| In this paper,we studied the effects of salt stress(0 mmol·L-1,50 mmol·L-1,100 mmol·L-1,150 mmol·L-1 NaCl)on seed germination,growth and physiological charateristics of Isatis indigotica seedlings which from five different areas(Hebei Baoding(HB);Neimeng Baotou(NB);Liaoning Tieling(LT);Anhui Bozhou(AH);Shandong Binzhou(SB)).Using pattern of pot and solution culture,the effects of different nitrogen forms and different nitrogen levels(3.75 mmol-L-1,7.5 mmol·L-1,11.25 mmol-L-1,15 mmol-L-1,18.75 mmol·L-1)on growth,physiological characteristics and nutrient and bioactive ingredients of 1.indigotica seedlings under salt stress was analyzed,and the expression of related genes of 1.indigotica seedlings supplied with different nitrogen forms under salt stress were also investigated.The salt-tolerant 1.indigotica cultivation screened in advanced salt stress experiment was used as research material.Results were as follows.1.Salt tolerance among the five populations showed significant difference.The germination rate,germination potential,germination index and vitality index decreased with the increase of NaCl concentration.50 mmol·L-1 NaCl promoted the fresh weight,hypocotyl and radicle length of I.indigotica seedlings from HB and SB areas.Germination rate,Germination potential,vigor index,hypocotyl and radical length,fresh weight of I.indigotica seedlings from five areas decreased significantly under 100 mmol·L-1 NaCl stress,and MDA content increased greatly.According to the comprehensive appraisal,the salt resistance of I.indigotica seeds from five different areas was in the following order:HB>SB>LT>NB>AH.(2)Mild salt stress promoted the growth of I.indigotica seedlings from HB and SB areas.The fresh and dry weights of leaf and root and water content of I.indigotica seedlings from LT and AH areas were decreased under salt stress.The net photosynthetic rate,stomatal conductance,transpiration rate and chlorophyll content of I.indigotica seedlings from HB and SB areas were enhanced initially and then reduced under salt stress,while such indexes of seedlings from LT and AH areas declined remarkably with salt concentration increasing.With the improvement of NaCl concentration,the activities of SOD and POD and osmoregulation substance contents such as soluble protein and proline of I.indigotica seedlings from five areas increased first and decreased thereafter,peaking at 100 mmol·L-1 NaCl.Besides,MDA content increased gradually with the salt concentration increasing.The salinity tolerance of I.indigotica seedlings from five areas was significantly different,and 100 mmol·L-1 NaCl was the salt tolerant limit of I.indigotica seedlings.By comprehensively evaluating,among five different areas,the I.indigotica seedlings from HB and SB had a strong tolerance and adaptability under salt stress,and the tolerance of seedlings from NB area was lower than that from above two areas.However,the seedlings of I.indigotica from LT and AH areas were intolerant to salt stress.2.The inhibitory effect of salt stress on growth of I.indigotica seedlings can be significanty relieved with nitrate nutrition,and the biomass of I.indigotica seedlings depressed with ammonium nutrition whether under salt stress or common nitrogen treatment.The activities of SOD,POD and CAT and MDA content decreased larger under salt stress with ammonium nutrition than when with nitrate.The proline content of I.indigotica seedlings increased greatly supplied with nitrate nutrition under salt atress.While,the proline contents of I.indigotica seedling had no significant difference between ammonium nutrition and nitrogen dificence treatment under salt stress.Whether during salt stress or common nitrogen treatment,the NR activity of I.indigotica seedling with nitrate was the highest among such three different nitrogen form treatments(nitrate nutrtion,ammonium nutrition and ammonium nitrate nutrition),while that of ammonium nutrition was the lowest.The GS activity showed no significant difference among different nitrogen form treatments.The salt stress had greater influence on GOGAT activity with ammonium nitrate nutrition than other nitrogen form treatment,and the influence on GDH was lager with ammonium nutrition.The nitrate contents in I.indigotica leaves with nitrate nutrtion was significnatly higher than other treatments under salt stress and non-stress conditon.3.The net photosynthetic rate and stomatal conductance of I.indigotica leaves was depressed greatly under salt stress.The reduction of net photosynthetic rate,stomatal conductance,transpiration rate and intercellular CO2 concentration of I.indigotica seedlings were greater with ammonium nutrition than nitrate nutrition under salt stress.Compared with control,the content of chlorophyll and carotenoid of I.indigotica seedlings with nitrate nutrtion had no significant changes under salt stress.However,that of seedling supplied with ammonium nutrition changed greatly.4.Salt stress promted the content of soluble sugar and soluble protein of I.indigotica seedlings.Among different nitrogen form treatments,the flavoid and amino acid content of I.indigotica seedlings under salt stress and non-stress condition showed a large difference in different tissues.Nitrate nutrtion was beneficial to the accumulation of flavoid and amino acid in leaves,and ammonium nitrate nutrition increased the flavoid content in root.However,the ammonium nutrition reduced the flavoid content dramatically in both leaves and roots.Compared with nitrogen deficiency treatment,the total alkaloid in leaves and(R,S)-epigoitrin in roots of I.indigotica seedlings under salt stress increased observably owing to the nitrogen supply.5.The tryptophan synthase gene and iron superoxide dismutase were isolated from I.indigotica which were named as IiTSA and IiFeSOD recpectively.The full-length cDNA of IiTSA is 1074 bp in length,and the coding sequence consist 936 bp.The predicated protein of IiTSA is 311 amino acids long.The above result is consistent with the pvevious study of our laboratory.The IiFeSOD cDNA is 882 bp in length,and the coding sequence consist 834 bp,and the predicated protein of IiFeSOD is 277 amino acids long.The multiple sequence alignments and phylogenetic analysis revealed that the amino acid sequence of IiFeSOD has high homology with cruciferae plants.Real-time quantity PCR(QPCR)resulted that the expression of IiTSA decreased remarkably after salt stress.Under salt stress,IiTSA with nitrate nutrtion and ammonium nitrate nutrition was upregualted compared with nitrogen deficiency treatment.However,the expression of IiTSA showed no significant difference between ammonium nutrition and nitrogen deficiency treatment under salt stress.The nitrate and ammonium nitrate nutrition were conducive to the down-regulation of IiFeSOD under salt stress.6.The biomasses of I.indigotica seedlings increased greatly at T4(18.75 mmol·L-1)-level.Both the higher and lower nitrogen application can promote the activities of antioxidant enzyme in I.indigotica seedlings under salt stress.Moreover,the former can significantly decrease the accumulation of MDA and alleviate the salty damage effectively.With the improvement of nitrogen concentration,the activities of key enzymes-NR,GS involved in nitrogen assimilation,the contents of soluble protein and epigoitrin in roots of salt-stressed I.indigotica seedlings initially increased and then decreased.The regulation of different nitrogen levels between antioxidant enzyme and key enzyme involved in nitrogen assimilation,biomass and epigoitrin accumulation showed significant differences. |
PDF Full Text Request