Xiqing Ma’s research while affiliated with China Agricultural University and other places

Soil salinity is a significant environmental challenge that adversely affects plant yield and quality. Zoysiagrass (Zoysia japonica), a member of the Gramineae family, is highly salt-tolerant, making it an excellent model for studying salt stress response mechanisms. We performed physiological and transcriptomic analyses on two contrasting Zoysiagrass germplasm accessions under high salt conditions. The salt-tolerant germplasm ST68 demonstrated superior growth phenotypes, higher chlorophyll and relative water content, greater photochemical efficiency, and lower relative electrolyte leakage and sodium ion content compared to the salt-sensitive germplasm SS9. Transcriptomic analysis revealed differential expression in pathways involved in photosynthesis, flavonoid biosynthesis, cell wall macromolecule catabolism, phosphate ion homeostasis, and reactive oxygen species response in the tolerant vs the sensitive line under salt stress. Notably, the ZjHEMA gene, which encodes glutamyl-tRNA reductase, a rate-limiting enzyme in chlorophyll biosynthesis, was identified as a key regulator due to its significant upregulation under salt stress in the salt-tolerant germplasm, compared to the sensitive one. Overexpression of the salt-responsive glutamyl-tRNA reductase gene, associated with chlorophyll metabolism in Zoysiagrass, in Arabidopsis led to increased salt tolerance, as evidenced by elevated chlorophyll content, relative water content, and photochemical efficiency compared to wild-type plants. Our findings offer new insights into the mechanisms of salt tolerance in Zoysiagrass, laying a foundation for breeding salt-tolerant germplasm.

Alfalfa (Medicago sativa L.), a kind of high-quality perennial legume forage, is widely distributed in the northern regions of China. In recent years, low temperatures have frequently occurred and limited alfalfa productivity and survival in early spring and late fall. However, the underlying molecular mechanisms of alfalfa response to cold tolerance are not well-documented. In this study, dormancy and non-dormancy alfalfa standard varieties were characterized under low-temperature stress. Our analysis revealed that plant height of the dormancy genotype was strongly inhibited by low temperature; flavonoids content, and higher expression of flavonoids biosynthesis genes (chalcone synthase, leucoanthocyanidin dioxygenase, and flavonoid 3'-monooxygenase) may play essential roles in response to low-temperature stress in dormancy genotype alfalfa. Further analyses revealed that receptor-like kinase family genes (such as cysteine-rich RLK10, lectin protein kinase, and S-locus glycoprotein like kinase), RNA and protein synthesis genes (RNA polymerases, ribosomal protein, and protein phosphatase 2C family protein), and proteasome degradation pathway genes (such as F-box family protein, RING/U-box superfamily protein, and zinc finger family protein) also highly upregulated and contributed to cold tolerance phenotype in dormancy genotype alfalfa. This will provide new insights into future studies for cold tolerance in alfalfa and offer new target genes for further functional characterization and genetic improvement of alfalfa.

Forage germplasm resources are the basis of the original innovation in grass industry and forage breeding, and strategically important to ensure the national security of food, ecology and seed industry. After more than 20 years’ rapid development and establishment in forage germplasm resources in China, it has made remarkable achievements in collection and preservation, evaluation and identification, and exploration of gene resources.

The color of bracts generally turns yellow or black from green during cereal grain development. However, the impact of these phenotypic changes on photosynthetic physiology during black bract formation remains unclear. Two oat cultivars (Avena sativa L.), ‘Triple Crown’ and ‘Qinghai 444’, with yellow and black bracts, respectively, were found to both have green bracts at the heading stage, but started to turn black at the flowering stage and become blackened at the milk stage for ‘Qinghai 444’. Their photosynthetic characteristics were analyzed and compared, and the key genes, proteins and regulatory pathways affecting photosynthetic physiology were determined in ‘Triple Crown’ and ‘Qinghai 444’ bracts. The results show that the actual PSII photochemical efficiency and PSII electron transfer rate of ‘Qinghai 444’ bracts had no significant changes at the heading and milk stages but decreased significantly (p < 0.05) at the flowering stage compared with ‘Triple Crown’. The chlorophyll content decreased, the LHCII involved in the assembly of supercomplexes in the thylakoid membrane was inhibited, and the expression of Lhcb1 and Lhcb5 was downregulated at the flowering stage. During this critical stage, the expression of Bh4 and C4H was upregulated, and the biosynthetic pathway of p-coumaric acid using tyrosine and phenylalanine as precursors was also enhanced. Moreover, the key upregulated genes (CHS, CHI and F3H) of anthocyanin biosynthesis might complement the impaired PSII activity until recovered at the milk stage. These findings provide a new insight into how photosynthesis alters during the process of oat bract color transition to black.

Background Alfalfa ( Medicago sativa L.) is a perennial legume extensively planted throughout the world as a high nutritive value livestock forage. Flowering time is an important agronomic trait that contributes to the production of alfalfa hay and seeds. However, the underlying molecular mechanisms of flowering time regulation in alfalfa are not well understood. Results In this study, an early-flowering alfalfa genotype 80 and a late-flowering alfalfa genotype 195 were characterized for the flowering phenotype. Our analysis revealed that the lower jasmonate (JA) content in new leaves and the downregulation of JA biosynthetic genes (i.e. lipoxygenase, the 12-oxophytodienoate reductase-like protein, and salicylic acid carboxyl methyltransferase) may play essential roles in the early-flowering phenotype of genotype 80. Further research indicated that genes encode pathogenesis-related proteins [e.g. leucine rich repeat (LRR) family proteins, receptor-like proteins, and toll-interleukin-like receptor (TIR)-nucleotide-binding site (NBS)-LRR class proteins] and members of the signaling receptor kinase family [LRR proteins, kinases domain of unknown function 26 (DUF26) and wheat leucine-rich repeat receptor-like kinase10 (LRK10)-like kinases] are related to early flowering in alfalfa. Additionally, those involved in secondary metabolism (2-oxoglutarate/Fe (II)-dependent dioxygenases and UDP-glycosyltransferase) and the proteasome degradation pathway [really interesting new gene (RING)/U-box superfamily proteins and F-box family proteins] are also related to early flowering in alfalfa. Conclusions Integrated phenotypical, physiological, and transcriptomic analyses demonstrate that hormone biosynthesis and signaling pathways, pathogenesis-related genes, signaling receptor kinase family genes, secondary metabolism genes, and proteasome degradation pathway genes are responsible for the early flowering phenotype in alfalfa. This will provide new insights into future studies of flowering time in alfalfa and inform genetic improvement strategies for optimizing this important trait.

Cyclophilins (CYPs), a class of proteins with a conserved peptidyl-prolyl cis-trans isomerase domain, are widely involved in the regulation of plant growth and development, as well as in the response to abiotic stresses including cold. In our previous study, we identified an Arabidopsis gain-of-function mutant ROC1S58F with enhanced cold-tolerance and enhanced expression of jasmonic acid (JA) and oxidative stress responsive genes. Here, we show the underlying molecular mechanisms for the improved cold tolerance observed in the ROC1S58F mutant. Compared to the WT, the ROC1S58F mutant showed an increased survival rates and a reduced level of electrolyte leakage and endogenous JA content under the freezing treatment. Correspondingly, the JA biosynthesis genes (AtAOC1 and AtOPR3) and signaling genes (AtJAZ5, AtJAZ10 and AtMYB15) are down-regulated in the ROC1S58F mutant compared with the WT. Moreover, both the transcripts and activities of the ROS-scavenging enzymes (SOD/POD/MDHAR) increased in cold-stressed ROC1S58F mutant, which might mitigate the ROS-induced oxidative stress and contribute to the mutant freezing tolerance. Taken together, our findings indicate that AtROC1S58F confers Arabidopsis freezing tolerance by modulating JA signaling and antioxidant metabolism jointly. This research thus provides a molecular mechanism for AtROC1S58F-conferred freezing resistance in Arabidopsis and offers guidance for crop breeding towards an improved cold tolerance.

Deterioration during seed storage generally causes seed vigour declining. However, the mechanism of deterioration occurred still not clear. Seeds and embryos of oat (Avena sativa L.) were selected to analyze the relation of physiological and metabolic reactions with DEGs by using RNA-seq. Oat seed vigour declined during seeds aged 0 day (CK), 16 days (CD16) and 32 days (CD32). The changes of MDA and H2O2 contents, antioxidant enzymes activities of APX, DHAR, MDHAR and GR related with AsA-GSH cycle in embryos illustrated that seed vigour declined to the minimum at CD32. Transcriptomic analysis showed a total of 11335 and 8274 DEGs were identified at CD16 and CD32 compared with CK respectively, of which 4070 were overlapped. When seed vigour declined to the moderate level (CD16), the accumulation of H2O2 caused by the inhibition of complex I in ETC could be alleviated with AsA-GSH cycle. RNA-seq and qRT-PCR results both showed alternative oxidase in alternate respiratory pathway was upregulated which would maintain seed respiration. However, as seed vigour was at the lowest level (CD32), blocked ETC caused by down-regulation of complex III, including Ubiquinol-cytochrome C reductase complex 14kD subunit and Ubiquinol-cytochrome C reductase, UQCRX/QCR9 like, were more seriously and H2O2 scavenging was limited by the inactive AsA-GSH cycle. It could be suggested that the function of AsA-GSH would play a key role for regulating the physiological responses of ETC in embryos during seed ageing. These results would provide an insight into embryo for the transcriptomic information during oat seed ageing.

Cyclophilins (CYPs) belonging to the immunophilin family are present in all organisms and widely distributed in various cells associated with the activity of peptidyl-prolyl cis/trans isomerase. Plant CYPs are members of a multi-gene family and are involved in a series of biological processes. However, little is known about their structure, evolution, developmental expression and functional analysis in Medicago truncatula. In this study, a total of 33 CYP genes were identified and found to be unevenly distributed on eight chromosomes. Among them, 21 are single-domain and 12 are multi-domain proteins, and most were predicted to be localized in the cytosol, nucleus or chloroplast. Phylogenetic and gene structure analysis revealed seven segmental gene pairs, indicating that segmental duplication probably made a large contribution to the expansion of MtCYP gene family. Furthermore, gene expression analysis revealed that about 10 MtCYP genes (were) highly expressed involved in vegetative and reproduction tissues in M. truncatula, and MsCYP20-3B was mainly upregulated in stems, leaves and flower buds in alfalfa (Medicago sativa). Overexpression of MsCYP20-3B was shown to regulate axillary shoot development associated with higher jasmonic acid and abscisic acid contents in M. truncatula. Our study suggests the importance of the CYP genes family in development, reproduction and stress responses, and provides a reference for future studies and application of CYP genes for alfalfa genetic improvement.

Mitochondria are the source of reactive oxygen species (ROS) in plant cells and play a central role in the mitochondrial electron transport chain (ETC) and tricarboxylic acid cycle (TCA) cycles; however, ROS production and regulation for seed germination, seedling growth, as well as mitochondrial responses to abiotic stress, are not clear. This study was conducted to obtain basic information on seed germination, embryo mitochondrial antioxidant responses, and protein profile changes in artificial aging in oat seeds (Avena sativa L.) exposed to exogenous nitric oxide (NO) treatment. The results showed that the accumulation of H2O2 in mitochondria increased significantly in aged seeds. Artificial aging can lead to a loss of seed vigor, which was shown by a decline in seed germination and the extension of mean germination time (MGT). Seedling growth was also inhibited. Some enzymes, including catalase (CAT), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR), maintained a lower level in the ascorbate-glutathione (AsA-GSH) scavenging system. Proteomic analysis revealed that the expression of some proteins related to the TCA cycle were down-regulated and several enzymes related to mitochondrial ETC were up-regulated. With the application of 0.05 mM NO in aged oat seeds, a protective effect was observed, demonstrated by an improvement in seed vigor and increased H2O2 scavenging ability in mitochondria. There were also higher activities of CAT, GR, MDHAR, and DHAR in the AsA-GSH scavenging system, enhanced TCA cycle-related enzymes (malate dehydrogenase, succinate-CoA ligase, fumarate hydratase), and activated alternative pathways, as the cytochrome pathway was inhibited. Therefore, our results indicated that seedling growth and seed germinability could retain a certain level in aged oat seeds, predominantly depending on the lower NO regulation of the TCA cycle and AsA-GSH. Thus, it could be concluded that the application of 0.05 mM NO in aged oat seeds improved seed vigor by enhancing the mitochondrial TCA cycle and activating alternative pathways for improvement.