Mechanism Involved In Drought/Salt Tolorance Improvement In Anfalfa Due To Symbiotic Interaction With Rhizobium

Abiotic stress such as drought and salinity is the adverse environmentalfactors reducing agriculture production. Drought and soil salinity are moreserious in China. Since alfalfa is relatively tolerant to drought and salinity, it canbe planted on arid or saline soil for harvesting high quality of forage. Thus, moreand more plant biologists focus on the mechanism of how alfalfa plants respondto drought/salt stress. There are few publications on how symbioticnitrogen-fixation affects alfalfa’s response to drought and salinity. We conductedgreenhouse experiments to study physiological and molecular responses ofalfalfa during abiotic stress treatments, with an interest in understanding hownodulation or symbiotic nitrogen-fixation affects plant stress response. Plantswere grown in pots and cones filled with quartz sand which allows us to easilyapply and control stress treatments. Alfalfa plants were subjected to drought,high salt and other treatments. Survival rate and other physiological changes were examined, such as osmotican accumulation, root fluxes of Ca2+, K+and H+.Stress-responsive genes were identified using microarray. Threedrought-responsive genes were studied in detail for their expression patterns andregulation. The major findings are:1. Nodulated alfalfa was more tolerant to abiotic stress such as drought, salt,high temperature, and physical wounding. The stress resistance ranked fromstrong to weak as plants with active nodules, plants with inactive nodules, andplants without nodules.The improved stress resistance in nodulated alfalfa may be resulted from:better nitrogen supply from nitrogen fixation, immune protection fromrhizobium-alfalfa interaction, and other biochemical composition changes(determining physiological changes observed).The greater stress tolerance acquired from nodulation could be sustainedfor a long time. This was demonstrated that plants, after removal of nodules, stillshowed better stress tolerance compared to the plants that were never nodulated.2. Physiological analyses showed that better stress tolerance in alfalfa withactive nodules was associated with greater abilities of osmotic adjustment,anti-oxidation, and intracellular homeostasis maintenance.3. Maintenance of intracellular homeostasis was associated with betterregulation of root ion content and fluxes, and improved osmotic adjustment. 4. The microarray study revealed that dehydration stress (mimickingdrought) is a complex process involving a large set of genes. The number ofdehydration responsive genes was less in the plants at early stage of stresstreatment (when stress was mild) and increased at late stages when stressbecame severe. During early dehydration, more genes were downregulated andfewer genes were upregulated when compared alfalfa with active nodules to theones with inactive nodules. However, more upregulated and less downregulatedgenes were found in alfalfa with active nodules compared to the ones withinactive nodules or without nodules at late stages of dehydration.Among the dehydration responsive genes (total5006), five expressionpatterns were identified. Interestingly,446genes that were found upregulated inalfalfa with active nodules were down regulated in alfalfa with inactive nodulesor no nodules. Seventy nine genes that were down regulated in alfalfa withactive nodule were upregulated in alfalfa with inactive nodules and no nodules.In particular,25genes in the phenylpropanoid pathway (lignin biosynthesis)were highly upregulated in nodulated alfalfa plants during dehydration stress.They encode proteins, EC4.3.1.24, EC2.3.1.91, EC2.3.1.92, EC1.14.13.11,EC6.2.1.2, EC3.2.1.21, and EC1.11.1.7in the pathway, suggesting improvedlignifications may contribute to the better drought tolerance in nodulated alfalfa.5. Using semi-quantitative PCR, we studied expression of3dehydration responsive genes, TC81048, TC83565and TC87183, in detail. These genes wereupregulated in non-nodulated alfalfa under dehydration. While they wereconfirmed as upregulated in nodulated alfalfa also, nodulation enhanced theexpression of TC83565but decreased expression of TC87183under stress.Nodulation had little effect on TC81048expression under dehydration. Alfalfacultivar SemiP showed the greatest upregulation of these genes duringdehydration stress in a multi-cultivar comparison experiment.In summary, our results indicated that nodulation could improve alfalfaplant tolerance to drought/salt stress. The improved tolerance may be a result ofdifferential regulation of stress responsive genes, leading to cellular andphysiological changes that help plant adapt to adverse growth conditions.