Central-cell-produced attractants control fertilization recovery.

Animal fertilization relies on hundreds of sperm racing toward the egg, whereas, in angiosperms, only two sperm cells are delivered by a pollen tube to the female gametes (egg cell and central cell) for double fertilization. However, unsuccessful fertilization under this one-pollen-tube design can be detrimental to seed production and plant survival. To mitigate this risk, unfertilized-gamete-controlled extra pollen tube entry has been evolved to bring more sperm cells and salvage fertilization. Despite its importance, the underlying molecular mechanism of this phenomenon remains unclear. In this study, we report that, in Arabidopsis, the central cell secretes peptides SALVAGER1 and SALVAGER2 in a directional manner to attract pollen tubes when the synergid-dependent attraction fails or is terminated by pollen tubes carrying infertile sperm cells. Moreover, loss of SALs impairs the fertilization recovery capacity of the ovules. Therefore, this research uncovers a female gamete-attraction system that salvages seed production for reproductive assurance.

Homeostasis of flavonoids and triterpenoids most likely modulates starch metabolism for pollen tube penetration in rice.

In angiosperms, the timely delivery of sperm cell nuclei by pollen tube (PT) to the ovule is vital for double fertilization. Penetration of PT into maternal stigma tissue is a critical step for sperm cell nuclei delivery, yet little is known about the process. Here, a male-specific and sporophytic mutant xt6, where PTs are able to germinate but unable to penetrate the stigma tissue, is reported in Oryza sativa. Through genetic study, the causative gene was identified as Chalcone synthase (OsCHS1), encoding the first enzyme in flavonoid biosynthesis. Indeed, flavonols were undetected in mutant pollen grains and PTs, indicating that the mutation abolished flavonoid biosynthesis. Nevertheless, the phenotype cannot be rescued by exogenous application of quercetin and kaempferol as reported in maize and petunia, suggesting a different mechanism exists in rice. Further analysis showed that loss of OsCHS1 function disrupted the homeostasis of flavonoid and triterpenoid metabolism and led to the accumulation of triterpenoid, which inhibits significantly α-amylase activity, amyloplast hydrolysis and monosaccharide content in xt6, these ultimately impaired tricarboxylic acid (TCA) cycle, reduced ATP content and lowered the turgor pressure as well. Our findings reveal a new mechanism that OsCHS1 modulates starch hydrolysis and glycometabolism through modulating the metabolic homeostasis of flavonoids and triterpenoids which affects α-amylase activity to maintain PT penetration in rice, which contributes to a better understanding of the function of CHS1 in crop fertility and breeding.

Osmoregulation determines sperm cell geometry and integrity for double fertilization in flowering plants.

Distinct from the motile flagellated sperm of animals and early land plants, the non-motile sperm cells of flowering plants are carried in the pollen grain to the female pistil. After pollination, a pair of sperm cells are delivered into the embryo sac by pollen tube growth and rupture. Unlike other walled plant cells with an equilibrium between internal turgor pressure and mechanical constraints of the cell walls, sperm cells wrapped inside the cytoplasm of a pollen vegetative cell have only thin and discontinuous cell walls. The sperm cells are uniquely ellipsoid in shape, although it is unclear how they maintain this shape within the pollen tubes and after release. In this study, we found that genetic disruption of three endomembrane-associated cation/H+ exchangers specifically causes sperm cells to become spheroidal in hydrated pollens of Arabidopsis. Moreover, the released mutant sperm cells are vulnerable and rupture before double fertilization, leading to failed seed set, which can be partially rescued by depletion of the sperm-expressed vacuolar water channel. These results suggest a critical role of cell-autonomous osmoregulation in adjusting the sperm cell shape for successful double fertilization in flowering plants.

POD1-SUN-CRT3 chaperone complex guards the ER sorting of LRR receptor kinases in Arabidopsis.

Protein sorting in the secretory pathway is essential for cellular compartmentalization and homeostasis in eukaryotic cells. The endoplasmic reticulum (ER) is the biosynthetic and folding factory of secretory cargo proteins. The cargo transport from the ER to the Golgi is highly selective, but the molecular mechanism for the sorting specificity is unclear. Here, we report that three ER membrane localized proteins, SUN3, SUN4 and SUN5, regulate ER sorting of leucine-rich repeat receptor kinases (LRR-RKs) to the plasma membrane. The triple mutant sun3/4/5 displays mis-sorting of these cargo proteins to acidic compartments and therefore impairs the growth of pollen tubes and the whole plant. Furthermore, the extracellular LRR domain of LRR-RKs is responsible for the correct sorting. Together, this study reports a mechanism that is important for the sorting of cell surface receptors.

Nucleolar histone deacetylases HDT1, HDT2, and HDT3 regulate plant reproductive development.

Nucleolus is a membrane-less organelle where ribosomes are assembled, and ribosomal RNAs (rRNAs) transcribed and processed. The assembled ribosomes composed of ribosomal proteins and rRNAs synthesize proteins for cell survival. In plants, the loss of nucleolar ribosomal proteins often causes gametophytically or embryonically lethality. The amount of rRNAs are under stringent regulation according to demand and partially switched off by epigenetic modifications. However, the molecular mechanism for the selective activation or silencing is still unclear, and the transcriptional coordination of rRNAs and ribosomal proteins is also unknown. Here, we report the critical role of three Arabidopsis nucleolar proteins HDT1, HDT2, and HDT3 in fertility and transcription of rDNAs and rRNA processing-related genes through histone acetylation. This study highlights the important roles of transcriptional repression of ribosome biogenesis-related genes for plant reproductive development.

Plasma membrane H+ -ATPases-mediated cytosolic proton gradient regulates pollen tube growth.

The polar growth of pollen tubes is essential for the delivery of sperm cells during fertilization in angiosperms. How this polar growth is regulated has been a long-standing question. An in vitro pharmacological assay previously implicated proton flux in pollen tube growth, although genetic and cellular supporting evidence was lacking. Here, we report that protons form a gradient from the pollen tube tip to the shank region and this gradient is generated by three members of Arabidopsis H+ -ATPases (AHAs). Genetic analysis suggested that these AHAs are essential for pollen tube growth, thus providing new insight into the regulation of polar growth.

Integration of ovular signals and exocytosis of a Ca2+ channel by MLOs in pollen tube guidance.

The spatiotemporal regulation of Ca2+ channels at the plasma membrane in response to extracellular signals is critical for development, stress response and reproduction, but is poorly understood. During flowering-plant reproduction, pollen tubes grow directionally to the ovule, which is guided by ovule-derived signals and dependent on Ca2+ dynamics. However, it is unknown how ovular signals are integrated with cytosolic Ca2+ dynamics in the pollen tube. Here, we show that MILDEW RESISTANCE LOCUS O 5 (MLO5), MLO9 and MLO15 are required for pollen tube responses to ovular signals in Arabidopsis thaliana. Phenotypically distinct from the ovule-bypass phenotype of previously identified mutants, mlo5 mlo9 double-mutant and mlo5 mlo9 mlo15 triple-mutant pollen tubes twist and pile up after sensing the ovular cues. Molecular studies reveal that MLO5 and MLO9 selectively recruit Ca2+ channel CNGC18-containing vesicles to the plasma membrane through the R-SNARE proteins VAMP721 and VAMP722 in trans mode. This study identifies members of the conserved seven transmembrane MLO family (expressed in the pollen tube) as tethering factors for Ca2+ channels, reveals a novel mechanism of molecular integration of extracellular ovular cues and selective exocytosis, and sheds light on the general regulation of MLO proteins in cell responses to environmental stimuli.

LOT regulates TGN biogenesis and Golgi structure in plants.

Trans-Golgi Network (TGN) is an essential organelle in eukaryotic cells. It acts not only as the sorting station of trafficking cargoes, but also as a signaling hub. In plant cells, TGN simultaneously takes the role of early endosome (EE) and contributes to the endocytic recycling. We recently characterized the first Golgi-localized protein Loss of TGNs (LOT) that is critical for TGN biogenesis and demonstrated its role during pollen tube growth in Arabidopsis. We also showed that the homozygous lot plant is dwarf and smaller than the wild type plant. As LOT is a single-copy gene and shows ubiquitous expression pattern, knowledge of its role in vegetative tissues, besides the pollen, is important for understanding the regulation of TGN/EE dynamics and signaling in plant development. Here, in this short communication, we present data to show that LOT also regulates TGN formation and Golgi structure in root meristem cells, and is critical for the elongation of hypocotyl and stamen filament.

Golgi-localized LOT regulates trans-Golgi network biogenesis and pollen tube growth.

The trans-Golgi network (TGN) is an essential tubular-vesicular organelle derived from the Golgi and functions as an independent sorting and trafficking hub within the cell. However, the molecular regulation of TGN biogenesis remains enigmatic. Here we identified an Arabidopsis mutant loss of TGN (lot) that is defective in TGN formation and sterile due to impaired pollen tube growth in the style. The mutation leads to overstacking of the Golgi cisternae and significant reduction in the number of TGNs and vesicles surrounding the Golgi in pollen, which is corroborated by the dispersed cytosolic distribution of TGN-localized proteins. Consistently, deposition of extracellular pectin and plasma membrane localization of kinases and phosphoinositide species are also impaired. Subcellular localization analysis suggests that LOT is localized on the periphery of the Golgi cisternae, but the mutation does not affect the localization of Golgi-resident proteins. Furthermore, the yeast complementation result suggests that LOT could functionally act as a component of the guanine nucleotide exchange factor (GEF) complex of small Rab GTPase Ypt6. Taken together, these findings suggest that LOT is a critical player for TGN biogenesis in the plant lineage.

Analysis of Peroxisome Biogenesis in Pollen by Confocal Microscopy and Transmission Electron Microscopy.

Peroxisome is an essential single-membrane bound organelle in most eukaryotic cells and functions in diverse cellular processes. De novo formation, division, and turnover of peroxisomes contribute to its biogenesis, morphology, and population regulation. In plants, peroxisome plays multiple roles, including metabolism, development, and stress response. Defective peroxisome biogenesis and development retard plant growth, adaption, and reproduction. Through tracing the subcellular localization of fluorescent reporter tagged matrix protein of peroxisome, fluorescence microscopy is a reliable and fast way to detect peroxisome biogenesis. Further fine-structural observation of peroxisome by TEM enables researchers to observe the detailed ultrastructure of its morphology and spatial contact with other organelles. Pollen grain is a specialized structure where two small sperm cells are enclosed in the cytoplasm of a large vegetative cell. Two features make pollen grain a good system to study peroxisome biogenesis: indispensable requirement of peroxisome for germination on the stigma and homogeneity. Here, we describe the methods of studying peroxisome biogenesis in Arabidopsis pollen grains by fluorescent live-imaging with confocal laser scanning microscopy (CLSM) and by DAB-staining based transmission electron microscopy (TEM).

Transmission Electron Microscopy (TEM) to Study Histology of Pollen and Pollen Tubes.

Here, we describe methods of transmission electron microscopy (TEM) based on conventional chemical fixation and high-pressure freezing (HPF) and freeze-substitution (FS) to examine the ultrastructure of Arabidopsis pollen grains and pollen tubes. Compared to other plant samples, such as root or leaf, pollen grains have thick pollen coat and cell wall which limit the permeation of fixative. Thus, it is difficult to obtain high-quality ultrastructural images of pollen. Moreover, pollen tube is very soft and the traditional procedure is too harsh to get an intact pollen tube sample. Up to now, there is no available mature protocol for TEM sample preparation of Arabidopsis pollen tube. Here, we describe the details and step-by-step procedures of chemical fixation, HPF, FS, and resin-embedding protocols for Arabidopsis pollen and pollen tube. In addition, we also provide a method on how to get longitudinal sections of pollen tubes.

The Arabidopsis Receptor Kinase ZAR1 Is Required for Zygote Asymmetric Division and Its Daughter Cell Fate.

Asymmetric division of zygote is critical for pattern formation during early embryogenesis in plants and animals. It requires integration of the intrinsic and extrinsic cues prior to and/or after fertilization. How these cues are translated into developmental signals is poorly understood. Here through genetic screen for mutations affecting early embryogenesis, we identified an Arabidopsis mutant, zygotic arrest 1 (zar1), in which zygote asymmetric division and the cell fate of its daughter cells were impaired. ZAR1 encodes a member of the RLK/Pelle kinase family. We demonstrated that ZAR1 physically interacts with Calmodulin and the heterotrimeric G protein Gß, and ZAR1 kinase is activated by their binding as well. ZAR1 is specifically expressed micropylarly in the embryo sac at eight-nucleate stage and then in central cell, egg cell and synergids in the mature embryo sac. After fertilization, ZAR1 is accumulated in zygote and endosperm. The disruption of ZAR1 and AGB1 results in short basal cell and an apical cell with basal cell fate. These data suggest that ZAR1 functions as a membrane integrator for extrinsic cues, Ca2+ signal and G protein signaling to regulate the division of zygote and the cell fate of its daughter cells in Arabidopsis.

A receptor heteromer mediates the male perception of female attractants in plants.

Sexual reproduction requires recognition between the male and female gametes. In flowering plants, the immobile sperms are delivered to the ovule-enclosed female gametophyte by guided pollen tube growth. Although the female gametophyte-secreted peptides have been identified to be the chemotactic attractant to the pollen tube, the male receptor(s) is still unknown. Here we identify a cell-surface receptor heteromer, MDIS1-MIK, on the pollen tube that perceives female attractant LURE1 in Arabidopsis thaliana. MDIS1, MIK1 and MIK2 are plasma-membrane-localized receptor-like kinases with extracellular leucine-rich repeats and an intracellular kinase domain. LURE1 specifically binds the extracellular domains of MDIS1, MIK1 and MIK2, whereas mdis1 and mik1 mik2 mutant pollen tubes respond less sensitively to LURE1. Furthermore, LURE1 triggers dimerization of the receptors and activates the kinase activity of MIK1. Importantly, transformation of AtMDIS1 to the sister species Capsella rubella can partially break down the reproductive isolation barrier. Our findings reveal a new mechanism of the male perception of the female attracting signals.

Functional characterization of the CKRC1/TAA1 gene and dissection of hormonal actions in the Arabidopsis root.

Cytokinin (CK) influences many aspects of plant growth and development, and its function often involves intricate interactions with other phytohormones such as auxin and ethylene. However, the molecular mechanisms underlying the role of CK and its interactions with other growth regulators are still poorly understood. Here we describe the isolation and characterization of the Arabidopsis CK-induced root curling 1 (ckrc1) mutant. CKRC1 encodes a previously identified tryptophan aminotransferase (TAA1) involved in the indole-3-pyruvic acid (IPA) pathway of indole-3-acetic acid (IAA) biosynthesis. The ckrc1 mutant exhibits a defective root gravitropic response (GR) and an increased resistance to CK in primary root growth. These defects can be rescued by exogenous auxin or IPA. Furthermore, we show that CK up-regulates CKRC1/TAA1 expression but inhibits polar auxin transport in roots in an AHK3/ARR1/12-dependent and ethylene-independent manner. Our results suggest that CK regulates root growth and development not only by down-regulating polar auxin transport, but also by stimulating local auxin biosynthesis.

Dual-rhodamine urea derivative, a novel chemidosimeter for Hg(II) and its application in imaging Hg(II) in living cells.

The synthesis and spectral properties of a chemidosimeter 1,4-di[2-(6-ethylamino-3-ethylimino-2,7-dimethyl-3H-xanthen-9-yl) benzoic acid (aminomethyl)-3-phenylthiourea] benzene (1) for Hg(II) ions are reported, and it has been demonstrated that 1 can be used as a fluorescent probe for monitoring Hg(II) ions in living cells.

Novel mode of action of polybia-MPI, a novel antimicrobial peptide, in multi-drug resistant leukemic cells.

As the frequent emergency of resistant tumor cells during treatment, the development of new agents with new modes of action attracts a great deal of interest. Polybia-MPI was a short cationic alpha-helical amphiphilic peptide that has selective toxicity toward cancer cells but no hemolytic activity. Its target selectivity is based on the binding preference to membranes containing anionic phospholipids by electrostatic driving. Its ability to make PI and trypan blue permeate into tumor cells at the same rate (within minutes), suggests a killing mechanism that involves plasma membrane perturbation. SEM and confocal microscopy experiments verified that the cell died as a result of acute injury and bursting, suggesting necrosis. As compared to the conventional chemotherapy, polybia-MPI targets at the cell membrane rather than enters into the cell to exert its action. So it is difficult for tumor cells to develop resistance to polybia-MPI during treatment and its action is not affected by the common multi-drug resistant mechanism. Although this is an initial study that looked at its in vitro activity rather than the in vivo activity, with the increasing resistance of conventional chemotherapy, polybia-MPI may offer a novel therapeutic strategy in the treatment of multi-drug resistant cancer.