Redundant function of the Arabidopsis phosphatidylinositol 4-phosphate 5-kinase genes PIP5K4-6 is essential for pollen germination.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is a key enzyme producing the signaling lipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 ] in eukaryotes. Although PIP5K genes are reported to be involved in pollen tube germination and growth, the essential roles of PIP5K in these processes remain unclear. Here, we performed a comprehensive genetic analysis of the Arabidopsis thaliana PIP5K4, PIP5K5, and PIP5K6 genes and revealed that their redundant function is essential for pollen germination. Pollen with the pip5k4pip5k5pip5k6 triple mutation was sterile, while pollen germination efficiency and pollen tube growth were reduced in the pip5k6 single mutant and further reduced in the pip5k4pip5k6 and pip5k5pip5k6 double mutants. YFP-fusion proteins, PIP5K4-YFP, PIP5K5-YFP, and PIP5K6-YFP, which could rescue the sterility of the triple mutant pollen, preferentially localized to the tricolpate aperture area and the future germination site on the plasma membrane prior to germination. Triple mutant pollen grains under the germination condition, in which spatiotemporal localization of the PtdIns(4,5)P2 fluorescent marker protein 2xmCHERRY-2xPHPLC as seen in the wild type was abolished, exhibited swelling and rupture of the pollen wall, but neither the conspicuous protruding site nor site-specific deposition of cell wall materials for germination. These data indicate that PIP5K4-6 and their product PtdIns(4,5)P2 are essential for pollen germination, possibly through the establishment of the germination polarity in a pollen grain.

Antagonistic RALF peptides control an intergeneric hybridization barrier on Brassicaceae stigmas.

Pollen-pistil interactions establish interspecific/intergeneric pre-zygotic hybridization barriers in plants. The rejection of undesired pollen at the stigma is crucial to avoid outcrossing but can be overcome with the support of mentor pollen. The mechanisms underlying this hybridization barrier are largely unknown. Here, in Arabidopsis, we demonstrate that receptor-like kinases FERONIA/CURVY1/ANJEA/HERCULES RECEPTOR KINASE 1 and cell wall proteins LRX3/4/5 interact on papilla cell surfaces with autocrine stigmatic RALF1/22/23/33 peptide ligands (sRALFs) to establish a lock that blocks the penetration of undesired pollen tubes. Compatible pollen-derived RALF10/11/12/13/25/26/30 peptides (pRALFs) act as a key, outcompeting sRALFs and enabling pollen tube penetration. By treating Arabidopsis stigmas with synthetic pRALFs, we unlock the barrier, facilitating pollen tube penetration from distantly related Brassicaceae species and resulting in interspecific/intergeneric hybrid embryo formation. Therefore, we uncover a "lock-and-key" system governing the hybridization breadth of interspecific/intergeneric crosses in Brassicaceae. Manipulating this system holds promise for facilitating broad hybridization in crops.

Distinct chromatin signatures in the Arabidopsis male gametophyte.

Epigenetic reprogramming in the germline contributes to the erasure of epigenetic inheritance across generations in mammals but remains poorly characterized in plants. Here we profiled histone modifications throughout Arabidopsis male germline development. We find that the sperm cell has widespread apparent chromatin bivalency, which is established by the acquisition of H3K27me3 or H3K4me3 at pre-existing H3K4me3 or H3K27me3 regions, respectively. These bivalent domains are associated with a distinct transcriptional status. Somatic H3K27me3 is generally reduced in sperm, while dramatic loss of H3K27me3 is observed at only ~700 developmental genes. The incorporation of the histone variant H3.10 facilitates the establishment of sperm chromatin identity without a strong impact on resetting of somatic H3K27me3. Vegetative nuclei harbor thousands of specific H3K27me3 domains at repressed genes, while pollination-related genes are highly expressed and marked by gene body H3K4me3. Our work highlights putative chromatin bivalency and restricted resetting of H3K27me3 at developmental regulators as key features in plant pluripotent sperm.

RALF peptide signaling controls the polytubey block in Arabidopsis.

Fertilization of an egg by multiple sperm (polyspermy) leads to lethal genome imbalance and chromosome segregation defects. In Arabidopsis thaliana, the block to polyspermy is facilitated by a mechanism that prevents polytubey (the arrival of multiple pollen tubes to one ovule). We show here that FERONIA, ANJEA, and HERCULES RECEPTOR KINASE 1 receptor-like kinases located at the septum interact with pollen tube-specific RALF6, 7, 16, 36, and 37 peptide ligands to establish this polytubey block. The same combination of RALF (rapid alkalinization factor) peptides and receptor complexes controls pollen tube reception and rupture inside the targeted ovule. Pollen tube rupture releases the polytubey block at the septum, which allows the emergence of secondary pollen tubes upon fertilization failure. Thus, orchestrated steps in the fertilization process in Arabidopsis are coordinated by the same signaling components to guarantee and optimize reproductive success.

From birth to function: Male gametophyte development in flowering plants.

Male germline development in flowering plants involves two distinct and successive phases, microsporogenesis and microgametogenesis, which involve one meiosis followed by two rounds of mitosis. Many aspects of distinctions after mitosis between the vegetative cell and the male germ cells are seen, from morphology to structure, and the differential functions of the two cell types in the male gametophyte are differentially needed and required for double fertilization. The two sperm cells, carriers of the hereditary substances, depend on the vegetative cell/pollen tube to be delivered to the female gametophyte for double fertilization. Thus, the intercellular communication and coordinated activity within the male gametophyte probably represent the most subtle regulation in flowering plants to guarantee the success of reproduction. This review will focus on what we have known about the differentiation process and the functional diversification of the vegetative cell and the male germ cell, the most crucial cell types for plant fertility and crop production.

VPS18-regulated vesicle trafficking controls the secretion of pectin and its modifying enzyme during pollen tube growth in Arabidopsis.

In eukaryotes, homotypic fusion and vacuolar protein sorting (HOPS) as well as class C core vacuole/endosome tethering (CORVET) are evolutionarily conserved membrane tethering complexes that play important roles in lysosomal/vacuolar trafficking. Whether HOPS and CORVET control endomembrane trafficking in pollen tubes, the fastest growing plant cells, remains largely elusive. In this study, we demonstrate that the four core components shared by the two complexes, Vacuole protein sorting 11 (VPS11), VPS16, VPS33, and VPS18, are all essential for pollen tube growth in Arabidopsis thaliana and thus for plant reproduction success. We used VPS18 as a representative core component of the complexes to show that the protein is localized to both multivesicular bodies (MVBs) and the tonoplast in a growing pollen tube. Mutant vps18 pollen tubes grew more slowly in vivo, resulting in a significant reduction in male transmission efficiency. Additional studies revealed that membrane fusion from MVBs to vacuoles is severely compromised in vps18 pollen tubes, corroborating the function of VPS18 in late endocytic trafficking. Furthermore, vps18 pollen tubes produce excessive exocytic vesicles at the apical zone and excessive amounts of pectin and pectin methylesterases in the cell wall. In conclusion, this study establishes an additional conserved role of HOPS/CORVET in homotypic membrane fusion during vacuole biogenesis in pollen tubes and reveals a feedback regulation of HOPS/CORVET in the secretion of cell wall modification enzymes of rapidly growing plant cells.

AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube guidance.

Reproductive isolation is a prerequisite to form and maintain a new species. Multiple prezygotic and postzygotic reproductive isolation barriers have been reported in plants. In the model plant, Arabidopsis thaliana conspecific pollen tube precedence controlled by AtLURE1/PRK6-mediated signaling has been recently reported as a major prezygotic reproductive isolation barrier. By accelerating emergence of own pollen tubes from the transmitting tract, A. thaliana ovules promote self-fertilization and thus prevent fertilization by a different species. Taking advantage of a septuple atlure1null mutant, we now report on the role of AtLURE1/PRK6-mediated signaling for micropylar pollen tube guidance. Compared with wild-type (WT) ovules, atlure1null ovules displayed remarkably reduced micropylar pollen tube attraction efficiencies in modified semi-in vivo A. thaliana ovule targeting assays. However, when prk6 mutant pollen tubes were applied, atlure1null ovules showed micropylar attraction efficiencies comparable to that of WT ovules. These findings indicate that AtLURE1/PRK6-mediated signaling regulates micropylar pollen tube attraction in addition to promoting emergence of own pollen tubes from the transmitting tract. Moreover, semi-in vivo ovule targeting competition assays with the same amount of pollen grains from both A. thaliana and Arabidopsis lyrata showed that A. thaliana WT and xiuqiu mutant ovules are mainly targeted by own pollen tubes and that atlure1null mutant ovules are also entered to a large extent by A. lyrata pollen tubes. Taken together, we report that AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube attraction representing an additional prezygotic isolation barrier.

Sperm cells are passive cargo of the pollen tube in plant fertilization.

Sperm cells of seed plants have lost their motility and are transported by the vegetative pollen tube cell for fertilization, but the extent to which they regulate their own transportation is a long-standing debate. Here we show that Arabidopsis lacking two bHLH transcription factors produces pollen without sperm cells. This abnormal pollen mostly behaves like the wild type and demonstrates that sperm cells are dispensable for normal pollen tube development.

Four closely-related RING-type E3 ligases, APD1-4, are involved in pollen mitosis II regulation in Arabidopsis.

Ubiquitination of proteins is one of the critical regulatory mechanisms in eukaryotes. In higher plants, protein ubiquitination plays an essential role in many biological processes, including hormone signaling, photomorphogenesis, and pathogen defense. However, the roles of protein ubiquitination in the reproductive process are not clear. In this study, we identified four plant-specific RING-finger genes designated Aberrant Pollen Development 1 (APD1) to APD4, as regulators of pollen mitosis II (PMII) in Arabidopsis thaliana (L.). The apd1 apd2 double mutant showed a significantly increased percentage of bicellular-like pollen at the mature pollen stage. Further downregulation of the APD3 and APD4 transcripts in apd1 apd2 by RNA interference (RNAi) resulted in more severe abnormal bicellular-like pollen phenotypes than in apd1 apd2, suggesting that cell division was defective in male gametogenesis. All of the four genes were expressed in multiple stages at different levels during male gametophyte development. Confocal analysis using green florescence fusion proteins (GFP) GFP-APD1 and GFP-APD2 showed that APDs are associated with intracellular membranes. Furthermore, APD2 had E2-dependent E3 ligase activity in vitro, and five APD2-interacting proteins were identified. Our results suggest that these four genes may be involved, redundantly, in regulating the PMII process during male gametogenesis.

Arabidopsis AtVPS15 plays essential roles in pollen germination possibly by interacting with AtVPS34.

VPS15 protein is a component of the phosphatidylinositol 3-kinase complex which plays a pivotal role in the development of yeast and mammalian cells. The knowledge about the function of its homologue in plants remains limited. Here we report that AtVPS15, a homologue of yeast VPS15p in Arabidopsis, plays an essential role in pollen germination. Homozygous T-DNA insertion mutants of AtVPS15 could not be obtained from the progenies of self-pollinated heterozygous mutants. Reciprocal crosses between atvps15 mutants and wild-type Arabidopsis revealed that the T-DNA insertion was not able to be transmitted by male gametophytes. DAPI staining, Alexander's stain and scanning electron microscopic analysis showed that atvps15 heterozygous plants produced pollen grains that were morphologically indistinguishable from wild-type pollen, whereas in vitro germination experiments revealed that germination of the pollen grains was defective. GUS staining analysis of transgenic plants expressing the GUS reporter gene driven by the AtVPS15 promoter showed that AtVPS15 was mainly expressed in pollen grains. Finally, DUALmembrane yeast two-hybrid analysis demonstrated that AtVPS15 might interact directly with AtVPS34. These results suggest that AtVPS15 is very important for pollen germination, possibly through modulation of the activity of PI3-kinase.

Distinguishing transgenic from non-transgenic Arabidopsis plants by (1)H NMR-based metabolic fingerprinting.

We have recently reported the construction of an nuclear magnetic resonance (NMR)-based metabonomics study platform, Automics. To examine the application of Automics in transgenic plants, we performed metabolic fingerprinting analysis, i.e., (1)H NMR spectroscopy and multivariate analysis, on wild-type and transgenic Arabidopsis. We found that it was possible to distinguish wild-type from four transgenic plants by PLS-DA following application of orthogonal signal correction (OSC). Scores plot following OSC clearly demonstrates significant variation between the transgenic and non-transgenic groups, suggesting that the metabolic changes among wild-type and transgenic lines are possibly associated with transgenic event. We also found that the major contributing metabolites were some specific amino acids (i.e., threonine and alanine), which could correspond to the insertion of the selective marker BAR gene in the transgenic plants. Our data suggests that NMR-based metabonomics is an efficient method to distinguish fingerprinting difference between wild-type and transgenic plants, and can potentially be applied in the bio-safety assessment of transgenic plants.

Arabidopsis AtBECLIN 1/AtAtg6/AtVps30 is essential for pollen germination and plant development.

Pollen germination on the surface of compatible stigmatic tissues is an essential step for plant fertilization. Here we report that the Arabidopsis mutant bcl1 is male sterile as a result of the failure of pollen germination. We show that the bcl1 mutant allele cannot be transmitted by male gametophytes and no homozygous bcl1 mutants were obtained. Analysis of pollen developmental stages indicates that the bcl1 mutation affects pollen germination but not pollen maturation. Molecular analysis demonstrates that the failure of pollen germination was caused by the disruption of AtBECLIN 1. AtBECLIN 1 is expressed predominantly in mature pollen and encodes a protein with significant homology to Beclin1/Atg6/Vps30 required for the processes of autophagy and vacuolar protein sorting (VPS) in yeast. We also show that AtBECLIN 1 is required for normal plant development, and that genes related to autophagy, VPS and the glycosylphosphatidylinositol anchor system, were affected by the deficiency of AtBECLIN 1.

A subgroup of MYB transcription factor genes undergoes highly conserved alternative splicing in Arabidopsis and rice.

MYB transcription factor genes play important roles in many developmental processes and in various defence responses of plants. Two Arabidopsis R2R3-type MYB genes, AtMYB59 and AtMYB48, were found to undergo similar alternative splicing. Both genes have four distinctively spliced transcripts that encode either MYB-related proteins or R2R3-MYB proteins. An extensive BLAST search of the GenBank database resulted in finding and cloning two rice homologues, both of which were also found to share a similar alternative splicing pattern. In a semi-quantitative study, the expression of one splice variant of AtMYB59 was found to be differentially regulated in treatments with different phytohormones and stresses. GFP fusion protein analysis revealed that both of the two predicted nuclear localization signals (NLSs) in the R3 domain are required for localizing to the nucleus. Promoter-GUS analysis in transgenic plants showed that 5'-UTR is sufficient for the translation initiation of type 3 transcripts (encoding R2R3-MYB proteins), but not for type 2 transcripts (encoding MYB-related proteins). Moreover, a new type of non-canonical intron, with the same nucleotide repeats at the 5' and 3' splice sites, was identified. Thirty-eight Arabidopsis and rice genes were found to have this type of non-canonical intron, most of which undergo alternative splicing. These data suggest that this subgroup of transcription factor genes may be involved in multiple biological processes and may be transcriptionally regulated by alternative splicing.