| As one of essential macronutrients for plant growth and development, phosphorus (P) is involved in almost all of plant life activities. However, the concentration of available Pi by roots in soil is usually about1μM, which can not satisfy the demand of plants to acquire sufficient Pi. In the main cultivation areas of maize, insufficient Pi in soil has been an important constraint for maize yield and increases the agriculture production cost. Although traditional breeding has made great achievements in genetic improvement maize, the low screening efficiecy and long screening cycle have been not met the production needs of elite maize. Therefore, breeding P-efficient maize genotypes and study the low-P tolerance mechanism of maize by transgenic technology is of theoretical and application significance.In this study, we preliminary identified TsCBFl over-expression maize with improved low phosphorus tolerance by sand culture experiment. According to the above results and drougt tolerance study of Zhang (2007) in TsCBFl over-expression maize, we selected three transgenic lines (L-7, L-9, L-19) to study phosphorus stress responses and the low-P tolerance mechanism. We measured various physiological and biochemical parameters and analyzed expression changes of phosphorus starvation responsive genes by real-time PCR.There were significant differences between responses to low-P stress of transgenic and control plants. Under sufficient-P condition (1000uM KH2PO4), there were no significant differences between transgenic and control plants either in shoots or roots biomass. Low-P stress (5uM KH2PO4) inhibited the plant growth and lead to a significant reduction of shoots and roots biomass. However, the shoots and roots biomasses of transgenic plants were signifiantly higher than control plants, respectively. When subjected to low-P treatment for18days, the P contents both in shoots and roots were reduced. However, the P contents in transgenic plants were significantly higher than control plants. And transgenic plants had a higher P use efficiency. Under low-P stress, transgenic plants have more lateral roots and longer total root length, which were more conducive for transgenic plants to obtain enough phosphorus nutrition. There were no significant differences between transgenic plants and control plants in P-uptake kinetics (Imax, Km, Cmin). Compared to control plants, transgenic plants had higher Apase activity both in leaves and roots under sufficient-P or low-P condition. These results indicated that transgenic plants had higher capacity of recycling phosphorus and stronger low-P tolerance. Photosynthetic characteristics of maize were adversely affected under P stress. The results showed that low-P stress decreased net photosynthetic rate, stomatal conductance and transpiration rate, but increased intercellular CO2concentration. However, the efficiency of light energy conversion and utilization of transgenic plants was higher than control plants, suggesting the superior tolerance to low-P stress. P starvation increased sucrose concentration and decreased fructose and glucose concentrations in leaves, while sucrose, fructose, and glucose concentrations in roots were all increased. Compared to control plants, transgenic plants had higher sucrose contents both in leaves and roots under P stress. Based on the results above, it could be concluded that the different capacity of recycling phosphorus and photosynthesis under P stress finally lead to stronger low-P tolerance of transgenic plants.The different responses of plants to low-P tolerance were essentially caused by the changes of gene expression regulation. We analyzed expression changes of phosphorus starvation responsive genes under P stress. The results showed that the expression level of purple acid phosphatase genes (PAP3, PAP4), RNase gene (RNS) and phosphate transporter gene (Phtl;1) were increased, beta-amylase gene (BAM1) and sucrose phosphate synthase gene (SPS) were up-regulated, while sucrose synthase gene (SUS1) was down-regulated. However, transgenic plants had higher expression level of BAM1than control plants. It showed that TsCBFl trancripional factor leaded to up-regulation of BAM1. The up-regulated BAM1was beneficial for the synthesis of beta-amylase, which can hydrolyze starch to generate maltose. The accumulation of maltose is conducive to the protection of functional protein, plasma membrane and photosynthetic electron transport chain from destruction.In this study, we also measured the related growth and development parameters of transgenic lines (L7, L9, and L19) and control plants by pot experiment. Low-P stress retarded maize heading, shedding and silking period, but transgenic plants were less affected than control plants. Plant height, ear height and tassel length were lower in low-P stress condition than those in sufficient P condition, while ear leaf length, ear leaf width and tassel branch number did not change remarkably. The plant height and ear height of L-7and L-19were significantly higher than control plants. Low-P stress reduced ear length, grain number, grain weight,100-grain weight. Compared to sufficient P conditions, under low-P stress the grain yield of L-7, L-9and L-19were reduced by16.4%,23.3%and16.7%, respectively, while the grain yield of control plants decreased by28.2%. The100-grain weight of transgenic plants was significantly greater than control plants. These results from pot experiment showed that transgenic plants have stronger low-P tolerance than control plants.In summary, we proved that TsCBFl over-expression maize had an improved low-P tolerance by sand culture experiment and pot experiment. The variety of the responses to low P stress of transgenic and control plants in hydroponic experiment was resulted from the capacity of recycling phosphorus and photosynthesis under P stress. We also studied the expression profiles of some P starvation responsive genes at transcriptional level and obtained some interesting results that might be important for future study of maize low-P tolerance mechanism. |
