Activation of CYCD7;1 in the central cell and early endosperm overcomes cell-cycle arrest in the Arabidopsis female gametophyte, and promotes early endosperm and embryo development.

In angiosperms, double fertilization of the egg and central cell of the megagametophyte leads to the development of the embryo and endosperm, respectively. Control of cell cycle progression in the megagametophyte is essential for successful fertilization and development. Central cell-targeted expression of the D-type cyclin CYCD7;1 (end CYCD7;1) using the imprinted FWA promoter overcomes cycle arrest of the central cell in the Arabidopsis female gametophyte in the unfertilized ovule, leading to multinucleate central cells at high frequency. Unlike FERTILIZATION-INDEPENDENT SEED (fis) mutants, but similar to lethal RETINOBLASTOMA-RELATED (rbr) mutants, no seed coat development is triggered. Unlike the case with loss of rbr, post-fertilization end CYCD7;1 in the endosperm enhances the number of nuclei during syncytial endosperm development and induces the partial abortion of developing seeds, associated with the enhanced size of the surviving seeds. The frequency of lethality was less than the frequency of multinucleate central cells, indicating that these aspects are not causally linked. These larger seeds contain larger embryos composed of more cells of wild-type size, surrounded by a seed coat composed of more cells. Seedlings arising from these larger seeds displayed faster seedling establishment and early growth. Similarly, two different embryo-lethal mutants also conferred enlarged seed size in surviving siblings, consistent with seed size increase being a general response to sibling lethality, although the cellular mechanisms were found to be distinct. Our data suggest that tight control of CYCD activity in the central cell and in the developing endosperm is required for optimal seed formation.

The role of the kinase OXI1 in cadmium- and copper-induced molecular responses in Arabidopsis thaliana.

The hypothesis that mitogen-activated protein kinase (MAPK) signalling is important in plant defences against metal stress has become accepted in recent years. To test the role of oxidative signal-inducible kinase (OXI1) in metal-induced oxidative signalling, the responses of oxi1 knockout lines to environmentally realistic cadmium (Cd) and copper (Cu) concentrations were compared with those of wild-type plants. A relationship between OXI1 and the activation of lipoxygenases and other initiators of oxylipin production was observed under these stress conditions, suggesting that lipoxygenase-1 may be a downstream component of OXI1 signalling. Metal-specific differences in OXI1 action were observed. For example, OXI1 was required for the up-regulation of antioxidative defences such as catalase in leaves and Fe-superoxide dismutase in roots, following exposure to Cu, processes that may involve the MEKK1-MKK2-WRKY25 cascade. Moreover, the induction of Cu/Zn superoxide dismutases in Cu-exposed leaves was regulated by OXI1 in a manner that involves fluctuations in the expression of miRNA398. These observations contrast markedly with the responses to Cd exposure, which also involves OXI1-independent pathways but rather involves changes in components mediating intracellular communication.

Site-specific phosphorylation profiling of Arabidopsis proteins by mass spectrometry and peptide chip analysis.

An estimated one-third of all proteins in higher eukaryotes are regulated by phosphorylation by protein kinases (PKs). Although plant genomes encode more than 1000 PKs, the substrates of only a small fraction of these kinases are known. By mass spectrometry of peptides from cytoplasmic- and nuclear-enriched fractions, we determined 303 in vivo phosphorylation sites in Arabidopsis proteins. Among 21 different PKs, 12 were phosphorylated in their activation loops, suggesting that they were in their active state. Immunoblotting and mutational analysis confirmed a tyrosine phosphorylation site in the activation loop of a GSK3/shaggy-like kinase. Analysis of phosphorylation motifs in the substrates suggested links between several of these PKs and many target sites. To perform quantitative phosphorylation analysis, peptide arrays were generated with peptides corresponding to in vivo phosphorylation sites. These peptide chips were used for kinome profiling of subcellular fractions as well as H 2O 2-treated Arabidopsis cells. Different peptide phosphorylation profiles indicated the presence of overlapping but distinct PK activities in cytosolic and nuclear compartments. Among different H 2O 2-induced PK targets, a peptide of the serine/arginine-rich (SR) splicing factor SCL30 was most strongly affected. SRPK4 (SR protein-specific kinase 4) and MAPKs (mitogen-activated PKs) were found to phosphorylate this peptide, as well as full-length SCL30. However, whereas SRPK4 was constitutively active, MAPKs were activated by H 2O 2. These results suggest that SCL30 is targeted by different PKs. Together, our data demonstrate that a combination of mass spectrometry with peptide chip phosphorylation profiling has a great potential to unravel phosphoproteome dynamics and to identify PK substrates.

Metal resistance in yeast mediated by the expression of a maize 20S proteasome alpha subunit.

Transformation of yeast cells with a maize cDNA ZmPAA, encoding a 20S proteasome alpha-subunit, conferred resistance to nickel, cadmium and cobalt. This resistance is not linked to a modification of the intracellular nickel content, as no accumulation of nickel was measured between yeast cells transformed with a void vector or the ZmPAA cDNA. The abundance of the ZmPAA mRNA was increased in the shoots of maize plants upon nickel treatment. These results suggest that the proteasome might be involved in nickel resistance by scavenging metal oxidized proteins both in plants and yeast.