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Journal of Plant Physiology ](]]]]) ]]]]]]

SHORT COMMUNICATION

The prohibitin genes in Arabidopsis thaliana:Expression in seeds, hormonal regulation and

possible role in cell cycle control during seed

germination

Juana G. De Diegoa, F. David Rodr gueza, Jose Luis Rodr guez Lorenzoa,Emilio Cervantesb,

aDepartamento de Bioqumica y Biologa Molecular, Universidad de Salamanca, 37007, Salamanca, SpainbIRNASA-CSIC, partado 257, 37080, Salamanca, Spain

Received 14 February 2006; accepted 10 May 2006

KEYWORDS

Arabidopsis;Prohibitin;Seed germination;Transcriptional regu-lation 30 UTR

SummaryA fragment encoding a partial sequence of a prohibitin (Phb) gene was isolated. Theexpression of Phb mRNA and protein in seeds of wild type and mutant Arabidopsis

thalianaseeds is presented. Phb mRNA is abundant in wild-type seeds; thus, it mayhave sequence or structural characteristics responsible for this stability. The 3 0

untranslated region sequence of a Phb gene has interesting features. We found thatArabidopsisPhb does not interact with a retinoblastoma-related protein or E2F in ayeast two-hybrid system, thus suggesting that the plant protein may have notconserved such interaction, described for mammalian Phb. The possible role of Phbin cell cycle regulation during germination is discussed.&2006 Published by Elsevier GmbH.

Introduction

An important mechanism of control of G1-Stransition depends on the retinoblastoma (Rb)protein. Rb, in its hypophosphorilated form, binds

E2F transcription factors, inhibiting the transcrip-tion of genes required for S phase (Weinberg,1995). Rb was first discovered in human cells, andlater, in plant cells (Grafi et al., 1996;Xie et al.,1996) and interaction of plant RB with E2F has beendemonstrated (Ram rez-Parra et al., 1999).

Studies with animal prohibitins (Phbs) haveshown the interaction of Phb with Rb and suggestthat this interaction is important in the control ofG1-S transition and apoptosis (Wang et al., 1999,

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www.elsevier.de/jplph

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pp:13col:fig::NILED:SwarnaR:

PAGN:santhamma SCAN:

0176-1617/$ - see front matter&2006 Published by Elsevier GmbH.doi:10.1016/j.jplph.2006.05.002

Abbreviations: Phb, prohibitin; Rb, retinoblastoma protein;UTR, untranslated region; Y2H, yeast two hybridCorresponding author. Tel.: +34923219606; fax:

+34923219609.E-mail address:ecervant@usal.es (E. Cervantes).

http://www.elsevier.de/jplphhttp://localhost/var/www/apps/conversion/tmp/scratch_2/dx.doi.org/10.1016/j.jplph.2006.05.002mailto:ecervant@usal.esmailto:ecervant@usal.eshttp://localhost/var/www/apps/conversion/tmp/scratch_2/dx.doi.org/10.1016/j.jplph.2006.05.002http://www.elsevier.de/jplph

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2002a;Fusaro et al., 2003). Phb protein has beenfound in the nucleus, where it co-localizes with Rband E2F (Wang et al., 2002b). Also, localization ofPhb in the mitochondria has been shown, where Phbhas a function in the stabilization of proteins(Nijtmans et al., 2000). Phb may also be able tocontrol cell cycle progression due to sequences in

the 30untranslated region (UTR) of its mRNA thathave been responsible for polymorphisms asso-ciated with breast cancer (Campbell et al., 2003,Jupe et al., 2001;Spurdle et al., 2003).

The objective in this work was to investigate theregulation of Phb genes during germination in

Arabidopsis thalianaand their possible roles duringgermination.

Materials and methods

Plant material

A. thaliana, cv. Wassilewskija and mutantABC33were obtained form INRA-Versailles. Ecotype Co-lumbia and other mutants were from NASC. Seedswere sown in agar plates with a light/dark cycle(16/8) at 20 1C.

Cloning of Phb cDNA clones

A Phb partial sequence from A. thaliana was

obtained (de Diego et al., 2006). A cDNA containingthe complete coding sequence corresponding toPhb encoded in clone K17E12 in chromosome 3(At3g27280) was obtained, confirmed by sequen-cing, and used for the two-hybrid analysis.

Two-hybrid analysis of the PHB-RBinteraction

PHB encoding cDNAs where cloned in frame inthe pGBT8 binding vector and two-hybrid analyseswere performed following published protocols.cDNAs encoding ZmRb1 (Xie et al., 1996) or TmE2F

(Ram rez-Parra et al., 1999) were cloned in-framein pGADGH vector (Clontech). Yeast two-hybrid(Y2H) assays were carried out in the S. cerevisiaeHF7c strain, as described previously (Ram rez-Parraet al., 1999).

Results and discussion

Phb mRNA is highly abundant in dry seeds anddecreases slightly in the course of 24 h of imbibition

(Fig. 1). The amount of Phb mRNA in mutantspresented important variations. This result relatesto the levels of Phb mRNA with ethylene andgibberellic acid action and, in general, with thegrowth rate of the root in the young seedlings(Table 1).

The results of protein blot (Fig. 1) show constantlevels of Phb protein in wild-type seeds andmutants. This is in contrast to notable changes inmRNA abundance. The hormonal effects observedon Phb mRNA affect transcription and/or stabilityduring imbibition, but do not affect protein levels.

The two-hybrid system could not demonstrate an

interaction between Phb and Rb and E2F proteins.The UTR regions of the Arabidopsis Phb gene

(At3g27280) contain interesting aspects, includingsequence similarity to miRNA precursors (Griffiths-Jones, 2004)(Fig. 2). Interestingly, MIR398 expres-

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Prohibitin mRNA (black) and protein (white) expression

Figure 1. Prohibitin mRNA (black) and protein (white) expression. Values given in the y-axis corresponds to bandintensity in Western and Northern blots relative to controls (wt dry seed).

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sion during germination has been demonstrated(Martin et al., 2006).

The fact that mRNA is stable and abundant in dryseeds of the wild type needs to be explained bystructural or sequence properties. Strong second-ary structures and the affinity for other RNAmolecules may be responsible for this stability.The concentration of Phb mRNA is in inverserelationship with the growth rate of the root (Fig.

1andTable 1) suggesting that the mRNA encodingPhb may have anti-proliferative activity in plants,as reported in mammals (Jupe et al., 2001;Spurdleet al., 2003). In plants this, most probably, may berelated with hormonal control of translation.

Acknowledgments

The antibody antiprohibitin was provided by DrHillel Fromm (University of Leeds, UK). Juana deDiego and David Rodrguez thank Consejera de

Ciencia y Cultura de Castilla y Leon for grant SA/007-03. We thank Drs. Elena Ramrez-Parra andCrisanto Gutierrez for help in Y2H experiments andcommentaries to the manuscript.

References

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Table 1. Root length in seedlings ofeto1-1, etr1-1 and wild-type Columbia at 5 days after germination

Genotype Mean N Std. dev. Variance Min Max

Columbia 4.81 160 1.13 1.28 2 8etr1-1 4.89 160 1.08 1.18 2 8eto1-1 2.81 160 1.00 1.01 1 5

Total 4.3 480 2.00 4.03 1 10

3UTR 554 GAAGCCACCAAAGUGACCACAUAAAAAAGUUUUAUUUAAUUUAUUUACCAUGUUUU |||| |||||| || | ||| | ||| | | || |||

ath-MIR393a 5 GAAGGAUCCAAAGGGAUCGCAUUGAUCCUAAUUAAGG-UGAAUUCUCCCCAUAUUU

3UTR UAAUUUAUAUUUUAAAUAAUACAAGUACAACAAC---CUUGGUGUUGGA 454 | |||||| || | |||| || |||| || |||||| |||||

ath-MIR393a UC-UUUAUAAUUGGCA-AAUA-AAUCACAAAAAUUUGCUUGGUUUUGGA 105

Figure 2. Similarity between 30 UTR in a prohibitin gene (At3g27280) and a microRNA precursor of Arabidopsis (ath-

MIR393a). Detected with miRNA registry (http://www.sanger.ac.uk/Software/Rfam/mirna/search.shtml ). Underlinedis the processed miRNA. Similarities to other miRNA precursors were detected within the 5 0 UTR sequence.

Prohibitin gene expression in seeds of Arabidopsis thaliana 3

http://www.sanger.ac.uk/Software/Rfam/mirna/search.shtmlhttp://www.sanger.ac.uk/Software/Rfam/mirna/search.shtmlhttp://www.sanger.ac.uk/Software/Rfam/mirna/search.shtml