The Arabidopsis organelle-localized glycyl-tRNA synthetase encoded by EMBRYO DEFECTIVE DEVELOPMENT1 is required for organ patterning

Maria Greco Adriana Chiappetta Leonardo Bruno Maria Beatrice Bitonti 0 siology, Ponte Pietro Bucci, I-87036 Arcavacata di Rende In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent eApbigsetnreatcict mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in Leaves develop as planar organs, with a morphology that is specialized for photosynthesis. Development of a planar 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, leaf requires genetic networks that set up opposing adaxial and abaxial sides of the leaf, which leads to establishthrough a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, ment of dorsoventral polarity. While many genes have been identified that regulate adaxial and abaxial fate there is respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, little information on how this is integrated with cellular function. EMBRYO DEFECTIVE DEVELOPMENT1 (EDD1) is a was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron nuclear gene that encodes a plastid and mitochondrial localized glycyl-tRNA synthetase. Plants with partial loss of microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de EDD1 function have changes in patterning of margin and distal regions of the leaf. In combination with mutations in the novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of MYB domain transcription factor gene ASYMMETRIC LEAVES1 (AS1), partial loss of EDD1 function results in leaves interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd with reduced adaxial fate. EDD1 may influence leaf dorsoventral polarity through regulating the abaxial fate genes perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are KANADI1 (KAN1) and ETTIN (ETT)/AUXIN RESPONSE FACTOR3 (ARF3) since these genes are upregulated in the edd1 linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. as1 double mutant. SCABRA3 (SCA3), a nuclear gene that encodes the plastid RNA polymerase is also required for Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants. leaf adaxial fate in the absence of AS1. These results add a novel component to networks of genetic regulation of leaf development and suggest that organelles, particularly plastids, are required in leaf patterning. Potentially, signalling - initiating leaves develop into determinate, flattened structures absorb and accumulate metals from sediments (Sanchiz with morphologically distinct dorsoventral polarity. The adaxet al., 1990; Pergent-Martini, 1998; Maserti et al., 2005) thus ial (dorsal) side of the leaf is specialized for capture of light influencing metal bioavailability in the marine ecosystem. energy and the abaxial (ventral) side of the leaf is specialized For this reason, this seagrass is widely considered to be for gas exchange, so that leaf dorsovental polarity is optimized a metal bioindicator species (Maserti et al., 1988; Pergent for photosynthesis. et al., 1995; Lafabrie et al., 2007). Cd is one of most widespread heavy metals in both terrestrial and marine Although not essential for plant growth, in terrestrial Sanitz di Toppi and Gabrielli, 1999; Benavides et al., 2005; dantly in adaxial fate (McConnell et al., 2001; Otsuga et al., an inhibition of photosynthesis, respiration, and nitrogen 2001; Emery et al., 2003; Prigge et al., 2005). While loss of metabolism, as well as a reduction in water and mineral PHB and PHV has no phenotypic effect, loss of REV results uptake (Ouzonidou et al., 1997; Perfus-Barbeoch et al., 2000; in a reduced number of lateral branches and floral organs, as Shukla et al., 2003; Sobkowiak and Deckert, 2003). At the genetic level, in both animals and plants, Cd well as vascular patterning defects (Talbert et al., 1995; Zhong and Ye, 1999; Otsuga et al., 2001; Emery et al., 2003; Prigge et al., 2005). Loss of these three class III HD-ZIP genes in triple mutants results in radial organs (Emery et al., 2003; Prigge et al., 2005). Dominant mutations, which disrupt posttranscriptional regulation of REV, PHB, and PHV by mir165/166, result in radial, adaxialized leaves, and in the dominant phb-1d mutant PHB expression is expanded to the abaxial side of the leaf (McConnell et al., 2001; Emery et al., 2003; Kidner and Martienssen, 2004; Zhong and Ye, 2004). Class III HD-ZIP gene expression is also restricted to the adaxial side of developing leaves through a genetic pathway involving KANADI (KAN) family genes. KANADI genes, which encode GARP-domain transcription factors, are expressed on the abaxial side of lateral organs and act redundantly to promote abaxial fate. Loss of KANADI1 (KAN1) causes mild dorsoventral patterning defects such as upward curled leaves and precocious development of abaxial trichomes (Eshed et al., 2001, 2004; Kerstetter et al., 2001). The extent of leaf adaxialization is increased as more of the KANADI gene family members (KAN2, KAN3, and KAN4) are mutated (Eshed et al., 2001, 2004). In kan1 kan2, for example, ectopic patches of adaxial fate on the abaxial side of the leaf leads to ectopic abaxial lamina protrusions. In addition, ectopic expression of KAN1 throughout the leaf results in development of radial, abaxial organs consistent with a requirement for KAN1 for abaxial fate (Eshed et al., 2001; Kerstetter et al., 2001). Abaxial fate also requires the AUXIN RESPONSE FACTOR (ARF) family genes ETTIN (ETT)/AFR3 and ARF4. Loss of both ETT and ARF4 in the ett arf4 double mutant results in leaves resembling kan1 kan2 and kan mutants are enhanced by loss of either ETT or ARF4. KANADI and ETT/ARF4 appear to cooperate in specification of abaxial fate, and this could be in part mediated by protein interaction between KAN1 and ETT (Pekker et al., 2005; Kelley et al., 2012). Initiation of organ primordia involves the transition of cells from an indeterminate to a determinate cell fate. Two genes involved in this transition are the MYB domain transcription factor gene ASYMMETRIC LEAVES1 (AS1) and the LOB/ ASL-domain transcription factor gene ASYMMETRIC LEAVES2 (AS2) (Byrne et al., 2000, 2002; Iwakawa et al., 2002; Lin et al., 2003). AS1 is expressed throughout developing leaves whereas AS2 expression is restricted to the adaxial side of the leaf (Byrne et al., 2000; Iwakawa et al., 2002, 2007). AS1 and AS2 act as a heterodimer, which may serve to limit the activity (...truncated)