Phosphorus Stress Response Of Different Genotype Maize (Zea Mays L.) And The Study Of Roots Proteomics

As one of essential macronutrients for plant growth and development, phosphorus (P) not only is an important component of nuclear acid and biomembranes, but also plays an important role in an array of processes such as energy metabolism, photosynthesis, respiration, enzyme activation/inactivation, redox reactions, signal transduction, carbon metabolism etc. P is involved in almost all life activities, so its deficiency seriously affects the growth and yield of crops. Plants mainly absorb soluble phosphate (Pi) in soil by roots to satisfy the demand for P nutrition. Although total P is quite abundant in many soils, the concentration of available Pi by roots in soil is usually about 1μM, which cannot satisfy the demand of plants to acquire the sufficient Pi. The low availability of Pi in soil has been an important constraint for crop production in the agriculture. It is reported that crop yield on above 30% of the world's arable land is limited by Pi availability. To cope with the problem, P fertilizer use increased four- to five-fold betweenl960 and 2000, however, up to 80% of which might be fixed in the soil, and caused water eutrophication in some areas. If this situation continues, rock Pi reserves could be depleted in as little as 60-80 years. Insufficient of available Pi by roots in soil and lack of rock Pi reserves are potential crisis for agriculture in the future. Therefore, exploiting biological characteristics of crops and developing P-efficient cultivars, to acquire P in soil more efficiently, may be the most promising way of achieving sustainable agricultural development and solving the problem of P-deficit and environment pollution.Maize (Zea mays L.) occupies a pivotal position in China and even the world food system. In recent years, maize growth usually meets the problem of P-deficit, which is an important limiting factor for yield and increases its production cost. Maize plant is tall and large, and heterosis is utilized in agricultural production. Screening P-efficient maize genotypes according to biomass and yield is not only time-consuming and labor-intensive, but also easily influenced by environment. Development of plant biotechnology provides a new way to obtain P-efficient maize genotypes, while understanding the low-P tolerance mechanism of maize well is required. Therefore, the further study of low-P tolerance mechanism of maize is of important theoretical significance and practical value.In recent years, the studies of low-P tolerance mechanism in plants were active and have made great progress. It has been showed to be a quantitative trait controlled by multi-gene or QTLs, and some individual genes responsive to P-starvation in higher plants have been characterized. Some researches on transcriptome suggested that the response of plants to P-stress is a metabolic process controlled by a complicated regulation system. It is well known that protein is the bearer of cell functions, and the development of proteomics provides a more precise and intuitive way to analyze the gene expression. However, to the best of our knowledge, the analysis of roots response to P-stress at the proteome level has not been available for any plant by far.Previously, our lab obtained an array of maize inbred lines with different low-P tolerance using cellular engineering technology. During the process of low-P tolerance determination and conformity analysis for six generations, we found that the low-P tolerant trait of 99038 with good agronomic traits could be stably inherited, which is an elite material for maize P-efficiency breeding and the study of low-P tolerance mechanism. In the study, low-P tolerant maize inbred line 99038 and the original line Qi-319 were used to study the low-P tolerance metabolism. The root morphology, and physiological and biochemical characteristics in 99038 and Qi-319 under +P (1000μM KH₂PO₄) or -P (5μM KH₂PO₄) condition were studied; the proteome changes of Qi-319 roots responsive to P-starvation were analyzed systemically, and a large number of differentially expressed proteins were identified; the comparative proteome analyses for proteins isolated from roots of 99038 and Qi-319 under +P or -P condition were conducted, respectively, and differentially expressed proteins were analyzed combining with their difference of root morphology and physiological and biochemical characteristics. The aim is to investigate the main reasons for higher low-P tolerance of 99038, compared to Qi-319, and also offer the valuable information for P-efficiency breeding by genetic engineering.1. Analyses of root morphology and physiological and biochemical characteristics of low-P tolerant maize inbred line 99038 and control Qi-319During the process of observation for continuous multi-generation, we found out that plants from 99038 and Qi-319 showed no obvious difference on shoots but substantial difference on roots morphology, and roots of 99038 were more developed than Qi-319. It is well known that developed roots are especially important for P-acquisition because of its relative immobility. Total root length, the number of lateral root, root absorptive surface area and root volume corresponding to unit shoot weight, root-shoot ratio, the average length of lateral root and the three longest roots of 99038 seedlings were increased significantly, while average root diameter was decreased obviously, compared to Qi-319. These changes expanded the root surface-soil contact area and explored a larger volume of soil, which improved the capacity of P-acquisition of 99038. Under +P or -P condition, the determination of P content, concentration and distribution in 99038 and Qi-319 plants showed that under P-stress 99038 distributed the greater proportion of P to the roots, P content of roots in 99038 increased to 1.5 folds, P concentration in the roots of 99038 was higher significantly and root growth was less restrained, compared to Qi-319. APase activity assay showed that under P-stress APase activity of old leaves and root tips of both maize genotypes increased significantly. However, whether under -P or +P condition, APase activity of corresponding to the area of 99038 and Qi-319 showed no significant difference. Therefore, APase activity was not the main factor for different low-P tolerance of 99038 and Qi-319. Under +P or -P condition, the analysis of P-uptake kinetics also suggested that the I_max value of 99038 was higher and K_m, C_min values were lower, compared to Qi-319. Namely, 99038 is with P-efficiency trait. Analysis of 2-DE gel maps (pH 3-10) of proteins from roots of 99038 and Qi-319 plants grown under -P condition showed that 13.6% protein spots changed significantly in amount (P < 0.01) . Based on the results above, it could be concluded that the different expression of proteins in roots of 99038 and Qi-319 might result in the differences of root morphology and the capability of P-absorption in two genotype maize, which lead to higher low-P tolerance of 99038 than Qi-319. Simultaneously, the results also confirmed that cell engineering technology is of important value in P-efficiency breeding of maize.2. Proteomic analysis of roots growth and metabolic changes under P deficit in Qi-319 maize plantsThe analysis of root morphology and physiological and biochemical characteristics of Qi-319 and 99038 indicated that under P-stress root morphology and P-absorptive characteristics of them were significantly different, but their change tendencies were consistent. Under P-stress total root length, the number of lateral root, root absorptive surface area and root volume corresponding to unit shoot weight and root-shoot ratio of both genotypes were increased significantly, average root diameter was decreased obviously and the capacity of P-absorption were enhanced markedly etc., compared to +P condition. In order to understand the mechanism of maize roots responsive to P-deficiency, we conducted the comparative proteome analysis for proteins isolated from Qi-319 roots treated with 1000μM KH₂PO₄ (control) or 5μM KH₂PO₄ for 17 days. Approximate 1300 protein spots were detected by 2-DE, 254 (19.5 %) of which changed significantly in amount (P < 0.05; = 2 folds). One hundred and twenty differentially expressed proteins were identified by MALDI-TOF MS and classified according to Arabidopsis MATDB. The 120 identified proteins represented a large range of functional categories, including 44 proteins in metabolism, 13 proteins in protein fate, 6 proteins in protein synthesis, 5 proteins in energy, 9 proteins in secondary metabolism, 7 proteins in cellular organization, 11 proteins in defense/interaction with environment, 3 proteins in transport, 7 proteins in transcription /cell cycle/signal transduction and 15 unclassified or unknown proteins. Several proteins, such as tRNA isopentenyl transferase, GTP-binding nuclear protein RAN-B1, AraC typr: Arac protein, purine rich element binding protein B-like and protein kinases, were involved in the biosynthesis of phytohormone, cell cycle, signal transduction and transcriptional regulation. They might play important roles in linking the change of external P concentration and the metabolic adaptation of plants, to regulate gene expression facilitating P-homeostasis. Those results above suggested that the low-P tolerance of plants is involved in a complicated process related with multiple metabolic and signal pathways at proteome level and also offered the valuable information for further characterization of function and regulation of P starvation responsive genes in plant P-nutrition.3. Difference analysis of roots proteome in 99038 and Qi-319In order to understand P-efficiency mechanism of 99038 and the different expression of which proteins resulting in the differences of root morphology and capacity of P-acquisition between both genotypes, we conducted comparative proteomic analysis for roots of Qi-319 and 99038 treated with 5μM or 1000μM KH₂PO₄ using pH 3-6 and pH 5-8 strips. Under +P condition, 1260 proteins were detected on 2-DE gels, 135 (10.7 %) of which were significantly different in amount (P < 0.05; = 2 folds); under -P condition, 1637 proteins were detected, 196 (12.0 %) of which were significantly different in amount. Therefore, whether under -P or +P condition, the roots proteome of 99038 and Qi-319 were significantly different. Seventy nine (+P) and 108 (-P) differently expressed proteins were identified by MALDI-TOF MS. They represented a large range of functional categories, respectively. Differently expressed proteins were involved in carbon metabolism, phytohormone synthesis, cell cycle, signal transduction etc., such as citrate synthase, 6-phosphogluconate dehydrogenase, pyruvate phosphate dikinase, UDP-glucose pyrophosphorylase, C-24 sterol methyltransferase,β-glucosidase, GTP-binding nuclear protein RAN-B1, importin a, MCM6, PP2A, 14-3-3, MAPK1, CDC48 and so on, which might play important roles in regulating root morphology and cell cycle, sensing the change of external Pi concentration etc.. Based on functional analyses of differently expressed proteins identified, combining the differences of root morphology and P-absorption ability between 99038 and Qi-319, we presumed that these differently expressed proteins were very likely to result in the difference of root morphology and P-absorption ability by participating in the regulation of carbon metabolism, phytohormone signal pathways or cell cycle. Moreover, in all differently expressed proteins identified, 18 protein species with the same or similar function changed in the same pattern under +P or -P condition, including cell cycle regulation related proteins, several carbon metabolism related proteins etc., different expressions of which in roots of both genotypes were thought to be caused by genotypic differences. Therefore, it is presumed that there were great genotypic differences in carbon metabolism and cell cycle regulation between 99038 and Qi-319. Based on the results above, the more developed roots and higher low-P tolerance of 99038 than those of Qi-319 were related with its carbon metabolism and cell proliferation regulation, which were more advantageous for root cell division and growth. Simultaneously, these data also promoted our understanding on the role of some P-starvation responsive genes in maize P-efficiency control.In this study, the analyses of maize low-P tolerance mechanism were conducted on root morphology, physiology and proteome with low P tolerant inbred line 99038 and original inbred line Qi-319. The results showed the characteristics of P-efficient maize inbred lines in root morphology, P uptake kinetics and proteome, reflected the effects of low-P stress on the growth and development, and proteome of roots, confirmed that P-efficient maize breeding materials could be obtained by cellular engineering technology, promoted our understanding on mechanisms of maize low-P tolerance and P-efficiency genetic control. All those offered the valuable information for further study in functions of P starvation responsive genes in plant P-nutrition, and also provided ideas and objects for establishing the experiment designs for improving low-P tolerance of crops by genetic engineering, thereby contributing to resolve the status of P-deficit and low utilization of P-fertilizer in the agricultural production of China and even the world.