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Keywords:

  • NAC domain;
  • abscisic acid;
  • stress;
  • Arabidopsis thaliana;
  • transcriptional activation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

Arabidopsis thaliana RD26 cDNA, isolated from dehydrated plants, encodes a NAC protein. Expression of the RD26 gene was induced not only by drought but also by abscisic acid (ABA) and high salinity. The RD26 protein is localized in the nucleus and its C terminal has transcriptional activity. Transgenic plants overexpressing RD26 were highly sensitive to ABA, while RD26-repressed plants were insensitive. The results of microarray analysis showed that ABA- and stress-inducible genes are upregulated in the RD26-overexpressed plants and repressed in the RD26-repressed plants. Furthermore, RD26 activated a promoter of its target gene in Arabidopsis protoplasts. These results indicate that RD26 functions as a transcriptional activator in ABA-inducible gene expression under abiotic stress in plants.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

Plants as sessile organisms are directly exposed to environmental stresses such as drought, cold and high salinity. The mechanism of the molecular response of higher plants to these stresses has been investigated by studying the genes upregulated under stress conditions (Bray, 1997; Ingram and Bartels, 1996; Shinozaki and Yamaguchi-Shinozaki, 1997; Zhu, 2002). We isolated 25 independent cDNAs for drought-inducible genes in Arabidopsis thaliana by using the technique of differential screening (Kiyosue et al., 1994; Taji et al., 1999; Yamaguchi-Shinozaki et al., 1992). Sequence analysis of these genes showed that the proteins encoded by these genes, for instance, a group II LEA protein (Iwasaki et al., 1997), cysteine protease (Koizumi et al., 1993), and plasma membrane intrinsic protein (Yamaguchi-Shinozaki et al., 1992), may function to protect cells from dehydration. In addition, regulatory genes such as Ca2+-binding proteins have also been identified (Takahashi et al., 2000). Recently, further gene-expression profiling using cDNA microarrays or gene chips revealed many other genes that are regulated by cold, drought or salt stress (Kawasaki et al., 2001; Seki et al., 2001).

Although not all genes responsive to drought stress require abscisic acid (ABA) mediation, a large number of drought-inducible genes is induced by exogeneous ABA (Busk and Pages, 1998; Rock, 2000; Shinozaki and Yamaguchi-Shinozaki, 1997, 2000). Drought, salt and low-temperature stresses induce accumulation of ABA that plays a crucial role in adaptation to abiotic stress (Finkelstein et al., 2002; Leung and Giraudat, 1998; Zeevaart and Creelman, 1988). Many ABA-inducible genes contain a conserved, ABA-responsive, cis-acting element named ABA-responsive element (ABRE; PyACGTGGC) in their promoter regions (Guiltinan et al., 1990; Mundy et al., 1990; Yamaguchi-Shinozaki et al., 1990). ABREs are recognized by transcription factors containing a basic leucine zipper structure (Choi et al., 2000; Finkelstein and Lynch, 2000; Hobo et al., 1999; Uno et al., 2000). Other transcription factors such as the B3 domain, MYC and MYB proteins are also involved in ABA-responsive signal transduction (reviewed by Finkelstein et al., 2002; Shinozaki et al., 2003).

NACs are genes encoding a polypeptide containing a plant-specific highly conserved N-terminal domain, NAC (for NAM, ATAF1, 2, and CUC2) reported by Aida et al. (1997). In the Arabidopsis genome, about 100 putative members of NAC genes have been identified (Ooka et al., 2003; Riechmann et al., 2000). Among them, NAM of petunia (Souer et al., 1996) and CUC2 of Arabidopsis (Aida et al., 1997) have been shown to participate in the development and maintenance of shoot apical meristem (SAM). Recently, two additional NAC genes, CUC1 and CUC3 have been reported to be involved in SAM development (Takada et al., 2001; Vroemen et al., 2003). Another Arabidopsis NAC gene, NAP (Sablowski and Meyerowitz, 1998), has been isolated as a target of Apetala3 and suggested to control cell expansion in specific flower organs. NAC1 was shown to be involved in auxin signaling in lateral root formation (Xie et al., 2000). Other NAC genes have been found to be upregulated during senescence (John et al., 1997) or by wounding and bacterial infection in potato (Collinge and Boller, 2001; Hegedus et al., 2003; Mysore et al., 2002). Furthermore, some NAC proteins have been shown to mediate viral resistance (Ren et al., 2000; Xie et al., 1999). Thus, members of the NAC family seem to play various roles not only in plant development but also in the recognition of environmental stimuli.

In this study, we show that a drought-inducible gene, RD26, encodes a NAC transcription factor. To investigate the in vivo functions of RD26, we generated transgenic Arabidopsis plants constitutively overexpressing or repressing RD26. Here, we show that RD26-overexpressing lines are hypersensitive to ABA and that many ABA- and abiotic stress-responsive genes were upregulated. On the contrary, RD26-repressed plants were less sensitive to ABA and the expression of ABA-responsive genes was repressed in the plants. Here, we suggest that RD26 participates in a novel ABA-dependent stress-signaling pathway.

RD26 encodes a NAC domain protein

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

The RD26 cDNA encodes a protein of 297 amino acids and is a member of the plant-specific NAC gene family. The N-terminal NAC domain contains predicted nuclear localization signals (NLS) between amino acid 76–88 and 113–179 (Figure 1a). A database search showed that there are 109 NAC genes in the Arabidopsis genome (Riechmann et al., 2000). The evolutionary relationship among 67 NAC proteins that showed the closest homology to RD26 was analyzed using the amino acid sequence of the NAC domain (Figure 1b). The phylogenetic analysis revealed that RD26 belongs to a group that includes ATAF1 and ATAF2. Northern blot and microarray analysis using the Agilent 22K Oligo DNA Microarray (K. Maruyama, JIRCAS, Tsukuba, Ibaraki, Japan unpublished data) showed that seven NAC genes that belong to the ATAF subfamily are responsive to ABA, dehydration and NaCl treatments. As none of the other NAC genes except for five genes, shown in Figure 1(b), were induced by these treatments (data not shown), only members of the ATAF subfamily may function in the ABA-dependent stress-signaling pathway. Although the C-terminal sequences of NAC proteins are divergent, two genes closest to RD26 show relatively high homology (∼30%) even in the C-terminal region (data not shown). The ANAC gene that has been reported to interact with a RING-H2 domain protein (Greve et al., 2003) is one of the closest homologues of RD26.

image

Figure 1. Nucleotide sequence and phylogenetic tree of the RD26 gene. (a) Nucleotide and amino acid sequence of the RD26 gene. The NAC domain is boxed, and the putative nuclear localization signal (NLS) indicated in Kikuchi et al. (2000) is shaded. Open triangles represent introns. The first intron is 109 nucleotides in length, with its 5′ splice junction at position 282 of the cDNA, and the second intron is 83 nucleotides, with its 5′ splice junction at position 552. (b) Phylogenetic tree of 67 NAC genes. NAC domain sequences were aligned using the Clustal X program with the following parameter sets: gap open penalty = 5.00, gap extension penalty = 0.05. The alignment was finally adjusted manually. A phylogenetic tree was constructed by the neighbor-joining method using the mega software (Saitou and Nei, 1987). The confidence level of monophyletic groups was estimated by bootstrap analysis of 1000 replicates. *Stress response analyzed by microarray analysis using the Agilent 22K Oligo DNA Microarray (K. Maruyama, unpublished data). A, ABA; D, drought; N, NaCl; C, cold.

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Expression of RD26

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

The RD26 cDNA was isolated by screening the cDNA library that had been prepared from Arabidopsis rosette plants dehydrated for 10 h, and the accumulation of RD26 mRNA was shown to be induced by dehydration (Yamaguchi-Shinozaki et al., 1992). We further analyzed the effects of various environmental stresses on the expression of the RD26 genes by RNA gel blot analysis. As shown in Figure 2(a), the RD26 gene was induced within 30 min after dehydration, NaCl and ABA treatments, and the mRNA level was continuously increased up to 24 h. RD26 was also responsive to methyl jasmonate, H2O2 and Rose Bengal, which are known to generate reactive oxygen species (ROS) (Green and Fluhr, 1995; Narusaka et al., 2003b). In the ABA-deficient aba2 mutant, the accumulation of RD26 mRNA during dehydration was markedly decreased suggesting that the dehydration-responsive expression of RD26 is regulated mainly by ABA (Figure 2b). On the contrary, in the NaCl-treated aba2 mutant, significant induction of RD26 was still observed (Figure 2b), implying the existence of an ABA-independent pathway of NaCl signaling for RD26 expression.

image

Figure 2. Expression of RD26 gene. (a) RNA gel blot analysis of expression of the RD26 gene under stress conditions. Each lane was loaded 5 μg of total RNA from 3-week-old Arabidopsis plants that had been dehydrated on Whatman 3MM paper (dry), transferred from agar plates to hydroponic growth in 250 mm NaCl, 50 μm ABA, 50 μm methyl jasmonate (MeJA), 50 μm salicylic acid (SA), 50 μm GA3, 20 mm H2O2, 25 μm paraquat, 10 mm Rose Bengal, or water (H2O), or transferred to and grown at 4°C (cold) for indicated times. The membranes were hybridized with [32P]-labeled 3′-specific region of RD26 cDNA as a probe. The amount of RNA loaded in each lane is represented by the amount of rRNA shown below or at the right of the blots. The degree of dehydration is shown by the percentage of leaf weight (FW) at each time point. Each data point represents the average of quadruplicate measurements (n = 4 each). SD: standard deviation. (b) RD26 expression in response to drought and high salinity in aba2 mutant or wild type (Columbia). Three-week-old plants were exposed to dehydration or high salinity described as above. (c) Histochemical analysis of RD26 promoter::GUS transgenic Arabidopsis. Three-week-old plants were grown under normal conditions (upper) or exposed to dehydration for 5 h (lower). (d) Distribution of cis-acting elements in the regulatory regions (1.5 kb upstream of 5′ end of full-length cDNA sequences) of RD26 gene. DNA sequences similar to the reported c/s-acting element are shown as follows: open circles, ABRE; closed diamonds, MYB recognition site; open diamonds, MYC recognition site; filled square, DRE; open triangles, W-box; filled triangle, as1; filled box, TATA.

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The more detailed spatial expression pattern of RD26 was determined by histochemical β-glucuronidase (GUS) staining of transgenic plants that harbored a RD26 promoter::GUS reporter construct. Figure 2(c) shows the expression of the RD26 promoter::GUS fusion gene in transgenic Arabidopsis plants raised under normal and water-deficit conditions. Weak expression of the RD26 promoter::GUS fusion gene was observed in several regions of rosette plants raised under normal growth conditions (Figure 2c, upper). By contrast, the dehydration of transgenic Arabidopsis plants strongly induced GUS expression in all organs and tissues (Figure 2c, lower), indicating that the RD26 promoter functions in all vegetative tissues of Arabidopsis plants during dehydration.

Figure 2(d) summarizes the sequences of various cis-acting elements observed in the promoter region of RD26. We found four ABREs, one dehydration responsive element (DRE), two recognition sites for MYB and one recognition site for MYC (reviewed by Busk and Pages, 1998; Finkelstein et al., 2002; Shinozaki et al., 2003). The RD26 promoter also includes a W-box, a recognition site of WRKY transcription factors. Members of the WRKY transcription-factor family appear to be involved in the regulation of various plant-specific physiological processes such as pathogen defense, senescence and trichome development (Eulgem et al., 2000). Additionally, the RD26 promoter has two AS-1 motifs known as oxidative stress-responsive elements (Lam et al., 1989). Therefore, in the ROS-responsive promoters, these sequences may function as cis-acting elements (Garreton et al., 2002).

Nuclear localization of GFP–RD26 fusion protein

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

The detection of putative NLS implies that RD26 may be localized to the nucleus. To examine the localization of the RD26 protein, we transiently expressed GFP–RD26 fusion protein in the epidermal cells of onion and observed the tissue by confocal microscopy. GFP–RD26 was localized in the nucleus (Figure 3a, i–iii), while GFP–ΔRD26 that lacks the NAC domain of RD26 was localized in the cytoplasm and nucleus (3a, iv–vi). These data showed that the NAC domain of RD26 is necessary for nuclear localization of RD26.

image

Figure 3. Transactivation activity and nuclear localization of the RD26 protein. (a) Nuclear localization of the RD26 protein. GFP::RD26 (i-iii) or GFP::ΔRD26 (a.a. 1–64 corresponding to NAC domain, iv–vi) fusion proteins were transiently expressed in onion epidermal cells and analyzed by confocal microscopy. (i, iv) Fluorescent images of GFP; (ii, v) fluorescent images stained with propidium iodide (N, nucleus), and (iii, vi) merged images. (b) Transactivational analysis of RD26 in yeast. Fusion proteins of GAL4 DNA-binding domain and different portions of RD26 were expressed in yeast strain AH109. The transformed yeast culture was dropped onto SD plates with or without histidine and the necessary amino acids. The plates were incubated for 3 days and applied to β-gal assay.

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The C-terminal domain of RD26 has transactivation activity

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

Several NAC family genes have been suggested to function as transcriptional factors (Aida et al., 1999; Duval et al., 2002; Xie et al., 1999, 2000). We examined the transactivation activity of RD26 using a yeast system. GAL4 DNA binding domain–RD26 fusion proteins were expressed in yeast and assayed for their ability to activate transcription from the GAL4 upstream activation sequence and promote yeast growth in the absence of histidine (Figure 3b). The full-length and C-terminal part of RD26 showed obvious activation capacity (Figure 3b, rows 2 and 4), while the N-terminal part that included the NAC domain sequence did not (Figure 3b, rows 1 and 3). The region of amino acid 164–272 still promoted growth as vigorously as that observed with the intact C-terminus (Figure 3b, row 5). However, further deletion of this region completely abolished transactivation activity (Figure 3b, row 6), suggesting that the region of amino acid 217–272 is a potential transactivation domain.

Growth phenotypes of RD26-overexpressed transgenic lines

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

To analyze the biological functions of RD26, we generated transgenic Arabidopsis plants in which RD26 was overexpressed (35S::RD26). The coding region of RD26 was fused to the 35S promoter of the cauliflower mosaic virus and the omega sequence of the tobacco mosaic virus, which elevates the translation level of the transgene. We examined the expression levels of the transgene in the 35S::RD26 plants (RD26-overexpressed plants hereafter) by RNA gel blot analysis using an RD26-specific probe (Figure 4a).

image

Figure 4. Growth phenotype of 35S::RD26 plants. (a) RNA gel blot analysis of RD26 expression in vector control and 35S::RD26 lines. Four micrograms of total RNA was applied to each lane. (b) Three-week-old seedlings of transgenic plants grown on germination medium (GM) plate containing kanamycin. Bars = 0.5 cm. (c) Eight-week-old plants. Three-week-old seedlings were transferred from GM agar plates to soil. Bars = 2 cm.

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The growth of the transgenic plants was compared with that of the wild-type plants at 3 weeks after sowing (Figure 4b). The RD26-overexpressed plants growing on germination medium (GM) agar plates had slightly chlorotic leaves with a larger leaf blade and shorter petiole than the wild-type plants (Figure 4b). When grown on soil, the RD26-overexpressed plants expressing a higher level of the transgene were much smaller than the wild-type plants, and had markedly reduced floral apical dominance, shorter bolts (Figure 4c), fewer flowers, and reduced seed yield (data not shown). The S3 line that expressed a lower level of transgene showed an intermediate phenotype (Figure 4b,c), suggesting that these morphological phenotypes were caused by overexpression of RD26.

RD26-overexpressed plants were hypersensitive to ABA

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

To evaluate the effect of RD26 overexpression on the ABA sensitivity, we germinated the RD26-overexpressed plants on or transferred them to GM media containing various concentrations of ABA. During the germination process, no obvious difference was observed between the RD26-overexpressed plants and vector control plants (data not shown). These seedlings were transferred to GM agar media containing various concentrations of ABA. Upon transfer to a medium supplemented with ABA, the seedlings of the RD26-overexpressed plants became bleached and their root growth was inhibited to a greater extent than that of the vector control plants (Figure 5a). On the medium containing 1.0 μm ABA, root growth of vector control plants was 82% of that in the medium without ABA, whereas that of the RD26-overexpressed plants was 54% of that in the medium without ABA (Figure 5b). On the medium containing 3.0 μm ABA, the root growth of the RD26-overexpressed plants was reduced to 45%. The root growth of the vector control plants in the medium containing ABA was 72% of that in the medium without ABA. These results demonstrate the hypersensitivity of the RD26-overexpressed plants to ABA.

image

Figure 5. Abscisic acid (ABA) sensitivity of 35S::RD26 (RD26-overexpressed) plants. Four-day-old seedlings grown on germination medium (GM) agar plate containing 30 mg l−1 kanamycin were transferred to GM plates containing various concentrations of ABA. (a) Growth of seedlings on GM agar plate containing indicated concentration of ABA. (b) ABA dose response of root growth. Two RD26-overexpressed lines (S4, S10) were used. Experiments were performed in triplicate (n = 6 each). The error bars represent standard deviations.

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Although we also tested the sensitivity of the plants to osmotic stresses such as high-salinity and drought, no significant tolerance was observed in our experimental conditions (data not shown).

RD26 protein transactivates the promoter of target gene in vivo

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

Using a yeast system we found that the RD26 protein may act as a transcription activator. To test this hypothesis, we preliminarily checked upregulated genes in the RD26-overexpressed plants using the ∼7000 Arabidopsis full-length cDNA microarray (Seki et al., 2002), and found that the most strongly upregulated gene in the RD26-overexpressed plants encoded a glyoxalase I family protein (GLY) (data not shown). We also carried out RNA gel blot analysis to confirm this result. The GLY gene was upregulated in both the ABA-untreated and treated RD26-overexpressed plants (Figure 6a, lanes 1–6). Dehydration, NaCl and ABA treatments also induced expression of this gene (Figure 6b), and the induction was slower than that of RD26 (Figures 2a and 6b).

image

Figure 6. RD26 transactivates the expression of GLY gene in vivo. (a) RNA gel blot analysis of GLY gene expression in vector control, 35S::RD26 lines (S4, S10) and 35S::RD26SRDX lines (R2, R18) treated with (+) or without (−) 50 μm abscisic acid (ABA) for 5 h. Each lane contained 5 μg of total RNA. (b) RNA gel blot analysis of expression of GLY gene. To each lane was applied 5 μg of total RNA from 3-week-old wild-type plants that had been exposed to dry, NaCl, ABA or cold stress for 2 or 10 h. Stress conditions were as described in Figure 2. (c) Schemes of gene constructs used in cotransfection experiments. The effector constructs contain the CaMV 35S promoter and TMV Ω sequence fused to RD26 cDNA. The reporter constructs contain the 1000-bp fragments of the GLY promoter fused to a GUS construct. (d) The nucleotide sequence of the promoter region of the GLY gene. Numbers refer to the nucleotides, beginning at the 5′ end of the GLY mRNA determined by the full-length cDNA (Seki et al., 2002). A putative TATA box is underlined. The sequences homologous to ABRE (T/CACGTG) are underlined with thick lines. The core binding motifs of NAC genes (Tran et al., 2004) are boxed. (e) Activation of the GLY promoter::GUS fusion gene by RD26 protein using Arabidopsis protoplasts. Arabidopsis protoplasts were transfected by the reporter plasmids with different sets of effector plasmids: pBI 35SΩ vector as a control (VC) and 35SΩ::RD26 (RD26). The transfected protoplasts were incubated for 24 h with (closed bars) or without (open bars) 100 μm ABA. Co-transfection of a constitutively expressed luciferase (LUC) gene allowed normalization of expression in independent experiments. Bars indicate the fold GUS activity compared with each reporter activity with pBI 35SΩ vector in the absence of ABA. The data show the results of three independent transfections ±SD.

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To determine whether the RD26 protein is capable of transactivating a promoter of the GLY gene, we performed transactivation experiments using protoplasts prepared from Arabidopsis T87 cultured cells. Protoplasts were cotransfected with a GUS reporter construct that had a deletion in the promoter region of the GLY gene and an effector plasmid containing RD26 cDNA fused to the 35S promoter and Ω sequence (Figure 6c,d). Expression of the GUS reporter gene containing the −410 or −215 bp region of the GLY promoter was moderately activated by adding ABA (Figure 6e). Co-expression of the RD26 protein strongly transactivated the −410 or −215 bp GLY promoter, and the expression level of the GUS gene was markedly elevated by ABA treatment (Figure 6e). However, the −86 bp promoter region was not activated by ABA nor was it activated by co-expression of the RD26 protein (Figure 6e). These results suggest that the RD26 protein functions as a transcription activator involved in the ABA-responsive expression of the GLY gene.

Construction of RD26-repressed plants – loss-of-function phenotype of RD26

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

Although we obtained two RD26 T-DNA insertion mutant lines, no obvious change in the phenotype was observed in our experimental conditions (data not shown). As reviewed previously (Zhang, 2003), the negative results may be attributed to the potential functional redundancy among NAC genes that belong to the ATAF subfamily. Actually, as shown in Figure 1(b), there were at least six NAC genes responsive to abiotic stress and ABA treatment other than RD26, suggesting that they work redundantly. Therefore, to minimize the effects of phenotypic masking caused by potential functional redundancy, we used a recently established dominant repression technique exploiting a repression domain, SRDX, derived from the EAR-motif of superman, a TFIIIA-type zinc finger repressor (Hiratsu et al., 2003). By using several well-studied transcription factors such as EIN3, PAP1 and AtMYB23, Hiratsu et al. (2003) demonstrated that translational fusion between these genes and the repression domain suppressed the expression of their target genes and caused loss-of-function phenotypes. Furthermore, ectopic expression of the CUC1 chimeric repressor, which had the repression domain, resulted in cup-shaped or fused cotyledons, the phenotype of cuc1/cuc2 double mutant (Aida et al., 1997); this demonstrated that the repression domain can convert a transcriptional activator into a strong suppressor which produces loss-of-function phenotypes even in the presence of redundant transcription factors (Hiratsu et al., 2003).

To generate RD26-repressed plants, we fused the RD26 coding region to the 12 amino acid-repression domain of SRDX (LDLDLELRLGFA) in frame and expressed the chimeric RD26 repressor under the control of the 35S promoter (Figure 7a). None of the 42 T1 35S::RD26SRDX seedlings that we analyzed had the cup-shaped or fused cotyledons observed in the CUC1-repressed plants (35S::CUC1SRDX; Hiratsu et al., 2003), suggesting that the overexpression of RD26SRDX confers an RD26-dependent phenotype. The transgenic plants expressing the RD26SRDX chimeric suppressor had longer petiole and smaller leaf blades than vector control plants (Figure 7b). After the transfer to soil, the 35S::RD26SRDX plants (RD26-repressed plants hereafter) grew normally (data not shown). The morphological changes in the leaves of the RD26-repressed plants were the inverse of those in the RD26-overexpressed plants that had larger leaf blades and shorter petioles (Figure 4). Furthermore, the expression of the GLY gene, the target of RD26, was repressed in the RD26-repressed plants (Figure 6a, lanes 7–10). These observations indicate that RD26 was efficiently repressed by the EAR motif and produced the loss-of-function phenotype.

image

Figure 7. Growth phenotype of 35S::RD26SRDX (RD26-repressed) plants. (a) A Schematic diagram of construct used for expression of the chimeric RD26 repressors with modified version of the EAR-motif repression domain (SRDX) consisting 12 peptides. The chimeric RD26 repressor was fused to the CaMV 35S promoter and the TMV Ω sequence. (b) RNA gel blot analysis of RD26 expression in vector control (VC) and 35S::RD26SRDX lines (R2, R18). Five micrograms of total RNA was applied to each lane. (c) Three-week-old seedlings of transgenic plants grown on germination medium (GM) plates containing kanamycin. Bars = 0.5 cm.

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RD26-repressed plants were insensitive to ABA

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

As RD26 has been shown to function as a transcriptional activator in ABA signaling, we analyzed the sensitivity of the RD26-repressed plants to ABA. As observed in the experiments of the RD26-overexpressed plants, there was no obvious difference in sensitivity between the RD26-repressed plants and vector control plants during the germination process (data not shown). Then seedlings of the 35S::RD26SRDX (RD26-repressed), 35S::RD26 (RD26-overexpressed), and vector control plants were transferred to GM agar plates containing 10 μm of ABA. One week after the transfer, the RD26-repressed plants grew normally on the ABA-containing plates, but the RD26-overexpressed plants and vector control plants became bleached (data not shown). After 3 weeks, the RD26-repressed plants were still green and continued to grow on the ABA plates, while the vector control plants showed suppressed growth or wilted (Figure 8). In contrast to the RD26-repressed plants, the RD26-overexpressed plants became bleached earlier than the vector control plants (data not shown), and were completely bleached 3 weeks after transfer (Figure 8). These results indicate clearly that RD26 acts positively in ABA signaling.

image

Figure 8. Abscisic acid (ABA) sensitivity of 35S::RD26SRDX (RD26-repressed) plants. Four-day-old seedlings grown on germination medium (GM) agar plates containing 30 mg l−1 kanamycin were transferred to GM plates with or without 10 μm of ABA and grown for 2 weeks. R2 and R18, RD26-repressed lines; VC, vector control; S4, RD26-overexpressed line.

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Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

To identify the target genes of the RD26 protein, we used the Agilent Arabidopsis 2 Oligo Microarray (Agilent Technologies, Inc., Palo Alto, CA, USA) which covers over 21 000 Arabidopsis genes. RNAs prepared from 2-week-old seedlings of the RD26-overexpressed, RD26-repressed and vector control plants with or without ABA treatment (100 μm ABA for 5 h) were used for microarray analysis. In the microarray analysis, RD26 cDNA was highly overexpressed in 35S::RD26 plants (ratio to non-treated control plants: 11.8) and 35S::RD26SRDX plants (ratio to non-treated control plants: 13.7; ratio to ABA-treated control plants: 4.3) (Table 1). Genes whose expression levels were significantly higher in the RD26-overexpressed plants are summarized in Table 1. Twenty genes were upregulated in the RD26-overexpressed plants and 15 of them were downregulated in ABA-treated RD26-repressed plants, suggesting that RD26 regulates the expression of these genes. In addition, most of the upregulated genes were responsive to ABA and abiotic stresses (Table 1). The overexpression of RD20 in the RD26-overexpressed plants is notable. RD20 encodes a Ca2+-binding protein that has been shown to respond to ABA and dehydration, and is supposed to function in ABA-mediated stress-signal transduction (Takahashi et al., 2000). The GLY gene, demonstrated as the direct target of RD26 in Figure 6, is also listed. Glyoxalase is one of the glutathione-dependent detoxification enzymes (Dixon et al., 1998). The glutathione transferase and aldo/keto reductase family gene upregulated in the RD26-overexpressed plants has also been shown to play a role in detoxification of ROS (Oberschall et al., 2000). Interestingly, many defense- and senescence-related genes were found among the genes upregulated in the RD26-overexpressed plants, for example, SAG13, a marker gene of senescence (Lohman et al., 1994). The ELI3-2 gene encoding a cinnamil-alcohol dehydrogenase is known not only as a defense-related gene (Kiedrowski et al., 1993), but also as a senescence-associated gene, SAG25 (Quirino et al., 1999). Most of the remaining genes are related to the metabolism of cellular components. These genes may be involved in morphological changes in transgenic plants.

Table 1.  Genes upregulated or downregulated in the RD26-overexpressed or RD26-repressed plants identified by microarray analysis
GeneAccession no.Experiment 1aExperiment 2bExperiment 3cStressd response
RatioeP-valueRatioeP-valueRatioeP-value
  1. aExperiment 1: upregulated in non-treated 35S::RD26 plants (non-35S::RD26/non-vector).

  2. bExperiment 2: downregulated in ABA-treated 35S::RD26SRDX plants (ABA-vector/ABA-35S:: RD26SRDX).

  3. cExperiment 3: downregulated in non-treated 35S::RD26SRDX plants (non-vector/non-35S::RD26SRDX).

  4. dStress response analyzed by microarray analysis (Seki et al., 2001; K. Maruyama, unpublished data). D, drought; N, high salinity; A, ABA; C, cold.

  5. eValues represent the mean of two replicates for two experiments (i.e. individual RNA preparations from two different lines).

  6. fUpregulated in 35S::RD26SRDX plants.

Signal transduction
 RD26At4g2741011.85.1E-32(4.3)f8.9E-22(13.7)f7.5E-31A D N C
 RD20At2g333803.32.8E-151.51.2E-023.21.6E-07A D N
Detoxification
 Glyoxalase I family protein (GLY)At1g801603.94.7E-181.51.5E-03  A D N
 Glutathione transferase, putativeAt1g597002.53.2E-116.69.2E-229.21.6E-26 
 Aldo/keto reductase familyAt2g377602.57.8E-123.61.2E-16  A D
Senescence
 Putative tropinone reductase (SAG13)At2g293504.78.7E-163.93.0E-16  A N
 Putative proteinAt3g625503.09.7E-14    N C
 Glycosyl hydrolase family 1At1g028502.85.1E-123.01.4E-131.96.7E-03D N
 Amino acid permease IAt1g583602.72.1E-122.23.5E-103.04.5E-14A D N C
Defense
 Cinnamyl-alcohol dehydrogenase (ELI3-2)At4g379904.26.2E-143.71.5E-12  A D N
 Coronatine-responsive tyrosine transaminase-like proteinAt4g236003.62.2E-177.01.1E-25  A D N
 Lysine-ketoglutarate reductase/saccharopine dehydrogenaseAt4g331502.72.7E-111.52.8E-032.43.9E-10A D N
Others (metabolism)
 Xyloglucan endotransglycosylase (XTR7)At4g141305.03.6E-192.41.4E-06  A D N
 UDP-glycosyltransferase, putativeAt4g154903.99.0E-195.21.6E-21  A D N
 Stress-responsive protein, putativeAt1g293953.75.3E-17    A D N C
 Starch excess protein-relatedAt4g244503.75.8E-13     
 Nicotianamine synthase, putativeAt1g092403.33.7E-154.57.9E-144.83.1E-12 
 Amino acid transporter family proteinAt1g082302.66.9E-111.66.5E-03   
Unknown
 Hypothetical proteinAt2g284003.63.8E-12    A D N
 Expressed proteinAt1g678603.62.1E-122.01.6E-051.88.8E-02 
 Putative proteinAt3g552403.15.7E-15     

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

We characterized RD26, a new member of the NAC-domain gene family. It is one of the RD (responsive to dehydration) genes we isolated by differential screening (Taji et al., 1999; Yamaguchi-Shinozaki et al., 1992). We found that RD26 protein functions as a transcriptional activator not only in yeast (Figure 3) but also in Arabidopsis protoplasts (Figure 6). Deletion analysis showed that the region of amino acid 214–272 is necessary for transcriptional activation, although this region has no characteristic structure. Furthermore, we confirmed that the RD26 protein contains a functional NLS in the NAC domain, suggesting that the RD26 functions in the nucleus (Figure 2a). These data are consistent with the previous observations that the other NAC genes function as transcriptional activators (Aida et al., 1999; Duval et al., 2002; Xie et al., 1999, 2000).

The expression of RD26 was induced strongly by dehydration, high salinity and ABA treatments. The dehydration-responsive expression of RD26 was found to be regulated mainly by ABA, but ABA is not needed in the response of RD26 to NaCl. The 1.5-kb promoter region of RD26 contains four ABREs, one MYC recognition site, two MYB recognition sites, and one DRE. ABRE and MYC and MYB recognition sites are involved in ABA-responsive gene expression (reviewed by Busk and Pages, 1998; Finkelstein et al., 2002; Shinozaki et al., 2003). Therefore, these cis-acting elements regulate the ABA-dependent expression of RD26. RD26 was also responsive to jasmonic acid and ROS-related reagents. We found two W-boxes and two AS-1 motifs in the RD26 promoter. These cis-elements may act as defense- and oxidative stress-responsive cis-acting elements, respectively, suggesting that they may regulate the biotic stress responses of RD26.

To investigate the in vivo functions of RD26, we generated transgenic Arabidopsis plants overexpressing intact RD26 or RD26SRDX chimeric repressor. Considering the potential functional redundancy of NAC genes, an overexpression approach would be better than a loss-of-function approach such as knockout, antisense, and RNA interference. Especially, the dominant repression technique using the EAR-motif repression domain is a unique and powerful tool for the functional analysis of redundant transcription factors (Hiratsu et al., 2003). The overexpression and repression of RD26 resulted in an opposite phenotype (Figure 4 and 7). The seedlings of the RD26-overexpressed plants had larger leaf blades and shorter petioles while the RD26-repressed plants had smaller leaf blades and longer petioles. Typically, salt-stressed plants become smaller and have shorter petioles like the RD26-overexpressed plants (Burssens et al., 2000), suggesting that stress responsive signaling is constitutively activated in the RD26-overexpressed plants.

Direct analysis of the responses of transgenic plants to exogenous ABA revealed that the overexpression of RD26 increases the sensitivity of both seedlings and roots to ABA whereas the growth of RD26-repressed plants was not inhibited by a high concentration of ABA (Figures 5 and 8). These findings suggest that RD26 functions as a transcriptional activator in ABA signal transduction in vegetative tissue.

To investigate the target genes of RD26, we performed microarray analysis using the RD26-overexpressed plants. As might be expected, many ABA- and drought-responsive genes were upregulated in the RD26-overexpressed plants, and most of them were repressed in the ABA-treated RD26-repressed plants. For example, the RD20 gene suggested to function in ABA-mediated stress-signal transduction (Takahashi et al., 2000) was overexpressed in the RD26-overexpressed plants. Genes involved in the antioxidant defense system, such as the GLY gene, a glutathione transferase gene, and an aldo/keto reductase family gene were also upregulated in the RD26-overexpressed plants. Overexpression of defense- and senescence-responsive genes in the RD26-overexpressed plants is also notable. In both senescence and pathogen infection, the generation of several ROS plays a role in signal transduction or physiological processes (Overmyer et al., 2003). Recently, increasing evidence indicates that water stress-induced ABA accumulation triggers the generation of ROS, which, in turn, leads to the upregulation of the antioxidant defense system (Jiang and Zhang, 2003). Although, it is not yet clear whether such a stress-signal transduction system exists, the expression of RD26 was also induced by methyl jasmonate, H2O2 and Rose Bengal. Thus, RD26 is considered to play a key role in the crosstalk among defense, senescence, and ABA-mediated stress-signaling pathways.

We further demonstrated that RD26 transactivates its target gene in Arabidopsis protoplasts. The promoter of the GLY gene, upregulated in the RD26-overexpressed plants, was used for the transactivation experiment (Figure 6). The −410 and −215 bp GLY promoter fragments were activated moderately by ABA treatment and strongly by co-expression of the RD26 protein. However, the −86 bp GLY promoter region did not respond to the ABA treatment or the RD26 co-expression. The −410 bp GLY promoter contains two ABREs which interact with bZIP proteins such as rice TRAB1, Arabidopsis ABI5 and AREB/ABF proteins. However, deletion analysis of the GLY promoter showed that elimination of one of the ABREs did not affect the activation of the GLY promoter by ABA treatment or co-expression of RD26. A single copy of ABRE is not sufficient for ABA-responsive transcription, and at least one coupling element in the promoter regions is necessary for expression of ABA-inducible genes (Hobo et al., 1999; Narusaka et al., 2003a). These data suggest that the GLY gene may be expressed independently of the ABREs. Recently, RD26 and the closest homologues, At3g15500 and At1g52890, were found to recognize the ERD1 promoter (Tran et al., 2004) and the ‘CACG’ to be the core-binding motifs of these three proteins using a yeast system (Tran et al., 2004). Although the GLY promoter contains two CACG motifs, deletion analysis that a −86 bp GLY promoter fragment that included the two CACG motifs could not be activated by the RD26 protein. This result suggests that the −86 bp GLY promoter contains an additional cis-acting element recognized by RD26 together with the CACG core sequence, or that RD26 recognizes a sequence in the GLY promoter different from that identified in the ERD1 promoter.

In conclusion, the RD26 gene encoding a NAC-related transcription factor is responsive not only to dehydration, but also to NaCl, ABA and jasmonic acid treatments. The transgenic plants overexpressing RD26 cDNA were hypersensitive to ABA, and inversely, the transgenic plants with RD26 repressed were insensitive to ABA. The expressions of many ABA- and stress-induced genes including RD20 and GLY genes were upregulated in plants overexpressing RD26 and repressed in plants with RD26 repressed. In Arabidopsis protoplasts, RD26 activated a promoter of the GLY gene that was upregulated in plants overexpressing RD26. Thus, these results provide strong evidence for the involvement of RD26 in stress-responsive ABA signaling and the presence of a novel ABA-dependent signaling pathway.

Constructs and transformation of Arabidopsis

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

The 35S::RD26 plasmid was constructed by amplifying the entire coding region of RD26 by PCR with BamHI linker primers and cloned into a binary vector pBE2113Not (Liu et al., 1998) in the sense orientation. The RD26 promoter::GUS plasmid was constructed by amplifying a 2-kb DNA fragment upstream of the RD26 coding region by PCR with an upstream SalI linker primer and a downstream BamHI linker primer and cloned into the XbaI/BamHI site of pBI101.1. Arabidopsis plants were transfected with Agrobacterium tumefaciens strain GV3101 by the vacuum infiltration method (Bechtold et al., 1993).

RNA gel blot analysis

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

Three-week-old plants were harvested from GM agar plates, and then dehydrated on Whatman 3MM paper at room temperature with approximately 60% humidity under dim light. Plants subjected to treatment with hormones and salt stress were grown hydroponically in a solution containing 250 mm NaCl, 100 μm ABA, 50 μm IAA, 50 μm GA3, 50 μm 1-aminocyclopropane-1-carboxylic acid (ACC), 50 μm methyl jasmonate, 20 mm H2O2 and 5% glucose or water, under dim light. Cold treatment was applied under dim light by exposure of plants grown at 22°C to a temperature of 4°C. Plants subjected to the stress treatments for various periods were frozen in liquid nitrogen. Total RNA was isolated according to the method described by Nagy et al. (1988) and subjected to RNA gel blot analysis as described previously (Taji et al., 2002).

Transient expression of the sGFP protein in onion epidermal cells

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

The RD26 protein was fused to the C terminal of GFP by constructing a plasmid pGFP3BX which includes an sGFP coding region without a stop codon, and new cloning sites downstream of the sGFP coding region. The PCR fragment of sGFP (Chiu et al., 1996) amplified with an upstream BamHI linker primer 5′-CCCGGATCCATGGTGAGCAAGGGCGAG-3′ and a downstream noSTOP-BglII/XhoI linker primer 5′-CTGCAGCCCGGGCGGCCGCTCGAGATCTGTACAGCTC-3′ was digested with a BamHI and NotI, and replaced with a BamHI–NotI fragment of p35S-sGFP (Nakashima et al., 1997). The entire region or C terminal region (encoding amino acids 163–298, 487–894) of the RD26 coding sequence was amplified by PCR with BamHI linker primers and cloned into the BglII site of pGFP3BX in sense orientation to produce GFP–RD26 fusion proteins. These DNA constructs were introduced into onion epidermal cells using a pneumatic particle gun (PDS-1000/He; Bio-Rad Laboratory, Tokyo, Japan) as described previously (Ito and Shinozaki, 2002). After incubation at 22°C for 8–12 h, the tissues were stained with propidium iodide (10 μg ml−1) and the GFP fluorescence was observed in whole mounts under a confocal laser-scanning microscope (Zeiss LSM510; Carl Zeiss, Jena, Germany) as described previously (Ito and Shinozaki, 2002).

Transactivation analysis in yeast

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

RD26 was examined for the presence of an activation domain using a yeast assay system. The complete region or partial region of the RD26 coding sequence was cloned into the DNA-binding domain vector pGBKT7 (Clontech, Palo Alto, CA, USA), so that a GAL4 DNA-binding domain-RD26 fusion protein would be produced when the yeast cells were transformed. We used the yeast strain AH109 harboring the LacZ and HIS3 reporter genes. The transformed yeast culture was dropped onto SD plates with or without histidine. The plates were incubated for 3 days and applied to a β-gal assay.

Transient expression assay using Arabidopsis protoplasts

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

Arabidopsis protoplasts were transfected by a method described previously (Ueda et al., 2001), with minor modifications. Arabidopsis ecotype Columbia T87 suspension-cultured cells (Axelos et al., 1992) were incubated in an enzyme solution [0.4 m mannitol, 1% (w/v) cellulase ‘Onozuka’ R-10 (Yakult, Tokyo, Japan), 0.25% (w/v) macerozyme R-10 (Yakult), 8 mm CaCl2, and 5 mm MES, pH 5.6] for 2 h at 25–28°C under gentle agitation and then passed through three layers of nylon mesh (108 μm pore). Protoplasts were collected by centrifugation at 600 g for 5 min, washed twice with solution A (0.4 m mannitol, 70 mm CaCl2 and 5 mm MES, pH 5.6) and resuspended in MaMg solution (0.4 m mannitol, 15 mm MgCl2 and 5 mm MES, pH 5.6). After the addition of plasmid DNA to 150 μl of protoplast solution, 65 μl of DNA uptake solution containing 40% (w/v) polyethylene glycol 3350, 0.4 m mannitol and 100 mm Ca(NO3)2 was added. The mixture was placed at room temperature for 20–60 min and then diluted with 5 ml of 0.4 m mannitol, 125 mm CaCl2, 5 mm KCl, 5 mm glucose and 1.5 mm MES, pH 5.6. Protoplasts were collected by centrifugation at 600 g for 5 min, resuspended in Murashige and Skoog (MS) cell culture medium (Murashige and Skoog, 1962) supplemented with 0.4 m mannitol, and incubated at 22°C for 16–20 h in the dark. GUS and LUC activities were assayed as described previously (Urao et al., 1996), with a multilabel counter (Wallac AVROsx 1420; Perkin Elmer, Boston, MA, USA).

Microarray analysis

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References

Two-week-old seedlings of the 35S::RD26 (RD26-overexpressed), 35S::RD26SRDX (RD26-repressed), and vector control plants grown on the GMK plate were harvested directly or after ABA treatment for 5 h as described in Figure 2, and were subjected to microarray experiments using the Agilent Arabidopsis 2 Oligo Microarray (Agilent Technologies, Inc.). Total RNA isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) was used for the preparation of Cy5-labeled and Cy3-labeled cDNA probes. All microarray experiments including data analysis were carried out as described previously (Hughes et al., 2001). The reproducibility of microarray analysis was assessed by dye swap in each experiment.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. RD26 encodes a NAC domain protein
  6. Expression of RD26
  7. Nuclear localization of GFP–RD26 fusion protein
  8. The C-terminal domain of RD26 has transactivation activity
  9. Growth phenotypes of RD26-overexpressed transgenic lines
  10. RD26-overexpressed plants were hypersensitive to ABA
  11. RD26 protein transactivates the promoter of target gene in vivo
  12. Construction of RD26-repressed plants – loss-of-function phenotype of RD26
  13. RD26-repressed plants were insensitive to ABA
  14. Identification of target genes of RD26 using the Agilent 22K Oligo DNA Microarray
  15. Discussion
  16. Experimental procedures
  17. Plant materials
  18. Constructs and transformation of Arabidopsis
  19. RNA gel blot analysis
  20. Transient expression of the sGFP protein in onion epidermal cells
  21. Transactivation analysis in yeast
  22. Transient expression assay using Arabidopsis protoplasts
  23. Microarray analysis
  24. Acknowledgements
  25. References
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