Juanjuan Feng

Transformation of β-Lycopene Cyclase Genes from Salicornia europaea and Arabidopsis Conferred Salt Tolerance in Arabidopsis and Tobacco

Inhibition of lycopene cyclization decreased the salt tolerance of the euhalophyte Salicornia europaea L. We isolated a β-lycopene cyclase gene SeLCY from S. europaea and transformed it into Arabidopsis with stable expression. Transgenic Arabidopsis on post-germination exhibited enhanced tolerance to oxidative and salt stress. After 8 and 21 d recovery from 200 mM NaCl treatment, transgenic lines had a higher survival ratio than wild-type (WT) plants. Three-week-old transgenic plants treated with 200 mM NaCl showed better growth than the WT with higher photosystem activity and less H(2)O(2) accumulation. Determination of endogenous pigments of Arabidopsis treated with 200 mM NaCl for 0, 2 or 4 d demonstrated that the transgenic plants retained higher contents of carotenoids than the WT. Furthermore, to compare the difference between SeLCY and AtLCY from Arabidopsis, we used viral vector mediating ectopic expression of SeLCY and AtLCY in Nicotiana benthamiana. Although LCY genes transformation increased the salt tolerance in tobacco, there is no significant difference between SeLCY- and AtLCY-transformed plants. These findings indicate that SeLCY transgenic Arabidopsis improved salt tolerance by increasing synthesis of carotenoids, which impairs reactive oxygen species and protects the photosynthesis system under salt stress, and as a single gene, SeLCY functionally showed no advantage for salt tolerance improvement compared with AtLCY.

Coordination of carbon fixation and nitrogen metabolism in Salicornia europaea under salinity: Comparative proteomic analysis on chloroplast proteins

Halophyte, like Salicornia europaea, could make full use of marginal saline land for carbon fixation. How the photosynthesis of S. europaea is regulated under high salinity implicates a significant aspect to exploit this pioneer plant in future. Measurement of photosynthesis parameters demonstrated the reduction of photosynthesis for the 0 and 800u2009mM NaCl treated plants are more likely due to non‐stomatal limitation, which might be caused by changes in the enzymes associated with photosynthesis. Different salinity induced ultrastructure changes other than photosynthetic apparatus damage, suggesting the photosynthesis of S. europaea might be affected via biochemical regulation. Comparative proteomics analysis of chloroplast proteins by 2‐D gel electrophoresis reproducibly detected 90 differentially expressed proteins, among which 66 proteins were identified by nanoLC MS/MS. Further study of thylakoid membrane proteins by Blue‐Native PAGE proved the increase in abundance of light reaction proteins under salinity. Analysis of gene expression patterns of 12 selected proteins provides evidence for the correlations between transcription and proteomics data. Based on our results, a putative model of photosynthesis regulatory network figured out proper coordination of carbon fixation and nitrogen metabolism in chloroplast of S. europaea under salinity, which provided subcellular level insight into salt tolerance mechanism in S. europaea.

High-throughput deep sequencing reveals that microRNAs play important roles in salt tolerance of euhalophyte Salicornia europaea.

BackgroundmicroRNAs (miRNAs) are implicated in plant development processes and play pivotal roles in plant adaptation to environmental stresses. Salicornia europaea, a salt mash euhalophyte, is a suitable model plant to study salt adaptation mechanisms. S. europaea is also a vegetable, forage, and oilseed that can be used for saline land reclamation and biofuel precursor production on marginal lands. Despite its importance, no miRNA has been identified from S. europaea thus far.ResultsDeep sequencing was performed to investigate small RNA transcriptome of S. europaea. Two hundred and ten conserved miRNAs comprising 51 families and 31 novel miRNAs (including seven miRNA star sequences) belonging to 30 families were identified. About half (13 out of 31) of the novel miRNAs were only detected in salt-treated samples. The expression of 43 conserved and 13 novel miRNAs significantly changed in response to salinity. In addition, 53 conserved and 13 novel miRNAs were differentially expressed between the shoots and roots. Furthermore, 306 and 195 S. europaea unigenes were predicted to be targets of 41 conserved and 29 novel miRNA families, respectively. These targets encoded a wide range of proteins, and genes involved in transcription regulation constituted the largest category. Four of these genes encoding laccase, F-box family protein, SAC3/GANP family protein, and NADPH cytochrome P-450 reductase were validated using 5′-RACE.ConclusionsOur results indicate that specific miRNAs are tightly regulated by salinity in the shoots and/or roots of S. europaea, which may play important roles in salt tolerance of this euhalophyte. The S. europaea salt-responsive miRNAs and miRNAs that target transcription factors, nucleotide binding site-leucine-rich repeat proteins and enzymes involved in lignin biosynthesis as well as carbon and nitrogen metabolism may be applied in genetic engineering of crops with high stress tolerance, and genetic modification of biofuel crops with high biomass and regulatable lignin biosynthesis.

Virus‐induced gene silencing reveals control of reactive oxygen species accumulation and salt tolerance in tomato by γ‐aminobutyric acid metabolic pathway

γ-Aminobutyric acid (GABA) accumulates in many plant species in response to environmental stress. However, the physiological function of GABA or its metabolic pathway (GABA shunt) in plants remains largely unclear. Here, the genes, including glutamate decarboxylases (SlGADs), GABAu2005transaminases (SlGABA-Ts) and succinic semialdehyde dehydrogenase (SlSSADH), controlling three steps of the metabolic pathway of GABA, were studied through virus-induced gene silencing approach in tomato. Silencing of SlGADs (GABA biosynthetic genes) and SlGABA-Ts (GABA catabolic genes) led to increased accumulation of reactive oxygen species (ROS) as well as salt sensitivity under 200u2009mm NaCl treatment. Targeted quantitative analysis of metabolites revealed that GABA decreased and increased in the SlGADs- and SlGABA-Ts-silenced plants, respectively, whereas succinate (the final product of GABA metabolism) decreased in both silenced plants. Contrarily, SlSSADH-silenced plants, also defective in GABA degradation process, showed dwarf phenotype, curled leaves and enhanced accumulation of ROS in normal conditions, suggesting the involvement of a bypath for succinic semialdehyde catabolism to γ-hydroxybutyrate as reported previously in Arabidopsis, were less sensitive to salt stress. These results suggest that GABA shunt is involved in salt tolerance of tomato, probably by affecting the homeostasis of metabolites such as succinate and γ-hydroxybutyrate and subsequent ROS accumulation under salt stress.

Chloroplast-targeted Hsp90 plays essential roles in plastid development and embryogenesis in Arabidopsis possibly linking with VIPP1

The Arabidopsis genome contains seven members of Hsp90. Mutations in plastid AtHsp90.5 were reported to cause defects in chloroplast development and embryogenesis. However, the exact function of plastid AtHsp90.5 has not yet been defined. In this study, albino seedlings were found among AtHsp90.5 transformed Arabidopsis, which were revealed to be AtHsp90.5 co-suppressed plants. The accumulation of photosynthetic super-complexes in the albinos was decreased, and expression of genes involved in photosynthesis was significantly down-regulated. AtHsp90.5 T-DNA insertion mutants were embryo-lethal with embryo arrested at the heart stage. Further investigation showed AtHsp90.5 expression was up-regulated in the siliques at 4 days post anthesis (DPA). Confocal microscopy proved AtHsp90.5 was located in the chloroplasts. Plastid development in the AtHsp90.5 mutants and co-suppressed plants was seriously impaired, and few thylakoid membranes were observed, indicating the involvement of AtHsp90.5 in chloroplast biogenesis. AtHsp90.5 was found to interact with vesicle-inducing protein in plastids 1 (VIPP1) by bimolecular fluorescence complementation system. The ratio between VIPP1 oligomers and monomers in AtHsp90.5 co-suppressed plants drastically shifted toward the oligomeric state. Our study confirmed that AtHsp90.5 is vital for chloroplast biogenesis and embryogenesis. Further evidence also suggested that AtHsp90.5 may help in the disassembly of VIPP1 for thylakoid membrane formation and/or maintenance.

Lignin engineering through laccase modification: a promising field for energy plant improvement.

Laccase (p-diphenol:dioxygen oxidoreductase, EC 1.10.3.2) is a member of the multicopper oxidases and catalyzes the one-electron oxidation of a wide range of substrates, coupled with the reduction of oxygen to water. It is widely distributed in bacteria, fungi, plants and insects. Laccases are encoded by multigene family, and have been characterized mostly from fungi till now, with abundant industrial applications in pulp and paper, textile, food industries, organic synthesis, bioremediation and nanobiotechnology, while limited researches have been performed in plants, and no application has been reported. Plant laccases share the common molecular architecture and reaction mechanism with fungal ones, despite of difference in redox potential and pH optima. Plant laccases are implicated in lignin biosynthesis since genetic evidence was derived from the Arabidopsis LAC4 and LAC17. Manipulation of plant laccases has been considered as a promising and innovative strategy in plant biomass engineering for desirable lignin content and/or composition, since lignin is the major recalcitrant component to saccharification in biofuel production from lignocellulose, and therefore directly limits the fermentation yields. Moreover, plant laccases have been reported to be involved in wound healing, maintenance of cell wall structure and integrity, and plant responses to environmental stresses. Here, we summarize the properties and functions of plant laccase, and discuss the potential of biotechnological application, thus providing a new insight into plant laccase, an old enzyme with a promising beginning in lignocellulose biofuel production.

Transcriptome analysis of Salicornia europaea under saline conditions revealed the adaptive primary metabolic pathways as early events to facilitate salt adaptation.

Background Halophytes such as Salicornia europaea have evolved to exhibit unique mechanisms controlled by complex networks and regulated by numerous genes and interactions to adapt to habitats with high salinity. However, these mechanisms remain unknown. Methods To investigate the mechanism by which halophytes tolerate salt based on changes in the whole transcriptome, we performed transcriptome sequencing and functional annotation by database search. Using the unigene database, we conducted digital gene expression analysis of S. europaea at various time points after these materials were treated with NaCl. We also quantified ion uptakes. Gene functional enrichment analysis was performed to determine the important pathways involved in this process. Results A total of 57,151 unigenes with lengths of >300 bp were assembled, in which 57.5% of these unigenes were functionally annotated. Differentially expressed genes indicated that cell wall metabolism and lignin biosynthetic pathways were significantly enriched in S. europaea to promote the development of the xylem under saline conditions. This result is consistent with the increase in sodium uptake as ions pass through the xylem. Given that PSII efficiency remained unaltered, salt treatment activated the expression of electron transfer-related genes encoded by the chloroplast chromosome. Chlorophyll biosynthesis was also inhibited, indicating the energy-efficient state of the electron transfer system of S. europaea. Conclusions The key function of adjusting important primary metabolic pathways in salt adaption was identified by analyzing the changes in the transcriptome of S. europaea. These pathways could involve unique salt tolerance mechanisms in halophytes. This study also provided information as the basis of future investigations on salt response genes in S. europaea. Ample gene resources were also provided to improve the genes responsible for the salt tolerance ability of crops.

Na+/H+ exchanger 1 participates in tobacco disease defence against Phytophthora parasitica var. nicotianae by affecting vacuolar pH and priming the antioxidative system

Summary NbNHX1 affects the cellular pH and oxidation state by regulating the vacuolar H+ flux, which primes the antioxidative system associated with Phytophthora parasitica var. nicotianae resistance in tobacco.

Comparative transcriptome combined with morpho-physiological analyses revealed key factors for differential cadmium accumulation in two contrasting sweet sorghum genotypes

Summary Cadmium (Cd) is a widespread soil contaminant threatening human health. As an ideal energy plant, sweet sorghum (Sorghum bicolor (L.) Moench) has great potential in phytoremediation of Cd‐polluted soils, although the molecular mechanisms are largely unknown. In this study, key factors responsible for differential Cd accumulation between two contrasting sweet sorghum genotypes (high‐Cd accumulation one H18, and low‐Cd accumulation one L69) were investigated. H18 exhibited a much higher ability of Cd uptake and translocation than L69. Furthermore, Cd uptake through symplasmic pathway and Cd concentrations in xylem sap were both higher in H18 than those in L69. Root anatomy observation found the endodermal apoplasmic barriers were much stronger in L69, which may restrict the Cd loading into xylem. The molecular mechanisms underlying these morpho‐physiological traits were further dissected by comparative transcriptome analysis. Many genes involved in cell wall modification and heavy metal transport were found to be Cd‐responsive DEGs and/or DEGs between these two genotypes. KEGG pathway analysis found phenylpropanoid biosynthesis pathway was over‐represented, indicating this pathway may play important roles in differential Cd accumulation between two genotypes. Based on these results, a schematic representation of main processes involved in differential Cd uptake and translocation in H18 and L69 is proposed, which suggests that higher Cd accumulation in H18 depends on a multilevel coordination of efficient Cd uptake and transport, including efficient root uptake and xylem loading, less root cell wall binding, and weaker endodermal apoplasmic barriers.

H+‐pyrophosphatase from Salicornia europaea confers tolerance to simultaneously occurring salt stress and nitrogen deficiency in Arabidopsis and wheat

High salinity and nitrogen (N) deficiency in soil are two key factors limiting crop productivity, and they usually occur simultaneously. Here we firstly found that H(+) -PPase is involved in salt-stimulated NO3 (-) uptake in the euhalophyte Salicornia europaea. Then, two genes (named SeVP1 and SeVP2) encoding H(+) -PPase from S.u2009europaea were characterized. The expression of SeVP1 and SeVP2 was induced by salt stress and N starvation. Both SeVP1 or SeVP2 transgenic Arabidopsis and wheat plants outperformed the wild types (WTs) when high salt and low N occur simultaneously. The transgenic Arabidopsis plants maintained higher K(+) /Na(+) ratio in leaves and exhibited increased NO3 (-) uptake, inorganic pyrophosphate-dependent vacuolar nitrate efflux and assimilation capacity under this double stresses. Furthermore, they had more soluble sugars in shoots and roots and less starch accumulation in shoots than WT. These performances can be explained by the up-regulated expression of ion, nitrate and sugar transporter genes in transgenic plants. Taken together, our results suggest that up-regulation of H(+) -PPase favours the transport of photosynthates to root, which could promote root growth and integrate N and carbon metabolism in plant. This work provides potential strategies for improving crop yields challenged by increasing soil salinization and shrinking farmland.