Identification and Characterization of Regulatory Pathways Controlling Dormancy Under Lower Temperature in Alfalfa (Medicago sativa L.).

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.

Transcriptomic analysis reveals the changes of energy production and AsA-GSH cycle in oat embryos during seed ageing.

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.

Gibberellin-Stimulation of Rhizome Elongation and Differential GA-Responsive Proteomic Changes in Two Grass Species.

Rapid and extensive rhizome development is a desirable trait for perennial grass growth and adaptation to environmental stresses. The objective of this study was to determine proteomic changes and associated metabolic pathways of gibberellin (GA) -regulation of rhizome elongation in two perennial grass species differing in rhizome development. Plants of a short-rhizome bunch-type tall fescue (TF; Festuca arundinacea; 'BR') and an extensive rhizomatous Kentucky bluegrass (KB; Poa pratensis; 'Baron') were treated with 10 µM GA3 in hydroponic culture in growth chambers. The average rhizome length in KB was significantly longer than that in TF regardless of GA3 treatment, and increased significantly with GA3 treatment, to a greater extent than that in TF. Comparative proteomic analysis using two-dimensional electrophoresis and mass spectrometry was performed to further investigate proteins and associated metabolic pathways imparting increased rhizome elongation by GA. A total of 37 and 38 differentially expressed proteins in response to GA3 treatment were identified in TF and KB plants, respectively, which were mainly involved in photosynthesis, energy and amino acid metabolism, protein synthesis, defense and cell development processes. Accelerated rhizome elongation in KB by GA could be mainly associated with the increased abundance of proteins involved in energy metabolism (glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, and ATP synthase), amino acid metabolism (S-adenosylmethionine and adenosylhomocysteinase), protein synthesis (HSP90, elongation factor Tu and eukaryotic translation initiation factor 5A), cell-wall development (cell dividion cycle protein, alpha tubulin-2A and actin), and signal transduction (calreticulin). These proteins could be used as candidate proteins for further analysis of molecular mechanisms controlling rhizome growth.

Functional characterization and hormonal regulation of the PHEOPHYTINASE gene LpPPH controlling leaf senescence in perennial ryegrass.

Chlorophyll (Chl) degradation occurs naturally during leaf maturation and senescence, and can be induced by stresses, both processes involving the regulation of plant hormones. The objective of this study was to determine the functional roles and hormonal regulation of a gene encoding pheophytin pheophorbide hydrolyase (PPH) that catabolizes Chl degradation during leaf senescence in perennial grass species. A PPH gene, LpPPH, was cloned from perennial ryegrass (Lolium perenne L.). LpPPH was localized in the chloroplast. Overexpressing LpPPH accelerated Chl degradation in wild tobacco, and rescued the stay-green phenotype of the Arabidopsis pph null mutant. The expression level of LpPPH was positively related to the extent of leaf senescence. Exogenous application of abscisic acid (ABA) and ethephon (an ethylene-releasing agent) accelerated the decline in Chl content in leaves of perennial ryegrass, whereas cytokinin (CK) and aminoethoxyvinylglycine (AVG; an ethylene biosynthesis inhibitor) treatments suppressed leaf senescence, corresponding to the up- or down-regulation of LpPPH expression. The promoters of five orthologous PPH genes were predicted to share conserved cis-elements potentially recognized by transcription factors in the ABA and CK pathways. Taken together, the results suggested that LpPPH-mediated Chl breakdown could be regulated positively by ABA and ethylene, and negatively by CK, and LpPPH could be a direct downstream target gene of transcription factors in the ABA and CK signaling pathways.