Xian Sheng Zhang

Arabidopsis COBRA-LIKE 10, a GPI-anchored protein, mediates directional growth of pollen tubes

Successful reproduction of flowering plants requires constant communication between female tissues and growing pollen tubes. Female cells secrete molecules and peptides as nutrients or guidance cues for fast and directional tube growth, which is executed by dynamic changes of intracellular activities within pollen tubes. Compared with the extensive interest in female cues and intracellular activities of pollen tubes, how female cues are sensed and interpreted intracellularly in pollen is poorly understood. We show here that COBL10, a glycosylphosphatidylinositol (GPI)-anchored protein, is one component of this pollen tube internal machinery. Mutations in COBL10 caused gametophytic male sterility due to reduced pollen tube growth and compromised directional sensing in the female transmitting tract. Deposition of the apical pectin cap and cellulose microfibrils was disrupted in cobl10 pollen tubes. Pollen tube localization of COBL10 at the apical plasma membrane is critical for its function and relies on proper GPI processing and its C-terminal hydrophobic residues. GPI-anchored proteins are widespread cell sensors in mammals, especially during egg-sperm communication. Our results that COBL10 is critical for directional growth of pollen tubes suggest that they play critical roles in cell-cell communications in plants.

Type-B ARABIDOPSIS RESPONSE REGULATORs Is Critical to the Specification of Shoot Stem Cell Niche by Dual Regulation of WUSCHEL

Type-B ARRs specify the shoot stem cell niche by directly activating WUS transcription and repressing the expression of YUCs that indirectly promote WUS induction. Plants are known for their capacity to regenerate the whole body through de novo formation of apical meristems from a mass of proliferating cells named callus. Exogenous cytokinin and auxin determine cell fate for the establishment of the stem cell niche, which is the vital step of shoot regeneration, but the underlying mechanisms remain unclear. Here, we show that type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), critical components of cytokinin signaling, activate the transcription of WUSCHEL (WUS), which encodes a key regulator for maintaining stem cells. In parallel, type-B ARRs inhibit auxin accumulation by repressing the expression of YUCCAs, which encode a key enzyme for auxin biosynthesis, indirectly promoting WUS induction. Both pathways are essential for de novo regeneration of the shoot stem cell niche. In addition, the dual regulation of type-B ARRs on WUS transcription is required for the maintenance of the shoot apical meristem in planta. Thus, our results reveal a long-standing missing link between cytokinin signaling and WUS regulator, and the findings provide critical information for understanding cell fate specification.

ABNORMAL POLLEN TUBE GUIDANCE1, an Endoplasmic Reticulum-Localized Mannosyltransferase Homolog of GLYCOSYLPHOSPHATIDYLINOSITOL10 in Yeast and PHOSPHATIDYLINOSITOL GLYCAN ANCHOR BIOSYNTHESIS B in Human, Is Required for Arabidopsis Pollen Tube Micropylar Guidance and Embryo Development

An Arabidopsis mannosyltransferase modulates micropylar guidance of pollen tube and early embryo development by processing and targeting of GPI-anchored proteins. 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.

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.

Disrupted actin dynamics trigger an increment in the reactive oxygen species levels in the Arabidopsis root under salt stress

AbstractChanges 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 200xa0mM NaCl treatment disrupted the dynamics of the actin filaments within 10xa0min 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.n 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.

The tae-miR408-Mediated Control of TaTOC1 Genes Transcription Is Required for the Regulation of Heading Time in Wheat

A wheat microRNA promotes heading by negatively regulating expression of homologs of the clock gene TOC1. Timing of flowering is not only an interesting topic in developmental biology, but it also plays a significant role in agriculture for its effects on the maturation time of seed. The hexaploid wheat (Triticum aestivum) is one of the most important crop species whose flowering time, i.e. heading time, greatly influences yield. However, it remains unclear whether and how microRNAs regulate heading time in it. In our current study, we identified the tae-miR408 in wheat and its targets in vivo, including Triticum aestivum TIMING OF CAB EXPRESSION-A1 (TaTOC-A1), TaTOC-B1, and TaTOC-D1. The tae-miR408 levels were reciprocal to those of TaTOC1s under long-day and short-day conditions. Wheat plants with a knockdown of TaTOC1s via RNA interference and overexpression of tae-miR408 showed early-heading phenotype. Furthermore, TaTOC1s expression was down-regulated by the tae-miR408 in the hexaploid wheat. In addition, other important agronomic traits in wheat, such as plant height and flag leaf angle, were regulated by both tae-miR408 and TaTOC1s. Thus, our results suggested that the tae-miR408 functions in the wheat heading time by mediating TaTOC1s expression, and the study provides important new information on the mechanism underlying heading time regulation in wheat.

The Arabidopsis KINβγ 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. 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.

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

AbstractMain conclusionMaize 1,491 small secreted peptides were identified, which were classified according to the character of peptide sequences. PartialSSPgene expressions in reproductive tissues were determined by qRT-PCR.n 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.

Endogenous auxin biosynthesis and de novo root organogenesis.

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Thioredoxin-Mediated ROS Homeostasis Explains Natural Variation in Plant Regeneration

Thioredoxin-dependent redox modification regulates plant regeneration via modulation of ROS homeostasis. Plant regeneration is fundamental to basic research and agricultural applications. The regeneration capacity of plants varies largely in different genotypes, but the reason for this variation remains elusive. Here, we identified a novel thioredoxin DCC1 in determining the capacity of shoot regeneration among Arabidopsis (Arabidopsis thaliana) natural variation. Loss of function of DCC1 resulted in inhibited shoot regeneration. DCC1 was expressed mainly in the inner tissues of the callus and encoded a functional thioredoxin that was localized in the mitochondria. DCC1 protein interacted directly with CARBONIC ANHYDRASE2 (CA2), which is an essential subunit of the respiratory chain NADH dehydrogenase complex (Complex I). DCC1 regulated Complex I activity via redox modification of CA2 protein. Mutation of DCC1 or CA2 led to reduced Complex I activity and triggered mitochondrial reactive oxygen species (ROS) production. The increased ROS level regulated shoot regeneration by repressing expression of the genes involved in multiple pathways. Furthermore, linkage disequilibrium analysis indicated that DCC1 was a major determinant of the natural variation in shoot regeneration among Arabidopsis ecotypes. Thus, our study uncovers a novel regulatory mechanism by which thioredoxin-dependent redox modification regulates de novo shoot initiation via the modulation of ROS homeostasis and provides new insights into improving the capacity of plant regeneration.