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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.

Background Tiller number is a factor determining panicle number and grain yield in wheat (Triticum aestivum). Auxin plays an important role in the regulation of branch production. PIN-FORMED 1 (PIN1), an auxin efflux carrier, plays a role in the regulation of tiller number in rice (Oryza sativa); however, little is known on the roles of PIN1 in wheat. Results Nine homologs of TaPIN1 genes were identified in wheat, of which TaPIN1-6 genes showed higher expression in the stem apex and young leaf in wheat, and the TaPIN1-6a protein was localized in the plasma membrane. The down-expression of TaPIN1s increased the tiller number in TaPIN1-RNA interference (TaPIN1-RNAi) transgenic wheat plants, indicating that auxin might mediate the axillary bud production. By contrast, the spikelet number, grain number per panicle, and the 1000-grain weight were decreased in the TaPIN1-RNAi transgenic wheat plants compared with those in the wild type. In summary, a reduction of TaPIN1s expression increased the tiller number and grain yield per plant of wheat. Conclusions Phylogenetic analysis and protein structure of nine TaPIN1 proteins were analyzed, and subcellular localization of TaPIN1-6a was located in the plasma membrane. Knock-down expression of TaPIN1 genes increased the tiller number of transgenic wheat lines. Our study suggests that TaPIN1s is required for the regulation of grain yield in wheat.

Noncoding RNA (ncRNA), such as microRNA (miRNAs), long ncRNAs (lncRNAs), and circular RNAs (circRNAs) play roles in plant growth, development, and response to environmental cues via diverse manners...

Background N6-Methyladenosine (m6A) is the most widespread RNA modification that plays roles in the regulation of genes and genome stability. YT521-B homology (YTH) domain-containing RNA-binding proteins are important RNA binding proteins that affect the fate of m6A-containing RNA by binding m6A. Little is known about the YTH genes in common wheat (Triticum aestivum L.), one of the most important crops for humans. Results A total of 39 TaYTH genes were identified in common wheat, which are comprised of 13 homologous triads, and could be mapped in 18 out of the 21 chromosomes. A phylogenetic analysis revealed that the TaYTHs could be divided into two groups: YTHDF (TaDF) and YTHDC (TaDC). The TaYTHs in the same group share similar motif distributions and domain organizations, which indicates functional similarity between the closely related TaYTHs. The TaDF proteins share only one domain, which is the YTH domain. In contrast, the TaDCs possess three C3H1-type zinc finger repeats at their N-termini in addition to their central YTH domain. In TaDFs, the predicated aromatic cage pocket that binds the methylysine residue of m6A is composed of tryptophan, tryptophan, and tryptophan (WWW). In contrast, the aromatic cage pocket in the TaDCs is composed of tryptophan, tryptophan, and tyrosine (WWY). In addition to the general aspartic acid or asparagine residue used to form a hydrogen bond with N1 of m6A, histidine might be utilized in some TaDFb proteins. An analysis of the expression using both online RNA-Seq data and quantitative real-time PCR verification revealed that the TaDFa and TaDFb genes are highly expressed in various tissues/organs compared with that of TaDFcs and TaDCs. In addition, the expression of the TaYTH genes is changed in response to various abiotic stresses. Conclusions In this study, we identified 39 TaYTH genes from common wheat. The phylogenetic structure, chromosome distribution, and patterns of expression of these genes and their protein structures were analyzed. Our results provide a foundation for the functional analysis of TaYTHs in the future.

Plant genomes encode numerous proteins with obscure features (POFs) that lack recognized domains or motifs. However, there is little functional information for POFs even in Arabidopsis because biochemical, physiological, and genetic assay are required for the functional annotations of POFs. Here, we identified a small gene family, the endoplasmic reticulum-localized POF (ERP) family, in Arabidopsis. Phylogenetic analysis revealed that the number of ERP family members was conserved in the plant kingdom, suggesting strong selective pressure was imposed on ERP family during plant evolution. No recognizable domains were identified in the predicted ERP proteins, except for the N-terminal signal peptide. ERPs were found to be widely expressed during Arabidopsis development and showed endoplasmic reticulum localization. It was reported that ERP1 is an inositol-1,4,5-trisphosphate 5-phosphatase (5PTase), but ERP1 could not substitute for At5PTase12 in precocious pollen germination, indicating that ERP1 did not have the similar functions as At5PTase12 in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] metabolism. Further studies are needed to dissect the functions of ERP family proteins in Arabidopsis development.

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.

In flowering plants, pollen germination on the stigma and pollen tube growth in pistil tissues are critical for sexual plant reproduction, which are involved in the interactions between pollen/pollen tube and pistil tissues. GPI-anchored proteins (GPI-APs) are located on the external surface of the plasma membrane and function in various processes of sexual plant reproduction. The evidences suggest that GPI-APs participate in endosome machinery, Ca2+ oscillations, the development of the transmitting tract, the maintenance of the integrity of pollen tube, the enhancement of interactions of the receptor-like kinase (RLK) and ligand, and guidance of the growth of pollen tube, and so on. In this review, we will summarize the recent progress on the roles of GPI-APs in the interactions between pollen/pollen tube and pistil tissues during pollination, such as pollen germination on the stigma, pollen tube growth in the transmitting tract, pollen tube guidance to the ovule, and pollen tube reception in the embryo sac. We will also discuss the future outlook of GPI-APs in the interactions between pollen/pollen tube and pistil tissues.

The male–female interactions in pollination mediate pollen hydration and germination, pollen tube growth and fertilization. Reactive oxygen species (ROS) derived from both male and female tissues play regulatory roles for the communication between the pollen/pollen tube and female tissues at various stages, such as pollen hydration and germination on the stigma, pollen tube growth in the pistil and pollen tube reception in the female gametophyte. In this minireview, we primarily summarize the recent progress on the roles of ROS signaling in male–female interactions during pollination and discuss several ROS-regulated downstream signaling pathways for these interactions. Furthermore, several ROS-involved downstream pathways are outlined, such as Ca2+ signaling, cell wall cytomechanics, the redox modification of CRP, and cell PCD. At the end, we address the roles of ROS in pollen tube guidance and fertilization as future questions that merit study.

Pollen hydration is a critical step that determines pollen germination on the stigma. KINβγ is a plant-specific subunit of the SNF1-related protein kinase 1 complex (SnRK1 complex). In pollen of the Arabidopsis kinβγ 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 kinβγ mutant pollen by RNA-seq and found the expression of inward shaker K+ channel SPIK was down-regulated in the kinβγ 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 inflorescence architecture of grass crops affects the number of kernels and final grain yield. Great progress has been made in genetic analysis of rice inflorescence development in the past decades. However, the advances in wheat largely lag behind those in rice due to the repetitive and polyploid genomes of wheat. In view of the similar branching patterns and developmental characteristics between rice and wheat, the studies on inflorescence architecture in rice will facilitate related studies in wheat in the future. Here, we review the developmental regulation of inflorescences in rice and wheat and highlight several pathways that potentially regulate the inflorescence architecture of wheat.

Plant genomes encode numerous proteins with obscure features (POFs) that lack recognized domains or motifs. However, there is little functional information for POFs even in Arabidopsis because biochemical, physiological, and genetic assay are required for the functional annotations of POFs. Here, we identified a small gene family, the endoplasmic reticulum-localized POF (ERP) family, in Arabidopsis. Phylogenetic analysis revealed that the number of ERP family members was conserved in the plant kingdom, suggesting strong selective pressure was imposed on ERP family during plant evolution. No recognizable domains were identified in the predicted ERP proteins, except for the N-terminal signal peptide. ERPs were found to be widely expressed during Arabidopsis development and showed endoplasmic reticulum localization. It was reported that ERP1 is an inositol-1,4,5-trisphosphate 5-phosphatase (5PTase), but ERP1 could not substitute for At5PTase12 in precocious pollen germination, indicating that ERP1 did not have the similar functions as At5PTase12 in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] metabolism. Further studies are needed to dissect the functions of ERP family proteins in Arabidopsis development.

Highlight RID1 interacts with GFA1 to direct and regulate the splicing and expression of the genes required for female gametophyte development in Arabidopsis., ROOT INITIATION DEFECTIVE 1 (RID1) is an Arabidopsis DEAH/RHA RNA helicase. It functions in hypocotyl de-differentiation, de novo meristem formation, and cell specification of the mature female gametophyte (FG). However, it is unclear how RID1 regulates FG development. In this study, we observed that mutations to RID1 disrupted the developmental synchrony and retarded the progression of FG development. RID1 exhibited RNA helicase activity, with a preference for unwinding double-stranded RNA in the 3′ to 5′ direction. Furthermore, we found that RID1 interacts with GAMETOPHYTIC FACTOR 1 (GFA1), which is an integral protein of the spliceosome component U5 small nuclear ribonucleoprotein (snRNP) particle. Substitution of specific RID1 amino acids (Y266F and T267I) inhibited the interaction with GFA1. In addition, the mutated RID1 could not complement the seed-abortion phenotype of the rid1 mutant. The rid1 and gfa1 mutants exhibited similar abnormalities in pre-mRNA splicing and down-regulated expression of some genes involved in FG development. Our results suggest that an interaction between RID1 and the U5 snRNP complex regulates essential pre-mRNA splicing of the genes required for FG development. This study provides new information regarding the mechanism underlying the FG developmental process.

Pollen hydration is a critical step that determines pollen germination on the stigma. KINβγ is a plant-specific subunit of the SNF1-related protein kinase 1 complex (SnRK1 complex). In pollen of the Arabidopsis kinβγ 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 kinβγ mutant pollen by RNA-seq and found the expression of inward shaker K

Background Phytohormone synergies and signaling interdependency are important topics in plant developmental biology. Physiological and genetic experimental evidence for phytohormone crosstalk has been accumulating and a genome-scale enzyme correlation model representing the Arabidopsis metabolic pathway has been published. However, an integrated molecular characterization of phytohormone crosstalk is still not available. Results A novel modeling methodology and advanced computational approaches were used to construct an enzyme-based Arabidopsis phytohormone crosstalk network (EAPCN) at the biosynthesis level. The EAPCN provided the structural connectivity architecture of phytohormone biosynthesis pathways and revealed a surprising result; that enzymes localized at the highly connected nodes formed a consecutive metabolic route. Furthermore, our analysis revealed that the transcription factors (TFs) that regulate enzyme-encoding genes in the consecutive metabolic route formed structures, which we describe as circular control units operating at the transcriptional level. Furthermore, the downstream TFs in phytohormone signal transduction pathways were found to be involved in the circular control units that included the TFs regulating enzyme-encoding genes. In addition, multiple functional enzymes in the EAPCN were found to be involved in ion and pH homeostasis, environmental signal perception, cellular redox homeostasis, and circadian clocks. Last, publicly available transcriptional profiles and a protein expression map of the Arabidopsis root apical meristem were used as a case study to validate the proposed framework. Conclusions Our results revealed multiple scales of coupled mechanisms in that hormonal crosstalk networks that play a central role in coordinating internal developmental processes with environmental signals, and give a broader view of Arabidopsis phytohormone crosstalk. We also uncovered potential key regulators that can be further analyzed in future studies. Electronic supplementary material The online version of this article (doi:10.1186/s12918-016-0333-9) contains supplementary material, which is available to authorized users.

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. KINβγ 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 KINβγ was a cytoplasm- and nucleus-localized protein in the vegetative cells of pollen grains in Arabidopsis. The pollen of the Arabidopsis kinβγ mutant could not germinate on stigma, although it germinated normally in vitro. Further analysis revealed the hydration of kinβγ 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 kinβγ 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 kinβγ 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 KINβγ 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., Author Summary After landing on the stigma, pollen grains germinate and create pollen tubes following adhesion and hydration processes, during which pollen–stigma interactions determine whether the pollen grains can germinate on the stigma. In recent years, the interaction mechanisms between the pollen and stigma have been studied extensively at the cellular and molecular level in self-incompatibility systems. However, few studies have focused on pollen–stigma interactions during self-compatible pollination. Arabidopsis thaliana provides an excellent system to study the interaction mechanisms between the pollen and stigma during self-compatible pollination. KINβγ is a plant-specific subunit of the SNF1-related protein kinase 1 complex. In this study, we identified an Arabidopsis kinβγ mutant showing defective pollen germination on the surface of the stigma but not on the culture medium, which resulted from the compromised hydration of pollen on the stigma. Further analysis revealed that the biogenesis of mitochondria and peroxisomes was impaired in this mutant, which reduced the levels of reactive oxygen species (ROS) in pollen. Application of H2O2 recovered the capability of pollen to undergo hydration in vitro. These results suggest that ROS signaling is involved in the regulation of pollen–stigma interactions during pollination. This study provides new insights into the mechanism underlying pollen–stigma interactions in self-compatible plant species.

The reorganization of actin filaments (AFs) and vacuoles in guard cells is involved in the regulation of stomatal movement. However, it remains unclear whether there is any interaction between the reorganization of AFs and vacuolar changes during stomatal movement. Here, we report the relationship between the reorganization of AFs and vacuolar fusion revealed in pharmacological experiments, and characterizing stomatal opening in actin-related protein 2 (arp2) and arp3 mutants. Our results show that cytochalasin-D-induced depolymerization or phalloidin-induced stabilization of AFs leads to an increase in small unfused vacuoles during stomatal opening in wild-type (WT) Arabidopsis plants. Light-induced stomatal opening is retarded and vacuolar fusion in guard cells is impaired in the mutants, in which the reorganization and the dynamic parameters of AFs are aberrant compared with those of the WT. In WT, AFs tightly surround the small separated vacuoles, forming a ring that encircles the boundary membranes of vacuoles partly fused during stomatal opening. In contrast, in the mutants, most AFs and actin patches accumulate abnormally around the nuclei of the guard cells, which probably further impair vacuolar fusion and retard stomatal opening. Our results suggest that the reorganization of AFs regulates vacuolar fusion in guard cells during stomatal opening.

Angiosperm stigma supports compatible pollen germination and tube growth, resulting in fertilization and seed production. Stigmas are mainly divided into two types, dry and wet, according to the absence or presence of exudates on their surfaces. Here, we used 2DE and MS to identify proteins specifically and preferentially expressed in the stigmas of maize (Zea Mays, dry stigma) and tobacco (Nicotiana tabacum, wet stigma), as well as proteins rinsed from the surface of the tobacco stigma. We found that the specifically and preferentially expressed proteins in maize and tobacco stigmas share similar distributions in functional categories. However, these proteins showed important difference between dry and wet stigmas in a few aspects, such as protein homology in "signal transduction" and "lipid metabolism," relative expression levels of proteins containing signal peptides and proteins in "defense and stress response." These different features might be related to the specific structures and functions of dry and wet stigmas. The possible roles of some stigma-expressed proteins were discussed. Our results provide important information on functions of proteins in dry and wet stigmas and reveal aspects of conservation and divergence between dry and wet stigmas at the proteomic level.

Changes in actin dynamics represent the primary response of the plant cell to extracellular signaling. Recent studies have now revealed that actin remodeling is involved in abiotic stress tolerance in plants. In our current study, the relationship between the changes in actin dynamics and the reactive oxygen species (ROS) level at the initial stages of salt stress was investigated in the elongation zone of the Arabidopsis root tip. We found that a 200 mM NaCl treatment disrupted the dynamics of the actin filaments within 10 min and increased the ROS levels in the elongation zone cells of the Arabidopsis root tip. We further found that the NADPH oxidase activity inhibitor, diphenyleneiodonium, treatment blocked this ROS increase under salt stress conditions. The roles of actin dynamics and the NADPH oxidases in ROS generation were further analyzed using the actin-specific agents, latrunculin B (Lat-B) and jasplakinolide (Jasp), and mutants of Arabidopsis NADPH oxidase AtrbohC. Lat-B and Jasp promote actin depolymerization and polymerization, respectively, and both were found to enhance the ROS levels following NaCl treatment. However, this response was abolished in the atrbohC mutants. Our present results thus demonstrate that actin dynamics are involved in regulating the ROS level in Arabidopsis root under salt stress conditions. Key message Salt stress disrupts the dynamics of the actin filaments in Arabidopsis in the short term which are involved in regulating the ROS levels that arise under salt stress conditions via the actions of the AtrbohC.

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 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. Electronic supplementary material The online version of this article (doi:10.1007/s11103-011-9806-9) contains supplementary material, which is available to authorized users.

Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.

Inflorescence regeneration in vitro provides a simplified approach for the study of inflorescence development. In this study, high frequency of regenerated inflorescences was established using Arabidopsis stage-10 pistil as the explants on the inducing medium containing the 2 mg/L zeatin and 0.01 mg/L indole-3-acetic acid. TERMINAL FLOWER 1 (TFL1) expression was detected in callus at 6 days after transferred to inducing medium, and LEAFY (LFY) expression was detectable subsequently, suggesting that both genes play important roles as they function on inflorescence development in the plant. To investigate the formation of the stem cell organizing center, we examined the WUSCHEL (WUS) and CLAVATA3 (CLV3) expression within callus during inflorescence regeneration. WUS signals start to accumulate on callus at 4 days after induction, and then, the CLV3 signals are induced on callus at 5 days on the inflorescence-inducing medium. The expression domain of WUS is below that of CLV3, indicating that the patterns of the organizing center and stem cell formation are similar to that in zygotic and somatic embryogenesis. However, more cells of the organizing center were observed within callus than pro-embryo, suggesting that inflorescence differentiation requires more cells of the organizing center. Furthermore, it was found that the WUS expression is controlled by the ratio of cytokinin with auxin. The results suggest that other factors besides WUS and CLV3 are required for inflorescence regeneration.

Pollination is one of the most important steps during fertilization and sexual reproduction in plants, and numerous cell-cell interaction events occur between the pistil and the pollen grain/tube during this process. The pollen-stigma interaction is a highly selective process which leads to compatible or incompatible pollination. Previous studies in Solanaceae, Papaveraceae, and Brassicaceae provided some important insights into pollen-stigma recognition in self-incompatible systems. In recent years, considerable data have been available regarding pollen-stigma interaction during self-compatible pollination. In this review, we focus on discussing current knowledge on stigma factors that regulate pollen-stigma interaction in self-compatible systems in comparison with self-incompatible systems.

Cytokinin is an essential regulator of numerous plant growth and developmental processes. However, less is known about the mechanisms of cytokinin-regulated floral development. In the present study, we found that flower-specific elevation of cytokinin through transgenic expression of an Arabidopsis ATP/ADP isopentenyltransferase 4 (AtIPT4) gene under the control of the APETALA1 (AP1) promoter lead to floral developmental alterations. These changes included promotion of the number of flowers and abnormal development of flowers, which were correlated with enlarged inflorescence and flower meristems. Genome-wide expression profiling revealed that a large number of genes were responsive to increased cytokinin levels, including this first report that the expression of CUP-SHAPED COTYLEDON2 (CUC2) and CUC3 is elevated by cytokinin. Further analysis showed that mutation of cuc2 or cuc3 attenuates the phenotypes caused by the AP1Colon, two colonsAtIPT4 transgene. Mutation of the cytokinin receptors Arabidopsis histidine kinase 2 (AHK2) and AHK3 nearly abolished the transgene phenotypes and the enhanced CUC2 and CUC3 transcription induced by cytokinin. Our results indicate that the overproduced cytokinin in flower primordia results in alteration of floral development mainly through AHK2 and AHK3 signaling leading to increased expression of the effector genes CUC2 and CUC3. Thus, it is likely that the CUC2 and CUC3 expression mediated by AHK2 and AHK3 signaling may play roles in regulation of flower development.

Vacuoles and actin filaments are important cytoarchitectures involved in guard cell function. The changes in the morphology and number of vacuoles and the regulation of ion channel activity in tonoplast of guard cells are essential for stomatal movement. A number of studies have investigated the regulation of ion channels in animal and plant cells; however, little is known about the regulating mechanism for vacuolar dynamics in stomatal movement. Actin filaments of guard cells are remodelling with the changes in the stomatal aperture; however, the dynamic functions of actin filaments in stomatal movement remain elusive. In this paper, we summarize the recent developments in the understanding of the dynamics of actin filaments and vacuoles of guard cells during stomatal movement. All relevant studies suggest that actin filaments might be involved in stomatal movement by regulating vacuolar dynamics and the ion channels in tonoplast. The future study could be focused on the linker protein mediating the interaction between actin filaments and tonoplast, which will provide insights into the interactive function of actin and vacuole in stomatal movement regulation.

NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), a key enzyme in sorbitol metabolism, plays an important role in regulating sink strength and determining the quality of apple fruit. Understanding the tissue and subcellular localization of NAD-SDH is helpful for understanding sorbitol metabolism in the apple. In this study, two NAD-SDH cDNA sequences were isolated from apple fruits (Malus domestica Borkh cv. Starkrimson) and named MdSDH5 and MdSDH6. Immunohistochemical analysis revealed that NAD-SDH is distributed in both the flesh and the vascular tissue of the fruit, and the vascular tissue and mesophyll tissue in the young and old leaves, indicating that it is a ubiquitous protein expressed in both sink and source organs. Immunogold electron microscopy analysis demonstrated that NAD-SDH is localized mainly in the cytoplasm and chloroplast of the fruit and leaves. The chloroplast localization of NAD-SDH was confirmed by the transient expression of MdSDH5-GFP and MdSDH6-GFP in the mesophyll protoplast of Arabidopsis. NAD-SDH was also found in electron opaque deposits of vacuoles in young and mature leaves. These data show that NAD-SDH has different subcellular localizations in fruit and leaves, indicating that it might play a different role in sorbitol metabolism in different tissues of apple.

The ARGOS gene in Arabidopsis plays a key role in controlling plant organ size. To determine the function of it's ortholog in rice, a putative ARGOS orthologous gene from rice tissues was isolated and designated as OsARGOS. This gene has only one copy in the rice genome. OsARGOS transcripts were detected in most of rice tissues, particularly in the young tissues, and its expression was induced in rice seedlings by the application of either auxin or cytokinin. Arabidopsis plants expressing OsARGOS led to larger organs, such as leaves and siliques, compared with wild-type plants. Interestingly, the root growth was also enhanced in these transgenic Arabidopsis plants. Therefore, the biomass of the transgenic plants was significantly increased. Further analysis revealed that, different from the ARGOS and ARGOS-LIKE genes in Arabidopsis, the OsARGOS gene enlarged organ by an increase in both cell number and cell size. In addition, the transcript levels of several organ size-associated genes regulating either cell division or cell growth were upregulated in the transgenic Arabidopsis plants. We also transferred the OsARGOS gene to rice, but the transgenic plants did not show any changes in organ size compared with the control plants. It is likely that the function of OsARGOS in organ size control depends on other size regulators in rice. The expression of OsARGOS in Arabidopsis may activate the signaling pathways that control cell proliferation and cell expansion during the course of plant growth and development. Since the expression of OsARGOS causes organ enlargement, the potential application of this gene through genetic engineering may significantly improve the production of biomass in agricultural practice.

Actin filaments in guard cells and their dynamics function in regulating stomatal movement. In this study, the array and distribution of actin filaments in guard cells during stomatal movement were studied with two vital labeling, microinjection of alexa-phalloidin in Vicia faba and expression of GFP-mTn in tobacco. We found that the random array of actin filaments in the most of the closed stomata changed to a ring-like array after stomatal open. And actin filaments, which were throughout the cytoplasm of guard cells of closed stomata (even distribution), were mainly found in the cortical cytoplasm in the case of open stomata (cortical distribution). These results revealed that the random array and even distribution of actin filaments in guard cells may be required for keeping the closed stomata; similarly, the ring-like array and cortical distribution of actin filaments function in sustaining open stomata. Furthermore, we found that actin depolymerization, the trait of moving stomata, facilitates the transformation of actin array and distribution with stomatal movement. So, the depolymerization of actin filaments was favorable for the changes of actin array and distribution in guard cells and thus facilitated stomatal movement.

An alfalfa (Medicago sativa) full length cDNA, designated MsNHX1, was isolated by RT-PCR according to the homologous region of plant Na+/H+ antiporter genes. Southern blot analysis revealed that there was a small antiporter gene family in alfalfa genome. Sequence analysis indicated that MsNHX1 shared high homologies with other vacuolar Na+/H+ antiporters. Transient transfection of MsNHX1-GFP fusion gene indicated that MsNHX1 was targeted to vacuolar membrane. The transcription of MsNHX1 was up-regulated after high salinity treatment. The expression of MsNHX1 in yeast partly suppressed the salt-sensitive phenotype of yeast Na+/H+ antiporter mutant, indicating that MsNHX1 is a functional Na+/H+ antiporter of alfalfa. The physiological analysis demonstrated that the expression of MsNHX1 in Arabidopsis enhanced the resistance of transgenic plants to salt stress. These results suggest that MsNHX1 is a vacuolar Na+/H+ antiporter and functions in salt tolerance of plants.

Stomatal movement is important for plants to exchange gas with environment. The regulation of stomatal movement allows optimizing photosynthesis and transpiration. Changes in vacuolar volume in guard cells are known to participate in this regulation. However, little has been known about the mechanism underlying the regulation of rapid changes in guard cell vacuolar volume. Here, we report that dynamic changes in the complex vacuolar membrane system play a role in the rapid changes of vacuolar volume in Vicia faba guard cells. The guard cells contained a great number of small vacuoles and various vacuolar membrane structures when stomata closed. The small vacuoles and complex membrane systems fused with each other or with the bigger vacuoles to generate large vacuoles during stomatal opening. Conversely, the large vacuoles split into smaller vacuoles and generated many complex membrane structures in the closing stomata. Vacuole fusion inhibitor, (2s,3s)-trans-epoxy-succinyl-l-leucylamido-3-methylbutane ethyl ester, inhibited stomatal opening significantly. Furthermore, an Arabidopsis (Arabidopsis thaliana) mutation of the SGR3 gene, which has a defect in vacuolar fusion, also led to retardation of stomatal opening. All these results suggest that the dynamic changes of the tonoplast are essential for enhancing stomatal movement.

The perception and response of pollen tubes to the female guidance signals are crucial for directional pollen tube growth inside female tissues, which leads to successful reproduction. In pursuing the mechanisms underlying this biological process, we identified the Arabidopsis (Arabidopsis thaliana) abnormal pollen tube guidance1 (aptg1) mutant, whose pollen tubes showed compromised micropylar guidance. In addition to its male defect, the aptg1 mutant showed embryo lethality. APTG1 encodes a putative mannosyltransferase homolog to human PHOSPHATIDYLINOSITOL GLYCAN ANCHOR BIOSYNTHESIS B and yeast (Saccharomyces cerevisiae) GLYCOSYLPHOSPHATIDYLINOSITOL10 (GPI10), both of which are involved in the biosynthesis of GPI anchors. We found that APTG1 was expressed in most plant tissues, including mature pollen, pollen tubes, mature embryo sacs, and developing embryos. By fluorescence colabeling, we showed that APTG1 was localized in the endoplasmic reticulum, where GPI anchors are synthesized. Disruption of APTG1 affected the localization of COBRA-LIKE10, a GPI-anchored protein important for pollen tube growth and guidance. The results shown here demonstrate that APTG1 is involved in both vegetative and reproductive development in Arabidopsis, likely through processing and proper targeting of GPI-anchored proteins.

It is difficult to derive all qualitative proteomic and metabolomic experimental data in male (pollen tube) and female (pistil) reproductive tissues during pollination because of the limited sensitivity of current technology. In this study, genome-scale enzyme correlation network models for plants (Arabidopsis/maize) were constructed by analyzing the enzymes and metabolic routes from a global perspective. Then, we developed a data-driven computational pipeline using the “guilt by association” principle to analyze the transcriptional coexpression profiles of enzymatic genes in the consecutive steps for metabolic routes in the fast-growing pollen tube and stigma during pollination. The analysis identified an inferred pattern of pollen tube-stigma ethanol coupling. When the pollen tube elongates in the transmitting tissue (TT) of the pistil, this elongation triggers the mobilization of energy from glycolysis in the TT cells of the pistil. Energy-rich metabolites (ethanol) are secreted that can be taken up by the pollen tube, where these metabolites are incorporated into the pollen tube's tricarboxylic acid (TCA) cycle, which leads to enhanced ATP production for facilitating pollen tube growth. In addition, our analysis also provided evidence for the cooperation of kaempferol, dTDP-alpha-L-rhamnose and cell-wall-related proteins; phosphatidic-acid-mediated Ca2+ oscillations and cytoskeleton; and glutamate degradation IV for γ-aminobutyric acid (GABA) signaling activation in Arabidopsis and maize stigmas to provide the signals and materials required for pollen tube tip growth. In particular, the “guilt by association” computational pipeline and the genome-scale enzyme correlation network models (GECN) developed in this study was initiated with experimental “omics” data, followed by data analysis and data integration to determine correlations, and could provide a new platform to assist inachieving a deeper understanding of the co-regulation and inter-regulation model in plant research.

The reorganization of actin filaments (AFs) and vacuoles in guard cells is involved in the regulation of stomatal movement. However, it remains unclear whether there is any interaction between the reorganization of AFs and vacuolar changes during stomatal movement. Here, we report the relationship between the reorganization of AFs and vacuolar fusion revealed in pharmacological experiments, and characterizing stomatal opening in actin-related protein 2 (arp2) and arp3 mutants. Our results show that cytochalasin-D-induced depolymerization or phalloidin-induced stabilization of AFs leads to an increase in small unfused vacuoles during stomatal opening in wild-type (WT) Arabidopsis plants. Light-induced stomatal opening is retarded and vacuolar fusion in guard cells is impaired in the mutants, in which the reorganization and the dynamic parameters of AFs are aberrant compared with those of the WT. In WT, AFs tightly surround the small separated vacuoles, forming a ring that encircles the boundary membranes of vacuoles partly fused during stomatal opening. In contrast, in the mutants, most AFs and actin patches accumulate abnormally around the nuclei of the guard cells, which probably further impair vacuolar fusion and retard stomatal opening. Our results suggest that the reorganization of AFs regulates vacuolar fusion in guard cells during stomatal opening.

After flower pollination, a programmed process called abscission occurs in which unwanted floral organs are actively shed from the main plant body. We found that a member of the DOF (for DNA binding with one finger) transcription factor family, Arabidopsis (Arabidopsis thaliana) DOF4.7, was expressed robustly in the abscission zone. The Arabidopsis 35S::AtDOF4.7 lines with constitutive expression of AtDOF4.7 exhibited an ethylene-independent floral organ abscission deficiency. In these lines, anatomical analyses showed that the formation of the abscission zone was normal. However, dissolution of the middle lamella failed to separate between the cell walls. AtDOF4.7 was identified as a nucleus-localized transcription factor. This protein had both in vitro and in vivo binding activity to typical DOF cis-elements in the promoter of an abscission-related polygalacturonase (PG) gene, PGAZAT. Overexpression of AtDOF4.7 resulted in down-regulation of PGAZAT. AtDOF4.7 interacted with another abscission-related transcription factor, Arabidopsis ZINC FINGER PROTEIN2. Taken together, our results suggest that AtDOF4.7 participates in the control of abscission as part of the transcription complex that directly regulates the expression of cell wall hydrolysis enzymes.

Plant helicases are known to be involved in salinity and low-temperature tolerance. However, a functional involvement of helicases in the antioxidative response of plants has not been described. We have isolated a DEAD-box-containing cDNA sequence from Medicago sativa (alfalfa) that is a homolog of the pea DNA helicase 45 (PDH45) and named it M. sativa helicase 1 (MH1). Transient transfection of 35S::MH1-GFP to onion epidermis revealed that MH1 was localized in the nucleus. Expression of MH1 was detected in roots, stems and leaves of alfalfa. Furthermore, real-time PCR analysis revealed that mannitol, NaCl, methyl viologen and abscisic acid induced the expression of MH1. The ectopic expression of MH1 in Arabidopsis improved seed germination and plant growth under drought, salt and oxidative stress. The capacity for osmotic adjustment, superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities and proline content were also elevated in the transgenic Arabidopsis plants. Our results suggest that MH1 responds to reactive oxygen species (ROS) and functions in drought and salt stress tolerance by enhancing the capacities for ROS scavenging and osmotic adjustment.

Experimental evidences support that the circadian rhythm regulates the transcription levels of genes encoding the enzymes involved in plant metabolism. However, there is no paper to refer the correlation of the circadian rhythms and the metabolic processes for facilitating pollen tube growth. In this study, we found that many central components of the circadian clock were highly enriched and specifically present in the in vivo grown Arabidopsis pollen tubes. Our analysis also identified the significant differentially expressed genes encoding co-expressed enzymes in the consecutive steps of fatty acid β-oxidation II, pentose phosphate pathway (oxidative branch) and phosphatidic acid biosynthesis pathway in the in vivo grown Arabidopsis pollen tubes during pollination. Thus, it is implicated that the circadian rhythms of pollen tube may be adjusted and have a greater probability of the direct or indirect functional relationship with enhanced intracellular Ca(2+) dynamics and ATP production for facilitating pollen tube growth in vivo.

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.

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.

There is increasing evidence for considerable interlinking between the responses to heat stress and oxidative stress, and recent researches suggest heat shock transcription factors (Hsfs) play an important role in linking heat shock with oxidative stress signals. In this paper, we present evidence that AtHsfA2 modulated expression of stress responsive genes and enhanced tolerance to heat and oxidative stress in Arabidopsis. Using Northern blot and quantitative RT-PCR analysis, we demonstrated that the expression of AtHsfA2 was induced by not only HS but also oxidative stress. By functional analysis of AtHsfA2 knockout mutants and AtHsfA2 overexpressing transgenic plants, we also demonstrated that the mutants displayed reduced the basal and acquired thermotolerance as well as oxidative stress tolerance but the overexpression lines displayed increased tolerance to these stress. The phenotypes correlated with the expression of some Hsps and APX1, ion leakage, H202 level and degree of oxidative injuries. These results showed that, by modulated expression of stress responsive genes, AtHsfA2 enhanced tolerance to heat and oxidative stress in Arabidopsis. So we suggest that AtHsfA2 plays an important role in linking heat shock with oxidative stress signals.