Overlapping functions and protein-protein interactions of LRR-extensins in Arabidopsis.

Plant cell growth requires the coordinated expansion of the protoplast and the cell wall, which is controlled by an elaborate system of cell wall integrity (CWI) sensors linking the different cellular compartments. LRR-eXtensins (LRXs) are cell wall-attached extracellular regulators of cell wall formation and high-affinity binding sites for RALF (Rapid ALkalinization Factor) peptide hormones that trigger diverse physiological processes related to cell growth. LRXs function in CWI sensing and in the case of LRX4 of Arabidopsis thaliana, this activity was shown to involve interaction with the transmembrane Catharanthus roseus Receptor-Like Kinase1-Like (CrRLK1L) protein FERONIA (FER). Here, we demonstrate that binding of RALF1 and FER is common to most tested LRXs of vegetative tissue, including LRX1, the main LRX protein of root hairs. Consequently, an lrx1-lrx5 quintuple mutant line develops shoot and root phenotypes reminiscent of the fer-4 knock-out mutant. The previously observed membrane-association of LRXs, however, is FER-independent, suggesting that LRXs bind not only FER but also other membrane-localized proteins to establish a physical link between intra- and extracellular compartments. Despite evolutionary diversification of various LRX proteins, overexpression of several chimeric LRX constructs causes cross-complementation of lrx mutants, indicative of comparable functions among members of this protein family. Suppressors of the pollen-growth defects induced by mutations in the CrRLK1Ls ANXUR1/2 also alleviate lrx1 lrx2-induced mutant root hair phenotypes. This suggests functional similarity of LRX-CrRLK1L signaling processes in very different cell types and indicates that LRX proteins are components of conserved processes regulating cell growth.

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 Ca²âº/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.

Disruption of the pollen-expressed FERONIA homologs ANXUR1 and ANXUR2 triggers pollen tube discharge.

The precise delivery of male to female gametes during reproduction in eukaryotes requires complex signal exchanges and a flawless communication between male and female tissues. In angiosperms, molecular mechanisms have recently been revealed that are crucial for the dialog between male (pollen tube) and female gametophytes required for successful sperm delivery. When pollen tubes reach the female gametophyte, they arrest growth, burst and discharge their sperm cells. These processes are under the control of the female gametophyte via the receptor-like serine-threonine kinase (RLK) FERONIA (FER). However, the male signaling components that control the sperm delivery remain elusive. Here, we show that ANXUR1 and ANXUR2 (ANX1, ANX2), which encode the closest homologs of the FER-RLK in Arabidopsis, are preferentially expressed in pollen. Moreover, ANX1-YFP and ANX2-YFP fusion proteins display polar localization to the plasma membrane at the tip of the pollen tube. Finally, genetic analyses demonstrate that ANX1 and ANX2 function redundantly to control the timing of pollen tube discharge as anx1 anx2 double-mutant pollen tubes cease their growth and burst in vitro and fail to reach the female gametophytes in vivo. We propose that ANX-RLKs constitutively inhibit pollen tube rupture and sperm discharge at the tip of growing pollen tubes to sustain their growth within maternal tissues until they reach the female gametophytes. Upon arrival, the female FER-dependent signaling cascade is activated to mediate pollen tube reception and fertilization, while male ANX-dependent signaling is deactivated, enabling the pollen tube to rupture and deliver its sperm cells to effect fertilization.