Diel Magnesium Fluctuations Manipulated By OsMGT3 In Chloroplasts Contribute To Photosynthetic Carbon Assimilation In Rice

Photosynthesis is the source of the energy and materials required for normal growth and development of plants.Photosynthesis shows diel changes,which has been largely attributed to external light/dark conditions,as well as internal gene expressions and post-translational modifications of critical enzymes.It would be helpful to develop the new strategies for improving photosynthesis through understanding the mechanisms underlying its dynamic changes.As a structure component of chlorophyll and a cofactor of photosynthetic enzyme,magnesium(Mg)plays an important role in photosynthesis.However,it is not clear whether Mg is involved in the diel regulation of photosynthesis.In order to explore the regulation mechanisms of Mg on the diurnal changes of photosynthesis,this study firstly revealed the effects of Mg deficiency on photosynthetic light and dark reactions in plants through the dynamic detection of photosynthetic parameters in rice leaves treated with Mg deficiency.Thereafter,chloroplast Mg content and Ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco)activity were dynamically monitored to reveal the physiological mechanisms for diel regulation of photosynthesis in rice.Finally,a candidate Mg transporter gene from rice chloroplast,OsMGT3,was found from the chloroplast Mg uptake inhibition experiment and transcriptomic analysis,and its functions related to diel regulation of photosynthesis were analyzed.The main results were summarized as follows:(1)Under long-term Mg deficiency conditions,a remarkable growth retardation was observed in rice,which was accompanied by a decrease in dry weight,chlorophyll concentration and photosynthetic light and dark reactions.A time-course(15 d)Mg depletion experiment revealed that in the early stages of Mg depletion(-Mg),net photosynthetic rate(A_n)and maximum rate of Rubisco carboxylation(Rubisco activity in vivo;V_c,max)gradually decreased in the youngest fully-expanded rice leaves,without any changes in the parameters observed for light reactions,such as maximum efficiency of PSII photochemistry(F_v/F_m),electron transport rate(ETR),chlorophyll concentration(SPAD value),transpiration rate or stomatal conductance.After 15 d of-Mg,A_n and V_c,max in rice leaves decreased by 30.2%and 27.7%,respectively,suggesting that the photosynthetic dark reaction is more sensitive to Mg deficiency.(2)Under short-term Mg deficiency conditions,a dynamic monitoring test was conducted to monitor the dynamics of V_c,max and chloroplast Mg content.Over the observed daily cycles,photosynthetic CO₂ fixation and chloroplast Mg content of rice leaves exhibited clear rhythmmic fluctuations with peaks occurring at the onset of the light phase and troughs at the end of daylight.Furthermore,both parameters were influenced by Mg depletion through weakening of wave amplitude,suggesting that diel oscillation of Mg content in chloroplasts is closely related to Rubisco activity in vivo.Exogenous addition of Mg²⁺to Rubisco extracted from Mg deficient leaves led to recovery of Rubisco activity in vitro to the level observed in Mg sufficient(+Mg)leaves.In contrast,Rubisco content was not impacted by either daily cycles or Mg depletion.These results suggest that Mg might regulate Rubsico activity in vivo through daily fluctuations in chloroplast Mg content.(3)Addition of Mg transport inhibiter cobalt hexaminide(III)(CHA)significantly reduced Mg uptake in chloroplasts,supporting the idea that Mg transporter family members(Cor A/MGT)might be responsible for Mg²⁺uptake in chloroplasts.RNA-seq analysis was carried out and the transcript abundance of all MGT members in rice was assessed.Among MGTs,OsMGT3 showed an extremely high transcript level in shoots,and its transcript level was remarkably suppressed by Mg depletion.Quantitative RT-PCR results revealed that the expression of OsMGT3 in the leaf blades was much higher than those in the leaf sheaths,shoot basal regions and roots.OsMGT3 displayed a clear rhythmic expression pattern in leaf blades and the amplitude of this rhythmic expression pattern was significantly suppressed by Mg depletion.Tissue-specificity of OsMGT3 expression showed that strong signals were detected in the mesophyll cells of both leaf blades and sheaths.Subcellular localization of OsMGT3 showed that OsMGT3 protein was localized to the chloroplast envelope.(4)Functional complementary test using Mg transport defective mutant MM281showed that OsMGT3 was capable of Mg²⁺influx transport in bacteria.Knockout of OsMGT3 decreased chloroplast Mg uptake and resulted in a significant reduction in the amplitude of the Mg fluctuations in chloroplasts.Furthermore,knockout of OsMGT3decreased free Mg²⁺concentration in chloroplast stroma and not in nucleus through free Mg²⁺indicator MARIO.These results demonstrate that OsMGT3 is mainly responsible for free Mg²⁺oscillations in chloroplast stroma.(5)Phenotypic comparision of the wild type(WT)and knockout mutants showed that a remarkable growth retardation was observed in the osmgt3 mutants,which was accompanied by a decrease in dry weight,tiller number and plant height.Among them,dry weight decreased by 45.1%to 55.7%.There were no obvious differences in SPAD,F_v/F_m,stomatal conductance,or transpiration rate between WT and mutant plants,yet A_n and V_c,max were much lower in the two osmgt3 mutants compared to WTs.Mesophyll-specific overexpression of OsMGT3 displayed significant improvements in growth performance,which were accompanied by increases in biomass,A_n,day time V_c,max and Mg²⁺uptake in chloroplasts.Overexpression of OsMGT3 did not alter F_v/F_m,transpiration rate,stomatal conductance,night-time Mg uptake and V_c,max.These results suggest that OsMGT3primarily contributes to diel regulation of Rubisco activity in chloroplasts.In summary,the study demonstrates that there are diel oscillations of Rubisco activity and Mg content in rice chloroplasts,and these oscillations are modulated by the rhythmically expressed Mg²⁺transporter gene OsMGT3 on the chloroplast envelopes.OsMGT3 facilitates daily influx of free Mg²⁺to chloroplasts and participates in the regulation of Rubisco activity for efficient photosynthesis.The results partially elucidate the physiological and molecular mechanisms underlying the diel regulation of photosynthetic CO₂ assimilation in plants,and thus provide a potential route for improving crop photosynthetic efficiency and yield.