Studies On Physiological And Biochemical Responses Of Two Perennial Halophytes: Kalidium Foliatum And Halostachys Caspica To Salt Stress And Salt-tolerant Genes Cloning And Expression

Salt stress is the primary effect that limits and decreases the output of crops in many parts of the world, particularly irrigated land. Overly high soil salinity can cause salina and limit the utility of soil. Up to date, improving the salt tolerance of crops, keeping steady-state growth and increasing the output in the saline environment are more emphasized and some progress has been made by biotechnological strategies.Plants exposed to salt stress undergo changes in their environment. The ability of plants to tolerate salt is determined by multiple biochemical pathways that facilitate retention and/or acquisition of water, protect chloroplast functions, and maintain ion homeostasis and so on. Essential pathways include those that lead to synthesis of osmotically active metabolites, specific proteins, and certain free radical scavenging enzymes and chaperones that control ion and water flux.Under salt stress, high Na⁺ is toxic to most plants. Important mechanisms that contribute to Na⁺ tolerance are Na⁺ efflux transporters and Na⁺ transporters that mediate Na⁺ sequestration into vacuoles. Because Na⁺ inhibits the activities of many enzymes, it is important to prevent Na⁺ from accumulating to a high level in the cytoplasm or in organelles other than the vacuole. Na⁺ compartmentation is an economical means of preventing Na⁺ from doing harm to the cytosol and Na⁺ can be used as an osmolyte in the vacuole to help to achieve osmotic homeostasis. The vacuolar Na⁺/H⁺ antiporters mediate Na⁺ uptake into vacuoles, which is driven by the vacuolar proton gradient established by the vacuolar (V-type) proton ATPase and H⁺-pyrophosphatase.In our first study, the experiment was done to compare the salt tolerance of two halophytes: Kalidium foliatum and Halostachys caspica during seed germination. The results showed that the two species were both very salt-tolerant plants and Halostachys caspica was more salt-tolerant than Kalidium foliatum. NaCl and iso-osmotic PEG had inhibitory effect on the germination of two halophytic seeds, and the inhibitory level of PEG was greater than that of iso-osmotic NaCl. Inhibitory effects of NaCl and water stress mainly showed lowering the seed germination rate, delaying initial time of seed germination and extending the time of seed germination. Low concentration of NaCl solution could promote the growth on the shoot of seed germination. The growing length of roots and shoots of two halophytes treated by 100 mmol·L^-1 NaCl reached the highest and the growing condition on shoots was best. Then some physiological and biochemical parameters including the content of Na⁺, K⁺, Ca²⁺, proline and plant leaf tissue water potential, the assays of membrane lipid peroxidation and antioxidant enzymes (SOD and POD) were determined with the two halophytic plants on salinity. The results showed Na⁺ and proline were both effective osmoprotecants, Kalidium foliatum and Halostachys caspica were able to take up water by maintaining a high osmotic potential through the accumulation of inorganic ions and proline. And if was thought because of long time environmental evolution,some very salt-tolerant halophytes grew better in optimal salt environment and grew badly in no salt and high salt environment. The experiments showed the growth of the two species need salt. A relative low salt concentration environment was optimal environment for the growth of the two plants, however no salt and high concentration salt environment were stress environment.In this work, we isolated the three kind cDNAs of the vacuolar membrane proton-translocating inorganic pyrophosphatase (H⁺-PPase) and the B subunit of the vacuolar-type H⁺-ATPase and betaine aldehyde dehydrogenase from Kalidium foliatum and Halostachys caspica by RT-PCR and race. (1) obtaining H⁺-pyrophosphatase genes of the two species, the two genes were named as KfVP1 and HcVP1, respectively. The KfVP1 and HcVP1 cDNAs contained an uninterrupted open reading frame(ORF) of 2292 bp, coding for a polypeptide of 764 amino acids with predicted transmembrane segments. GenBank accession numbers were EF114315 and EF471358. (2) obtaining the B subunit of the vacuolar-type H⁺-ATPase genes of the two species, the two genes were named as KfVHA-B and HcVHA-B, respectively. The KfVHA-B and HcVHA-B cDNAs contained a complete open reading frame(ORF) of 1467 bp, coding for a polypeptide of 488 amino acids. GenBank accession numbers were. EF114316 and EF471357. (3) obtaining betaine aldehyde dehydrogenase genes from Kfoliatum and H.caspica. The two genes were named as KfBADH and HcBADH, respectively. The KfBADH and HcBADH cDNAs contained an complete open reading frame of 1503 bp, coding for a polypeptide of 500 amino acids. GenBank accession numbers were DQ923617 and EF471356.Semi-quantitative gene expression analysis showed that 5 genes of Kfoliatum (KfVP1, KfVHA-B and KfNHX1 genes existing in the vacuolar membrane of Kfoliatum and KfBADH and P5CS genes catalizing to synthesize betain and proline) were all induced by salt. The expression levels of BADH and P5CS mRNA in plants treated with different NaCl concentration (100, 300, 500, 700 mmol·L^-1) for 4days were higher than that in the control plants, and increasing with increased salt concentration. Proline was much more accumulated in the fleshy leaves of K.foliatum with increased salt concentration concided with the expression pattern of K.foliatum P5CS, suggesting that proline and betain as two effective osmolytes had important functions for Kalidium foliatum during stress. KfVP1, KfVHA-B and KfNHX1 mRNAs were not high or were inhibited without salt, however expression of KfNHX1 was the highest at 300 mmol·L^-1 NaCl; KfVP1 mRNA was the strongest at 100 mmol·L^-1 NaCl and decreasing with increased salt concentration; KfVHA-B mRNA was no different at 0 and 100 mmol·L^-1 of NaCl, however persistently high in the concentration of 300, 500 and 700 mmol·L^-1 NaCl respectively. If might be the vacuolar H⁺-pyrophosphatase and vacuolar ATPase could continue to provide energy for Na⁺/H⁺ antiporters on salt stress.Green fluorescent protein (GFP) gene is a new reporter gene and is widely used in plant research. For exploring the expression and localization of KfVP1 and KfNHX1, we constructed the two fusion plant expression vectors of pCAMBIA1301-1-KfdtVPl-GFP and pCAMBIA1301-1-KfdtNHX1-GFP. These plasmids including PCAMBIA1301-1-GFP as negative control were transformed into onion epidermal cells by particle bombardment, then the transient expression in onion epidermal cells during 16-24 h was observed by fluorescence microscopy. The results suggested that KfVP1 and KfNHX1 proteins might be localized on the vacuolar membrane (at least the membrane system).Three plant expression vectors for KfVP1 and KfNHX1 were constructed, named pCAMBIA1301-1-KfVP1, pCAMBIA1301-1-KfNHX1 and pBin438-KfNHX1, and T₀ transgenic tobaccos were obtained by transformation pCAMBIA1301-1-KfVP1 and pCAMBIA1301-1-KfNHX1. Salt-tolerant experiment and determination of leaf cell membrane damage as the leakage of electrolytes on tobaccos treated with 400 mmol·L^-1 NaCl for 12d showed KfVP1 has definitely salt-tolerant function. We will continue genetic transformation by Agrobacterium tumefaciens in model plants: tobacco and Arabidopsis thaliana with both genes of KfVP1 and KfNHX1 and single gene KfVP1 and KfNHX1, respectively, in order to compare salt-tolerant difference.This study could provide primary reference for salt tolerance of Kfoliatum and H.caspica comprehensively. It was very important significance to improve salt tolerance in crops and trees by making good use of gene resources of the two species.