Hahb-4, a sunflower homeobox-leucine zipper gene, is a developmental

regulator and confers drought tolerance to Arabidopsis thaliana plants

Carlos Alberto Dezar, Gabriela Marisa Gagoz, Daniel Hector Gonzalez & Raquel Lıa Chan*Facultad de Bioquımica y Ciencias Biologicas, Universidad Nacional del Litoral, CC 242 Paraje El Pozo, 3000

Santa Fe, Argentina

Received 2 December 2004; accepted 27 March 2005

Key words: drought tolerance, HD-Zip, homeodomain-leucine zipper, inducible promoter, plant home-odomain, sunflower, transcription factor, water stress

Abstract

Homeodomain-leucine zipper proteins constitute a family of transcription factors found only in plants.Hahb-4 is a member of Helianthus annuus (sunflower) subfamily I. It is regulated at the transcriptional levelby water availability and abscisic acid. In order to establish if this gene plays a functional role in droughtresponses, transgenic Arabidopsis thaliana plants that overexpress Hahb-4 under the control of the 35SCauliflower Mosaic Virus promoter were obtained. Transformed plants show a specific phenotype: theydevelop shorter stems and internodes, rounder leaves and more compact inflorescences than their non-transformed counterparts. Shorter stems and internodes are due to a lower rate in cell elongation ratherthan to a stop in cell division. Transgenic plants were more tolerant to water stress conditions, showingimproved development, a healthier appearance and higher survival rates than wild-type plants. Indeed,either under normal or drought conditions, they produce approximately the same seed weight per plant aswild-type plants under normal growth conditions. Plants transformed with a construct that bears the Hahb-4 promoter fused to gusA show reporter gene expression in defined cell-types and developmental stages andare induced by drought and abscisic acid. Since Hahb-4 is a transcription factor, we propose that it mayparticipate in the regulation of the expression of genes involved in developmental responses of plants todesiccation.

Introduction

One of the major environmental factors limitingplant productivity is lack of water. Althoughconventional breeding and marker-assisted selec-tion are currently being used to develop varietiesmore tolerant to water stress, these methods aretime and resource consuming, and do not alwaysgive the expected results. Genetic engineering is an

attractive alternative to improve water stresstolerance in plants.

Plants respond to water stress with the expres-sion of a specific set of genes, which allow them toadapt to altered environmental conditions (Al-moguera et al., 1993; Coca et al., 1996; Bray, 1997;Shinozaki & Yamaguchi-Shinozaki, 1997). Thehormone abscisic acid (ABA) plays an importantrole in a subset of these responses (Skriver &Mundy, 1990; Shinozaki & Yamaguchi-Shinozaki,1997; Leung & Giraudat, 1998).

Efforts to improve abiotic stress tolerance bymeans of genetic engineering have relied on theoverexpression of genes involved in one of the

*Author for correspondenceE-mail: rchan@fbcb.unl.edu.ar

zPresent address: Area de Microbiologıa, IBR, Suipacha531, 2000 Rosario, Argentina.

Transgenic Research (2005) 14:429–440 � Springer 2005DOI 10.1007/s11248-005-5076-0

various adaptation transduction signal pathways(Kasuga et al., 1999; Kim et al., 2001; Polidoros etal., 2001; Hsieh et al., 2002a; Tilahun et al., 2003;Kasukabe et al., 2004; Pellegrineschi et al., 2004;Umezawa et al., 2004). Within these overexpressedgenes, transcription factors have been shown toproduce phenotypic changes, many of which areinvolved in stress tolerance. This type of proteinscan drive a complex alteration in plant metabolismand architecture enhancing or decreasing theexpression of a big number of their target genes(Kasuga et al., 1999; Hsieh et al., 2002b; Kasugaet al., 2004; Yi et al., 2004).

Within transcription factors, HD-Zip proteins,unique to plants, have been proposed to beinvolved in regulating developmental processesassociated with the response of plants to environ-mental conditions (Carabelli et al., 1993; Schenaet al., 1993; Chan et al., 1998). These proteinscontain a homeodomain associated with a leucinezipper, a coiled-coil structure involved in dimeri-sation (Ruberti et al., 1991; Mattsson et al., 1992;Schena & Davis, 1992; Gonzalez et al., 1997) andbind DNA efficiently only as dimers (Sessa et al.,1993; Palena et al., 1999).

Previous studies indicated that the sunflowergene encoding the HD-Zip protein Hahb-4 is up-regulated by drought and ABA in roots, stems,and leaves (Gago et al., 2002), suggesting that itmay function in the signalling cascade that con-trols a subset of the ABA-mediated responses ofsunflower to water stress.

In order to investigate the function of Hahb-4and its possible role in conferring drought toler-ance, we decided to overexpress this factor inArabidopsis thaliana plants. Assuming that mostbasic processes are conserved in dicot plants, wehave made use of this heterologous system toevaluate the potential use of Hahb-4 as a biotech-nological tool.

We have observed that Arabidopsis transgenicplants that overexpress Hahb-4 have a character-istic phenotype that affects stem and internodelength, leaf shape and form, inflorescence devel-opment and growth rate. Under water stressconditions, transgenic plants show an enhancedtolerance during vegetative developmental stagesboth in soil and on culture medium. This factopens the possibility of using Hahb-4 as a bio-technological tool to improve water stress toler-ance in agricultural crops.

Materials and methods

Plant material and growth conditions

Arabidopsis thaliana Heyhn. ecotype Columbia(Col-0) was purchased from Lehle Seeds (Tucson,AZ). Plants were grown in soil in a growthchamber at 22–24�C under long-day photoperiods(16 h of illumination by a mixture of cool-whiteand GroLux fluorescent lamps) at an intensity ofapproximately 200 lE m)2 s)l. Plants used for thedifferent treatments were grown in 8 cm diame-ter · 7 cm height pots during the time indicated inthe figures. Since transgenic plants expressingHahb-4 show phenotypic characteristics thatresemble those of the Landsberg erecta ecotype,genotypic analysis using SSLP with several pairs ofoligonucleotides, as suggested in the ArabidopsisBiological Resource Center (ABRC) web page,was performed to assess their Columbia back-ground.

Water stress treatments

Water stress treatment in soil was carried out asfollows: to a 35 cm plastic square tray, a definedamount of water was added (1, 1.5 or 2 1). Sixteen8 · 7 cm pots, each with 100 g soil, were placed inthe tray. One or three seeds, depending on theexperiment, were sowed in each pot and the trayswere transferred to culture conditions as describedabove. Additional water was added only whensevere damage of non-transformed plants wasobserved. Plants were analysed after rewatering.

For Hahb-4 promoter analysis, 14 day-old-plants grown in Petri dishes were placed on filterpaper during 2–3 h until water stress was clearlyobserved. Then, total RNA was extracted asdescribed below. Control plants were kept in tubeswith water. To analyse induction by ABA, theplants were placed in 100 lM ABA for the sameperiods of time and then harvested for RNAisolation.

Genetic constructs

The cDNA corresponding to the Hahb-4 genecloned in vector pUC119 as previously describedwas amplified with oligonucleotides T1(5¢-GCGGGATCCACCATGTCTCTTCAACAA-GTA) and T2 (5¢-GCCGAGCTCTTAGAACT

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CCAACCACTTTTG), restricted with BamHI andSacI and cloned in plasmid pBI121 digested withthe same enzymes. In this way, only the codingregion of Hahb-4 (nucleotides representing startand stop colons are shown in bold in T1 and T2,respectively) was inserted between the 35S pro-moter and the nos termination sequence with anextra ACC triplet just before the ATG to improvetranslation efficiency (Futterer & Hohn, 1996).E. coli DH5a cells were transformed with thisconstruct and, once positive clones were obtainedand sequenced, Agrobacterium tumefaciens cellswere transformed (Hofgen & Willmitzer, 1988)with the same plasmid.

A 1015-bp region upstream of the transcriptioninitiation site of Hahb-4 was amplified by PCRwith oligonucleotides PROM1 (5¢-GCGGTCGAC-ACCTGGCACATCGTATCTT-3¢) and PROM2(5¢-CGCGGATCCGAGGGTTTGATAAGTGA-T-3¢) using genomic sunflower DNA as template.This DNA fragment was restricted with SalI andBamHI and cloned in plasmid pB1101.3 previouslydigested with the same enzymes. In this way, thepromoter region of Hahb-4 controls the expressionof the reporter gusA gene when plants are trans-formed with this construct.

Transformation and identification of transformedplants

Transformed Agrobacterium tumefaciens strainGV2260 was used to obtain transgenic Arabidop-sis plants by the floral dip procedure (Clough &Bent, 1998). Transformed plants were selected onthe basis of kanamycin resistance and positivePCR carried out on genomic DNA with oligonu-cleotides T1 and T2 described below. To assessHahb-4 expression, northern blot analysis wasperformed on T2 transformants. Three positiveindependent lines (arising from two differenttransformation experiments) were developedfurther and homozygous T3 and T4 plants wereused to analyse the expression levels ofHahb-4 andthe phenotype of transgenic plants. Plants trans-formed with pBI101.3 or pBI121, used as negativeor positive controls, respectively, or with a con-struction bearing the Hahb-4 promoter regionfused to the reporter gene gusA, were obtained ina similar way. In all cases three independenthomozygous transformed lines (T3 and/or T4)were analysed.

RNA isolation and analysis

Total RNA was isolated as described by Carpenterand Simon (1998). For northern blot analysis,specific amounts of RNA were electrophoresedthrough 1.5% (w/v) agarose/6% formaldehyde gels.The integrity of the RNA and uniformity of RNAloading were verified by ethidium bromide staining.RNA was transferred to Hybond-N nylon mem-branes (AmershamCorp.) andhybridised overnightat 65�C to 32P-labeled probes in buffer containing6 · SSC, 0.1% (w/v) polyvinylpyrrolidone, 0.1%(w/v) BSA, 0.1% (w/v) Ficoll, 0.2% (w/v) SDS, and10% (w/v) polyethylene glycol 8000. Filters werewashed with 2· SSC plus 0.1% (w/v) SDS at 65�C(4 times, 15 min each), 0.1· SSC plus 0.1% (w/v)SDS at 37�C during 15 min, dried and exposed toKodak BioMax MS films. To check the amount oftotal RNA loaded in each lane, filters were thenre-probed with a 25S rDNA from Vicia faba undersimilar conditions as those described above, exceptthat hybridisation was performed at 62�C and thewash with 0.1· SSC was omitted. For Hahb-4, theprobe used was an SpeI/EcoRI cDNA fragment(from +424 to +674), corresponding to the 3¢-noncoding region plus the last 177 nucleotides of thecoding region, which does not include the HD-Zipdomain. For Athb-7 and -12, full-length cDNAclones (RAFL05-20-M16 and RAFL11-01-J18)obtained from the RIKFN BRC ExperimentalPlant Division, Tsukuba, Japan, were used. ForGST8 (clone 116M6T7), RAB18 (clone 1251M19T7), UBQ10 (clone 193N23T7) and RD21A(clone 187D5T7), EST clones obtained from theArabidopsis Biological Resource Center (ABRC)were used. An EST clone from RD22 (SQ069b10)was obtained from the Kazusa DNA ResearchInstitute (Japan). For DREB2A and RD29A, genespecific probes were obtained by PCR. For gusAdetection, a full-length probe was obtained byrestriction of pBI101.3 with BamHI/SacI. In thiscase, hybridisation was performed at 68�C to avoidunspecific reactions.

DNA isolation and Southern blot analysis

Arabidopsis genomic DNA was isolated accordingto the method described by Doyle and Doyle(1987). The DNA (10 lg) was digested overnightwith HindIII electrophoresed through 0.7% (w/v)agarose gels and transferred to Hybond-N nylon

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membranes (Amersham Corp.). Southern blotanalysis was carried out essentially as describedin Ausubel et al. (1983) using as probe the samefragment described for northern analysis. Filterswere washed with 2· SSC plus 0.1% (w/v) SDS at65�C (4 times, 15 min each) and 0.1· SSC plus0.1% (w/v) SDS at 37�C during 15 min, dried andexposed to Kodak BioMax MS films.

Histochemical GUS staining

In situ assays of GUS activity were performed asdescribed by Jefferson et al. (1987). Whole plantswere immersed in a 1 mM 5-bromo-4-chloro-3-indolyl-b-glucuronic acid solution in 100 mMsodium phosphate pH 7.0 and 0.1% Triton X-100 and, after applying vacuum for 5 min, theywere incubated at 37�C overnight. Chlorophyllwas cleared from the plant tissues by immersingthem in 70% ethanol.

Microscopic analysis

Tissue preparation for staining was carried outessentially as described byDixon andKlessig (1995)with slight modifications. Plant material was fixedfor 3 h in phosphate buffer/12% glutaraldehyde atroom temperature, washed twice with phosphatebuffer (Na2HPO3, pH 7.1) for 20 min and kept at4�C in fresh phosphate buffer. The tissue was thendehydrated through an ethanol series and embed-ded in Histoplast (Biopack). Sections (8–10 lmthick) were mounted on slides coated with 50 lg/mlpoly-D-lysine (Sigma Chemical Co., St. Louis, MO)in 10 mM Tris–HCl, pH 8.0 and dried overnight at37�C. After removing the paraffin with xylene,sections were rehydrated by an ethanol series andstained with Fast Green. After a brief wash withH2O/ethanol/xylol, sections were dried at roomtemperature for 3 days. Digital images were gener-ated using a Coolpix 995 digital camera (Nikon)mounted on an optical microscope, and processedwith Adobe Photoshop 5.0.

Results

Hahb-4 overexpressing Arabidopsis lines

To investigate the in vivo functions of Hahb-4, weused an overexpression approach. The coding

region of Hahb-4 was fused to the 35S promoterof Cauliflower Mosaic Virus, and the constructwas used to transform Arabidopsis plants. Severalhomozygous lines were recovered and, afterpreliminary analysis, three transgenic independentlines, named 35S:Hahb4-A, -B and -C, respec-tively, were selected for more detailed analysis.Figure 1a shows a northern blot hybridised withan Hahb-4 probe, where total RNA from wild typeor transgenic plants was analysed. The probe didnot hybridise with RNA extracted from wild-typeplants, indicating no cross-reaction with membersof the Arabidopsis HD-Zip family. Strong signals

Figure 1. Expression of Hahb-4 in Arabidopsis transgenicplants. (a) Northern blot analysis of transgenic Arabidopsisplants. Total RNA (10 lg) was extracted from wild-type(WT) and three independent transgenic plants (A, B and C)overexpressing Hahb-4. Probes specific for Hahb-4 or rRNAwere used. (b) Southern blot analysis of transgenic Arabidop-sis plants. Total genomic DNA (10 lg) from wild type (firstlane) or transgenic plants A, B and C (lanes 2, 3, and 4,respectively) was isolated, digested, electrophoresed and anal-ysed as described in ‘Materials and methods’. Lambda phageDNA digested with HindIII was used as molecular weightmarker.

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of similar intensity were observed with RNA fromthe different transgenic plants suggesting that theyexpress the Hahb-4 mRNA at high levels. South-ern blot analysis (Figure 1a) indicated the presenceof one copy of the transgene in lines A and C,while two copies seem to be present in line B. Thisdifference in copy number does not seem toinfluence significantly expression levels, since thethree lines (as well as others that were analysed,not shown) show similar Hahb-4 transcript levels.

Phenotype of Hahb-4 overexpression lines

Compared with wild-type plants, 35S:Hahb4transgenic plants exhibited a characteristic pheno-type when cultured under standard irrigationconditions. When grown in soil, the rosette oftransgenic plants was smaller, leaf petioles wereshorter, and leaves were rounder in shape (Fig-ure 2a and b). Transgenic plants also showedreduced stem elongation rate compared to wild-type plants (Figure 2c). This gives rise to a morecompact inflorescence as shown in Figure 2d.Flower bud formation occurs with a difference ofapproximately 2 days between wild type andtransgenic plants: 18 days after germination forwild-type plants and 20 days for transgenic coun-terparts. In both cases, the number of rosetteleaves was the same. A delay of 5 days in anthesiswas also observed in transformed plants withrespect to non-transformed plants. The differencebetween wild type and transgenic plants becameless evident upon progression of the reproductivestage of development, with transgenic plantshaving about 87% of the wild-type height whenseed maturation begun (approximately 50 daysafter germination). The difference in stem andinternode length between transgenic and non-transformed plants is probably due to a slowerlongitudinal enlargement of cells. In Figure 3,longitudinal sections of internodes from the twogenotypes are shown. It can be appreciated thatcells from transgenic plants are considerablysmaller than their wild type counterparts.

Interestingly, seed production was not affectedin transgenic plants with respect to non-trans-formed plants. In five independent experiments,the total weight of dried seeds produced by plantsof both genotypes was measured. The observeddifference in weight was smaller than the standarddeviation calculated in each experiment performed

with 20 individuals of each genotype. Significantdifferences were observed between experimentsperformed with either one or three plants perpot, but both genotypes behaved similarly. Thisindicates that no loss in seed productivity is causedby the transgene, as it has been observed withother transcription factors when they were over-expressed under the control of the 35S promoter(Kesuga et al., 1999; Hsieh et al., 2002). Thisagrees with the fact that the phenotypic differenceis reduced when plants reach the reproductivestage.

Transgenic Arabidopsis plants are more tolerant towater deficit than wild-type plants

The strong induction of Hahb-4 under water stressled us to evaluate the capacity for droughtresistance of transgenic plants during vegetativedevelopment. For this purpose, transgenic andwild-type Arabidopsis plants were grown in potsintercalated in the same tray. After cold treatmentfor dormancy break, the pots were watered byadding either 1, 1.5 or 2 l of water to the trays andthen transferred to a culture chamber. Since plantswere not further watered until severe damage wasobserved in controls, these treatments producedwater stress at different developmental stages. Thefirst symptom of water deficit was a reduction ingrowth rate. In fact, wild-type plants under theseconditions showed a phenotype that resembledtransgenic plants under normal conditions (i.e.reduction in leaf size, delayed inflorescence, stemelongation and flowering) suggesting that the effectof Hahb-4 expression is to enhance a naturaldefence mechanism present in a wild-type plant.Upon prolonged water deficit, damage becameevident in wild-type plants. After 20/25 days, itwas observed that leaves of wild-type plantsinitially irrigated with 1 l water became wiltedand curled, whereas transgenic plants were notaffected (Figure 4a and c). Similar observations,but at later stages of development, were done whenthe initial water supply was 1.5 l (Figure 4b and d)or 2 l (Figure 4e). To examine the survival rates ofnon-transformed and transgenic plants under con-ditions of severe water deficit, the drought treat-ment was extended 5 days until severe damage wasvisible. At this stage, plants were watered and,2 days after that, survivors were observed andcounted in the two populations. Compared with

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the survival rate of non-transformed plants, trans-genic plants were clearly more resistant at the threedevelopmental stages. Drought resistance wasmore pronounced when the stress was applied atearlier developmental stages (2/3-week-old plants)than in the reproductive stage (4/5-week-oldplants). Table 1 illustrates experiments done withthe three independent lines subjected to waterstress as described in ‘Materials and methods’. Inall cases the percentage of plants surviving undersevere stress conditions was considerably higher

for transgenic plants than for non-transformedplants. Small variations were observed betweenexperiments, probably due to differences in the soilquality and its initial water content. This isreflected in higher survival rates observed forwild-type plants in a given experiment which werealways correlated with a similar increase in sur-vival rate for transformed plants present in thesame fray, independently of the line tested.Consequently, significant differences in survivalrates were observed between transformed and

Figure 2. Phenotype of 35S:Hahb4 transgenic plants. (a) Leaves from Hahb-4 overexpressing transgenic plants and non-trans-formed plants at 21 days of development (left) and 45 days of development (right). (b) Rosette size and leaf shape of transgenicand non-transformed plants during the vegetative stage. (c) Rosette size, leaf shape and inflorescence stern of transgenic and non-transformed plants during the reproductive stage. (d) Compact inflorescence observed in transgenic plants.

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non-transformed plants in all experiments, withmean values of 80 ± 15% for transformed plantsversus 19 ± 11% for wild-type plants.

We have also analysed seed production insurviving transgenic plants subjected to drought.Seeds from 100 transformed plants in five inde-pendent drought experiments were collected indi-vidually and seed weight after dehydration wasdetermined. Total seed weight was unaffected intransformed plants that were watered after a longperiod of water stress with respect to plants thatdid not suffer drought.

Increased drought resistance was alsoobserved in plants grown in Petri dishes. Tosubject plants to water deficit, transgenic or wild-type plants were grown in Petri dishes whosecover was larger than normal in diameter andplaced in the culture chamber under low humidityconditions. Evaporation of water under theseconditions was slow but constant and the MS-agar medium progressively dried. Twenty fivedays after germination, wild-type plants showedretarded growth with respect to transgenic plantssubjected to the same treatment (Figure 4f and g).In addition, leaves of wild-type plants showedvisible damage while transgenic plants remainedhealthy. These results indicate that overexpressionof Hahb-4 can significantly improve water deficittolerance in Arabidopsis plants.

Expression of drought-related genes is unaffected inHahb-4 overexpressing plants

Since Hahb-4 belongs to the sunflower HD-Zipfamily, we tested the expression levels of its closestArabidopsis homologues, Athb-7 and -12, intransgenic and wild-type plants. As deduced from

northern blot experiments, neither Athb-7 norAthb-12 transcript levels were significantly affected(Figure 5). This result enhances the conclusionthat Hahb-4 overexpression is directly responsiblefor the phenotype observed and for water stresstolerance.

Figure 3. Microscopic views of internode cells of 35S:Hahb4overexpressing plants and control plants. Tissue sections wereprepared as described in ‘Materials and methods’ and stainedwith fast green. Scale bar: 100 lm.

Figure 4. Drought tolerance of 35S:Hahb4 transgenic plants.(a, b and c) Transgenic lines A, B and C (TG) and wild-type(WT) plants were grown on soil in the same container, plantswere re-watered when damage was observed. The photo-graphs were taken 2 days after rewatering. (d) A closer viewof 3-week-old transgenic plants subjected to a strong waterdeficit stress (the same plants as in ‘b’ without rewatering). (e)A group of 45-day-old plants previously subjected to waterdeficit stress and re-watered (transgenic plants are on the leftand non-transformed plants on the right). (f and g) Trans-genic (f) or wild-type (g) plants were grown in Petri disheswith slightly larger covers on MS medium as described in‘Materials and methods’. Photographs were taken after30 days.

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In order to investigate the mechanism of actionof Hahb-4, we also analysed expression levels ofseveral genes that are induced by water stress,some having in their promoter region the targetDNA sequence bound by Hahb-4 in vitro. We haveprepared probes for RAB18 (At5g66400), RD22(At5g25610), DREB2A (At5g05410), RD29A(At5g52310), RD21A (At1g47128), GST8(At1g78380) and UBQ10 (At4g05320), the lastthree having the target sequence of Hahb-4 in theirpromoters, to investigate this point. Notably,neither of the genes under study showed significantchanges in RNA steady-state levels in Hahb-4overexpressing plants compared to control plants(data not shown). This indicates that the pheno-type conferred by Hahb-4 originates from changesin gene expression that do not include the usualdrought-responsive genes regulated by other tran-scription factors.

Expression patterns driven by the Hahb-4 promoter

The phenotype described here indicates that Hahb-4 is involved in water deficit stress responses,conferring tolerance especially during vegetativestages of plant development. It has been previouslyobserved that this gene is expressed in roots,seedlings and leaves of sunflower and regulated bydrought and ABA (Gago et al., 2002). Thesestudies have been performed with the RNAseprotection technique and northern blot hybridisa-tion. To assess the physiological relevance of theresults presented here, we investigated the spatialand temporal expression patterns conferred by theHahb-4 promoter region. For this purpose, wehave cloned a 1015 bp fragment, containingsequences upstream of the +1 transcription initi-ation site of Hahb-4, in front of the gusA reportergene. We have obtained several independenttransgenic Arabidopsis homozygous lines with thisconstruct, and analysed three by histochemicalb-glucuronidase (GUS) staining. GUS activity wasalready clearly detectable 20 h after germinationand in the emerging radicle some hours later (notshown). Expression was also observed in vegeta-tive tissues including hypocotyls, stems and leavesuntil approximately 40 days after germination(Figure 6a). Cotyledons, as well as young andmature leaves were strongly stained in all the linesanalysed but expression decreased after 20–25 days of germination (Figure 6a and b). Rootsexhibited strong staining at the tips, in the vascularcylinder and in the nascent secondary roots(Figure 6c). GUS activity was not detected inreproductive organs (not shown). We were alsointerested in testing if the Hahb-4 promoter isinducible by drought and ABA. For this purpose,we analysed gusA expression in transgenic plantssubjected to drought stress or treated with 100 lMABA. Expression levels, analysed by northern blothybridisation using a gusA probe, were comparedwith those of untreated Hahb-4 promoter-gusAplants or of plants transformed with the T-DNAregion of plasmid pBI121, which contains the gusAgene under the control of the strong 35SCaMVpromoter (Figure 6d). Hahb-4 promoter activity isstrongly induced by ABA and drought stress,reaching in both cases transcriptional levels similarto those of the 35S Cauliflower Mosaic Viruspromoter. We conclude that the Hahb-4 promoteris recognised in Arabidopsis plants and the

Figure 5. Arabidopsis Athb-7 and Athb-12 expression is notaffected by overexpression of Hahb-4. Total RNA (10 lg) wasextracted from 2-week-old wild-type or transgenic plants,electrophoresed, blotted onto nylon membranes, and probedwith 32P-labelled Athb-7 or Athb-12 cDNA. The same filterwas hybridised with an rRNA probe as a control for RNAloading and transfer (lower panel).

Table 1. Survival rates of transgenic plants

Transgenic line Survival Total %

Drought tolerance in 4-week-old plants

35S:Hahb4 A 15 18 83

WT 1 22 4.5

35S:Hahb4 B 24 24 100

WT 5 24 21

35S:Hahb4 C 8 22 36

WT 0 24 0

Number of 4-week-old plants overexpressing Hahb-4 survivingexposure to water stress. Each set of transgenic plants is com-pared with non-transformed plants that shared the tray withthem.

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expression profile of the reporter gene agrees withthat observed in sunflower for the Hahb-4 mRNA.The expression profile conferred by the promoteralso agrees with the effect of Hahb-4 overexpres-sion, which is more pronounced at earlier devel-opmental stages, during vegetative development.Otherwise, this activity is strongly induced bywater stress or ABA treatment, leading us to inferthat regulation of this gene occurred at thetranscriptional level.

Discussion

HD-Zip proteins, unique to plants, are proposedas good candidates to trigger developmentalresponses to environmental conditions, a charac-teristic feature of plants. Several authors havereported that expression of members of the HD-Zip family of transcription factors is regulated bydifferent external factors for example illuminationor water stress (Schena & Davis, 1992; Carabelli etal., 1993; Schena et al., 1993; Soderman et al.,1994, 1996; Lee & Chun, 1998).

Recently, Hahb-4 has been shown to encode anHD-Zip factor that belongs to the sunflower HD-Zip I subfamily. This gene is expressed at earlystages of plant development and its expression isinduced by water deficit and ABA (Gago et al.,2002). The stress inducibility of Hahb-4 suggestedthat it may have a potential role in stress-respon-sive signalling and, possibly, in conferring droughttolerance to plants. To address this question, weused an overexpression approach choosing Ara-bidopsis as a model system. The expression levelsof Hahb-4 in the 35S:Hahb4 transgenic lines usedin our study are higher with respect to theirArabidopsis counterparts under control conditionsand to its own expression level in sunflower.Transgenic plants showed a slightly retardeddevelopment during the vegetative stage. Shorterstems and petioles and smaller leaves indicate thatHahb-4 expression produces a decrease in cellenlargement. Developmental retardation was alsoobserved when non-transformed plants were sub-jected to water stress deficit, indicating that thephenotype caused by the overexpression of Hahb-4is similar to the normal plant response to drought.Another interpretation of these observations, spe-cially the growth retardation, is that they resultfrom the constitutive expression of Hahb-4 due tothe presence of the 35S promoter.

Experiments with non-constitutive promoters(rd29 and the Hahb-4 own promoter) are currentlyunder way. Preliminary results indicate thatwater-stress tolerance is also conferred by theseconstructions but to a lesser extent, while devel-opmental differences are almost absent in irrigatedplants (not shown). Additional analysis of thesetransgenic plants will also be helpful in determin-ing the relationship between developmentalchanges and water-stress tolerance. The fact thatsevere abnormalities were not observed and that

Figure 6. Expression pattern of Hahb-4. (a) Arabidopsisplants transformed with the promoter region of Hahb-4 fusedto gusA. From left to right: 2-day, 10-day, and 20-day-oldplants. (b) Cotyledons of histochemically stained transformedplants. (c) Detail of roots of 20-day-old plants. (d) Inductionof gusA expression by drought-stress and ABA. Total RNA(10 lg) was extracted from 3-week-old plants transformedwith pBI101.3 or with the Hahb-4 promoter-gusA fusion andkept under control conditions or subjected to water stress or100 lM ABA. RNA was electrophoresed, blotted onto nylonmembranes, and probed with a 32P-labelled gusA cDNA asdetailed in ‘Materials and methods’. The same filter washybridised with an rRNA probe as a control for RNAloading and transfer (lower panel).

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the effect of Hahb-4 on development is coincidentwith the expression patterns directed by its ownpromoter suggest, however, that the observationsmade are related to its in vivo function rather thanto its aberrant expression in incorrect tissues orcells. Additionally, transcriptional levels of Athb-7and -12 are unaffected in transgenic plants, and asimilar phenotype to that conferred byHahb-4 wasnot observed comparing the transgenic plantsdescribed in this work with plants transformedwith either of two other members of the sunflowerHD-Zip family (Hahb-1 and -10), nor when plantswere transformed with pBI121 as control (data notshown).

The phenotype of 35S:Hahb4 plants includestolerance to prolonged water deficit, observed inplants of different ages. Again, these observationsare coincident with the expression characteristicsof Hahb-4, which is strongly induced by waterstress in sunflower and in Arabidopsis carryingpromoter-gusA fusions. These facts point to theexistence of conserved regulatory mechanisms inthe response to drought in both species.

Phylogenetic analysis indicates that Athb-7 and-12 are the Arabidopsis genes more related toHahb-4. However, they are more related to eachother than to Hahb-4, suggesting that they havearisen from a recent duplication event. It isnoteworthy that both genes are also induced bywater stress and ABA (Soderman et al., 1994,1996; Lee & Chun, 1998). Outside the HD-Zipdomain, however, there is no significant homologybetween the encoded proteins and Hahb-4. Thisfact, and the low percentage of identity at theamino acid level within the homeodomain, makesit difficult to ascertain whether Hahb-4 is anorthologue of any of the Arabidopsis genes.Another important difference between Hahb-4and the Arabidopsis proteins is that while the firstone is able to bind the sequence CAAT(A/T)ATTG in vitro (Palena et al., 1999), Athb-7and -12 do not bind this or related sequences(Johannesson et al., 2001). This may indicate arequirement for post-translational modificationsof the Arabidopsis proteins. Recently, it has beenreported that both genes, Athb-7 and -12, act asnegative regulators of plant growth (Hjellstromet al., 2003; Olsson et al., 2004). Transgenic plantsthat express a reporter gene under the control ofthe Athb-7 promoter were obtained (Olsson et al.,2004). Expression patterns seem to be quite

different from the ones conferred by the Hahb-4promoter, suggesting that the latter is not theorthologue of the Arabidopsis genes. Anothergene from the same family, Athb-6, is also inducedby the same treatments but to a lesser extent(Soderman et al., 1999). Transgenic Arabidopsisplants overexpressing this gene have been obtainedby Himmelbach et al. (2002), who reported thatthey show increased ABA insensitivity. We did notobserve differences in ABA-dependent inhibitionof germination in the three independent lines oftransgenic plants that overexpress Hahb-4 withrespect to non-transformed plants in a range ofconcentrations from 0.5 to 1.5 lM ABA (data notshown), indicating that Athb-6 and Hahb-4 do notshare the same mechanism of action.

Changes in gene expression play a central rolein the plant adaptive responses to water stress.Many genes whose expression is regulated bystress, generally termed RD (responsive to dehy-dration), ERD (early responsive to dehydration) orLEA (late embryogenesis abundant protein), aswell as transcription factors that interact withthem, have been isolated (Shinozaki & Yamagu-chi-Shinozaki, 2000; Uno et al., 2000). The mech-anism of action and role of these genes seems to beconserved between species. In some cases, trans-genic plants overexpressing these genes or tran-scription factors show enhanced tolerance to waterdeficit and salt stress, but sometimes also negativephenotypic characteristics, such as severe growthretardation and/or deficiencies in seed production(Xu et al., 1996; Kasuga et al., 1999; Haake et al.,2002; Hsieh et al., 2002; Kang et al., 2002). In thiscontext, it is interesting that transgenic plants thatoverexpress Hahb-4 produce the same weight ofseeds compared with non-transformed plantsunder normal conditions, and that seed produc-tion is not affected when they are subjected toextreme water deficit stress.

Among the genes involved in water deficitstress responses, transcription factors are thoughtto be ‘master switches’. At present, numerousfactors from different families were shown to berelated with these responses. Most of these factorsact by directly or indirectly inducing the above-mentioned drought-responsive genes. Since theproduct of Hahb-4 is a transcription factor, itcan be postulated that this protein also regulates aset of genes involved in producing the observedphenotype. The lack of response of typical

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drought-responsive genes in Hahb-4 overexpress-ing plants, however, leads us to conclude that themechanism of action of the sunflower gene isdifferent from that of other transcription factors.We speculate that Hahb-4 may be speciallyinvolved in producing developmental changes thatallow the plant to survive under water deficitconditions. Two Arabidopsis mutants weredescribed, ERECTA (ER) and corymbosa2(crm2), where the mutation results in a compactinflorescence and short pedicels similar to thoseobserved in the Hahb-4 overexpressing lines (Tioriiet al., 1996; Suzuki et al., 2002). It is possible thatthe genes responsible for these mutations partici-pate in similar transduction pathways as thoseaffected by Hahb-4 overexpression. The identifica-tion of the genes whose expression is modifiedwhen Hahb-4 is overexpressed will allow to inferthe mechanisms used by the plant to increase itsdrought tolerance. Microarray analysis of plantsexpressing Hahb-4 will hopefully allow us to betterunderstand the mechanism followed by this tran-scription factor to confer drought tolerance toArabidopsis plants.

Great effort to improve plant tolerance todrought, high salinity and other abiotic stresseshave been pursued by breeding and genetic engi-neering. The success was limited owing to thegenetic complexity of the stress response. Theimproved understanding of the role of novel genesin stress adaptation will provide the basis foreffective engineering strategies leading to greaterstress tolerance. The results reported here allow usto envisage a biotechnological use of Hahb-4 forthe production of water deficit tolerant plants.

Acknowledgements

We thank Dr E. Zabaleta for technical advicewith plant transformation and helpful sugges-tions and Drs Marta and Susana Gattuso forassistance with the microscopic analysis. We alsothank Dr Kimitaka Yakura, Kanazawa Univer-sity, Japan, for sending us a Vicia faba rDNAclone and the RIKEN BRC Experimental PlantDivision, the Arabidopsis Biological ResourceCenter and the Kazusa DNA Research Institutefor providing us cDNA clones.

This work was supported by grants fromCONICET, ANPCyT, Fundacion Antorchas and

Universidad Nacional del Litoral. RLC andDHG are members of CONICET; CAD andGMG are fellows of the same Institution.

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