Function Identification Of A Na~+/Pi Transporter Gene (ThNPT) And Cloning And Function Analysis Of Flavin-containing Monooxygenase (FMO) Genes From T.halophila

With the development of modern techniques of plant molecular biology,great and widespread studies have been carried out in the mechanisms of plant adoptions to abiotic stress,especially in signal sensation and transduction,regulations of transcript, ion homeostasis,ROS scavenging systems and compatible solutes synthesis mechanisms in model plant Arabidopsis and Rice.As a typical glycophyte, Arabidopsis can't reveal all salt tolerance mechanisms,especially some specialized mechanisms in halophytes.Salt cress(Thellungiella halophila),a relative species of the Arabidopsis,is a classical halophyte with high salt tolerance.T.halophila produces neither salt glands nor other complex morphological alterations either before or after salt adaptation.It appears that the salt-tolerance is more dependent on its basic biochemical and physiological mechanisms,and salt cress has desirable genetic traits: high cDNA sequence similarity to that of Arabidopsis,small genome size and easy transformation,so it becomes the prospective model plant for studying salinity tolerance.In the present study,a novel Flavin-containing monooxygenase(FMO)-like gene, designated as TsFMO1,was cloned from T.halophila,and the functions and expression models of TsFMO1 and a Na⁺/Pi transporter gene(ThNPT) cloned in our lab before were further studied.Function analysis of T.halophila Na⁺/Pi transporter gene(ThNPT)A full-length cDNA sequence encoding T.halophila Na⁺/Pi transporter gene was cloned by our lab.The deduced 510 amino acid sequence contains 10 trans-membrane domains and it was located in chloroplast by expressing N terminal GFP fusion protein in tobacco cells.Heterologous expression of truncated ThNPT cDNA in yeast mutant PAM2 can transport Pi from medium into yeast cells and Na⁺ at low concentration(500μM) could activate the Pi transport activity.In this work,expression pattern of ThNPT was analyzed by RT-PCR and Real-time RT-PCR.The results showed that:(1) the expression level of ThNPT in rosette leaves was the highest,cauline leaf also had high expression,the stems,silique and open flowers also had gene expression and the expression in the root was the lowest;(2) the gene was significantly up-regulated in the leaves when exposed to NaCl or 42℃stress and down-regulated under 4℃stress;(3) the expression in leaf was induced by light.We could conclude that ThNPT was a gene mainly expressed in leaf and induced by light,heat and salt stress.Intact chloroplasts of T.halophila were isolated to determine the Pi transport activity under different pH,and different concentrations of Na⁺ and Pi,respectively. The results showed that the optimum pH of the Pi transport activity of chloroplasts is pH6.5,25mM Na⁺ could activate the Pi transport activity most significantly.The ThNPT over-expression and RNAi vectors were constructed,and transgenic T.halophila and Arabidopsis were got,separately.The Pi transport activity and Pi content of chloroplasts in over-expression transgenic T.halophila plants were higher than those wild types(WT),but there were no significant differences between RNAi transgenic plants and WT in Pi transport activity and Pi content of chloroplasts.The accumulation of Na⁺ in chloroplasts was measured using Sodium Green fluorescent indicator and laser scanning confocal microscopy.Higher fluorescent signals(i.e.higher Na⁺ concentration) were observed in chloroplasts of over-expression transgenic T.halophila plants.It was found that the over-expression transgenic T.halophila plants accumulated significantly higher Na⁺ than WT in chloroplasts,but there was no obvious difference on Na⁺ accumulation between the WT and RNAi transgenic plants chloroplasts.These results suggested that the over expression of ThNPT enhanced the accumilations of Na⁺ and Pi in chloroplasts.We found that the photosynthesis rates of over-expression transgenic T.halophila plants were enhanced.Eight week-old T.halophila seedlings were hydrophilic cultured for 2 weeks in normal,Pi deprived Hoagland solutions or Hoagland solutions added with 100mM NaCl,respectively.The results indicated that:In Hoagland solutions,the content of Na⁺ in leaves of the over-expression transgenic T.halophila plants was significantly higher than that of WT;in Hoagland solutions added with 100mM NaCl,the content of Pi in leaves of the over-expression transgenic T.halophila plants was significantly higher than that of WT;while in Pi deprived Hoagland solutions,the content of Na⁺ in leaves of the over-expression transgenic T.halophila plants was significantly higher than that of WT.The photosynthesis rates were also measured and the results showed that:In Hoagland solutions,the photosynthesis rates of the over-expression transgenic T.halophila plants was significantly higher than that of WT;in Hoagland solutions added with 100mM NaCl,the photosynthesis rates of all plants were enhanced and the photosynthesis rates of the over-expression transgenic T.halophila plants were significantly higher than that of WT;while in Pi deprived Hoagland solutions,the photosynthesis rates of all plants were decreased,and there was no obvious difference on the photosynthesis rates between the WT and over-expression transgenic plants.It could be concluded that:(1) the over-expression transgenic T.halophila plants can improve the photosynthesis rates and accumulate more Na⁺ in leaves than WT;(2) the content of Pi in leaves of the over-expression transgenic T.halophila plants was increased when 100 mM NaCl was added in the Hoagland solution;(3) the photosynthesis rates of T.halophila can be enhanced by low concentration of NaCl. We inferred that ThNPT might change the ion balance and Pi content of the chloroplast,which influenced the photosynthesis rate.The expression of ThNPT was significantly up-regulated in the leaves when exposed to NaCl stress.T.halophila is a classical halophyte with high salt tolerance, over-expression and RNAi ThNPT in T.halophila can not find the function of ThNPT in plant stress tolerance.Because ThNPT and its homolog from Arabidopsis have high similarity up to 86%at cDNA level,ThNPT was over expressed and its homolog from Arabidopsis was suppressed in Arabidopsis.The stress tolerance of transgenic Arabidopsis was analyzed to confer the putative function of ThNPT in plant stress tolerance.The results showed that:On the MS medium containing 150mM or 200 mM NaCl,the ThNPT over-expression transgenic Arabidopsis plants showed the lower percentage of seed germination than WT;the growth,root length and fresh weight of ThNPT over-expression transgenic Arabidopsis plants were lower than those of WT; after cultured on MS medium containing the 150mM or 200 mM NaCl,there was no obvious difference on seed germination and seedling growth between the WT and RNAi transgenic plants.The ThNPT over-expression transgenic Arabidopsis plants showed the higher root length and fresh weight than WT after 10 day on MS medium plates,while there was no obvious difference on root length and fresh weight between the WT and over-expression transgenic plants on Pi deprived MS medium plates.We inferred that the over-expression transgenic Arabidopsis plants might accumulate more Na⁺ in leaves than WT,and their sensitivity of salt stress was increased.Cloning and function analysis of T.halophila Flavin-containing monooxygenase, TsFMO1A novel Flavin-containing monooxygenase(FMO)-like gene,designated as TsFMO1,was cloned from Thellungiella halophila by reverse transcriptase PCR (RT-PCR) coupled with rapid amplification of cDNA ends(RACE) approach according to the sequence of putative Arabidopsis Flavin-containing monooxygenase (At1G62570).This cDNA Sequence included 1743bp(Accession no.EF011646 in genbank) containing an open reading frame(ORF) of 1386 bp,a 5′untranslated region (UTR) of 84 bp and a 3′untranslated region of 272 bp.The deduced amino acid sequence was 461 amino acids in length which contained the putative FAD and NADP binding motifs.In order to determine whether TsFMO1 has similar functions as Yeast FMO, TsFMO1 was used to rescue the yeast FMO mutant YHR176W which exhibited retarded growth in the presence of DTT.The growth rate of YHR176W (pYES-TsFMO1) was similar with that of the BY4741(pYES),which suggested that TsFMO1 could rescue the retardant growth of yeast FMO mutant to DTT.This result indicated that TsFMO1 was a functional flavin-containing monooxygenase like Yeast FMO.RT-PCR analysis indicated that the TsFMO1 was constitutively expressed in the roots and rosette leaves,stems and open flowers under normal growth conditions,the expression level of TsFMO1 in rosette leaves was the highest,moderate in flower, root and stem and the lowest in silique and cauline leaf.The expressions of the TsFMO1 under different environmental stress conditions were analyzed by quantitative real-time RT-PCR.The results showed that the transcript level of TsFMO1 in leaf was quickly and significantly(more than two folds) up-regulated by high salt stress,ABA,4℃and 42℃treatment,respectively;the transcript level of TsFMO1 in root was down-regulated under PEG and H₂O₂ treatment,respectively. The over-expression and RNAi vectors were constructed,and transgenic T.halophila and Arabidopsis were got,respectively.The accumulation of Reactive Oxygen Species(ROS) in cells was measured using H₂DCFDA fluorescent indicator and laser scanning confocal microscopy.Higher fluorescent signals(i.e.higher ROS concentration) were observed in protoplasts of over-expression transgenic T.halophila plants.It was found that the over-expression transgenic T.halophila plants accumulated significantly higher ROS than WT protoplasts,but there was no obvious difference on ROS accumulation between the WT and RNAi transgenic plants protoplasts.The results indicated that over expression of TsFMO1 enhanced the accumilations of ROS in protoplasts,and provided direct evidence for the relationship between TsFMO1 and ROS generation.As TsFMO1 and its homolog from Arabidopsis have high similarity up to 83%at cDNA level,TsFMO1 was over expressed and its homolog from Arabidopsis was suppressed in Arabidopsis.The stress tolerance of transgenic Arabidopsis was analyzed to confer the putative function of TsFMO1 in plant stress tolerance.On the MS medium containing 0,50,100,150 and 200mM NaCl,there was no significant difference on seed germination and seedling growth between the WT and RNAi transgenic plants,while the TsFMO1 over-expression transgenic Arabidopsis plants showed higher percentage of seed germination and fresh weight biomass than WT. There was no significant difference on seed germination and seedling growth between the WT and over-expression transgenic plants when exposed to Mannitol and H₂O₂ stress.Based on the results above we concluded that ThNPT was mainly expressed in leaves,and the expression was up-regulated by light,NaCl and 42℃,respectively.Pi transport activity of intact chloroplasts was analyzed and Na⁺ stimulated Pi transport activity was detected.The Pi transport activity,Pi content of chloroplasts and net photosynthesis rates in over-expression transgenic T.halophila plants were higher than that of wild types(WT),but there were no significant differences between RNAi transgenie plants and WT in Pi transport activity,Pi content of chloroplasts and net photosynthesis rates.We inferred that ThNPT might be a multiply member gene family,and the Pi transport activity of each member might overlap.Our work may help reveal the biological role of the Na⁺ and Pi homeostasis between cytoplasm and chloroplast under high salinity and low Pi stress. A novel Flavin-containing monooxygenase(FMO)-like gene,designated as TsFMO1,was cloned from Thellungiella halophila according to the sequence of putative Arabidopsis Flavin-containing monooxygenase(At1G62570).TsFMO1 was found to complement and rescue the retardant growth to DTT of yeast fmo mutant strain,indicating that TsFMO1 was a functional flavin-containing monooxygenase like Yeast FMO.This gene was mainly constructively expressed in many tissues and regulated by salt stress,ABA,4℃,42℃,PEG and H₂O₂.Over expression of TsFMO1 in T.halophila enhanced the accumilations of ROS in protoplasts,and over expression of TsFMO1 in Arabidopsis improved the salt tolerance under low concentration of NaCl.These works may help us understand the difference in the salt tolerance mechanisms between nonhalophytes(Arabidopsis) and halophytes(T.halophila),and provide the valuable information for establishing the experiment designs of crop salt tolerance improvement.