Mutagenesis reveals that the rice OsMPT3 gene is an important osmotic regulatory factor

Plant mitochondrial phosphate transporters regulate phosphate transport and ATP synthesis. Determining whether they function in abiotic stress response process would shed light on their response to salt stress. We used the CRISPR/Cas9 gene-editing system to mutagenize two mitochondrial phosphate transporters, OsMPT3;1 and OsMPT3;2, to investigate their regulatory roles under salt stress. Two cas9(CRISPR-associated protein9)-free homozygous mutants, mpt33 and mpt30, were confirmed to be stable. Both OsMPT3;1 and OsMPT3;2 were markedly induced by salt stress, and their mutagenesis strongly inhibited growth and development, especially under salt stress. Mutagenesis sharply reduced the accumulation of ATP, phosphate, calcium, soluble sugar, and proline and increased osmotic potential, malondialdehyde, and Na^+ /K^+ ratio under salt stress. Both mutants demonstrate normal growth and development in the presence of ATP, revealing high sensitivity to exogenous ATP under salt stress. The mutants showed lowered rates of Na^+ efflux but also of K^+ and Ca^(2+) influx under salt stress. Mutagenesis of OsMPT3;2 altered the enrichment profiles of differentially expressed genes involved mainly in synthesis of secondary metabolites, metabolism of glycolysis, pyruvate, tricarboxylic acid cycle, in response to salt stress. The mutant displayed significant accumulation differences in 14 metabolites involved in 17 metabolic pathways, and strongly up-regulated the accumulation of glutamine, a precursor in proline synthesis, under salt stress. These findings suggest that the OsMPT3 gene modulates phosphate transport and energy supply for ATP synthesis and triggers changes in accumulation of ions and metabolites participating in osmotic regulation in rice under salt stress, thus increasing rice salt tolerance. This study demonstrates the effective application of CRISPR/Cas9 gene-editing to the investigation of plant functional genes.

Tomato expressing Arabidopsis glutaredoxin gene AtGRXS17 confers tolerance to chilling stress via modulating cold responsive components

Chilling stress is a production constraint of tomato,a tropical origin,chilling-sensitive horticultural crop.The development of chilling tolerant tomato thus has significant potential to impact tomato production.Glutaredoxins(GRXs)are ubiquitous oxidoreductases,which utilize the reducing power of glutathione to reduce disulfide bonds of substrate proteins and maintain cellular redox homeostasis.Here,we report that tomato expressing Arabidopsis GRX gene AtGRXS17 conferred tolerance to chilling stress without adverse effects on growth and development.AtGRXS17-expressing tomato plants displayed lower ion leakage,higher maximal photochemical efficiency of photosystem II(Fv/Fm)and increased accumulation of soluble sugar compared with wild-type plants after the chilling stress challenge.Furthermore,chilling tolerance was correlated with increased antioxidant enzyme activities and reduced H2O2 accumulation.At the same time,temporal expression patterns of the endogenous C-repeat/DRE-binding factor 1(SlCBF1)and CBF mediated-cold regulated genes were not altered in AtGRXS17-expressing plants when compared with wild-type plants,and proline concentrations remained unchanged relative to wild-type plants under chilling stress.Green fluorescent protein-AtGRXS17 fusion proteins,which were initially localized in the cytoplasm,migrated into the nucleus during chilling stress,reflecting a possible role of AtGRXS17 in nuclear signaling of chilling stress responses.Together,our findings demonstrate that genetically engineered tomato plants expressing AtGRXS17 can enhance chilling tolerance and suggest a genetic engineering strategy to improve chilling tolerance without yield penalty across different crop species.

Vacuolar phosphate transporters account for variation in phosphate accumulation in Astragalus sinicus cultivars

Astragalus sinicus is a commonly used legume green manure that fixes atmospheric N2 and accumulates mineral nutrients and organic substances that are beneficial to soils and subsequent crops.However,little is known about genotypic variation in,and molecular mechanisms of,Pi(phosphate)uptake and storage in A.sinicus.We recorded the morphological responses of six A.sinicus cultivars from four regions of China to external Pi application and measured their Pi accumulation.We identified full-length transcripts of Pi-signaling and Pi-homeostasis regulators by sequencing and measured the expression level of these genes by qRT-PCR.The major components in Pi signaling and Pi homeostasis were largely conserved between A.sinicus and the model species rice and Arabidopsis.Different A.sinicus varieties responded differently to low-phosphorus(P)stress,and their Pi accumulation was positively correlated with the expression of vacuolar Pi influx gene(SYG1/PHO81/XPR1-MAJOR FACILITATOR SUPERFAMILY(SPX-MFS)-TYPE PROTEIN)AsSPXMFS2 and negatively correlated with the expression of the vacuolar Pi efflux gene(VACUOLAR Pi EFFLUX TRANSPORTER)AsVPE1.We identified key Pi-signaling and Pihomeostasis regulators in A.sinicus.The expression of vacuolar Pi transporter genes could be used as an index to select A.sinicus accessions with high Pi accumulation.

NIGT1 represses plant growth and mitigates phosphate starvation signaling to balance the growth response tradeoff in rice

Inorganic phosphate(Pi)availability is an important factor which affects the growth and yield of crops,thus an appropriate and effective response to Pi fluctuation is critical.However,how crops orchestrate Pi signaling and growth under Pi starvation conditions to optimize the growth defense tradeoff remains unclear.Here we show that a Pi starvationinduced transcription factor NIGT1(NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1)controls plant growth and prevents a hyper-response to Pi starvation by directly repressing the expression of growth-related and Pisignaling genes to achieve a balance between growth and response under a varying Pi environment.NIGT1 directly binds to the promoters of Pi starvation signaling marker genes,like IPS1,mi R827,and SPX2,under Pi-deficient conditions to mitigate the Pi-starvation responsive(PSR).It also directly represses the expression of vacuolar Pi efflux transporter genes VPE1/2 to regulate plant Pi homeostasis.We further demonstrate that NIGT1 constrains shoot growth by repressing the expression of growth-related regulatory genes,including brassinolide signal transduction master regulator BZR1,cell division regulator CYCB1;1,and DNA replication regulator PSF3.Our findings reveal the function of NIGT1 in orchestrating plant growth and Pi starvation signaling,and also provide evidence that NIGT1 acts as a safeguard to avoid hyper-response during Pi starvation stress in rice.

Identification of long chain isoprenoid hydrocarbons from pyrolytic product of Dunaliella

In the pyrolytic research on Dunaliella salina cultured under natural conditions, abundant long chain regular isoprenoid hydrocarbons have been first detected and identified from algal pyrolytic product. Specially, the discovery of high abundant 2,6,10,14,18-pentamethyle-icosane indicated that regular iC25 probably originated from specific holophilic algae in hyper-

Construction, characterization and application of a genomewide promoter library in Saccharomyces cerevisiae

Promoters are critical elements to control gene expression but could behave differently under various growth conditions. Here we report the construction of a genome-wide promoter library, in which each native promoter in Saccharomyces cerevisiae was cloned upstream of a yellow fluorescent protein （YFP） reporter gene. Nine libraries were arbitrarily defined and assembled in bacteria. The resulting pools of promoters could be prepared and transformed into a yeast strain either as centromeric plasmids or integrated into a genomic locus upon enzymatic treatment. Using fluorescence activated cell sorting, we classified the yeast strains based on YFP fluorescence intensity and arbitrarily divided the entire library into 12 bins, representing weak to strong promoters. Several strong promoters were identified from the most active bins and their activities were assayed under different growth conditions. Finally, these promoters were applied to drive the expression of genes in xylose utilization to improve fermentation efficiency. Together, this library could provide a quick solution to identify and utilize desired promoters under user-defined growth conditions.

Dissecting PCNA function with a systematically designed mutant library in yeast

Proliferating cell nuclear antigen (PCNA), encoded by POL30 in Saccharomyces cerevisiae, is a key component of DNA metabolism. Here, a library consisting of 304 PCNA mutants was designed and constructed to probe the contribution of each residue to the biological function of PCNA. Five regions with elevated sen sitivity to DNA damaging reagents were identified using high-throughput phe no type screening. Using a series of genetic and biochemical analyses, we demonstrated that one particular mutant, K168A, has defects in the DNA damage tolerance (DDT) pathway by disrupting the interaction between PCNA and Rad5. Subsequent domain analysis showed that the PCNA-Rad5 interaction is a prerequisite for the function of Rad5 in DDT. Our study not only provides a resource in the form of a library of versatile mutants to study the functions of PCNA, but also reveals a key residue on PCNA (K168) which highlights the importance of the PCNA-Rad5 interaction in the template switching (TS) pathway.

Distinct roles of alternative oxidase pathway during the greening process of etiolated algae

The vital function of mitochondrial alternative oxidase(AOX) pathway in optimizing photosynthesis during plant de-etiolation has been well recognized. However, whether and how AOX impacts the chloroplast biogenesis in algal cells remains unclear. In the present study, the role of AOX in regulating the reassembly of chloroplast in algal cells was investigated by treating Auxenochlorella protothecoides with salicylhydroxamic acid(SHAM), the specific inhibitor to AOX, in the heterotrophy to autotrophy transition process. Several lines of evidences including delayed chlorophyll accumulation, lagged reorganization of chloroplast structure, altered PSI/PSII stoichiometry, and declined photosynthetic activities in SHAM treated cells indicated that the impairment in AOX activity dramatically hindered the development of functioning chloroplast in algal cells. Besides, the cellular ROS levels and activities of antioxidant enzymes were increased by SHAM treatment, and the perturbation on the balance of NAD+/NADH and NADP+/NADPH ratios was also observed in A. protothecoides lacking AOX activity, indicating that AOX was essential in promoting ROS scavenging and keeping the redox homeostasis for algal chloroplast development during greening. Overall, our study revealed the essentiality of mitochondrial AOX pathway in sustaining algal photosynthetic performance and provided novel insights into the physiological roles of AOX on the biogenesis of photosynthetic organelle in algae.

Hydrocarbons pyrolysed from nannoplanktonic algae:An experimental organism system for study on the origin of petroleum and natural gas

Enukuania huxleyi is marine nanoplanktonic alga with calcify scale called coccolith om its cell surface.This ubiquitous species with the Lafgest biomass in marice forms gigantic blooms covering thousands

Effects of hydrolysis and bacterial degradation in the cells on hydrocarbons and biomarkers generated by pyrolysis of green alga Chlorella protothecoides

The relative contents of normal alkanes and isoprenoid alkanes from pyrolysed products of green alga Chlorella protothecoides increased when the algal cell subject to hydrolysis and bacterial degradation were used. Their value of C 23 -/C 24 + and the rate of Pr/C 17 increased greatly whereas the contents of C 20 alkenes were remarkably low compared with that from untreated sample (as control). These results are more analogous to the formation process of hydrocarbons derived from algae in natural geological environment.