Mechanism Of Maize Root System And Mycorrhizal Symbioses In Response To Low Phosphorus Stress

Phosphorus（P）is a key nutritional element necessary for plant growth and development.However,most of the soil P exists in the form of insoluble organic forms,which are easily adsorbed and have low mobility,leading to scarce availability of P in the soil.Scarcity of available P in soils can cause slow plant growth,delayed flowering,low fruit set,and eventually yield reduction.Maize（Zea mays L.）,an important grain crop,feed crop,industrial materials and energy material,is one of the most widely planted crops in the world with the highest total production.However,P deficiency in the soil is one of the major factors limiting maize growth,yield and quality.To solve this problem,a large amount of phosphate fertilizer has been widely used to meet the P needs of plants.However,phosphate fertilizer is expensive and have negative impacts on the environment and damage to soil beneficial microorganisms.Most importantly,phosphate ore is a non-renewable resource.The application of P fertilizer alone to alleviate P scarcity is not only a temporary solution to the problem but also a drain on humanity’s future.In fact,plants have evolved a series of strategies to deal with low P stress,especially the root system,which is an important organ for plant nutrient uptake.Roots can resist low P stress through changes in root morphology and structure,root exudates,and the formation of arbuscular mycorrhizal（AM）fungi.AM fungal mycelium can directly absorb P in the soil and transport it to plant roots,induce plant roots to secrete organic acids and acid phosphatase,activate the transformation of soil organic P,and change root architecture to expand root absorption area.These mechanisms can improve the efficiency of plant absorption of soil P and help alleviate plant low P stress.The absorption of available P in soil by plants mainly depends on the absorption of P transporters（PHT）against the concentration gradient.Most of the PHT1 family are high-affinity P transporters,which play an important role in the absorption of P by plant roots.Their gene expression is induced by low P stress and inoculation of AM fungi,and is involved in the direct absorption of P by roots and the indirect absorption of mycorrhizal fungi.Currently,13 members of the Maize PHT1 family have been identified.However,the response of different crops or even different genotypes of the same crop to low P stress may differ.Therefore,identifying and promoting the cultivation of P-efficient maize genotypes is an important strategy to reduce fertilizer application and achieve sustainable agricultural development.Based on the screening and identifying P-efficient maize genotypes,fluorescence quantitative PCR technology was used to explore the expression of PHT1family P transport genes in P-efficient maize under low P stress and inoculation with AM fungi.This revealed the P absorption mechanism of maize under low P stress and inoculation with AM fungi,providing theoretical support for the breeding of low P tolerant,efficient,and high-yield maize varieties.Based on the previous research on maize root phenotypes in our group,20representative genotypes with contrasting root system architectures were selected.These genotypes were tested using the semi-hydroponic root phenotyping system under conditions of low P（10μM）or optimal P（200μM）.The response and tolerance of these 20 genotypes to low P stress were analyzed,and 5 representative genotypes with different P efficiency and root architecture were screened out（Chapter 2）.Using rhizobox technology,the genetic stability and plasticity of root architectural traits were determined by comparing the results in the semi-hydroponic system with soil environments under suitable P conditions（Chapter3）.In a subsequent study of two genotypes,Shengrui 999（P efficient）and Zhongke 11（P inefficient）,under conditions of low P（no P addition）and optimal P(200 mg kg^-1)with or without inoculation of AMF（Funneliformis mosseae BGC NM02A）,the alleviative effect of AMF on low P stress in maize was investigated（Chapter 4）.An modified rhizobox system with 4μm mesh for separating the mycelium from the root system was used to set up six P and AM fungal inoculation combinations,exploring the regulatory mechanism of AM fungal-mediated PHT1 family P transporter genes in maize in response to low P stress using Real-time PCR technology（Chapter 5）.The main results of the research are as follows:1.Response of maize genotypes with different root system architecture to low P stress and identification of P effectsThe results of the semi-hydroponic experiment revealed that the above-ground part and root systems of maize plants had opposite responses to low P stress.Low P stress inhibited the growth of the above-ground part of maize but altered the morphological characteristics of the root system and promoted the growth of lateral roots.Compared with the P-adequate treatment,low P stress significantly inhibited the growth of the aerial parts of the 20 maize genotypes.The plant height was reduced by 12.4%,and shoot dry weight was reduced by20.0%.However,low P stress promoted root growth.Compared with optimal P treatment,root dry weight was increased by 20.6%,and root-shoot ratio was significantly increased by48.7%under P deficit.In terms of root-related traits,the main root depth was decreased by5.21%,but the total root length was increased by 17.8%,the maximum root width was increased by 29.3%,and the root diameter was increased by 1.79%.In terms of P-related traits,P content in shoots and roots of low P stress decreased by 78.9%and 70%,respectively,and the P absorption efficiency decreased by 80.1%,but the P-utilization efficiency was significantly increased by 2.75 times.This study also revealed that there were significant differences in the responses of maize genotypes with contrasting root systems to low P stress（Chapter 2）.By analyzing the differences and correlations in P levels and genotypes of the 18 traits tested,9 traits were selected and converted into their corresponding low-P-tolerance coefficients（LPTC）,and then the principal component analysis（PCA）was used to analyze the differences in P treatment and genotype.The main components were extracted for LPTC of these 9 traits.Finally,according to the scores of the 20 maize genotypes on the extracted 3 principal components,the 20 maize genotypes were clustered into 3 categories,including 2 P-efficient genotypes and 9 P-inefficient genotypes,and the rest 9 intermediate types.Under low P stress,the P-efficient genotypes had stronger ability to regulate root structure and distribute P and photosynthetic products in plant organs,which enabled better adaption to the low P environment.2.Study on the stability and plasticity of maize genotypes to differernt cultivation environmentFive maize genotypes with different P effects were selected,and the plasticity and stability of maize in different culture systems were studied using Rhizobox technology.The research results indicated that plant height and root traits（total root length,root diameter,specific root length）had significant plasticity in different culture systems,but above-ground and root biomass had strong stability.Shengrui 999 and Zhongke 11 had stronger stability in the above traits than the other three genotypes,and there were significant differences in root architecture and P effects between Shengrui 999 and Zhongke 11.3.Alleviation of low P stress in maize inculated with AM fungiIn the absence of AM fungi inoculation,low P stress significantly inhibited the growth of above-ground and root morphological indicators in maize,and also had a significant inhibitory effect on physiological indicators such as photosynthetic rate,aboveground and root P content,and root P concentration;Once again,it has been confirmed that Shengrui999 has stronger adaptability to low P environments than Zhongke 11.Under low P conditions,inoculation with AM fungi significantly promoted the growth of maize above-ground and root systems,increased photosynthetic rate,phosphorus concentration,P content,and P absorption efficiency,thereby effectively alleviating maize low P stress.Under low P conditions,the dependence of P inefficient Zhongke 11 on AM is higher than that of P efficient Shengrui 999,but the opposite is true under suitable P conditions4.Regulatory mechanism of AM fungal-mediated P transporter genes in maize response to low P stressThe results showed that low P stress（LP/LP and LP/HP）significantly inhibited above-ground morphology,dry weight,root configuration related traits,root dry weight,and physiological traits（except for root diameter）,such as P content,P absorption efficiency,photosynthetic rate,and chlorophyll content.However it significantly promoted acid phosphatase activity in the rhizosphere soil;Under low P stress,inoculation with AM fungi significantly increased soil acid phosphatase activity,rhizosphere soil available P content,P absorption efficiency,chlorophyll content,and photosynthetic rate,and promoted above-ground and root growth,effectively alleviating low P stress.The inoculation effect of various traits under LP/HP treatment was better than that under LP/LP treatment.For the soil in the lower mycelial compartment,inoculation with AM fungi significantly increased the acid phosphatase activity.However,it only significantly increased the available P content in the HP/HP and LP/HP treatments.This indicates that extracellular hyphae can activate soil insoluble P through their secretion of acidic phosphate.The Real-time PCR analysis of root P transport genes showed that all 12 P transport genes outside of ZmPht1;10 are expressed in the maize root.among which 7 genes（ZmPht1;2,ZmPht1;3,ZmPht1;7,ZmPht1;1,ZmPht1;11,ZmPht1;12）are P starvation responsive genes involved in the direct pathway of P absorption in maize;Compared with HP/HP treatment,low P stress（LP/LP and LP/HP）significantly upregulated the relative expression levels of seven PHT1 P transport genes,but inoculation with AM fungi significantly downregulated the relative expression levels of these genes.Three P transport genes（ZmPht1;1,ZmPht1;4,ZmPht1;6）are mycorrhizal induced P transport genes,mainly involved in the absorption of P by maize roots in the mycorrhizal pathway.The correlation analysis results showed that under low P treatment,ZmPht1;7 is significantly positively correlated with shoot P concentration（P<0.05）,ZmPht1;8 is significantly positively correlated with root P concentration（P<0.05）,ZmPht1;3（P<0.01）and ZmPht1;7（P<0.05）are significantly positively correlated with P acquisition efficiency（P<0.05）;Under low P stress,inoculation with AM fungi（LP/LP+AM and LP/HP+AM）significantly upregulated the relative expression levels of these three genes.In addition,ZmPht1;9 and ZmPht1;13was significantly upregulated only after LP/HP+AM treatment,so it can be inferred that these two P transport genes are mycorrhizal induced and their expression is influenced by the P concentration in the soil exposed to the extracellular mycelium.The correlation analysis results showed that ZmPht1;6 showed a significant negative correlation with root P concentration in LP/LP+AM treatment（P<0.05）,ZmPht1;9 was significantly positively correlated with root P concentration in LP/LP+AM（P<0.05）,but significantly negatively correlated with root P concentration under LP/HP+AM（P<0.05）.In summary,low P stress can inhibit the overall plant growth of maize,but it can be combatted by adjusting root system configuration,allocating and reusing absorbed P,secreting acid phosphatase,and forming symbiosis with AM fungi.Additionally,there are significant genotypic differences in response to low P stress.Therefore,the screening of P-efficient genotypes and the inoculation of AM fungi are crucial for achieving sustainable agricultural development.Furthermore,the effect of mycelia on soil with different P concentrations was separately studied through rhizobox compartment technology.The differences of P transport genes expression under low P stress and inoculation conditions,as well as their relationship with P-related traits and biomass,were studied through molecular biology technology.This provides theoretical support for revealing the molecular mechanism of P absorption in maize and improving its P absorption efficiency.