Báo cáo khoa học: CAP2 enhances germination of transgenic tobacco seeds at high temperature and promotes heat stress tolerance in yeast

CAP2 enhances germination of transgenic tobacco seeds at high temperature and promotes heat stress tolerance in yeast Rakesh Kumar Shukla*, Vineeta Tripathi, Deepti Jain, Rajiv Kumar Yadav and Debasis Chattopadhyay

National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, India

Keywords CAP2; chickpea; heat stress; tobacco; yeast

Correspondence D. Chattopadhyay, National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India Fax: +91 11 26741658 Tel: +91 11 26735189 E-mail: debasis_chattopadhyay@nipgr.res.in

*Present address Central Institute of Medicinal and Aromatic Plants, Lucknow, India

(Received 16 June 2009, revised 13 July 2009, accepted 16 July 2009)

doi:10.1111/j.1742-4658.2009.07219.x

from chickpea (Cicer arietinum)

We reported earlier that ectopic expression of CAP2, a single AP2 domain containing transcription activator in tobacco improves growth and development, and tolerance to dehydration and salt stress, of the transgenic plants. Here, we report that, in addition, the CAP2-transgenic tobacco seeds also exhibit higher germination efﬁ- ciency at high temperature and show higher expression levels of genes for tobacco heat shock proteins and a heat shock factor. CAP2 was able to activate the 5¢-upstream activating sequence of tobacco heat shock factor. Surprisingly, expression of CAP2 cDNA in Saccharomyces cerevisiae also enhanced heat tolerance, with increased expression of the gene for yeast heat shock factor 1 (Hsf1) and its target, the gene for yeast heat shock pro- tein 104 (Hsp104). Sequence analysis of the Hsf1 promoter revealed the presence of a dehydration-responsive element ⁄ C-repeat-like element (DRE/ CRE). Recombinant CAP2 protein bound to the DRE/CRE in the Hsf1 promoter in a gel shift assay and transactivated the Hsf1 promoter–His reporter construct. The full-length CAP2 protein was required to provide thermotolerance in yeast. If these ﬁndings are taken together, our results suggest that CAP2 is involved in the heat stress response and provides an example of functioning of a plant transcription factor in yeast, highlighting the strong evolutionary conservation of the stress response mechanism.

Introduction

Abbreviations ApiAP2, AP2 integrase DNA-binding domain; AtDREB2A, Arabidopsis thaliana DREB2A; CAP2, a single AP2 domain containing transcription activator from chickpea (Cicer arietinum); DRE, dehydration-responsive element; DREB, dehydration-responsive element-binding protein; EST, expressed sequence tag; HSF, heat shock factor; HSP, heat shock protein; NtHsfA4, Nicotiana tabacum HsfA4; UAS, upstream activating sequence.

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stresses Like all organisms, higher plants have evolved mecha- nisms to respond to different environmental stresses, including heat stress to protect cellular homeostasis at high temperatures. Dehydration-responsive element- binding (DREB) ⁄ C-repeat-binding factor proteins were shown to regulate the expression of a large number of genes in response to drought, salt and low-temperature stresses [1]. This transcription activator subfamily, with a single DNA-binding domain, belongs to the AP2 ⁄ ethylene-responsive element-binding protein family of proteins. In Arabidopsis, expression of all three DREB1 genes is induced by cold stress but not by drought and salt. Of eight DREB2 homologues in Arabidopsis, only two (DREB2A and DREB2B) genes respond to [2]. Overexpression of drought and salt AtDREB1A from the constitutive promoter resulted in

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CAP2 expression enhances heat stress tolerance

emerged from a common ancestral protein with an independent lineage [11,12]. In this article, we describe CAP2 function in plants and yeasts that indicates func- tional evolutionary conservation of AP2 proteins.

Results

Constitutively upregulated genes in CAP2-expressing tobacco

line and the

tolerance to drought, salinity and freezing stresses in Arabidopsis, but caused growth retardation. However, overexpression of full-length cDNA of AtDREB2A neither produced any stress tolerance nor induced expression of stress-responsive genes [3,4]. Later, a neg- ative regulatory domain was identiﬁed in Arabidopsis thaliana DREB2A (AtDREB2A), and AtDREB2ACA, a deletion mutant lacking the negative element, was shown to be constitutively active [5]. Overexpression of both AtDREB1A and AtDREB2ACA caused expres- sion of some of the heat shock proteins (HSPs). How- ever, expression of only HsfA3, out of 21 heat shock in Arabidopsis, was upregulated in factors (HSFs) AtDREB2ACA-expressing plants. AtDREB2A itself is expressed rapidly and transiently in heat shock, and its importance was demonstrated by acquired thermo- tolerance of AtDREB2ACA-expressing plants and attenuation of the basal thermotolerance of atdreb2A knockout plants [6,7]. DREB2A has been shown to be for heat-induced expression of HsfA3. essential AtDREB2A and AtDREB2B directly bind to and acti- vate the HsfA3 promoter, and thereby regulate the expression of heat shock genes [8]. Zea mays DREB2A was also shown to enhance heat stress tolerance and induce HsfA3 expression [9].

We have previously shown that CAP2 expression enhances growth and drought and salt tolerance in tobacco. To identify the genes upregulated in trans- genic plants by CAP2 overexpression, we constructed a subtracted cDNA library with 10-day-old seedlings of one overexpressing control plant. Repeated rounds of cDNA subtraction followed by reverse northern analysis identiﬁed expressed sequence tags (ESTs) representing 34 genes (other than for CAP2 itself) that were upregulated more than two- fold. Apart from the genes involved in dehydration and auxin signaling, as expected from the phenotype a number of ESTs representing genes involved in the heat stress response, such as Nicotiana tabacum HsfA4 and the class I small HSPs Hsp70 and Hsp90, were found to be upregulated in the transgenic tobacco plants (Table 1). The expression result was validated by northern analysis of some of the identiﬁed ESTs (Fig. 1A). The result provided a genetic basis for the phenotype of transgenic plants, and also indicated a probable role of CAP2 in heat stress tolerance.

elements showed signiﬁcant improvement

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Interestingly, only one gene, encoding the HSF N. tabacum HsfA4 (NtAsfA4) (UniProt: Q9SXK9), was found to be expressed almost ﬁve-fold in the CAP2-expressing tobacco in comparison with the con- trol plants. To further investigate the effect of CAP2 on N. tabacium HsfA4 expression, we cloned the 1.3-kb-long 5¢-upstream activating sequence (UAS) of NtHsfA4 (GenBank accession number: FI276782) by identiﬁed two genome walking. Sequence analysis potential CAP2-binding (CCGAC ⁄ RYC- GAC), similar to the HvCBF1-binding site or that present in the Arabidopsis COR15A gene promoter, required for cold, drought and abscisic acid response, at 830 and 900 bp upstream of the translational start site of NtHsfA4 [13,14]. A 1598 bp upstream activator including the 285 bp 5¢-UTR of NtHsfA4, sequence, was fused to the GUS reporter gene by replacing the CaMV35S promoter of plasmid pCAMBIA1305.1. The construct was mobilized into the vector control and CAP2-expressing tobacco explants (line L44) through Agrobacterium-mediated transformation. Hygromycin- resistant shootlets emerging from transformed callus We reported earlier that CAP2, a single AP2 domain containing the transcription activator from chickpea (Cicer arietinum), promoted drought and salinity stress tolerance when expressed under the 35S promoter in tobacco. Although CAP2 possesses an AP2 domain similar to AtDREB2A, it is smaller in size and consti- tutively active. In contrast to plants overexpressing Arabidopsis DREBs, CAP2-expressing plants showed improved root branching and overall growth and devel- opment. Moreover, expression of CAP2 is induced in response to auxin treatment, and CAP2-expressing plants show higher basal levels of some auxin-respon- sive and drought ⁄ salt response genes, suggesting its involvement in both abiotic stress and auxin signaling [10]. Here, we report that CAP2-expressing tobacco seeds in thermo- tolerance. Subsequently, an expression study of the upregulated genes in CAP2-expressing tobacco with respect to control plants showed relatively higher levels of gene expression of tobacco HSPs and an HSF. Surprisingly, expression of CAP2 also improved ther- motolerance in Saccharomyces cerevisiae. Recently, a sensitive sequence proﬁle search showed that apicom- plexans, such as the malarial parasite Plasmodium and the cattle parasite Theileria, possess a lineage-speciﬁc expansion of a novel family of proteins with a version of the AP2 integrase DNA-binding domain (ApiAP2), supporting the idea that the plant AP2 proteins have

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CAP2 expression enhances heat stress tolerance

Table 1. ESTs constitutively expressing more than two-fold in CAP2-transgenic tobacco line L44 in comparison to the vector control plant. E-value, the Expect value obtained by BLAST search.

GenBank accession number

Annotation

E-value

Fold expression

GenBank match

FE192514 FE192588 FE192524 FE192525 FE192477 FE192491 FE192530 FE192536 FE192544 FE192523 FE192587 FE192556 FE192553 FE192558 FE192567 FE192569 FE192579 FE192581 FE192474 FE192488 FE192580 FE192498 FE192504 FE192473 FE192510 FE192545 FE192493 FE192541 FE192509 FE192566 FE192568 FE192466 FE192468 FE192467

ABW99093 AAU03363 AAF42974 BAE97400 O49954 BAB16430 Q43497 AAD00255 AAK84479 ABY57764 ABN08957 BAA96501 CAA41415 CAA70705 ACB05668 AAQ93011 CAA54803 AAR01500 Q8RX97 P46942 P27082 ABK94990 AAR83875 CAB39974 BAG16715 ABX76301 AAD33072 ABR26184 CAA78365 P31542 AAR8386 BAD13498 BAD13499 BAA83711

Myoinositol 3-phosphate Wound stress protein S-Adenosyl-L-methionine synthetase HSP90 Glycine dehydrogenase Probable phospholipid hydroperoxide glutathione Monodehydroascorbate reductase Solanum tuberosum ci21A gene product Putative auxin growth promoter protein Extracellular calcium-sensing receptor H+-transporting two-sector ATPase Cysteine protease Thioredoxin Guanine nucleotide-binding protein subunit Cyclophilin FtsH-like protein precursor Shaggy-related protein kinase NtK-1 Cytosolic class I small HSP1A Ferritin-1, chloroplast precursor NtFer1 ATP-dependent RNA helicase-like Superoxide dismutase Unknown (Populus trichocarpa) Mary Storys protein Glyceraldehyde-3-phosphate dehydrogenase Protein disulﬁde isomerase HSP70 Secretory peroxidase Aminotransferase 2 Tobacco pre-pro cysteine proteinase ATP-dependent Clp protease Elicitor-inducible protein EIG-J7 ERD10B ⁄ Dehydrin ERD10C ⁄ Dehydrin N. tabacum HSFA4

4e-11 1e-43 5e-66 1e-37 1e-53 6e-32 4e-25 3e-21 6e-21 2e-31 2e-12 2e-19 2e-51 1e-20 1e-19 9e-44 6e-45 1e-38 4e-07 3e-32 8e-40 4e-29 8e-32 1e-49 2e-26 9e-33 4e-45 2e-32 1e-23 6e-46 3e-45 6e-05 2e-23 2e-92

8.86 2.82 3.16 4.15 2.23 2.35 3.32 2.17 7.25 2.39 2.25 2.81 2.82 2.58 2.62 2.32 2.19 8.85 2.96 4.41 2.13 2.09 2.97 2.56 3.13 5.82 2.38 2.3 2.02 2.93 2.9 4.3 3.5 4.8

were assayed for GUS activity. The activity was nor- malized by the relative abundance of mRNA of the hygromycin resistance gene quantitated by quantitative RT-PCR. GUS activity in the CAP2-expressing shoot- lets was more than 13-fold higher than that in the shootlets derived from the control plants, showing that CAP2 can activate transcription from the NtHsfA4 5¢-UAS (Fig. 1B).

CAP2 enhances germination efﬁciency in transgenic tobacco at high temperature

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As CAP2-expressing tobacco plants exhibit enhanced expression of genes implicated in heat stress tolerance, we investigated the expression of the CAP2 gene in chickpeas under heat stress. Northern analysis showed that CAP2 expression quickly reached a peak within 30 min of stress, was maintained for up to 1 h, started to decline from 3 h, and reached a basal level within 5 h (Fig. 2A). A transient but signiﬁcant increase in CAP2 expression in chickpeas under heat stress and constitutive expression of heat stress tolerance genes in CAP2-expressing tobacco plants led us to investigate whether CAP2 can enhance the germination efﬁciency of tobacco seeds at higher temperatures. We germi- nated the seeds of previously mentioned transgenic tobacco lines expressing CAP2 along with the vector control seeds on ﬁlter paper soaked with germination medium. A germination frequency of 90–92% was observed for all the lines within 8 days under control conditions (25 (cid:2)C). However, at a higher temperature (38 (cid:2)C), all of the CAP2-transgenic lines germinated at a much higher frequency (75–87% in three replicates; total of 150 seeds in each) than the vector control

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CAP2 expression enhances heat stress tolerance

L105

Con

L38 L44

A

FE192514 (myo-Inositol-1-phosphate synthase)

FE192553 (Thioredoxin H-typeI)

FE192525 (Hsp90)

FE192545 (Hsp70)

FE192544 (Auxin growth promoter protein)

FE192558 (G protein beta subunit)

FE192467 (NtHsfA4)

rRNA

B

20 15 10 5 0

y t i v i t c a S U G d l o F

Vec Con

CAP2

Fig. 1. (A) RNA blot analysis of selected ESTs to validate the expression of genes in CAP2-expressing tobacco lines. Twenty micrograms of total RNA isolated from 7-day-old control (Con) or three CAP2-expressing transgenic tobacco lines were analyzed by northern analysis with the indicated radiolabeled tobacco ESTs. Ribosomal RNA is shown as a loading control. (B) Activity of a GUS reporter construct fused to the NtHsfA4 5¢-upstream activating sequence in transgenic tobacco plants harboring empty vector or CAP2. The promoter–GUS construct in a plant vector with a hygro- mycin resistance gene was introduced through Agrobacterium in the explants of tobacco plants already transformed with control vector or CAP2. Fold GUS activity in the CAP2-expressing plants relative to that in the control plants is presented. Expression of the hygromycin resistance gene was used for normalization. The data show the average of 15 transgenic shootlets of each sample.

seeds (8 ± 3%) after 18 days of sowing (Fig. 2B). This experiment showed that CAP2-expressing tobacco seeds can tolerate ⁄ adapt to higher temperature. At a much higher temperature (45 (cid:2)C), seeds of none of the lines germinated (not shown). 30 (cid:2)C (Fig. 3A). When transferred to 39 (cid:2)C, CAP2- transformed yeast cells grew at a considerably higher rate than the wild-type and vector control cells, showing enhanced thermotolerance (Fig. 3B). CAP2-transfor- med yeast cells did not show improved tolerance when galactose was replaced by glucose in the growth med- ium, indicating that heat stress tolerance was dependent on expression of the transgene (Fig. 3C). To investigate the mechanism of thermotolerance induced by CAP2 in yeast, we analyzed the expression proﬁle of yeast Hsp104 in wild-type and CAP2-expressing cells. The lacking Hsp104 is sensitive to heat shock. mutant Hsp104 is constitutively expressed at a basal level in yeast, and is quickly induced by heat shock. Northern analysis showed that steady-state expression of Hsp104 in the CAP2-expressing strain was much higher than that of the wild-type strain, although accumulation of Hsp104 transcript upon heat treatment did not increase proportionately in CAP2-expressing cells, probably owing to a high turnover rate after a threshold level (Fig. 3D). Expression of Hsp104 in heat stress is regu- lated by the yeast heat shock transcription factor Hsf1 (UniProt: P10961). Hsf1 is the only HSF in yeast, and is required for multiple functions. The hsf1 knockout mutant is not viable. Under normal growth conditions, Hsf1 is constitutively bound to the heat shock element in the Hsp104 promoter. Shifting the growth conditions to higher temperatures increases the accumulation of Hsf1 on the Hsp104 promoter [16]. Therefore, we fur- ther investigated the expression of yeast Hsf1 in CAP2- transformed yeast cells. In wild-type yeast, we could detect an induced level of Hsf1 expression within 15 min of heat shock, whereas in CAP2-expressing cells, there was a high constitutive level of Hsf1 that increased further alike its expression pattern in the wild-type strain after heat shock (Fig. 3E). The heat shock- mediated further increase in Hsf1 level indicated that CAP2 did not interfere with heat-mediated transcrip- tional activation of the Hsf1 promoter.

CAP2 enhances thermotolerance in yeast

thermosensitivity of a yeast mutant

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Arabidopsis HSP101 was previously shown to suppress the lacking HSP104. CAP2 expression in tobacco caused increased expression of some HSPs and an HSF. CAP2-expressing tobacco plants showed higher activity of the 5¢-UAS of NtHsfA4. In order to test whether CAP2 can promote a similar phenotype in a distant eukaryote such as yeast, we introduced CAP2 cDNA under a galactose-inducible promoter into a budding yeast, S. cerevisiae BCY123 [15]. The transformed yeast cells grew equally as well as the wild-type and vector-transformed yeast strains at To investigate the mechanism of induction of Hsf1 expression in CAP2-expressing yeast cells, we inserted 750 bp of genomic DNA 5¢-upstream of the translation start point of the Hsf1 gene in front of the auxotropic selection marker HIS in the plasmid pHIS2.1 (Clon- Tech, Palo Alto, CA, USA). This construct was cointro- duced with another plasmid (modiﬁed pGAD, with a LEU selection marker), where CAP2 cDNA was cloned under the constitutive yeast alcohol dehydrogenase (Adh1) promoter in the his)leu) strain AH109 of S. cere- visiae (ClonTech). The transformed yeast cells were able to grow on HIS)LEU) medium, whereas cells from a similar experiment without CAP2 cDNA could not grow on the same medium (Fig. 4A). This result

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CAP2 expression enhances heat stress tolerance

0.5

1.0

3.0

5.0 (h)

0 A

CAP2

rRNA

B

V ector

L105

L44

L38

25 °C

38 °C

Fig. 2. (A) Expression pattern of CAP2 during heat stress (45 (cid:2)C) in chickpea seedlings. Seven-day-old soil-grown chickpea seedlings were transferred to 45 (cid:2)C for the indicated period. 20 lg of total RNA analyzed in agarose gel and blotted on nylon membrane was hybridized with a cDNA fragment representing the C-terminal part of CAP2 (Experimental procedures) used as a radiolabeled probe. Ethidium bromide- stained rRNA was used as an equivalent loading control. (B) Effect of heat stress on the germination of tobacco seeds of the vector control and CAP2-transgenic lines (CAP2L38, CAP2L44, and CAP2L105). Seeds were sown on ﬁlter paper soaked with germination medium and kept at 25 (cid:2)C (control condition) for 8 days (left panel) and at 38 (cid:2)C for 18 days (right panel). A 16 h light period and 60% relative humidity were maintained. One representative experiment of three replicates of 150 seeds for each line is shown.

mutant construct and CAP2-expressing plasmid did not enable the his)leu) strain AH109 to grow on HIS)LEU) medium (Fig. 4C). CAP2-mediated activation of the Hsf1 promoter, speciﬁcally through the DRE sequence, together with the gel shift assay suggested that CAP2 activated Hsf1 expression by directly binding to a C-repeat element present in its promoter, and thereby enhanced thermotolerance in yeast.

CAP2 requires the C-terminal domain to promote thermotolerance

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The AP2 domain has been shown to be required for DNA binding for the AP2 family proteins. In order to determine the role of different domains in CAP2 func- tion in yeast, three deletion constructs were made, by serial deletion of 40 amino acids (D162), 60 amino acids (D142) and 80 amino acids (D122) from the C-terminus of CAP2, and expressed in yeast. Equal expression of the CAP2 deletion mutant proteins was checked by protein blot (Fig. 5A). In contrast to the full-length protein, none of the truncated constructs could enhance thermotolerance in yeast, indicating that almost the full-length protein is required for CAP2 function (Fig. 5B). To further investigate the ability of suggested that CAP2 was able to activate the Hsf1 pro- moter and that accumulation of Hsf1 transcript in CAP2-expressing cell is most likely due to transcrip- tional activation of the Hsf1 promoter, although the possibility of stabilization of Hsf1 mRNA cannot be the Hsf1 promoter ignored. Sequence analysis of revealed the presence of a variant dehydration-respon- sive element (DRE) ‘TACCGACTA’ with a conserved core sequence ‘CCGAC’ at 525 bp 5¢-upstream of the translation start site. To investigate whether CAP2-med- iated activation of the Hsf1 promoter was direct, we used a 39 bp DNA element from this region, including the DRE, as a probe in a gel shift assay. CAP2 protein fused with glutathione-S-transferase expressed in Escherichia coli directly interacted with the probe, producing signiﬁcant gel shift, which was competed out with excess cold probe. A point mutation in the core sequence (M1-DRE) of the probe prevented CAP2 bind- ing, whereas replacement of two bases (M2-DRE) ﬂank- ing the core DRE sequence did not compromise the binding (Fig. 4B). To demonstrate the speciﬁcity of Hsf1 promoter activation by CAP2, a replacement mutation was created in the 5¢-UAS in the DRE sequence, as in M1-DRE, and introduced in front of the HIS selection marker as before. Cointroduction of the

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CAP2 expression enhances heat stress tolerance

A

Galactose

BCY pYES CAP2(1) CAP2(2)

B Galactose BCY pYES CAP2-1 CAP2-2

C Dextrose BCY pYES CAP2-1 CAP2-2

10–2

10–3

10–4

10–5

30 °C

39 °C

BCY -CAP2

BCY

D

min Hsp104

rRNA

BCY

BCY -CAP2

BCY -CAP2 162

E

min

Hsf1

rRNA rRNA

Fig. 3. CAP2 enhances heat stress tolerance in yeast. S. cerevisiae BCY123 cells harboring empty vector (pYES2.1) or vector carrying CAP2 cDNA were grown after the indicated dilutions on medium containing galactose (A, B) or dextrose (C) at 30 (cid:2)C (A) or at 39 (cid:2)C (B, C). Two independent CAP2 transformants are shown. (D) RNA blot analysis of yeast Hsp104 and Hsf1 (E) in S. cerevisiae BCY123 and the cells expressing full-length CAP2 or truncated CAP2D162 cDNA upon exposure to heat stress (39 (cid:2)C) for the time periods indicated. 20 lg of total RNA analyzed in agarose gel and blotted on nylon membrane was hybridized with DNA fragments representing yeast Hsp104 and Hsf1 used as radiolabeled probes. Ethidium bromide-stained rRNA was used as an equivalent loading control.

the Hsf1 transcript level

ing seed germination efﬁciency at high temperature. AtDREB2A and Z. mays DREB2A have been shown to enhance tolerance to salt, dehydration and, recently, heat stresses in transgenic plants. Whereas AtDREB2A requires inactivation of a negative element in its C-ter- minus, Z. mays DREB2A and CAP2 are constitutively active. Our data provide additional evidence that proteins with DREB2-like AP2 domains have a role in enhancing heat stress tolerance.

the truncated CAP2 protein (D162) to activate Hsf1 transcription, we analyzed the abundance of Hsf1 tran- scripts in yeast cells expressing CAP2D162, i.e. the truncated protein lacking the last 40 amino acids. Figure 3E shows that in CAPTD162-expressing yeast cells was much lower than that in CAP2-expressing cells, and was closer to that in the control wild-type cells, suggesting that CAP2- mediated thermotolerance of yeast cells is mediated through activation of yeast HSF by the full-length CAP2 protein.

Discussion

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Previously we have demonstrated that CAP2, an AP2 family transcription factor from a legume crop, chick- pea, is involved in growth and in the responses to abi- otic stresses such as salt and dehydration. In this study, we have shown that CAP2 is capable of enhanc- We have presented a list of genes that are constitu- tively expressed more than two-fold in CAP2-express- ing tobacco plants, as identiﬁed from a subtractive cDNA library. A complete gene expression analysis using microarrays would have provided detailed infor- mation on CAP2 target genes. Nonetheless, CAP2 showed a clear difference from AtDREB2 in inducing the expression of some auxin-responsive genes, which is expected from the phenotype of the CAP2-over- expressing plants. Although the list of upregulated

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CAP2 expression enhances heat stress tolerance

functions of CAP2 and AtDREB2A, the two proteins share a common set of pathways.

genes identiﬁed in this study may be incomplete, CAP2 and AtDREB2ACA share some common target genes e.g. those encoding cyclophillin, dehydrins, HSP70, cysteine protease, and ferritin-1 precursor. CAP2 pro- moted the expression of cytosolic small HSPs, and AtDREB2A increased the expression of chloroplast- targeted small HSP. Another point of difference is that CAP2 expression increased the expression of NtHsfA4, whereas AtDREB2A regulates only A. thaliana HsfA3 [7]. We have demonstrated that the 5¢-UAS of NtHsfA4 possesses two potential CAP2-binding sites, and CAP2 was able to activate the 5¢-UAS of NtHsfA4. Alto- it appears that, apart from some distinctive gether,

A

pGCAP2(1) +

pGCAP2(2) +

pGCAP2(2) + pyHsf1 HIS

-

py s

H f1 HIS -

py s

H f1 HIS -

pGCAP2(1) + f1

S

pyHs

-HI

pGCAP2

pyHsf1-HIS

pGCAP2

pyHsf1-HIS

YPDA

SD-HIS–LEU–

B

TTCAATGA TTCAATGA TTCAATGA

AP2 proteins were thought to be plant-speciﬁc pro- teins, because of their absence in the animal kingdom. Recent studies with bioinformatic search tools have identiﬁed AP2 integrase domains in bacteria, cyano- bacteria, ciliates, and bacteriophages, and a version of ApiAP2 in apicomplexans. Plant AP2 proteins bind GC-rich cis-elements (GCC box ⁄ C-repeat) of their tar- get genes and so do the Tetrahymena AP2 and the ApiAP2 proteins [12,17,18]. We searched the S. cerevi- sae protein database with the plant consensus AP2 domain sequence as query for the existence of similar proteins through psi-blastp. The search did not result in any signiﬁcant similarity. However, a search with the ApiAP2 domain identiﬁed a family of proteins with a moderate level of sequence similarity within the putative AP2 domain. The overall similarity between the ApiAP2 and the yeast proteins is very low (E-value: 0.53). The overall similarity between the plant AP2 family proteins outside the AP2 domain is also low. A search in the non- redundant protein database identiﬁed two Oryza sativa proteins with similar sequence homology. Alignment of the putative AP2 domains of these proteins is shown in Fig. 6. Although the ApiAP2 and yeast proteins possess weak homology with the plant proteins, they maintain some of the conserved or similar amino acids. The same size and similar sequence of the yeast proteins indicate that they may have evolved through gene duplication.

1 2 3 4 5 6

The existence of different forms of AP2 domains out- side the plant kingdom raises the possibility of transfer of this domain from unicellular organisms to plants.

LANES LANES:- 1- DRE 2- DRE + GST 3- DRE + CAP2 4- DRE + COLD-DRE (100X) + CAP2 5- M1-DRE + CAP2 6- M2-DRE + CAP2

C

located at

pGCAP2 + pyM1Hsf1-HIS

pGCAP2 p + pyHsf1-HIS

pGCAP2 p + pyM1Hsf1-HIS

pGCAP2 + pyHsf1-HIS

pyHsf1-HIS

pGCAP2

pyHsf1-HIS

pGCAP2

YPDA

SD-HIS–LEU–

Fig. 4. (A) Activation of the yeast Hsf1 promoter by CAP2. S. cere- visiae AH109 (his)leu)) was transformed with the constructs as described. CAP2 cDNA was cloned in a vector (with a LEU marker) under a constitutive alcohol dehydrogenase promoter, and intro- duced into AH109 cells harboring the HIS marker under the yeast Hsf1 promoter. The transformants were grown at 30 (cid:2)C on YPDA (left) or synthetic drop-out medium lacking histidine and leucine. Two individual transformants carrying CAP2 are shown. (B) Gel shift assay demonstrating that CAP2 directly binds the DRE ⁄ the yeast Hsf1 promoter. A 39 bp (DRE) C-repeat sequence of the Hsf1 promoter was used as a probe with glutathi- one-S-transferase-fused recombinant CAP2 protein expressed and puriﬁed from E. coli. Two other probes with substitution point mutations (M1-DRE and M2-DRE) were also used. Mutated bases are indicated by gray letters. (C) A similar experiment as in (A) was conducted with AH109 cells harboring the HIS marker under the yeast Hsf1 promoter with a mutated DRE sequence (see text). S. cerevisiae AH109 cells cotransfected with CAP2 under a constit- utive alcohol dehydrogenase promoter and HIS marker under origi- nal (pyHsf1-HIS) or mutated (pyM1Hsf1-HIS) yeast Hsf1 promoters were grown at 30 (cid:2)C on YPDA and synthetic drop-out medium lacking leucine and histidine.

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CAP2 expression enhances heat stress tolerance

AP2

202aa

support for genetic and functional dissection of proteins of same family in a relatively simple model system.

A CAP2

162aa

142aa

Experimental procedures

122aa

162 142 122

Vector

122

162

142

CAP2

Protein loading

Plant materials and growth conditions

B

Galactose BCY pYES CAP2 122 142 162

10–2

10–3

10–4

10–5

39 °C

The construction of CAP2-transgenic tobacco plants has been described previously [10]. Tobacco plants (N. tabacum var. Xanthi) were grown on 1 ⁄ 2MS agar plates containing soilrite ⁄ vermiculite (1 : 1) at 25(cid:2) C and 60% humidity, under 16 h per day growth conditions. For RNA blot anal- ysis, 1-week-old CAP2-expressing or vector control Nico- tiana seedlings were used. Germination efﬁciency was determined with T1 seeds from vector ⁄ CAP2-transgenic lines. Seeds were sown on ﬁlter paper soaked with germina- tion medium, and incubated for 18 days at 38(cid:2) C or 45(cid:2) C. The other growth conditions remained the same as control conditions. Chickpea (C. arietinum) cv. PUSABGD72 was used in this study. Seedlings were grown as described previ- ously [20]. For heat stress, 7-day-old soil-grown chickpea seedlings were exposed to 45(cid:2) C in the growth chamber. Control seedlings were maintained at 24(cid:2) C.

Fig. 5. (A) Construction and equal expression (by western blot) of CAP2 deletion mutants. Whole cell extracts (20 lg) were analyzed and hybridized using polyclonal antibody against CAP2. Equal pro- tein loading was shown by staining the proteins blotted on nylon membrane by Ponceau S. (B) Thermotolerance assay of the CAP2 deletion constructs in yeast. The assay was performed as in Fig. 3. S. cerevisiae BCY123 cells harboring full-length or truncated versions of CAP2 were exposed to heat stress as described.

Yeast strains and culture conditions

independent

Yeast (S. cerevisiae) strain BCY123 (MATa, Can1, ade2, leu2-3, 112, pep::his+, prb1::leu2+, trp1, Ura3-52, his3, bar1::HisG+, lys2::pGAL1 ⁄ 10-GAL4+) was used in the study. YPG medium consisted of 2% Difco yeast extract, 1% bacto-tryptone, 2% galactose, and 1% rafﬁnose. Syn- thetic drop-out medium contained 0.67% yeast nitrogen base, 2% galactose, 1% rafﬁnose, and all standard amino acids except for the mentioned component. The YPG med- ium containing dextrose ⁄ glucose in place of galactose and 40 mgÆL)1 adenine is referred to as YPDA medium. Yeast transformations were performed using lithium acetate, as described by Ito et al. [21]. pYES2.1 (Invitrogen, Carlsbad, CA, USA) was used as expression vector. The normal growth temperature was 30(cid:2) C. For CAP2 domain map- ping, the truncation containing amino acids 1–162 was named D162. The truncation containing amino acids 1–142 was named D142. The truncation containing amino acid 1–122 was named D122. The full-length and truncated con- structs of CAP2 were cloned in the XhoI–XbaI site of pYES2.1 under the control of the inducible GAL1 pro- moter, using the following primers: CAP2-PYESF, 5¢- CCCTCGAGATGTTAGTGAAAAGCCATCATAAAGG-3¢; CAP2-PYESR, 5¢-GCTCTAGATCCTCCAACAGATTAT CCATTATG-3¢; CAP2T1-PYEST1R, 5¢-GCTCTAGATCA TGAGAATTGGATACCTCTG-3¢; CAP2T2-PYESR, 5¢- GCTCTAGATCAATTGTTGTCTTTACAGAACC-3¢; and CAP2T3-PYESR, 5¢-GCTCTAGATTCAATGTCAACATC CTCAGCTGCA-3¢.

The hypothesis of lateral transfer of AP2 domains from bacteria or ciliates to plants is also supported by the fact that most of the plant AP2 proteins are intronless [18,19]. However, the weak similarity between plant AP2, ApiAP2 and yeast proteins suggests that they may have undergone lineage-speciﬁc expansions. It should be noted here that AtDREB2A and AtDREB2B are interrupted by single intron, whereas CAP2 is intronless, and its protein size is much less and closer to the sizes of the yeast proteins shown in Fig. 6. These facts suggest that AtDREB2A and CAP2 may have different lineages, and thus their modes of function might be different, as seen from the difference in their overexpression phenotypes [3,10].

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S. cerevisiae has served as a suitable model system for studying stress response mechanisms in plants for dec- ades. The compatibility of these two systems has been demonstrated by complementation of yeast mutants with homologous plant genes. Our results provide evidence that a plant AP2 domain protein can be func- tional in budding yeast. Our results also highlight the potential agronomic importance of CAP2, and provide

R. K. Shukla et al.

CAP2 expression enhances heat stress tolerance

Fig. 6. Amino acid sequence alignment of AP2 domains of apicomplexans with hypo- thetical proteins of S. cerevisiae and O. sati- va. The dark background indicates identical amino acids, and the gray background indi- cates amino acids with similar character. GenBank accession numbers with the pres- ent annotations of the proteins are given below.

Investigations on the thermotolerance of yeast cells were performed on either YPG or YPD ⁄ agar plates as mentioned above. Cells were grown in appropriate liquid medium to mid-log phase, plated after suitable dilution, and incubated at normal (30 (cid:2)C) or at high (39 (cid:2)C) temperature. For gene expression studies, S. cerevisiae BCY123 with or without CAP2 was grown in YPDA liquid medium at 30 (cid:2)C up to mid-log phase. The cells were precipitated and resuspended in prewarmed medium at the temperature mentioned in the text.

(637–1026 bp) of

5¢-TTCTTGAACAAACAGTACCTACTAGGACTGTTT CAATGA-3¢; and M1R, 5¢-TCATTGAAACAGTCCTAG TAGGTACTGTTTGTTCAAGAA-3¢. Mutagenesis reac- tions were carried out on double-stranded plasmid DNA using Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA, USA), with the following parameters: 95 (cid:2)C for 30 s; and 16 cycles of 95 (cid:2)C for 30 s, 58 (cid:2)C for 1 min, and 72 (cid:2)C for 7 min. The bacteria-derived template (double-stranded plasmid DNA used as template for the PCR reaction) was digested with the methylation-speciﬁc restriction enzyme DpnI at 37 (cid:2)C for 6 h, and the digested PCR product was transformed into E. coli DH5a cells. The mutation and the ﬁdelity of the rest of the construct were conﬁrmed by DNA sequencing. The protein blot was performed using poly- clonal antibody against full-length CAP2 protein. Whole cell extracts (20 lg) were analyzed by 12% PAGE and transferred to nitrocellulose membranes. Western blot was performed with a 1 : 100 dilution of primary antibody and a 1 : 10 000 dilution of horseradish peroxidase-conjugated secondary antibody. The blotted proteins were visualized using an ECL kit (GE Healthcare, Little Chalfont, UK).

The NtHsfA4 5¢-UAS was cloned with a ClonTech Genome Walker Kit (BD Biosciences Clontech), using the gene-speciﬁc primers 5¢-CACACAAATTTGTTGCAATTT CAC-3¢ and 5¢-AACCCATACACAGACAACTTGCTC-3¢, and the adaptor-speciﬁc primers provided with the kit.

RNA and protein gel blot analysis, cDNA library, mutagenesis, and genome walking

Total RNA was extracted using TRIZOL from tobacco and chickpea seedlings. RNA gel blot analyses were per- formed with 20 lg of total RNA, as described previously [10,20]. An EcoRV–SspI fragment the CAP2 cDNA was used to make radiolabeled probe. Yeast heat-responsive genes Hsp104 and Hsf1 were cloned from the yeast genomic DNA using the primer pairs Hsp104F (5¢-AATCAATAATTGAGAAGGGCCG-3¢) and Hsp104R (5¢-TTTGTATTTAGCACCTGCGG-3¢), and YHsf1 (5¢- CGCCCACTGTATAAGTTTCG-3¢) and YHSF2 (5¢-ATA ATGCTGCAAATACAGGG-3¢), respectively. The ampli- ﬁed products were sequenced and used as probes for north- ern blots. The subtracted cDNA library was constructed with mRNA isolated from 1-week-old seedlings of tobacco harboring vector with or without CAP2 using a ClonTech PCR select cDNA subtraction kit (BD Biosciences Clon- tech, Palo Alto, CA, USA), and reverse northern analysis was performed as described in Boominathan et al. [20]. For RNA gel blot analyses of the tobacco genes, corresponding radiolabeled ESTs were used as probes.

For the reporter assay, the 1683 bp 5¢-UAS including the 285 bp 5¢-UTR of NtHsfA4 was cloned between the HindIII and NcoI sites of pCAMBIA1305.1. The constructs were chemically mobilized into Agrobacterium tumefaciens strain GV3101. Agrobacterium-mediated transformation of tobacco explants was carried out using standard protocols.

For in vitro mutagenesis in the yM1Hsf1 construct, the forward and reverse primers used were as follows: M1L,

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GUS assay

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CAP2 expression enhances heat stress tolerance

2 Shinozaki K, Yamaguchi-Shinozaki K & Seki M (2003) Regulatory network of gene expression in the drought and cold stress responses. Curr Opin Plant Biol 6, 410– 417.

[22]. The

3 Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S,

The transgenic shootlets were selected by growing the trans- formed explants in 10 lgÆmL)1 hygromycin. GUS activity was assayed using a standard procedure with the substrate 4-methylumbelliferyl-b-d-glucuronide average activity of 15 shootlets from each source is presented.

Yamaguchi-Shinozaki K & Shinozaki K (1998) Two transcription factors, DREB1 and DREB2, with an EREBP ⁄ AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respec- tively, in Arabidopsis. Plant Cell 10, 1391–1406.

4 Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K & Shinozaki K (1999) Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress- inducible transcription factor. Nat Biotechnol 17, 287– 291.

5 Sakuma Y, Maruyama K, Osakabe Y, Qin F, Seki M, Shinozaki K & Yamaguchi-Shinozaki K (2006) Func- tional analysis of an Arabidopsis transcription factor, DREB2A, involved in drought-responsive gene expres- sion. Plant Cell 18, 1292–1309.

6 Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M,

Oono Y, Kamiya A, Nakajima M, Enju A, Sakurai T et al. (2002) Monitoring the expression proﬁles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. Plant J 31, 279–292.

7 Sakuma Y, Maruyama K, Qin F, Osakabe Y, Shinozaki K & Yamaguchi-Shinozaki K (2006) Dual function of an Arabidopsis transcription factor DREB2A in water- stress-responsive and heat-stress-responsive gene expres- sion. Proc Natl Acad Sci USA, 103, 18822–18827. 8 Schramm F, Larkindale J, Kiehlmann E, Ganguli A,

trp1-901,

LYS2::GAL1UAS-GAL1TATA-HIS3,

Gel mobility shift assays were performed as described previ- ously [10], with a 39 bp monomer sequence containing the DRE (TACCGACTA) in the YHsf1 promoter as probe. The oligonucleotides used to amplify the sequence were HsfP1 (5¢-TTCTTGAACAAACAGTACCGACTAGGAC TGTTTCA-3¢) and HsfP2 (5¢-TCATTGAAACAGTCCTA GTCGGTACTGTTTGTTCA-3¢). Mutations in the probe sequences are shown in the ﬁgures. For promoter activation assays, the YHsf1 promoter was cloned in the pHIS2 vector with the primer pair yhsfp1 (5¢-CGGAATTCTCGAAAA CGTGCGAAACAAATC-3¢) and yhsfp2 (5¢-CGAGCTCG the EcoRI and AAGATAATGTGGTGAATGGG-3¢) at SacI restriction sites. The resulting construct was named pyHsf1-HIS. The mutation in the M1Hsf1 construct has been described previously. The CAP2 ORF was cloned between two HindIII sites of the pGAD (BD Biosciences ClonTech) vector with LEU as selection marker, and the primer pair CAP2pADF (5¢-AAGCTTATGTTAGTGAAA AGCCATCATAAAGG-3¢) and CAP2pADR (5¢-AAGCT TTCACAGTCACACTTAAGGATAACGAAAGA-3¢), so that the SV40 activation domain of pGAD was removed and CAP2 was expressed constitutively from the alcohol resulting construct was dehydrogenase promoter. The named pGCAP2. Both pGCAP2 and pyHsf1-HIS2 (or pyM1Hsf1-HIS2) were introduced into S. cerevisiae AH109 leu2-3, 112, ura3-52, his3-200, gal4D, (MATa, GAL2UAS- gal80D, GAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA-lacZ, MEL1), and the transformants were selected on HIS)LEU) synthetic drop-out medium.

Gel mobility shift and promoter activation assay

Acknowledgements

Englich G, Vierling E & von Koskull-Do¨ ring P (2008) A cascade of transcription factor DREB2A and heat stress transcription factor HsfA3 regulates the heat stress response of Arabidopsis. Plant J 53, 264–274. 9 Qin F, Kakimoto M, Sakuma Y, Maruyama K, Osaka- be Y, Tran LS, Shinozaki K & Yamaguchi-Shinozaki K (2007) Regulation and functional analysis of ZmDREB2A in response to drought and heat stresses in Zea mays L. Plant J 50, 54–69.

10 Shukla RK, Raha S, Tripathi V & Chattopadhyay D (2006) Expression of CAP2, an APETALA2-family transcription factor from chickpea, enhances growth and tolerance to dehydration and salt stress in trans- genic tobacco. Plant Physiol 142, 113–123.

This project is supported by a seed grant from NIPGR and a grant from the Department of Biotechnology, Government of India. R. K. Shukla is the recipient of a Research Fellowship from NIPGR. V. Tripathi, D. Jain and R. K. Yadav acknowledge the Council for Scientiﬁc and Industrial Research, Government of India, for fellowships.

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