Abscisic acid and desiccation-dependent expression of a novel putative SNF5-type chromatin-remodeling gene in Pisum sativum

Plant Physiology and Biochemistry 45 (2007) 427e435 www.elsevier.com/locate/plaphy

Research article

Abscisic acid and desiccation-dependent expression of a novel putative SNF5-type chromatin-remodeling gene in Pisum sativum Gabino Rı´os1,2, Andre´s P. Gagete1, Josefa Castillo3, Ana Berbel4, Luis Franco, M. Isabel Rodrigo* Department of Biochemistry and Molecular Biology, University of Valencia, Dr Moliner 50, E-46100 Burjassot, Valencia, Spain Received 10 July 2006; accepted 16 March 2007 Available online 21 March 2007

Abstract Snf5-like proteins are components of multiprotein chromatin remodeling complexes involved in the ATP-dependent alteration of DNAehistone contacts. Mostly described in yeast and animals, the only plant SNF5-like gene characterized so far has been BSH from Arabidopsis thaliana (L.) Heynh. We report the cloning and characterization of expression of a SNF5-like gene from pea (Pisum sativum L. cv. Lincoln), which has been designated PsSNF5. Southern analysis showed a single copy of the gene in the pea genome. The cDNA contained a 723 bp open reading frame encoding a 240 amino acid protein of 27.4 kDa with a potential nuclear localization signal. PsSNF5 protein sequence closely resembled BSH, with which it showed an overall amino acid identity of 78.5%. Two-hybrid experiments showed that PsSNF5 is functionally interchangeable with Arabidopsis BSH in the interactions with other components of the remodeling complex. Phylogenetic analysis demonstrated that PsSNF5 clustered with translated expressed sequence tags from other Leguminosae, hypothetically coding for new Snf5-like proteins. RTe PCR expression analysis demonstrated that the PsSNF5 gene is constitutively expressed in all the tissues examined, with minor differences in expression level in different tissues. Nevertheless, expression analysis revealed that PsSNF5 was up-regulated in the last stages of embryo development, when water content decreases. Moreover, abscisic acid and drought stress induced PsSNF5 accumulation in germinating embryos and vegetative tissues, suggesting that chromatin remodeling induced by PsSNF5-containing complexes might contribute to the response to that phytohormone. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Pisum sativum; Abscisic acid; Desiccation; SNF5; Chromatin remodeling complex

Abbreviations: ABA, abscisic acid; cDNA, complementary DNA; CRC, chromatin remodeling complex; DAF, days after fertilization; EST, expressed sequence tag; HAI, hours after imbibition; NUE, near upstream elements; ORF, open reading frame; RACE, rapid amplification of cDNA ends; RTe PCR, reverse transcriptionepolymerase chain reaction; UTR, untranslated region. * Corresponding author. Tel.: þ34 96 354 3013; fax: þ34 96 354 4635. E-mail address: [email protected] (M.I. Rodrigo). 1 These authors contributed equally to this work. 2 Present address: Instituto Valenciano de Investigaciones Agrarias, IVIA, E-46113 Moncada, Spain. 3 Present address: Divisio´n de Hepatologı´a y Terapia Ge´nica, CIMA, Universidad de Navarra, E-31008 Pamplona, Spain. 4 Present address: Instituto de Biologı´a Molecular y Celular de Plantas, CSIC, E-46022 Valencia, Spain. 0981-9428/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2007.03.022

1. Introduction Nucleosomes consist in an octamer of histones wrapped by DNA. This highly structured organization, which further folds to build up eukaryotic chromatin, potentially impairs DNA dynamics. Chromatin remodeling complexes (CRCs) are involved in the ATP-dependent alteration of DNAehistone contacts in nucleosomes allowing the progression of transcriptional, replicative or repair machineries along specific loci. Classification of CRCs attends to the identity of the subunit performing ATP hydrolysis, which in fact constitutes a wide family with numerous subfamilies. The first CRC described, the Saccharomyces cerevisiae SWI/SNF complex, containing

428

G. Rı´os et al. / Plant Physiology and Biochemistry 45 (2007) 427e435

the Swi2/Snf2 ATPase, still is one of the best known CRCs. It is a 2 MDa multisubunit assembly with 11 components, including the core proteins Swi2/Snf2, Swi3 and Snf5 [5,22,30]. Despite the obvious functional relevance of Swi2/Snf2 ATPases, Snf5-like proteins are causing an increasing interest. As asserted by the analysis of two yeast conditional mutants, in addition to a role in the structural assembly of SWI/SNF complex, Snf5 protein turned out to affect chromatin remodeling activity in vivo [12]. Moreover published work describing the interaction of yeast Snf5 with acidic transcriptional activators [20,23] and the human hSNF5/INI1 with the transcription factor c-MYC [10], points to optional gene activation mechanisms: On one hand interaction of Snf5 with its partner could mediate the targeting of SWI/SNF-like complexes to specific promoters; on the other hand, Snf5-like subunit from a SWI/SNF complex previously located on an inducible gene could recruit transcriptional factors acting as inductors. Further genetic approaches to Snf5 function linked the homozygous deletions of SNF5/INI1 to embryo lethality in mice [13,15], and to aggressive malignant rhabdoid tumors in children, pointing to a role as tumor suppressor [31]. The importance of SWI/SNF complex function is highlighted by the evolutionary conservation of these proteins in all the eukaryotic organisms, including plants. Indeed, Arabidopsis thaliana database sequence analysis predicts the presence of about 40 SNF2-like, four SWI3-like, two SWP73-like and one SNF5-like gene (Plant Chromatin Database, http:// www.chromdb.org/). So far, few of these genes and coded proteins have been characterized and numerous biochemical and functional issues remain unknown. The first characterization of a plant SNF5-like gene by Brzeski et al. [3] showed that antisense silencing of Arabidopsis BSH induces sexual sterility in addition to a characteristic ‘‘bushy’’ phenotype. Moreover, knock-out mutations of AtSWI3A and AtSWI3B, two out of the four SWI3-like genes from Arabidopsis, led to embryo lethality, whereas AtSWI3C and AtSWI3D disruption caused developmental defects and variable levels of sexual sterility [25]. Taken together, these results argue for a relevant function of SWI/SNF complex in embryo development and seed viability in the model grass Arabidopsis thaliana. A wider knowledge on developmental issues affected by chromatin remodeling complexes will require the molecular characterization of SWI/SNF elements in other plant species, particularly those that accumulate storage products in their eatable seeds. The phytohormone ABA is necessary both for regulation of late seed development events that enable an embryo to survive desiccation and for the response to environmental stresses such as drought, salt and cold [4]. The involvement of chromatin remodeling in ABA-dependent synthesis of a seed specific protein has been shown in detail in phas gene from bean (Phaseolus vulgaris). This gene, which encodes b-phaseolin (a 7S seed storage protein), contains a positioned nucleosome on the transcription start site in vegetative tissue in which it is inactive; displacement of this nucleosome favored by the binding of the acidic activator PvALF in the presence of ABA facilitates activation of phas expression during embryogenesis [16,17]. Further independent reports show chromatin related

genes subject to ABA and drought transcriptional control, instead of mediators of ABA signal: On one hand, the H1 histone variant H1-S of tomato (Lycopersicon esculentum) is targeted to the nucleus and accumulates in chromatin in response to water-deficit stress and ABA [28]; on the other hand, we had previously shown that p16 from pea (Pisum sativum) is a chromatin associated protein accumulated in embryonic tissue during seed desiccation progression, synthesized by processing of a larger protein, p54, and coded by psp54 gene which is induced by ABA and several stresses in seedlings [6e8]. New and surprising links of ABA and chromatin have been recently uncovered by the finding of the ABA receptor FCA in Arabidopsis [24], which was previously described as an interactor of the chromatin remodeling subunit AtSWI3B [26], and the involvement of Arabidopsis histone deacetylase AtHD2C in the ABA response [29]. The improvement of available genetic tools and a better understanding of molecular mechanisms underlying gene activation and repression, might help to elucidate new connections between the poorly known chromatin remodeling phenomena in plants and the much more exhaustively studied water deficit and ABA signaling. In order to inquire into the role of CRCs in the desiccation-dependent synthesis of seed specific proteins, we started an approach to clone chromatin remodeling elements in plants of agronomic interest. This paper describes the presence in Pisum sativum of a SNF5-like gene, and the study of its properties suggests that chromatin remodeling induced by the corresponding complex might be involved in ABA response. 2. Methods 2.1. Plant material and treatments The culture of pea (Pisum sativum L. cv. Lincoln) and plant material collection were carried out as previously described by Castillo et al. [7]. Briefly, seeds imbibed in water for 20 h at 4  C were placed on moistened filter paper in the bottom of 14 cm petri-dishes and germinated at 28  C in the dark. For ABA or sorbitol treatments, after 18 h or 72 h of germination, 100 ml of 100 mM ABA or 1.5 M sorbitol were applied onto each seedling. For mock-treatments (controls), equal volumes of water were applied. When seedlings were to be dried, they were transferred to dry filter paper. After all treatments the samples were allowed to stand for 24 h in the dark. To obtain adult plants, the seedlings were cultured on moist vermiculite in a greenhouse. ABA treatment of leaves was performed by spraying with 100 mM ABA. After 24 h, the leaves were excised, frozen in liquid nitrogen and stored at 80  C until use. Plants used for control were submitted to a similar treatment, but water was used instead of ABA. Drying of plants was achieved by placing them on dry filter paper for 24 h. The leaves were then excised and processed as before. 2.2. cDNA and genomic cloning In order to clone the pea SNF5-like gene, cDNA sequences for Arabidopsis thaliana BSH, Zea mays CHE101 (Plant Chromatin

G. Rı´os et al. / Plant Physiology and Biochemistry 45 (2007) 427e435

Database, http://www.chromdb.org/), Oriza sativa CHE701 (Plant Chromatin Database), and expressed sequence tags (EST) from Solanum tuberosum (CV431089), Lycopersicon esculentum (AJ832061), Medicago truncatula (BF637348), Glycine max (BU925858), Antirrhinum majus (AJ789235), Vitis vinifera (CF511160), Liriodendron tulipifera (CK751743), Malus domestica (CV084345), Populus balsamifera (BU876760), Mesembryanthemum crystallinum (BF479808) and Stevia rebaudiana (BG526399) were aligned by ClustalW (http://www.ebi.ac. uk/clustalw) [9]. Regions with the highest conservation were identified and two degenerated oligonucleotides DSNF1fw and DSNF2rev were designed. Reverse transcribed RNA from pea embryos (see below) was employed as template in a PCR reaction with the previous primers under the following conditions: 94  C for 5 min followed by 35 cycles of amplification (94  C for 30 s, 60  C for 30 s, 72  C for 30 s) and a final step at 72  C for 5 min. The PCR reaction yielded a product of about 280 bp, which was cloned into the pGEM-T vector (Promega) and sequenced, being highly similar to BSH and the ESTs shown above. The 50 /30 RACE kit (Roche) was used to isolate the 50 and 30 ends of PsSNF5. All reactions were performed according to the manufacturer’s instructions, on total RNA obtained from pea embryos at 35 DAF. The primer PSSNF1rev was employed to synthesize the 50 first-strand cDNA, followed by amplification with the nested primers PSSNF2rev and PSSNF3rev to obtain a clean band in an agarose gel. Amplification of 30 end required the use of these three nested primers: PSSNF4fw, PSSNF5fw and PSSNF6fw. The 50 and 30 amplified products were purified, cloned into pGEM-T and sequenced. After comparing and aligning the three partial sequences containing PsSNF5, the full-length cDNA of the gene was deduced and obtained through RTePCR with primers PASSNF1fw and PASSNF2rev, and subsequently cloned into NcoI/EcoRI sites of plasmid pAS2-1 (Clontech). The sequence of the employed primers is shown in Table 1. 2.3. Southern blot analysis Genomic DNA was prepared from 2 g young leaves as described by Michaels et al. [19]. Aliquots of 25 mg were digested overnight at 37  C with BclI, EcoRI and EcoRV, Table 1 Sequence of primers designed for PCR reactions Primer name

50 e30 sequence

DSNF1fw DSNF2rev PSSNF1rev PSSNF2rev PSSNF3rev PSSNF4fw PSSNF5fw PSSNF6fw PASSNF1fw PASSNF2rev Act1111fw Act1286rev BSTH1 BSTH2

GA(T/C)GC(G/C)TT(C/T)ACTTGGAACCC GCAAA(C/T)TC(C/T)TCAGG(G/A)TCACT CTCCGGCATACATATCTTGACCCTC GGTGGAGGCAGCTTCAAATCCTTC CGCAAACACCACAACCTCAGAATCC GGATTCTGAGGTTGTGGTGTTTGCG GAAGGATTTGAAGCTGCCTCCACC GAGGGTCAAGATATGTATGCCGGAG ATAAGATCTCCATGGGGATGAAAACCCCTATTTCT CCCCGAATTCACCTGAAATTTCTCTC CGAAGGTTGTAGCACCACCAGAGAG TCCCTTGATCCAACTCTCAGGCTCC GGTGAATTCATGAAGGGTTTAGTGTC CGAGGATCCTCACCTTGCATGTCTCT

429

respectively, which do not cut the cloned cDNA and then fractionated by 0.7% agarose gel electrophoresis, and transferred onto a nylon membrane (Hybond-Nþ, Amersham Pharmacia). The probe was labeled with [a-32P]dCTP using a random primer labeling kit (High Prime, Roche). The procedure of Southern blotting was based on a standard method [1]. 2.4. Sequence and phylogenetic analysis The molecular weight and isoelectric point of PsSNF5 protein were calculated by using the DNAMAN program. The deduced amino acid sequence of PsSNF5 was compared to other SNF5-like sequences of higher plants and yeast. The sequences were aligned online by ClustalW [9]. Phylogenetic analysis was performed using programs from the PHYLIP group, PHYLogeny Inference Package, Version 3.6 [11] at http://bioweb.pasteur.fr/seqanal/phylogeny/phylip-uk.html. A distance matrix was computed according to the Dayhoff PAM model by the program Protdist and then it was used as input for the program Neighbor, where the UPGMA method of clustering was selected. The phylogenetic tree was drawn with Phylodendron TreePrint program at http://www.es.embnet. org/Services. 2.5. Protein interaction assays in the yeast two-hybrid system A full-length PsSNF5 cDNA was amplified by PCR using primers PASSNF1fw and PASSNF2rev carrying restriction sites NcoI and EcoRI (Table 1), cloned into the pAS2-1 vector containing the GAL4 DNA binding domain (Clontech) and sequenced (PsSNF5-BD plasmid). Arabidopsis BSH gene was PCR-amplified with primers BSTH1 and BSTH2, cloned into pAS2-1 on EcoRI and BamHI sites and sequenced (BSH-BD). On the other hand, pACT2 vectors carrying the GAL4 activation domain fused to AtSWI3A (AtSWI3A-AD) and AtSWI3B (AtSWI3B-AD) were obtained by G.R. in C. Koncz’ laboratory by screening an Arabidopsis cell suspension library using BSH-BD as bait. The Saccharomyces cerevisiae strain Y190 was cotransformed with the following pairs of plasmids: PsSNF5-BD/AtSWI3A-AD, PsSNF5-BD/AtSWI3B-AD, BSH-BD/AtSWI3A-AD, BSH-BD/AtSWI3B-AD, as well as with the control pairs PsSNF5-BD/pACT2, BSHBD/pACT2, pAS2-1/AtSWI3A-AD, pAS2-1/AtSWI3B-AD and pAS2-1/pACT2. All transformants were grown in selection medium containing 100 mM 3-amino-1,2,4-triazole for the growth assay without histidine, and the level of b-galactosidase activity was monitored in three independent clones per transformant by the replica filter lift method described in the Clontech yeast protocol handbook. 2.6. RTePCR Total RNA from different tissues was obtained from 2 g material following standard procedures [1]. Subsequently, RTePCR was carried out to analyze the expression of PsSNF5 at different stages of embryo development, seed germination,

G. Rı´os et al. / Plant Physiology and Biochemistry 45 (2007) 427e435

430

different tissues and stresses. Briefly, 1 mg of RNA was reverse transcribed by using Superscript II Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. The cDNA was properly diluted (between five- and hundredfold), and 1 ml was subjected to PCR with the PsSNF5 specific primers PSSNF4fw and PSSNF1rev, amplifying a 143 bp product, under the following temperature profile: 94  C for 5 min, then 34 cycles of amplification (94  C for 30 s, 66  C for 30 s, 72  C for 30 s) and a final step at 72  C for 5 min. Simultaneously, a 185 bp actin cDNA fragment (U81047) was amplified with specific primers Act1111fw and Act1286rev (see Table 1) as a reference control for the RTePCR, by using the same PCR conditions but with 30 rounds of amplification instead of 34. The program ImageJ was used for quantification and all experiments were performed for duplicate.

that were subsequently aligned in order to search for highly similar stretches. The degenerated primers DSNF1fw and DSNF2rev, amplifying a 278 bp internal sequence on most plant EST, were successfully employed on cDNA from pea embryonic tissue. The cloned sequence yielded new primers used to obtain the full length cDNA of PsSNF5 by 50 and 30 -RACE protocols. Confirmation of the sequence was achieved by PCR amplification of the whole ORF with primers PASSNF1fw and PASSNF2rev, located on the translation start and stop codons respectively, and carrying restriction sites suitable for cloning into pAS2-1 plasmid. The full sequence of PsSNF5 and deduced protein (GenBank accession number DQ539420) are shown in Fig. 1. The 50 UTR is 158 bp long, carrying only one additional ATG at position 143, with no chance to initiate a coding frame due to the immediate stop codon placed at 140. The fact that several stop codons in the three frames are spread along the 50 UTR indicates that the whole PsSNF5 protein coding region is contained into the cloned cDNA, and that the underlined ATG is indeed the only believable translation start codon of the gene. The translation initiation context is mostly matching the plant consensus, with two purines (A) located at 3 and þ4 [14].

3. Results 3.1. Cloning of the PsSNF5 cDNA In order to clone a putative pea SNF5-like gene, we obtained plant BSH-related sequences from the EST databases 1

CAACCAATAGTGGGGATGTAATTGCAAAATCAGAAGTACTGTTAGCGGTACCCGCGGAGA

61

ATTGAAAATTGGAACAAGAAATTATTTGGTTTCGCCGTGTTCCATCATCATCACCATTGA

121 1

* * TTGAAGCGTAGTGGAAGCCATAAGAGTTGAGGTTGAAGATGAAAACCCCTATTTCTGGTT M K T P I S G

181 8

TCTACAGAAACCCCGTCAAGTTCAGAATGCCAACTTCGGAAAATCTCGTTCCCGTTCGAC F Y R N P V K F R M P T S E N L V P V R

241 28

TCGACATCGAAATCGACGGACATAGATACAAAGATGCCTTCACATGGAACCCTTCTGATC L D I E I D G H R Y K D A F T W N P S D

301 48

CGGATTCTGAGGTTGTGGTGTTTGCGAAAAGGACTGTGAAGGATTTGAAGCTGCCTCCAC P D S E V V V F A K R T V K D L K L P P

361 68

CTTTTGTTACTCAGATTGCTCAGTCTATTCAATCGCAGTTAGCAGAGTTTCGATCCTATG P F V T Q I A Q S I Q S Q L A E F R S Y

421 88

AGGGTCAAGATATGTATGCCGGAGAGAAGATTATTCCCATTAAGCTTGATCTTCGTGTAA E G Q D M Y A G E K I I P I K L D L R V

481 108

ATCATACGCTTGTAAAGGACCAATTTTTATGGGACTTGAACAACTTCGATAGTGATCCTG N H T L V K D Q F L W D L N N F D S D P

541 128

AAGAGTTTGCAAGGACCTTCTGCAGAGACATGGGCATTGAGGATCCTGAGGTTGGACCAG E E F A R T F C R D M G I E D P E V G P

601 148

CTGTCGCCTTTGCCATCAGAGAACAGCTTTACGAGATTGCAATTCAAAGCGTGGTTTCAG A V A F A I R E Q L Y E I A I Q S V V S

661 168

CAAGAGAAAGCAGACTAAGCAAGAAGGGTCGTCGAGGGGCTGATTTTACTCCAGTCAGTA A R E S R L S K K G R R G A D F T P V S

721 188

AAGGAGGCGCCGTAGCAGTGGACTTGGTTAAGTTATTTGGTATTAAATCAAGTGTTGTTC K G G A V A V D L V K L F G I K S S V V

781 208

GGAAAAGAAAGGAGTGGGACGTATATGAACCCATTGTGGATCTTCTATCCAACGAAGAAG R K R K E W D V Y E P I V D L L S N E E

841 228

TTGATGTCCTTGAAGCAAAGGAAGAGAGAAATTTCAGGTGAATTGGCTCTGTAGTCAGTT V D V L E A K E E R N F R

901

ATGTCTTGTCTCAGATAGTTCAAAAGAGTGTATATTTTTAATGTTGTAGTTTGAAGTTAT

961

TTACATGTATACTAGATGGTTTTATAAGACCAAAAATCATTAGTTCGAAAAAAAAAAAAA

I1

I2

I3 I4 I5 I6 I7 I8

I9

Fig. 1. Nucleotide and deduced amino acid sequences of PsSNF5 cDNA. The asterisks label two purines (A) located at 3 and þ4 around the initiation codon. The location of the nine introns is marked by black arrows and translational start and stop codons are underlined. The putative polyadenylation signals are underlined with dashed lines. Bold letters represent a putative nuclear localization signal on the protein sequence.

G. Rı´os et al. / Plant Physiology and Biochemistry 45 (2007) 427e435

A recent re-evaluation of plant polyadenylation signals found no high-consensus signal patterns on cis near upstream elements (NUE) and far upstream elements (FUE), based on Arabidopsis thaliana 30 UTRs [18]. Indeed, the most typical element with sequence AATAAA, was only found in 10% of the analyzed mRNAs. Despite AATAAA consensus is not present in PsSNF5 30 UTR, the underlined sequences TTTTAT, TTATAA and AAAAAT are also frequently found on Arabidopsis NUE regions (Fig. 1). 3.2. Gene structure of PsSNF5 In contrast to yeast and some animal organisms, Arabidopsis genome contains only one SNF5-like gene. To analyze whether PsSNF5 is present as a single copy gene in pea genome, Southern blot analysis was performed. Hybridization of genomic DNA digested with the enzymes BclI, EcoRI and EcoRV against a PsSNF5 cDNA probe rendered just a single band after digesting with BclI and EcoRV. After EcoRI digestion an additional faint band of ca. 6 kb is observed (Fig. 2). To determine the structure of the PsSNF5 gene, genomic sequence was obtained from clones isolated by genomic PCR using gene specific primers designed after the PsSNF5 full-length cDNA. The gene contains nine introns which are fully coincident in position on the cDNA sequence with the ones from BSH gene from Arabidopsis thaliana (Fig. 1). However intron sizes, 79, 72, 102, 2295, 110, 111, 96, 126 and 193 bp, showed complete divergence with their BSH counterparts. (kb) 23.0 9.4

BclI

4.4 2.2 2.0

-

Restriction analysis of genomic DNA showed that there is an EcoRI site within the fourth intron and this explains the appearance of the two bands in the Southern blot of Fig. 2. Consequently, pea genome seems to contain a unique SNF5 related gene. 3.3. Characterization of the deduced PsSNF5 protein The predicted PsSNF5 protein displayed a sequence of 240 amino acid residues (Fig. 1), with a calculated molecular mass of 27.4 kDa and an isoelectric point of 5.30. A putative nuclear targeting signal composed of four basic residues (RKRK) is found at position 208. In order to elaborate a phylogenetic tree of plant SNF5 related proteins, a yeast Snf5 protein fragment containing the conserved SNF5 domain (residues 423 to 700), Pisum sativum PsSNF5 protein, Arabidopsis thaliana BSH, Zea mays CHE101 (Plant Chromatin Database, http:// www.chromdb.org/), Oriza sativa CHE701 (Plant Chromatin Database), and translated ESTs from Solanum tuberosum (CV431089), Lycopersicon esculentum (AJ832061), Medicago truncatula (BF637348, partial protein) and Glycine max (BU925858, partial protein) were aligned by ClustalW and treated as described in Section 2. The phylogenetic analysis clearly clustered the pea PsSNF5 with the other Leguminosae partial proteins from Medicago and Glycine, with which it showed an amino acid identity of 94.7% and 92.1% respectively (Fig. 3). High identity rates were likewise found with respect to Arabidopsis BSH (78.5%), and the Snf5-like proteins from Solanum tuberosum (77.5%), Lycopersicon esculentum (77.4%), Oriza sativa (64.0%) and Zea mays (61.5%).

EcoRV

-

6.4

EcoRI

431

Zea mays

0.1

Oriza sativa Medicago truncatula Pisum sativum Glycine max Solanum tuberosum Lycopersicon esculentum

0.5

-

Arabidopsis thaliana Saccharomyces cerevisiae

Fig. 2. Southern blot analysis of the PsSNF5 gene. Genomic DNA from leaves (25 mg) was digested with restriction enzymes BclI, EcoRI and EcoRV, blotted and hybridized with a probe containing the PsSNF5 coding sequence. Size markers are shown on the left.

Fig. 3. Phylogenetic analysis of plant SNF5-like amino acid sequences. The following sequences were compared: PsSNF5 from pea (DQ539420), indicated in bold, BSH from A. thaliana (U88061), CHE101 from Z. mays, CHE701 from O. sativa, translated ESTs from S. tuberosum and L. esculentum (CV431089 and AJ832061 respectively), partial proteins from M. truncatula and G. max (BF637348 and BU925858 respectively) and Snf5 from S. cerevisiae. The sequences were aligned using ClustalW program and analyzed as described in Section 2.

G. Rı´os et al. / Plant Physiology and Biochemistry 45 (2007) 427e435

432

Plant Snf5-like are proteins nearly 4 times shorter than the yeast counterpart (Fig. 4A), containing solely the conserved region that defines the SNF5 Pfam family [2]. A particular domain in the N-terminal half of yeast Snf5, absent in its plant relatives, is a glutamine and proline rich region which has been reported to bind acidic transcriptional activators, to hypothetically contribute to the targeting of SWI/SNF complexes to specific genes. The absence of this and other stretches in the uniform group of plant Snf5-like proteins address new questions about specificity and functional diversification. The highly conserved SNF5 domain contains a direct repeat present in every Snf5-like protein characterized so far. A comparison of first and second repeats of yeast Snf5, BSH and PsSNF5, shown in Fig. 4B, highlights the inter- and intramolecular conservation of certain residues and the coincidence of related amino acids in a relevant rate of positions along the sequence. The identity percentage between the first and second repeated motif in PsSNF5 reaches 30.0%, far away from the similarity values between different species. 3.4. PsSNF5 interacts with Arabidopsis AtSWI3A and AtSWI3B Despite the high degree of sequence similarity between PsSNF5 and other well described SNF5 proteins, as the Arabidopsis BSH, some functional or structural evidence is required to identify the pea protein as a bona fide component of plant CRCs. Due to the experimental difficulties to assay chromatin remodeling activities of plant SWI/SNF complexes, which indeed have not yet been purified to date, a simpler and more feasible procedure, involving the yeast two-hybrid system, may be employed to assess the heterologous interaction of the Pisum protein with some of the already known subunits of plant CRCs. Out the four SWI3-like Arabidopsis proteins, only AtSWI3A and AtSWI3B interact with BSH as revealed by the yeast two-hybrid approach [25]. In order to test whether PsSNF5 could structurally replace BSH within its protein

A

Q/P

complex, we transformed the yeast strain Y190 with vector pAS2-1 containing the GAL4 DNA binding domain fused in frame to either PsSNF5 or BSH, and pACT2 vector containing the GAL4 activation domain fused to either AtSWI3A or AtSWI3B. The lacZ and HIS3 genes under the control of the GAL4-regulated promoter of GAL1 were used as reporter genes for the yeast two-hybrid experiment. Both the b-galactosidase assay showing lacZ expression, observed by blue-colored colonies (Fig. 5), and histidine starvation experiments confirming HIS3 expression (not shown), gave identical results. Self-activation assays of PsSNF5-BD and BSH-BD in combination with empty vector AD (Fig. 5, left) were negative. Similarly, AtSWI3A-AD and AtSWI3B-AD did not activate lacZ expression in the absence of a partner (Fig. 5, left). However, PsSNF5-BD and BSH-BD produced an intense blue spot when combined with AtSWI3A-AD and AtSWI3B-AD (Fig. 5, right). Therefore, when assayed with the yeast twohybrid method, PsSNF5 is able to form heterodimers with Arabidopsis AtSWI3A and AtSWI3B at a level similar to that of Arabidopsis BSH, confirming that PsSNF5 and BSH are structurally interchangeable. 3.5. PsSNF5 expression responds to embryo development, ABA and drought stress As an initial approach to the characterization of PsSNF5 role in pea, we investigated the tissue dependent expression of its gene. As shown in Fig. 6A, no relevant alteration of PsSNF5 mRNA levels was observed by RTePCR when analyzing selected samples from roots, stems, leaves and flowers. PCR amplification of reverse transcribed total RNA produced a major DNA fragment with the expected size for each tested organ, which in fact was smaller than the PCR product of the same primers on genomic DNA (data not shown). Such a difference, due to the presence of the third intron, served to discard any contribution of genomic DNA contamination to the intensity of the band. This result is consistent with the reported

SNF5 Snf5p

0

100

200

BSH PsSNF5

B

PsSNF5 rep1 BSH rep1 Snf5 rep1 PsSNF5 rep2 BSH rep2 Snf5 rep2

MPTSENLVPVRLDIEIDGH--RYKDAFTWNPSDPDSEVVVFAKRTVKDLKLPPPFVT----QIAQSIQSQLAEFR MPTAENLVPIRLDIQFEGQ--RYKDAFTWNPSDPDNEVVIFAKRTVKDLKLPYAFVT----QIAQSIQSQLSDFR NETSEQLVPIRLEFDQDRDRFFLRDTLLWNKNDKLIKIEDFVDDMLRDYRFEDATREQHIDTICQSIQEQIQEFQ MYAGEKIIPIKLDLRVNHT--LVKDQFLWDLNNFDSDPEEFARTFCRDMGIEDPEVGP---AVAFAIREQLYEIA MYTGEKIIPIKLDLRVNHT--LIKDQFLWDLNNFESDPEEFARTLCKDLGVEDPEVGP---AVAFAIREQLYEIA LGGDDLRIRIKLDIVVGQN--QLIDQFEWEISNSDNCPEEFAESMCQELELPGEFVT----AIAHSIREQVHMYH

Fig. 4. Comparison of PsSNF5 protein with Snf5 from yeast and BSH from A. thaliana. (A) Schematic representation and motif organization of the three proteins. The SNF5 domain is indicated by a black box and the Gln and Pro rich region in the N-terminal region of Snf5p by a gray box. (B) Alignment of first and second internal repeats (rep1 and rep2 respectively) which constitute SNF5 domain. Identical residues are highlighted in black. Residues of amino acids with closely similar properties are shaded in gray.

G. Rı´os et al. / Plant Physiology and Biochemistry 45 (2007) 427e435

Fig. 5. Analysis of the interactions of PsSNF5 with AtSWI3A and AtSWI3B by yeast two-hybrid assays. b-Galactosidase filter lift assays of pair-wise interactions between pea and Arabidopsis proteins are shown. Blue-colored spots indicate protein interactions. The pairs BSHeAtSWI3A, and BSHeAtSWI3B are included as positive controls. Negative control assays including one or both empty vectors are shown at the left. BD stands for the GAL4 DNA binding domain and AD, for the GAL4 activation domain.

ubiquitous expression of BSH in Arabidopsis thaliana [3]. The current lack of information on SNF5-like expression in plant embryonic tissues led us to extract RNA from isolated pea seed tissues and embryo axes at different developmental stages. PsSNF5 expression showed increased values at the final stages of seed maturation, at 20 and 35 days after fertilization (DAF, Fig. 6B). Imbibition of seeds did not result in the disappearance of PsSNF5 transcripts in axes and cotyledons (see Fig. 6C,D, lanes 0 HAIdhours after imbibition). This means that PsSNF5 mRNA is stored in the dry seed. Nevertheless, a drastic reduction of PsSNF5 mRNA levels in axes

433

occurs concomitantly with germination, but the transcription is resumed by 72 HAI. A somewhat different situation takes place in cotyledons, in which the PsSNF5 mRNA is present still at 18 HAI and the resumption of transcription by 72 HAI is more evident. We have previously described a comparable behavior in the case of psp54 gene, which codes for a bicupin precursor [6]. As the latter gene is induced by ABA, present results may suggest the involvement of ABA signaling in PsSNF5 expression. To test this possibility, 18 HAI embryonic axes (during germination) and 72 HAI seedlings (after germination) were subjected to ABA, osmotic and drought stress for 24 h, collected and their levels of PsSNF5 mRNA compared with normally germinated seeds. ABA, sorbitol and drought treatments increased PsSNF5 expression in both cases, especially in seedlings (Fig. 6E), confirming the regulatory role of ABA on PsSNF5. In order to test the response of vegetative tissues to this phytohormone, leaves were similarly subjected to ABA and drought stress. RTePCR of total RNA from these samples confirmed the positive effect of both treatments on PsSNF5 transcription also in an adult tissue (Fig. 6F). 4. Discussion Despite the extensive knowledge on chromatin remodeling activities and molecular complexes from yeast and

Fig. 6. Expression analysis of the PsSNF5 gene by RTePCR. RNA was extracted and used for generation of the corresponding cDNAs. The presence of PsSNF5 mRNA was detected by PCR amplification of a 143 bp product with primers PSSNF4fw and PSSNF1rev. A 185 bp fragment from actin cDNA was amplified as an internal control using the primers Act1111fw and Act1286rev. The RTePCR analysis was performed in different plant organs (A); during seed development, at different days after fertilization (B); in embryonic axes (C) and cotyledons (D) at different hours after imbibition of seeds; after different treatments with abscisic acid (ABA), desiccation (DES) and sorbitol (SOR) on 18 HAI and 72 HAI (E) and leaves (F). Control samples for the stress experiments (lanes C) were similarly treated, but water was used instead (see Section 2). The intensity of the bands was determined with the program ImageJ and divided by the intensity of the actin band in the same lane. The resulting values were normalized to the control ones and this normalized intensity is given below each lane.

434

G. Rı´os et al. / Plant Physiology and Biochemistry 45 (2007) 427e435

animal models, few molecular approaches to the chromatin remodeling phenomena have been initiated in plants, almost only in the model organism Arabidopsis thaliana. Those studies have related SNF5-like, SNF2-like, and SWI3-like proteins from Arabidopsis to specific vegetal processes as flowering initiation, flower organ identity, embryo development and gametophyte formation, as well as to general mechanisms as DNA recombination, gene silencing and transcriptional modulation. Our goal was to isolate SWI/ SNF-related genes as a starting approach to the functional characterization of chromatin remodeling complexes in plants of agricultural interest and we selected pea as the material of choice. Specifically, our interest was mainly focused on the genes expressed during seed maturation and germination, whose transcription obeys a complex program, involving ABA signaling in some instances. Interestingly, studies reporting the contribution of ABA and chromatin remodeling activities on seed dependent activation of phas gene from Phaseolus vulgaris [16,17,21], already suggest an important role for SWI/SNF-like complexes in legume nutrient accumulation. This work represents the second characterization of a plant SNF5-like protein after BSH from Arabidopsis thaliana [3], and the first one in a legume, even when multiple SNF5-like sequences may be found in plant EST collections and the Plant Chromatin Database (http://www.chromdb.org/). The analysis of pea PsSNF5 protein confirmed its postulated similarity with other plant homologues, which are small proteins with the minimal SNF5 domain that characterize the Pfam SNF5 family [2]. The high conservativeness of this domain implies that almost its entire structure is essential for its biological function. The lack of the glutamine and proline rich domain involved in the interaction with acidic activators found in yeast Snf5p and of other sequence features suggests certain functional divergence. Indeed the Arabidopsis BSH is only partially able to rescue the growth defects on galactose caused by disruption of SNF5 in yeast [3]. The existence of a unique SNF5-like gene in both Arabidopsis thaliana and Pisum sativum (Fig. 2) carries to an extreme the simplicity of SNF5 contribution to plant remodeling complexes, whose diversity seems to be mostly conferred by SNF2 and SWI3-like subunits. The presence in databases of partial EST from Medicago truncatula and Glycine max allowed the clustering of the Pisum sativum sequence within a Leguminosae group in the phylogenetic tree (Fig. 3). The identity rate between PsSNF5 and the deduced partial protein from Medicago and Glycine were the highest obtained due to their evolutionary closeness. In fact, relatedness between SNF5 proteins used to elaborate the tree matched properly the genetic distance between species. In a more detailed view to PsSNF5 features we observed that pea protein reproduces the typical direct repeat present in the canonical SNF5 domain. The similarity between first and second repeated motif within PsSNF5 is much lower than the relatedness between first or second motifs from different species, which is in fact expected due to the ancestral origin of such a duplication event (Fig. 4).

The heterologous interaction of PsSNF5 protein with the Arabidopsis SWI3-like proteins AtSWI3A and AtSWI3B, roughly similar to that revealed for the homologous Arabidopsis BSHeAtSWI3A/B interactions by the yeast two-hybrid system (Fig. 5), allows the classification of PsSNF5 as a member of the SNF5 family of chromatin remodeling factors not only on the basis of sequence similarity to other members of the family, but also from a functional point of view. Our data confirm the high degree of structural conservativeness between plant SWI/SNF remodeling complexes. The expression analyses described in this paper indicate that PsSNF5 is transcribed during seed maturation and that its mRNA levels are maintained during seed imbibition (Fig. 6BeD). Although no definitive conclusions can be drawn in the absence of data on the protein level, these facts may suggest a role for PsSNF5 protein, and consequently for its remodeling activity, in processes occurring during embryogenesis and germination. These processes may likely include the regulation of genes concerned with seed storage protein accumulation or seed desiccation and those with nutrient mobilization, for instance the genes coding for proteases required for the degradation of storage proteins in axes and cotyledons. The absence of PsSNF5 transcription during germination does not mean that SWI/SNF-governed remodeling is not required during this period, as we do not know whether the protein persists. At any rate, the transcription recovery observed during postgerminative periods ought to obey either to a reposition or to an increase of the remodeling machinery necessary for controlling gene expression. For instance, in cotyledons it may be required to initiate the senescence program, which involves protease activation and protein degradation coincidently with seed nutrient mobilization. On the other hand, the active transcription of the gene observed both during embryogenesis and in adult tissues (Fig. 6A and B) suggests the involvement of Pisum SWI/SNF complex in general or constitutive gene expression programs on the whole plant life cycle. The fact that ABA and hydric stress cause in both seedlings (72 HAI) and adult tissues and in a lesser extent in embryos (18 HAI) the induction of PsSNF5 gene (Fig. 6E and F) might imply an additional role for SWI/ SNF chromatin remodeling in the transmission of ABA signal and, therefore, in drought protection. A recent paper describes the ABA receptor FCA in Arabidopsis [24], which interestingly turned out to interact with the SWI/SNF subunit AtSWI3A and AtSWI3B [25], offering an attractive direct model for ABA dependent gene regulation through chromatin remodeling. Our results place ABA signal and SWI/SNF complex accumulation at different but consecutive transcriptional levels, that is PsSNF5 overproduces as a consequence of ABA signal transduction, instead of a direct biochemical interaction, which in fact support the idea that ABA response requires an additional SWI/SNF complex supply. In this sense, the increased transcription induced by sorbitol and desiccation in seedlings and adult tissues (Fig. 6E and F) suggests that ABA-independent signaling pathways, perhaps acting in synergy with those dependent on the hormone, play a role in the expression of PsSNF5 gene.

G. Rı´os et al. / Plant Physiology and Biochemistry 45 (2007) 427e435

Microarray online analysis by the AtGenExpress Visualization Tool [27] allows the systematic study of the developmental stage and treatment-dependent expression of the Arabidopsis BSH gene. This analysis offers a way to find shared transcriptional features between Pisum PsSNF5 and Arabidopsis BSH. This tool showed a higher expression of BSH in the shoot apex; however, no induction by ABA on seedlings or seeds may be observed. Similarly, osmotic, salt and drought stresses do not affect BSH expression, which, moreover, does not accumulate during seed development. Consequently despite their high sequence and structure similarity, PsSNF5 and BSH show major differences at the transcriptional regulation level, suggesting new and interesting roles for the Pisum sativum gene. Acknowledgments We thank Csaba Koncz for his helpful and stimulating supervision during the German years of G.R., as well as for the facilities to use his Arabidopsis cell suspension library. This work was supported by Grant BFU2004-03616 from the Ministerio de Ciencia y Tecnologı´a, Spain, by Grant ACOM06/132 from the Conselleria d’Empresa, Universitat i Cie`ncia, Generalitat Valenciana, Spain and a Marie Curie European Reintegration Grant (ERG) from the European Sixth Framework Programme to G.R. and L.F. (MERG-CT-2004510702). References [1] F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl, Current protocols in molecular biology, John Wiley, New York, 1995. [2] A. Bateman, L. Coin, R. Durbin, R.D. Finn, V. Hollich, S. Griffiths-Jones, A. Khanna, M. Marshall, S. Moxon, E.L. Sonnhammer, D.J. Studholme, C. Yeats, S.R. Eddy, The Pfam protein families database, Nucleic Acids Res. 32 (2004) 138e141. [3] J. Brzeski, W. Podstolski, K. Olczak, A. Jerzmanowski, Identification and analysis of the Arabidopsis thaliana BSH gene, a member of the SNF5 gene family, Nucleic Acids Res. 27 (1999) 2393e2399. [4] P.K. Busk, M. Pages, Regulation of abscisic acid-induced transcription, Plant Mol. Biol. 37 (1998) 425e435. [5] B.R. Cairns, Y.-J. Kim, M.H. Sayre, B.C. Laurent, R.D. Kornberg, A multisubunit complex containing the SWI1/ADR6, SWI2/SNF2, SWI3, SNF5, and SNF6 gene products isolated from yeast, Proc. Natl. Acad. Sci. USA 91 (1994) 1950e1954. [6] J. Castillo, A. Genoves, L. Franco, M.I. Rodrigo, A multifunctional bicupin serves as precursor for a chromosomal protein of Pisum sativum seeds, J. Exp. Bot. 56 (2005) 3159e3169. [7] J. Castillo, M.I. Rodrigo, J.A. Marquez, A. Zuniga, L. Franco, A pea nuclear protein that is induced by dehydration belongs to the vicilin superfamily, Eur. J. Biochem. 267 (2000) 2156e2165. [8] J. Castillo, A. Zuniga, L. Franco, M.I. Rodrigo, A chromatin-associated protein from pea seeds preferentially binds histones H3 and H4, Eur. J. Biochem. 269 (2002) 4641e4648. [9] R. Chenna, H. Sugawara, T. Koike, R. Lopez, T.J. Gibson, D.G. Higgins, J.D. Thompson, Multiple sequence alignment with the Clustal series of programs, Nucleic Acids Res. 31 (2003) 3497e3500. [10] S.W. Cheng, K.P. Davies, E. Yung, R.J. Beltran, J. Yu, G.V. Kalpana, c-MYC interacts with INI1/hSNF5 and requires the SWI/SNF complex for transactivation function, Nat. Genet. 22 (1999) 102e105.

435

[11] J. Felsenstein, Confidence limits on phylogenies: an approach using the bootstrap, Evolution 39 (1985) 783e791. [12] F. Geng, Y. Cao, B.C. Laurent, Essential roles of Snf5p in Snf-Swi chromatin remodeling in vivo, Mol. Cell. Biol. 21 (2001) 4311e4320. [13] C.J. Guidi, A.T. Sands, B.P. Zambrowicz, T.K. Turner, D.A. Demers, W. Webster, T.W. Smith, A.N. Imbalzano, S.N. Jones, Disruption of Ini1 leads to peri-implantation lethality and tumorigenesis in mice, Mol. Cell. Biol. 21 (2001) 3598e3603. [14] C.P. Joshi, H. Zhou, X. Huang, V.L. Chiang, Context sequences of translation initiation codon in plants, Plant Mol. Biol. 35 (1997) 993e1001. [15] A. Klochendler-Yeivin, L. Fiette, J. Barra, C. Muchardt, C. Babinet, M. Yaniv, The murine SNF5/INI1 chromatin remodeling factor is essential for embryonic development and tumor suppression, EMBO Rep. 1 (2000) 500e506. [16] G. Li, K.J. Bishop, M.B. Chandrasekharan, T.C. Hall, Beta-phaseolin gene activation is a two-step process: PvALF- facilitated chromatin modification followed by abscisic acid-mediated gene activation, Proc. Natl. Acad. Sci. USA 96 (1999) 7104e7109. [17] G. Li, S.P. Chandler, A.P. Wolffe, T.C. Hall, Architectural specificity in chromatin structure at the TATA box in vivo: nucleosome displacement upon beta-phaseolin gene activation, Proc. Natl. Acad. Sci. USA 95 (1998) 4772e4777. [18] J.C. Loke, E.A. Stahlberg, D.G. Strenski, B.J. Haas, P.C. Wood, Q.Q. Li, Compilation of mRNA polyadenylation signals in Arabidopsis revealed a new signal element and potential secondary structures, Plant Physiol. 138 (2005) 1457e1468. [19] S.D. Michaels, M.C. John, R.M. Amasino, Removal of polysaccharides from plan DNA by ethanol precipitation, Biotechniques 17 (1994) 274e275. [20] K.E. Neely, A.H. Hassan, C.E. Brown, L. Howe, J.L. Workman, Transcription activator interactions with multiple SWI/SNF subunits, Mol. Cell. Biol. 22 (2002) 1615e1625. [21] D.W.-K. Ng, M.B. Chandrasekharan, T.C. Hall, Ordered histone modifications are associated with transcriptional poising and activation of the phaseolin promoter, Plant Cell 18 (2006) 119e132. [22] M.L. Phelan, S. Sif, G.J. Narlikar, R.E. Kingston, Reconstitution of a core chromatin remodeling complex from SWI/SNF subunits, Mol. Cell 3 (1999) 247e253. [23] P. Prochasson, K.E. Neely, A.H. Hassan, B. Li, J.L. Workman, Targeting activity is required for SWI/SNF function in vivo and is accomplished through two partially redundant activator-interaction domains, Mol. Cell 12 (2003) 983e990. [24] F.A. Razem, A. El-Kereamy, S.R. Abrams, R.D. Hill, The RNA-binding protein FCA is an abscisic acid receptor, Nature 439 (2006) 290e294. [25] T.J. Sarnowski, G. Rı´os, J. Jasik, S. Swiezewski, S. Kaczanowski, Y. Li, A. Kwiatkowska, K. Pawlikowska, M. Kozbial, P. Kozbial, C. Koncz, A. Jerzmanowski, SWI3 subunits of putative SWI/SNF chromatin-remodeling complexes play distinct roles during Arabidopsis development, Plant Cell 17 (2005) 2454e2472. [26] T.J. Sarnowski, S. Swiezewski, K. Pawlikowska, S. Kaczanowski, A. Jerzmanowski, AtSWI3B, an Arabidopsis homolog of SWI3, a core subunit of yeast Swi/Snf chromatin remodeling complex, interacts with FCA, a regulator of flowering time, Nucleic Acids Res. 30 (2002) 3412e3421. [27] M. Schmid, T.S. Davison, S.R. Henz, U.J. Pape, M. Demar, M. Vingron, B. Scho¨lkopf, D. Weigel, J.U. Lohmann, A gene expression map of Arabidopsis thaliana development, Nat. Genet. 37 (2005) 501e506. [28] G.S. Scippa, A. Griffiths, D. Chiatante, E.A. Bray, The H1 histone variant of tomato, H1-S, is targeted to the nucleus and accumulates in chromatin in response to water-deficit stress, Planta 211 (2000) 173e181. [29] S. Sridha, K. Wu, Identification of AtHD2C as a novel regulator of abscisic acid responses in Arabidopsis, Plant J. 46 (2006) 124e133. [30] P. Sudarsanam, V.R. Iyer, P.O. Brown, F. Winston, Proc. Natl. Acad. Sci. USA 97, Whole-genome expression analysis of snf/swi mutants of Saccharomyces cerevisiae (2000) 3364e3369. [31] I. Versteege, N. Sevenet, J. Lange, M.F. Rousseau-Merck, P. Ambros, R. Handgretinger, A. Aurias, O. Delattre, Truncating mutations of hSNF5/INI1 in aggressive paediatric cancer, Nature 394 (1998) 203e206.