Effects Of Overexpression Of ZmPP2AA1 And ZmNF-YA1 On Tolerance To Abiotic Stress In Maize

Abiotic stresses,for example,drought,salt damage,heat/chilling stress and deficiency of nutrient,seriously affect the growth and development of plants.After perceiving stress signals,plants activate series of signal transduction and respond to stresses.Phosphorylation/dephosphorylation and transcription factors play an important role in mediating stress signal transduction.This study analyzed the molecular characteristics and function of two genes in the response to abiotic stresses in maize.One gene encoding protein phosphatase 2A structural A subunit(ZmPP2AAl)responded to low phosphate(Pi)stress.The other gene belonging to transcript factor NF-YA gene family(ZmNF-YA1)responded to drought,salt stress and ion toxicity.Overexpression of ZmPP2AA1 improves low phosphate tolerance in maizeMaize is an important crop,fodder and industrial energy raw materials worldwide.Phosphorus(P)is an essential macronutrient for plants.Despite its abundance in natural soil,inorganic phosphateis often limiting for plants,that severely affects the crop growth and yield.Phosphate fertilizer application could remiss phosphorus deficiency in soil.However,the fertilization not only raises the production costs,but also pollutes the environment.Therefor,studying the mechanisms of resistance to low phosphorus in maize and exploring the biological characteristics of crops to develop P-efficient cultivar have significant meaning in agriculture,economics and environment.Root system architecture(RSA)is particularly important for phosphorus acquirement.Transgenic maize with valuable genes by genetic engineering is an important way to obtain P-efficient cultivar.Several studies have reported that engineering a crop with modifiedRSA could improve the resistance of plants to phosphorus deficiency.Preliminary investigation of the transcriptional response of maize roots to Pi starvation revealed that two genes encoding the protein phosphatase 2A(PP2A)subunits were up-regulated by low Pi availability.The phosphoproteome profiles of maize axial roots under low phosphate(LP)stress also revealed that the catalytic subunit isoform 2 of PP2A displayed dynamic temporal patterns induced by low Pi availability.These resultssuggest that,theexpression changes of PP2Ain Pi-deprived maize roots is involved in the low Pi signal instruction.In this study,ZmPP2AA1 was significantly up-regulated by low Pi availability in maize roots.To discover the physiological functions and mechanisms of ZmPP2AA1 in the regulation ofmaize development and the tolerance to LP,we producedZmPP2AA1 overexpression and RNAi(ZmPP2AA1 OE and ZmPP2AA1 RNAi)transgenic lines in maize elite inbred line Qi-319.The phenotype and physiological-biochemical characteristics of transgenic lines and WT cultured under sufficient phosphate(SP)or LP conditions were observed and determinated.The roots of the ZmPP2AA1 OE plants were curled,especially primary root,which were not observed in the roots of the WT and ZmPP2AA1 RNAi seedlings.Compared with the WT,the PR length was reduced in the ZmPP2AA1 OE plants,while that in the ZmPP2AA1 RNAi lines was increased.The lateral root density and lateral root length of the ZmPP2AA1 OE plants were significantly increased compared with the WT plants,whereas there was a significant decrease in the ZmPP2AA1 RNAi lines.Together,the results suggest that ZmPP2AA1 regulates primary root growth and the gravitropic response,and it positively regulateslateralroot formation and elongation.Modification of the RSA is a typical developmental response to Pi deficiency.After subjecting to SP or LP conditions for 15 d,the hydroponically grown WT and ZmPP2AA1 transgenic plants were collected for root morphology analysis.The results showed that overexpression of ZmPP2AA1 increased axial root number and stimulated lateral root formation,forming a highly branched root system,and significantly increased the effective absorptive surface area.The axial root number of ZmPP2AA1 RNAi lines was comparable to that of WT,but the axial root length was increased,lateral root density and the effective absorptive surface area were decreased.ZmPP2AA1 is involved in the regulation of modification of RSA by Pi deficiency.Under the LP conditions,the anthocyanin accumulation of ZmPP2AA1 OE plants was delayed and decreased,the shoot biomass and root biomass were increased.The shoot and root biomasses of ZmPP2AA1 RNAi lines were significantly lower than those of WT under both SP and LP conditions.Compared with the WT,the phosphorus content and concentration of ZmPP2AA1 OE plants were increased and those of ZmPP2AA1 RNAi lines were decreased when all plants suffered Pi starvation.The different Pi accumulation of genotypes might be caused by the different Pi uptake capacities.Regardless of Pi treatment,ZmPP2AA1OE lines had the highest Imax values.Compared with the WT,ZmPP2AA1 OE seedlings exhibited significantly lower Km and Cmin parameters whereas the ZmPP2AA1 RNAi seedlings exhibited remarkably higher values under LP conditions.Therefor,when sufferred low Pi environment,ZmPP2AA1 OE roots had higher affinities to Pi,better Pi uptake capacities,and faster Pi uptake rates.More accumulation of Pi in plants under LP conditions is extremely beneficial for the growth of the overexpressing plants.ZmPP2AA1 overexpressing maize plants had shortened anthesis-silking interval,more tassel braches and produced higher yields than the WT and RNAi plants under Pi starvation conditions.In total,overexpression of ZmPP2AA1improved the tolerance to LP in maize.The regulation of ZmPP2AA1to LP toletance might be related to the alteration of auxin content and distribution in root systemAuxin plays a key role in root development and mediates the Pi starvation effects on the RSA.The root tips of the ZmPP2AA1 OE seedlings with short and curly axial roots accumulated more free auxin than the other two maize seedling genotypes.This indicated that the changes in axial root development might be attributable to the variation in auxin contentor auxin distribution.ZmPP2AA1OE plants were more sensitive.to.auxin.transport inhibitor NPA.These results indicated thatZmPP2AAl might change the auxin accumulation in root by changing auxin transport,which regulated the maize root growth and development.In conclusion,under LP conditions,ZmPP2AA1 overexpressing maize plants showed superior performance,formed a shallow and highly branched root system,increased the Pi uptake capacity,accumulated more Pi and the tolerance to LP was significantly improved.When suffering long Pi starvation,the reproductive development was less affected and the yield was higher in the ZmPP2AA1 OE plants.ZmNF-YA1 regulates plant growth and stress resistance in maizeCultivating crop varieties with strong resistance to stressfulenvironment has significant meaning for agricultural production.NF-Y transcription factor is involved in the regulation of plant growth and development,and plays an important biological role in plant responses to abiotic stresses such as drought,high or low temperature,and salt stress.The primary work about maize transcriptomics found that one member of NF-Y transcription factor gene family encoding A subunit(ZmNF-YA1)in drought-tolerant inbred lines responded to drought stress.In this work,the expression patterns of ZmNF-YA1 in different organs and under several abiotic stresses of maize were analyzed.The effect of this gene on maize growth and development was studied by using ZmNF-YA1 overexpression and Mu transposon insertion mutant materials,and the biological function of this gene in regulating resistance to abiotic stress was also studied.ZmNF-YA1 expressed widely in maize organs at different developmental stages,and had high expression levels in leaves,pollinated grains and anthers,suggesting that the gene might play a regulatory role in the growth and development of these organs.The expression level of ZmNF-YA1 was upregulated by osmotic stress,salt stress and heat stress after 2 hours in maize leaves,and the increased expression levels maintained until 3 days,suggesting that ZmNF-YA1 responded to these abiotic stresses and might be involved in these stresses signal induction.We observed and analyzed the phenotypes of hydroponics cultured ZmNF-YA1 overexpression lines,Mu transposon insertion mutants and their corresponding wild-type control inbred lines DH4866 and W22.In the early developmental stages,the axial roots of the ZmNF-YA1 overexpressing lines were shorter than that of the wild type DH4866,and there was no difference in shoots.As thefaster growth rate ofZmNF-YA1 overexpression lines,the plant height was higher than that of the wild type,leading toincreased shoot biomass.The ZmNF-YA1 overexpression lines had more lateral roots,to form a more developed root system in the later development stages.The growth and development of Mu transposon insertion mutantswere retarded.The plant height,leave wide,leave length and leave area were siginificantly lower than those of W22,so did the root system.These results indicate that ZmNF-YA1 is a possitive regulator of growth and development of shoot and root in maize.The responses ofmaize seedlings to osmotic stress,salt stress and ion toxicity were studied.The plant height and biomass were less inhibited by these stresses in overexpression lines compared with DH4866,and the Mu transposon insertion mutants were more sensitive than W22.These results suggest that the variation of ZmNF-YA1 could affect the tolerance to osmotic stress,salt stress and ion toxicity in maize.ZmNF-YA1 plays roles in the response of maize to stress and may be involved in the resistance mechanism.In summary,the variations of ZmNF-YA1 gene expression in maize could regulate the plant growth and root development,and could affect the tolerance to abiotic stress such as osmotic stress,salt stress and ion toxicity.Overexpression of ZmNF-YA1 improves the drought stress resistance in maizeSeedlings at five leaf stage were subjected to drought lethaltreatment and then rewatered.Most of the ZmNF-YA1 overexpression lines could resume growth,but only few DH4866 plants slowly recovered and Mu transposon insertion mutant plants almost completely died.The results suggest that ZmNF-YA1 overexpression lines have improved tolerance to drought stress.The potted plants in the early flowering stage-were subjected to drought treatment.The results showed that the growth status of overexpression lines were better than that of DH4866,the root system was more developed,the degree of cell membrane damage and the membrane lipid peroxidation wereto a lesser extent,indicating that ZmNF-YA1 overexpressing maize was less inhibited by drought stress.This might be due to increased osmotic regulators such as soluble sugar and free proline in the overexpression lines,maintaining a lower leaf osmotic potential,helping to increase the water absorption capacity and water holding ability to maintain a high relative water content.In addition,under drought stress,overexpression lines had a higher level of antioxidant protective enzyme activity,which could help the scavenging of free radicals such as reactive oxygen species.Therefore,overexpression of ZmNF-YA1 effectively increased the drought tolerance of maize compared with wild type.In the Wang Baomei's work in our laboratory,overexpression of ZmNF-YB16 promoted the growth of shoots and roots in maize plants,increased the osmotic regulators and antioxidant enzymes under drought stress,and improved the drought tolerance of plants.These phenotypes are similar to overexpressing ZmNF-YA1 maize plants.Wang Baomei using yeast two-hybrid system found that ZmNF-YA1 interacted with ZmNF-YC17,and ZmNF-YB16 interacted with ZmNF-YC17.In this study,the interaction between ZmNF-YA1 and ZmNF-YC17 in vivo was verified by immunoprecipitation.Therefore,we speculate that ZmNF-YA1 may form a complex with ZmNF-YB16/-YC17 to work together to regulate the growth and development of maize and the response to drought stress.Therefore,overexpression of ZmNF-YA1 gene enhances maize resistance to drought stress,which provides candidate genes and operates plansfor maize resistance improvement.