Characterization of the phosphoproteome of mature Arabidopsis pollen

Successful pollination depends on cell-cell communication and rapid cellular responses. In Arabidopsis, the pollen grain lands on a dry stigma, where it hydrates, germinates and grows a pollen tube that delivers the sperm cells to the female gametophyte to effect double fertilization. Various studies have emphasized that a mature, dehydrated pollen grain contains all the transcripts and proteins required for germination and initial pollen tube growth. Therefore, it is important to explore the role of post-translational modifications (here phosphorylation), through which many processes induced by pollination are probably controlled. We report here a phosphoproteomic study conducted on mature Arabidopsis pollen grains with the aim of identifying potential targets of phosphorylation. Using three enrichment chromatographies, a broad coverage of pollen phosphoproteins with 962 phosphorylated peptides corresponding to 598 phosphoproteins was obtained. Additionally, 609 confirmed phosphorylation sites were successfully mapped. Two hundred and seven of 240 phosphoproteins that were absent from the PhosPhAt database containing the empirical Arabidopsis phosphoproteome showed highly enriched expression in pollen. Gene ontology (GO) enrichment analysis of these 240 phosphoproteins shows an over-representation of GO categories crucial for pollen tube growth, suggesting that phosphorylation regulates later processes of pollen development. Moreover, motif analyses of pollen phosphopeptides showed an over-representation of motifs specific for Ca²⁺/calmodulin-dependent protein kinases, mitogen-activated protein kinases, and binding motifs for 14-3-3 proteins. Lastly, one tyrosine phosphorylation site was identified, validating the TDY dual phosphorylation motif of mitogen-activated protein kinases (MPK8/MPK15). This study provides a solid basis to further explore the role of phosphorylation during pollen development

Genome-wide transcriptomic analysis of the sporophyte of the moss Physcomitrella patens

Bryophytes, the most basal of the extant land plants, diverged at least 450 million years ago. A major feature of these plants is the biphasic alternation of generations between a dominant haploid gametophyte and a minor diploid sporophyte phase. These dramatic differences in form and function occur in a constant genetic background, raising the question of whether the switch from gametophyte-to-sporophyte development reflects major changes in the spectrum of genes being expressed or alternatively whether only limited changes in gene expression occur and the differences in plant form are due to differences in how the gene products are put together. This study performed replicated microarray analyses of RNA from several thousand dissected and developmentally staged sporophytes of the moss Physcomitrella patens, allowing analysis of the transcriptomes of the sporophyte and early gametophyte, as well as the early stages of moss sporophyte development. The data indicate that more significant changes in transcript profile occur during the switch from gametophyte to sporophyte than recently reported, with over 12% of the entire transcriptome of P. patens being altered during this major developmental transition. Analysis of the types of genes contributing to these differences supports the view of the early sporophyte being energetically and nutritionally dependent on the gametophyte, provides a profile of homologues to genes involved in angiosperm stomatal development and physiology which suggests a deeply conserved mechanism of stomatal control, and identifies a novel series of transcription factors associated with moss sporophyte development