Investigation Of The Mechanism Of High Photosynthetic Efficiency Of Maize Mutant Qi319-96 Under Low Phosphorus Stress And Creation Of Transgenic Cotton New Germplasm For Salt And Drought Tolerance

1.Investigation of the Mechanism of High Photosynthetic Efficiency of Maize Mutant Qi319-96 under Low Phosphorus StressPromotion of photosynthesis efficiency plays an important role in increasing crop yield.Identifying the relationship between plant phosphorus efficiency and photosynthesis is the key to agricultural sustainable development.We identified a low-phosphate-tolerant maize mutant(Qi319-96)that has a higher photosynthetic efficiency than wild-type Qi319 under low phosphorus conditions.The purpose of this study was to clarify the molecular mechanisms of the high photosynthetic efficiency of Qi319-96 under low phosphorus conditions.The mutant Qi319-96 and wild-type Qi319 were treated with 1000μM(+P,Pi-sufficient)or 5μM(-P,Pi-deficient)KH2PO4.Then,comparative proteomics,genomics,and physiology analyses were performed.The increased low phosphorus tolerance of mutant Qi319-96 is due to its ability to increase the internal P-use efficiencyLow phosphorus levels limit plant growth and metabolism.Under low phosphorus conditions,although the total phosphorus contents of Qi319 and Qi319-96 were not significantly different,the Pi content of Qi319-96 was 52.94%higher than the Pi content of Qi319.Qi319-96 had increased chlorophyll levels and increased photosynthesis.The contents of starch and sucrose were also different between Qi319 and Qi319-96,where Qi319-96 generated more sucrose than its Qi319 counterpart.Physiological results showed that Qi319-96 was more efficient at remodeling membrane lipid composition and had increased levels of V-A-TPase activity,which enhanced the recycling of Pi and increased the Pi levels of Qi319-96.This resulted in improved chlorophyll biosynthesis and higher enzymatic activities of several Calvin cycle-related and "CO2 pump"-related enzymes,which led to increased photosynthetic performance of Qi319-96 under low phosphorus stress.Our results clearly indicated that the difference in internal P-use efficiency is the main explanation for the higher tolerance to low phosphorus conditions in the mutant compared with the wild-type maize.Furthermore,this study suggested that inbred lines of maize with low phosphorus tolerant traits could be obtained effectively through cellular engineering.miR395 and miR399,located in Qi319-96,may participate in the regulation of Pi levelsMicroRNAs(niRNAs)are a type of small RNA that can regulate the expression of target genes.MicroRNAs participate in a variety of physiological processes,such as the regulation of plant growth and the stress resistance.We compared miRNA expression levels in the leaves of Qi319 and Qi319-96 maize under sufficient and deficient phosphorus conditions using small RNA high-throughput sequencing technologies.We discovered that 34 members of eight known miRNA families along with 23 new miRNAs were differentially expressed under-P condition,while 23 members of ten known miRNA families and 40 new miRNAs were differentially expressed under the +P condition.In addition,the expression levels of some target genes were significantly different between Qi319 and Qi319-96 maize in the-P condition.Therefore,we concluded that miR395 and miR399 expression in the leaves of mutant Qi319-96 may participate in the regulation of Pi levels.Better photosystem and carboxylation system performance under low phosphorus are due to Qi319-96 has better photosynthetic performanceLow phosphorus stress reduces the Pn and Gs of maize leaves but increases the Ci.The decrease in Pn was due to non-stomatal factors.Fv/Fm,ΦPsⅡ,RC/CS,ABSo/CS,ETo/Cs,TRo/CS,PsII performance(Ψ0),PSI performance(△Ir/Io),photosystem performance(Φ(PSⅠ/PSⅡ))and RuBPcase carboxylase activity of carboxylation were decreased,while DIo/CS,Vj,and Vi were increased under low phosphorus conditions.These data suggested that phosphorus starvation caused damage to the electron transport chain,which reduced the electron transfer ability of the photosynthesis pathway.This led to increased thermal dissipation from the light energy absorption and reduced leaf ATP content,which resulted in a lower carbon assimilation rate.We measured these parameters in both inbred lines of maize and found that Qi319-96 had better PsⅡ performance(Ψo),PsI performance(△Ir/Io),photosystem performance(Φ(PSⅠ/PSⅡ))and higher RuBPcase carboxylase carboxylation activity.This resulted in increased ATP levels and carbon dioxide assimilation ability under low phosphorus conditions.Differentially expressed proteins in the chloroplasts of the Qi319-96 contribute to low phosphorus tolerance by promoting the stability,coordination and efficiency of the photosynthetic systemThe light reactions of photosynthesis occur in the chloroplasts,where plant growth substances and other metabolites are synthesized.Therefore,chloroplast activity is directly related to plant growth and the abiotic stress response.To determine the mechanism for increased carbon assimilation ability in Qi319-96 compared with Qi319,we compared the chloroplast proteomes of Qi319-96 and Qi319.In our diagram of two-dimensional gel electrophoresis analysis,we determined that 24 proteins from 27 significant differentially expressed proteins of Qi319-96 were increased compared with Qi319 under low phosphorus conditions.These proteins can be divided into four categories.The first category is comprised of photosynthesis proteins,including ribulose-1,5-bishosphate carboxylase/oxygenase,ATP synthase CF1-β subunits,ATP synthase CF1-α subunits,and photosystem II oxygen-evolving enhancer protein.The second category of proteins is the related stability proteins of the photosynthesis system,including photosystem II stability/assembly factor HCF136,FtsH(filamentation temperature-sensitive)protein,and chlorophyll a-b binding protein.The third category of proteins is the electron transport chain related proteins,including cytochrome b6/f complex iron-sulfur subunits and Zea Mays Leaf ferredoxin-NADP+ reductase.The fourth category of proteins are peptidyl-prolyl cis-trans isomerase,pathogenesis-related proteins,and so on.These proteins promote the stability,coordination and efficiency of the photosynthetic system.Our data supports the idea that Qi319-96 had a higher electron transfer performance and carbon assimilation under phosphorus starvation conditions.Taken together,we investigated possible mechanisms to explain the high photosynthetic efficiency of Qi319-96 under low phosphorus conditions using comparative proteomics,genomics,and physiology analyses of the mutant Qi319-96.The increase in low phosphorus tolerance of the mutant Qi319-96 was likely due to increased intracellular Pi availability.Qi319-96 had better photosystem and carboxylation system performance under low phosphorus.This study provides new information concerning the mechanism of maize photosynthesis system response to low phosphorus stress and provides a new strategy for maize improvement of high photosynthetic efficiency under low phosphorus stress.2.Creation of Transgenic Cotton New Germplasm for Salt and Drought ToleranceDrought and salt stress affect the growth,yield and fiber quality of cotton.To improve yield and fiber quality of cotton and to maintain cotton production capacity under drought and/or salt stress conditions,it is urgent to cultivate drought-tolerant and/or salt-tolerant cotton varieties.In this work,we overexpressed the TsVP gene from Thellungiella salsuginea(coding for H+-PPase),the AtNHXl gene from Arabidopsis thaliana(coding for Na+/H+ Antiporter),and the ZmPLCl gene from Zea mays(coding for PI-PLC)in cotton.We tested the salt resistance and drought resistance of these transgenic cottons by selection in the greenhouse combined with a multi-point field test over a period of years.Finally,we obtained genetically modified cotton strains with high resistance to salt and drought resistance and fostered a batch of new material cotton resistance to salt and drought.Expression of the TsVP gene in cotton improves salinity tolerance and increases seed cotton yield in a saline fieldThe emergence and survival of cotton seedlings is crucial for cotton cultivation in saline fields.Limited seed cotton yield in saline fields is also a matter of concern for farmers.In this study,to evaluate the application value of the genetically modified cotton,the time of emergence,emergence rate,survival rate,carbon assimilation capacity during bud stage,seed cotton yield,and cotton fiber quality were determined in saline field trials using TsVP-overexpressing transgenic cotton and wild-type cotton plants.In a saline field,TsVP-overexpressing transgenic cotton emerged two days earlier than wild-type plants and displayed a greater emergence rate(67.46%)and a greater survival rate(51.08%).The results from our field trials in 2013 showed that the photosynthetic CO2 assimilation rates and electron transfer efficiency of transgenic lines were significantly greater than those of wild-type plants.The seed cotton yield of TsVP-overexpressing transgenic cotton plants increased by an average of 14.81%compared with that of wild-type plants,and cotton fiber quality also improved.In greenhouse conditions,TsVP-overexpressing plants also required a shorter time to reach 50%emergence than wild-type plants when the NaCl concentration was greater than 100 mM.This research indicates that TsVP overexpression has the potential to improve seed cotton yield in saline fields.Co-overexpression of the AtNHX1 and TsVP in cotton improves salinity tolerance and increases seed cotton yield in a saline fieldTo improve the salt resistance of cotton,AtNHX1 and TsVP were co-overexpressed in cotton GK35.Although the leaf relative water content(RWC)of all cotton plants was reduced under salt stress,the leaf relative water content of AT3 was higher than that of the wild type.The overexpression of TsVP and AtNHX1 enhanced the water retention capacity of cotton leaf cells.This phenomenon can be explained by increased solutes,such as K+,Na+ and Ca2+,in the cells of transgenic plants.AT3 produced 35.79%and 23.87%more total number of cationic moles than wild-type plants.The saturation osmotic potential of the leaves was also lower by 23.88%and 22.01%than the wild type under greenhouse cultivation and in a saline field,respectively.The transgenic leaf cells have a relatively low saturation osmotic potential and thus result in increased water absorption and water-holding capacity in these plants,which therefore have a higher relative water content.A higher relative water content benefits transgenic plants in that they can better perform photosynthesis after exposure to high salinity conditions.We found that transgenic cotton plants exerted increased CO2 assimilation,had a higher net photosynthetic rate(Pn),and had higher stomatal conductance(Gs)than wild-type plants in greenhouse and saline fields.In a saline field,transgenic cotton emerged two days earlier than wild-type plantsand displayed a greater emergence rate(53.22%)and survival rate(49.45%).The seed cotton yield of transgenic cotton plants increased by an average of 22.46%compared with the seed cotton yield of wild-type plants.Therefore,our results show that overexpression of AtNHX1 and TsVP in genetically modified cotton grow well under salt stress.This may be because of a greater accumulation of K+,Na+ and Ca2+in the leaves,which could be beneficial to maintain ionic equilibrium and cell osmotic potential under salt stress.This would result in higher relative water content and higher carbon assimilation in the leaves.In addition,we determined that the AtNHXl-TsVP gene has the potential to improve seed cotton yield in saline fields and could be applied to improve seed cotton yield in a saline field.Expression of Maize phospholipase Cl gene(ZmPLC1)in cotton improves the drought resistance and transgenic cotton seed cotton yield under drought stressPhosphoinositide-specific phospholipase C(PI-PLC)plays an important role in a variety of physiological process,including drought tolerance.We obtained cotton that overexpressed ZmPLC1 via agrobacterium-mediated transformation.Southern blot analyses,qRT-PCR and PI-PLC activity assays were performed to confirm transformants.The results showed that the ZmPLCl gene was integrated into the cotton genome and was expressed in transgenic cotton.We studied the drought stress tolerance of transgenic cotton plants in the cotton seedling,bud flowering and flowering stages.Our results showed that transgenic cotton plants had higher relative water content,better osmotic adjustment,increased photosynthesis rate,lower ion leakage,smaller lipid membrane peroxidation,and higher seed cotton yield than wild type cotton under the drought stress.In the Xinjiang field,compared with wild-type plants,transgenic cotton plants had better carbon assimilation rate in bud flowering,and higher seed cotton yield under natural drought condition.Our results showed that overexpression of ZmPLC1 could significantly enhance the tolerance to drought stress,which may be due to the accumulation of more solute and ABA in cells under drought stress.Taken together,our data shows that the overexpression of TsVP,TsVP-AtNHX1,or ZmPLC1 in cotton can enhance the stress resistance and seed cotton yield both in the greenhouse and in drought or saline fields.Therefore,the TsVP,TsVP-AtNHX1,and ZmPLCl genes have the potential to improve salt tolerance or drought tolerance in cotton.