Search

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

Nitric oxide (NO), a bioactive signaling molecule, serves as an antioxidant and anti-stress agent under abiotic stress. A hydroponics experiment was conducted to investigate the effects of sodium nitroprusside (SNP), a NO donor, on tomato seedlings exposed to 50 μmol L −1 CuCl 2 . The results show that copper is primarily stored in the soluble cell sap fraction in the roots, especially after treatment with Cu+SNP treatment, which accounted for 66.2% of the total copper content. The copper concentration gradually decreased from the roots to the leaves. In the leaves, exogenous NO induces the storage of excess copper in the cell walls. Copper stress decreases the proportion of copper integrated with pectates and proteins, but exogenous NO remarkably reverses this trend. The alleviating effect of NO is blocked by hemoglobin. Thus, exogenous NO is likely involved in the regulation of the subcellular copper concentrations and its chemical forms under copper stress. Although exogenous NO inhibited the absorption and transport of excess copper to some extent, the copper accumulation in tomato seedlings significantly increased under copper stress. The use of exogenous NO to enhance copper tolerance in some plants is a promising method for copper remediation.

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.

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.

The anthocyanin pigment extracted from green-wheat-bran was studied to identify its antioxidant activity. The antioxidant activities of the pigment were evaluated by anti-lipid peroxidation, total antioxidant activity (TAA), superoxide anion radical scavenging activity (SARSA), active oxygen scavenging activity (AOSA), and DPPH (1, 1-diphenyl-2 picrylhydrazyl free radical) radical scavenging activity. The results showed that the pigment had higher antioxidant activity and TAA, SARSA, AOSA and DPPH•scavenging activities at a certain concentration than Vc (antiscorbutic vitamin, vitamin C), and the capacity increased with the increase of pigment concentration. Its TAA was 51.06 U mL^(-1), 1.73 times of Vc, and SARSA 18025.21 U mU^(-1), 2.26% higher than Vc, and AOSA 3776.31 U mL^(-1), 1.24 times of Vc. As to the DPPH•scavenging activity of the pigment, there was a trend that higher concentration performed higher activity significantly improved with the company of Vc. The pigment showed significant antioxidant activities evaluated by different assays. Results will provide a better understanding on antioxidant activity of green wheat and allow the screening or breeding of green wheat varieties with higher antioxidant activity for food processing.

Simple sequence repeat (SSR) or microsatellite marker is a valuable tool for several purposes, such as mapping, fingerprinting, and breeding. In the present study, an inter-simple sequence repeat (ISSR)-PCR technique was applied for developing SSR markers in non-heading Chinese cabbage ( Brassica rapa ). A total of 190 SSRs were obtained. Among these, AG or CT (54.7%) was the most frequent repeat, followed by AC or GT (31.6%) of the microsatellites. The average number of the SSRs length array was 16 and 10 times, respectively. Based on the determined SSR sequences, 143 SSR primer pairs were designed to evaluate their transferabilities among the related species of Brassica. The number of alleles produced per marker averaged 2.91, and the polymorphism information content (PIC) value ranged from 0 to 0.863 with an average of 0.540. Monomorphism was observed in 16 primer pairs. The transferability percentage in CC genome was higher than in BB genome. More loci occurred in the BBCC genome. This result supported the hypothesis that BB genome was divergent from A and C genomes, and AA and CC genomes were relatively close. The polymorphic primers can be exploited for further evolution, fingerprinting, and variety identification.

To improve agronomic traits of partial waxy wheat, crossing between Chinese Baihuomai and wheat cultivars PH85-16, Jinan 17, and Yannong 15 was performed. The progeny plants were further backcrossed to these cultivars as recurrent parents for five generations. To get homozygous plants with the null allele at the Wx-D1 locus, self-pollination was carried out in the BC(5)F(1) generation. Through another three generations, 6 partial waxy wheat lines were obtained, which had similar agronomic performance as their recurrent parents and carried the null allele at the Wx-D1 locus. In each generation, the Wx-D1 locus was identified by a PCR-based DNA marker and the agronomic traits were examined in progeny plants. The results from this study indicate that the use of backcrossing with a PCR-based DNA marker was useful in waxy wheat breeding. These partial waxy wheat lines can be used in field production.

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.

The grain color of wheat (Triticum aestivum L.) is an important characteristic in crop production. Dihydroflavonol 4-reductase genes (DFR) encode the key enzyme dihydroflavonol 4-reductase, which is involved in the pigmentation of plant tissues. To investigate the molecular mechanism of anthocyanin deposition in grains of wheat, we determined the expression of the wheat DFR gene in purple grains of cultivar Heimai 76. The results showed that DFR transcripts were localized in the seed coat of purple grains rather than in the pericarp, whereas anthocyanins were accumulated in both tissues of purple grains, suggesting that anthocyanin deposition was mainly regulated at the transcriptional level. Overexpression of the TaDFR-A gene in Arabidopsis showed that TaDFR-A was responsible for the pigmentation of Arabidopsis plant tissues, indicating TaDFR-A gene has the same role in Arabidopsis. (Managing editor: Li-Hui ZHAO)

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.

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.