A cytosolic NAD+-dependent GPDH from maize (ZmGPDH1) is involved in conferring salt and osmotic stress tolerance.

BACKGROUND: Plant glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to produce glycerol-3-phosphate (G-3-P), and plays a key role in glycerolipid metabolism as well as stress responses. RESULTS: In this study, we report the cloning, enzymatic and physiological characterization of a cytosolic NAD+-dependent GPDH from maize. The prokaryotic expression of ZmGPDH1 in E.coli showed that the enzyme encoded by ZmGPDH1 was capable of catalyzing the reduction of DHAP in the presence of NADH. The functional complementation analysis revealed that ZmGPDH1 was able to restore the production of glycerol-3-phosphate and glycerol in AtGPDHc-deficient mutants. Furthermore, overexpression of ZmGPDH1 remarkably enhanced the tolerance of Arabidopsis to salinity/osmotic stress by enhancing the glycerol production, the antioxidant enzymes activities (SOD, CAT, APX) and by maintaining the cellular redox homeostasis (NADH/NAD+, ASA/DHA, GSH/GSSG). ZmGPDH1 OE Arabidopsis plants also exhibited reduced leaf water loss and stomatal aperture under salt and osmotic stresses. Quantitative real-time RT-PCR analyses revealed that overexpression of ZmGPDH1 promoted the transcripts accumulation of genes involved in cellular redox homeostasis and ROS-scavenging system. CONCLUSIONS: Together, these data suggested that ZmGPDH1 is involved in conferring salinity and osmotic tolerance in Arabidopsis through modulation of glycerol synthesis, stomatal closure, cellular redox and ROS homeostasis.

Genome-wide characterization and expression profiling of diacylglycerol acyltransferase genes from maize.

Diacylglycerol acyltransferase (DGAT) catalyzes the only rate-limiting step in the pathway of plant oil (TAG) biosynthesis and is involved in plant development. In this study, five DGAT family members were identified from maize genome database. Phylogenetic analysis classified the ZmDGATs into type-I, II, and III clusters. Conserved functional domain analysis revealed that the proteins encoded by ZmDGAT1 contained conserved MBOAT domains, while two ZmDGAT2-encoding proteins harbored LPLAT domains. qRT-PCR analysis showed that ZmDGAT genes exhibited very high relative expression in developing seeds, especially at the early stage of seed development. Under various abiotic stress conditions, differential responses of ZmDGAT genes were observed. An overall significant induction of ZmDGAT genes under cold stress in leaves and a quick and strong response to osmotic stresses in roots were highlighted. This study provides useful information for understanding the roles of DGATs in oil accumulation and stress responses in higher plants.

Inhibition of NADPH oxidase increases defense enzyme activities and improves maize seed germination under Pb stress.

Contaminated soil accumulated high levels of Pb, which shows great risk to human health and crop growth. To alleviate Pb impaired seed germination and seedling growth, effects of three methods were compared. Here, effects of the heavy metal chelator EDTA, reactive oxygen species (ROS) scavenger (e.g. dimethylthiourea/DMTU, glutathione and melatonin), and specific inhibitors of NADPH oxidase or NOX (e.g. imidazole/IMZ and diphenylene iodonium/DPI), on maize seed germination and seedling growth were examined under Pb stress. IMZ and DPI increased seed germination by 1-2-fold under Pb stress, compared with less than 50% for ROS scavengers, while EDTA decreased germination. Pb-induced H2O2 accumulation was reduced more dramatically by IMZ than DMTU. Compared with DMTU, Pb-impaired SOD and CAT enzyme can be reversed more significantly by IMZ. Thus, inhibiting the NOX was more efficient than using ROS scavengers for improving seed germination under Pb stress. Compared with EDTA and ROS scavenger, IMZ and DPI treatment cannot protect seedling growth under Pb stress. In addition, IMZ is cheap and highly efficient, making it suitable for improving seed germination in Pb-polluted soil.

Omethoate treatment mitigates high salt stress inhibited maize seed germination.

Omethoate (OM) is a highly toxic organophophate insecticide, which is resistant to biodegradation in the environment and is widely used for pest control in agriculture. The effect of OM on maize seed germination was evaluated under salt stress. Salt (800mM) greatly reduced germination of maize seed and this could be reversed by OM. Additionally, H2O2 treatment further improved the effect of OM on seed germination. Higher H2O2 content was measured in OM treated seed compared to those with salt stress alone. Dimethylthiourea (DTMU), a specific scavenger of reactive oxygen species (ROS), inhibited the effect of OM on seed germination, as did IMZ (imidazole), an inhibitor of NADPH oxidase. Abscisic acid (ABA) inhibited the effect of OM on seed germination, whereas fluridone, a specific inhibitor of ABA biosynthesis, enhanced the effect of OM. Taken together, these findings suggest a role of ROS and ABA in the promotion of maize seed germination by OM under salt stress.

[Effects of Enhanced Ultraviolet B Irradiation on Photosynthetic and Antioxidant System of Sorghum Seedlings].

The UV-B radiation on the surface of our planet has been enhanced due to gradual thinning of ozone layer. The change of solar spectrum UV-B radiation will cause damage to all kinds of terrestrial plants at certain degree. In this paper, taking breeding sorghum (Sorghum bicolor (L.Moench))variety Longza No.5 as sample, 40 µW·cm-2 UV-B radiation treatment was conducted on sorghum seedlings at two-leaf and one-heart stage and different time courses; then after a 2 d recovering, photosynthetic parameters were measured with a photosynthetic apparatus; the activities of antioxidant enzymes were detected as well. Our results revealed that, as the dosages of UV-B increasing, leaf browning injury was aggravated, plants dwarfing and significantly were reduced fresh weight and dry weight were observed; anthocyanin content was significantly increased; chlorophyll and carotenoid content significantly were reduced and net photosynthetic rate and chlorophyll fluorescence parameters were decreased. Meanwhile, with the increase in UV-B dosages, stomatal conductance, intercellular CO2 concentration and transpiration rate showed "down - up - down" trend; the activities of SOD and GR presented "down - up" changes; activities of POD and CAT demonstrated "down - up - down", and APX, GPX showed an "up - down - up" pattern. It is worth to note that, under the four-dose treatment, a sharp decline in net photosynthesis in sorghum seedlings was observed at 6 h UV-B treatment (equals to 2.4 J·m-2), and an obvious turning point was also found for other photosynthetic parameters and activities of antioxidant enzymes at the same time point. In summary, the results indicated that the enhanced UV-B radiation directly accounted for the damages in photosynthesis system including photosynthetic pigment content, net photosynthetic rate and chlorophyll fluorescence parameters of sorghum; the antioxidant system showed different responses to UV-B radiation below or above 6 h treatment: ASA-GSH cycle was more sensitive to low-dose UV-B radiation, while high-dose UV-B radiation not only undermined the photosynthesis system, but also triggered plant enzymatic and non-enzymatic antioxidant systems, resulting in leaf browning and necrosis,biomass accumulation reduction, plant dwarfing and even death.

Molecular regulation of lipid metabolism in Suaeda salsa.

Suaeda salsa is remarkable for its high oil content and abundant unsaturated fatty acids. In this study, the regulatory networks on fatty acid and lipid metabolism were constructed by combining the de novo transcriptome and lipidome data. Differentially expressed genes (DEGs) associated with lipids biosynthesis pathways were identified in the S. salsa transcriptome. DEGs involved in fatty acid and glycerolipids were generally up-regulated in leaf tissues. DEGs for TAG assembly were enriched in developing seeds, while DEGs in phospholipid metabolic pathways were enriched in root tissues. Polar lipids were extracted from S. salsa tissues and analyzed by lipidomics. The proportion of galactolipid MGDG was the highest in S. salsa leaves. The molar percentage of PG was high in the developing seeds, and the other main phospholipids had higher molar percentage in roots of S. salsa. The predominant C36:6 molecular species indicates that S. salsa is a typical 18:3 plant. The combined transcriptomic and lipidomic data revealed that different tissues of S. salsa were featured with DEGs associated with specific lipid metabolic pathways, therefore, represented unique lipid profiles. This study will be helpful on understanding lipid metabolism pathway and exploring the key genes involved in lipid synthesis in S. salsa.

Involvement of Phospholipase C in Photosynthesis and Growth of Maize Seedlings.

Phospholipase C is an enzyme that catalyzes the hydrolysis of glycerophospholipids and can be classified as phosphoinositide-specific PLC (PI-PLC) and non-specific PLC (NPC), depending on its hydrolytic substrate. In maize, the function of phospholipase C has not been well characterized. In this study, the phospholipase C inhibitor neomycin sulfate (NS, 100 mM) was applied to maize seedlings to investigate the function of maize PLC. Under the treatment of neomycin sulfate, the growth and development of maize seedlings were impaired, and the leaves were gradually etiolated and wilted. The analysis of physiological and biochemical parameters revealed that inhibition of phospholipase C affected photosynthesis, photosynthetic pigment accumulation, carbon metabolism and the stability of the cell membrane. High-throughput RNA-seq was conducted, and differentially expressed genes (DEGS) were found significantly enriched in photosynthesis and carbon metabolism pathways. When phospholipase C activity was inhibited, the expression of genes related to photosynthetic pigment accumulation was decreased, which led to lowered chlorophyll. Most of the genes related to PSI, PSII and TCA cycles were down-regulated and the net photosynthesis was decreased. Meanwhile, genes related to starch and sucrose metabolism, the pentose phosphate pathway and the glycolysis/gluconeogenesis pathway were up-regulated, which explained the reduction of starch and total soluble sugar content in the leaves of maize seedlings. These findings suggest that phospholipase C plays a key role in photosynthesis and the growth and development of maize seedlings.

The Adjustment of Membrane Lipid Metabolism Pathways in Maize Roots Under Saline-Alkaline Stress.

Plants are frequently confronted by diverse environmental stress, and the membrane lipids remodeling and signaling are essential for modulating the stress responses. Saline-alkaline stress is a major osmotic stress affecting the growth and development of crops. In this study, an integrated transcriptomic and lipidomic analysis was performed, and the metabolic changes of membrane lipid metabolism in maize (Zea mays) roots under saline-alkaline stress were investigated. The results revealed that phospholipids were major membrane lipids in maize roots, and phosphatidylcholine (PC) accounts for approximately 40% of the total lipids. Under 100 mmol NaHCO3 treatment, the level of PC decreased significantly (11-16%) and the parallel transcriptomic analysis showed an increased expression of genes encoding phospholipase A and phospholipase D/non-specific phospholipase C, which suggested an activated PC turnover under saline-alkaline stress. The plastidic galactolipid synthesis was also activated, and an abnormal generation of C34:6 galactolipids in 18:3 plants maize implied a plausible contribution from the prokaryotic pathway, which could be partially supported by the up-regulated expression of three putative plastid-localized phosphatidic acid phosphatase/lipid phosphate phosphatase. A comprehensive gene-metabolite network was constructed, and the regulation of membrane lipid metabolism under saline-alkaline stress in maize was discussed.

Membrane Lipids' Metabolism and Transcriptional Regulation in Maize Roots Under Cold Stress.

Low temperature is one of the major abiotic stresses that restrict the growth and development of maize seedlings. Membrane lipid metabolism and remodeling are key strategies for plants to cope with temperature stresses. In this study, an integrated lipidomic and transcriptomic analysis was performed to explore the metabolic changes of membrane lipids in the roots of maize seedlings under cold stress (5°C). The results revealed that major extraplastidic phospholipids [phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), and phosphatidylinositol (PI)] were dominant membrane lipids in maize root tissues, accounting for more than 70% of the total lipids. In the transcriptome data of maize roots under cold stress, a total of 189 lipid-related differentially expressed genes (DEGs) were annotated and classified into various lipid metabolism pathways, and most of the DEGs were enriched in the "Eukaryotic phospholipid synthesis" (12%), "Fatty acid elongation" (12%), and "Phospholipid signaling" (13%) pathways. Under low temperature stress, the molar percentage of the most abundant phospholipid PC decreased around 10%. The significantly up-regulated expression of genes encoding phospholipase [phospholipase D (PLD)] and phosphatase PAP/LPP genes implied that PC turnover was triggered by cold stress mainly via the PLD pathway. Consequently, as the central product of PC turnover, the level of PA increased drastically (63.2%) compared with the control. The gene-metabolite network and co-expression network were constructed with the prominent lipid-related DEGs to illustrate the modular regulation of metabolic changes of membrane lipids. This study will help to explicate membrane lipid remodeling and the molecular regulation mechanism in field crops encountering low temperature stress.

Characterisation of genes involved in galactolipids and sulfolipids metabolism in maize and Arabidopsis and their differential responses to phosphate deficiency.

Galactolipids (MGDG and DGDG) and sulfolipids (SQDG) are key components of plastidic membranes, and play important roles in plant development and photosynthesis. In this study, the whole families of MGD, DGD and SQD were identified in maize genome, and were designated as ZmMGD1-3, ZmDGD1-5 and ZmSQD1-5 respectively. Based on the phylogenetic analyses, maize and Arabidopsis MGDs, DGDs and SQDs were clearly divided into two major categories (Type A and Type B) along with their orthologous genes from other plant species. Under low-phosphorus condition, the expression of Type B MGD, DGD and SQD genes of maize and Arabidopsis were significantly elevated in both leaf and root tissues. The lipid analysis was also conducted, and an overall increase in non-phosphorus lipids (MGDG, DGDG and SQDG), and a decrease in phosphorus lipids (PC, PE and PA) were observed in maize leaves and roots under phosphate deficiency. Several maize MGD and SQD genes were found involved in various abiotic stress responses. These findings will help for better understanding the specific functions of MGDs, DGDs and SQDs in 18:3 plants and for the generation of improved crops adapted to phosphate starvation and other abiotic stresses.

Extraction of acetanilides in rice using ionic liquid-based matrix solid phase dispersion-solvent flotation.

Ionic liquid-based matrix solid phase dispersion-solvent flotation coupled with high performance liquid chromatography was developed for the determination of the acetanilide herbicides, including metazachlor, propanil, alachlor, propisochlor, pretilachlor, and butachlor in rice samples. Some experimental parameters, including the type of dispersant, the mass ratio of dispersant to sample, pH of sample solution, the type of extraction solvent, the type of ionic liquid, flotation time, and flow rate of N2 were optimized. The average recoveries of the acetanilide herbicides at spiked concentrations of 50, 125, and 250 µg/kg ranged from 89.4% to 108.7%, and relative standard deviations were equal to or lower than 7.1%, the limits of quantification were in the range of 38.0 to 84.7 µg/kg.

Glycerol-3-phosphate dehydrogenase (GPDH) gene family in Zea mays L.: Identification, subcellular localization, and transcriptional responses to abiotic stresses.

Glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the formation of glycerol-3-phosphate, and plays an essential role in glycerolipid metabolism and in response to various stresses in different species. In this study, six ZmGPDH genes were obtained by a thorough search against maize genome, and designated as ZmGPDH1-6, respectively. The structural and evolutionary analyses showed that the ZmGPDHs family had typical conserved domains and similar protein structures as the known GPDHs from other plant species. ZmGPDHs were divided into NAD+-dependent type A form (ZmGPDH1-5) and FAD-dependent type B form (ZmGPDH6) based on their N-terminal sequences. Four full length ZmGPDHs were fused with GFP fusion proteins, and their subcellular localization was determined. ZmGPDH1 and ZmGPDH3 were located to the cytosol and mainly recruited to the surface of endoplasmic reticulum (ER), whereas ZmGPDH4 and ZmGPDH5 were located in the chloroplast. The transcriptional analysis of the ZmGPDHs in different maize tissues revealed relatively high level of transcripts accumulation of ZmGPDHs in roots and early stage developing seeds. Furthermore, we examined the transcriptional responses of the six GPDH genes in maize under various abiotic stresses, including salt, drought, alkali and cold, and significant induction of ZmGPDHs under osmotic stresses was observed. Together, this work will provide useful information for deciphering the roles of GPDHs in plant development and abiotic stress responses.

Can antioxidant's reactive oxygen species (ROS) scavenging capacity contribute to aged seed recovery? Contrasting effect of melatonin, ascorbate and glutathione on germination ability of aged maize seeds.

It is well known that antioxidants such as AA (reduced ascorbate), glutathione (GSH) (reduced glutathione) and melatonin can delay seed ageing. Can they recover aged seed? Artificial aged maize seeds were obtained and their reduced germination rate (GR) and high lipid peroxidation were recorded. Exogenous melatonin was applied on these aged seeds and enhanced GR was observed. However, treatment with other antioxidants such as AA, GSH or DMTU (dimethyl thiourea) did not significantly improve or even reduce the GR of aged seeds. In addition, melatonin improved germination ability of theses aged seeds can be significantly impaired by DDC (diethyldithiocarbamic acid, a specific inhibitor of superoxide dismutase or superoxide dismutase (SOD)) and ATZ (aminotriazol, a specific inhibitor of catalase or CAT). In a further study, we found that melatonin but not other antioxidants (AA, GSH and DMTU) significantly induced CAT and SOD activities of aged seeds after imbibition. Accordingly, melatonin significantly reduced lipid peroxidation in aged seeds than that of other antioxidants. Taken together, these data suggest that melatonin induced antioxidant enzyme but not its direct reactive oxygen species (ROS) scavenging capacity contributing to recovery of aged maize seeds.

Alleviation of the effects of saline-alkaline stress on maize seedlings by regulation of active oxygen metabolism by Trichoderma asperellum.

This study investigated the influence of Trichoderma asperellum on active oxygen production in maize seedlings under saline-alkaline stress conditions. Two maize cultivars were tested: 'Jiangyu 417' ('JY417'), which can tolerate saline-alkaline stress; and, 'Xianyu 335' ('XY335'), which is sensitive to saline-alkaline stress. The seedlings were grown on natural saline-alkaline soil (pH 9.30) in plastic pots. To each liter of saline-alkaline soil, 200 mL of T. asperellum spore suspension was applied; three fungal suspensions were used, namely, 1 × 103, 1 × 106, and 1 × 109 spores/L. A control with only the vehicle applied was also established, along with a second control in which untreated meadow soil (pH 8.23) was used. Root and leaf samples were collected when the seedlings had three heart-shaped leaves and the fourth was in the developmental phase. Physical and biochemical parameters related to oxidation resistance were assessed. The results indicated that the 'JY417' and 'XY335' seedlings showed different degrees of oxidative damage and differences in their antioxidant defense systems under saline-alkaline stress. As the spore density of the fungal suspension increased, the K+ and Ca2+ contents in the seedlings increased, but Na+ content decreased. Moreover, fungal treatment promoted the synthesis or accumulation of osmolytes, which enhanced the water absorbing capacity of the cells, increased antioxidant enzyme activities, enhanced the content of non-enzyme antioxidants, and reduced the accumulation of reactive oxygen species. Fungal treatment alleviated oxidative damage caused by the saline-alkaline stress in roots and leaves of the seedlings. The application of T. asperellum overcame the inhibitory effect of saline-alkaline soil stress on the growth of maize seedlings. In the present experiment, application with 1 × 109 spores/L gave the optimal results.

Biochemical and Transcriptional Regulation of Membrane Lipid Metabolism in Maize Leaves under Low Temperature.

Membrane lipid modulation is one of the major strategies plants have developed for cold acclimation. In this study, a combined lipidomic and transcriptomic analysis was conducted, and the changes in glycerolipids contents and species, and transcriptional regulation of lipid metabolism in maize leaves under low temperature treatment (5°C) were investigated. The lipidomic analysis showed an increase in the phospholipid phosphatidic acid (PA) and a decrease in phosphatidylcholine (PC). And an increase in digalactosyldiacylglycerol and a decrease in monogalactosyldiacylglycerol of the galactolipid class. The results implied an enhanced turnover of PC to PA to serve as precursors for galactolipid synthesis under following low temperature treatment. The analysis of changes in abundance of various lipid molecular species suggested major alterations of different pathways of plastidic lipids synthesis in maize under cold treatment. The synchronous transcriptomic analysis revealed that genes involved in phospholipid and galactolipid synthesis pathways were significantly up-regulated, and a comprehensive gene-metabolite network was generated illustrating activated membrane lipids adjustment in maize leaves following cold treatment. This study will help to understand the regulation of glycerolipids metabolism at both biochemical and molecular biological levels in 18:3 plants and to decipher the roles played by lipid remodeling in cold response in major field crop maize.

Can heavy metal pollution defend seed germination against heat stress? Effect of heavy metals (Cu(2+), Cd(2+) and Hg(2+)) on maize seed germination under high temperature.

Heavy metal pollution, as well as greenhouse effect, has become a serious threat today. Both heavy metal and heat stresses can arrest seed germination. What response can be expected for seed germination under both stress conditions? Here, the effects of heavy metals (Cu(2+), Cd(2+) and Hg(2+)) on maize seed germination were investigated at 20 °C and 40 °C. Compared with 20 °C, heat stress induced thermodormancy. However, this thermodormancy could be significantly alleviated by the addition of a low concentration of heavy metals. Heavy metals, as well as heat stress induced H2O2 accumulation in germinating seeds. Interestingly, this low concentration of heavy metal that promoted seed germination could be partly blocked by DMTU (a specific ROS scavenger), irrespective of temperature. Accordingly, H2O2 addition reinforced this promoting effect on seed germination, which was induced by a low concentration of heavy metal. Furthermore, we found that the NADPH oxidase derived ROS was required for seed germination promoted by the heavy metals. Subsequently, treatment of seeds with fluridone (a specific inhibitor of ABA) or ABA significantly alleviated or aggravated thermodormancy, respectively. However, this alleviation or aggravation could be partly attenuated by a low concentration of heavy metals. In addition, germination that was inhibited by high concentrations of heavy metals was also partly reversed by fluridone. The obtained results support the idea that heavy metal-mediated ROS and hormone interaction can finally affect the thermodormancy release or not.

[Alleviation of salt stress during maize seed germination by presoaking with exogenous sugar].

The maize variety Kenyu 6 was used to study the effects of exogenous glucose (Glc) and sucrose (Suc) on salt tolerance of maize seeds at germination stage under 150 mmol · L(-1) NaCl treatment. Results showed that under salt stress condition, 0.5 mmol · L(-1) exogenous Glc and Suc presoaking could promote seed germination and early seedling growth. Compared with the salt treatment, Glc presoaking increased the shoot length, radicle length and corresponding dry mass up to 1.5, 1.3, 2.1 and 1.8 times, and those of the Suc presoaking treatment increased up to 1.7, 1.3. 2.7 and 1.9 times, respectively. Exogenous Glc and Suc presoaking resulted in decreased levels of thiobarbituric acid reactive substances (TBARS) and hydrogen peroxide (H2O2) content of maize shoot under salt stress, which were lowered by 24.9% and 20.6% respectively. Exogenous Glc and Suc presoaking could increase the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutathione reductase (GR) and induce glucose-6-phosphate dehydrogenase (G6PDH) activity of maize shoot under salt stress. Compared with the salt treatment. Glc presoaking increased the activity of SOD, APX, GPX, GR and G6PDH by 66.2%, 62.9%, 32.0%, 38.5% and 50.5%, and those of the Suc presoaking increased by 67.5%, 59.8%, 30.0%, 38.5% and 50.4%, respectively. Glc and Suc presoaking also significantly increased the contents of ascorbic acid (ASA) and glutathione (GSH), ASA/DHA and GSH/GSSG. The G6PDH activity was found closely related with the strong antioxidation capacity induced by exogenous sugars. In addition, Glc and Suc presoaking enhanced K+/Na+ in maize shoot by 1.3 and 1.4 times of water soaking salt treatment, respectively. These results indicated that exogenous Glc and Suc presoaking could improve antioxidation capacity of maize seeds and maintain the in vivo K+/Na+ ion balance to alleviate the inhibitory effect of salt stress on maize seed germination.