Comparison Of Salinity-Tolerance And Photosynthetic Characteristics Among Parents And Their Somatic Hybrid Descendants In Soybean

Salt stress is one of the major abiotic stresses that depress plants growth and drastically limit crop production.To study the mechanisms for plant tolerance to salt stress,salinity-resistant traits of a wild soybean species Glycine cyrtoloba ACC547 were introduced to a cultivated soybean G.max Melrose using asymmetric somatic hybridization.The capacity for salt tolerance was compared in the parents and their hybrid descendants,and a wild-soybean-special-ndhH gene was discovered with these materials and the possible function of this gene in the tolerance of soybean to salt stress was studied.The main results obtained were shown as follow:Glycine cyrtoloba ACC547,with high tolerance to salinity,is a wild soybean species native to saline soils in Australian beach.G.max Melrose is a high-yielding soybean cultivar but susceptible to salinity.A series of regenerated lines were obtained from the fused protoplast using asymmetric somatic hybridization.RAPD(random amplified polymorphic DNA)and AFLP(amplified fragment length polymorphism)analysis revealed that these regenerated plants were hybrids between ACC547 and Melrose.The hybrid descendants possessed several morphological features of their parents and some of them also possessed higher salinity-tolerant traits of the wild soybean detecting with salinity-tolerance experiments.It was shown that two of the stable hybrid descendants(S111-9 and S113-6)and their wild parent had higher salinity-tolerance in comparison with the cultivated soybean.Salt stress dramatically depressed the growth of cultivated soybean whose leaves lost green with necrotic lesions,biomass and photosynthetic rate decreased markedly.Both necrotic lesions in leaves and decline in biomass are much lighter in hybrid descendants than in cultivated soybean,but more serious than in wild soybean.However,the decline in photosynthetic rate was not significantly different between hybrid descendants and their wild parent.These results suggested that the descendants inherited partially tolerance to salt stress from the wild species.In the late time of saline treatment,it had no significant difference in root and stem Na~+ and Cl~-concentrations between the wild and cultivated soybean,but the latter owned much higher concentrations of the two ions and much lower K~+ content and K~+/Na~+ ratio in leaves.These results indicated that the partial reason for salt tolerance in wild species was the exclusion of Na~+ and Cl~-from the whole plant and especially from the leaves,the enhancement in K~+ selectivity of leaves,the alleviation in damage of salt to leaves,and the coupled maintenance in photosynthesis.Although possessing higher leaf K~+ content and K~+/Na~+ ratio than their cultivated parent,the descendances showed higher Na~+ and Cl~- contents in both leaves and whole plant than their wild parent with no significant difference from the cultivated soybean.It was suggested that the higher adaptation of the descendances to salt stress might not be due to the capacity for excluding toxic ions from their bodies, but to their tolerance to salinity.One of the AFLP bands which the descendants obtained from their wild soybean parent--ASH1 was found to have highly similar sequence to ndhH,a gene for the H-subunit of chloroplast NAD(P)H dehydrogenase(NDH).PCR analysis with special primers for ASH1 demonstrated that ASH1 represented an ACC547-special ndhH gene,and the whole sequence of this gene was obtained through PCR amplification and DNA sequencing.Multi-alignment of the deduced protein sequences indicated only 5 amino acids in ACC547-special NDH-H different from soybean NDH-H,and the ACC547-special NDH-H lacked oneβcorner in related site in three-dimensional model.In wild soybean,salt stress induced the synthesis of an approximately 45kD thylakoid-membrane-bound protein which was similar to ACC547-special NDH-H in molecular weight.Under salt-stress conditions,the post-illumination transient increase in chlorophyll fluorescence,the rate of p700~+ rereduction,and PSI activity detected with different electron donors and accepters,were all much higher in wild than in cultivated soybean,indicating that NDH-mediated cyclic electron flow around PS I(CEF1)was accelerated by salt stress more greatly in wild than in cultivated soybean.Based on these results,we suspected ACC547-special ndhH could be induced by salinity to express with high level,and its product might be a part of a special NDH complex which mediated efficient CEF1 under salt stress.Moreover,salt stress also prompted CEF1 activity in the descendants due to its derivation of ndhH gene from their wild parent,which might make great contributions to the high salinity-tolerance of the descendants.Under salt tress,Ms-delayed light emission(ms-DLE)was markedly strengthened, non-photochemical quenching of chlorophyll fluorescence(NPQ),particularly high-energy state non-photochemical quenching(qE)were still maintained at a high level,less reactive oxygen species (ROS)was generated in the leaves,chloroplast remained the integrated ultrastructure,and there was higher PSⅡmaximal photochemical efficiency(Fv/Fm)in the descendants and their wild parent. Comparatively,salt treatment significantly decreased ms-DLE,NPQ,qE and Fv/Fm,enhanced the accumulation of ROS,and disrupted the chloroplast structure in Melrose.These results suggested that the higher CEF1 activity via ACC547-special NDH in the wild and hybrid soybeans contributed to the generation and maintenance of proton gradient across thylakoid membranes(△pH)and hence to the efficient thermal dissipation of excess light energy,which protected photosynthetic apparatus from ROS damage.However,this protective mechanism functioned less due to the absence of the efficient CEF1 in cultivated soybean.In addition,light-induced ATP synthesis in leaves was higher in the descendants and their wild parent than in their cultivated parent under salt stress,which indicated that the higher CEF1 activity accelerated the generation of extra ATP in the wild and hybrid soybeans.We also discovered that most of Na~+ were localized in vacuoles in wild soybean,and further study was required to elucidate whether the extra ATP participated the compartmentation of Na~+ into vacuoles.