PerspectivePlant Biology

Complex regulation of plant sex by peptides

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Science  22 Dec 2017:
Vol. 358, Issue 6370, pp. 1544-1545
DOI: 10.1126/science.aar4190

Most of our food—whether fruit or grain—is the direct result of flowering plant fertilization. As this also underlies the transmission of genetic information over generations, it is essential to understand the molecular events controlling plant reproduction. During fertilization, pollen (the male gametophyte, or reproductive cell) forms a pollen tube which is guided toward the ovule (the female gametophyte), where, upon bursting, it will deliver its sperm cells (1). All steps—from the perception of compatible pollens by the flower pistil (the female reproductive organ) to pollen tube growth, guidance and, ultimately, rupture—are controlled by a complex molecular dialogue involving numerous plant receptor kinases and endogenous signaling molecules, including secreted peptides (2). On pages 1596 and 1600 of this issue, Ge et al. (3) and Mecchia et al. (4), respectively, characterize signaling events between the male and female gametophytes, which offer exciting insights into the molecular mechanisms controlling plant fertilization and seed setting.

The Catharanthus roseus RLK1-like (CrRLK1L) receptor kinases (of which there are 17 in the model plant Arabidopsis thaliana) play critical roles in the interaction between the pollen and the ovule where the pollen will release sperm cells, thus allowing fertilization and subsequent seed development (5). The founding member of this receptor family, FERONIA (FER), is involved in numerous processes including fertilization, immunity, and growth, and endogenous RAPID ALKALINIZATION FACTOR (RALF) peptides are its ligands (6, 7). RALF peptides (of which there are at least 36 in A. thaliana) may also be ligands for other CrRLK1L family members. The FER paralogs ANXUR1 (ANX1) and ANX2 are key regulators of pollen tube growth and integrity in A. thaliana (8, 9). However, no ligand had been reported for them until now.

Based on gene expression profiles, Ge et al. and Mecchia et al. identified RALF4 and RALF19 as being preferentially expressed in mature pollen grains and tubes (3, 4). Notably, simultaneous loss-of-function mutations of RALF4 and RALF19 led to male-specific fertilization defects associated with premature pollen tube bursting (3, 4)—a phenotype also observed in anx1 and anx2 double mutants (810). Interestingly, genetic analyses indicated that ANX1 functions upstream of RALF4 and RALF19 (4). Consistently, Ge et al. showed that RALF4 and RALF19 directly bind to ANX1 and ANX2 ectodomains (the region of the receptors in the extracellular space) with high, nanomolar affinities (3), strongly implicating RALF4 and RALF19 as ligands for ANX1 and ANX2 in an autocrine (i.e., signals from the same cell type) manner to control pollen tube integrity so that it does not burst prematurely.

Surprisingly, Ge et al. identified two additional CrRLK1Ls, BUDDHA'S PAPER SEAL 1 (BUPS1) and BUPS2, that are preferentially expressed in mature pollen grains and tubes, and in which mutations similarly affected male-specific fertility and pollen tube integrity (3). BUPS1 and BUPS2 ectodomains can also bind RALF4 and RALF19 with affinities similar to those of ANX1 and ANX2 ectodomains. Furthermore, BUPS1 and BUPS2 could form complexes with ANX1 and ANX2 (3), suggesting that they form a receptor complex for RALF4 and RALF19 at the plasma membrane of pollen tubes (see the figure).

The ectodomains of CrRLK1Ls contain malectin-like domains, which show homology to the animal carbohydrate-binding protein malectin. Thus, CrRLK1Ls are proposed to be sensors of the carbohydraterich cell wall (5). Their role in fertilization is consistent with the requirement of adequate cell wall mechanical properties to enable rapid anisotropic pollen tube growth while withstanding the high turgor pressure that exists within the cell (11). Furthermore, ANX1 overexpression results in the hyperaccumulation of cell wall materials, resulting in repression of pollen tube growth (10). A potential link between the properties of the cell wall and ANX1 and ANX2 function is provided by Mecchia et al. They identified several pollen-expressed LEUCINE-RICH REPEAT EXTENSIN (LRX) proteins—extracellular, cell wall–bound proteins with a leucine-rich repeat and an extensin domain—as being required for pollen tube integrity (4). In particular, triple loss-of-function mutants of LRX8, LRX9, and LRX10 or LRX11 abolish the ability of exogenous RALF4 to repress pollen tube growth (4), indicating that these pollen-expressed LRXs are required for RALF4 signaling. Accordingly, the authors showed that LRX8 binds RALF4 with nanomolar affinity, and they postulated that these molecular interactions may enable the pollen tube to sense cell wall changes that accompany rapid cell elongation (see the figure).

Controlling the pollen tube

During pollen tube growth through the pistil, pollen-derived RALF4 and RALF19 positively regulate pollen tube integrity. Upon arrival at the ovule, synergid cell–expressed RALF34 outcompetes RALF4 and RALF19 and induces pollen tube rupture to enable the release of sperm cells for fertilization.

GRAPHIC: V. ALTOUNIAN/SCIENCE

Given the demonstrated biochemical and genetic roles of ANX1, ANX2, BUPS1, and BUPS2 in RALF4 and RALF19 sensing (3, 4), it will be interesting to test whether pollen-expressed LRXs form a supramolecular heteromeric ligand-binding complex with these CrRLK1Ls, or, alternatively, whether LRXs enable the stepwise transfer of RALF4 and RALF19 to ANX1 and ANX2 and/or to BUPS1 and BUPS2.

Recent studies illustrate that antagonistic secreted endogenous peptides can differentially regulate plant receptor kinases (12). In particular, RALF23 and RALF17 differentially regulate immune signaling mediated by FER (7). Interestingly, Ge et al. hypothesized that pollen tube integrity mediated by the autocrine RALF4 and RALF19 signaling may be inactivated by paracrine signals (i.e., signals from different cell types) to enable pollen tube rupture upon reception by the ovule. They thus chemically synthesized RALF peptides that are preferentially expressed in ovules, and found that RALF34, most likely secreted from the synergid cells (part of the ovule), induced pollen tube bursting (3). Notably, RALF34 binds to the ectodomain of BUPS1, BUPS2, ANX1, and ANX2, and RALF34 outcompetes RALF4 and RALF19 binding to BUPS1 and ANX1. Additional synergid-derived RALF peptides are likely involved in such regulation, as no fertilization defects could be observed when RALF34 alone was genetically inactivated (3). Together, this indicates that ovule-produced RALF34 is a paracrine signal that disrupts RALF4- and RALF19-mediated pollen tube integrity to enable the release of sperm cells. Whether RALF34 similarly affects RALF4- and RALF19-LRX binding remains to be tested.

FER is required to generate an appropriate “ovule environment” for pollen tube reception and rupture (13); thus, it will be interesting to identify whether a similar antagonistic regulation by RALF peptides also occurs. Whether RALF34 production and secretion by the ovule is FER-dependent will also be an interesting hypothesis to test. RALF34 was recently shown to regulate lateral root initiation (14), and it is possible that this process may engage in a similar (paracrine) antagonistic mechanism with other RALF peptides binding to as yet uncharacterized CrRLK1Ls.

Ge et al. and Mecchia et al. advance our understanding of the complex signaling mechanisms underlying plant fertilization. In addition, the reported antagonistic role of RALF34 further illustrates the important molecular dialogue occurring between the male and female gametophytes. Excitingly, these studies have far-reaching implications beyond plant reproduction. Indeed, it is becoming increasingly clear that several CrRLK1Ls, including ANX1 and ANX2, regulate many aspects of a plant's life, ranging from growth to development and responses to the environment (5, 7, 15). Therefore, it will be interesting to test whether these CrRLK1Ls also form heteromeric complexes with additional CrRLK1Ls and/or LRXs to perceive specific RALF ligands. Future structural studies should finally clarify the molecular basis of RALF perception by this important family of malectin-domain–containing receptor kinases, which regulate a myriad of processes in plants.

References and Notes

Acknowledgments: Our research is supported by the Gatsby Charitable Foundation and the European Research Council (grant “PHOSPHinnATE”). M.S. is supported by the Deutsche Forschungsgemeinschaft (STE 2448/1).
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