Small heat-shock proteins and leaf cooling capacity account for the unusual heat tolerance of the...

Small heat-shock proteins and leaf cooling capacityaccount for the unusual heat tolerance of the central spikeleaves in Agave tequilana var. Weberpce_2035 1791..1803

ROSARIO LUJÁN*, FERNANDO LLEDÍAS*, LUZ MARÍA MARTÍNEZ, RITA BARRETO, GLADYS I. CASSAB &JORGE NIETO-SOTELO

Departmento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, P.O.Box 510-3, Cuernavaca, Mor. Mexico 62250

ABSTRACT

Agaves are perennial crassulacean acid metabolism (CAM)plants distributed in tropical and subtropical arid environ-ments, features that are attractive for studying the heat-shock response. In agaves, the stress response can beanalysed easily during leaf development, as they form aspirally shaped rosette, having the meristem surrounded byfolded leaves in the centre (spike) and the unfolded andmore mature leaves in the periphery. Here, we report thatthe spike of Agave tequilana is the most thermotolerantpart of the rosette withstanding shocks of up to 55 °C. Thisfinding was inconsistent with the patterns of heat-shockprotein (Hsp) gene expression, as maximal accumulationof Hsp transcripts was at 44 °C in all sectors (spike, inner,middle and outer). However, levels of small HSP (sHSP)-CIand sHSP-CII proteins were conspicuously higher in spikeleaves at all temperatures correlating with their thermotol-erance. In addition, spike leaves showed a higher stomataldensity and abated more efficiently their temperatureseveral degrees below that of air. We propose that thegreater capacity for leaf cooling during the day in responseto heat stress, and the elevated levels of sHSPs, constitutepart of a set of strategies that protect the SAM and foldedleaves of A. tequilana from high temperatures.

Key-words: HSP101; HSP90; HSP70; oxidative stress;protein carbonyl formation; ubiquitin.

INTRODUCTION

Agave tequilana Weber var. azul is one of the more than200 species of Agavaceae found in Mexico and theofficial species utilized to produce tequila (Cedeño 1995).Members of the Agavaceae family are widely distributed inthe North American deserts and well adapted to arid andsemiarid regions (Gentry 2003). As most succulent plants,agaves have undergone morphological and physiological

adaptations to survive under adverse environmental condi-tions. An important adaptation of agaves is the use of cras-sulacean acid metabolism (CAM). CAM plants reducewater loss by opening the stomata at night when the tem-perature is lower, decreasing the evapourative demandduring the hot daylight hours (Larcher 1995).

The thick cuticle overlying the epidermis of agave plantsand its low absorbance to short-wave radiation apparentlyprevent high temperature damage to their leaves (Nobel &Smith 1983). In two succulents (Agave deserti and Opuntiaficus-indica), acclimation to extreme heat takes place withinhours in response to mild heat stress and, during hot days,heat resistance is greater in the night than in the morning(Nobel 1988). An effective form of heat protection at thecellular level is provided by the synthesis of heat-shockproteins (HSPs) (Lindquist & Craig 1988). HSPs playimportant roles in heat tolerance as molecular chaperonesstabilizing chromatin structure, proteins and membranes,and promoting repair mechanisms through the refolding ofproteins during and following exposure to stress (Wanget al. 2004). Although HSPs disappear gradually after fewhours of heat stress, some of them have functions duringnormal growth. Homeostatic roles of HSPs include theproper folding of nascent polypeptides, their assembly,translocation and degradation (Lindquist & Craig 1988).Plants synthesize six major families of HSPs: the HSP100(ClpB) family; the HSP90 family; the HSP70 (DnaK)family; the chaperonine family (GroEL and HSP60); thesmall HSP family (sHSP); and calnexin and calreticulin(Miernyk 1999; Wang et al. 2004).

The tolerance of different Agave species to high tempera-ture, as measured by uptake of a vital stain, is within therange of 57 to 65 °C (Nobel & Smith 1983). Interestingly, A.tequilana is cultivated in regions where minimum tempera-tures seldom drop below –4 °C and maximum temperaturesare 36 °C or below (Nobel et al. 1998). Apparently, the cli-matic restriction for cultivation of A. tequilana shows apositive correlation with the avoidance of carbon loss atday/night temperatures of 35 °C/25 °C and above, and notwith protoplasmic heat resistance (Nobel et al. 1998). Thishypothesis is based in the observation that A. tequilanachlorenchyma cells are viable to treatments up to 55 °C

Correspondence: J. Nieto-Sotelo. Fax: +(52) 777 313 9988; e-mail:[email protected]

*These authors contributed equally to this work.

Plant, Cell and Environment (2009) 32, 1791–1803 doi: 10.1111/j.1365-3040.2009.02035.x

© 2009 Blackwell Publishing Ltd 1791

(Nobel et al. 1998). Paradoxically, CO2 uptake by leaves isdramatically reduced when the plants are grown at day/night temperatures of only 35 °C/25 °C (Nobel et al. 1998).A previous study described the accumulation of HSPs in A.deserti and two other desert succulents in response to heat(Kee & Nobel 1986). However, no molecular characteriza-tion of Hsp genes from agaves has been done to date. In thesame token, correlative studies between the levels and iden-tity of HSPs and resistance to high temperatures have notbeen reported for members of this plant family.

Several interesting features of agaves make them goodmodel systems for the study of the heat-shock response:they are perennial and succulent, during maturation shift toCAM metabolism, and are extremely tolerant to high tem-peratures both at the cellular and the whole-plant levels.Moreover, the arrangement of the agave leaves around therosette axis, produces a spirally shaped gradient wheredevelopmentally immature leaves are found at the centreand the most mature ones at the periphery (Nobel 1988).Therefore, agaves offer a unique opportunity for the studyof the role of HSPs in the heat-shock response during thedevelopment of the leaves of an individual plant.

Here, we describe the sequence of several cDNAs encod-ing proteins of the sHSP and HSP90 families from A. tequi-lana. The study of their expression in response to hightemperature shocks, together with that of Hsp101, Hsp70-1,Hsp21, Ubiquitin and Cab genes, indicated that, in A. tequi-lana plants, the differential tolerance to heat stress – amongthe different sectors of the rosette – is not dependent on thetranscriptional control of the Hsp gene expression. Thespike leaves showed an unmatched basal thermotolerance,consistent with their lower degree of protein ubiquitinyland carbonyl formation, indicators of irreversible proteindamage. Moreover, we found that spike leaves have thecapacity to reduce their temperature during the day inresponse to extreme heat shocks, display the highest sto-matal density of the rosette and present large amounts ofsHSPs (CI and CII) both at optimal temperature of growthand in response to heat shock.We concluded that heat stressresistance in A. tequilana depends on a large array of devel-opmentally regulated strategies encompassing leaf coolingpotential and sHSP accumulation.

MATERIALS AND METHODS

Plant materials and growth conditions

Agave tequilana specimens were obtained from the TequilaSauza factory (‘El Indio’ ranch) in Tequila, Jalisco, Mexico,and maintained in greenhouse conditions at a maximumdaily temperature of 32 °C and minimum of 20 °C undernatural photoperiod. Plants were irrigated twice a weekwith the nutrient solution described by Broughton and Dil-worth (Broughton & Dilworth 1971) supplemented with10 mm potassium nitrate and 2 mm ammonium nitrate.Agave seedlings propagated in vitro under sterile condi-tions were obtained from Agroforte, Tapachula, Chiapas,Mexico. Agave seedlings were grown in magenta boxes

and/or 50 mL capped test tubes supplemented withMurashige and Skoog medium (Murashige & Skoog 1962)containing 0.1% (w/v) activated carbon, 4.0% (w/v)sucrose, and 1.2% (w/v) agar. Seedlings were maintained ina growth chamber at 25 °C under 16 h light and 8 h darkphotoperiods and lamp intensities of 70 mmol m-2 s-1 ofphotosynthetic photon flow (400–700 nm).

Isolation of total RNA and mRNA fromagave plants

Agave seedlings were heat-shocked at 40 °C for 2 h in anilluminated growth chamber [70 mmol m-2 s-1 of photosyn-thetic photon flow (400–700 nm)]. Non-shocked controlplants were kept at 25 °C in a similar growth chamber.TotalRNA from leaves from heat-shocked and control seedlingswas prepared as described by Rochester and colleagues(Rochester, Winter & Shah 1986). Polyadenylated mRNAwas isolated using the PolyATract mRNA isolation kit(Promega Corporation, Madison, WI, USA).

Construction and screening of a cDNA library

A leaf cDNA library from heat-shocked seedlings was con-structed by using the SMART cDNA library constructionsystem (Clontech, Mountain View, CA, USA). For the syn-thesis of the first-strand cDNA, PowerScript reverse tran-scriptase (Clontech) and 1 mg of total RNA were used.cDNA was amplified by long-distance polymerase chainreaction (LD PCR). After digestion with SfiI, the cDNAwas fractionated by size using a Chroma Spin™ 400 column(Clontech), ligated to l-TriplEx2 vector, and packaged withthe ZAP cDNA Gigapack III kit (Stratagene, La Jolla,CA, USA). A total of 2 x 108 PFU were obtained of which96% were recombinant. Ten thousand recombinants werescreened by differential hybridization with single-strandedcDNA probes complementary to mRNA from either heat-shock treated (40 °C for 2 h) or untreated (25 °C) seedlings.The cDNA probes were prepared using 300 ng of polyA+

RNA, PowerScript™ reverse transcriptase, oligo dT and32P-2′-deoxycytidine-5′-triphosphate.

Sequencing of DNA and DNAsequence analyses

The nucleotide sequence of the cDNAs was determined withthe BigDye Terminator V1.1 Cycle Sequencing Kit on anApplied Biosystems ABI PRISM 377 DNA Sequencer.Databank searches were performed through the NationalCenter for Biotechnology Information (NCBI) web site(http://www.ncbi.nlm.nih.gov) using the Basic Local Align-ment Search Tool (BLAST). All cDNA sequences obtainedwere deposited in GenBank and their accession numbers arefound in Table 1. Molecular weight of A. tequilana sHSPswas calculated with the Compute pI/MW program availableat the ExPASy Proteomics Server (http:/www.expasy.org/).HSP90 and sHSP orthologs from Arabidopsis and rice

1792 R. Luján et al.

© 2009 Blackwell Publishing Ltd, Plant, Cell and Environment, 32, 1791–1803

were obtained from the NCBI website. HSP90 and sHSPsequences from poplar (release v1.1) were obtained fromthe DOE Joint Genome Institute website (http://align.genome.jp/). To visually estimate similarities among relatedHSP sequences, multiple sequence alignments were madewith the T-Coffee: Advanced program (Poirot, O’Toole &Notredame 2003) available at the Centre National de laRecherche Scientifique web site (http://www.igs.cnrs-mrs.fr/Tcoffee/tcoffee_cgi/index.cgi). To construct phylogenetictrees, a multiple sequence alignment was first performedwith the CLUSTALW program, available at the KyotoUniversity Bioinformatics Center web site (http://align.genome.jp/). Pairwise alignment was made with the slow/accurate option. A rooted phylogenetic tree with branchlengths was obtained using the neighbor-joining method(N-J). GenBank accesion numbers for all sequences consid-ered in the phylogenetic analyses are found in SupportingInformation Table S1.

Analysis of RNA gel blots

Equal amounts of total RNA were electrophoresed on1.2% agarose gels as described (Ausebel et al. 1989). Afterblotting to Hybond-N+ (Amersham Corporation,ArlingtonHeights, IL, USA), RNA was ultraviolet-cross-linked to themembrane. Prehybridization and hybridization were per-formed as described (Church & Gilbert 1984). 32P-LabelledDNA probes were prepared with the ReadyPrime™ II kit(Amersham Corporation).The cDNA inserts of clones 6.21,1.1, 6.18 and 1.7 (see Table 1) were used as probes fordetection of Hsp90A1, Hsp18.4B, Hsp17.7 and Cab tran-scripts, respectively. Probes for Hsp101, Hsp70-1, Hsp21and Ubiquitin transcripts were amplified by PCR using oli-gonucleotides derived from EST reads ajswflant001_f04,

ajswble03018_f03, aamhhppib018_e10 and ajswble02019_h02, respectively. These sequences were kindly providedby Drs. Aída Martínez Hernández (ColPos) and JuneSimpson (CINVESTAV-Irapuato) and are part of theAgaveEST-DB project (Martínez-Hernández & Simpsonunpublished data). Quantitation of transcript levels wasmade by densitometry of the autoradiograms using NIHImage 1.62 software (http://rsb.info.nih.gov/nih-image/download.html).

Heat-shock experiments to study Hsp mRNAand protein levels

One-year-old A. tequilana greenhouse-grown plants wereused. Plants were first transferred for two days to a growthroom [200 mmol m-2 s-1 of photosynthetic photon flow (400–700 nm)] that was set at 25 °C and a 16 h light/8 h darkphotoperiod. After this adjustment period, plants wereshocked during the day (around noon time) for 2 h in illu-minated incubators [70 mmol m-2 s-1 of photosyntheticphoton flow (400–700 nm)] preset at 28, 30, 32, 34, 36, 38, 40,42,44,46,48,50,52,54 or 55 °C.The two most external leavesof the rosette were collected after temperature treatments,frozen in liquid nitrogen and stored at –70 °C until used forRNA isolation. To estimate the levels of Hsp mRNAs, as afunction of the developmental stage of A. tequilana leaves,3-year-old plants containing 14 unfolded leaves were used.Plants were pre-adjusted for 2 d in the growth roomdescribed earlier.After pre-adjustment,plants were shockedat 28, 44, 48 or 54 °C for 2 h in illuminated growth chambersaround noon time. Leaves from four different sectors of therosette (spike, inner,middle and outer) were harvested sepa-rately, frozen in liquid nitrogen and stored at –70 °C untilused for RNA isolation or protein extraction.

Table 1. Agave tequilana cDNA clonesisolated and characterized in this workClone no. Encoded protein Class MW (Da) Accession number

1.1 HSP18.4B CI 18 438 DQ5157741.3 HSP18.3B CI 18 337 DQ5157751.8 HSP18.4A CI 18 427 DQ5157722.18 HSP17.5 CI 17 513 DQ5157732.34 HSP18.1 CI 18 160 DQ5157765.4 HSP18.4C CI 18 366 DQ5157776.17 HSP18.5 CI 18 466 DQ5157786.26 HSP18.4D CI 18 390 DQ5157817.1 HSP18.3B CI 18 337 DQ5157827.4 HSP18.1 CI 18 160 DQ5157837.7 HSP18.3A CI 18 393 DQ5157847.8 HSP18.1 CI 18 160 DQ5157856.18 HSP17.7 CII 17 749 DQ5157796.21 HSP90A1 Cyt.a b DQ5157801.7 CABc – b DQ525854

The table indicates, for each clone, the name, class, size in Daltons and GenBank accessionnumber of the predicted protein.aCytoplasmic.bBecause sequences were derived from partial cDNAs, no data is available for the full-lengthpredicted protein.cChlorophyll a/b-binding protein.

Heat tolerance in a tropical CAM plant 1793


Heat-shock resistance assays

To evaluate the viability after heat shock, 3-year-oldgreenhouse-grown plants were first pre-adjusted for 2 d asdescribed in the ‘Heat-shock experiments to study HspmRNA and protein levels’ section. Following pre-adjustment, plants were transferred to illuminated growthchambers [70 mmol m-2 s-1 of photosynthetic photon flow(400–700 nm)] around noon time. Growth chambers werepreset at 28, 44, 48 or 54 °C. Treatments were for 2 h. Toestimate the unfolding of leaves following the treatments,the most recently unfolded leaf of each plant at the time oftreatment was identified with a tag as a reference. Before,and immediately after the temperature treatments, eachplant was photographed and the number of leaves and theirlengths recorded. Resistance was evaluated after returningthe plants to a greenhouse that was maintained at maximal/minimal temperature of 32/20 °C with a natural photope-riod. Each plant was measured at 9, 21, 45 and 90 d afterrecovery. The percentage of leaf damage was calculatedafter multiplying by 100 the ratio of the length occupied bynecrotic tissue of each leaf/total length of the leaf. This wassimple to estimate using a measuring tape as, typically, alllesions provoked by heat shock expanded evenly from thetip towards the base of the leaf. Pictures from each plantwere taken at each time point of recovery. The number ofleaves that unfolded following the heat-shock treatmentswas also recorded.

Measurement of leaf temperature in responseto heat shocks

Leaf temperature of agave plants was measured with aBAT-10 digital thermometer (Physitemp, Clifton, NJ, USA)and a type T (copper constantan) thermocouple (modelMT-4). To measure leaf temperatures, thermocouple wascarefully inserted in the upper third and central sectionof each leaf ensuring that the tip remained between theadaxial and abaxial faces of the leaf. Plants were treated asdescribed in the ‘Heat-shock experiments to study HspmRNA and protein levels’ section.

Stomatal density measurements

To measure stomatal density, free-hand sections of A. tequi-lana leaves were made with a razor blade. Cuts were madeparallel to the epidermis both from the adaxial and abaxialsides of the leaf. Because of the elaborate anatomy of thestomatal complex in agaves (Blunden, Yi & Jewers 1973;Alvarez de Zayas 1985), it is difficult to make an accuratedetermination of stomatal density in A. tequilana by justlooking at the leaf surface under the microscope. Thus,tissue was fixed in 1 mL of FAA (50% ethanol, 5% glacialacetic acid, 10% formaldehyde, 35% water) for 48 h at roomtemperature.After fixation, sections were quickly washed indistilled water, blotted dry and stained for 20 min in I2-KI(1% KI, 1% I2, in distilled water) to visualize the starchgrains found in the guard cells. To count the stomata,

pictures of the guard cells found in the subepidermal layerwere taken with a Nikon D1 digital camera (Nikon Co.,Tokyo, Japan) coupled to a dissecting microscope (NikonSMZ 1500). Stomatal density was expressed as stomatamm-2.

Extraction of total proteins and Westernblot analysis

Total proteins were extracted with a protocol that will bepublished elsewhere (Lledías & Nieto-Sotelo unpublisheddata).This method prevents the degradation of A. tequilanaproteins and removes contaminants such as waxes, polysac-charides, pigments, etc. Equal amounts (5 mg) of totalprotein were loaded on sodium dodecyl sulphate–polyacrylamide gel electrophoresis mini gels. After separa-tion, proteins were transferred to nitrocellulose membranes(Hybond-C Extra, Amersham Biosciences). Membraneswere blocked in 4% low-fat milk in phosphate-bufferedsaline 0.1% Tween 20 (PBS-T) buffer overnight at 4 °C.Incubation with primary antibody was for 1.5 h at roomtemperature. After washing three times with PBS-T themembrane was incubated with secondary anti-rabbit-HRPantibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA,USA, cat. SC-2317) at a 1:5000 dilution for 1.25 h at roomtemperature. Finally, the membrane was washed three timeswith PBS-T and developed with ECL reagent (Amershamcat. RPN2109) and exposed to X-ray films (Kodak cat.6040331). The following primary rabbit-antibodies wereused to detect HSPs: anti-Arabidopsis full-length HSP17.6(class I sHSP); anti-Arabidopsis full-length HSP17.7 (classII sHSP); and anti-Arabidopsis N-terminal 145 aa fromHSP101 (cat. # AS07254,AS07255 and AS07253 from Agris-era, Vännäs, Sweden) at 1:128 000, 1:32 000 and 1:16 000dilution, respectively. HSP70 was detected with a commer-cial antibody raised against recombinant human HSP70(HSP72) (Stressgen Bioreagents,Victoria, BC, Canada) at a1:2500 dilution. HSP90 was detected with anti-ArabidopsisHSP90 (at-115) antibody (cat. # sc-33755, Santa Cruz Bio-technology, Inc.) at 1:200 dilution. Ubiquitin was detectedwith anti-ubiquitin antibody (cat. # sc-9133 Santa Cruz Bio-technology, Inc.) at 1:1000 dilution. Profilin was detectedwith an anti-profilin antibody (Vidali et al. 1995) at a 1:5000dilution. Carbonyl content in proteins was estimated with aprotein slot-blot procedure followed by an immunochemi-cal protocol (OxyBlot Protein Oxidation detection kit, fromChemicon International, Temecula, CA, USA) that detects2,4-dinitrophenylhidrazone after reacting samples with 2,4-dinitrophenylhydrazine (DNPH). Quantitation of carbonylformation was made by densitometry of the autoradio-grams using NIH Image 1.62 software.

RESULTS

Isolation and characterization of A. tequilanacDNA clones encoding sHSP and HSP90

Because no genomic, cDNA or EST sequences encodingHSPs had been reported from Agavaceae or closely related



families, a cDNA library of A. tequilana was prepared usingtotal RNA isolated from leaves of in vitro grown seedlingsexposed to heat shock. The library was screened by differ-ential hybridization using cDNAs from control (25 °C) orheat-shocked plants (40 °C) (see Materials and Methods).DNA sequencing and Blast analysis against the GenBankdatabase allowed the identification of 13 cDNA clonesencoding sHSPs, one clone encoding an HSP90 homologueand one clone encoding the constitutively expressed chlo-rophyll a/b-binding protein (CAB) (Table 1). Based ontheir amino acid sequence, predicted size and similarity toother plant sHSPs, 12 A. tequilana sHsp cDNA clones wereidentified as encoding sHSP of the CI subfamily, whereasone was more similar to the CII subfamily. cDNAs encodingidentical amino acid sequences were given the same name(i.e. three clones HSP18.1 and two clones HSP18.3B).Because substantial differences in the nucleotide sequencesof these five cDNA clones were found, we postulate thatthey might represent allelic variations of the same gene orproducts of duplicated genes.

A phylogenetic analysis was performed to define moreclearly the relationships between the A. tequilana sHSPsand other CI and CII subfamily plant sHSPs. As a compari-son, all annotated sequences from Arabidopsis, rice andpoplar, corresponding to these subfamilies, were used. Thederived phylogenetic tree (Fig. 1) showed that eight A.tequilana sequences (Agave18.4B, Agave18.4C, Agave18.5,Agave18.3A, Agave18.4D, Agave18.4A, Agave18.1 andAgave18.3B) formed an evolutionary distinct lineage. Theclosest relatives to this group were Pt18.3A-CI, Pt18.3D-CI,and Pt18.5-CI from poplar and Agave17.5 from A. tequi-lana. The analysis indicated that two main clusters formclass CI sHSPs. Arabidopsis CI-type sHSPs assembled inboth clusters. In contrast, all CI subfamily sHSPs fromA. tequilana, poplar and rice assembled in only one cluster.The only exceptions were rice Os16.93-CI and poplarPt17.4B-CI that formed independent branches rooted inthe early ancestor of the CI subfamily. We can infer thatmembers of the two main clusters arose by a gene duplica-tion event in the angiosperm ancestor of the monocots. Aposterior event caused the loss of one of the duplicatedcopies in the lineages leading to Agave, rice and poplar,whereas in the Arabidopsis lineage, both copies were con-served. In the four species, further duplication events of theremaining copies seemed to give rise to the whole subfam-ily. Because, the A. tequilana sHSP-CI sequences were notobtained from a full genome annotation project, it is apossibility that A. tequilana members of the rice sHSP-CIcluster have not been identified. The same tree showed thatA. tequilana HSP17.7 forms part of class CII of the sHSPfamily. The alignment of class CI and class CII A.tequilanasequences, to their closest plant relatives, clearly showedthe presence of the conserved N-terminal domain, thea-crystallin CR-I and CR-II domains, and the b-10 strand(Supporting Information Figs S1 & S2). The only sequencethat did not show all of these features was A. tequilanaHSP17.5 that is predicted to contain a deletion in theb-10 strand. This structure was found to be important

for oligomerization (van Montfort et al. 2001; Scharf,Siddique & Vierling 2001).

Clone 6.21 from A. tequilana contained a partial cDNAencoding HSP90. The phylogenetic analysis revealed thatA. tequilana HSP90 is a member of the cytoplasmic/nuclearA subfamily and its closest relatives were A. thalianaHSP90A1 and poplar PtHSP90A1/A2 forming a separateclade from the ER (B), plastid (C1), and mitochondrial(C2)-associated HSP90 subfamilies (Fig. 2). The alignmentof the predicted sequences from the cytoplasmic subfamilyshowed that A. tequilana HSP90 contains the block Jdomain, one of 10 highly conserved domains in HSP90family members, and the C-terminal pentapeptide that isimportant for binding to the TPR (tetratrico peptide

Pt15.9-CIPt17.8B-CI

Pt18.3A-CIPt18.3D-CI

Pt18.5-CIAgave18.4B-CI

Agave18.4C-CIAgave18.5-CIAgave18.3A-CIAgave18.4D-CIAgave18.4A-CIAgave18.1-CIAgave18.3B-CI

Agave17.5-CIPt18.0-CI

At17.6A-CIAt17.8-CI

At17.6B-CIPt17.5A-CI

Pt17.5B-CIPt18.2A-CI

Pt18.3-CIPt17.8A-CI

Pt17.6B-CIPt18.3B-CI

Pt18.1-CIOs16.9-CIOs16.92-CIOs16.91-CIOs17.4A-CI

Os17.4B-CIOs17.4C-CI

Os17.4D-CIAt17.4-CI

At17.6C-CIAt18.1-CI

Pt17.4B-CIOs16.93-CIPt17.5-CII

Agave17.7-CIIOs17.8-CII

At17.6-CIIAt17.7-CII

Os17.6C-CII

Figure 1. Phylogenetic analysis of small heat-shock proteins(sHSPs). All CI and CII sHSP sequences annotated in thegenome projects of Arabidopsis thaliana, Oriza sativa (rice)and Populus trichocarpa (poplar) were used to define theirphylogenetic relations to the A. tequilana sHSP’s described inthis work. Their complete sequences were aligned with theClustal W program and a phylogenetic tree was obtained withthe N-J method as described in Materials and Methods. Identityof sequences can be found in Supporting Information Table S1.Nomenclature for Arabidopsis, poplar and rice sHSP’s wasaccording to Waters, Aevermann & Sanders-Reed (2008).



repeat) of immunophillin and p60, members of the HSP90chaperone complex (Supporting Information Fig. S3). ThisC-terminal pentapeptide (MEEVD) is characteristic ofplant and animal HSP90 proteins (Krishna & Gloor 2001).We refer to the protein encoded by clone 6.21 as HSP90A1.

Accumulation of Hsp mRNAs, and tolerance inresponse to heat shock in A. tequilana plants

The transcript levels of all A. tequilana sHsps increased inthe leaves of whole plants in response to heat shock (datanot shown). In Fig. 3, a detailed analysis of transcriptabundance for Hsp18.4B (class I), Hsp17.7 (Class II) andHsp90A1 is depicted. Transcripts were barely detectable at28 °C, but increased in direct proportion to temperature,and reached a maximum at 42–44 °C. In contrast, Cabtranscript levels remained constant up to 50 °C, diminishingby 30 to 40% at 52 °C or above. The temperature for

short-term heat resistance for A. tequilana chlorenchymacells is 55 °C (Nobel et al. 1998) whereas for other agavespecies is 57 to 65 °C (Nobel & Smith 1983). This is muchhigher than the temperature at which accumulation of A.tequilana Hsp transcripts is maximal (shown earlier). Wethus investigated the heat tolerance of A. tequilana at thewhole-plant level and after long-term recovery (90 d).Three-year old A. tequilana plants were heat-shocked for2 h at 28, 44, 48 or 54 °C and the damage to the leavesestimated after 9, 21, 45 and 90 d of recovery. As shown inSupporting Information Fig. S4g–h, all plants remainedviable even after exposure to 54 °C. However, at this tem-perature, heavy damage occurred only in leaves that werealready unfolded at the time of heat shock (Fig. 4 and Sup-porting Information Fig. S4g–h). The damage was moresevere in leaves localized in the outer sector of the rosettethan in those in the inner sector. Ninety days after a 54 °Cheat shock all leaves from the outer sector were necrotic,whereas only 50% leaf damage occurred in the inner sector

PtHSP90A3

PtHSP90A2

PtHSP90A1

PtHSP90A4

PtHSP90A5

PtHSP90A6

PtHSP90A7

PtHSP90B1

PtHSP90C1-1

PtHSP90C1-2

PtHSP90C2

AgaveHSP90A1

AtHSP90A1

AtHSP90A3

AtHSP90A2

AtHSP90A4

AtHSP90B1

AtHSP90C1

AtHSP90C2

OsHSP90A1

OsHSP90A2

OsHSP90A3

OsHSP90A4

OsHSP90C1

OsHSP90C2

OsHSP90B1

Figure 2. Phylogenetic analysis of HSP90 proteins. The 174C-terminal amino acids from A. tequilana HSP90A1 were alignedwith the corresponding segments of all HSP90 sequencesannotated in the genome projects of Arabidopsis, poplar and ricewith the Clustal W program. A phylogenetic tree was obtainedwith the N-J method as described in Materials and Methods.Identity of sequences can be found in Supporting InformationTable S2. Nomenclature for Arabidopsis, poplar and rice HSP90sequences was according to Chen, Zhong & Monteiro (2006).

T (°C): 28 30 32 34 36 38 40 42 44 46 48 50 52 54 55

Hsp18.4B

Hsp17.7

Hsp90A1

Cab

Figure 3. Expression of Hsp genes in response to heat shock inA. tequilana leaves. Plants were exposed for 2 h at the indicatedtemperatures and the two most external leaves of the rosette(most mature sector) were analysed with the indicated probes byRNA hybridization.

–20

0

20

40

60

80

100

120

Lea

f d

amag

e (%

)

28 44 48 54

Temperature (°C)

MiddleInner Outer

Figure 4. Effect of heat shock on leaf viability in A. tequilana.Damage to leaves was from 3-year-old plants that were alreadyunfolded before treatments. Three-year old plants were incubatedfor 2 h at 28, 44, 48 or 54 °C, and damage was evaluated 90 dafter temperature treatments. In order to carry a comparativeanalysis of leaf damage, the rosette was divided in three partsaccording to the degree of maturity of the leaves: inner; middle;and outer sectors. Data are means and standard deviation of thedata from 12 leaves from each sector.



leaves (Fig. 4). On a given leaf, the damage was first visibleat the tip propagating evenly towards its base in a timeframe of several weeks. In contrast, the spike (Fig. S4i),containing the tightly overlapped immature leaves that sur-round the central meristem, and all its leaves that unfoldedafter the thermal treatment, showed no visible damage evenafter 90 d (Table 2). Similar results were obtained in plantstreated for 6 h at 55 °C (data not shown). Interestingly, thenumber of leaves that unfolded from the spike in responseto the 54 °C heat shock increased significantly (Fig. 5). Theunfolding of leaves started during the first week followingthe 54 °C heat shock. On average, five leaves unfolded oneach plant 90 d after a 54 °C shock (Fig. 5). The unfoldingrate of leaves at 44 or 48 °C was not different to the oneobserved in those treated at 28 °C.

Leaf temperatures of A. tequilana plants duringheat shock

To understand the basis of the extreme heat tolerance of thespike leaves, we measured the actual temperature of allleaves during the heat-shock treatments. Plants were incu-bated for 2 h in an illuminated growth chamber preset at 46,

50, 54 or 55 °C and leaf temperatures were recorded everyminute. When exposed to air temperatures of 54 or 55 °C,the spike and the inner sector leaves decreased their tem-perature more efficiently than the rest of the leaves. On theaverage, spike and inner sector leaves were 5 and 6 °Cbelow ambient temperature at the end of the 54 and 55 °Cshocks, respectively, whereas leaves from the middle andouter sectors were around 2 °C below ambient under thesame conditions (Fig. 6). When shocked at 46 or 50 °C, allleaves were around 1.5 or 2.5 °C below ambient, respec-tively (Fig. 6). In response to a 54 °C heat shock for 2 h, theleaf from the outer sector equilibrated with the surroundingair after 19 min and reached its first maximum at 52.8 °C(see Supporting Information Fig. S5). In contrast, the spikereached its maximum of 51 °C after 29 min. Surprisingly,the spike leaves slowly lowered their temperature a fewminutes after reaching the maximum temperature. As seenin the example of Supporting Information Fig. S5a, thespike dropped its temperature to 50 °C at 30 min, to 49 °Cat 45 min and to 48 °C at 52 min, remaining at 48 °C untilthe end of the 2 h heat shock. In contrast, leaves from theouter sector showed very small oscillations in temperature,maintaining a value similar to their first maximum after the2 h incubation (Supporting Information Fig. S5b).

Anatomical differences within the rosette ofA. tequilana

To further explore whether the heat tolerance and leaftemperature differences between the different leaf sectorsin the rosette were because of the differences in theiranatomy, stomatal density was studied. Stomatal densitywas twice as high in the adaxial and abaxial sides of thespike leaves relative to those in the periphery (Fig. 7). Inner

Table 2. Effect of heat shock on leaves that unfolded afterthermal treatments

Thermal treatment (°C) Leaf damage (%)

28 0.03 � 0.0444 0 � 048 0 � 054 0.04 � 0.06

Evaluation of damage was made after 90 d of recovery. Data aremeans � S.D. (standard deviation) of four replicates. n ranged from8 to 19 per treatment.

Uu

nfo

lded

leav

es /

pla

nt

–1

0

1

2

3

4

5

6

Temperature (°C)28 44 48 54

9 d 21 d 45 d 90 d

Figure 5. Unfolding of A. tequilana leaves in response to heatshock. Three-year-old plants were heat-shocked, as described inFig. 4, and leaf unfolding was evaluated 9, 21, 45 and 90 d afterrecovery. Data are means and standard deviation of the datafrom four replicates.

Tem

per

atu

re (

°C)

–1

0

1

2

3

4

5

6

7

8

9

46 50 54 55Air temperature (°C)

MiddleInner Outer

Figure 6. Difference in leaf temperature, relative to air, ofleaves from whole plants exposed to heat shock for 2 h at theindicated temperature. Leaf temperature was measured with athermocouple, as described in Materials and Methods. Data aremeans and standard deviation of the data collected from sixdifferent leaves.



and middle sector leaves showed intermediate stomataldensities on both sides relative to spike and outer leaves.

Levels of Hsp mRNA in different sectors of therosette in A. tequilana

As shown in Fig. 6, spike and inner sector leaves abatedtemperature 5 °C relative to ambient when shocked at54 °C. The spike leaves showed no visible damage whenexposed to 54 °C for 2 h (Table 2). Moreover, the innersector leaves, already unfolded before the treatments, suf-fered 50% damage after 90 d, less than observed in leavesfrom the middle and outer sectors (Fig. 4 and SupportingInformation Fig. S4). Clearly, these results indicated thatthe elevated heat tolerance of the spike was not solelycaused by its great capacity to depress ambient tempera-ture, as the inner sector leaves showed a similar responsebut did not show the same level of heat resistance. More-over, the actual temperature in the spike leaves, whenexposed to air at 54 °C, was around 49 °C, which was stillhigher than 44 °C, temperature at which the highest accu-mulation of Hsp transcripts takes place in the outer leaves(Fig. 3). We hypothesized that additional factors couldexplain these data, such as differences in the capacity toexpress Hsp genes at air temperatures above 44 °C and/ordifferences in the constitutive levels of HSPs at optimaltemperature. To test these ideas, transcript levels forHsp101, Hsp90A1, Hsp70-1, Hsp21, Hsp18.4B, Hsp17.7,Ubiquitin and Cab were measured in four different sectorsof the rosette. Figure 8 showed that the accumulation ofHsp mRNAs in the rosette in response to 2 h treatmentsat 28 °C was very low in all sectors with the exceptionof Hsp70-1 whose levels were high, especially in the spikeand inner sector. At 44 °C Hsp101, Hsp90A1, Hsp21 andHsp18.4B transcripts were more abundant in the spike

leaves. In contrast, Hsp70-1 and Hsp17.7 transcriptswere more abundant in the outer sector. After reachinga maximum accumulation at 44 °C, the levels of Hsptranscripts in all parts of the rosette were lower at moreelevated temperatures. Nonetheless, levels of all Hsp tran-scripts and Ubiquitin were higher in the middle and outersectors after 48 and 54 °C heat shocks. Ubiquitin mRNAabundance at 28 °C was higher in the unfolded leaves thanin the spike. At 44, 48 and 54 °C Ubiquitin transcript levelswere higher in the middle and outer sectors. Cab transcriptabundance slowly decreased in spike, inner and middlesector leaves as the heat-shock temperature increased. Inthe outer sector Cab transcripts decreased at a steeper rateafter 44, 48 and 54 °C heat shocks.

Levels of HSP and protein carbonyl content indifferent sectors of the rosette in A. tequilana

The analyses presented earlier did not show a clear cor-relation between Hsp transcript levels and heat shockresistance in the rosette. However, the study of Ubiquitintranscript levels helped to support the idea that thesensitive sectors (M and O) suffered a stronger proteindamage that required proteasomal-dependent degrada-tion. As an alternative approach to understand theobserved differences in thermotolerance within the rosettewe measured protein levels of HSP101, HSP90, HSP70,sHSP-CI, sHSP-CII and ubiquitin. As a loading control,the levels of profilin were estimated. The amount ofHSP101, HSP90 and HSP70 was always high at all

S I M O

Sto

mat

ald

ensi

ty(s

tom

ata

mm

–2)

0

10

20

30

40

50

60

70

80

90

Rosette sector

Adaxial Abaxial

Figure 7. Stomatal density in the leaves from different rosettesectors in A. tequilana. The stomatal density of adaxial andabaxial sides of the leaves of 3-year-old plants was evaluatedafter I2-KI staining, as described in the Materials and Methodssection. Data are means and standard deviation of the datacollected from the analysis of six to eight different leaves.

28 44 48 54T (°C):

Sector: S I M O S I M O S I M O S I M O

Hsp90A1

Hsp101

Hsp70-1

Hsp21

Hsp18.4B

Hsp17.7

Ubiquitin

Cab

Figure 8. Levels of Hsp gene transcripts in the leaves of A.tequilana plants that received a single heat shock. Whole plantswere incubated for 2 h at the indicated temperatures and theleaves from the spike (S), inner (I) middle (M) and outer (O)sectors were harvested. Equal amounts of total RNA wereloaded in denaturing-agarose gels. Levels of Hsp101, Hsp90A1,Hsp70-1, Hsp21, Hsp18.4B (class I), Hsp17.7 (class II), Ubiquitinand Cab mRNAs were measured by RNA hybridization asdescribed in Materials and Methods.



temperatures in the O and M sectors (Fig. 9). In the Isector, HSP101 was barely detectable at all temperatures,HSP90 levels were moderate at 28, 48 and 54 °C, butbelow detection at 44 °C, and HSP70 high at 28, 48 and54 °C, and low at 44 °C. In the S leaves, on the other hand,HSP101, HSP90, and HSP70 levels were undetectableat 28, 44 and 48 °C, whereas at 54 °C they reached amaximum with levels almost comparable with those inthe O sector. In contrast to the high MW HSPs, sHSP-CIand sHSP-CII levels were consistently more abundant in Sleaves at all temperatures. Levels of sHSP-CI were slightlyinduced at 44 °C in I, M and O leaves and below detectionat 48 and 54 °C. In I, M and O leaves sHSP-CII levelswere high at 28 °C, just detectable at 44 °C and, similarlyto sHSP-CI, undetectable at 48 °C and 54 °C. Proteinubiquitinylation increased in a gradient from the centre(S) to the periphery (O) at all temperatures. In O leavesmaximum levels were observed at 44 °C, in M leaves at48 °C, and in S and I leaves at 54 °C. The levels of profilinwere similar in all sectors at 28, 44, and 48 °C. At 54 °Clevels in M and O sector were lower than in S and I.

To further study whether the most sensitive sectors (Mand O) suffered a stronger protein damage as a result ofoxidative stress that may occur in response to high tempera-ture, we measured the carbonyl content in proteins. Carbo-nyl formation is an irreversible protein modification ofproteins caused by reactive oxygen species (ROS), as theycause the oxidation of amino acid residue side chains intotheir aldehyde or ketone derivatives (Halliwell & Gut-teridge 2007). This modification may cause the permanentloss of protein function, and it is considered a key indicatorof severe oxidative damage and disease-derived proteindysfunction (Fagan, Sleczka & Sohar 1999). Consistent withthe ubiquitinylation pattern, protein carbonyl contentgradually increased from the centre (S) to the periphery(O) at all temperatures (Fig. 10). In all sectors proteincarbonyl content increased in direct proportion withtemperature.

DISCUSSION

The extraordinary tolerance to extreme high temperaturesof the spike in A. tequilana showed a close correspondencewith its higher capacity to reduce leaf temperature inresponse to this environmental insult, perhaps as a result ofthe induction of transpirational cooling during the day. Ahigher stomatal density, combined with the ability to openthe stomata in response to heat shock during the day, seemsto be part of a large number of adaptations that help this

28 44 48 54T (°C):

Sector: S I M O S I M O S I M O S I M O

HSP101

HSP90

HSP70

sHSP-CI

sHSP-CII

Ubiquitin

Profilin

Figure 9. Levels of heat-shock proteins (HSPs) in the leaves ofA. tequilana plants that received a single heat shock. Equalamounts (5 mg) of total protein were loaded on sodium dodecylsulphate–polyacrylamide gel electrophoresis mini gels. Levels ofHSP101, HSP90, HSP70, HSP21, sHSP-CI, sHSP-CII, ubiquitin,and profilin proteins were estimated by immunochemistry, asdescribed in Materials and Methods. The labelling of the rosettesectors corresponds to that shown in Fig. 8. Arrow in theubiquitin panel indicates the border between stacking andseparating gel.

0

1

2

3

4

5

6

7

28 44 48 54

Temperature (°C)

Pro

tein

car

bo

nyl

co

nte

nt

(arb

itra

ry u

nit

s 10

4 ) MiddleInner OuterSpike

Figure 10. Carbonyl content in total proteins extracted from A.tequilana leaves after a single heat shock. Equal amounts of totalprotein (5 mg) obtained from different sectors of the rosette afterwhole plants received a heat shock for 2 h at the indicatedtemperatures were used to measure protein carbonyl content asdescribed in Materials and Methods. Values are expressed inarbitrary units. Data are means and standard deviation of thedata collected from three replicates. The labelling of the rosettesectors corresponds to that shown in Fig. 8.



species to resist high temperatures.These strategies contrib-uted to the survival of the less mature leaves and the shootapical meristem of the plant. At a first approximation,transpirational cooling during the day does not seem to beexpected for a CAM plant, which normally avoids waterloss by closing their stomata during the day, when tempera-ture is high (Larcher 1995). However, examples in the lit-erature indicate that CAM metabolism is a more dynamicprocess. In Citrullus colocynthis, a succulent in the Cucur-bitaceae family, leaf overheating can be effectively pre-vented by transpirational cooling, as long as sufficient wateris available; in this way, plants remain 4–6 °C, and inextreme cases even 10–15 °C, cooler than the air (Lange1959). In Agave deserti plants, the normal pattern ofstomata opening during the night is changed to openingduring the day in response to watering under laboratoryconditions (Hartsock & Nobel 1976). In the same species, asthe seedlings age, the late-afternoon CO2 uptake alsodecreased, being minor in 1-year-old plants and absent inolder plants (Nobel 1985). In A. tequilana, stomatal openingin greenhouse-grown plants follows the CAM pattern(Nobel & Valenzuela 1987). However, although CO2 uptakeand water vapour conductance were more predominant atnight, A. tequilana plants also show minor positive peaks ofCO2 uptake and water vapour conductance between 1200and 1800 h (Nobel & Valenzuela 1987). Thus, agaves haveevolved mechanisms that allow transpirational coolingduring the hottest part of the day. It remains to be shown if,as predicted, spike and inner sector leaves are more capableof carrying out this mechanism.

Another important response to heat shock in A. tequilanaat the whole plant level was the acceleration in the rate ofleaf unfolding from the spike. The unfolding of leaves infield-grown A. tequilana plants shows a positive correlationwith the environmental productivity index (EPI), as it ishigher between June and November (rainy season) andlower between December and May (dry season) (Nobel &Valenzuela 1987). Unfolding of leaves in response toextreme heat shock might be a developmental responseunrelated to EPI that may require sensing the death ofthe marginal leaves of the rosette. The mechanism by whichthis occurs, independently of EPI changes, remains to beinvestigated.

The comparative study of the levels of Hsp transcripts inresponse to heat shock, along the different sectors of therosette, gave no clue to the basis for the high heat toleranceof the spike leaves (Fig. 8). These leaves appeared to sufferless damage caused by the high temperature, as ubiquitiny-lation and carbonylation of proteins was less prominentthan in the more mature sectors (Figs 9 & 10). In contrast,the comparative study of HSP levels allowed us to find astrong positive correlation between sHSP-CI and sHSP-CIIlevels and basal thermotolerance (Fig. 9). The spike leavesshowed higher levels of sHSP-CI and sHSP-CII at optimaltemperature and after heat shock. These sHSPs were com-pletely absent in unfolded leaves at 48 and 54 °C (Fig. 9).The sHSPs accumulate in response to heat stress anddevelopmental signals such as embryogenesis, meristem

formation, germination and fruit development (Pla et al.1998; Wehmeyer & Vierling 2000; Sun, van Montagu &Verbruggen 2002). Both in vitro and in vivo assays indicatethat sHSPs function as molecular chaperones, preventingprotein aggregation and maintaining other heat-denaturedproteins in a folding-competent state (Lee et al. 1997; Löwet al. 2000). Although the functional analysis of sHSPs hasbeen the focus of many studies, their role during the devel-opment of plants is still not well understood. Recently, apositive correlation between HSP22 protein levels and heatstress resistance has been found in pea mitochondria (Stup-nikova et al. 2006). We think that the elevated levels ofsHSPs in the spike leaves of A. tequilana help them toprevent and protect other proteins from damage in case ofsudden exposure to high temperature and, perhaps, otherstress conditions.

Similarly to the sHSPs, the large molecular weight HSPsaccumulate during plant development, as in developing andmature embryos (Hong & Vierling 2001; Nieto-Sotelo et al.2002). HSP101 is required for induced thermotolerance andfor the high basal thermotolerance of young seedlings inArabidopsis and maize (Hong & Vierling 2001; Nieto-Sotelo et al. 2002). We observed that the levels of HSP101,HSP90 and HSP70, in the spike leaves of A. tequilana, werevery low or below detection at 28, 44 and 48 °C, whereasreaching maximum accumulation in response to a 54 °Cheat shock. In contrast, the less heat-tolerant part of therosette (middle and outer sectors) showed higher levels ofHSP101, HSP90 and HSP70 at all temperatures (Fig. 9).Theincrease in HSP101, HSP90 and HSP70 might be a reflec-tion of the accumulation of abnormal proteins in the moremature tissues, as shown by the protein ubiquitinylation andcarbonylation assays (Figs 9 & 10). Damaged proteins arethought to be players in the cycle of regulation of the heat-shock transcription factor (HSF). Under optimal conditionsHSP90 and HSP70 repress HSF activity. When damagedproteins accumulate, they become targets of the chaperonesand as a result HSF is free to activate the heat shockresponse (Morimoto 1998). The presence of HSP101,HSP70, and sHSPs in spike leaves at 54 °C may potentiatethe solubilization of aggregates, known to be removed bythe synergistic action of this triad of HSPs in bacteria,yeasts, and plants (Mogk et al. 2003; Cashikar, Duennwald& Lindquist 2005; Lee et al. 2005), thus increasing thermo-tolerance. In contrast, the absence of sHSP in inner, middle,and outer sectors at 48 and 54 °C (Fig. 9) might cause ahandicap to the solubilization of protein aggregates thataccumulate during severe heat shock, in spite of the highlevels of HSP101 and HSP70. Because the patterns of HSPaccumulation were not a mere reflection of the levels oftheir corresponding transcripts (Figs 8 & 9), we hypothesizethat HSP abundance is developmentally regulated as leavesunfold and mature. This mechanism remains to be studiedand we can only speculate that the differential control ofHSP synthesis and/or stability could result in the observeddifferences in HSP levels between sectors.

Notwithstanding the large body of work relating to thedevelopmental accumulation of HSPs, to our knowledge no



examples of the differential accumulation of HSPs amongsimilar organs of the same individual have been described.This finding is different to the more frequent observationthat HSP levels change with the age (calendar age) of agiven organ or organism as it develops, as it has beenobserved in plants and animals (Thompson, Bowsher &Tobin 1998; Lund et al. 2002; Calabrese et al. 2004). We donot know whether, in A. tequilana, differences in sHSPlevels may also occur in leaves of similar relative positionswithin the rosette as a function of the age of the wholeplant.We could predict that as A. tequilana ages, its capacityto accumulate sHSP also declines, as has been observedfor HSP70 in other organisms. However, this remains to beanalysed.

Part of the cellular damage caused during heat stress isbecause of the toxic effects caused by ROS (Davidson et al.1996; Locato et al. 2008). ROS are derivatives of the dioxy-gen molecule produced by its partial reduction or by thecapture of energy by one of its unpaired electrons (Mittler2002; Halliwell & Gutteridge 2007). As ROS are highlyreactive, they severely attack proteins by increasing theircarbonyl content (Halliwell & Gutteridge 2007). As withHSP70 and protein ubiquitinylation, the increased carbonylcontent in proteins of the outer leaves correlated with theirhigher sensitivity to heat stress (Figs 4, 9 & 10). On thecontrary, the lower level of carbonylated proteins in thespike leaves seemed to be an indication that a strongerantioxidant defense system existed in this sector of therosette and/or that a reduced level of ROS was generatedboth under optimal conditions and as a result of heatstress. The large amounts of sHSPs that accumulated in thespike leaves may have contributed to a decrease in ROSproduction.

If sHSPs are so important for stress survival of A. tequi-lana leaves, why did not outer leaves accumulate high levelsof sHSPs as the spike leaves did? Perhaps their presencemay have a costly consequence on fitness and growth asnatural selection has resulted in a tightly regulated controlof sHSP levels as leaves unfold and reach maturity. Theincreased metabolic and photosynthetic activities ofunfolded leaves may favor a higher energy expenditure ongrowth related processes than on stress resistance. Alterna-tively, the presence of sHSPs may be antagonistic to factorsthat regulate growth, as has been observed in Arabidopsis,where overexpression of Hsc70-1 decreased growth anddevelopment (Sung & Guy 2003).

The differential accumulation of sHSP within the rosetteseems to be part of a developmental program in whichouter-sector leaves maintain very low levels of sHSPsthrough post-transcriptional or post-translational regula-tion. This developmental process, along with a decrease instomatal density and transpirational cooling during the dayin response to high temperature, could explain the lowerheat tolerance of the unfolded leaves, especially those ofthe outer sector. In contrast, spike leaves show higher sHSPlevels, perhaps because factors that promote their degrada-tion are suppressed and/or factors that promote their trans-latability are set in place. The ability of spike leaves to

reduce their temperature, in response to elevated airtemperatures, and the developmental control of stomataldensity are strategies that facilitate the survival of thewhole plant by reducing the irreversible damage to pro-teins at extreme temperatures. In combination, these devel-opmentally regulated programs may protect the shootapical meristem and leaf primordia enclosed in the spike,thus assuring the survival of the plant.

ACKNOWLEDGMENTS

We thank Dr I. del Real and M.C.R. Ayala (Sauza-PedroDomecq) for providing A. tequilana plants and their com-mitted support to the project; Dr G. Ponce Romero foradvice on I2-KI staining of guard cells; Drs G. Guillén and F.Sánchez (IBt-UNAM) for kindly providing anti-profilinantibodies; P. Gaytán and E. López for help on oligonucle-otide synthesis; R. Hernández for help on DNA sequencing;A. Martínez Valle, J.M. Hurtado and A. Ocadiz for com-puter assistance. This work was supported by a grant fromPedro Domecq (P-150) to G.I.C. and J.N.-S.

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Received 30 May 2009; received in revised form 13 August 2009;accepted for publication 14 August 2009

SUPPORTING INFORMATION

Additional Supporting Information may be found in theonline version of this article:

Figure S1. Alignment of the complete sequences A. tequi-lana class I sHSPs to their closest plant homologs with the



use of the T-Coffee: Advanced program (Poirot et al. 2003).Amino acid positions are relative to A. tequilana HSP18.4B(Agave18.4B-CI) sequence. Blue line indicates consensusN-terminal region. Black 5 bar shows the conserveda-crystallin domain of sHSPs. Yellow lines show consensusI and II regions. Red line shows b-10 strand important foroligomerization (van Montfort et al. 2001).Figure S2. Alignment of the complete sequence A. tequi-lana class II sHSP (Agave17.7-CII) to its closest planthomologs with the use of the T Coffee: Advanced program(Poirot et al. 2003). Amino acid positions are relative to A.tequilana HSP17.7 sequence (Agave17.7-CII).The blue lineshows the consensus N-terminal domain. Black bar showsthe conserved a-crystallin domain of sHSPs. The yellowlines show consensus I and II regions within the a-crystallindomain. The red line shows the b-10 strand important foroligomerization (van Montfort et al. 2001).Figure S3. Alignment of the C-terminal region of A. tequi-lana HSP90A1 protein to its closest plant homologs withthe use of the T-Coffee: Advanced program (Poirot et al.2003). Amino acid positions are relative to Arabidopsisthaliana HSP90 (AtHSP90A2) sequence. Black bar showsthe Block J, one out of ten conserved regions in proteins ofthe HSP90 family. Pink bar shows the C-terminal pentapep-tide, critical for binding to tetratrico peptide repeat (TPR)of immunophilin and p60, members of the HSP90 chaper-one complex.Figure S4. High temperature tolerance of whole A. tequi-lana plants. Three-year-old plants were incubated for 2 h at28 °C [(a) and (b)], 44 °C [(c) and (d)], 48 °C [(e) and (f)] or54 °C [(g), (h)]. Photographs of the same plant were takenat time 0 [(a), (c), (e) and (g)] or 90 [(b), (d), (f) and (h)]

days after temperature treatments. A close-up of the spike(S) is shown in (i).Figure S5. Kinetics of leaf temperatures in the spike(A) and in an outer leaf (B) of a 3-year-old A. tequilanaplant after heat shock for 2 h at 54 °C in an illuminatedincubator during the middle part of the day. Temperaturesof the leaves and of air in the incubator were measuredwith a thermocouple, as described in Materials andMethods.Table S1. Small HSP sequences used for the phylogeneticanalysis. Full length sequences encoding type CI or CIIsHSP from Arabidopsis thaliana, Triticum aestivum orOriza sativa were retrieved from NCBI (http://www.ncbi.nlm.nih.gov/). The corresponding sequences from Populustrichocarpa (release v1.1) were obtained from the DOEJoint Genome Institute website (http://genome.jgipsf.org/Poptr1_1/Poptr1_1.home.html).Table S2. HSP90 sequences used for the phylogeneticanalysis. Accessions containing C-terminal sequencesencoding HSP90 from Arabidopsis thaliana or Oriza sativathat shared a significant similarity to the C-terminal 174 aasegment of AgaveHSP83 were retrieved from NCBI (http://www.ncbi.nlm.nih.gov/).The corresponding sequences fromPopulus trichocarpa (release v1.1) were obtained fromDOE Joint Genome Institute website (http://genome.jgipsf.org/Poptr1_1/Poptr1_1.home.html).

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