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First published online July 14, 2006; 10.1104/pp.106.081752 Plant Physiology 142:113-123 (2006) © 2006 American Society of Plant Biologists Expression of CAP2, an APETALA2-Family Transcription Factor from Chickpea, Enhances Growth and Tolerance to Dehydration and Salt Stress in Transgenic Tobacco1,[W]National Centre for Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India
The APETALA2 (AP2) domain defines a large family of DNA-binding proteins that play important roles in plant morphology, development, and stress response. We describe isolation and characterization of a gene (CAP2) from chickpea (Cicer arietinum) encoding a novel AP2-family transcription factor. Recombinant CAP2 protein bound specifically to C-repeat/dehydration-responsive element in gel-shift assay and transactivated reporter genes in yeast (Saccharomyces cerevisiae) one-hybrid assay. CAP2 appeared to be a single/low copy intronless gene, and the protein product localized in the nucleus. Transcript level of CAP2 increased by dehydration and by treatment with sodium chloride, abscisic acid, and auxin, but not by treatment with low temperature, salicylic acid, and jasmonic acid. The 35S promoter-driven expression of CAP2 in tobacco (Nicotiana tabacum) caused drastic increase in the leaf cell size, and, thereby, in leaf surface area and number of lateral roots. Transgenic plants demonstrated more tolerance to dehydration and salt stress than the wild-type plants. Transgenic plants expressed higher steady-state transcript levels of abiotic stress-response genes NtERD10B and NtERD10C and auxin-response genes IAA4.2 and IAA2.5. Taken together, our results indicated a mutual interrelation between plant growth-development and abiotic stress-response pathways and a probable involvement of CAP2 in both the signaling pathways.
The APETALA2 (AP2)/ethylene-responsive factor (ERF) family of proteins regulates diverse processes of plant development and metabolism, such as vegetative and reproductive development, cell proliferation, secondary metabolism, biotic and abiotic stress responses, and responses to different plant hormones. These transcription regulators are characterized by the presence of approximately 60-amino acid-long AP2/ERF DNA-binding domains that directly interact with GC-rich cis-acting elements (GCC box/C-repeat) in the promoter of their target genes. Proteins belonging to the AP2 subgroup contain two copies of the DNA-binding domain (BD) separated by a spacer region (Meyerowitz, 1994  ; Okamuro et al., 1997). In Arabidopsis (Arabidopsis thaliana), the homeotic gene AP2 is best known for its central role in establishment of floral meristem, determination of sepal and petal identity, and expression of other floral homeotic genes (Irish and Sussex, 1990; Bowman et al., 1993). Loss-of-function AP2 mutation causes a relative increase in the seed mass (Ohto et al., 2005). AP2 gene also expresses at RNA level in the nonfloral organs, suggesting its involvement in the development processes of other organs as well; however, AP2-mutant plants do not show apparent defects in vegetative development (Jofuku et al., 1994). Another homeotic gene of this subgroup, AINTEGUMENTA (ANT), is critical in regulation of ovule and female gametophyte development (Klucher et al., 1996). BABY BOOM (BBM) expresses preferentially in developing embryo and seeds in Arabidopsis and Brassica napus. Its ectopic expression induces pleiotropic phenotypes such as spontaneous formation of somatic embryo, neoplastic growth, and hormone-free regeneration of explants (Boutilier et al., 2002). PLETHORA1 and PLETHORA2 (PLT1 and PLT2) genes encoding repeated AP2 domain proteins are transcribed in response to auxin in the basal embryo region, embryonic root primordium, and later in root meristem stem cells, and are essential in maintaining the position of the stem cell niche in the Arabidopsis root (Aida et al., 2004). ERF/EREBPs, referred to as the ethylene-responsive element binding proteins, were first identified in Nicotiana and possess a single copy of the AP2 domain (Ohme-Takagi and Shinshi, 1995). ESR1 gene (enhancer of shoot regeneration), encoding a single AP2-domain protein, when overexpressed promotes cytokinin-independent shoot regeneration from Arabidopsis root explants (Banno et al., 2001). Semidominant expression of TINY, a similar protein, suppresses cell proliferation during vegetative and floral organogenesis (Wilson et al., 1996). Genetic and molecular studies with several other proteins related to AP2 (RAP2) also indicated that the AP2/ERF family of proteins functions in several tissues and organs and plays important roles in general plant development (Okamuro et al., 1997).
Transcriptional activation of the osmotic stress-response genes is regulated by abscisic acid (ABA)-dependent and -independent signal transduction pathways and by their mutual cooperation. Promoter analysis of these genes has identified ABA-dependent and ABA-independent cis-acting elements (Ingram and Bartels, 1996
The plant hormone auxin regulates a number of cellular and developmental processes depending on the context. It plays critical roles in patterning different tissues and organs, for example, promotion or inhibition of organ development according to tropic responses, and maintenance of the positions of the root and shoot stem cells during embryogenesis, initiation, and emergence of lateral roots. It also influences cell division, cell growth, and differentiation. At the molecular level, auxin exerts its effect by regulating expression of numerous auxin-responsive genes (e.g. auxin/indole-3-acetic acid [AUX/IAA]). A family of transcription factors (auxin-responsive factor [ARF]) binds to the auxin-response element in the promoters of auxin-inducible genes to promote auxin-mediated gene induction response (Leyser, 2001
Cross interactions of signal transduction pathways keep balance between the growth and development of the plants and their responses to different external stimuli. Mechanical wounding induces genes related to abiotic stress and hormonal responses (Cheong et al., 2002
In this study, we report full-length cloning and characterization of a chickpea (Cicer arietinum) expressed sequence tag (EST), earlier reported by us, that is induced by dehydration (Boominathan et al., 2004
CAP2 Encodes an AP2/ERF Family Protein
The 2 x 105 plaque-forming unit of a cDNA library constructed with mRNA isolated from 6-d-old chickpea seedlings dehydrated for 5 h was screened with a chickpea EST (GenBank accession no. CD051361), which was induced by 5 h of dehydration treatment (Boominathan et al., 2004
Stress-Induced Expression of CAP2 Expression pattern of CAP2 mRNA was analyzed using RNA-gel blot (Fig. 2A ). A CAP2 cDNA fragment representing the C terminus of the protein was used as a probe. In the whole seedling, very low levels of CAP2 mRNA expression can be detected in normal growth conditions, indicating its requirement in normal developmental process. The steady-state transcript of CAP2 quickly reached the maximum level within one-half hour of treatment with 150 mM NaCl and maintained the level up to 5 h. Under dehydration, CAP2 mRNA began to accumulate within 30 min and reached the highest level within 3 h and remained the same at 5 h. Accumulation of CAP2 transcript in root reached its maximum level within 30 min under salt stress and remained the same up to 3 h, but drastically reduced to almost a basal level at 5 h. In stem, the CAP2 transcript accumulation followed the same course as in the root, but reduction at 5 h was not so drastic. Accumulation of CAP2 transcript in leaf, under salt stress, was totally different and interesting. It appeared that down-regulation of CAP2 transcription in root and stem at 5 h under salt stress was accompanied by up-regulation in leaf (Fig. 2B), which indicated that either the mechanism of CAP2 induction in leaf in salt stress is different from that in root and stem, or a high threshold amount of salt accumulation in leaf is required to induce CAP2 expression. Expression of CAP2 in root and stem is similar in response to dehydration. There was no detectable expression in leaf in response to the same treatment (Fig. 2C). Treatment with 100 µM ABA transiently induced CAP2 mRNA accumulation in the whole seedling after 1 h, then declined and maintained at a very low level up to 5 h. Cold (4°C), wound, salicylic acid (5 mM), and jasmonic acid (100 µM) treatments could not induce detectable CAP2 mRNA expression (data not shown).
CAP2 Protein Binds in Vitro to C-Repeat/DRE and Activates Transcription in Yeast
A gel-shift experiment was conducted to demonstrate that the CAP2 protein binds specifically to C-repeat/DRE. The dimer of a 23-bp stretch from the promoter of Arabidopsis RD29A gene containing C-repeat/DRE (TACCGACAT) was used as the probe (Fig. 3A
). The full-length ORF of CAP2 cDNA was cloned in pGEX4T-2 vector (Amersham) and expressed in Escherichia coli DH5
Transactivation property of CAP2 was demonstrated by yeast (Saccharomyces cerevisiae) one-hybrid assay. CAP2 ORF was cloned at NdeI-EcoRI sites of the yeast expression vector pGBKT7 (CLONTECH) to express the protein as a fusion to GAL4 DNA-BD. Yeast reporter strain PJ694-A carrying HIS3, ADE2, and LacZ reporter genes under GAL4 promoter was transformed with the BD-CAP2 construct. Colonies transformed with BD-CAP2 construct grew on selective medium lacking His and Ade unlike the wild type and vector-transformed colonies (Fig. 3D). BD-CAP2-transformed cells also showed higher  -galactosidase activity (Fig. 3E).
Southern transfer of chickpea genomic DNA digested separately with various restriction enzymes was hybridized at high stringency with 3' terminus of CAP2 cDNA as a probe. A small number of bands were observed in each of the digests, indicating the CAP2 gene is either single or low copy number (Fig. 4A
). A PCR reaction was performed with a pair of primers designed from the 5' and 3' ends of CAP2 cDNA and chickpea genomic DNA as template. The sequence of the PCR product entirely matched the CAP2 cDNA sequence, indicating CAP2 gene does not have introns. To determine the intracellular localization of CAP2 protein, CAP2 ORF was cloned at the XbaI site of the vector pBI121 (CLONTECH) to express CAP2 protein fused with
Morphological Features of CAP2 Expressing Transgenic Tobacco Seedlings To establish the functional significance of the CAP2 gene, the complete ORF was cloned in the plant expression vector pBI121 by replacing the GUS gene and mobilized into tobacco plants using Agrobacterium-mediated transformation. Preliminary screening of the kanamycin-resistant transgenic lines was performed by PCR amplification of genomic DNA with CAP2-specific primers. We have grown 12 independently transformed lines to maturity; however, three randomly selected lines were taken for subsequent analyses. All three transgenic lines (CAP2L38, CAP2L44, and CAP2L105) carried a single copy of the transgene, as confirmed by Southern analyses and constitutively expressed CAP2 as determined by northern analyses; however, expression in CAP2L105 was more than 3-fold lower than that of other two lines (Fig. 5A ).
T1 seedlings of the transgenic lines were morphologically different from the wild-type plants (Fig. 5B). No difference was observed between the transgenic lines and the wild-type seeds in the rate and period taken for germination. Average surface area of the third and fourth leaves of 10 seedlings (16 d after germination in soil) from each transgenic line was measured (25.92 ± 2.14 mm2) and found to be more than 2-fold of that of the wild type (11.11 ± 2.36 mm2). Similarly, average fresh weight of the transgenic seedlings of the same age was almost 3-fold that of wild-type seedlings (35.73 ± 2.90 mg and 12.30 ± 2.15 mg, respectively). The leaves of the transgenic lines remain larger up to senescence stage (Supplemental Fig. 1; Fig. 5E). We further explored the reason behind the increase in the leaf area in the transgenic plants. Ventral epidermal peels from both sides of the midrib of the leaf base, middle lamina, and leaf tip of the wild-type and transgenic lines were compared for cell size. Six samples from each leaf and leaves from four plants of each line were taken. Calculated average cell size (from 24 samples of each line) of the ventral epidermal surface of the fourth leaf from the bottom of 16-d-old transgenic plants was 192.5% of the average cell size from the same area of the wild-type leaf; however, these results do not exclude effects on the cell division (Fig. 5D). Examination of the root growth revealed that 16-d-old transgenic seedlings developed a significantly higher number of lateral roots (average 2.5) compared to no visibly developed lateral roots in the wild-type seedlings (Fig. 5B). In comparison to the wild-type plants, transgenic lines showed contrastingly higher root and leaf growth, which is evident from the comparison of soil grown 50-d- and 90-d-old tobacco plants (Fig. 5, C and E). Average flowering time of the CAP2-expressing plants is 85 to 90 d in comparison to 125 to 130 d for the wild-type plants in normal growth conditions. Though the expression of the transgene in CAP2L105 is much less compared to that in two other lines, no significant difference in leaf size and/or in the number of lateral roots was observed among the transgenic lines. Expression level of CAP2 in the line L105 was probably sufficient for the morphological phenotype.
Plant hormone auxin induces many developmental effects, including lateral root formation and leaf expansion, by regulating ARFs that bind to auxin-responsive element. Loss-of-function mutation of ARF7 and ARF19 together caused several phenotypes in Arabidopsis, including drastic reduction in the number of lateral roots and decrease in the leaf cell expansion. Both ARF7 and ARF19 transcripts are accumulated in response to auxin and in turn induce expression of early auxin-response genes like Aux/IAA (Wilmoth et al., 2005
Expression of CAP2 gene increased in dehydration and exposure to high salinity, and CAP2 protein binds to DRE. T1 seedlings of the transgenic lines were therefore analyzed for salinity tolerance. Eight-day-old seedlings were transferred to medium containing 250 mM NaCl and allowed to grow for 6 d. Chlorosis was apparent in the wild-type seedlings from the second day and started appearing in transgenic seedlings from the fourth day. After 6 d, one-half of the wild-type seedlings died following complete bleaching, and the rest were showing extensive chlorosis. A total of 30% to 40% of seedlings of lines CAP2L44 and CAP2L38 and 20% of line CAP2L105 were green even after 6 d of exposure, and other seedlings showed moderate chlorosis (Fig. 6A
). CAP2-expressing transgenic lines were evaluated for dehydration-stress tolerance by germinating and then growing the T1 seedlings on medium containing 0.4 M mannitol for 20 d (Fig. 6B). The germination rate for line 44 after 8 d was 78%, followed by 65% and 60% for the lines 38 and 105, respectively, while for the wild type it was only 42%. However, after 20 d, germination of the wild-type seedlings reached almost 60%. As there is a drastic difference between the fresh weights of the wild-type and the transgenic seedlings under normal growth conditions, effect of the dehydration stress was presented as percent relative fresh weight, i.e. fresh weight of the seedlings grown in experimental conditions relative to the fresh weight of the seedlings of the same line grown in control conditions (Fig. 6C). The relative fresh weights of the transgenic lines were significantly higher in comparison to that of the wild type, as reflected by the quantitative estimation. To assess the effect of CAP2 expression on stress tolerance of the greenhouse-grown seedlings, 3-week-old wild-type and transgenic seedlings were irrigated with 200 mM sodium chloride solution for 1 week and then with water for 1 week for recovery. The transgenic lines showed much better recovery than the wild-type plants (Fig. 6D). Improvement of dehydration tolerance in the greenhouse-grown transgenic seedlings was evaluated. Three-week-old seedlings were not watered for 2 weeks. The wild-type plants showed more bleaching and loss of turgor in comparison to the transgenic plants (Fig. 6E). The same experiments were performed on matured plants. The 50-d-old wild-type and transgenic plants were exposed to salt stress by irrigation of 200 mM sodium chloride solution in 4-d intervals for 16 d and then with water for 2 more weeks. For dehydration stress, 75-d-old wild-type and transgenic plants were not irrigated for 2 months. It is evident from the figures of the representative plants under the experiments (Fig. 6, F and G) that the CAP2-expressing plants are more tolerant to salt and dehydration stresses in comparison to the wild-type plants. Transfer of Arabidopsis DREB1A in tobacco under the control of constitutive 35S or inducible rd29A promoter resulted in improved drought and cold stress tolerance and constitutive expression of NtERD10, a family of genes encoding group 2 LEA proteins (Kasuga et al., 2004
AP2 defines a large family of plant-specific DNA-binding proteins that regulate diverse plant developmental processes, including stress response. We screened a cDNA library to obtain a full-length clone of a chickpea EST induced by dehydration and cloned a cDNA that encodes an AP2 family transcription factor. CAP2 ORF is relatively small in comparison to most of the well-studied AP2/ERF family members. Like CAP2, a number of small ORFs containing AP2-domain have been reported in GenBank from different plants. Two of them are AAP83131 and AAT12423 from another legume, soybean, that show high homology to CAP2 along the whole stretch of sequences. Detailed functional study on these small AP2 proteins may open up new areas in plant developmental process.
CAP2 transcription, like DREB2A of Arabidopsis and rice (Oryza sativa), is activated by dehydration and salt and not by cold, and the induction is early. But, unlike AtDREB2A, CAP2 transcription is activated by ABA. This and the fact that CAP2 transcription is also activated by auxin indicate that CAP2 and DREB2A may have different roles and are regulated by different or additional pathways. Sequence analyses of the region 5' to the transcription initiation site of CAP2 revealed the presence of a number of abiotic stress-responsive cis-acting elements, such as ABRE, MYB2 consensus, MYC consensus, etc. Among the other significant elements, there are auxin-responsive SAUR elements (CATATG; Xu et al., 1997
CAP2 satisfied the definition of a transcription factor by directly binding to DNA in a sequence-specific manner, trans-activating reporter genes, and being localized in nucleus. However, NtERD10B, NtERD10C, IAA4.2, and IAA2.5, the constitutively expressing genes in the transgenic lines, may not be the direct targets of CAP2, even though NtERD10B and NtERD10C are supposed to have DRE in their promoters (Kasuga et al., 2004
CAP2 expression imparted tolerance to dehydration and salinity to the tobacco plants. The tolerance was possibly due to expression of genes like ERD10B and ERD10C coding for LEA proteins of tobacco. Constitutive expression of AtDREB1A in Arabidopsis and in tobacco caused similar expression of RD29A and NtERD10 genes, respectively, but in contrast to CAP2-transgenic plants, resulted in stunted growth (Liu et al., 1998
Biogenesis of lateral root is affected by the interplay of various environmental and endogenous factors, including biosynthesis and distribution of phytohormones like auxin, ABA, and ethylene. It is also evident that lateral root formation is promoted by salt stress. Mutations in the signaling pathways of the phytohormones also influence the formation of lateral roots. Mutations in genes ABI3 (ABA insensitive 3) and ARF8 compromise with lateral root formation and growth (Ruegger et al., 1998 We are aware that CAP2 expression in the transgenic tobacco was driven by 35S promoter, commonly used for strong and constitutive expression, and there may be a possibility that the phenotypes shown by the transgenic plants are due to high expression level of transgene. However, no significant morphological differences were observed in the low expressing line (CAP2L105) from the two high expressing lines. Nevertheless, CAP2, an AP2/ERF family transcription factor from chickpea, is structurally different from the other family members of other plants and shows a unique expression profile in response to different stresses and phytohormones. Expression of CAP2 in transgenic tobacco caused increase in the leaf size and number of lateral roots and simultaneously promoted tolerance to salt and osmotic stress, which highlighted its potential agronomic importance as well as provided preliminary evidences that CAP2 protein might be a common point of cross talk between growth and development and abiotic stress response.
Plant Materials and Treatment
Chickpea (Cicer arietinum) cv BGD72 was used in this study. Seedlings were grown and treated for dehydration and with ABA, as described in Boominathan et al. (2004)
Chickpea cDNA library was constructed using CLONTECH SMART cDNA library kit with 4 µg of total RNA isolated from dehydrated (uprooted from soil for 5 h) chickpea seedlings (6 d after germination). The library was packaged in Packgene lambda packaging extract (Promega). The library was amplified once and obtained a titer of 1.15 x 1011 pfu. A chickpea EST (GenBank accession no. CD051361; Boominathan et al., 2004
Wild-type and T1 seeds of transgenic tobacco lines were surface sterilized, germinated, and grown essentially as described by Mukhopadhyay et al. (2004) For cellular localization of CAP2 protein, stem peels from transgenic tobacco plants expressing CAP2 protein fused to GUS were used. Histochemical localization of GUS activity was analyzed after incubating the samples in X-Gluc buffer (50 mM sodium phosphate buffer, pH 7, 10 mM EDTA, 0.1% Triton X 100, 5 mM potassium ferro cyanide, and 2 mg/mL 5-bromo-4-chloro-3-indolyl glucuronide) at 37°C for 12 h. The same sample was stained with Orcein to confirm the position of the nucleus.
Isolation of total RNA and RNA gel-blot hybridizations were carried out as described by Boominathan et al. (2004)
CAP2 protein coding sequence was amplified by PCR with primers flanked by restriction sites for BamHI and EcoRI to clone in pGEX4T2 in frame with GST cDNA. GST-CAP2 protein was produced by inducing the Escherichia coli BL21(DE3) cells harboring pGEX4T2-GSTCAP2 with 0.5 mM isopropyl
CAP2 protein coding sequence was cloned in yeast (Saccharomyces cerevisiae) expression vector pGBKT7 (CLONTECH) at NdeI-EcoRI site to express CAP2 protein fused to GAL4 DNA-BD. The construct was transformed into an auxotropic yeast strain PJ69-4A (Cagne et al., 2000 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers DQ321719, AAP83131, and AAT12423.
The authors acknowledge the contribution of Dr. Manoj Prasad of the National Centre for Plant Genome Research in preparation of the manuscript. Received April 7, 2006; accepted July 11, 2006.
1 This work was supported by the National Centre for Plant Genome Research and a grant from the Department of Biotechnology, Government of India (DBT). R.K.S. and V.T. acknowledge Council for Scientific and Industrial Research and S.R. acknowledges DBT for fellowships.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Debasis Chattopadhyay (debasis_chattopadhyay@yahoo.co.in).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.106.081752 * Corresponding author; e-mail debasis_chattopadhyay{at}yahoo.co.in; fax 091–11–26716658.
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ABSTRACT
RESULTS
-helix. In silico analyses of the CAP2 protein sequence indicated a number of posttranslational modifications, as it contains five potential casein kinase 2 phosphorylation sites (at amino acids 14, 31, 129, 133, and 148); four potential protein kinase C phosphorylation sites (at amino acids 38, 47, 133, and 145); two potential N-glycosylation sites (at amino acids 127 and 174); and six potential myristoylation sites (at amino acids 46, 56, 92, 99, 144, and 193). There are other amino acids (Lys-4, Trp-22, Lys-23, Asn-26) also in the N terminus found to be invariant with most other AP2 family proteins. The basic amino acid-rich region at the N terminus may function for nuclear localization. We compared the amino acid sequence of CAP2 with other known AP2 family proteins. Only two proteins of similar size (GenBank accession nos. 
