5
Differential expression of two winter wheat alpha-tubulin genes during cold acclimation Nikolai K. Christov a, * , Ryozo Imai b , Yaroslav Blume c a AgroBioInstitute, Dragan Tsankov 8, Sofia 1164, Bulgaria b Crop Cold Tolerance Research Team, National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohira-ku, Sapporo 062-8555, Japan c Institute of Cell Biology and Genetic Engineering, Natl Academy of Sciences of Ukraine, acad. Zabolotnogo str., 148, Kiev, 03680, Ukraine Abstract Overwintering crops, such as winter wheat, display significant increase in freezing tolerance during a period of cold acclimation (CA). To gain better understanding of molecular mechanisms of CA, it is important to unravel functions and regulations of CA-associated genes. Differ- ential screening of a cDNA library constructed from cold-acclimated crown tissue of winter wheat identified an alpha-tubulin cDNA clone named wca18g11 that showed elevated expression upon cold acclimation. Nucleotide sequence analysis showed that the clone encoded a group 3 alpha-tubulin. Reverse transcription real-time PCR analysis of the expression of both wca18g11 clone and its closest paralogs of the wheat tubulin A-2 homeologous group during the course of cold acclimation revealed that both genes were differentially regulated with distinct ex- pression patterns. The involvement of the two alpha-tubulin genes in cold acclimation and signal transduction is discussed. Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Alpha-tubulin; WCS120; Cold acclimation; Wheat; Triticum aestivum; Abiotic stress 1. Introduction Cold acclimation (CA) is the process that allows hardy plants to develop essential tolerance for winter survival through multiple levels of biochemical and cell biological changes. Dynamic alteration in gene expression occurs during CA process. Reports have identified CA induced genes from many plant species including winter wheat (Pearce, 1999). Study of the regulatory and signaling elements that control this coordinated gene expression is essential for understanding CA and the genetic basis of cold tolerance. Microtubules are key elements of the cytoskeleton and their role in signaling and regulation is being recently extensively studied. Their intimate relationship with the plasma membrane, the major platform for signal perception and transduction (Gilroy and Trewavas, 2001; Wasteneys and Galway, 2003), suggests that microtubules are downstream targets of various signaling pathways. The identification of large number of RNA-binding and regulatory proteins in Arabidopsis that were shown to bind to microtubules (Chuong et al., 2004) and the very recent find- ing that a cold acclimation induced wheat receptor kinase interacts with a member of the alpha-tubulin family (Tardif et al., 2007), further supports this hypothesis. Signaling cas- cades have been shown to involve changes in cytoskeleton organization. In animal cells, microtubules have been proposed to transmit signals from the receptor to the nucleus since they span the distance from the plasma membrane to the nucleus (Gundersen and Cook, 1999). Inside the cells, the cytoskeleton can serve as a template where components that transduce extracellular signals interact. Therefore, the state of the cytoskeleton in plant cells can be critical in activat- ing and transporting signal molecules to a site where they interact (Gundersen and Cook, 1999; Camilleri et al., 2002). These structures, composed of alpha- and beta-tubulins, undergo a transient disorganization followed by major rear- rangements in root cortical cells during cold acclimation (Jian et al., 1989). Most freezing-resistant cultivars of wheat Abbreviations: CA, cold acclimation; NA, non-acclimation; DA, de-accli- mation; SNP, single nucleotide polymorphism; UTR, untranslated region. * Corresponding author. Tel.: þ359 2 963 5409; fax: þ359 2 963 5408. E-mail address: [email protected] (N.K. Christov). 1065-6995/$ - see front matter Ó 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.11.014 Cell Biology International 32 (2008) 574e578 www.elsevier.com/locate/cellbi

Differential expression of two winter wheat alpha-tubulin genes during cold acclimation

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Page 1: Differential expression of two winter wheat alpha-tubulin genes during cold acclimation

Cell Biology International 32 (2008) 574e578www.elsevier.com/locate/cellbi

Differential expression of two winter wheat alpha-tubulin genesduring cold acclimation

Nikolai K. Christov a,*, Ryozo Imai b, Yaroslav Blume c

a AgroBioInstitute, Dragan Tsankov 8, Sofia 1164, Bulgariab Crop Cold Tolerance Research Team, National Agricultural Research Center for Hokkaido Region,

Hitsujigaoka 1, Toyohira-ku, Sapporo 062-8555, Japanc Institute of Cell Biology and Genetic Engineering, Natl Academy of Sciences of Ukraine, acad. Zabolotnogo str., 148, Kiev, 03680, Ukraine

Abstract

Overwintering crops, such as winter wheat, display significant increase in freezing tolerance during a period of cold acclimation (CA). Togain better understanding of molecular mechanisms of CA, it is important to unravel functions and regulations of CA-associated genes. Differ-ential screening of a cDNA library constructed from cold-acclimated crown tissue of winter wheat identified an alpha-tubulin cDNA clonenamed wca18g11 that showed elevated expression upon cold acclimation. Nucleotide sequence analysis showed that the clone encoded a group3 alpha-tubulin. Reverse transcription real-time PCR analysis of the expression of both wca18g11 clone and its closest paralogs of the wheattubulin A-2 homeologous group during the course of cold acclimation revealed that both genes were differentially regulated with distinct ex-pression patterns. The involvement of the two alpha-tubulin genes in cold acclimation and signal transduction is discussed.� 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Keywords: Alpha-tubulin; WCS120; Cold acclimation; Wheat; Triticum aestivum; Abiotic stress

1. Introduction

Cold acclimation (CA) is the process that allows hardyplants to develop essential tolerance for winter survivalthrough multiple levels of biochemical and cell biologicalchanges. Dynamic alteration in gene expression occurs duringCA process. Reports have identified CA induced genes frommany plant species including winter wheat (Pearce, 1999).Study of the regulatory and signaling elements that controlthis coordinated gene expression is essential for understandingCA and the genetic basis of cold tolerance. Microtubules arekey elements of the cytoskeleton and their role in signalingand regulation is being recently extensively studied. Theirintimate relationship with the plasma membrane, the majorplatform for signal perception and transduction (Gilroy andTrewavas, 2001; Wasteneys and Galway, 2003), suggests

Abbreviations: CA, cold acclimation; NA, non-acclimation; DA, de-accli-

mation; SNP, single nucleotide polymorphism; UTR, untranslated region.

* Corresponding author. Tel.: þ359 2 963 5409; fax: þ359 2 963 5408.

E-mail address: [email protected] (N.K. Christov).

1065-6995/$ - see front matter � 2007 International Federation for Cell Biology.

doi:10.1016/j.cellbi.2007.11.014

that microtubules are downstream targets of various signalingpathways. The identification of large number of RNA-bindingand regulatory proteins in Arabidopsis that were shown to bindto microtubules (Chuong et al., 2004) and the very recent find-ing that a cold acclimation induced wheat receptor kinaseinteracts with a member of the alpha-tubulin family (Tardifet al., 2007), further supports this hypothesis. Signaling cas-cades have been shown to involve changes in cytoskeletonorganization. In animal cells, microtubules have beenproposed to transmit signals from the receptor to the nucleussince they span the distance from the plasma membrane tothe nucleus (Gundersen and Cook, 1999). Inside the cells,the cytoskeleton can serve as a template where componentsthat transduce extracellular signals interact. Therefore, thestate of the cytoskeleton in plant cells can be critical in activat-ing and transporting signal molecules to a site where theyinteract (Gundersen and Cook, 1999; Camilleri et al., 2002).These structures, composed of alpha- and beta-tubulins,undergo a transient disorganization followed by major rear-rangements in root cortical cells during cold acclimation(Jian et al., 1989). Most freezing-resistant cultivars of wheat

Published by Elsevier Ltd. All rights reserved.

Page 2: Differential expression of two winter wheat alpha-tubulin genes during cold acclimation

575N.K. Christov et al. / Cell Biology International 32 (2008) 574e578

show transient and partial disassembly of microtubules duringthe early phase of cold acclimation, whereas the sensitivecultivar lacks this disassembly (Abdrakhamanova et al.,2003). The finding that, artificially induced disassembly ofmicrotubules in freezing-sensitive cultivars, led to enhancedfreezing tolerance supports the idea that transient disassemblyof microtubules is involved in the cold acclimation process(Abdrakhamanova et al., 2003). The microtubules appear toplay a central role in cold signaling and cold acclimation. Atthe early phase of cold acclimation, flexibility and re-organization of the microtubule cytoskeleton are needed,while completion of the process requires the formation ofcold-stable microtubules, which ensure growth even whenthe temperature is not optimal. The molecular basis of micro-tubule cold resistance seems to involve the differential induc-tion of tubulin isotypes during cold acclimation (Kerr andCarter, 1990; Abdrakhamanova et al., 2003).

To investigate the role of wheat microtubules in cold signaltransduction we assessed the expression of two alpha-tubulingenes during cold acclimation. In this study, we report identi-fication of a CA induced alpha-tubulin clone wca18g11belonging to the A-3 subfamily by differential screening ofwheat CA crown tissue cDNA library. The changes in thelevels of mRNA expression of both wca18g11 clone and itsclosest paralogs of the wheat tubulin A-2 homeologous groupin response to cold acclimation were analyzed by reversetranscription real-time PCR.

2. Materials and methods

2.1. Plant materials and cold acclimation treatment

Surface sterilized seeds of winter wheat (Triticum aestivumL. cv. Chihoku) were germinated on wet paper and wereplanted in commercial soil mix. Plants were grown at 22 �C/18 �C (16 h light/8 h dark) in a growth chamber for 14 daysprior to the initiation of cold acclimation. Cold acclimationwas performed at 6 �C/2 �C (8 h light/16 h dark) for anadditional 2 weeks. Plants were subsequently de-acclimatedat 22 �C/18 �C (16 h light/8 h dark) for 3 days. Meristematiccrown tissue from pools of 30e40 plants was harvested, frozenimmediately in liquid nitrogen and stored at �80 �C until usedfor RNA extraction. Total RNA was isolated from wheat crowntissues using TRIzol reagent (Invitrogen) following themanufacturer’s protocol.

2.2. cDNA isolation and sequence analyses

A cDNA library from 14 days cold-acclimated crown tissueof winter wheat was constructed by using ZAP Express cDNAGigapack� Gold cloning kit (Stratagene, USA), according tothe manufacturer’s instruction. Approximately 107 plaque-forming units (pfu) of the library were in vivo excised in E.coli strain XLOLR to give pBK-CMV cDNA phagemids.Screening of cold regulated cDNA clones in the library wasperformed using a macroarray-based differential screening

method following the protocol described in Christova et al.(2006)

DNA sequencing was performed with a DNA sequencer373A (Applied Biosystems, San Jose, CA) using a Big DyeTerminator Cycle Sequencing Kit v1.1 (Applied Biosystems).Sequencing data analyses were performed with STADENPAKAGE software (Staden, 1996), the sequence alignmentswere made by CLUSTALX (Chenna et al., 2003) and visual-ized by GENEDOC (Nicholas et al., 1997).

2.3. cDNA synthesis

First strand cDNA was synthesized by reverse transcriptionfrom total RNA. Two micrograms of total RNA were used forDNase treatment with the DNase I (Fermenats) following themanufacturer’s protocol. One microgram of DNase treatedtotal RNA was used for cDNA synthesis with oligo dT18primer and Enhanced Avian RT First Strand Synthesis Kit(Sigma). The reverse transcription reaction was performedfor 1 h at 50 �C according to the manufacturer’s instruction.

2.4. Primer design and real-time PCR

Real-time PCR was performed on a Bio-Rad iCYCLER iQreal-time PCR machine (Biorad). The PCR reaction consistedof 2� iQ� SYBR� Green Supermix (Biorad), 25 pmol ofeach primer, and 5 ml of cDNA as template in a final reactionvolume of 25 ml. The PCR conditions were 95 �C for 3 minand 40 cycles of 95 �C for 15 s, 52 �C for 15 s and 60 �Cfor 1.5 min each. Gene specific primers for each studiedgene were designed to amplify 150e170 bp at the 30 UTR ofthe gene of interest using the PerlPrimer software (Marshall,2004). The primers used and the length of the fragmentamplified are shown in Table 1.

The threshold was determined as the first cycle, above thebackground fluorescence that all samples were in the exponen-tial amplification phase. After the real-time PCR experimentwas completed, a dissociation curve was conducted in orderto determine if only one product was amplified. A singlepeak in the dissociation curve implied that only the gene ofinterest was amplified. The DDCT method (Livak and Schmitt-gen, 2001) was used to determine the relative expression of thegene of interest by using Biorad Gene Expression Macro�Version 1.1. where the wact 2 was used as housekeepingreference gene and the relative expression for each studiedgene was calculated against the non-acclimated sample.

3. Results and discussion

3.1. Cloning and sequence analysis of an alpha-tubulincDNA clone that is accumulated incold-acclimated crown

A cDNA library constructed from cold-acclimated crowntissue was screened with labeled cDNA from 14 days cold-acclimated and non-acclimated crown tissues. The macroar-ray-based differential screening identified a clone named

Page 3: Differential expression of two winter wheat alpha-tubulin genes during cold acclimation

Table 1

Primers and length of the amplified product in the real-time PCR experiments

Gene Name Sequence Primer length (nt.) Product length (bp)

wca18g11 18g11_utr_f 50 GATGAGTACTAGAGCAGCTGAG 30 22 162

18g11_utr_r 50 CGGATAACATGCATATTGACCA 30 22

wtub a2 (DQ435658) wtub_a2_f 50 GAGTATTAAGCCTGCCTCCT 30 20 145

wtub_a2_r 50 CAAGGTTCTTACAACACAACAG 30 22

wcs120 (M93342) wcs120_utr_f 50 CTACCCTTGCAGAATAATAACC 30 22 169

wcs120_utr_r 50 AACGAATTTGCACTACAGAG 30 20

wact2 (CJ902800) wact2_intr_rt_f 50 GTATGGTCAAGGCTGGTTTT 30 20 150

wact2_intr_rt_r 50 GAGGATACCCCTTTGGATTG 30 20

Wact2 is used as a reference gene and wcs120 was included as a control for the quality of CA treatment and reverse transcription.

576 N.K. Christov et al. / Cell Biology International 32 (2008) 574e578

wca18g11 showing elevated expression in cold-acclimatedcrown tissue. The nucleotide sequencing and BLAST searchesrevealed that wca18g11 encoded a homologous group 3 alpha-tubulin. The clone shared 98% nucleotide sequence identity toTa_TUBA-3-2 (DQ435662) (Table 2). Pairwise alignment(Fig. 1) revealed the presence of six translationally silentSNPs in addition to the alternative poliadenylation site,suggesting that the cDNA clone represented different alleleof the Ta_TUBA-3B gene. The nucleotide identity ofwca18g11 with the other members of the same homeologousset, Ta_TUBA-3-3 (DQ435664) and Ta_TUBA-3-1(DQ435663), was 93% and 92%, respectively. The alpha-tubulin cDNA clone also shared high degree of nucleic acidsequence identity with the members of the closest paralogousgroup, 87% with Ta_TUBA-2-1 (DQ435659) and 88% withboth Ta_TUBA-2-2 (DQ435661) and Ta_TUBA-2-3(DQ435658) (Table 2). This high degree of sequence identity,including the UTR sequences, raised the possibility of cross-hybridization of Ta_TUBA-2 homeologous group cDNAs tothe wca18g11 clone. To explore this possibility, a memberof the Ta_TUBA-2 subfamily was also included in the reversetranscription real-time PCR analysis.

3.2. Real-time RT-PCR expression analyses of twoalpha-tubulin genes in response to cold acclimation

Since the cDNA clone wca18g11 was isolated by a differen-tial screening between NA and CA plant tissues, the relativeexpression profile of the two members of wheat alpha-tubulingene family was investigated in plants undergoing cold accli-mation. The act 2 was used as reference gene and the

Table 2

Nucleic acid sequence identity among homeologous groups 2 and 3 T. aestivum a

wca18g11 (%) TUBA-3-2 (%) TUBA-3-1 (%) T

wca18g11 100

TUBA-3-2 98 100

TUBA-3-1 92 92 100

TUBA-3-3 93 94 93 1

TUBA-2-1 87 87 89

TUBA-2-2 88 88 89

TUBA-2-3 88 88 89

The values represent the overall sequence identity including the 50 and 30 UTR.

expression was calculated relative to NA sample. Fig. 2 illus-trates the relative transcript levels of the two alpha-tubulingenes and the stress marker gene wcs120. The transcript levelof wcs120 increased rapidly more than 100-fold within the firstday, remained high throughout the CA and declined after 3days of DA at 22 �C, which validated our experimental condi-tions. The expression of wca18g11 (Ta_TUBA-3-2) graduallydeclined until 6 days of CA, partially recovered at 14 daysand was elevated up to 2-fold after 3 days of DA. TheTa_TUBA-2-3 gene showed a distinct expression pattern. Itsexpression was strongly depressed after 1 day of CA, gradu-ally recovered up to 6 days, then was 12-fold elevated at 14days of CA and continued to increase up to 64-fold after 3days of DA (Fig. 2). These results indicate that the identifica-tion of wca18g11 as a CA induced clone in the macroarray-based differential screening was a result of cross-hybridizationwith the Ta_TUBA-2-3 message. The expression patterns ofboth alpha-tubulin genes reported here differ from those re-cently obtained by semi-quantitative RT-PCR for the samegenes (Ridha Farajalla and Gulick, 2007). The results of thetwo studies are not directly comparable. The major differencesinclude the use of spring cultivar by Ridha Farajalla andGulick (2007) vs. winter cultivar in the present study andthe different reference genes employed. However, strong in-duction of TA_TUBA-2-3 expression at later stages of CA isevident in both studies and it is tempting to speculate thatthis elevated expression, reported to happen after 36 days ofCA in the spring cultivar (Ridha Farajalla and Gulick,2007), occurred at earlier stage (14 days) in the winter one(Fig. 2). It was recently reported that a CA inducible recep-tor-like kinase specifically interacts with Ta_TUBA-2-3

-tubulins

UBA-3-3 (%) TUBA-2-1 (%) TUBA-2-2 (%) TUBA-2-3 (%)

00

88 100

89 95 100

89 95 96 100

Page 4: Differential expression of two winter wheat alpha-tubulin genes during cold acclimation

Fig. 1. Pairwise alignment of wca18g11 clone to the Ta_TUBA-3-2 (DQ435662) nucleotide sequence. The conserved nucleotides are shown as white letters on

black background.

577N.K. Christov et al. / Cell Biology International 32 (2008) 574e578

protein (Tardif et al., 2007). Therefore, a more detailed analy-sis of the expression of Ta_TUBA-2-3 and other members ofwheat alpha-tubulin family in spring and winter cultivars, aswell as, winter cultivars with different levels of freezing toler-ance may shed light on their function in cold acclimation andthe cold signal transduction.

Fig. 2. Relative expression of wca18g11/Ta_TUBA-3-2 (DQ435662) (black

bars), Ta_TUBA-2-3 (DQ435658) (grey bars) and wcs120 (M93342) (open

bars). Total RNA was isolated and transcribed into cDNA, and relative tran-

script levels were measured using real-time PCR analysis. The real-time

PCR experiment was repeated in three parallels to ensure statistical relevance.

Acknowledgement

This research was partially supported by a NATO grant Nr.LST-CLG-979536.

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