plant-sample-preparation-for-nucleosidenucleotide-content-measurement

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which ...

Nanjing Agricultural University

Our results show that methyl jasmonate induces plasma membrane H (+) -ATPase activity and subsequently influences the apoplastic pH of trichoblasts to maintain a cell wall pH environment appropriate for root hair development. Root hairs, which arise from root epidermal cells, are tubular structures that increase the efficiency of water absorption and nutrient uptake. Plant hormones are critical regulators of root hair development. In this study, we investigated the regulatory role of the plasma membrane (PM) H(+)-ATPase in methyl jasmonate (MeJA)-induced root hair formation. We found that MeJA had a pronounced effect on the promotion of root hair formation in lettuce seedlings, but that this effect was blocked by the PM H(+)-ATPase inhibitor vanadate. Furthermore, MeJA treatment increased PM H(+)-ATPase activity in parallel with H(+) efflux from the root tips of lettuce seedlings and rhizosphere acidification. Our results also showed that MeJA-induced root hair formation was accompanied by hydrogen peroxide accumulation. The apoplastic acidification acted in concert with reactive oxygen species to modulate root hair formation. Our results suggest that the effect of MeJA on root hair formation is mediated by modulation of PM H(+)-ATPase activity.

Plasma membrane H(+)-ATPase is involved in methyl jasmonate-induced root hair formation in lettuce (Lactuca sativa L.) seedlings.

Fifty-seven wheat cultivars were used to investigate the differences in arsenic (As) accumulation, efflux and translocation among wheat cultivars and their relationship with arsenate (As(V)) tolerance under hydroponic condition. The relationship between wheat root As accumulation, As(V) uptake, arsenite (As(III)) efflux and As(V) tolerance of 14 wheat cultivars were also investigated. The results showed there were significant (p<0.001) differences in As(V) tolerance, As accumulation and translocation among 57 wheat cultivars. Arsenate tolerance of wheat seedlings was positively correlated with As(V) uptake (p<0.05), root As concentration (p<0.001), but negatively correlated (p<0.05) with TFs and relative As(III) efflux. No significant correlation between As(III) efflux and As(V) tolerance was found (p=0.442). 56-83% of total As taken up by roots was extruded to nutrient solution. Root As concentration was positively correlated with As(V) uptake (not significant, p=0.100), negatively correlated (p<0.001) with relative As(III) efflux, whereas not significantly correlated (p=0.773) with As(III) efflux. The results indicated that As(V) tolerant wheat cultivars have much higher capacity of root As accumulation. Arsenic detoxification in root cells is important for wheat seedlings under As(V) exposure. 

Variation in arsenic accumulation and translocation among wheat cultivars: the relationship between arsenic accumulation, efflux by wheat roots and arsenate tolerance of wheat seedlings.

Lamina joint inclination or leaf angle (the angle between the leaf blade and vertical culm) is a major trait of rice plant architecture. The plant hormone brassinosteroid (BR) is the main regulator of this trait, while other plant hormones, including ethylene, gibberellin, and auxin, also influence leaf angle. In this study, we found that methyl jasmonate (MeJA) also participates in regulating lamina joint inclination. MeJA decreased lamina joint inclination and inhibited the BR-induced increase in lamina joint inclination. Furthermore, addition of a BR synthesis inhibitor increased the extent of change in lamina joint inclination in response to treatment with a low concentration of MeJA (0.05 or 0.5mgL(-1)), but it did not alter the lamina joint inclination of plants treated with a high concentration of MeJA (5mgL(-1)). Further studies showed that MeJA treatment significantly repressed the expression of BR biosynthesis-related genes and decreased endogenous BRs levels. In addition, the lamina joint inclination in the OsBRI1 mutant d61-1 was less sensitive to MeJA compared with its wild type counterpart, and lithium chloride-induced inactivation of GSK3-like kinase, a negative regulator of BR signaling, partly rescued the MeJA-induced reduction in lamina joint inclination. Further studies showed that MeJA treatment reduced the mRNA levels of BR signaling and target genes. These results indicate that MeJA-inhibition of lamina joint inclination may depend on BR biosynthesis and the BR signaling pathway.

Methyl jasmonate inhibits lamina joint inclination by repressing brassinosteroid biosynthesis and signaling in rice.

Root hairs are plastic in response to nutrient supply, but relatively little is known about their development under low ammonium (NH4(+)) conditions. This study showed that reducing NH4(+) for 3 days in wild-type Arabidopsis seedlings resulted in drastic elongation of root hairs. To investigate the possible mediation of ethylene and auxin in this process, seedlings were treated with 2,3,5-triiodobenzoic acid (TIBA, auxin transport inhibitor), 1-naphthylphthalamic acid (NPA, auxin transport inhibitor), p-chlorophenoxy isobutyric acid (PCIB, auxin action inhibitor), aminoethoxyvinylglycine (AVG, chemical inhibitor of ethylene biosynthesis), or silver ions (Ag(+), ethylene perception antagonist) under low NH4(+) conditions. Our results showed that TIBA, NPA and PCIB did not inhibit root hair elongation under low NH4(+) conditions, while AVG and Ag(+) completely inhibited low NH4(+)-induced root hair elongation. This suggested that low NH4(+)-induced root hair elongation was dependent on the ethylene pathway, but not the auxin pathway. Further genetic studies revealed that root hair elongation in auxin-insensitive mutants was sensitive to low NH4(+) treatment, but elongation was less sensitive in ethylene-insensitive mutants than wild-type plants. In addition, low NH4(+)-induced root hair elongation was accompanied by reactive oxygen species (ROS) accumulation. Diphenylene iodonium (DPI, NADPH oxidase inhibitor) and dimethylthiourea (DMTU, ROS scavenger) inhibited low NH4(+)-induced root hair elongation, suggesting that ROS were involved in this process. Moreover, ethylene acted together with ROS to modulate root hair elongation under low NH4(+) conditions. These results demonstrate that a signaling pathway involving ethylene and ROS participates in regulation of root hair elongation when Arabidopsis seedlings are subjected to low NH4(+) conditions. 

An ethylene and ROS-dependent pathway is involved in low ammonium-induced root hair elongation in Arabidopsis seedlings.

Excessive use of nitrogen (N) fertilizer has increased ammonium (NH ) accumulation in many paddy soils to levels that reduce rice vegetative biomass and yield. Based on studies of NH toxicity in rice (Oryza sativa, Nanjing 44) seedlings cultured in agar medium, we found that NH concentrations above 0.75 mM inhibited the growth of rice and caused NH accumulation in both shoots and roots. Use of excessive NH also induced rhizosphere acidification and inhibited the absorption of K, Ca, Mg, Fe and Zn in rice seedlings. Under excessive NH conditions, exogenous γ-aminobutyric acid (GABA) treatment limited NH accumulation in rice seedlings, reduced NH toxicity symptoms and promoted plant growth. GABA addition also reduced rhizosphere acidification and alleviated the inhibition of Ca, Mg, Fe and Zn absorption caused by excessive NH . Furthermore, we found that the activity of glutamine synthetase/NADH-glutamate synthase (GS; EC 6.3.1.2/NADH-GOGAT; EC1.4.1.14) in root increased gradually as the NH concentration increased. However, when the concentration of NH is more than 3 mM, GABA treatment inhibited NH -induced increases in GS/NADH-GOGAT activity. The inhibition of ammonium assimilation may restore the elongation of seminal rice roots repressed by high NH . These results suggest that mitigation of ammonium accumulation and assimilation is essential for GABA-dependent alleviation of ammonium toxicity in rice seedlings.

γ-Aminobutyric acid addition alleviates ammonium toxicity by limiting ammonium accumulation in rice (Oryza sativa) seedlings.

Salt stress is a main abiotic stress that limits agricultural productivity in many parts of the world. To investigate whether poly-γ-glutamic acid (γ-PGA) can alleviate the negative effects of salt stress on wheat, a foliar application of 400 mg/L γ-PGA was applied to wheat seedlings, which were then subjected to 150 mM NaCl. Our results showed that after application of γ-PGA, the plant height, the plant weight, and the antioxidant enzymes including superoxide dismutase, peroxidase, and catalase were significantly increased compared with the treatment of 150 mM NaCl alone. Meanwhile, γ-PGA application also resulted in high accumulation of K and decreased storage of Na in wheat leaves. These results suggest that γ-PGA treatment may improve salt tolerance of wheat by diminishing ionic imbalances and enhancing antioxidant capacity. Our results indicate that exogenous γ-PGA could alleviate the damage caused by salt stress.

Exogenously applied poly-γ-glutamic acid alleviates salt stress in wheat seedlings by modulating ion balance and the antioxidant system.

Over the years, germinated adlay products have been used as both food source and folk medicine. This study investigated the changes of total phenolic content (TPC), total flavonoid content (TFC), antioxidant activities, and phenolic acid profiles of adlay seed during germination.

Changes of the phenolic compounds and antioxidant activities in germinated adlay seeds.

This study explored the impact of re-acetylation on induced electric field (IEF)-assisted hydrolysis of chitosan. Chitosan with different degree of deacetylation (DD) exhibited different charge content but similar electrical conductivity and output voltage of the electrolyte system, thus theoretically resulting in equivalent electro-processing efficiency. However, molecular and rheological measurements suggested that the hydrolysis rate decreased with an increase of DD. Re-acetylation had no significant effect on the molecular weight and viscosity of chitosan but damaged its granular and crystal structure, making chitosan chains more susceptible to the acid. The IEF-assisted hydrolysis of dissolved chitosan confirmed that the structural compactness of chitosan played a more important role on the hydrolysis than the charge content. This study extended current knowledge regarding the effect of charge content and structural compactness on IEF-assisted hydrolysis process, which contributed to the applications of IEF in biopolymer modification.

Effect of re-acetylation on the acid hydrolysis of chitosan under an induced electric field.

Verticillium wilt of cotton is a serious soil-borne disease that causes a substantial reduction in cotton yields. A previous study showed that the endophytic fungus Chaetomium globosum CEF-082 could control Verticillium wilt of cotton, and induce a defense response in cotton plants. However, the comprehensive molecular mechanism governing this response is not yet clear.

Transcriptome analysis reveals the defense mechanism of cotton against Verticillium dahliae in the presence of the biocontrol fungus Chaetomium globosum CEF-082.

Verticillium wilt caused by  is a threatening disease of cotton, causing economic loss worldwide. In this study, nine endophytic  strains isolated from cotton roots exhibited inhibitory activity against  strain VD-080 in a dual culture assay.  HNH7 and  HNH9 were chosen for further experiments based on their high antagonistic activity. The secondary metabolites of HNH7 and HNH9 also inhibited the growth of VD-080. Genetic marker-assisted detection revealed the presence of bacillibactin, surfactin, bacillomycin and fengycin encoding genes in the genome of HNH7 and HNH9 and their corresponding gene products were validated through LC-MS. Scanning electron microscopy revealed mycelial disintegration, curling and shrinkage of VD-080 hyphae after treatment with methanolic extracts of the isolated endophytes. Furthermore, a significant reduction in verticillium wilt severity was noticed in cotton plants treated with HNH7 and HNH9 as compared to control treatments. Moreover, the expression of defense-linked genes, viz., , , , ,  and , was considerably higher in plants treated with endophytic  strains and inoculated with VD-080 as compared to control.

Antagonistic Potential of Novel Endophytic  Strains and Mediation of Plant Defense against Verticillium Wilt in Upland Cotton.

γ-Aminobutyric acid (GABA) is a widely distributed non-protein amino acid mediated the regulation of nitrate uptake and Al tolerance in plants. However, there are few reports about the involvement of GABA in the regulation of iron (Fe) acquisition and translocation. Here, we show that GABA regulates Fe homeostasis in rice seedlings. Exogenous GABA decreased the chlorophyll concentration in leaves, with or without Fe supply. Over-expression of glutamate decarboxylase () gene, coding a crucial enzyme of GABA production, elevated endogenous GABA content and caused more leaf chlorosis than wild type (Nipponbare). GABA inhibited Fe transportation from roots to shoots and GABA application elevated the expression levels of Fe deficiency (FD)-related genes under conditions of Fe-sufficiency (FS), suggesting that GABA is a regulator of Fe translocation. Using Perls' blue staining, we found that more ferric iron (Fe) was deposited in the epidermal cells of roots treated with GABA compared with control roots. Anatomic section analysis showed that GABA treatment induced more aerenchyma formation compared with the control. Aerenchyma facilitated the oxidization of soluble ferrous iron (Fe) into insoluble Fe, resulted in Fe precipitation in the epidermis, and inhibited the transportation of Fe from roots to shoots.

Na Yang

College of Life Sciences

Nanjing Agricultural University

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Na Yang

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Nanjing Agricultural University

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

College of Life Sciences