Physiological And Molecular Mechanisms Of Exogenous Potassium In Mitigating Negative Effects By Salt Stress On Tobacco Photosynthetic Apparatus

Photosynthesis is the material and energy basis of plant productivity,and increasing plant photosynthetic rate can significantly improve crop and herbage yield under saline-alkali stress.While exogenous potassium can alleviate the decrease of the photosynthetic rate caused by salt stress and repair the damage to the photosynthetic mechanism.Especially,exogenous potassium improves the stomatal conductance of plants under salt stress,enhances photophosphorylation of plants,and promotes the production of more ATP by chloroplasts,but its molecular mechanism is not clear.This study used the model plant Nicotiana tabacum L.as experimental material.Transcriptome data,35S overexpression and CRISPR-Cas9 gene editing technology were used to obtain overexpression and knockout thylakoid membrane potassium channel protein(TPK3)mutant.From plant physiology,biochemistry,genetics,biophysics and molecular biology,the phenotypic characteristics of mutants and wild types under salt stress and the role of potassium ion and potassium ion channel protein in regulating photosynthetic efficiency were analyzed and explored.The physiological and molecular mechanisms of exogenous potassium ions in alleviating the negative effects of salt stress on tobacco photosynthetic apparatus were elucidated.The results are as follows:(1)A new method for estimating the PGR5-dependent cyclic electron transport(CEF)was proposed,which provides a methodology basis for the role of exogenous potassium ions in regulating cyclic electron flow under salt stress.(2)Under salt stress,the growth of tobacco seedlings was slow,and the leaf area decreased,while the concentration of sodium ions increased,and potassium ions decreased.Salt stress decreased the photochemical yield(YII)of PSⅡ,the quantum yield(YNPQ)and the photochemical quenching coefficient(q P)of thylakoid membraneΔp H heat dissipation in tobacco leaves under light,while the quantum yield(YNO)dependent on basal dissipation and chlorophyll fluorescence emission loss significantly increased.It shows that salt stress reduces the linear electron transport(LEF)rate in the process of photosynthetic electron transport,increases the proportion of excess light energy in the form of antenna heat dissipation or basic heat dissipation,and the whole photosynthetic electron transport chain is in the state of over-reduction.(3)Salt stress decreased the maximum photochemical efficiency(Fv/Fm),chlorophyll content,photosynthetic performance index(PI),net photosynthetic rate and transpiration rate of PSⅡ in tobacco leaves.Chlorophyll synthesis decreased,the transcription levels of antenna proteins and D1 proteins in chloroplasts decreased significantly,and the photosynthetic apparatus was damaged.According to the analysis of chlorophyll a fluorescence kinetics curve(OJIP),the relative variable fluorescence yield at O,K and J sites decreased significantly,while the relative fluorescence yield at I sites increased,indicating that the PSⅡ oxygen-release complex(OEC)and reaction centre were damaged,and salt stress inhibited the electron transfer from the donor side to the recipient side of PSⅡ.The ability of the primary quinone receptor(QA)to transfer electrons to the secondary quinone receptor(QB)is also impaired,and the ability of plastoquinone(PQ)to accept electrons is reduced.(4)The contents of hydrogen peroxide(H2O2)and superoxide anion(O2~-)increased under salt stress,and the activity of superoxide dismutase(SOD),the contents of proline,abscisic acid and calcium increased compared with those of CK,indicating that salt stress-activated calcium and abscisic acid signals and the proline mechanism in leaves.The activities of peroxidase(POD)and ascorbate peroxidase(APX)decreased,indicating that most of the antioxidant systems in plants were damaged.At the same time,the outbreak of ROS led to a decrease in PSI activity(ΔI/Io)and PSI key protein gene psa A.(5)Exogenous potassium supplementation can alleviate the damage of leaves caused by salt stress by alleviating chlorophyll degradation and promoting chlorophyll synthesis,increasing the gene expression of key proteins,regulating abscisic acid signal,calcium signal and antioxidant mechanism.The expression of thylakoid membrane potassium channel gene TPK3 is upregulated,and the matrix[K~+]is increased,so that more K~+ions can quickly enter the lumen(lumen)to counteract the proton diffusion potential formed by protons leaving through ATP synthetase,which is beneficial to the synthesis of ATP.At the same time,the expression of ATP synthase gene ATPb was upregulated,which promoted the synthesis of ATP and the enhancement of cyclic electron transfer(CEF).These mechanisms promoted the synthesis of ATP,which was beneficial to the process of carbon assimilation under stress and avoided the highly reduced state of the electron transport chain.The expression of the PSⅡ donor side,receptor side and PSI reaction centre protein genes was upregulated,including Psb27,Psb A and other protein genes involved in PSⅡ assembly to maintain the integrity of the photosynthetic device.The use of potassium ion inhibitors reverse proved these important roles of potassium ions under salt stress.(6)In order to further examine the relationship between exogenous potassium ions and thylakoid membrane potassium channels,TPK3 transgenic mutant tobacco lines were constructed.After salt stress treatment of seedlings with overexpression of TPK3(OE)and inhibition of expression of TPK3(KO),it was found that the photosynthetic rate and pigment content of OE leaves were higher than those of wild type(WT).Still,the pigment content of KO leaves decreased and their ability to capture light energy decreased.Increased OEC activity and D1 protein expression in OE leaves promoted PSⅡ assembly,enhanced CEF and protected PSI.Salt stress inhibited PSⅡ and PSI activities in KO leaves,hindered electron transport from QA to QB on the PSⅡ receptor side,damaged OEC activity and destroyed the stability of the thylakoid membrane.(7)Using tobacco plants with overexpression of TPK3 as materials,it was found that under salt stress,the oxygen free radical(ROS)of tobacco leaves overexpressing TPK3 was lower than that of WT,while the peroxidation of tobacco leaves with overexpression of TPK3was intensified,and the antioxidant capacity decreased,but it could improve the adaptability to salt stress by regulating cytoplasmic osmotic potential.Under salt stress,there are many transcription factors in the leaves of transgenic plants with overexpression of TPK3,which are closely related to D1 protein and PSI activity.In summary,this study creatively determined the specific damage sites of NaCl stress on the photosynthetic apparatus of tobacco leaves and the mechanism of leaf response to salt stress,as well as the mechanism of potassium ion alleviating the negative effects of salt stress on photosynthetic apparatus,and analyzed the key role of thylakoid membrane potassium channel TPK3 in maintaining ion balance inside and outside thylakoid membrane from the point of view of molecular biology.It not only provides an important reference for revealing the effect mechanism of salt stress on plant growth and development and photosynthetic efficiency but also provides a new reference direction for improving plant salt tolerance.