Isolation and characterization of class A4 heat shock transcription

factor from alfalfa

Jeremy N. Friedberg a,1, Stephen R. Bowley a, Bryan D. McKersie a,2, William B. Gurley b,Eva Czarnecka-Verner b,*

a Department of Plant Agriculture, Crop Science Building, University of Guelph, Guelph, Ont. N1G 2W1, Canadab Microbiology and Cell Science Department, Program of Plant Molecular and Cellular Biology, University of Florida,

Bldg. 981, Gainesville, FL 32611-0700, USA

Received 4 October 2005; received in revised form 31 March 2006; accepted 10 April 2006

Available online 17 May 2006

Abstract

Plant heat shock transcription factors (HSFs) regulate transcription of heat shock (HS) genes. In Arabidopsis thaliana, 21 HSFs have been

classified into groups A–C. Members of class A act as typical transcriptional activators, whereas B HSFs function as coactivators or repressors

depending on promoter context. The function of class C HSFs is still unclear. Here, we present the isolation and characterization of the first HSF

from alfalfa (Medicago sativa L.) and designate it MsHSFA4 based on amino acid sequence analysis. The MsHSFA4 gene was determined to be

single copy and was detected at two separate genetic loci in the tetraploid Medicago sativa. Overexpression of MsHSFA4 in tobacco mesophyll

protoplasts resulted in weak transcriptional activity, similar to that exhibited by Arabidopsis AtHSFA4a. The MsHSFA4 proximal promoter contains

three putative HSE elements, and the gene itself is activated both by heat and cold stress.

# 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Heat shock factor; C-terminal region; Promoter; Chromosomal localization

www.elsevier.com/locate/plantsci

Plant Science 171 (2006) 332–344

1. Introduction

Plants respond to the environmental stress by increased

production of molecular chaperones that protect the cell from

irreparable damage. These proteins produced in response to

elevated temperatures are known as heat shock proteins (HSPs).

The induction of HSPs is universal, very rapid, and controlled at

the transcriptional level by HSFs that bind to heat shock

response DNA elements (HSEs) present in heat shock gene

promoters (reviewed by [1]). The HSE is composed of a

palindromic penta-nucleotide repeat of the core sequence 50-nGAAn-30, or its complement 50-nTTCn-30 [2,3]. The optimal

core sequence for plants was shown to be 50-aGAAg-30, or 50-cTTCt-30 [4]. Plants differ from most other organisms by the

* Corresponding author. Tel.: +1 352 392 2400; fax: +1 352 392 5922.

E-mail address: evaczar@ufl.edu (E. Czarnecka-Verner).1 Present address: BASF Plant Science, 26 Davis Dr., Research Triangle Park,

NC 27709-3528, USA.2 Present address: Vive Technologies Inc., 32 St. Clair Gardens, Toronto,

Ont., Canada M6E 3V4.

0168-9452/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.plantsci.2006.04.007

presence of an extensive network of HSFs. Originally, these

were classified into two [5–8], and later three HSF classes [9]

based on the structural and functional characteristics of HSF

proteins. While all class A HSFs activate transcription [10–13],

they exhibit a large range in the activity, possibly reflecting a

diversity of function [14,15]. Some HSFs play a key role in the

early heat shock response and thermotolerance [16,17], while

others function in a secondary role to boost the activity of other

class A HSFs [18]. Transcriptional activation depends on the

AHA domains present in the C-terminal regions (CTR) of the

class A HSFs [8,10,15,19]. AHA motifs were not found in class

B HSFs. As a result, class B HSFs do not activate transcription

on their own, but rather seem to be specialized as active

repressors of transcription in certain circumstances [20],

perhaps as attenuators of the heat shock (HS) response

[13,21]. Studies in Arabidopsis suggest that the A and B class

HSFs have differential roles in the early versus late phases of

the HS response [16].

Impairment of class B HSFs coincided with the loss of ability

to transiently shutdown transcription of HSF target genes [16].

Interestingly, class B HSFs can also act as coactivators through

J.N. Friedberg et al. / Plant Science 171 (2006) 332–344 333

synergistic mechanisms that may involve interactions of their C-

terminal domains with general transcription factor TFIIB [20]

and coactivators such as HAC1 [22]. The function of class C

HSFs still remains largely unexplained. There are only two

known members of this class, and surprisingly, although

Arabidopsis AtHSFC1 lacks obvious AHA motifs, it seems to

weakly activate transcription in plant and yeast systems [15].

HSF proteins have a highly conserved structural motifs [7,9]

interspaced with regions that vary significantly in size and

sequence. The DNA binding domain (DBD) has the greatest

conservation and has become the defining feature of the HSFs.

It is located in the N-terminus and contains a helix-turn-helix

structure [23] related to the HNF3/forkhead winged helix

family of DNA binding motifs [24]. The DBD is connected to

the oligomerization domain (OD) by a flexible linker domain

[25]. In plant HSFs, the length of the flexible linker seems to be

longer for B HSFs than for the class A HSFs [9]. HSF binds to

DNA most commonly as a trimer [26]. The trimerization

domain forms a triple-stranded, a-helical coiled coil analogous

to the one present in influenza hemagglutinin [27], with two

subdomains of hydrophobic heptapeptide repeats, HR-A and

HR-B, separated by a break in the helicity of the OD [28].

Structural features of the ODs considered together with the

parsimony analysis of DBDs [6] support differentiation of plant

HSFs into various classes [5,9]. In human HSFs, the third

hydrophobic heptapeptide repeat HR-C masks the OD and a

nuclear localization sequence (NLS) resulting in an inactive,

monomeric form of HSF localized in the cytoplasm [29,30].

Plant class B HSFs do not seem to have a functional HR-C [5,9]

and are present in the nucleus under HS and non-HS conditions

[18]. The intracellular distribution of plant A HSFs depends on

the overall balance of nuclear import and export processes that

are directed by the strength and accessibility of the NLS and

nuclear export sequence (NES) [15,31]. Transcriptional

activation of class A HSFs is attributed to AHA motifs located

in the C-terminal region (CTR) of class A HSFs [10,13].

Detailed analysis of these motifs revealed the importance of

aromatic, leucine and proline residues, as well as the synergism

between adjacent AHA motifs [19,20]. These AHA motifs have

been shown to recruit members of the transcriptional

preinitiation complex (PIC) such as TBP and TFIIB [20].

The combination of AHA and NES constitutes a signature

domain that has facilitated the recognition of over 60 new class

A HSFs among the expressed sequence tag (EST) database of

plants [15].

The HS response has been well documented in a wide variety

of organisms subjected to elevated temperatures. Remarkably,

several groups have reported the induction of HSP gene

expression during low temperature stress [32–35], including

freezing [36]. There are indications that at least members of the

HSP90 [33], HSP70 [32,35] and some low molecular weight

(17–21 kDa) HSP families [34,36–38] are involved in

protection against chilling injury and acquisition of freezing

tolerance. HS and HSP accumulation can provide cross-

adaptation to cold stress. For example, the increase of HSP17.6

and HSP21 expression has been correlated with the protection

of tomato fruits from chilling injury [37,38]. Heat treatments

prior to chilling reduced the chilling-induced inhibition of rice

seedling radicle growth [39] and rates of ion leakage and

chloroplast aggregation in petioles of African violet [40].

Evidence for HSPs providing protection from both heat and

chilling injury is provided by experiments in E. coli when

CsHSP17.5 from chestnut conveyed increased bacterial cell

viability under heat stress and increased survivability at low

temperatures [41]. The same cytosolic CsHSP17.5 was shown

to play an important role in the acquisition of freezing tolerance

by protecting the cold-labile lactate dehydrogenase enzyme

from freezing-induced inactivation [42]. The mechanisms of

cryo-protection provided by HSPs still need to be elaborated,

but molecular chaperone activity as a protection against freeze-

induced protein denaturation has been suggested [43]. The

accumulation of HSPs during episodes of chilling clearly

suggests that HSPs may enhance a plants’ ability to cope with

low temperature stress. However, it is still not known if HSFs

are responsible for the induction of HSPs under the low

temperature stress. Although, HSFs are responsible for heat-

induced expression of HSPs, their role in chilling-induced

promoter activation has not been documented.

Here, we present the isolation and characterization of the

first HSF cloned from Medicago sativa L. (alfalfa). We confirm

its ability to function as a transcriptional activator and find the

gene encoding MsHSFA4 to be induced by heat and cold

stresses. The novel finding of cold activated transcription of a

HSF may be physiologically significant since alfalfa is

susceptible to low temperature stress and winter kill.

2. Materials and methods

2.1. cDNA library screening

The cDNA library was kindly provided by Drs. Suzanne

Cunningham and Jeffrey Volenec from Purdue University.

PolyA+ RNA was extracted from field-acclimated alfalfa (cv.

Norseman) bud tissue harvested in September, November and

December. For library construction, this RNA was pooled and

used as template for cDNA synthesis and cloning into the

lambda ZAP vector from the Lambda ZapTM library kit

(Stratagene, La Jolla, CA) according to the protocol supplied by

the manufacturer. Screening of the library was performed

according to methods detailed in Current Methods in Molecular

Biology [44] and the protocols supplied by Stratagene. The

open reading frame (ORF) of the Arabidopsis thaliana

AtHSFA4a cDNA (GenBank accession no. AAC31792) was

used as a heterologous probe for library screening. The

AtHSFA4a ORF was amplified from plasmid DNA [13] by

polymerase chain reaction (PCR) and subsequently radio-

actively labeled with 32P using the PrimIt II Labeling KitTM

(Stratagene). The AtHSFA4a probe had a specific activity of

2.3 � 109 cpm/mg. One million phage plaques were tested in

the first library screen. Putative positive candidates were then

subjected to three rounds of plaque purification resulting in a

pure plaque containing MsHSF. The MsHSF cDNA recovered

from the lambda ZAP vector was sequenced from both

directions using the T3 and T7 universal primers by Lab

J.N. Friedberg et al. / Plant Science 171 (2006) 332–344334

Service, University of Guelph, and was designated MsHSFA4

(GenBank accession no. AF235958). The transcription start site

for MsHSFA4 was based on sequences derived from cDNA

clones and confirmed using a series of RT-PCR reactions with

mRNA template from control and heat stressed tissues and

primer pairs strategically located within the 50 region. The 50

end of MsHSFA4 mRNA was then independently determined

by primer extension assays (data not shown).

2.2. Genomic DNA libraries construction and screening

Alfalfa (cv. N442) genomic DNA was isolated from 1 g of

pooled mature and developing leaf tissue using the Trebuchet

genomic DNA extraction protocol (Bowley lab, University of

Guelph; protocol available upon request), dissolved in 100 ml

of distilled water and incubated overnight at 40 8C. Genomic

libraries were built using the BD GenomeWalkerTM Universal

Kit (ClonTech, Palo Alto, CA) according to the manufacturer’s

instructions. Genomic DNA was digested in four separate

reactions by restriction enzymes EcoR V, Dra I, Pvu II and Ssp I,

and adapter fragments containing unique primer sites were

ligated onto the ends of the digested genomic DNA from each

reaction. This process generated four genomic libraries that

were used as templates for two rounds of polymerase chain

reactions (PCR). This first amplification utilized a gene-specific

downstream primer and upstream adapter primer, followed by a

second amplification with nested primers. PCR fragments were

cloned using the TOPO TA cloning kit from Invitrogen. The

genomic DNA inserts were sequenced and 50 flanking

sequences were analyzed for promoter consensus elements

using the following programs:

PLACE (http://www.dna.affrc.go.jp/PLACE/fasta.html),

PROSCAN (http://bimas.dcrt.nih.gov/molbio/proscan/),

Promoter 2.0 (http://www.cbs.dtu.dk/services/Promoter/),

TESS (http://www.cbil.upenn.edu/tess/) and TRANSFAC

(http://www.gene-regulation.com/pub/databases.html).

2.3. Temperature stress treatments and RNA extractions

Approximately 500 mg of shoot tips (first three expanding

leaves) were harvested from alfalfa cultivar N442 and

incubated in 100 ml of temperature-acclimated SHK media

(derived from SH media [45] modified with 10 mM K2SO4),

with continuous rotation at 175 rpm in temperature-controlled

shakers. The treatments were continued for 0.5 and 4 h at the

following temperatures: heat shock at 41 8C, cold stress at 4 8Cand control at 28 8C. The samples were then immediately

frozen in liquid N2 and stored at�80 8C. An additional control

consisted of shoot meristems harvested from plants and

directly frozen in liquid N2. Frozen tissue samples were ground

under liquid N2 and total RNA was extracted using the

RNaqueous RNA Extraction Kit (Ambion, Austin, TX) in

conjunction with Plant RNA Isolation Aid (Ambion). Aliquots

of 20 mg of RNA were stored at �80 8C. Total RNA was

quantified using RiboGreen fluorescent RNA stain (Molecular

Probes, Eugene, OR) and detected with a Shimadzu RF-5000

spectrofluorophotometer with excitation at 480 nm and emi-

ssion at 580 nm.

2.4. Preparation of the digoxigenin-labeled DNA probe

and Northern blot hybridizations

The entire ORF of the alfalfa MsHSFA4 was used to generate

a digoxigenin (DIG)-labeled probe for Northern blot hybridi-

zations. The MsHSFA4 clone was digested using EcoR I and

Xho I restriction enzymes, and the 1.2 kb ORF insert was

isolated by purification on agarose gels. The MsHSFA4 insert

was used as a template in the DIG PCR reaction that was carried

out according to the procedure detailed in the DIG PCR Probe

Synthesis Kit (Roche Biochemicals, Laval, Quebec) with

primers specific to the full length ORF.

For Northern blots, 20 mg aliquots of total RNA were

electrophoresed on a 1.2% (w/v) agarose gel prepared with 1�MOPS buffer and 5.4% formaldehyde. The RNA was capillary-

transferred overnight in 10� SSC to a positively charged nylon

membrane (Roche Biochemicals) and then fixed to the

membrane via UV crosslinking. The membrane was prehy-

bridized for 2 h at 37 8C in EasyhybeTM (Roche Biochemicals)

prehybridization solution and then incubated overnight in a

rotating hybridization oven at 50 8C with DIG-labeled

MsHSFA4 probe. Subsequently, membranes were washed

twice at room temperature for 15 min each in 2� stringency

wash solution (2� SSC, 1% SDS (w/v)), followed by washes at

65 8C in 0.2� stringency wash solution (0.2� SSC, 1% SDS

(w/v)) and further processed according to protocols provided by

Roche Biochemicals. The membrane was placed on an acetate

sheet, overlaid with the chemiluminescent substrate solution

and heat-sealed. Hybridized blots were displayed by exposure

of the membrane to Kodak XGB X-ray film.

2.5. Protoplast transformations, GUS assays and

constructs

Protoplast transformation and evaluation of b-glucuroni-

dase (GUS) expression was performed as described pre-

viously [13]. Protoplast assays were repeated two to three

times and were conducted with triplicate replications. GUS

reporter activities were normalized against luciferase activity

as an internal standard. Two types of GUS reporter constructs

were used: 9� HSE/GUS for the full length HSF constructs

with the native HSF DBD, and 10� Gal4 DBS/GUS for

chimeric constructs containing CTR of HSFs fused to the

yeast Gal4 DBD. In either case, the multiple DNA recognition

sites were fused upstream to a minimal 35S CaMV promoter.

The effector constructs for testing with the 9� HSE/GUS

reporter consisted of (1) the full length MsHSFA4 ORF from

the first methionine to the last coding amino acid residues

cloned behind the yeast Gal4 leader sequence, and (2) the

complete cDNA of MsHSFA4, including its long 50

untranslated region (50UTR), cloned in the same Gal4 leader

vector. The full length Arabidopsis AtHSFA1a and AtHSFA4a

were used as positive examples of transcriptionally active

HSF genes, and vectors containing only a Gal4 leader

J.N. Friedberg et al. / Plant Science 171 (2006) 332–344 335

sequence, or additionally encoding T7 tag peptide, served as

negative controls.

The second group of effector constructs tested with the 10�Gal4 DBS/GUS reporter had yeast Gal4 transcription factor

DBD (aa 1-147) present, either alone or fused to the alfalfa

MsHSFA4 CTR (aa 189-402, plus its native stop codon and

most of the 30 untranslated region). The CTR (aa 189-401) of

Arabidopsis AtHSFA4a was used as a positive control.

2.6. Fluorescent in situ hybridization

This protocol was performed following instructions of the

Cereal Genetics Laboratory In Situ Hybridization Manual

(USDA, 1999) using germinating alfalfa seed (cv. ABI 700). A

biotin-labeled probe was prepared from a plasmid containing

Fig. 1. A comparison of class A4 HSF genes at the nucleotide level. A diagram of

included, and black bars represent either 50 or 30 UTRs. Black horizontal lines indicat

in the 50UTR of AtHSFA4c. +1, transcriptional start site; UTR, untranslated region; AT

the size (bp) of the corresponding feature of the cDNA or genomic clone. The black tr

DBD. ‘‘TY/GF’’ represent aa residues that flank the intron insertion site in the six

MsHSFA4 that flank a unique intron insertion site in the gene located downstream fr

shows variability in number of nucleotides, the exact aa positioning of the intron seem

nucleotide identities for seven class A4 HSFs (B). Percent values were obtained u

the MsHSFA4 cDNA by nick translation with the BioNick

Labeling System (Invitrogen) according to the manufacturer’s

instructions.

Briefly, alfalfa seeds were germinated and 2–3 mm root tips

were excised, treated with cellulase and pectolyase enzymes

(Sigma), placed on glass slides and gently broken up with

forceps, and subsequently fixed. After denaturation in formamide

and a series of ethanol washes, tissue on cover slides was

subjected to hybridizations with the MsHSFA4 cDNA biotiny-

lated probe solution and subsequently incubated in Fluorescent

Avidin DCS solution (Vector Laboratories) followed by washes

with SSC. This process was performed twice and then the slides

were incubated overnight at 4 8C in the dark and viewed the

following day. Images were captured at 1000� magnification

under oil immersion using a digital camera connected to a Leitz

seven class A4 HSFs (A). Grey bars indicate coding regions with stop codons

e 30 UTRs of undetermined length for HSF genomic clones and the 406 bp intron

G, translation start site; ORF, open reading frame. Numbers on the bars indicate

iangle and a short vertical line denote the position of an intron located within the

displayed HSFs and in most other known HSFs. ‘‘VD/PE’’ are aa residues in

om the customary one. Although exon 1 (grey box from ATG to black triangle)

s to be the same for all plant HSFs, with the exception of MsHSFA4. Analysis of

sing the Vector NTI program (Invitrogen).

J.N. Friedberg et al. / Plant Science 171 (2006) 332–344336

Fig. 2. Amino acid sequence comparison of plant class A4 HSFs. (A) The diagram of a typical class A4 HSF protein illustrates conserved regions. DBD, DNA

binding domain; OD, oligomerization domain; NLS, nuclear localization signal; AHA1 and AHA2, transcriptional activation motifs; NES, nuclear export

sequence; CTR, C-terminal regulatory region spans amino acid sequences from OD to the end of the HSF protein. (B) Amino acid sequence alignment of seven

classes of A4 plant HSFs: (1) Medicago sativa MsHSFA4 (GenBank accession no. AAF37579); (2) Phaseolus acutifolius PaHSFA4 (GenBank accession no.

AAL12248), (3) Nicotiana tabacum NtHSFA4, formerly known as NtHSF2 (GenBank accession no. BAA83711); (4) Arabidopsis thaliana AtHSFA4a, formerly

J.N. Friedberg et al. / Plant Science 171 (2006) 332–344 337

Fig. 3. HSF relatedness tree from CLUSTALW analysis of the HSF DNA binding domains. Only class A HSFs were used in the analysis. Although previous HSF

surveys [9] distinguished nine classes of A HSFs, this analysis puts AtHSFA5/4b (GenBank accession no. CAB10177) in class A4 alongside of ZmHSFA4 (GenBank

accession no. CAA58117), OsHSFA9 (GenBank accession no. AAQ23063) and OsHSFA10 (GenBank accession no. AAQ23064). Here, HaHSFA9 (GenBank

accession no. AAM43804) is placed in the HSFA2 subfamily.

phase contrast fluorescent microscope. Images of chromosome

spreads were captured using Northern Eclipse Image Analysis

Software (Empix Imaging, Mississauga, Ont., Canada). Indivi-

dual chromosomes [46,47] were karyotyped using MicroMea-

sure software (version 3.3, Aaron Reeves & Jim Tear, Colorado

State University).

3. Results

3.1. Alfalfa HSFA4 homologue

Sequencing of the positive cDNA clone isolated from the

alfalfa bud cDNA library revealed that it was approximately 2 kb

in length. BLASTx nucleotide analysis identified this clone to

exhibit a high degree of similarity to known genes encoding plant

HSFs, specifically, Medicago truncatula MtHSFA4 (GenBank

accession no. AC152407), Phaseolus acutifolius PaHSFA4

(GenBank accession no. AY052627), Lotus corniculatus var.

japonicus LjHSFA4 (GenBank accession no. AP004978),

Nicotiana tabacum NtHSFA4, formerly known as NtHSF2

(GenBank accession no. AB014484), Arabidopsis thaliana

AtHSFA4a, known previously as AtHSF21 (GenBank accession

nos. U68561 and AL021711) and Arabidopsis thaliana

AtHSFA4c (GenBank accession no. AB012245) (Fig. 1).

Comparison of the MsHSFA4 nucleotide sequence to PaHSFA4

known as AtHSF21 ([13], GenBank accession no. AAC31792); (5) Arabidopsis th

LjHSFA4 (GenBank accession no. AP004978) and Medicago truncatula MtHS

homology and correspond to textures in the diagram shown in A. Amino acid residu

represent hydrophobic heptapeptide repeats A and B shown in register. Two vertical

present in HSF C-terminal regions. The black triangle on the left indicates the most

its position within MsHSFA4 (see text). (C) Amino acid sequence identities of se

program.

and LjHSFA4 (all three Fabaceae) revealed a high degree of

similarity in both, the overall nucleotide sequence of the ORFs

(83% and 75% identical, respectively) and regions encoding the

DBD (respectively, 88 and 85% identical) (Fig. 1B). Tobacco

NtHSFA4 (Solanaceae) showed 80% similarity in the region

encoding the DBD and 69% over the entire ORF. In contrast, not

so closely related Arabidopsis AtHSFA4a and AtHSFA4c

(Brassicaceae) displayed only 62 and 52% similarity to

MsHSFA4 in the overall ORF sequence, with significantly

higher similarities (78 and 73%) in regions encoding DBDs. Not

surprisingly, MsHSFA4 and MtHSFA4 share the greatest

identity, especially in the nucleotide sequences of the ORF

region (89%) and the DBD region (97%). This high degree of

conservation indicates relatively recent divergence between both

Medicago species and an overall evolutionary need for a high

degree of amino acid conservation of the DBD.

A schematic representation of HSP genes related to

MsHSFA4 is shown in Fig. 1. Most have 50UTRs ranging

from approximately 250 bp to over 700 bp. The 50UTR for

MtHSFA4 was predicted based on the high degree of sequence

similarity with MsHSFA4 (not shown), and the 50UTR for

LjHSFA4 was based on the location of a putative promoter

sequence (TATAA and HS elements). Interestingly, the related

class A4 Spl7/OsHSF10 gene of rice also has an extensive

50UTR (approximately 600 bp), which contains an intron of

aliana AtHSFA4c (GenBank accession no. BAB09213); (6) Lotus japonicus

FA4 (GenBank accession no. ABE77517). Boxes indicate regions of high

es, written in bold type face are aligned in positions ‘‘d’’ and ‘‘a’’ of the OD and

boxes encompass highly conserved amino acid sequences of unknown function

common position of an intron within the DBD; the right triangle corresponds to

ven classes of A4 HSFs. Percent values were obtained using the Vector NTI

J.N. Friedberg et al. / Plant Science 171 (2006) 332–344338

243 bp [48]. A similar case is observed for the AtHSFA4c gene

that has a 50UTR of 724 bp with an intron of 406 bp.

The MsHSFA4 cDNA encodes a predicted protein of 402

amino acids with a molecular weight of 46.2 kDa (GenBank

accession no. AAF37579). The full-length protein sequence

shows high amino acid similarity to LjHSFA4, PaHSFA4,

NtHSFA4, AtHSFA4a and AtHSFA4c of 67, 67, 50, 46 and

43%, respectively (Fig. 2C). When only the DBD amino acid

sequence of HSFs was considered, the similarity increased to

77–86% among these six HSFs. Overall, the MsHSFA4 protein

showed the highest degree of similarity to the barrel medic

MtHSFA4 ORF (90%) and DBD (95%), with a very significant

conservation of the C-terminal transcriptional regulatory region

(87% identity) (Fig. 2C). In contrast, amino acid similarity to

Arabidopsis AtHSFA4 CTRs was only 27–29%. CLUSTALW

[49] neighbor joining analysis of the MsHSFA4 cDNA

(GenBank accession no. AF235958) and available plant full-

length HSF ORF sequences (data not shown) and DBDs (Fig. 3)

confirmed the placement of alfalfa MsHSF in class A4 HSFs,

alongside of barrel medic, tepary bean, tobacco, tale cress, rice

and maize A4 HSFs.

Conserved domains of the MsHSFA4 protein are dia-

grammed in Fig. 2 and include: (1) the DBD located between aa

residues 11 and 103, (2) a typical class A HSF OD (aa 127-184)

consisting of HR-A and HR-B, (3) downstream-localized NLS

(aa 202-205), (4) two transcriptional activation AHA motifs (aa

257-262 and 340-345) and (5) NES located around QMGHV

sequence (aa 392-396). CTR is immediately adjacent to the OD

and encompasses NLS, AHA motifs and NES.

3.2. MsHSFA4 genomic clone and promoter sequence

Isolation and sequencing of the MsHSFA4 genomic clone

(GenBank accession no. AF494082) revealed this gene to be

2358 bp in length and have two exons of 231 and 978 bp that

flank a single intron of 104 bp (Fig. 4A). The intron is located

within the DBD; however, it is shifted 6 aa residues downstream

(D77/P78; Figs. 4A and 1A) from its position in other plant

HSFs (Y71/G72; Fig. 1A) [9]. The intron/exon boundries

contain the canonical 50/30 sequence (GT/AG) that commonly

denotes splice junctions.

The genomic clone contains 278 bp of DNA upstream of the

50 UTR. Within this region, a number of promoter elements

were identified; specifically, a putative TATA box, two TATA-

proximal heat shock elements (sites I and II), a third upstream

HSE III and several HSE core tri-nucleotide elements present in

both orientations (Fig. 4B). These isolated core sequences lack

flanking sequence identity required to meet the definition of

active head/tail modules described by Nover et al. [9], albeit

some show partial conservation with the optimal plant HSE

core consensus [4].

From the current sequence database, we selected and

analyzed genomic sequence data available for class A4 HSF

from Medicago truncatula. The promoter region of MtHSFA4

exhibits 89% homology with that of MsHSFA4. CLUSTALW

alignment of the MsHSFA4 and MtHSFA4 promoter regions

(Fig. 4C) indicated that, although most of the HSE cores

were conserved in sequence and location within promoters,

some point mutations and deletion/insertions of the promoter

sequences occurred. In general, the MtHSFA4 promoter is

less conserved than the MsHSFA4 promoter with respect to

the identity of HSE consensus sequence. The MtHSFA4

promoter, compared to MsHSFA4, displayed seven single or

double nucleotide differences within the HSE cores, as well

as the lack of five nucleotides within the putative HSE site II.

Although the differences occurring in the MtHSFA4 promoter

weakened the overall homology of cores to the consensus,

the missing nucleotides within HSE II seemed to preserve

the register of HSE arrays and brought six of them closer

together, still making it possible for two hypothetical

HSF trimers (designated by head-to-toe arrows) to bind

(Fig. 4D).

3.3. MsHSFA4 gene copy number and chromosomal

localization

Southern analysis using a DNA probe to the MsHSFA4 ORF

revealed a single strong band and a possible faint second band

(data not shown) suggesting that MsHSFA4 is a single copy

gene. Chromosome spreads exhibited the expected 32 visible

chromosomes (8 homologues), confirming the source plants as

tetraploid alfalfa. Chromosomal localization of MsHSFA4

through fluorescent in situ hybridization revealed that this gene

exists in single copy number, with four copies in a tetraploid

genome located on chromosome 6, or 7. Among the four

homologous chromosomes, MsHSFA4 resides at one of two

genetic loci (Fig. 5). The first locus is found near the telomere,

and the second is found near the centromere.

3.4. Transcriptional activity of MsHSFA4 in tobacco

protoplasts

The goal of this series of experiments was two-fold. The first

was to determine if the alfalfa HSFA4 gene possessed

transcriptional activity, and the second was to determine the

ability of the isolated CTR of MsHSFA4 to act as a typical

transcriptional activator outside of the HS-dependent mechan-

ism. These experiments were conducted using transient assays

in tobacco mesophyll protoplasts. The reporter consisted of

GUS gene under control of an artificial HS promoter consisting

of the minimal CaMV 35S promoter (TATAA) with nine typical

HSE motifs located upstream. The second objective was met by

separating the requirements for trimerization and HSE binding

from transcriptional activation and, thereby, alleviating the

need for HS-mediated induction. The CTR of MsHSFA4 was

fused to the yeast Gal4 DBD by cloning and tested for the

ability to activate a GUS reporter driven by a minimal CaMV

35S promoter with 10 Gal4 DNA binding sites (Gal4 DBS)

located upstream.

Two types of vectors were used as negative controls: one

with transcript corresponding to the Gal4 untranslated 50-leader

and the other additionally encoding the T7 epitope. Both of

these controls showed no transcriptional activity, indicating that

there was little to no background GUS activity in tobacco

J.N. Friedberg et al. / Plant Science 171 (2006) 332–344 339

Fig. 4. Alfalfa MsHSFA4 gene promoter analysis. (A) Diagram of the MsHSFA4 genomic sequence shows the organization of the gene. VD/PE are the last two amino

acid residues in exon 1 and the first two amino acid residues in exon 2 of MsHSFA4. Numbers indicate the size (bp) of specific regions. (B) The diagram illustrates the

location of the putative TATA box (rectangular box), transcriptional start site (+1) and distribution of HSE cores (semi-square boxes) within the MsHSFA4 promoter.

Triangles mark conservation to the HSE pentanucleotide core consensus sequence [4]. Brackets indicate three potentially functional HSE elements. HSE; heat shock

element. (C) Alignment of promoter sequences for two closely related Medicago HSFA4 genes using CLUSTALW. Grey box indicates a putative TATA box. Clear

boxes encompass conserved HSE core sequences, and arrows illustrate their head-to-toe orientations.

protoplasts (Fig. 6, lanes 2 and 1, respectively). In contrast,

Arabidopsis class A1 HSF, AtHSFA1a ORF, displayed very

high levels of GUS activity (Fig. 6, lane 3) and served as the

positive control for transcriptional activation. The AtHSFA4a

ORF construct showed approximately 20% of transcriptional

activity as compared to the AtHSFA1a ORF (Fig. 5, lane 4), a

finding consistent with previous experiments designating

AtHSFA4a as a weak transcriptional activator [13,14,20].

The alfalfa MsHSFA4 ORF displayed comparable levels of

activity to its homologue Arabidopsis AtHSFA4a (Fig. 6,

lane5). However, the MsHSFA4 full length cDNA, that

included a long 50 UTR sequence, was unable to activate the

9� HSE/GUS reporter construct, indicating that the 50 UTR

may potentially act as a negative regulator (Fig. 6, lane 6).

J.N. Friedberg et al. / Plant Science 171 (2006) 332–344340

Fig. 5. A digital image of fluorescent in situ hybridization of alfalfa (cv. ABI 700) chromosomes isolated from root tips using a probe for the MsHSFA4 cDNA. Top panel

shows a spread of chromosomes (red) showing four positive hybridization loci (white arrows—green spots). The bottom panel displays a series of chromosomes excised

from a number of chromosome spreads. The telomeric loci (T) can be seen in the top row, and the centromeric loci (C) can be seen in the bottom row (white arrows).

In transient expression assays that used the Gal4 DBS/GUS

reporter together with the T7 tag negative control showed, as

expected, no transcriptional activation (Fig. 6, lane 7); however,

the second negative control, the Gal4 DBD, exhibited a low

level of activity (base-line transcription) (Fig. 6, lane 8)

attributable to a weakly activating region (B region) located

within the first 25 aa residues of the DBD and shown to slightly

enhance transcriptional activation [50]. The Gal4DBD-AtHS-

FA4a CTR chimeric construct activated the GUS reporter to

levels approximately two to three-fold higher as compared to

the Gal4 DBD control (Fig. 6, lane 9). In addition, the

Gal4DBD fusion construct of the MsHSFA4 CTR induced the

activity of the 10� Gal4 DBS/GUS reporter to levels

approximately two-fold higher than the AtHSFA4a CTR

(Fig. 6, 10), indicating MsHSFA4 to be a bona fide

transcriptional activator.

3.5. Stress response

The induction of the MsHSFA4 transcript under temperature

stress conditions was analyzed by Northern blot hybridizations.

Equal amounts of total RNA isolated from alfalfa heat shocked

(41 8C), cold shocked (4 8C) and control (28 8C) shoot meristems

were analyzed on Northern blots (Fig. 7). The alfalfa MsHSFA4

probe displayed an approximately 2.0 kb transcript present after

0.5 h exposure to heat shock conditions (Fig. 7, lane 3). These

transcripts persisted even after 4 h of elevated temperature

(Fig. 7, lane 4). MsHSFA4 transcripts were generally not

detectable under control conditions (Fig. 7, lanes 1 and 2).

Surprisingly, the MsHSFA4 gene was also induced after

0.5 h of cold shock at 4 8C and transcripts were even more

detectable after 4 h of cold stress applied to alfalfa shoot

meristems (Fig. 7, lanes 5 and 6, respectively). Moreover, the

J.N. Friedberg et al. / Plant Science 171 (2006) 332–344 341

Fig. 6. The full length MsHSFA4 and its isolated CTR activate transcription of reporter genes when expressed in tobacco mesophyll protoplasts. Transcriptional

activities of HSF effector constructs containing their native DBDs were tested using the 9� HSE/GUS reporter and are shown in lanes 1–6. Transcriptional activities

of isolated HSF CTRs in fusion constructs with yeast acidic activator Gal4 DBD for Arabidopsis and Medicago sativa class A4 HSFs are presented in lanes 7 through

10. Transcriptional activity of HSF CTRs was tested using the 10� Gal4 DBS/GUS reporter. Basal transcriptional activity of the tobacco protoplast system is

presented in lanes 1 and 2 for the 9� HSE/GUS reporter, and in lane 7 for 10� Gal4 DBS/GUS. Base-line activity using Gal4 DBD alone is reflected in lane 8. GUS

activity was normalized to the activity of the internal luciferase (Luc) standard that was driven by a full-length ubiquitin promoter. ORF (open reading frame), G4L

(Gal 4 leader sequence), 50 UTR (50 untranslated leader of MsHSFA4), T7 (T7 tag peptide). Graphed values represent means � S.D., where n = 3.

Fig. 7. Heat and cold stress induced the expression of MsHSFA4 gene. Northern

blots containing equal amounts of total RNA isolated from shoot meristems from

either control or stressed alfalfa were hybridized to the DIG-labeled probe of

MsHSFA4 ORF generated by PCR. A non-treated (nt) control grown at 28 8C and

harvested is shown in line 1. Shoot tips incubated in the incubation medium at

28 8C for 4 h are considered a treated control (liq) and are presented in lane 2. Heat

stressed samples are located in lanes 3 (41 8C for 30 min) and 4 (heat shock at

41 8C for 4 h), and cold stressed in lanes 5 (4 8C for 30 min), 6 (cold shock at 4 8Cfor 4 h) and 7 (bud tissue from field acclimated plants harvested in November).

The accuracy of RNA loading was confirmed by using RNA fluorescent dye and

through normalizing against an ethidium bromide stained ribosomal 18S and 28S

RNA (rRNA) bands. Molecular weight markers are denoted on the left side of the

top panel. This experiment was replicated three times.

same transcript was detected in the alfalfa shoot meristems

RNA sample isolated from field acclimated alfalfa (cv. N442)

crown buds collected in November 1999 (Fig. 7, lane 7), raising

the possibility of a role for this transcriptional regulator of the

HS response during periods of low temperature.

4. Discussion

We have isolated the first heat shock transcription factor

from Medicago sativa. MsHSFA4 is a single copy gene, and the

predicted protein exhibits structural and functional domains

highly conserved in other eukaryotes and specifically in plants.

At both the DNA and protein levels, the sequence conservation

for the DBD is higher than for the ORF as a whole. As expected,

at the DNA level, the sequence conservation directly correlates

with the overall degree of phylogenetic relatedness among the

species examined. For example, MsHSFA4 is more similar to

orthologs in Medicago truncatula, Phaseolus acutifolius and

Lotus japonicus, species belonging to the same family

Fabaceae; whereas Arabidopsis, which exhibited the least

similarity of the seven sequences surveyed, is found in the

Brassicaceae, and tobacco belongs to the Solanaceae. As its

designation indicates, MsHSFA4 is best categorized as a

member of the class A4 HSFs (Fig. 3). The phylogenetic tree

shown in Fig. 3 was based on CLUSTALW neighbor joining

analysis of the aa sequences and indicates that MsHSFA4 is

solidly grouped within the A4 clade. In addition, four other

more distantly related HSFs are located on the same branch

(OsHSF9, OsHSF10, ZmHSFA4 and AtHSFA5/A4b). Of these,

rice HSFs showed the greatest diversity within the CTRs;

blocks of sequences were inserted between AHA1 and AHA2

motifs for OsHSF10, and one such insertion seemed to

eliminate the OsHSF10 AHA1 domain. Interestingly, rice

spotted leaf gene Spl7 (identical to OsHSF10), whose DBD

mutant spl7 (W40/C40) is responsible for the lesion-mimic

plant phenotype, was identified previously to be class A4 HSF

and implicated in protection against necrosis caused by

environmental stress [48].

Alfalfa chromosomes are small and relatively similar in size,

which makes it extremely difficult to identify different

chromosomes by in situ hybridization procedures. As a result,

relative length and arm ratios were the only tools that could be

used to differentiate between chromosomes. Thus, through

J.N. Friedberg et al. / Plant Science 171 (2006) 332–344342

repetitive analysis we concluded that MsHSFA4 is most likely

located on a single chromosome, either 6 or 7. These studies

have revealed two loci for this single copy gene. In an auto-

tetraploid, one expects to find a single copy gene at one locus

among the four homologous chromosomes. However, alfalfa

exhibits one locus near the telomere and the second locus near

the centromere. Cytologically, this type of locus orientation is

usually indicative of a large chromosomal inversion event.

However, since two homologues have the telomere locus and

the other two homologues have the centromere locus, a 2:2

locus ratio, as opposed to a 1:3 locus ratio, suggest that the

inversion event occurred before alfalfa’s fusion from diploid to

tetraploid. This is significant, since it suggests that alfalfa arose

from an ‘‘allo’’ doubling of chromosomes as opposed to the

current belief of an ‘‘auto’’ doubling of chromosomes. Thus, the

alfalfa genome may exist as a chimera, exhibiting character-

istics of diploid and tetraploid genetics, reminiscent of its

progenitor parents.

The MsHSFA4 protein contains all critical domains

important for it to function as a transcriptional activator, i.e.

DNA binding domain, oligomerization domain, nuclear

localization signal, nuclear export signal and two transcrip-

tional activation domains AHA1 and AHA2. MsHSFA4

contains a conserved amino acid sequence PVHSHS flanking

the C-terminus of the DBD that is also a feature of other class

A4 HSFs. Strikingly, the intron in the DNA encoding the

MsHSF DBD, that is located between amino acid residues TY-

GF in all other plant HSFs [9], is found in a different location, 6

aa residues downstream between VD–PE.

In general, the C-terminal halves of HSFs are the least

conserved portions of the protein; however, this region usually

contains recognizable transcriptional activation domains (AHA

motifs), a NES, and other blocks of conserved aa sequences. The

AHA2 transcription activation motif of MsHSFA4 shows strong

conservation to the consensus AHA, FWxxF/LF/I/L [15],

whereas AHA1 does not. Additionally, in the very extreme C-

terminus of MsHSFA4, there are two regions located upstream

from the NES that exhibit a high degree of sequence conservation

with other members of the A4 class. Perhaps these sequences

(EVQSERK and WWN) constitute additional hydrophilic and

hydrophobic patches for interactions with MsHSFA4 targets.

The transient expression study of MsHSFA4 in tobacco

mesophyll protoplasts demonstrated its capacity to activate HS

promoters as predicted by the presence of a typical HSF DBD,

an oligomerization domain and AHA motifs (Fig. 6). Its ability

to activate transcription was comparable to the Arabidopsis

thaliana A4 homologue, which is a much weaker transcrip-

tional activator than Arabidopsis thaliana A1a HSF, as

observed previously [14]. Transient expression assays using

Gal4 DBD fusion constructs showed that the weak activities of

Arabidopsis and alalfa HSFA4 were largely due to the presence

of weak AHA activation motifs located in the C-terminal half of

these proteins.

The expression studies in tobacco protoplasts raised the

possibility that the long native 50 UTR of MsHSFA4 may play a

regulatory role in expression, since the construct containing

these sequences had no transcriptional activity. Since the

protein product produced from the open reading frame was

functional, we can dismiss the possibility of post-translational

regulation. The inhibitory effect of the 50UTR may occur at the

level of transcription or translation, but there is insufficient data

to suggest its true mode of action. It is, however, possible that

the effect is caused by the context of the tobacco system as

opposed to alfalfa. Additional studies using the same

constructs, but in alfalfa protoplasts, are warranted.

Northern analysis revealed that the MsHSFA4 gene was not

expressed under non-stress conditions and induced by heat

shock, a pattern that is also seen in AtHSFA4a expression

(unpublished). However, MsHSFA4 appears to also be actively

transcribed under cold stress conditions in growth chambers

and under low temperature conditions in the field (Fig. 7).

Although the specific role of MsHSFA4 in activating heat shock

gene expression under cold stress conditions is unknown at the

present time, our results, in combination with similar reports in

the literature of HSP gene expression under low temperature

stress [32,33,38,43], suggest that this alfalfa HSF is an integral

component of the low temperature stress response. Further-

more, at least one report indicates that HSP expression may

enhance chilling tolerance in plants [51].

There are several transcription factors involved in cold

responsive mechanisms in plants. The CBF/DREB1 proteins, a

family of Arabidopsis transcription factors, have been

determined to control a regulon of cold-induced (COR) genes

that enhance plant freezing tolerance (reviewed by [52]). The

CBF transcription factors provide example that innate systems

exist in plants that respond specifically to cold stress and

regulate a set of genes that provide cellular protection during

low temperature stress. Our report begins to expand on cold

response mechanisms by involving aspects of the heat shock

response. Many HSP genes are up regulated in response to high

temperature stress and some to low temperature stress.

However, there is very little known about how HSFs are

induced by cold stress and how they may function to protect or

acclimate plants for coping with low temperature stress.

Interestingly, it has been shown in Drosophila that HSF can be

expressed in several alternatively spliced isoforms (dHSFb, c

and d). The ratio of dHSFb is increased upon heat shock, while

that of dHSFd is enhanced upon exposure to cold [53].

Our report provides the first evidence of plant HSF A4 gene

induction by a cold stress. It is necessary to further investigate

the activity of MsHSFA4 in the context of other HSP and HSF

expression since multiple HSFs tend to work together to

activate and attenuate the HS response. It is possible that

MsHSFA4 may regulate a subset of HSP and other genes that

have evolved to enhance survivability to chilling injury.

Acknowledgements

This project was supported in part by the National Science

and Engineering Research Council of Canada (NSERC) and

Ontario Forage Council. Additionally, this project was

supported in part by the Florida Institute of Food and

Agricultural Sciences (IFAS) and USDA Grant 9500959 to

W.B.G. and E.C.-V.

J.N. Friedberg et al. / Plant Science 171 (2006) 332–344 343

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