PALD encoding a lipid droplet-associated protein is critical for the accumulation of lipid droplets and pollen longevity in Arabidopsis.

Pollen longevity is critical for plant pollination and hybrid seed production, but few studies have focused on pollen longevity. In this study, we identified an Arabidopsis thaliana gene, Protein associated with lipid droplets (PALD), which is strongly expressed in pollen and critical for the regulation of pollen longevity. PALD was expressed specifically in mature pollen grains and the pollen tube, and its expression was upregulated under dry conditions. PALD encoded a lipid droplet (LD)-associated protein and its N terminus was critical for the LD localization of PALD. The number of LDs and diameter were reduced in pollen grains of the loss-of-function PALD mutants. The viability and germination of the mature pollen grains of the pald mutants were comparable with those of the wild-type, but after the pollen grains were stored under dry conditions, pollen germination and male transmission of the mutant were compromised compared with those of the wild-type. Our study suggests that PALD was required for the maintenance of LD quality in mature pollen grains and regulation of pollen longevity.

Pollen-specific gene SKU5-SIMILAR 13 enhances growth of pollen tubes in the transmitting tract in Arabidopsis.

Pollen tube growth and penetration in female tissues are essential for the transfer of sperm to the embryo sac during plant pollination. Despite its importance during pollination, little is known about the mechanisms that mediate pollen tube growth in female tissues. In this study, we identified an Arabidopsis thaliana pollen/pollen tube-specific gene, SKU5-SIMILAR 13 (SKS13), which was critical for the growth of pollen tubes in the transmitting tract. The SKS13 protein was distributed throughout the cytoplasm and pollen tube walls at the apical region. In comparison with wild-type pollen tubes, those of the sks13 mutants burst more frequently when grown in vitro. Additionally, the growth of sks13 pollen tubes was retarded in the transmitting tract, thereby resulting in decreased male fertility. The accumulation of pectin and cellulose in the cell wall of sks13 pollen tubes was altered, and the content of jasmonic acid (JA) in sks13 pollen was reduced. The pollen tubes treated with an inhibitor of JA biosynthesis grew much more slowly and had an altered distribution of pectin, which is similar to the pollen tube phenotypes of the SKS13 mutation. Our results suggest that SKS13 is essential for pollen tube growth in the transmitting tract by mediating the biosynthesis of JA that modifies the components of pollen tube cell walls.

Arabidopsis shaker pollen inward K+ channel SPIK functions in SnRK1 complex-regulated pollen hydration on the stigma.

Pollen hydration is a critical step that determines pollen germination on the stigma. KINßγ is a plant-specific subunit of the SNF1-related protein kinase 1 complex (SnRK1 complex). In pollen of the Arabidopsis kinßγ mutant, the levels of reactive oxygen species were decreased which lead to compromised hydration of the mutant pollen on the stigma. In this study, we analyzed gene expression in kinßγ mutant pollen by RNA-seq and found the expression of inward shaker K+ channel SPIK was down-regulated in the kinßγ pollen. Furthermore, we showed that the pollen hydration of the Arabidopsis spik mutant was defective on the wild-type stigma, although the mutant pollen demonstrated normal hydration in vitro. Additionally, the defective hydration of spik mutant pollen could not be rescued by the wild-type pollen on the stigma, indicating that the spik mutation deprived the capability of pollen absorption on the stigma. Our results suggest that the Arabidopsis SnRK1 complex regulates SPIK expression, which functions in determining pollen hydration on the stigma.

The Arabidopsis KINßγ Subunit of the SnRK1 Complex Regulates Pollen Hydration on the Stigma by Mediating the Level of Reactive Oxygen Species in Pollen.

Pollen-stigma interactions are essential for pollen germination. The highly regulated process of pollen germination includes pollen adhesion, hydration, and germination on the stigma. However, the internal signaling of pollen that regulates pollen-stigma interactions is poorly understood. KINßγ is a plant-specific subunit of the SNF1-related protein kinase 1 complex which plays important roles in the regulation of plant development. Here, we showed that KINßγ was a cytoplasm- and nucleus-localized protein in the vegetative cells of pollen grains in Arabidopsis. The pollen of the Arabidopsis kinßγ mutant could not germinate on stigma, although it germinated normally in vitro. Further analysis revealed the hydration of kinßγ mutant pollen on the stigma was compromised. However, adding water to the stigma promoted the germination of the mutant pollen in vivo, suggesting that the compromised hydration of the mutant pollen led to its defective germination. In kinßγ mutant pollen, the structure of the mitochondria and peroxisomes was destroyed, and their numbers were significantly reduced compared with those in the wild type. Furthermore, we found that the kinßγ mutant exhibited reduced levels of reactive oxygen species (ROS) in pollen. The addition of H2O2 in vitro partially compensated for the reduced water absorption of the mutant pollen, and reducing ROS levels in pollen by overexpressing Arabidopsis CATALASE 3 resulted in compromised hydration of pollen on the stigma. These results indicate that Arabidopsis KINßγ is critical for the regulation of ROS levels by mediating the biogenesis of mitochondria and peroxisomes in pollen, which is required for pollen-stigma interactions during pollination.

Identification of small secreted peptides (SSPs) in maize and expression analysis of partial SSP genes in reproductive tissues.

MAIN CONCLUSION: Maize 1,491 small secreted peptides were identified, which were classified according to the character of peptide sequences. Partial SSP gene expressions in reproductive tissues were determined by qRT-PCR. Small secreted peptides (SSPs) are important cell-cell communication messengers in plants. Most information on plant SSPs come from Arabidopsis thaliana and Oryza sativa, while little is known about the SSPs of other grass species such as maize (Zea mays). In this study, we identified 1,491 SSP genes from maize genomic sequences. These putative SSP genes were distributed throughout the ten maize chromosomes. Among them, 611 SSPs were classified into 198 superfamilies according to their conserved domains, and 725 SSPs with four or more cysteines at their C-termini shared similar cysteine arrangements with their counterparts in other plant species. Moreover, the SSPs requiring post-translational modification, as well as defensin-like (DEFL) proteins, were identified. Further, the expression levels of 110 SSP genes were analyzed in reproductive tissues, including male flower, pollen, silk, and ovary. Most of the genes encoding basal-layer antifungal peptide-like, small coat proteins-like, thioredoxin-like proteins, γ-thionins-like, and DEFL proteins showed high expression levels in the ovary and male flower compared with their levels in silk and mature pollen. The rapid alkalinization factor-like genes were highly expressed only in the mature ovary and mature pollen, and pollen Ole e 1-like genes showed low expression in silk. The results of this study provide basic information for further analysis of SSP functions in the reproductive process of maize.

Identification of genes specifically or preferentially expressed in maize silk reveals similarity and diversity in transcript abundance of different dry stigmas.

BACKGROUND: In plants, pollination is a critical step in reproduction. During pollination, constant communication between male pollen and the female stigma is required for pollen adhesion, germination, and tube growth. The detailed mechanisms of stigma-mediated reproductive processes, however, remain largely unknown. Maize (Zea mays L.), one of the world's most important crops, has been extensively used as a model species to study molecular mechanisms of pollen and stigma interaction. A comprehensive analysis of maize silk transcriptome may provide valuable information for investigating stigma functionality. A comparative analysis of expression profiles between maize silk and dry stigmas of other species might reveal conserved and diverse mechanisms that underlie stigma-mediated reproductive processes in various plant species. RESULTS: Transcript abundance profiles of mature silk, mature pollen, mature ovary, and seedling were investigated using RNA-seq. By comparing the transcriptomes of these tissues, we identified 1,427 genes specifically or preferentially expressed in maize silk. Bioinformatic analyses of these genes revealed many genes with known functions in plant reproduction as well as novel candidate genes that encode amino acid transporters, peptide and oligopeptide transporters, and cysteine-rich receptor-like kinases. In addition, comparison of gene sets specifically or preferentially expressed in stigmas of maize, rice (Oryza sativa L.), and Arabidopsis (Arabidopsis thaliana [L.] Heynh.) identified a number of homologous genes involved either in pollen adhesion, hydration, and germination or in initial growth and penetration of pollen tubes into the stigma surface. The comparison also indicated that maize shares a more similar profile and larger number of conserved genes with rice than with Arabidopsis, and that amino acid and lipid transport-related genes are distinctively overrepresented in maize. CONCLUSIONS: Many of the novel genes uncovered in this study are potentially involved in stigma-mediated reproductive processes, including genes encoding amino acid transporters, peptide and oligopeptide transporters, and cysteine-rich receptor-like kinases. The data also suggest that dry stigmas share similar mechanisms at early stages of pollen-stigma interaction. Compared with Arabidopsis, maize and rice appear to have more conserved functional mechanisms. Genes involved in amino acid and lipid transport may be responsible for mechanisms in the reproductive process that are unique to maize silk.

Metabolism and roles of phosphatidylinositol 3-phosphate in pollen development and pollen tube growth in Arabidopsis.

Phosphoinositides play important roles in eukaryotic cells, although they constitute a minor fraction of total cellular lipids. Specific kinases and phosphatases function on the regulation of phosphoinositide levels. Phosphatidylinositol 3-phosphate (PtdIns3P), a molecule of phosphoinositides regulates multiple aspects of plant growth and development. In this mini-review, we introduce and discuss the kinases and phosphatases involved in PtdIns3P metabolism and their roles in pollen development and pollen tube growth in Arabidopsis.

Arabidopsis AtVPS15 is essential for pollen development and germination through modulating phosphatidylinositol 3-phosphate formation.

Arabidopsis thaliana phosphatidylinositol 3-kinase (AtVPS34) functions in the development and germination of pollen by catalyzing the biosynthesis of phosphatidylinositol 3-phosphate (PI3P). In yeast, Vps15p is required for the membrane targeting and activity of Vps34. The expression of Arabidopsis thaliana VPS15 (AtVPS15), an ortholog of yeast Vps15, is mainly detected in pollen grains and pollen tubes. To determine its role in pollen development and pollen tube growth, we attempted to isolate the T-DNA insertion mutants of AtVPS15; however, homozygous lines of atvps15 were not obtained from the progeny of atvps15/+ heterozygotes. Genetic analysis revealed that the abnormal segregation is due to the failure of transmission of the atvps15 allele through pollen. Most pollen grains from the atvps15/+ genotype are viable, with normal exine structure and nuclei, but some mature pollen grains are characterized with unusual large vacuoles that are not observed in pollen grains from the wild AtVPS15 genotype. The germination ratio of pollen from the atvps15/+ genotype is about half when compared to that from the wild AtVPS15 genotype. When supplied with PI3P, in vitro pollen germination of the atvps15/+ genotype is greatly improved. Presumably, AtVPS15 functions in pollen development and germination by regulating PI3P biosynthesis in Arabidopsis.

[The ultracytochemical localization of ATPase activity in pollen tube and stigma of Fagopyrum esculentum after compatible and incompatible pollination].

Ultracytochemical localization of adenosine triphosphatase (ATPase) activity in stigmas, pollens and pollen tubes of Fagopyrum esculentum was performed with the cytochemical method of lead phosphate precipitation. The results were as follows: (1) Lower activities of ATPase appeared in stigma cells at 0.5 hour after compatible and incompatible pollination. Stigma surface and pollen grains attached on stigma showed higher ATPase activities after compatible pollination and lower or no activities after incompatible pollination at 0.5 hour. ATPase localized on endoplasmic reticula and sperm cell in pollen grain. (2) Lower ATPase activities appeared both in stigma cells and pollen tubes in style at 1.5 hours after incompatible pollination. Pollen tube stopped growing and the degeneration of its cytoplasma began; on the contrary, at 1.5 hours after compatible pollination, higher ATPase activities were detected both in stigma cells and pollen tube in style. ATPase localized mainly on plasmolemma, in cytoplasmic matrix of stigma cells and in mitochondria, dictyosome, plastid envelop, and on the wall of pollen tube as well. The present study indicated that the stop of pollen tube growth after self-incompatibility of Fagopyrum esculentum resulted not only from the nutrient starvation of pollen tube, but also from its metabolic disorder in incompatible style.

[The ultrastructures of storage protein accumulation in aleurone layer and cotyledons of Fagopyrum esculentum].

The ultrastructures of storage protein accumulation in cotyledons and aleurone layer in buckwheat (Fagopyrum esculentum Monch) were investigated by electron microscopy. (1) On 15 days after anthesis (DAA), there are many dictyosomes and vacuoles accumulating protein in cytoplasm of the outer layer endosperm cells. These cells containing abundant aleurone grains (1-2 microm in diameter) from vacuoles accumulated storage protein form aleurone layer where division of aleurone grain is also observed on 25 DAA. (2) On 20 DAA, protein begins to accumulate in vacuoles of cotyledon cells. At the early stage of protein deposition, vacuoles are becoming smaller ones by ingrowth of tonoplast or by pinch-off. A multitude of ribosomes, many dictyosomes and electron-dense vesicles (0.1-0.7 microm in diameter) coated with membrane are observed in cytoplasm of cotyledon cells at every stage of protein accumulation. Originated from the separation of saccules containing protein at the fringe of dictyosome, these vesicles (0.1-0.2 microm in diameter) probably increase their volume by fusion each other. They play the leading role in transporting storage protein into vacuoles that become PSVs in cotyledon cells of buckwheat. On 25 DAA, there are many PSVs (1-3 microm in diameter) and some vesicles (0.1-0.7 microm) filled with protein in mature cotyledon cells. These vesicles may be also play a part in accumulating storage protein in mature buckwheat seed.