(SHI) Family Transcription Factor Gene Regulates Awn Elongation and Pistil

Journal of Experimental Botany , Vol. 63, 63, No. No. 14, 2, pp. pp. 695–709, 5223–5232, 2012 2012 doi:10.1093/jxb/err313doi:10.1093/jxb/ers182 Advance Advance Access Access publication publication 12 4 November,July, 2012 2011 ThisThis paper is available online free of all access charges charges (see (see http://jxb.oxfordjournals.org/open_access.html http://jxb.oxfordjournals.org/open_access.html for for further further details)) details) RESEARCH PAPER InA SHORTPosidonia INTERNODES oceanica cadmium (SHI) family induces transcription changes factor in DNA gene methylationregulates awn and elongation chromatin and patterning pistil morphology in barley MariaTakahisa Greco, Yuo1 Adriana, Yuko Yamashita Chiappetta,1, Hiroyuki Leonardo Kanamori Bruno and2, Takashi Maria Matsumoto Beatrice Bitonti*2, Udda Lundqvist3, Kazuhiro Sato1, 4 5 1, DepartmentMasahiko ofIchii Ecology,, Stephen University A. Jobling of Calabria, and Laboratory Shin Taketa of Plant* Cyto-physiology, Ponte Pietro Bucci, I-87036 Arcavacata di Rende, Cosenza,1 Institute Italyof Plant Science and Resources, Okayama University, 2-20-1 Chuo, Kurashiki 710-0046, Japan *2 ToNational whom Institute correspondence for Agrobiological should be Sciences, addressed. Tsukuba E-mail: 305–8602, [email protected] Japan 3 Nordic Genetic Resource Center, Smedjevägen 3, PO Box 41, SE-230 53 Alnarp, Sweden 4 Received Faculty 29of MayAgriculture, 2011; Revised Kagawa 8 JulyUniversity, 2011; Accepted2393 Ikenobe, 18 August Miki, 2011Kagawa 761-0795, Japan 5 CSIRO Plant Industry, GPO Box1600, Canberra, ACT 2601, Australia * To whom correspondence should be addressed: E-mail: [email protected] Abstract Received 28 February 2012; Revised 16 May 2012; Accepted 22 May 2012 In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with itsAbstract effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd, The awn, an apical extension from the lemma of the spikelet, plays important roles in seed dispersal, burial, and through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, photosynthesis. Barley typically has long awns, but short-awn variants exist. The short awn 2 (lks2) gene, which pro- respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, duces awns about 50% shorter than normal, is a natural variant that is restricted to Eastern Asia. Positional cloning was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron revealed that Lks2 encodes a SHI-family transcription factor. Allelism tests showed that lks2 is allelic to unbranched microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de style 4 (ubs4) and breviaristatum-d (ari-d), for which the phenotypes are very short awn and sparse stigma hairs. The novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of gene identity was validated by 25 mutant alleles with lesions in the Lks2 gene. Of these, 17 affected either or both interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd conserved regions: the zinc-binding RING-finger motif and the IGGH domain. Lks2 is highly expressed in awns and perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are pistils. Histological observations of longitudinal awn sections showed that the lks2 short-awn phenotype resulted linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. from reduced cell number. Natural variants of lks2 were classified into three types, but all shared a single-nucleotide Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants. polymorphism (SNP) that causes a proline-to-leucine change at position 245 in the IGGH domain. All three lks2 natural Keyvariants words: were5-Methylcytosine-antibody, regarded as weak alleles cadmium-stress because their condition, awn and chromatinpistil phenotypes reconfiguration, are mildCHROMOMETHYLASE compared with those, of the DNA-methylation,25 mutant alleles. Methylation- Natural variants Sensitive of Amplification lks2 found Polymorphismin the east of (MSAP), China Posidoniaand the Himalayas oceanica (L.) had Delile. considerably different sequences in the regions flanking the critical SNP, suggesting independent origins. The available results suggest that the lks2 allele might have a selective advantage in the adaptation of barley to high-precipitation areas of Eastern Asia. IntroductionKey words: Hordeum vulgare L., IGGH domain, RING finger, stigma, varietal difference. In the Mediterranean coastal ecosystem, the endemic Although not essential for plant growth, in terrestrial seagrassIntroductionPosidonia oceanica (L.) Delile plays a relevant role plants,burial has Cd been is readily well studied absorbed in wild by roots tetraploid and translocatedwheat, which intohas by ensuring primary production, water oxygenation and aeriala pair of organs awns while,in each in spikelet; acquatic the plants, awn movement it is directly driven taken by the up providesBarley (Hordeum niches forvulgare some L.) animals, inflorescences besides are counteracting characterized bydaily leaves. humidity In plants, cycle propels Cd absorption the seeds induces into the complex ground (Elbaum changes coastalby stiff erosionbristles, throughcalled awns, its widespread one on the meadows tip of each (Ott, spikelet. 1980; atet al the., 2007). genetic, The biochemical awn also protects and physiological seeds from levelspredation which by PiazziBotanically,et al. the, 1999; awn is Alcoverro an apical etextension al., 2001). of the There distal isend also of ultimatelybirds and animals account (Grundbacher, for its toxicity 1963). (Valle Under and agricultural Ulmer, 1972; con- considerablethe lemma; it is evidence probably that a modifiedP. oceanica leaf bladeplants (Dahlgren are able et al to., Sanit di Toppi and Gabrielli, 1999; Benavides et al., 2005; ditions,z however, the long and stiff awn with barbs has persisted absorb1985). Awns and are accumulate also found in metals other grass from species sediments such as (Sanchiz wheat, Weberas a nuisanceet al. ,to 2006; farmers Liu becauseet al., they 2008). hinder The manual most obviousharvest- etrye, al. oats,, 1990; sorghum, Pergent-Martini, and rice. The 1998; awn Masertiis consideredet al. ,to 2005) play vari thus- symptoming. More ofimportantly Cd toxicity foris modern a reduction agriculture, in plant the growth injurious due awn to influencingous biological metal roles bioavailability(Grundbacher, 1963). in the In marinethe wild, ecosystem. awns with anreduces inhibition the feed of value photosynthesis, of straw for livestock respiration, (Takahashi, and nitrogen 1955). Forbarbs thisaid in reason, seed dispersal this seagrass by attaching is widely to animal considered fur (zoochory) to be metabolism,Consequently, asduring well domestication as a reduction and insubsequent water and varietal mineral dif- a(von metal Bothmer bioindicator et al., 1995). species The (Maserti importanceet al.of ,the 1988; awn Pergent in seed uptakeferentiation, (Ouzonidou much selectionet al., 1997; pressure Perfus-Barbeoch has been appliedet al.to ,reduce 2000; et al., 1995; Lafabrie et al., 2007). Cd is one of most Shukla et al., 2003; Sobkowiak and Deckert, 2003). widespreadAbbreviations: BAC, heavy bacterial metals artificial in chromosome; both terrestrial qRT-PCR, andreal-time marine quantitative reverseAt the transcription-PCR; genetic level, QTLs, in quantitative both animals trait loci; RACE, and rapid plants, Cd environments. amplification of cDNA ends; SNP, single-nucleotide polymorphism; UTR, untranslatedcan region. induce chromosomal aberrations, abnormalities in © The Author [2012]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. ªFor2011 Permissions, The Author(s). please e-mail: [email protected] This is an Open Access article distributed under the terms of the Creative Commons Commons Attribution Attribution Non-Commercial License License (http://creativecommons.org/licenses/ (http://creativecommons.org/licenses/by- nc/3.0),by-nc/2.0/uk/) which permitswhich permits unrestricted unrestricted non-commercial non-commercial use, distribution, use, distribution, and reproduction and reproduction in any in medium, any medium, provided provided the original the original work iswork properly is properly cited. cited. 5224 | Yuo et al. awn length (Takahashi, 1955). Despite such selection pressure, most barleys have retained rather long awns. This is in contrast to other small grain crops, particularly rice (Takahashi et al., 1986), where breeding has remarkably reduced awn length. This is probably because the awn in barley contributes significantly to photosynthesis (Kjack and Witters, 1974). In cross-sections, barley awns have a triangular shape with two zones of chlor- enchema cells and three well-developed vascular bundles inside and stomata on the surface (Reid,

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