Nitrate Uptake By Roots Of Wheat Seedlings, Nitrate Intracellular Compartmentation And Its Regulation

Nitrate is not only the main nitrogen source for majority plants, but also acts as signal substance to regulate the growth and morphogenesis of plants. Nitrate content usually fluctuates greatly in agricultural soils, and the nitrate supply in medium has significant influence in plant growth and nitrogen status inside plant body. Plants can accumulate much nitrate in shoots and roots, and the accumulation is enhanced by high external nitrate supply. Nitrate in plant can be divided into two pools, one is metabolic pool in cytosols and the other is storage pool in vacuoles. Though mechanisms of nitrate accumulation and regulation have attracted considerable attention, the intracellular partition of nitrate in vacuole and cytosol is still unclearly understood. This paper investigated the effects of nitrate supply on growth, total nitrogen content, nitrate content, nitrate intracellular activities, nitrate uptake and nitrate reductase activities of wheat seedlings, discussed the relationship between nitrate intracellular compartmentation and transtonoplast transport, uptake and reduction, explored nitrate transport with electrophysiological technique, and aimed to provide some useful information for the study of nitrate accumulation mechanisms and improving plant nitrogen use efficiency. The main results were as the following:1) Nitrate supply and plant growthBiomass of shoots and roots increased with the increase of external nitrate supply, but decreased respectively when medium nitrate was higher than 5¹0 mmo1·L^-1 and 1² mmo1·L^-1. Biomass of whole plant reached maximum when nitrate content and total nitrogen of wheat seedlings was 30⁴0 mmo1·kg-1 fw and 3%, respectively. Root to shoot ratio decreased with the increase of external nitrate supply. Nitrate starvation inhibited the growth of shoots, but stimulated root growth. Resupply of nitrate to N-depleted seedlings decreased root/shoot ratio, and shoot growth apparently increased.2) Nitrate supply and nitrate status in seedlingsBiomass of shoots and roots increased with the increase of external nitrate supply, and maintained stable when medium nitrate was higher than 10 mmo1·L^-1. Contents of nitrate and total nitrogen in seedlings showed good correlation, nitrate content increased with the increase of total nitrogen content. After stopping external nitrate supply, total nitrogen accumulation in shoots decreased and that in roots increased, while nitrate content in both shoots and roots decreased. After resupply of nitrate, total nitrogen and nitrate increased and then came to stable.3) Nitrate supply and intracellular nitrate compartmentaionWhen supplied with 0.05²5 mmo1·L^-1 nitrate, nitrate activity in vacuoles and cytosols was respectively 14.7⁸3.2 mmo1·L^-1 and 1.9⁶1 mmo1·L^-1. Nitrate activity of epidermal cells was lower than that in cortical cells. Compared with the cytosol, vacuole nitrate had better correlation with nitrate content measured by tissue analysis. During nitrogen starvation, nitrate activity in cytoplasm and vacuole decreased, but vacuole had bigger decrease size. Nitrate remobilization had correlationship with nitrate activity in vacuole nitrate activity.After nitrate resupply, vacuole nitrate activity in roots of N-depleted wheat seedlings increased obviously, while cytosol nitrate activity only had little change. Vacuole nitrate content reached stable after 5 days nitrate resupply, while cytoplasm nitrate contend reached only 1day after nitrate resupply. Accumulation rate of vacuole nitrate decreased with the decrease of nitrate activity in cytosol and vacuole nitrate.4) Nitrate supply and transtonoplast potential differencesWhen supplied with 0.05²5 mmo1·L^-1 nitrate, trans- plasma membrane potential differences was -65^-86 mV in epidermal cells and -62^-96 mV in cortical cells, trans tonoplast was respectively 6¹4 mV and 7²4 mV. Trans-tonoplast membrane potential decreased during nitrogen starvation, while increased during nitrogen resupply.5) Nitrate supply and nitrate reductase activityLeaf nitrate reductase activity had good relationship with leaf nitrate accumulation, while root nitrate reductase activity had no obvious relationship with nitrate accumulation. Nitrate reductase activity decreased during nitrogen starvation, while increased during nitrogen resupply. Root nitrate reductase activity had correlationship with remobilization rate of vacuole nitrate.6) Nitrate uptake by roots Nitrate uptake rate of 2-day-starved seedlings was higher than that of 5- or 10-day-starved seedlings. Vmax and Km of 2- and 5-day-starved Qingmai11 were higher than those of Yannong19. Nitrate efflux decreased during N starvation. Depolarization size of 2-day-starved seedlings was higher than others, and hyperpolarization was observed when 1-day-starved seedlings responded to nitrate. Depolarization and hyperpolarization of Qinmai11 membrane potential were higher than those of Yannong 19. When added nitrate, membrane potential sometimes hyperpolarized first and then depolarized. Measurements of double-barreled nitrate selective microelectrode indicated that the phenomenon might be caused by the location of microelectrode tip in vacuole.