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Role of the Pollen-expressed FERONIA-like Receptor-like Kinases during Plant Fertilization

Periodic Report Summary - PFERLS (Role of the pollen-expressed Feronia-like receptor-like kinases during plant fertilization)

Fertilisation relies on complex and specialised mechanisms that achieve the precise delivery of the male gamete to the female gamete and their subsequent union. In plants, the molecular mechanisms that mediate the discharge of sperm cells by the male gametophyte - pollen tube (PT) - into the female gametophyte are still a mystery although they are of great interest for the manipulation of breeding barriers in wide-crosses and the production of hybrids. Recently, it was shown that a molecular dialogue between the male and female gametophytes must occur for successful sperm discharge.

A receptor-like kinase called Feronia (FER) has been shown by Prof. U. Grossniklaus' group, Universitaet Zurich (UZH), Switzerland, the Host Laboratory, to be required for this dialogue in the female gametophyte. The French Research Fellow Aurelien Boisson-Dernier initiated this project in his former laboratory, i.e. Prof. J. Schroeder s group, University of California San Diego (UCSD), United States of America (USA) and recently (November 16th 2008) joined Prof. U. Grossniklaus' group to characterise pollen-expressed Feronia-like receptor-like kinases (PFERL-RLKs), which are excellent candidates to assume the role of the FER counterpart in the male gametophyte. The main objective of this project was to characterise in detail the function(s) of these two PFERL-RLKs.

The function of the uncharacterised CrRLK1L PFERL-RLKs (renamed ANXUR1 and ANXUR2) has been elucidated in Arabidopsis. Pollen-preferential expression and plasma membrane localisation at the tip of growing PTs have been demonstrated for ANX1 and ANX2. Surprisingly, careful examination of fertilisation behavior of double knock-outs plants for PFERL-RLKs indicated that disruption of both genes was not triggering PT overgrowth as initially suspected but actually triggered the opposite phenotype: double mutant PTs burst in vitro and in vivo shortly after germination preventing them from reaching and fertilising female gametophytes. These two redundant receptor-like kinases function to maintain cell wall (CW) integrity at the tip of growing PTs (Boisson-Dernier et al., 2009). This study has revealed a completely uncharacterised signalling pathway that allows coordination of CW remodelling and tip growth. CWs are critical to plant and microbial growth, herbivore nutrition and to the maintenance of ecosystems. Moreover, CWs are widely exploited in diverse human activities relating to food, industrial enzymology, fibres, textiles, paper and biofuels. Despite the importance of plant CWs, little is known about the molecular mechanisms involved in its synthesis and remodelling.

Then, we have shown that the reactive oxygen species (ROS) producing enzymes RbohH and RbohJ are part of the ANXUR-RLK dependent pathway that coordinate CW remodelling and PT growth. ANX1 and ANX2 function as CW integrity sensors that regulate positively the redundant ROS-producing RbohH and RbohJ enzymes to regulate ROS and calcium ion (Ca2+) homeostases. Consequently, this results in a proper and coordinated CW remodelling that maintains CW integrity, allowing the PT to grow efficiently (Boisson-Dernier et al., 2012, in preparation).

Domains that putatively bind carbohydrate-rich ligands have been identified in the extracellular region of the RLKs of the CrRLK1L family (Boisson-Dernier et al., 2011). This discovery led us to deviate from the original yeast two-hybrid approach proposed initially to identify the ligands of ANX1/ANX2. Instead a forward genetic screen for suppressor mutations of the anx1 anx2 sterility phenotype has been initiated. Thirty two 'strong' suppressors were confirmed to display rescue of PT growth in vivo despite carrying the anx1 anx2 mutations and some of these suppressors exhibit also rescue of anx1 anx2 PT growth in vitro. Next step will consist of characterising the corresponding suppressor mutations by an approach developed in the host laboratory based on next-generation sequencing (Lindner et al., 2012). Assessing ROS and Ca2+ levels as well as CW composition in these suppressor mutants will allow the positioning of the corresponding genes in the ANXUR-RLK dependent CW integrity pathway.

This approach will undoubtedly lead to the identification of new components of the ANXUR-RLK dependent CW integrity system, allowing the researcher to establish himself as a leading scientist in this novel niche. Plant CW integrity is an emerging exciting field as it can impact many areas of plant research: CW biology, cell growth, cell-cell communication, plant-microorganism interactions, plant responses to abiotic stresses and glycobiology.