GR1 and NTRA involved in pollen tube growth in the stigma of Arabidopsis.

MAIN CONCLUSION: Arabidopsis GR1 and NTRA function in pollen tube penetrating the stigma into the transmitting tract during pollination. During pollination, recognition between pollen (tube) and stigma mediates the hydration and germination of pollen, as well as the growth of the pollen tube on the stigma. Arabidopsis glutathione reductase 1 (GR1) and NADPH-dependent thioredoxin reductase A (NTRA) are involved in regulating cell redox hemostasis. Both GR1 and NTRA are expressed in pollen, but their roles in pollen germination and the growth of the pollen tube need further investigation. In this study, we performed pollination experiments and found that the Arabidopsis gr1/ + ntra/- and gr1/- ntra/ + double mutation compromised the transmission of male gametophytes. Pollen morphology and viability of the mutants did not show obvious abnormalities. Additionally, the pollen hydration and germination of the double mutants on solid pollen germination medium were comparable to those of the wild type. However, the pollen tubes with gr1 ntra double mutation were unable to penetrate the stigma and enter the transmitting tract when they grew on the surface of the stigma. Our results indicate that GR1 and NTRA play a role in regulating the interaction between the pollen tube and the stigma during pollination.

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.

Characterization of the ERP gene family in Arabidopsis thaliana.

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.

Phospholipase D engineering for improving the biocatalytic synthesis of phosphatidylserine.

Phosphatidylserine is widely used in food, health, chemical and pharmaceutical industries. The phospholipase D-mediated green synthesis of phosphatidylserine has attracted substantial attention in recent years. In this study, the phospholipase D was heterologously expressed in Bacillus subtilis, Pichia pastoris, and Corynebacterium glutamicum, respectively. The highest activity of phospholipase D was observed in C. glutamicum, which was 0.25 U/mL higher than these in B. subtilis (0.14 U/mL) and P. pastoris (0.22 U/mL). System engineering of three potential factors, including (1) signal peptides, (2) ribosome binding site, and (3) promoters, was attempted to improve the expression level of phospholipase D in C. glutamicum. The maximum phospholipase D activity reached 1.9 U/mL, which was 7.6-fold higher than that of the initial level. The enzyme displayed favorable transphosphatidylation activity and it could efficiently catalyze the substrates L-serine and soybean lecithin for synthesis of phosphatidylserine after optimizing the conversion reactions in detail. Under the optimum conditions (trichloromethane/enzyme solution 4:2, 8 mg/mL soybean lecithin, 40 mg/mL L-serine, and 15 mM CaCl2, with shaking under 40 °C for 10 h), the reaction process showed 48.6% of conversion rate and 1.94 g/L of accumulated phosphatidylserine concentration. The results highlight the use of heterologous expression, system engineering, and process optimization strategies to adapt a promising phospholipase D for efficient phosphatidylserine production in synthetic application.

Comparative Transcriptome Analysis Revealing the Effect of Light on Anthocyanin Biosynthesis in Purple Grains of Wheat.

To study the mechanism of anthocyanin-biosynthesis regulation, we examined light-regulated gene expression involved in anthocyanin biosynthesis in purple grains of wheat. Ten kinds of anthocyanins were identified from a purple-grained wheat cultivar by HPLC-ESI-MS/MS analysis. Libraries constructed from the total RNA of purple grains under light (L) or dark (D) conditions for 15 or 20 days were sequenced. In total, 1874 differentially expressed genes (DEGs) were identified in L20 vs L15, 1432 DEGs were identified in D20 vs D15, 862 DEGs were identified in D15 vs L15, and 1786 DEGs were identified in D20 vs L20. DEG functional enrichments suggested that light-signal transduction is critical to anthocyanin biosynthesis. The 911 DEGs referred to as light-regulated DEGs (LDEGs) involved a number of genes in anthocyanin biosynthesis, transcription regulation, sugar- and calcium-signaling pathways, and hormone metabolism. These findings laid the foundation for future studies on the regulatory mechanisms of anthocyanin biosynthesis in purple grains of wheat.

DNA METHYLTRANSFERASE1-mediated shoot regeneration is regulated by cytokinin-induced cell cycle in Arabidopsis.

DNA methylation plays a critical role in diverse biological processes of plants. Arabidopsis DNA METHYLTRANSFERASE1 (MET1) represses shoot regeneration by inhibiting WUSCHEL (WUS) expression, which is essential for shoot initiation. However, the upstream signals regulating MET1 expression during this process are unclear. We analyzed the signals regulating MET1 expression using a number of established strategies, such as genetic analysis, confocal microscopy, quantitative real-time PCR and chromatin immunoprecipitation. MET1 expression patterns underwent dynamic changes with the initiation of WUS during shoot regeneration. The cell cycle regulator E2FA was characterized as an upstream factor directly promoting MET1 expression. Moreover, cytokinin promoted MET1 expression partially by enhancing CYCD3 expression. Our findings reveal that MET1-mediated shoot regeneration is regulated by the cytokinin-induced cell cycle, and provide new insights into the regulation of DNA methylation in shoot regeneration.

Type-B ARRs Control Carpel Regeneration Through Mediating AGAMOUS Expression in Arabidopsis.

Plants are known for their capacity to regenerate organs, such as shoot, root and floral organs. Recently, a number of studies contributed to understanding the mechanisms of shoot and root regeneration. However, the mechanisms underlying floral organ regeneration are largely unknown. In this study, we established a carpel regeneration system in which two types of carpels were induced by exogenous cytokinin. For type I, all the floral organs in the regenerated inflorescence were transformed into carpels. For type II, carpels were generated directly from callus. The transcript level of AGAMOUS (AG), the carpel identity gene, was up-regulated during carpel induction. The expression signals of AG were detected in the initiating carpel primordia and regenerating carpels, and co-localized with those of two Type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), ARR1 and ARR10. Repression of either AG or type-B ARRs reduced carpel regeneration. Binding analyses showed that ARR1 and ARR10 directly bound to transcriptional regulatory regions of AG and positively regulated its expression. In addition, the expression of type-B ARRs overlapped with that of AG in the floral primordia in planta. Defects in type-B ARRs reduced the number of carpels. The results indicate that type-B ARRs control carpel regeneration through activating AG expression. Our results provide new information for understanding the mechanism of carpel formation.

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.

Microfilament Depolymerization Is a Pre-requisite for Stem Cell Formation During In vitro Shoot Regeneration in Arabidopsis.

De novo shoot regeneration is widely used in fundamental studies and agricultural applications. Actin microfilaments are involved in many aspects of plant cell division, cell morphogenesis and cell signal transduction. However, the function of actin microfilaments during de novo shoot regeneration is poorly understood. Here, we investigated the organization of actin microfilaments during this process and found that stem cell formation was associated with microfilament depolymerization. Furthermore, inhibition of microfilament depolymerization by phalloidin treatment or downregulation of actin depolymerizing factors (ADFs) restrained stem cell initiation and shoot regeneration. Inhibition of ADF expression affected the architecture of microfilaments during stem cell formation, and the polar transport and distribution of auxin were also disrupted. Together, our results demonstrate that organization of the microfilament cytoskeleton play important roles in stem cell formation and shoot meristem induction during shoot regeneration.

The Arabidopsis phytohormone crosstalk network involves a consecutive metabolic route and circular control units of transcription factors that regulate enzyme-encoding genes.

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.

Alleviating effects of exogenous NO on tomato seedlings under combined Cu and Cd stress.

To investigate the effect of NO on the different origin and regulation of oxidative stress of Cu and/or Cd, tomato seedlings were treated with Cu, Cd, or Cu + Cd in a nutrient solution culture system. The main effect of Cu(2+) was a significant reduction in root activity and nitrate reductase (NR) activity, which was similar to that under 50 µM Cd treatment, but promoted Cu accumulation. The supply of Cu under Cd treatment decreased Cd concentration, while not altered Cu concentration by contrast with Cu treatment, which is suggestive of a replacement of Cu(2+) with Cd(2+) and effective decrease in the boiotoxicity of 50 µM Cd(2+) to tomato seedlings. However, NO alleviated the restriction to NR activity significantly and made the biomass of tomato seedlings recover under Cd treatment, and also increased root activity under Cu and Cu + Cd treatment. Exogenous NO markedly reduced the absorption and transportation of Cu but did not obviously change the translocation of Cd to the aboveground parts under Cu + Cd treatment. Both metals induced lipid peroxidation via the decreasing activation of antioxidant enzymes. The antioxidant enzyme system worked differently under Cu, Cd, or Cu + Cd stress. The activities of peroxidase (POD) and catalase (CAT) were higher under single Cd stress than under the control. Meanwhile, Cu + Cd treatment decreased the activities of POD, superoxide dismutase (SOD), and ascorbic acid peroxidase (APX). Exogenous NO increased POD and SOD activities in the leaves and roots, and CAT activity in the roots under combined Cu and Cd stress. These results suggest that a different response and regulation mechanism that involves exogenous NO is present in tomato seedlings under Cu and Cd stress.

Expression of a putative alfalfa helicase increases tolerance to abiotic stress in Arabidopsis by enhancing the capacities for ROS scavenging and osmotic adjustment.

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.