number and grain weight were analysed after harvest (Figure 3). Pictures of all analysed transgenic and control plants 1 week before flowering are depicted in Figure S2. Analysis of transgenic plants revealed a small decrease in plant height for both types of transgenic plants when compared with control plants. No difference in leaf length was observed for plants transformed with pWRKY71–TaDREB3 construct. However, significant variation of leaf length was found for pCor39–TaDREB3 transgenic barley lines (Figure 3). The number of tillers at flowering was significantly smaller in transgenic barley transformed with pCor39–TaDREB3 construct, although the number of harvested spikes was roughly the same as for control plants. In contrast, the OsWRKY71 promoter constructs resulted in no differences in the number of tillers or spikes between transgenic and control plants (Figure 3). Both types of transgenic plants had a delay in flowering (Figure 4). The average delay in flowering time of transgenic barley plants versus control plants were 9.8 days for the TdCor39 promoter and 3.6 days for the OsWRKY71 promoter, which are much shorter than 3–6 weeks delay in flowering of barley T1 plants transformed with 2x35S–TaDREB3 construct (Morran et al., 2011). Interestingly, some null segregants (marked with stars in the figure) also showed up to 4 days delay in flowering time, indicating that transformation and regeneration processes might slightly influence plant development (Figure 4). There was no significant difference between the number of spikes in transgenic and control plants. However, the length of the spikes and the number of spikelets per spike were significantly smaller in one of the WRKY71 and two of the Cor39 lines. Line 16 (the WRKY71 promoter) also showed much stronger level of transgene expression than in the other two tested lines. The total grain weight per plant, grain number per spike and single seed weight were lower in transgenic barley transformed with the pCor39–TaDREB3 construct than for control plants. In contrast, grain yield from barley transformed with pWRKY71–TaDREB3 was the same or similar (L16) for both groups of control plants (Figure 3). Comparison of results obtained in this experiment (Experiment 2) with results of the similar experiment performed earlier on T1 transgenic barley plants with constitutive expression of TaDREB3 (Experiment 1) are shown in Table S1.

Figure 2.

Transgene copy number and constitutive levels of transgene expression in barley plants used for the phenotyping. (a) Copy number of TaDREB3 gene in T1 barley plants measured by quantitative PCR. (b) Basal levels of OsWRKY and TdCor39 promoter activity in transgenic barley plants measured by quantitative RT-PCR. Control—untransformed wild-type barley.

Figure 3.

A comparison of phenotypes and grain yields of transgenic and control (WT) barley plants. Results for the eight null segregants (L16-5, L2-1, L2-2, L2-7, L5-5, L5-7, L18-2 and L19-1) were combined together as a second control (Null). Seven WT plants and four to seven transgenic plants for each line were used in the experiment. Differences between transgenic lines and WT plants were tested in a Pearson's chi-squared test (*mean P-value <0.1).

Figure 4.

Delay in flowering time for T1 transgenic plants transformed with either pWRKY71–TaDREB3 or pCor39–TaDREB3 constructs. Flowering time of transgenic plants was compared with the average flowering time of seven control (WT) plants, which is represented as Day 0. Null segregants are marked with asterisks.

Frost tolerance tests of transgenic barley plants

Three-week-old T1 barley seedlings, 6–12 plants for each transgenic line, were used for frost survival tests in an automatic cold cabinet. Design and conditions of the experiment were described by Morran et al. (2011) and can be seen in the Figure 1S. Five transgenic lines (L5, L12, L18, L19 and L20) for the pCor39–TaDREB3 construct and three (L2, L5 and L16) for the pWRKY71–TaDREB3 construct were tested in three independent experiments. All transgenic lines demonstrated significantly higher survival rates than control (wild-type) plants (Figures 5, 6,S3). Transgene expression before the cold treatment and after 7 h of temperature decrease from 18 to 4 °C and following 5 h of incubation at 4 °C was assessed by Northern blot hybridization. Most of the transgenic plants demonstrated strong transgene activation by cold; some plants had relatively high basal levels of transgene expression, although the results of the Northern blot hybridization can only be considered as semi-quantitative. Plants, where transgene expression was not detected, were excluded from the survival rate data.

Figure 5.

Results of the frost test for barley plants transformed with pWRKY–TaDREB3 construct. (a) Survival rates for control plants (WT) and three independent transgenic lines. (b) Transgene activation in tested plants demonstrated by Northern blot hybridization: A—no stress; B—stress; C—WT plants; P—positive control (RNA isolated from pUbi-TaDREB3 plants).

Figure 6.

Results of the frost test for barley plants transformed with pCor39–TaDREB3 construct. (a) Survival rates of control plants (WT) and three independent transgenic lines. (b) Transgene activation in tested plants demonstrated by Northern blot hybridization: A—no stress; B—stress; C—WT plants; P—positive control (RNA isolated from pUbi-TaDREB3 plants).

To compare frost tolerance of transgenic lines with inducible and constitutive expression of the TaDREB3 gene, we used seeds of two homozygous T1 sublines (G330-2-4 and G330-5-3) transformed with pWRKY71–TaDREB3 construct and one homozygous T3 subline transformed with 2x35S–TaDREB3 (G62-11-4-1-19) in the same experiment (Figure 7). The subline G62-11-4-1-19 was selected as subline with the best developmental phenotype among the progeny of 3 lines with strong constitutive TaDREB3 expression (data not shown). Height of seedlings was measured 1 day before frost tolerance test. The plants with constitutive expression of TaDREB3 were significantly smaller than control plants. In contrast, size of transgenic barley plants where TaDREB3 expression was driven by OsWRKY71 promoter was the same or very similar to the size of control plants (Figure 7a). All three transgenic plants demonstrated significant improvement in frost tolerance of vegetative tissues (Figure 7a). These experiments demonstrate stability of transgene function in several generations. Although constitutive expression of transgene still gave the best result in respect of frost tolerance, no difference in flowering time and yield of transgenic lines with inducible expression of transgene compared to control plants compensate this small advantage. Furthermore, thorough analysis of the larger number of lines and sublines can potentially provide lines with even better characteristics compared to already selected lines.

Figure 7.

Results of the frost test for homozygous barley plants transformed with either pWRKY–TaDREB3 or 2x35S–TaDREB3 constructs. (a) Height of 3-week-old seedlings. (b) Survival rates for control plants (WT), two independent pWRKY–TaDREB3 lines (T2 generation: G330-5-3 and G330-2-4) and one p2x35S–TaDREB3 line (T4 generation: G62-11-4-1-19).

Three T1 plants for each construct (one plant per independent transgenic line) were used for the analysis of transgene expression. All plants, except L18-5, demonstrated a low-to-moderate basal level of activity and strong activation of the transgene by cold. The L18-5 had very high basal level of promoter activity and very small further activation of the TdCor39 promoter by cold (Figure 8). Overall expression was stronger in plants transformed with the pCor39–TaDREB3 construct. Expression of three cold-responsive genes, HvCor14b, HvDhn5 and HvA22, were tested in the same transgenic plants with the aim to compare levels of expression of the transgene and downstream target genes, thus indirectly confirming the presence of the functional TaDREB3 protein. All three genes were induced by cold in control plants. However, their induction by cold in transgenic plants was much stronger. As observed earlier for barley plants with constitutive expression of TaDREB3 (Morran et al., 2011), expression levels of the HvCor14b gene correlated well with levels of transgene expression, suggesting possible direct regulation of this gene by TaDREB3 (Figure 8). There was less correlation between the expression patterns of the transgene and the two other tested genes, HvDhn5 and HvA22, although the expression levels of both of the genes in all transgenic plants were higher than in control plants. Surprisingly, transgenic plants transformed with the pWRKY71–TaDREB3 construct showed an overall lower up-regulation of the transgene at the transcriptional level than the plants transformed with the pCor39–TaDREB3 construct but stronger up-regulation of downstream genes (Figure 8).

Figure 8.

Activation of transgene (TaDREB3) and three cold-responsive downstream genes in control (WT) and selected T1 transgenic barley plants demonstrated by Q-PCR; a—leaf samples collected before cold stress; b—leaf samples collected during stress (See Figure S1 for conditions of cold stress).

Spatial activity of TdCor39 and OsWRKY71 promoters

To assess tissue-specific activation of TdCor39 and OsWRKY71 promoters by cold, two lines for each construct (one flowering T1 plant per line, all with single copy of transgene) were incubated at a constant 4 °C and tissue samples were collected at 0, 2, 5 and 7 h of incubation. TaDREB3 expression was analysed by Q-PCR. Activation of both promoters by cold was observed in all tested tissues, although basal and inducible transgene expression levels were very different (Figure 9). The OsWRKY71 promoter was activated in all tested tissues with higher levels in leaves than in spikes and stems. Activation of the promoter in stems after 7 h of cold stress for both tested lines was less than two fold over the constitutive level of promoter activity. In contrast, activation of the TdCor39 promoter over basal level of activity in stem was stronger than activation of the OsWRKY71 promoter.

Figure 9.

Activation of the OsWRKY71 and TdCor39 promoters in leaf, stem and developing spike by incubation of plant at constant 4 °C demonstrated by Q-PCR. Two T1 lines for each promoter were used in the experiment.

Discussion

Constitutive over-expression of stress-related TFs often leads to an improvement in plant survival under stress (Hsieh et al., 2002; Kobayashi et al., 2008a,b; Oh et al., 2005, 2007; Takumi et al., 2008). However, strong constitutive over-expression of TFs frequently results in negative developmental phenotypes in transgenic plants (Hsieh et al., 2002; Ito et al., 2006; Jaglo-Ottosen et al., 1998; Kasuga et al., 1999; Liu et al., 1998; Morran et al., 2011). Stress-inducible promoters have been used to overcome this negative influence (James et al., 2008; Kasuga et al., 1999; Kasuga et al., 2004; Morran et al., 2011; Pellegrineschi et al., 2004) but the number of well-characterized cold-inducible promoters tested in grasses is limited (Ouellet et al., 1998; Santos et al., 2009; Soltész et al., 2012).

The core purpose of this study was to explore the use of promoters that responded to cold stress to drive the expression of stress-related TFs. The wheat DREB3 gene was selected for these studies because it was previously demonstrated that constitutive over-expression of this gene in barley under the 2x35S promoter significantly improved frost tolerance of transgenic seedlings. However, the transgenic plants showed stunted growth, delayed flowering and reduced grain yield (Morran et al., 2011). The aim of this research was to decrease the pleiotropic effects of TaDREB3 over-expression on barley development while enhancing frost tolerance by using stress-inducible promoters.

For this work, we compared two promoters from different cold-activated genes in combination with the same stress tolerance gene, TaDREB3, in transgenic barley plants. The activation of both OsWRKY71 and TdCor39 genes was induced by several abiotic stresses and wounding. One of these genes, OsWRKY71, belongs to the early stress-response genes from rice. It was demonstrated that this gene (designated as JRC0189 in Rabbani et al., 2003) is induced within 1 h by cold, drought and high salinity but induction by cold was stronger than for the other stresses (Rabbani et al., 2003). Expression of the OsWRKY71 gene is ABA independent (Rabbani et al., 2003). Here, we demonstrated that the OsWRKY71 promoter was activated by cold in stems, leaves and spikes of transgenic barley (Figure 9). The OsWRKY71 gene was also reported to be expressed in the aleurone cells of rice grains in the absence of stress (Zhang et al., 2004). Due to the low basal activity, moderate strength under stress, early activation and independence from the ABA-mediated pathway, the OsWRKY71 promoter was selected as one of candidate promoters for our study. Another promoter selected for this work belongs to the TdCor39 gene, a member of the cold adaptation family of WCS120-like genes. Initially, Cor39 was described by Guo et al. (1992) as a gene with low basal level of expression and strong induction by ABA, cold and dehydration. Under similar conditions of cold treatment, the TdCor39 gene reached a maximum level of expression several hours later than the OsWRKY71 (Rabbani et al., 2003). Under treatment at a constant 4 °C, both OsWRKY71 and TdCor39 genes remained active for at least 24 h. Our data (Figure 1) together with earlier published data for the OsWRKY71 gene (Chujo et al., 2008; Liu et al., 2007) suggest that expression of both genes can also be induced by wounding and/or pathogens. However, in contrast to activation by abiotic stresses, induction by wounding was rapid and not prolonged.

Our results demonstrated that the cold-inducible promoters reduced the negative pleiotropic phenotypes previously observed when TaDREB3 was constitutively expressed (Morran et al., 2011). In the case of the OsWRKY71 promoter, the detrimental phenotype was strongly reduced and partial improvement was observed when the TdCor39 promoter was applied (Figure 3). The negative influence of TaDREB3 expression correlated with the strength of promoter activity (quantified on the level of mRNA expression), which was higher overall for transgenic lines with the TdCor39 promoter and lower for transgenic lines with the OsWRKY71 promoter (Figure 8). Most of the tested T1 barley transgenic plants had a single copy of the transgene (Figure 2b). However, levels of both constitutive and inducible expression were very different (Figures 2b,c and 8). This difference in expression levels suggests a strong influence of the position of the insertion event in the genomic DNA. Among the possible causes of the variation in promoter activity are occasional distal enhancer/repressor elements and/or differences in accessibility to chromatin for transactivators. Nevertheless, such variability of promoter strength allows for selection of lines that show no yield penalty in the absence of stress in ‘good years’, but increased vegetative frost tolerance in years with extreme temperatures. However, the stability of transgene expression in the selected lines over multiple generations still needs to be evaluated.

Another interesting observation was the influence of the different promoter/gene constructs on the initiation of tillers. TaDREB3 driven by the OsWRKY71 promoter led to the initiation of the same or more tillers than in control plants, whereas use of the TdCor39 promoter led to a significant decrease in tiller number (Figure 3). This phenomenon can be explained by possible differences in the spatial patterns of background expression from the promoter. Reports from the literature suggest that the Cor39-like genes may be active in tissues that are responsible for tiller initiation. Immunohistochemical localization of the WCS120-like proteins demonstrated their abundance in the vascular transition zone of crown meristematic tissue (Houde et al., 1995), which is important for the regrowth of wheat seedlings after harsh winter conditions (Tanino and McKersie, 1985). However, it is not clear whether the expression of TaDREB3 under the TdCor39 promoter in shoot meristems and particularly in the vascular transition zone is responsible for the reduced or delayed initiation of tillers. The OsWRKY71 promoter may be inactive or weak in the tissues responsible for tiller initiation, and therefore, we see no major reduction in tiller number. However, the progeny of Line 16 where the OsWRKY71 promoter was used did show reduced tiller number and yield and there was a delay in flowering. This could be the result of the unusually strong transgene expression in this line.

The expression of three cold-inducible LEA/COR/DHN genes, HvCor14b, HvDHN8 and HvA22, was studied by Q-PCR to confirm the presence of the functional TaDREB3 protein in transgenic plants. These three genes were earlier found to be up-regulated in response to constitutive over-expression of TaDREB3 (Morran et al., 2011). As it was expected, all three target genes were up-regulated by cold treatment to much higher levels in transgenic plants than in controls (Figure 8). However, contrary to expectations, the weaker OsWRKY71 promoter resulted in stronger activation of two from three tested downstream genes than the more active TdCor39 promoter. The possible explanations for this result is different spatial patterns of transgene expression, and the presence/absence of modifying or modulating cofactors in particular tissues/cells that in combination with TaDREB3 could influence expression of particular downstream genes. Stronger activation of the transgene in plants with the weaker OsWRKY71 promoter suggests that ‘transcriptional’ strength of the promoter (transgene mRNA level) does not necessarily correlate to induction of downstream genes and, if used as criteria for promoter selection, should be used with caution.

Analysis of frost survival rates of 3-week-old transgenic barley seedlings was repeated several times for each promoter–TaDREB3 construct, and transgene activation by cold was confirmed by Northern blot hybridization in all experiments. Transgenic seedlings with both promoter–TaDREB3 constructs demonstrated significantly improved frost survival relative to control plants (Figures 5, 6 and S3). This represents a significant improvement in the tolerance of vegetative tissues to frost damage. The frost tolerance experiment was repeated using T2 progeny of two selected sublines and T4 progeny of our best so far subline with constitutive expression of TaDREB3. These experiment demonstrated stability of transgene function in several generations. Although constitutive expression of transgene produced the best result in respect of frost tolerance, the advantage was compensated with absence of any significant difference in flowering time and yield of control plants and transgenic lines with inducible transgene expression. Furthermore, thorough analysis of the larger number of lines and sublines can potentially provide with lines having even better characteristics than already selected lines.

In many regions, frost at flowering is the major cause of yield loss. For example, in Australia, damage caused by frost at flowering is estimated to cost the wheat and barley industries around $100 million annually. The overnight frost events during flowering stage can damage the sensitive reproductive tissues and cause near-total loss of grain. Both the OsWRKY71 and TdCor39 promoters were activated by cold in all tested tissues including developing spikes (Figure 9). Therefore, the best selected lines with good frost tolerance at vegetative stage of plant development and minimal differences in flowering time and grain yield from control plants will be used in frost tolerance tests at flowering.

In summary, the use of cold-inducible promoters reduced the severity of negative developmental phenotypes of transgenic barley compared with constitutive expression. However, the promoter of OsWRKY71 is recommended to use for cold-inducible expression of DREB genes. The more powerful TdCor39 promoter did not perform as well as the OsWRKY71 promoter in driving expression of the low-abundance TaDREB3 gene, but it may be useful for expressing genes encoding more abundant TFs.

Experimental procedures

Promoter cloning and plasmid construction

The full-length coding region of the TaCor39 cDNA (GeneBank accession AF058794) was isolated by PCR using a cDNA library obtained from spikes of drought-stressed wheat (Triticum aestivum cv. Chinese Spring) as a template. The TaCor39 cDNA was used as a probe to screen a BAC library prepared from genomic DNA of Triticum durum cv. Langdon (Cenci et al., 2003), as described by Kovalchuk et al. (2009). The T. durum homologue of the TaCor39 gene was identified by PCR using DNA of the selected BAC clone (#891 H17) as template and primers derived from the coding region of TaCor39 cDNA. The gene of the T. durum orthologue of TaCor39 was designated TdCor39. The TdCor39 promoter sequence was identified through the sequencing of the BAC clone as described by Kovalchuk et al. (2009).

A 2326-bp-long fragment of the promoter sequence upstream of the translation start codon of OsWRKY71 (Acc. AP004087; 76083-78409 bp of the BAC clone) was isolated by PCR using rice (Oryza sativa L. ssp. japonica cv. Nipponbare), genomic DNA as template. It was cloned into the pENTR-D-TOPO vector and verified by sequencing. New vectors were generated for plant transformation, where the 2x35S promoter was excised from the pMDC32 vector using HindIII–KpnI restriction sites (Curtis and Grossniklaus, 2003) and replaced with either a 3121-bp-long fragment of the TdCor39 promoter or 2326-bp-long fragment of the OsWRKY71 promoter. The vectors were designated pCor39 and pWRKY71, respectively. The coding region of TaDREB3 cDNA (Lopato et al., 2006) was cloned into the pENTR-D-TOPO vector (Invitrogen, Melbourne, Victoria, Australia). The cloned insert was verified by sequencing and subcloned into pCor39 and pWRKY71 vectors, resulting in pCor39–TaDREB3 and pWRKY71–TaDREB3 constructs.

Barley transformation and analysis of transgenic plants

The pCor39–TaDREB3 and pWRKY71–TaDREB3 constructs were transformed into barley (Hordeum vulgare L. cv. Golden Promise) using Agrobacterium-mediated transformation (Matthews et al., 2001; Tingay et al., 1997). Transgene integration was confirmed by PCR using the forward primer from the 3′ end of the promoter and the reverse primer from the 5′ end of the nos terminator. The transgene copy number was estimated in T1 progeny of selected transgenic lines using Q-PCR. The basal level of activity of the TdCor39 and OsWRKY71 promoters in unstressed leaves of transgenic T0 lines was demonstrated either by Northern blot hybridization or by Q-PCR.

Transgenic barley plants were grown in either a growth room (for cold and drought tests) or in a glasshouse (for characterization of plant phenotypes). Growth room temperatures were maintained at 24 °C during the 12 daylight hours and 18 °C during the night, and the average relative humidity was 50% during the day and 80% during the night. Wild-type plants were used as control. For experiments in hydroponics, seeds were initially germinated at room temperature in Petri dishes on wet filter paper and grown hydroponically in a base growth solution changed every 7 days containing 5 mm NH4NO3, 5 mm KNO3, 2 mm Ca(NO3)2·4H2O, 2 mm MgSO4·7H2O, 0.1 mm KH2PO4, 50 μm NaFe(III)EDTA, 50 μm H3BO3, 10 μm ZnSO4·7H2O, 5 μm MnCl2·4H2O, 0.5 μm CuSO4·5H2O and 0.1 μm Na2MoO3 (Shavrukov et al., 2010) at pH 5.5 in a glasshouse at 15 °C (night) to 23 °C (day) with a 14-h photoperiod. Two-week-old seedlings were either subjected to cold stress at 4 °C or to dehydration by withholding growth solution for 7 h. In the case where experiments were performed in pots, seeds were germinated directly in soil. For cold treatment, control and transgenic seedlings were grown first for 3 weeks in soil in a growth room and were then transferred to a cold cabinet (BINDER GmbH, Tuttlingen, Germany) and kept at constant temperatures of either 2 or 4 °C for up to 8 h. Leaves were collected for RNA isolation before stress was applied and during cold treatment at 2, 5 and 8 h.

For frost survival tests, 3-week-old seedlings were exposed to gradual temperature decreases to a minimum of −6 °C and then slowly returned to a maximum of 18 °C (Figure S1). Leaf tissue was collected before stress and after the temperature had decreased to 4 °C (Figure S1). When the temperature reached −5 °C, plants were sprayed with a 2 g/L solution of Snomax (York Snow, Victor, NY) to initiate simultaneous ice crystallization before the temperature reached −6 °C. The temperature then decreased to −6 °C for 10 h and treatment continued according to the regime described in the Figure S1. When the temperature returned to 18 °C, plants were transferred to the growth room to recover. The number of survived plants was estimated 2 weeks after recovery; survivors were repotted and transferred to a glasshouse for seed production. Homozygous T1 plants were selected in the pilot experiment, where 12 seedlings were germinated for each of selected T1 lines with two copies of transgene. The absence of null segregants was demonstrated by PCR using transgene-specific primers. Seeds of these T1 sublines were used to repeat frost tolerance experiment with the aim to compare these sublines with earlier published T3 subline of 2x35S–TaDREB3 transgenic plant (Morran et al., 2011).

RNA isolation and Northern blot hybridization

Collected plant material was immediately frozen in liquid nitrogen and stored at −80 °C until RNA extraction. Total RNA was isolated from wild-type and transgenic plant leaf tissue using the TRIzol kit (Invitrogen). RNA was electrophoretically separated on a 1.3% agarose gel containing 6% formaldehyde, transferred to nylon membrane and hybridized with 32P-labelled DNA probes according to the protocol described by Church and Gilbert (1984).

Quantitative PCR

Transgene copy number was estimated by efficiency-adjusted real-time quantitative PCR. A modified ΔΔCt method adjusted for amplification efficiency was used to determine the number of copies of the transgene per genome in each sample (Yuan et al., 2008). DNA was extracted from leaf tissue using a freeze-drying method described by Shavrukov et al. (2010). Prior to use in quantitative real-time PCR, each DNA sample was diluted with sterile deionized water to be within the copy-standard serial dilution range (12.5–200 ng/μL). For template loading normalization, PCRs were performed using primers and probes complimentary to single-copy endogenous reference genes. In the case of barley, primers and probes complimentary to a portion of the barley orthologue of the Puroindoline-b (Pin-b) gene from wheat were used (Li et al., 2004). The oligonucleotide sequences were as follows: forward 5′-ATTTTCCAGTCACCTGGCCC-3′; reverse 5′-TGCTATCTGGCTCAGCTGC-3′; and dual-labelled TaqMan probe 5′-CAL fluor Gold 540-ATGGTGGAAGGGCGGCTGTGA-BHQ1-3′.

For the various transgenes analysed, a portion of the hygromycin resistance gene (Hyg) was used as the target sequence. The complimentary oligonucleotide sequences were as follows: forward 5′-CGCTCGTCTGGCTAAGATCG-3′; reverse 5′-AGGGTGTCACGTTGCAAGAC-3′; and dual-labelled TaqMan probe 5′-FAM-TGCCTGAAACCGAACTGCCCGCTG-BHQ1-3′. Quantitative real-time PCRs were performed on LightCycler 480 thermal cycler. Each PCR comprised 1× IQ Supermix (Bio-Rad, SA, Australia), forward and reverse primers (400 nm each), dual-labelled probe (200 nm), DNA (2 μL) and deionized water to a total volume of 10 μL. The thermal cycling parameters were 95 °C for 3 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s with fluorescence readings acquired at each cycle on the yellow and green channels.

To allow for the calculation of transgene copy number from unknown DNA samples, a copy-standard serial dilution series was set up. Genomic DNA from a plant known to contain a single copy of the hygromycin gene in addition to the single-copy endogenous reference gene was extracted and diluted. Amounts of 400, 200, 100, 50 and 25 ng were used. Three replicate PCRs for each unknown sample and each diluted copy-standard sample were performed with each primer/probe set. Ct values were calculated using the supplied software for LightCycler 480 thermal cycler. The PCR efficiency for each primer/probe set was determined via analysis of the Ct values obtained from the diluted copy-standard series. Subsequently, all Ct values were adjusted using these PCR efficiency values prior to calculating ΔCtadjustedCtadjusted = Hyg Ctadjusted−reference gene Ctadjusted). The transgene copy number for each unknown sample was determined by calculating 2−ΔΔCtadjusted. (ΔΔCtadjusted is defined as the difference between the average ΔCtadjusted of the copy-standard series and the ΔCtadjusted of the unknown sample.) Calculated transgene copy numbers were rounded to the closest integer.

Quantitative RT-PCR analyses of the Cor39 gene expression in different tissues and under several stresses were performed as described by Burton et al. (2008). For gene expression analysis, the cDNA tissue series were prepared from different tissues of T. aestivum cv. Chinese Spring as described by Morran et al. (2011). The stress cDNA series for Q-PCR analysis were prepared from three to four leaves from each of 2–4 6-week-old plants of either T. aestivum cv. RAC875 or T. durum cv. Langdon subjected to each of the following stresses: cold stress at 4 °C (samples were collected at 0, 1, 4, 24 and 48 h after stress application), drought (samples were collected from well-watered plants growing in soil, and plants at different stages of drought until plants became strongly wilted (volumetric water content in soil was 3%)) and wounding with a metal brush (samples of T. durum were collected at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 7, 12, 24, 36 and 48 h after wounding). For ABA treatment, seeds were surface-sterilized and left to germinate for 2 days at 4 °C in Petri dishes on wet filter paper. After 2–3 days, seedlings were transplanted to two hydroponics growth units and were grown one week in a glasshouse. The growth solution (Shavrukov et al., 2010) was renewed the day before ABA treatment. ABA was dissolved in DMSO to make a 50 mm stock and 2 mL was added to growth solution making final ABA concentration of 200 μm. Control plants received 2 mL of DMSO. Aerial portions of three seedlings were harvested at each time point (0, 1, 2 and 4 h). The results of all experiments are based on 2–3 biological replicas.

The specific primers used in the Q-PCR for the analysis of the expression of transgene (TaDREB3) and downstream genes in transgenic barley plants were listed in Morran et al. (2011).

Acknowledgements

We thank Ursula Langridge, Ming Li and Alex Kovalchuk for assistance with growing plants in the glasshouse and collecting samples for analysis, and Natalia Tikhomirov and Margaret Pallotta for the BAC library screening. This work was supported by the Masters of Plant Biotechnology Project (PLANTSC 7229B WT) at the University of Adelaide, by the Australian Research Council, the Grains Research and Development Corporation, Government of South Australia and the DuPont Ag Biotechnology Company.

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