Yinong Xu

Decreased stability of photosystem I in dgd1 mutant of Arabidopsis thaliana.

The dgd1 mutant of Arabidopsis thaliana provides us with a powerful tool for revealing the specific role of digalactosyldiacylglycerol (DGDG) in photosynthesis. Blue‐native polyacrylamide gel electrophoresis analysis revealed that photosystem I (PSI) subunits are assembled into a PSI complex, and that a PSI subcomplex lacking stroma side subunits was also present. PSI subunits in the dgd1 mutant were decreased to a similar level compared with that in the wild type (WT) Arabidopsis. Further experiments showed that PSI subunits in the stroma side, PsaD and PsaE, in the dgd1 mutant were more susceptible to removal by chaotropic agents than those in the WT plant, indicating that the stability of PsaD and PsaE is impaired in the dgd1 mutant. These results provide evidence that DGDG is important for the stability of the PSI complex.

Hypocotyl-based Agrobacterium-mediated transformation of soybean (Glycine max) and application for RNA interference

An efficient system of gene transformation is necessary for soybean [Glycine max (L.) Merrill] functional genomics and gene modification by using RNA interference (RNAi) technology. To establish such system, we improved the conditions of tissue culture and transformation for increasing the frequency of adventitious shoots and decreasing the browning and necrosis of hypocotyls. Adding N6-benzylaminopurine (BAP) and silver nitrate in culture medium enhanced the shoot formation on hypocotyls. BAP increased the frequency of the hypocotyls containing adventitious shoots, while silver nitrate increased the number of shoots on the hypocotyls. As a result, the number of adventitious shoots on hypocotyls cultured in medium containing both BAP and silver nitrate was 5-fold higher than the controls. Adding antioxidants in co-cultivation medium resulted in a significant decrease in occurrence of browning and necrosis of hypocotyls and increase in levels of β-Glucuronidase (GUS) gene expression. Histochemical assays showed that the apical meristem of hypocotyls was the “target tissue” for Agrobacterium tumefaciens transformation of soybean. Gene silencing of functional gene by using RNAi technology was carried out under above conditions. A silencing construct containing an inverted-repeat fragment of the GmFAD2 gene was introduced into soybean by using the A. tumefaciens-mediated transformation. Several lines with high oleic acid were obtained, in which mean oleic acid content ranged from 71.5 to 81.9%. Our study demonstrates that this transgenic approach could be efficiently used to improve soybean quality and productivity through functional genomics.

Monogalactosyldiacylglycerol deficiency in tobacco inhibits the cytochrome b6f-mediated intersystem electron transport process and affects the photostability of the photosystem II apparatus

Monogalactosyldiacylglycerol (MGDG) is the most abundant lipid component of the thylakoid membrane. Although MGDG is believed to be important in sustaining the structure and function of the photosynthetic membrane, its exact role in photosynthesis in vivo requires further investigation. In this study, the transgenic tobacco plant M18, which has an MGDG deficiency of approximately 53%, and which contains many fewer thylakoid membranes and exhibits retarded growth and a chlorotic phenotype, was used to investigate the role of MGDG. Chlorophyll fluorescence analysis of the M18 line revealed that PSII activity was inhibited when the plants were exposed to light. The inactive linear electron transport found in M18 plants was mainly attributed to a block in the intersystem electron transport process that was revealed by P700 redox kinetics and PSI light response analysis. Immunoblotting and Blue Native SDS-PAGE analysis suggested that a reduction in the accumulation of cytochrome b6f in M18 plants is a direct structural effect of MGDG deficiency, and this is likely to be responsible for the inefficiency observed in intersystem electron transport. Although drastic impairments of PSII subunits were detected in M18 plants grown under normal conditions, further investigations of low-light-grown M18 plants indicated that the impairments are not direct structural effects. Instead, they are likely to result from the cumulative photodamage that occurs due to impaired photostability under long-term exposure to relatively high light levels. The study suggests that MGDG plays important roles in maintaining both the linear electron transport process and the photostability of the PSII apparatus.

Positional distribution of fatty acids on the glycerol backbone during the biosynthesis of glycerolipids in Ectocarpus fasciculatus

The biosynthesis of glycolipids in E. fasciculatus was studied by 14C label and chase. The fatty acids in sulphoquinovosyl diacylglycerol (SQDG) were almost 16-carbon and 18-carbon ones. In addition to the two fatty acids, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) contained 8.5 mol% and 31.0 mol% of eicosapentaenoic acid (20: 5), respectively, and this fatty acid was usually distributed in the sn-1 position of the glycerol backbone. When plants were incubated with [2−14C] acetate, differences existed in the positional distribution of the labeled fatty acids in sn-1 and sn-2 among the three glycerolipids. In SQDG., 14C-labeled fatty acids were distributed uniformly in the sn-1 and sn-2 positions. In DGDG., 14C-labeled fatty acids were mainly distributed in the sn-2 position. In MGDG., the radioactivity of fatty acids in sn-1 position was far greater than that in sn-2 position after a 30 min pulse label, and the difference in radioactivity between the two positions decreased rapidly. The above results indicated that differences in the positional distribution of 14C-labeled fatty acids between sn-1 and sn-2 positions might be related to 20: 5 and the biosynthesis of DGDG. Our results also suggested that E. fasciculatus had the same DGDG biosynthetic pathway as that in higher plants and galactosyl transferase was selective for MGDG.

RNA interference-based suppression of phosphoenolpyruvate carboxylase results in susceptibility of rapeseed to osmotic stress.

The diverse functions of phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) in C(3) plants are not as well understood as in C(4) plants. To investigate the functions of PEPCase in C(3) plants, rapeseed (Brassica napus L.) PEPCase gene (referred to as BNPE15) was silenced by the RNA interference (RNAi) technique. Under normal growth conditions, no significant difference in lipid content and fatty acid composition were found between wild-type (WT) and transgenic rapeseed plants. However, when these plants were subjected to osmotic stress induced by osmoticum polyethylene glycol (PEG-6000), membrane permeability and membrane lipid peroxidization in roots and leaves of transgenic plants were higher than those of WT plants. It suggested that transgenic plants are more susceptible to osmotic stress than WT plants. Taken together, the results showed that the suppression of PEPCase by RNAi leads to susceptibility to osmotic stress in rapeseed, and PEPCase is involved in the response of C(3) plants to environmental stress.

Cloning, Characterization and Functional Analysis of Two Type 1 Diacylglycerol Acyltransferases （DGAT1s） from Tetraena mongolica

Two cDNAs encoding putative type 1 acyl-CoA: diacylglycerol acyltransferases (DGAT1, EC 2.3.1.20), were cloned from Tetraena mongolica, an extreme xerophyte with high oil content in the stems. The 1 488-bp and 1 485-bp of the open reading frame (ORF) of the two cDNAs, designated as TmDGAT1a and TmDGAT1b, were both predicted to encode proteins of 495 and 494 amino acids, respectively. Southern blot analysis revealed that TmDGAT1a and TmDGAT1b both had low copy numbers in the T. mongolica genome. In addition to ubiquitous expression with different intensity in different tissues, including stems, leaves and roots, TmDGAT1a and TmDGAT1b, were found to be strongly induced by high salinity, drought and osmotic stress, resulting in a remarkable increase of triacylglycerol (TAG) accumulation in T. mongolica plantlets. TmDGAT1a and TmDGAT1b activities were confirmed in the yeast H1246 quadruple mutant (DGA1, LRO1, ARE1, ARE2) by restoring DGAT activity of the mutant host to produce TAG. Overexpression of TmDGAT1a and TmDGAT1b in soybean hairy roots as well as in T. mongolica calli both resulted in an increase in oil content (ranging from 37% to 108%), accompanied by altered fatty acid profiles.

Effects of Monogalactoglycerolipid Deficiency and Diacylglycerol Acyltransferase Overexpression on Oil Accumulation in Transgenic Tobacco

Engineering accumulation of triacylglycerol (TAG) in vegetative tissues has been recently proposed as a promising strategy for increasing plant oil production. However, little is known about regulatory mechanisms involved in increasing oil production in plant vegetative tissues. In this study, expression of NtMGD1 encoding a major biosynthetic enzyme for the chloroplast membrane lipid was inhibited by RNAi interference in tobacco. Furthermore, AtDGAT1, a rate-regulating gene involved in TAG biosynthesis, was ectopically overexpressed. Results showed that leaf TAG accumulations were significantly increased both by NtMGD1 RNAi and AtDGAT1 overexpression. However, combination of AtDGAT1 overexpression with NtMGD1 RNAi did not result in additive increase in TAG accumulation in leaves than AtDGAT1 overexpression or NtMGD1 RNAi alone. In addition, reduction of monogalactosyldiacylglycerol (MGDG) biosynthesis by NtMGD1 RNAi was relieved by AtDGAT1 overexpression. Expression of lipid transfer protein (LTP) was upregulated both by AtDGAT1 overexpression and NtMGD1 RNAi and correlated with increased oil accumulation in leaves. Our results indicated that fatty acids deesterified from chloroplast membrane galactolipids could be redirected into TAG. TAG is an energy-dense molecule that might act as a storage pool for carbohydrate. This membrane lipid remodeling may represent an adaptive response that enables plant cells to avoid toxic effects of free fatty acids.

Effect of Sodium Thiosulfate on the Depletion of Photosynthetic Apparatus in Cyanobacterium Synechocystis sp. PCC 6803 Cells Grown in the Presence of Glucose

Fluorescence spectroscopy at 77 K showed that the application of glucose lead to the depletion of phycobilisomes (PBS) and photosystems (PS) 2 and 1, and that PS2 was more sensitive to glucose than PS1. The application of sodium thiosulfate, an effective scavenger of reactive oxygen intermediates, counteracted the effects of glucose. Sodium thiosulfate effectively protected photosynthetic apparatus, PS2, PS1, and PBS against glucose-induced depletion. Sodium thiosulfate showed strong capability to inhibit the disappearance of chlorophyll induced by glucose. At a relatively low concentration of glucose, the application of sodium thiosulfate can even be helpful for the assembly of photosynthetic apparatus. Hence the reactive oxygen species might be involved in the depletion of the photosynthetic apparatus in the cyanobacterium Synechocystis sp. PCC 6803 cells grown in the presence of glucose.

Overexpression of the Transcription Factors GmSHN1 and GmSHN9 Differentially Regulates Wax and Cutin Biosynthesis, Alters Cuticle Properties, and Changes Leaf Phenotypes in Arabidopsis.

SHINE (SHN/WIN) clade proteins, transcription factors of the plant-specific APETALA 2/ethylene-responsive element binding factor (AP2/ERF) family, have been proven to be involved in wax and cutin biosynthesis. Glycine max is an important economic crop, but its molecular mechanism of wax biosynthesis is rarely characterized. In this study, 10 homologs of Arabidopsis SHN genes were identified from soybean. These homologs were different in gene structures and organ expression patterns. Constitutive expression of each of the soybean SHN genes in Arabidopsis led to different leaf phenotypes, as well as different levels of glossiness on leaf surfaces. Overexpression of GmSHN1 and GmSHN9 in Arabidopsis exhibited 7.8-fold and 9.9-fold up-regulation of leaf cuticle wax productions, respectively. C31 and C29 alkanes contributed most to the increased wax contents. Total cutin contents of leaves were increased 11.4-fold in GmSHN1 overexpressors and 5.7-fold in GmSHN9 overexpressors, mainly through increasing C16:0 di-OH and dioic acids. GmSHN1 and GmSHN9 also altered leaf cuticle membrane ultrastructure and increased water loss rate in transgenic Arabidopsis plants. Transcript levels of many wax and cutin biosynthesis and leaf development related genes were altered in GmSHN1 and GmSHN9 overexpressors. Overall, these results suggest that GmSHN1 and GmSHN9 may differentially regulate the leaf development process as well as wax and cutin biosynthesis.

Phosphatidylglycerol degradation is one crucial reason for the decrease of its concentration in wheat leaves under phosphate deprivation stress

Phosphatidylglycerol (PG) is of crucial importance for the organization and function of thylakoid membranes. The reason for a decrease of PG concentration in plants under phosphate deprivation stress still remains unclear. By comparing PG concentration and PG hydrolase activity of wheat leaves at different developmental stages, we show that when the first leaves are fully developed, the PG concentration and PG hydrolase activity in phosphate-deficient plants were similar to those of the controls. With age, there was a significant decrease in PG concentration combined with an increase in PG hydrolase activity for phosphate-deficient plants. The close correlation between the decrease in PG concentration and the increase in PG hydrolases activities suggests that PG hydrolysis was the most important factor responsible for the decrease in its concentration. The main hydrolysis products of PG are phosphatidic acid (PA), diacylglycerol (DAG) and free fatty acid (FFA). The application of an inhibitor, n-butanol, which blocks the formation of PA via the PLD pathway, to the in vitro enzyme reaction mixture may restrict PA and DAG production. Neomycin sulfate, a PLC inhibitor, was shown to exhibit significant inhibition in DAG generation. These results suggest that both PLD and PLC were responsible for PG degradation in the leaves of phosphate-deficient wheat. The possible role of PLA activity for PG degradation is also discussed.