Effect Of Low Phosphorus Stress On Phosphate Uptake And Transport, Levels And Distribution Of IAA And CTK In Maize

As an essential macronutrient for plant growth and development, phosphorus (P) not only is an important architectural component of plant cells, but also plays an important role in cell metabolism and signal transduction. However, low availability of phosphate (Pi) in soil has become a major constraint for plant growth and production, due to its low immobility. To adapt the low-P conditions, plants have evolved complex strategies including modifications of root architecture, improvement of Pi acquisition and recycling etc. In recent years, some progress has been made in the studies on regulation of Pi uptake in plants. Nonetheless, the relationship between Pi uptake and internal P concentrations (IPCs) of plants needs to be further explored; kinetic characteristics of Pi uptake by cells of mature leaf under low P stress and its physiological significance remain to be discovered; and knowledge concerning the role of phytohormones in low-P response of plants remain to be elucidated. In the present work, using maize (Zea mays L.) inbred line Qi319, the relationship between Pi uptake and IPCs of plants was analyzed, changes of kinetic characteristics of Pi uptake by cells of mature leaf under P deficiency were studied, and the physiological role of these changes in leaf P output was discussed. Furthermore, root architectural characteristics, levels and distribution of endogenous auxin and cytokinkin within root system, and effect of exogenous phytohormones and chemical inhibitor on root architecture were investigated in seedlings of Qi319 and 99037 (a low P-tolerant line derived from Qi319) under different P conditions. The objective of the present study is to reveal regulation mechanisms of Pi uptake and transport, and the role of auxin and cytokinnin in the low-P-induced alterations of root architecture in maize.1. Regulation of Pi uptake by maize roots(1) Relationship between Pi uptake and internal P concentrations of plantsSeedlings of maize imbed line Qi319 were grown under different P supplies (0.1-1,000μM KH₂PO₄) for 25 days, and the relationship among P supply level, IPCs, and Pi uptake of plants were investigated. The results indicated that IPCs of the plants increased with the elevation of P supply level, whereas Pi uptake rate (V_max) and affinity for Pi of the plants reduced. Correlation analysis showed that apparent V_max value of Pi uptake was reversely related to external P concentrations (EPCs), shoot and root P concentrations (SPCs and RPCs) of the plants within the entire P supply range (0.1-1,000μM) (P < 0.01). Among the P supply range from75μM to 1,000μM apparent K_m and C_min values of Pi uptake were positively related to EPCs. SPCs and RPCs respectively (P < 0.01). As P supply level ranged from 0.1μM to 75μM, there was no significant changes in K_m or C_min values. Partial correlation analysis revealed that these parameters (V_max, K_m and C_min) were only significantly correlated with SPCs and RPCs (P < 0.05), not EPCs (P > 0.05). Taken together, these results showed that Pi uptake by roots had a close relationship with the IPCs.Qi319 seedlings grown in nutrient solution containing 1,000μM KH₂PO₄ for 23 days were deprived of Pi for 10 days, and variations of IPCs and Pi uptake during P deprivation were investigated. The results indicated that SPCs and RPCs gradually decreased with the prolongation of P deprivation period, while affinity for Pi and Pi uptake rate (V_max) of the plants increased continually. Furthermore, significant correlations were also clearly showed between the parameters (V_max, K_m and C_min) and IPCs of shoots and roots in the phase of P deprivation (P < 0.01).Seedlings of Qi319 grown in nutrient solution without P were supplied with Pi through leaves. It was observed that within 3 days of the P recovery period, IPCs of the plants increased gradually. However, difference in IPCs between the shoots and roots was considerable: the SPCs had increased to a sufficient P level, while the RPCs, though increased at some extent, were still under severe P deficiency. Meanwhile, both Pi uptake capacity (V_max and affinity for Pi of roots in these seedlings decreased significantly compared to those without P supply (P < 0.01). These results showed that the Pi uptake status of roots have a relationship with SPCs of the plants in this P recovery period, and provided compelling evidence for the hypothesis that systemic P signal regulates Pi uptake of roots.(2) Estimation of Pi uptake status according to SPCs of maize plantsSignificant correlation between IPCs and parameters of Pi uptake kinetics suggested that Pi uptake by maize plants could be estimated by IPCs. In the present study, three equations with high determinants of coefficient (R²) were obtained based on the linear regressions of the kinetic parameters and SPCs. With these equations, values of K_m, C_min and V_max of Pi uptake by seedlings grown in different circumstances were calculated by SPCs and compared with the values determined by Pi depletion technique. For V_max, there was a parallel relationship between the values estimated by SPCs and determined by Pi depletion technique, only except the period of resupplying Pi to the Pi-starved seedlings over several days. For K_m and C_min. the values estimated by SPCs were consistent with those obtained from Pi depletion experiments. These results indicated that SPCs basically reflect the status of Pi uptake of plants in a linear manner, and it is possible to estimate the Pi uptake kinetic parameters according to SPCs, which is potential to be a novel and convenient method for estimating the Pi-uptake status of maize plants. This method would be helpful for applying P fertilizers finely and economically and achieving high yield of maize.2 Effect of low-P stress on P transport in maize mature leaf(1) After grown under 1000μM KH₂PO₄ for 23 days, Qi319 seedlings were treated under low P (0.1μM Pi) condition (0-14 days), and Pi concentrations of stem and mature leaf xylem saps as well as P contents of mature leaf phloem exudates were monitored. The results indicated that P which was imported to mature leaf through xylem decreased, whereas P output of the leaf through phloem was enhanced relatively under low-P conditions.(2) Pi uptake assay with plasma membrane vesicles revealed that apparent V_max value of Pi uptake by plasma membrane vesicles which were prepared from the mature leaves of seedlings treated under low P condition was greater than those of the control plants (P < 0.01). The result indicated that Pi uptake by cells of mature leaf was enhanced under P deficiency. To explore the physiological significance of variations of Pi uptake by cells of mature leaf under P deficiency, the maize mature leaf was applied locally with Pi solutions labeled by ³²P in the presence or absence of an inhibitory agent (NEM) for Pi transport across plasma membranes; and then, radioactivity of the leaf (laminas where ³²P was applied were cut out) and leaf phloem exudates were measured. The results indicated that in the presence of NEM, ³²P labeled Pi uptake by the leaf cells from leaf apoplasts was inhibited significantly (P< 0.01), and synchronously, radioactivity of the leaf phloem exudates decrease significantly compared with that in the absence of NEM (P < 0.05). showing a relationship between Pi uptake by leaf cells from apoplasts and P export of the leaf. Taken together, it could be concluded that under low-P stress capacity of Pi uptake by cells of mature leaf was enhanced, which would benefit leaf cells to absorb Pi from leaf apoplasts (including xylem apoplasts), and maintain the normal physiological activity of the cells and increase the P export of mature leaf.3 Roles of auxin and cytokinin in the low-P-induced alterations of root architectureThe postembryonic developmental program of plant root system is plastic, allowing changes in root architecture to adapt to environmental conditions including P availability. Insufficient of available Pi in soil is one of the important factors that strongly limit plant growth. Root architecture, to some extent, determines the capacity of plant Pi acquisition. Phytohormones, such as auxin and cytokinin, involve in the postembryonic development of root system, however, the roles of both hormornes and their mutual interaction in the low-P-induced alterations of root architecture have been unclear so far.The inbred line 99037 is developed from Qi319 by cellular engineering. Under different P supply levels, 99037 plants have longer lateral and axile roots (including primary and adventitious roots) and higher capacity of P uptake and accumulation than Qi319. In the field experiments, both total dry weight and seed yield of 99037 are greater than those of Qi319, respectively.Seedlings of 99037 and Qi319 were grown hydroponically under low (LP, 5μM Pi) or high (HP, 1,000μM Pi) P conditions for18 days, and the root architecture and levels and distribution of IAA and zeatin within root system were investigated. The results showed that under LP, zeatin concentrations in both young segments of axile roots (1 cm long from apex, YSARs) and mature segments of axile roots (behind YSARs) decreased (P < 0.05) compared to those under HP; IAA concentrations and the ratio of IAA to zeatin (IAA concentrations/zeatin concentrations) increased markedly in YSARs under LP, while reduced significantly in MSARs (P < 0.05), compared to those under HP. Synchronously, the number of aixle roots, and the density and average length of lateral roots decreased significantly (P < 0.05), whereas average length of axile roots increased significantly (P < 0.05) under LP, compared to those under HP. These results indicated that root architecture, levels and distribution of auxin and zeatin as well as the ratio of IAA to zeatin within root system were altered notably under LP, implying that variations of levels and distribution of IAA and zeatin as well as their mutual interaction within root system played roles in the low-P-induced alterations of root architecture.To validate this presumption, seedlings of both lines grown under LP or HP were treated with 6-BA and NAA solely or together, or an auxin porlar transport inhibitor (TIBA), and the alterations of root architecture of the plants were investigated. The results supported the presumption that variations of levels and distribution of auxin and cytokinin as well as interaction between them within root system played roles in alterations of root architecture under low-P conditions. In addition, this presumption was further supported by the differences in root architecture, level and distribution of IAA, ratio of IAA to zeatin, and the resistence of lateral roots to low-P stress and TIBA between 99037 and Qi319 root systems.In summary, levels and distribution of auxin and cytokinin as well as the ratio of both within root system vary notably under LP condition. And these variations and interaction between the two hormones play an important role in the low-P-induced alterations of maize root architecture. Seeking the cross-talk of phytohormone and phosphorus signaling network may help to understand the low P tolerance mechanism in maize.