Study On Adaptive Mechanisms And Expression Analysis Of Different Genes Of Sweet Sorghum Seedlings Under Saline-alkali Stress

Large amount areas of saline and alkaline soils limit crop growth and reduce agriculturalproductivity. Sorghum （Sorghum bicolor L. Moench） crop has been considered relativelymore salt tolerant than other cereals and has the potential as a grain and fodder crop in salineand alkaline soils. In order to study the adaptive mechanisms of sweet sorghum seedlingsresponded to saline-alkali stress （molar ratio of NaHCO₃:Na₂CO₃=5:1）, based on thescreening results of different varieties resisting the stress, the two contrasting varieties,314B（the sensitive） and M-81E （the resistant） were selected for the actual study. Undersaline-alkali stress, a series of issues were analyzed, including growth and development,osmoregulation, scaenging of reactive oxygen species （ROS）, sodium absorption anddistribution, changes and secretion of organic acid, ultrastructure of chloroplast andmitochondria, and leaf anatomical structure. Comparison of the changes and difference ofabove matters between two varieties, the adaptive mechanisms of sweet sorghum seedlingresponded to saline-alkali stress were generally stated. These results will attribute to screenresistant genotypes of sweet sorghum and formulate reasonable measures for planting crops atsoda saline-alkaline lands. Meanwhile, using SSH （suppression subtractive hybridization） kits,a group of ESTs of genes related to enduring the saline-alkali stress were obtained, that willprovide partial sequences to further acquire the overall length cDNA of some important genesand scientific basis on genetic improvement of sweet sorghum and gramineous plants for salttolerance. The major results were as follows:1. Under saline-alkali stress, the germination rate of sweet sorghum increased withincreasing concentration of treatment solutions, but salt toxicity rate was the opposite. Therewere differences among varieties. The degree of restraining the growth of radicle and germ,which come from saline-alkali stress was more serious than germination. On the basis of thegermination rate, germination percentage, germination index, relative-vigor index,average-root length, average-germination percentage and salt toxicity rate of7varieties, thetolerance of sweet sorghum was assessed with the method of subordinate function valuesanalysis. The results showed that the7varieties of sweet sorghum were ordered in sequencefrom the strong to weak: MN3739> M-81E> Berry> Rio> ES725>3222>Wray.2. After screening the tolerances in buds period and seedling stage of sweet sorghum, therewere various tolerant to saline-alkali stress among varieties. Then, the growth changes anddamage of plasma membrane in the six varieties of sweet sorghum seedlings was determinedwith condition of saline-alkali stress. The results showed that compared with the respectivecontrol group, under saline-alkali stress, the sweet sorghum seedlings was decreased the totalweight of plant, height, root vigor and total chlorophyll content, but increased the relativepermeability of plasma membranes and content of malonaldehyde. However, root-shoot ratioswere showed higher or lower among the different varieties. The assessment of the sweet sorghum tolerance to saline-alkali stress with the method of subordinate function valuesshowed that M-81was more tolerant to saline-alkali stress than314B.3. The osmotic regulation substances, antoxidant enzymes and NO contents of sweetsorghum seedlings treated with alkali solutions were detected. The results showed that undersaline-alkali stress, the sweet sorghum seedlings increased the content of soluble sugar,soluble protein, proline and betaine, also, the activities of peroxidase, catalase, andglutathione peroxidase were improved. But, the activities of superoxide were showed higheror lower between the two varieties. The osmotic regulation substances produced from314Bwere more than M-81E, yet, the activities of antoxidant enzymes of M-81E were high than314B. Therefore,314B possessed higher osmotic adjustment abilities, and M-81E ownedstronger scavenging ROS abilities. Under saline-alkali stress, the NO contents in shoots ofsweet sorghum were increased, but, the ones in roots were decreased.4. After sweet sorghum seedlings treated for3days with soda alkali solutions, the Na⁺contents of whole plant, leaves, sheath and roots in two genotypes almost entirely increased,yet, major Na⁺were accumulated in roots and sheath. Under saline-alkali stress, the contentsof K⁺and Ca²⁺in whole plant, leaves, sheath and roots decreased universally in twogenotypes. Consequently, the K⁺/Na⁺and Ca²⁺/Na⁺ratio decreased completely in eachtissue of two varieties, but, the K⁺/Na⁺transport selectivity （TSK，Na） and the Ca²⁺/Na⁺transport selectivity （TSCa，Na） in roots were obviously risen, that from roots to leaves, theselective abilities of sweet sorghum to transport K⁺and Ca²⁺were strong, and to transport Na⁺were weak.5. Both hydrolytic activities and proton transporting activities of V-H⁺-ATPase of rootswere stimulated under soda alkali stress, yet V-H⁺-PPase activities were decreased followingincreasing concentration of solutions. Na⁺/H⁺exchange activities were intensively promoted.When treated with high concentration solution, the Na⁺/H⁺exchange activities weremarkedly restrained, but the activities were still higher than control.6. The Na⁺secretion from leaves wasn’t main way for sweet sorghum to resist soda alkali,because under saline-alkali stress, the Na⁺concentration expelled from leaves was very little.It played an important role in sweet sorghum adapting to saline-alkali stress that the main Na⁺exclusion localization was in roots and sheath. Meanwhile, vacuolar Na⁺compartment wasalso major adaptive mechanism of sorghum to alkali stress. In roots, M-81E accumulatedmajor Na⁺, but314B in leaves. M-81E had a higher ability to vacuolar Na⁺compartment inroot cells that was in view of M-81E had higher activities of V-H⁺-ATPase and Na⁺/H⁺exchanger, the depressed degree of V-H⁺_-PPase activities was lesser than314B. And M-81Emaintained a higher level of K⁺uptake, which resulted in higher K⁺/Na⁺transport selectivitythan314B. These result also demonstrated that the most of Na⁺accumulated in root and vacuolar Na⁺compartment in root cells were conductive to sweet sorghum tolerance to sodaalkali stress.7. The organic acid contents in shoots and roots of sweet sorghum after treated soda alkalisolutions were decreased generally. Some organic acids which played important role inresisting stress weren’t accumulated. When treated with low concentration solutions, PEPCactivities were enhanced, but with high concentration solutions, the ones were decreased.Under saline-alkali stress, the organic acid contents had no direct correlation with PEPCactivities, but, the fall of organic acid was small with increasing PEPC activities, and the fallwas declined largely following with decreasing PEPC activities. When grown in soda alkalisolutions, sweet sorghum could rapidly cut down pH value of culture solution. Yet, theorganic acid secreted from root system was very little, thus, we concluded that the organicacid played a little part in regulating pH value outside roots. Both all kinds of organic acidsand total organic acid in shoots or roots were higher of M-81E than314B. Compare with314B, the degree of increasing PEPC activities was greater under low concentration solutions,and the degree of decreasing PEPC activities was littler under high concentration solutions.The pH value was from9.34to9.10in314B culture solution, yet, the value was from9.34to8.81in M-81E culture solution and M-81E showed stronger ability to regulate pH value ofoutside roots than314B.8. The growth and development of sweet sorghum seedlings were suppressed on conditionof alkali stress. The plant height and stem base perimeter were reduced dramatically （P<0.01）.The leaves located stem base and blade tips were flavescent, the leaf developments werelagged, and the leaf numbers become less. The leaf thickness, midrib thickness and biggervessels diameter of the two sweet sorghum seedlings were significantly decreased undersaline-alkali stress, while the upper stratum corneum thickness and lower stratum corneumthickness were significantly increased compared with the control group （P<0.01）. The upperepidermis thickness stayed mainly the same, but lower epidermis thickness were significantlyenhanced （P<0.05）. After treated with alkali solutions, the etiolated areas of leaves, the fall ofmidrib thickness plant height and stem base perimeter in314B were larger than in M-81E.9. The effects of saline-alkali stress on ultrastructure in chloroplast and mitochondria werestudied. The results showed that the damages appeared such as expansion of chloroplast, thematrix separated from the chloroplast envelope, dissociation of thylakoid membrane,loosening and expanding of grana lamella, and even deformation of some grana lamella. Thestarch grain and osmophilic globule number in chloroplast were significantly increased（P<0.01）. The mitochondria number become more and distributed largely near chloroplast. Inthe mitochondria, the number of ridge in the inner coat appeared to decrease and evendisappear. The chloroplast of314B seemed to be injured more serious than M-81E, for themore osmophilic globule number and greater degree of separated between matrix and the envelope.10. A suppression subtractive hybridization cDNA library was constructed which wasM-81E treated with soda alkali. Similarity analysis based on Blast software in GenBank wasperformed.81.1%（103） of the ESTs could be found encoding putative proteins.18.9%（24）of the ESTs were found no significant similarity with ESTs collected in GenBank. Functionof48ESTs （46.6%） were known, and55（53.4%） were unknown. Most ESTs of guessedputative proteins were found having similarity with ESTs from cDNA libraries which wereconstructed about the different expression of sweet sorghum grown in various stresses.11. According to the annotated function, classifying with GO and COGs, we found that agood deal of genes were induced to express excessively, involving related withphotosynthesis, the metabolisms of substances （such as carbohydrate, fat, amino acid,cozymaze and inorganic irons） and energy, structure of cell wall and membrane, aquaporin,signal transduction and transcription regulation.