Plant Growth Promoting Rhizobacteria Having 1-Aminocyclopropane-1-Carboxylic Acid Deaminase to Induce Salt Tolerance in Sunflower (<i>Helianthus annus L.</i>)

Soil salinity badly affects agriculture productivity through accumulation of salts in upper layers of soils. The harmful effects of salts in arable lands have influenced modern as well as ancient civilizations. A pot study was carried out to test the performance of two PGPR isolates (KS 8, KS 28) on sunflower (SMH-0917) under different salinity levels (8, 10 and 12 dS·m-1). These salinity levels were developed by adding calculated amount of salts (NaCl, Na2SO4, CaCl2 and MgSO4) with ratio of 3:4:2:1. The bacterial strains KS 8 and KS 28 were applied separately in two treatments while third treatment was co-inoculation (KS mix). Completely randomized experimental design (CRD) was used and data were collected at flowering stage about pre-decided plant growth parameters (plant height, shoot dry weight and root dry weight). The bacterial isolate KS 8 showed an increase of 26, 102% and 83% in plant height, shoot dry weight and root dry weight at EC 8 dS·m-1, while this improvement was 67%, 163% and 296% at EC 10 dS·m-1, however an increase of 100%, 74% and 382% was recorded over control respectively at EC 12 dS·m-1. Similarly isolate KS 28 exhibited an increase of 14%, 69% and 54% in plant height;shoot dry weight and root dry weight at EC 8 dS·m-1, whereas this improvement was 56%, 163% and 188% at EC 10 dS·m-1, while an increase of 60%, 41% and 282% was registered respectively over control at EC 12 dS·m-1. The increase due to mixture treatments was 4%, 41% and 16% in plant height, shoot dry weight and root dry weight at EC 8 dS·m-1, while an increase of 33%, 57% and 100% at EC 10 dS·m-1, whereas an improvement of 53%, 33% and 164% respectively was noted at EC 12 dS·m-1 over un-inoculated. The isolate KS 8 performed better than KS 28 and mixture treatment. These two PGPR strains could be used to mitigate the adverse impact caused by salinity stress on sunflower.

Isolation of a Tomato Protease that May Be Involved in Proteolysis of 1-Aminocyclopropane-l-Carboxylate Synthase

1-aminocyclopropane- 1-carboxylate （ACC） synthase is a principal enzyme that catalyses the committed step in phytohormone ethylene biosynthesis. Previous evidence indicates that the hypervariable C-terminus of ACC synthase is most likely to be processed proteolytically in vivo. However, the protease responsible has not been identified thus far. In the present study, we detected proteolytic activity against ACC synthase （LeACS2） in tomato （Lycopersicon esculentum Mill.） fruit extract based on a newly established in vitro assay system. Purification of the protease through DEAE, gel filtration and MonoQ chromatography resulted in considerable enrichment of a 64-kDa protein species. Subsequent biochemical analysis of the purified tomato protease revealed that the optimal conditions for its proteolytic activity were at pH 8.0 and at 37 ~C. In addition, the protease activity was blocked completely by the metalloprotease inhibitor 1,10-phenanthroline. The present study represents the first report on the isolation of an ACC synthase- processing protease from plant tissues.

番茄ACC合成酶反义cDNA转化及转基因番茄果实贮藏生理特性研究

ACC 合成酶 cDNA 被反向插入载体 PMON316,通过三亲交配,将表达质粒转移到农杆菌中,采用叶盘法转化番茄子叶。经卡那霉素筛选及分子杂交检测,表明,反义 ACC 合成酶基因已在 mRNA 水平表达。对转基因番茄果实的成熟生理指标测定结果说明,转基因番茄果实成熟受到严重抑制,乙烯的峰值仅为正常番茄的25%～30%,PG 活性有所下降,果实在室温下可贮藏1个多月,品质和其他性状与正常番茄相近。

Development of Alfalfa (Medicago sativa L.) Regeneration System and Agrobacterium-Mediated Genetic Transformation

The regeneration ability of four alfalfa （Medicago sativa L.） cultivars, Xinjiang Daye, Longdong, Gannong 1 and Gannong 3, was studied, and the effects of various cultivars, explant sources and medium recipes on regeneration were compared. The better callus forming frequency obtained from hypocotyls of Xinjiang Daye is 88.5% and regeneration frequency is 9.8% in our initial experiments. To further optimize regeneration system for genetic transformation, we therefore changed concentrations of plant growth regulators and supplemented with glutamine into callus-induction and shoot-regeneration media. Callus forming frequency and shoot differentiation frequency were increased to 100%. The time taken to generate transgenic plants （16 weeks） was shorter than that for previouse procedure （25 weeks） and regeneration frequency was promoted to 15.1%. The results show that addition of glutamine is particularly important for shortening period of regeneration and promoting regeneration frequency. For study of genetic transformation of alfalfa, Agrobacterium tumefaciens-mediated transformation of Xinjiang Daye was developed based on this optimized regeneration system. The plant expression vector carrying two glutamine synthetases （GS 1 and GS2） and △1-pyrroline-5-carboxylate synthetase （P5CS） gene was used for alfalfa in vitro transformation. Six transgenic alfalfa plantlets with resistance to PPT were obtained. The introduction of foreign genes into plants was assessed in the transformants by PCR analysis and Southern hybridizations.

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.

Functional mechanism study of the allelochemical myrigalone A identifies a group of ultrapotent inhibitors of ethylene biosynthesis in plants

Allelochemicals represent a class of natural products released by plants as root,leaf,and fruit exudates that interfere with the growth and survival of neighboring plants.Understanding how allelochemicals function to regulate plant responses may provide valuable new approaches to better control plant function.One such allelochemical,Myrigalone A(MyA)produced by Myrica gale,inhibits seed germination and seedling growth through an unknown mechanism.Here,we investigate MyA using the tractable modelDictyostelium discoideum and reveal that its activity depends on the conserved homolog of the plant ethylenesynthesis protein 1-aminocyclopropane-1-carboxylic acid oxidase(ACO).Furthermore,in silico modeling predicts the direct binding of MyA to ACO within the catalytic pocket.In D.discoideum,ablation of ACO mimics the MyA-dependent developmental delay,which is partially restored by exogenous ethylene,and MyA reduces ethylene production.In Arabidopsis thaliana,MyA treatment delays seed germination,and this effect is rescued by exogenous ethylene.It also mimics the effect of established ACO inhibitors on root and hypocotyl extension,blocks ethylenedependent root hair production,and reduces ethylene production.Finally,in silico binding analyses identify a rangeof highlypotentethylene inhibitorsthatblock ethylene-dependent responseand reduce ethyleneproduction in Arabidopsis.Thus,we demonstrate a molecular mechanism by which the allelochemical MyA reduces ethylene biosynthesis and identify a range of ultrapotent inhibitors of ethylene-regulated responses.

Arbuscular mycorrhizal fungi and endophytic fungi differentially modulate polyamines or proline of peach in response to soil flooding

Symbiotic fungi are involved in plant flooding tolerance,while the underlying mechanism is not yet known.Since polyamines(PAs)and proline are also associated with stress tolerance,it is hypothesized that the enhancement of stress resistance by symbiotic fungi is associated with changes in PAs and/or proline.The aim of this study was to analyze the effect of inoculation with Funneliformis mosseae and Serendipita indica on plant growth,PAs,and proline and the metabolisms in peach(Prunus persica)under flooding.Two-week flooding did not affect root colonization frequence of F.mosseae,while it promoted root colonization frequence of S.indica.Under flooding,plants inoculated with F.mosseae and S.indica maintained relatively higher growth rates than uninoculated plants.Funneliformis mosseae promoted root ornithine(Orn)contentration and arginine(Arg)and Orn decarboxylase activities under flooding,which promoted putrescine(Put),cadaverine(Cad),and spermidine(Spd)contentrations.Conversely,S.indica decreased contentrations of Arg,Orn,and agmatine and Arg decarboxylase activities,thus decreasing PA contentrations under flooding.Polyamines were negatively correlated with the expression of PA uptake transporter genes,PpPUT1 and PpPUT2,in peach.Polyamine transporter genes of F.mosseae(FmTPO)and S.indica(SiTPO)were regulated by flooding,of which FmTPO1 was positively correlated with Put,Cad,and Spd,along with positive correlations of Spd with SiTPO1,SiTPO2,and SiTPO4.Under flooding,F.mosseae decreased proline concentration,while S.indica increased proline concentration and correlated with expression of a△^(1)-pyrroline-5-carboxylate synthetase gene,PpP5CS2.It was thus concluded that F.mosseae modulated polyamine accumulation,while S.indica induced proline accumulation to tolerate flooding.

芹菜素对肥胖型小鼠脂肪组织AMPK信号通路的作用机制

目的：研究芹菜素对肥胖型db/db小鼠脂肪紊乱的作用及机制。方法：8周龄雄性db/db小鼠随机分为模型组、芹菜素组（50 mg·kg^-1·d^-1）,雄性C57BL/6J小鼠为正常组,药物干预4周后,检测小鼠体质量、空腹血糖（fasting blood glucose,FBG）,总胆固醇（total cholesterol,CHO）,甘油三酯（total triglyceride,TG）,游离脂肪酸（free fatty acids,FFA）,天冬氨酸氨基转移酶（aspartate aminotransferase,AST）,血肌酐（serum creatinine,SCr）,血尿素氮（blood urea nitrogen,BUN）;脂肪组织苏木素-伊红染色（hematoxylin-eosin staining,HE）观察脂肪细胞病理变化;实时荧光定量PCR（Real-time PCR）检测胆固醇应答元件结合蛋白1c（sterol regulatory element-binding protein 1c,SREBP1c）,脂肪酸合成酶（fatty acid synthetase,FAS）,过氧化物增殖物激活受体α（peroxisome proliferator activated receptorα,PPARα）基因表达;蛋白免疫印迹法（Western blot）检测腺苷酸活化蛋白激酶（phosphorylated adenosine monophosphate activated protein kinase,AMPK）和乙酰CoA羧化酶（acetyl-CoA carboxylase,ACC）磷酸化水平。结果：与模型组比较,芹菜素组小鼠体质量,FBG,CHO,TG,FFA明显降低（P〈0.05,P〈0.01）;HE染色脂肪细胞变小,脂质沉积减低;脂肪组织SREBP1c,FAS mRNA表达降低（P〈0.01）,p-AMPKα,p-ACC蛋白表达升高（P〈0.05,P〈0.01）。结论：芹菜素可以改善db/db小鼠脂肪代谢紊乱,可能是通过上调脂肪组织AMPK,ACC磷酸化水平,下调SREBP1c,FAS mRNA表达实现。

Exogenous ethylene influences flower opening of cut roses (Rosa hybrida) by regulating the genes encoding ethylene biosynthesis enzymes

The purpose of this paper is to investigate the differential responses of flower opening to ethylene in two cut rose cultivars, ‘Samantha’, whose opening process is promoted, and ‘Kardinal’, whose opening process is inhibited by ethylene. Ethylene production and 1- aminocyclopropane-1-carboxylate (ACC) synthase and oxidase activities were determined first. After ethylene treatment, ethylene production, ACC synthase (ACS) and ACC oxidase (ACO) activities in petals increased and peaked at the earlier stage (stage 3) in ‘Samantha’, and they were much more dramatically enhanced and peaked at the later stage (stage 4) in ‘Kardinal’ than control during vasing. cDNA fragments of three Rh-ACSs and one Rh- ACO genes were cloned and designated as Rh-ACS1, Rh-ACS2, Rh-ACS3 and Rh-ACO1 respectively. Northern blotting analysis revealed that, among three genes of ACS, ethylene-in- duced expression patterns of Rh-ACS3 gene corresponded to ACS activity and ethylene production in both cultivars. A more dramatic accumulation of Rh-ACS3 mRNA was induced by ethylene in ‘Kardinal’ than that of ‘Samantha’. As an ethylene action inhibitor, STS at concentration of 0.2 mmol/L generally inhib-ited the expression of Rh-ACSs and Rh-ACO in both cultivars, although it induced the expression of Rh-ACS3 transiently in ‘Kardinal’. Our results suggests that ‘Kardinal’ is more sensitive to ethylene than ‘Samantha’; and the changes of Rh-ACS3 expression caused by ethylene might be related to the acceleration of flower opening in ‘Samantha’ and the inhibition in ‘Kardinal’. Additional results indicated that three Rh-ACSs genes were differentially associated with flower opening and senescence as well as wounding.