Yangsheng Li

Use of isotope mass probes for metabolic analysis of the jasmonate biosynthetic pathway

In order to quantitatively study the jasmonate biosynthetic pathway, we chemically synthesized a pair of isotope mass probes and established a labeling protocol. The pair of mass probes used in our work were ω-bromoacetonylpyridinium bromide (BPB) and d(5)-ω-bromoacetonylpyridinium bromide (d(5)-BPB), which contain carboxylic acid reactive groups, isotopically labeled groups and permanent positive charges. High performance liquid chromatography (HPLC) and electrospray ionization quadrupole-time of flight mass spectrometry (ESI-QTOF-MS) were used for the detection of labeled standard mixtures and plant samples. In comparison to negative mode electrospray ionization detection of unlabeled analytes, the ESI signal of reverse charge labeled compounds was shown to improve by 20- to 80-fold. Accurate relative quantification was achieved as no isotopic effects of the different isotope labeled phytohormones during RP/SCX mixed-mode liquid chromatographic separation were observed. A data analysis method was established for analyzing metabolic pathways using our labeling strategy. We then applied our method and examined the jasmonate biosynthetic pathway of rice under salt stress and the premature senescence mutant. Here we found that under salt stress conditions, rice showed up-regulation in (13S)-hydroperoxyoctadecatrienoic acid (HOPT), cis-(+)-12-oxophytodienoic acid (OPDA), 3-oxo-2-(2-pentenyl)-cyclopentane-1-octanoic acid (OPC-8) and jasmonoyl-valine (JA-Val) levels, while α-linolenic acid (LA) and jasmonic acid (JA) showed down-regulation, and three components (HPOT, OPC-8 and JA-Val) were accumulated. The premature senescence mutant showed up-regulation in all major components of the jasmonate biosynthetic pathway with the exception of LA, and an accumulation of HPOT, OPC-6 and JA-Val. This study demonstrates that our chemical stable isotope labeling strategy can be used as a powerful tool for metabolic pathway analysis of phytohormones in plants.

Functional inactivation of UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) induces early leaf senescence and defence responses in rice

Highlight This study identified the novel gene UAP1 in rice. UAP1 is involved in early leaf senescence and defence responses.

Differential antioxidant responses to cold stress in cell suspension cultures of two subspecies of rice

Crops often encounter oxidative stresses due to changes of environmental conditions. Cold damage is one of the serious problems to agricultural production. In this study, cell suspension cultures of two subspecies of rice (Oryza sativa L.), Japonica cv. Nipponbare and indica cv. 9311, were developed to investigate their antioxidant responses to cold stress. The inherent morphology and surface features of cultured cells were examined by use of Cryo-scanning electron microscopy technique. The transcript of OsDREB2B, a cold-inducible transcription factor encoding gene, was strongly induced by cold stress in both cell lines. A significant accumulation of malondialdehyde, a product of lipid peroxidation, in cv. 9311 cells exposed to low temperature after 24xa0h demonstrated that they had higher levels of membrane damage compared with cv. Nipponbare under cold stress. In addition, responses of the antioxidant system to cold stress were genotype dependent. Compared to cv. 9311, cell suspension cultures of cv. Nipponbare exhibited elevated levels/contents of glutathione peroxidase, higher catalase, glutathione reductase, and glutathione. These would serve to protect cells against oxidative injury during the first few hours of chilling stress.

A Thioredoxin-Dependent Glutathione Peroxidase (OsGPX5) Is Required for Rice Normal Development and Salt Stress Tolerance

Crops often suffer from oxidative stress due to changes of the environmental conditions. Glutathione peroxidases (GPXs) are a family of enzymes that protect cells against oxidative damage caused by generation of excessive reactive oxygen species (ROS) in vivo. Compared to animal GPXs, the precise physiological role of plant GPXs remained poorly understood, particularly that of endoplasmic reticulum (ER)-type GPXs. Rice OsGPX5 has been reported to have dual subcellular compartments (ER and chloroplast), suggesting that it might play important roles in vivo. In the present study, transcriptional expression pattern analysis of OsGPX5 indicated that it displayed differential responses to various abiotic stresses or hormone treatments. Additionally, OsGPX5 was ubiquitously expressed in most of the tissues and organs analyzed and was found to accumulate particularly high transcript abundance in green tissues. Similar to other plant GPX homologs, the purified recombinant OsGPX5 protein showed activities of thioredoxin-dependent peroxidase, catalyzing the reduction of hydrogen peroxide and organic hydroperoxides. Furthermore, disruption of OsGPX5 via T-DNA insertion had adverse effects on the normal development of rice. The OsGPX5 mutant line exhibited lower germination potential, shorter length of 6-day-old seedlings, and a smaller number of filled grains compared to wild-type plants. Moreover, the seedlings of OsGPX5 mutant plants exhibited enhanced sensitivity to salt stress. Beyond being an efficient ROS and peroxide scavenger, OsGPX5 has therefore been proposed to participate in the interaction between ER stress and redox homeostasis, which is essential for the normal development of rice under stress conditions.

Reliable Selection and Holistic Stability Evaluation of Reference Genes for Rice Under 22 Different Experimental Conditions

Stable and uniform expression of reference genes across samples plays a key role in accurate normalization of gene expression by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). For rice study, there is still a lack of validation and recommendation of appropriate reference genes with high stability depending on experimental conditions. Eleven candidate reference genes potentially owning high stability were evaluated by geNorm and NormFinder for their expression stability in 22 various experimental conditions. Best combinations of multiple reference genes were recommended depending on experimental conditions, and the holistic stability of reference genes was also evaluated. Reference genes would become more variable and thus needed to be critically selected in experimental groups of tissues, heat, 6-benzylamino purine, and drought, but they were comparatively stable under cold, wound, and ultraviolet-B stresses. Triosephosphate isomerase (TI), profilin-2 (Profilin-2), ubiquitin-conjugating enzyme E2 (UBC), endothelial differentiation factor (Edf), and ADP-ribosylation factor (ARF) were stable in most of our experimental conditions. No universal reference gene showed good stability in all experimental conditions. To get accurate expression result, suitable combination of multiple reference genes for a specific experimental condition would be a better choice. This study provided an application guideline to select stable reference genes for rice gene expression study.

Development and characterization of microsatellite markers for rice leaffolder, Cnaphalocrocis medinalis (Guenée) and cross-species amplification in other Pyralididae

Rice leaffolder, Cnaphalocrocis medinalis (Guenée), is a destructive and widespread pest on rice. In this study, 20 microsatellite markers were isolated and characterized from C. medinalis partial genomic libraries using the method of fast isolation by AFLP of sequence containing repeats. Of these markers, 18 markers displayed polymorphisms. Polymorphisms were evaluated in 48 individuals from two natural populations. The number of alleles per locus ranged from 2 to 15, and the expected and observed heterozygosities ranged from 0.324 to 0.934 and from 0.304 to 0.917, respectively. Cross-species amplification was also performed to test the transferability of the 20 microsatellite markers and a moderate level of cross amplication was observed across the three species of Pyralididae (26.67xa0%). These microsatellite loci would facilitate the future study on population genetics and molecular genetics of rice leaffolder and would also be useful for study in Chilo suppressalis, Scirpophaga incertulas and Pyrausta nubilalis.

Nitrite Promotes the Growth and Decreases the Lignin Content of indica Rice Calli: A Comprehensive Transcriptome Analysis of Nitrite-Responsive Genes during In Vitro Culture of Rice

As both major macronutrients and signal molecules, nitrogen metabolites, such as nitrate and nitrite, play an important role in plant growth and development. In this study, the callus growth of indica rice cv. 9311 was significantly enhanced by nitrite, whereas the soluble protein content remained unchanged. The deep RNA sequencing technology (RNA-seq) showed that the transcriptional profiles of cv. 9311 calli were significantly changed after adding nitrite to the nitrate-free medium, and these nitrite-responsive genes were involved in a wide range of plant processes, particularly in the secondary metabolite pathways. Interestingly, most of the genes involved in phenylpropanoid-related pathways were coordinately down-regulated by nitrite, such as four cinnamoyl-CoA reductase, and these in turn resulted in the decrease of lignin content of indica calli. Furthermore, several candidate genes related to cell growth or stress responses were identified, such as genes coding for expansins, SMALL AUXIN UP RNA (SAUR) and HSP20s, and these suggested that nitrite could probably serve as a transcriptome signal to enhance the indica calli growth by regulation of various downstream genes expression. This study contributes to a better understanding of the function of nitrite during the process of plant tissue culture and could aid in the application of this technology to improved indica genetic transformation efficiency.

Comparative transcriptional profiling of two rice genotypes carrying SUB1A-1 but exhibiting differential tolerance to submergence

Submergence tolerance in rainfed lowland rice (Oryza sativa L.) is determined mainly by SUB1A-1, which confers the tolerance by regulating the ethylene- and gibberellin-mediated gene expression responsible for carbohydrate consumption, cell elongation and ethanolic fermentation. However, two indica rice genotypes, FR13A and Goda Heenati, both carrying this gene, exhibited differential tolerance to submergence. Comparative analysis of transcriptional profiling of the two genotypes revealed that many of antioxidant genes were more highly expressed in FR13A than in Goda Heenati under both submergence and control conditions, or only under submergence, whereas most of genes involved in biosynthesis and signalling of ethylene and GA and in anaerobic carbohydrate metabolism had comparable levels of expression between genotypes under the same conditions. H2O2 and malondialdehyde (MDA) assays demonstrated that Goda Heenati accumulated more H2O2 and had more MDA, a product of lipid peroxidation, than FR13A under submergence. These findings suggest that apart from SUB1A-mediated quiescence strategy, the detoxification of reactive oxygen species (ROS) is another important trait associated with submergence tolerance. The information obtained from this study helps in further understanding of the mechanism underlying submergence tolerance.

Silencing of D-Lactate Dehydrogenase Impedes Glyoxalase System and Leads to Methylglyoxal Accumulation and Growth Inhibition in Rice

D-Lactate is oxidized by two classes of D-lactate dehydrogenase (D-LDH), namely, NAD-dependent and NAD-independent D-LDHs. Little is known about the characteristics and biological functions of D-LDHs in rice. In this study, a functional NAD-independent D-LDH (LOC_Os07g06890) was identified in rice, as a result of alternative splicing events. Characterization of the expression profile, subcellular localization, and enzymatic properties of the functional OsD-LDH revealed that it is a mitochondrial cytochrome-c-dependent D-LDH with high affinity and catalytic efficiency. Functional analysis of OsD-LDH RNAi transgenic rice demonstrated that OsD-LDH participates in methylglyoxal metabolism by affecting the activity of the glyoxalase system and aldo-keto reductases. Under methylglyoxal treatment, silencing of OsD-LDH in rice resulted in the accumulation of methylglyoxal and D-lactate, the decrease of reduced glutathione in leaves, and ultimately severe growth inhibition. Moreover, the detached leaves of OsD-LDH RNAi plants were more sensitive to salt stress. However, the silencing of OsD-LDH did not affect the growth under photorespiration conditions. Our results provide new insights into the role of NAD-independent D-LDHs in rice.

Identification and Characterization of a Plastidic Adenine Nucleotide Uniporter (OsBT1-3) Required for Chloroplast Development in the Early Leaf Stage of Rice.

Chloroplast development is an important subject in botany. In this study, a rice (Oryza sativa) mutant exhibiting impairment in early chloroplast development (seedling leaf albino (sla)) was isolated from a filial generation via hybridization breeding. The sla mutant seedlings have an aberrant form of chloroplasts, which resulted in albinism at the first and second leaves; however, the leaf sheath was green. The mutant gradually turned green after the two-leaf stage, and the third leaf was a normal shade of green. Map-based cloning indicated that the gene OsBT1-3, which belongs to the mitochondrial carrier family (MCF), is responsible for the sla mutant phenotype. OsBT1-3 expression was high in the young leaves, decreased after the two-leaf stage, and was low in the sheath, and these findings are consistent with the recovery of a number of chloroplasts in the third leaf of sla mutant seedlings. The results also showed that OsBT1-3-yellow fluorescent protein (YFP) was targeted to the chloroplast, and a Western blot assay using a peptide-specific antibody indicated that OsBT1-3 localizes to the chloroplast envelope. We also demonstrated that OsBT1-3 functions as a unidirectional transporter of adenine nucleotides. Based on these findings, OsBT1-3 likely acts as a plastid nucleotide uniporter and is essential for chloroplast development in rice leaves at the young seedling stage.