Study On Ecophysiological Mechanisms Of Thellungiella Salsuginea At The Key Satges Of Growth And Development

Halophytes face many abiotic stressors such as salinity, light,temperature and water content caused by seasonal alteration during thewhole life span. Whether halophyte could adapt the changes at the keystages such as seed germination and flowering determined the surviral anddistribution of the species population. In this paper the eco-physiologicaladaptations of Thellungiella halphila to salinity, temperatures and seasonalchanges were studied with the aim to illustrate the adaptive mechanism andcertainty of its survival and distribution. The main results are as follows:Salinity is one of the major abiotic stresses that adversely affect plantgrowth and development. The occurrence of halophytes in inland saltmarshes may depend on their tolerance to salt stress at different stages ofplant development. Germination is a key stage in the life cycle of plants insaline environments as it determines whether or not plants can establishsuccessfully in certain areas. So, in the first experiment, effects of salinityon germination and seedling growth of T. salsuginea and Arabdopsisthaliana were compared with the aim to explore the salinity-resistantmechanisms of T. salsuginea. The seeds of T. salsuginea and A. thalianawere treated with0（control）,30,60,90,120,200,300,400and500mmol L^-1NaCl in discs after10days’ seed vernalization. Then the seedgermination and physiology indexes of seedlings grown for7days weredetermined.The results showed that salinity inhibited seed germination inboth species.The germination rate of both A. thaliana and T. salsugineaincreased gradually by all concentrations of NaCl in the initial2days,germination rate kept consitent in some extent after3days.30mmolL^-1NaCl treatments had no effect on seed germination rate of A. thaliana,but higher concentrations of NaCl markedly decreased seed germinationrate in both species. At30,60,90and120mmol L^-1NaCl, the germinationpercentage of A. thaliana was100%,99%,77%and28%of the control,however the germination percentage of T. salsuginea was91%、68%、26%and0%of the control. Seeds of both species did not germinate when theconcentration of NaCl was over200mmol L^-1. Surprisingly, theungerminated seeds of both species quickly germinated. Moreover, the totalseed germination rate recovering from200,300,400and500mmol L^-1NaCl in T. salsuginea was105%,104%,106%and99%, but in A.thaliana was94%、84%、82%、54%, respectively. Therefore, salinitypromotes the seed germination of T. salsuginea during the water recoverystage. The ungerminated seeds of A. thaliana suffered ion toxicity in highsalinity to some degree as evidenced by the low final germination rate afterungerminated seeds pretreated with NaCl were transferred to distilledwater. At the seedling growth stage, seedlings of T. salsuginea were more salttolerant than those of A. thaliana. For example, seedlings of T. salsugineahad better plant growth （root length, fresh and dry weight）, higherchlorophyll content, less MDA content and higher proline content and K+/Na+ratio under salinity. These characteristics indicate that T. salsuginea ismore salt-tolerant to salinity than A. thaliana at both seed germination andseedling stages.Generally, salt cress, Thellungiella salsuginea （Cruciferae）, germinatesin autumn, is vernalized at rosette seedling in winter and flowers afterlong-day irradiance in spring in its natural habitat. But little was knowabout how many days did T. salsuginea finish its vernalization andphotoperiod requirement, which restricts the insight into the molecularmechanisms of abiotic stresses sach as salt tolerance in labotory. Tofacilitate research on T. salsuginea, the present study focuses on seedvernalization and photoperiod for flowering induction. Seeds of T.salsuginea were vernalized at4°C in discs for0,2,4,6,8,10,12,14,16,18,20,22,24,26,28and30days respectively. After vernalization, seedswere placed in a growth chamber to observe the seed germination. After7days’germination, the seedling were planted in the pots in the samechamber to observe the flowering.The results showed that seedvernalization promoted the seed germination. Seed germination rateincreased with the prolonged duration of vernalization. The seed germination rate was90%of the control when the seeds were vernalizedfor30days and flowered after the the seedlings grown under suitable lightperiod. These results suggested that T. salsuginea can finish itsvernalization at stage of imbibed seeds. T. salsuginea still stayed atvegetative stage and could not flower when seeds were vernalized for lessthan6days. The flowering plants increased when seeds were vernalized for6-22days. And100%of T. salsuginea flowered when seeds werevernalized for22-40days. The time when the first flower appeared wasshortened by seed vernalization. Longer seed vernalizaiton, earlier the firstflower emergence. The earliest emergence of the first flower was the29thday after seeds were vernalized for30-40days. Expression of ThFLC,which is a repressor of flowering, was reduced by vernalization. Relativeexpression of ThFLC dropped from100%of control to24.3%of30d ofseed vernalization. Plants growing from seeds that had been vernalized for30d did not flower when day length was <9h, and day lengths>9hpromoted flowering. These results will facilitate the utilization of T.salsuginea as a model plant of abiotic stress to study the molecularmechanisms. The results also suggest that the dependency on vernalizationand long-day photoperiod is critical for the adaptation of T. salsuginea tothe local environment.T. salsuginea （Shangdong ecotype） germinates in autumn, vernalized asrosette seedlin in winter, flowers in March and ripen in May in the Yellow River Delta. This season is characteristic of low rainfall, quick temperaturerise and high salinity in the soil.Why does T. salsuginea complete its lifespan in this season and what is the adaptive mechanism? These questionsdeserve further research. In the third expreripent, seasonal ion content andeco-physiological characters variation in T. salsuginea in its habitat, YellowRiver delta saline and alkaline land, from January to May in2012were alsostudied. The results showed that Na⁺, Ca²⁺ and Cl^-content in the soilincreased from January to May, and was closely related to temperature andwater content in the soil. Na+content in T. salsuginea was highest inJanuary and February and decreased with time. K⁺、Ca²⁺、Mg²⁺、Cl^-、SO₄^2-content showed increasing tendency, in particular, Cl-content, with theincrease of temperature and the growth stage. The accumulation of K⁺、Ca²⁺、Mg²⁺、Cl^-、SO₄^2- was beneficial to osmaregulation in T. salsugineaunder salinity. K⁺, Mg²+and Ca²⁺ content in the root were lower than that inthe shoot because nutritional ion was in priority distribution to leaves,flowers and pods from roots. The eco-physiological variation in T.salsuginea showed that the effect of temperature was larger than that of ioncontent changes in the soil. T. salsuginea showed retarded growth withhigher accumulation of MDA, lower chlorophyte content. But T. salsugineaadjusted eco-physiological characters to the low temperature such as higherproline and soluble sugar content which protected T.salsuginea from theharm of low temperature and ion variation. These results suggested that compatible solute content is closely related to temperature, which is one ofthe adaptive mechanisms of T. salsuginea to low temperature and highsalinity.Based on the research of T. salsuginea, T. salsuginea could survive in200mmol L^-1NaCl. The main mechanism was compartmentalization ofexcessive salt ion into vacuoles, which is similar to heavy metalsuperaccumulator plant. Heavy metal pollution in Yellow River delta soil isgetting worse; T. salsuginea might be a potential heavy metalsuperaccumulator, which needs to study. In the fourth experiment, a potexperiment was used to investigate growth responses and eco-physiologicalcharacters of T. salsuginea under Cd, Cu and Pb treatments. The resultshowed that the root length and dry mass decreased and MDA accumulatedsignificantly with the increase of Cd, Cu and Pb concentrations. T.salsuginea showed apparent damage symptoms such as lower chlorophyllcontent, etiolated leaves, higher MDA accumulations and death ofseedlings under Cd treatment compared with Cu and Pb. T. salsuginea cangrow well under Pb treatment though Pb treatment concentration was thehighest. From the point of metal accumulation and BAF, T. salsugineaabsorbed Cd>Cu>Pb. These results indicate that T. salsuginea has certainpotential on remedying the soil polluted by Cd with low and middleconcentrations. The innovations of this thesis were shown as follows:1. In this study, the ungerminated seeds of T. salsuginea under highsalinity quickly germinated though the seed germination of T. salsugineawas a little more sensitive to salinity compared with A. thaliana. At theseedling stage, however, T. salsuginea showed better growth with higherchlorophyll content, less MDA content and higher proline content and K⁺/Na⁺ratio under salinity. These characteristics indicate that T. salsuginea ismore adaptive to salinity than A. thaliana at both seed germination andseedling stages and explain why A. thaliana is excuded from salinelocations and T. salsuginea can survive and form its populations in salinesoils.2. The seed vernalization and photoperiod of T. salsuginea were studiedfor the first time. The results showed that seed vernalization promoted theseed germination and that T. salsuginea flowered after seed vernalizationwhich showed that T. salsuginea is capable of vernalization at stage ofimbibed seeds. T. salsuginea still stayed at vegetative stage and couldn’tflower when seeds were vernalized less than6days. And100%of T.salsuginea flowered when seeds were vernalized for22-40days. The timewhen the first flower appeared was shortened by seed vernalization.Expression of ThFLC, which is a repressor of flowering, was markedlyreduced by vernalization. The requirement for vernalization of T. salsuginea is obligate instead of facultative. We also observed here that T.salsuginea generated flowers only when day length exceeded9h. Thenumber of flowers and dry mass per plant also increased with an increaseof day length. Dry mass per plant and number of flowers per plant were thehighest when day length exceeded14h. These results therefore confirmthat T. salsuginea is a long-day plant with a critical day length requirementof approximately9h. In addition, the time required to produce the firstflower decreased as day length exceeded9h. These results indicated that T.salsuginea is a typical long-day plant with9h photoperiod. Therequirement of vernalization and long days by T. salsuginea （Shandongecotype） is at least partially the product of adaptation to the localenvironment. These results facilitate greatly the future research onsalt-tolerant mechanisms of T. salsuginea.3. Soluble ion, proline, MDA and chlorophyll content of T. salsugineawere closely related to growth and development stages, ion content in thesoil and rainfall. For T. salsuginea, the effect of growth and developmentstages on the adaptation to stress environment was larger than that oftemperature and ion content changes in the soil. Under the natural location,T. salsuginea adjusted eco-physiological characters to low temperature,drought and salinity caused by the seasonal variances.4. T. salsuginea accumulated Cd to some degree, but did not survive inhigher concentration of Cd (130mg kg^-1). T. salsuginea finished its life cycle under Cu and Pb stress. Therefore, T. salsuginea was tolerant to Cuand Pb but not accumulated them. Conclusively, T. salsuginea can be usedin remedying the soil polluted by Cd with low and middle concentrations.