Drought-induced responses of organic osmolytes and proline metabolism during pre-flowering stage in leaves of peanut (Arachis hypogaea L.)

Peanut （Arachis hypogaea L.）, an improtant oil crop, usually encounters drought stress in the process of growth and development, especially at pre-flowering stage. In order to gain insight into the drought tolerance potentials based on osmolyte accumulation and metabolism of proline aspects of peanut, pot experiments were conducted with a split-plot design in Tai＇an, Shangdong Province, China in 2013 and 2014. Pre-flowering drought （PFD） stress and optinum irrigation （control, CK） were served as the main plots and the two peanut cultivars Shanhua 11 and Hua 17 served as sub-plots. Shanhua 11 was drought-tolerant cultivar and Hua 17 was drought-sensitive. The content of soluble sugars, soluble protein, free proline and other free amino acids, the activities of enzymes involved in proline metabolism, and malondialdehyde （MDA） content and ion leakage were all investigated in the two cultivars at pre-flowering stage. Results showed that PFD stress significantly increased the levels of soluble protein, free proline and free amino acid, and increased Al-pyrroline-5-car- boxylate synthetase （P-5-CS, EC 2.7.2.11） activity in the leaves of drought-tolerant and drought-sensitive cultivars. The activity of proline dehydrogenase （proDH） （EC 1.5.99.8） decreased under PFD stress in both cultivars. The leaves of the tolerant cultivar maintained higher increments of osmolyte levels, lower increments of MDA content and ion leakage, as well as a higher increased proportion of P-5-CS activity and higher inhibited proportion of proDH activity under water stress compared with the drought-sensitive cultivar. The study suggests that proline accumulation in peanut leaves under PFD can be explained by the higher enhanced activities of P-5-CS and higher inhibition of proDH. The results will provide useful information for genetic improvement of peanut under drought tolerance.

Gene Cloning and Expression of the Pyrroline-5-carboxylate Reductase Gene of Perennial Ryegrass(Lolium perenne)

Salt and drought limit the range of applications of perennial ryegrass(Lolium perenne L.), which is one of the important turf and forage grasses. Previous studies have suggested that pyrroline-5-carboxylate reductase(P5CR) might play a central role in proline accumulation in plants that are responsive to stresses. In the present study, the Lolium perenne L. pyrroline-5-carboxylate reductase(LpP 5CR) gene was cloned from leaves of the cultivar ‘Derby' using the RACE technique. The full-length LpP 5CR gene was 1 047 bp in length, which comprised an open reading frame(ORF) of 840 bp in size. Sequence alignment revealed that the putative Lp P5 CR had a 94.3% similarity to Ta P5 CR. q RT-PCR displayed that the mR NA levels of the LpP 5CR gene were almost the same as that in the roots, stems, and leaves of perennial ryegrass seedlings subjected to normal condition or NaC l treatment for 1 h. Moreover, the transcription level of LpP 5CR was up- or down- regulated with Na Cl, polyethylene glycol(PEG), cold, or abscisic acid(ABA) treatment for 3 to 48 h. In addition, confocal microscopy localized the GFP-Lp P5 CR fusion protein to the cytoplasm of onion epidermal cells. These findings suggest that LpP 5CR encodes a cytoplasmic P5 CR protein that plays an important role in the response of perennial ryegrass to various stresses.

Reference genes for quantitative real-time PCR analysis and quantitative expression of P5CS in Agropyron mongolicum under drought stress

Reference genes, stably expressing in different tissues and cells, are commonly used as the references in expression analysis. Selecting the optimum reference gene is crucial to the success of experiments. In this study, the expression stabilities of nine common reference genes, including ACT2, 18 S r RNA, APRT, EF-1α, RNA POL II, TUBα, TUBβ, GAPDH and TLF of Agropyron mongolicum, were studied under drought condition. Among them, 18 S r RNA was found to be the most optimum reference gene under drought stress by the analyzing of ge Norm and Norm Finder software. Quantitative expression levels of P5 CS using 18 S r RNA as the reference gene, and proline contents under drought stress in A. mongolicum were further operated, and we found the expression level of P5 CS gene and proline content had a significantly positive relationship（R^2=0.7763, P〈0.05）. This study established and validated 18 S r RNA as the reference genes in A. mongolicum under drought stress, providing a powerful tool for the quantitative expression analysis of drought genes in A. mongolicum.

African Swine Fever Virus Protein E199L Promotes Cell Autophagy through the Interaction of PYCR2

African swine fever virus(ASFV),as a member of the large DNA viruses,may regulate autophagy and apoptosis by inhibiting programmed cell death.However,the function of ASFV proteins has not been fully elucidated,especially the role of autophagy in ASFV infection.One of three Pyrroline-5-carboxylate reductases(PYCR),is primarily involved in conversion of glutamate to proline.Previous studies have shown that depletion of PYCR2 was related to the induction of autophagy.In the present study,we found for the first time that ASFV E199 L protein induced a complete autophagy process in Vero and HEK-293 T cells.Through co-immunoprecipitation coupled with mass spectrometry(Co IP-MS)analysis,we firstly identified that E199 L interact with PYCR2 in vitro.Importantly,our work provides evidence that E199 L down-regulated the expression of PYCR2,resulting in autophagy activation.Overall,our results demonstrate that ASFV E199 L protein induces complete autophagy through interaction with PYCR2 and down-regulate the expression level of PYCR2,which provide a valuable reference for the role of autophagy during ASFV infection and contribute to the functional clues of PYCR2.