Comparing nitrate storage and remobilization in two rice cultivars that differ in their nitrogen use efficiency

Xiaorong Fan 1 Lijun Jia 1 Yilin Li 1 Susan J. Smith 0 Anthony J. Miller 0 Qirong Shen 1 0 Crop Performance and Improvement Division, Rothamsted Research , Harpenden, Hertfordshire AL5 2JQ, UK 1 College of Resources and Environmental Sciences, Nanjing Agricultural University , Nanjing 210095, PR China Soil nitrogen (N) is available to rice crops as either nitrate or ammonium, but only nitrate can be accrued in cells and so factors that influence its storage and remobilization are important for N use efficiency (NUE). The hypothesis that the ability of rice crops to remobilize N storage pools is an indicator of NUE was tested. When two commonly grown Chinese rice cultivars, Nong Ken (NK) and Yang Dao (YD), were compared in soil and hydroponics, YD had significantly greater NUE for biomass production. The ability of each cultivar to remobilize nitrate storage pools 24 h after N supply withdrawal was compared. Although microelectrode measurements of the epidermal subcellular nitrate pools in leaves and roots showed similar patterns of vacuolar remobilization in both cultivars, whole-tissue analysis showed very little depletion of storage pools after 24 h. However, leaf epidermal cell cytosolic nitrate activities were significantly higher in YD when compared with NK. Before N starvation and growing in 10 mM nitrate, the xylem nitrate activity in YD was lower than that of NK. After 24 h of N starvation the xylem nitrate had decreased more in YD than in NK. Tissue analysis of stems showed that YD had accumulated significantly more nitrate than NK, and the remobilization pattern suggested that this store is important for both cultivars. Changes in nitrate reductase activity (NRA) and expression were measured. Growing in 10 mM nitrate, NRA was undetectable in roots of both cultivars, and the leaf total NRA of equivalent leaves was similar in NK and YD. When the N supply was withdrawn, after 24 h NRA in NK was reduced to 80% but no decrease was found in YD. The proportion of NRA in an active form in YD was significantly higher than that in NK under both nitrate supply and deprivation conditions. Checking NR gene expression showed that leaf expression of OsNia1 was faster to respond to nitrate deprivation than OsNia2 in both cultivars. These measurements are discussed in relation to cultivar differences and physiological markers for NUE in rice. Introduction Ammonium is the main nitrogen (N) form available to rice roots growing under anaerobic paddy conditions, but in aerobic and upland soils nitrate is the main N source. Rice (Oryza sativa L.) grows well in both mixed and single source nitrate and ammonium supplies (Chanh et al., 1981; Youngdahl et al., 1982). In common with most plants, rice can accumulate nitrate but not ammonium within its tissues and this N store may be important for later growth and grain filling. For cultivation, rice is usually first grown in aerobic nursery soil beds where nitrate is the main form of available N. Later japonica rice seedlings are transplanted into flooded soil that is anaerobic and the plant is then chiefly supplied with ammonium as an N source. Rice of the indica type can be continuously grown in aerobic soils and therefore has access to nitrate that may be stored in the leaves. Both these types of rice can accumulate nitrate in the leaf when supplied with this N form (Fan et al., 2005) but it was felt necessary to test if japonica rice has a greater capacity to store, and subsequently remobilize, vacuolar stored nitrate. The term N use efficiency (NUE) can be defined in several different ways and it is likely to be regulated by many different genes (reviewed by Gallais and Hirel, 2004; Good et al., 2004). During grain filling the ability of a plant to remobilize leaf-stored N is an important factor for NUE in crops, and has been strongly implicated in quantitative trait locus (QTL) studies with cereals (Mickelson et al., 2003). However, much less is known about NUE during the earlier vegetative stages of cereal development when biomass is accumulating. Yet this growth stage is important because it occurs when rainfall is heavy and leaching losses are maximal. In cells, vacuolar nitrate may provide a store that maintains cellular assimilation and thereby optimizes N utilization. Remobilization of vacuolar nitrate stores can be measured by removing all N supply from a growing plant (van der Leij et al., 1998) and in this work this treatment has been used to test the response of two rice cultivars that differ in their NUE. Leaf tissue or sap nitrate concentrations are used as indicators of a plants N status and this fact is exploited by farmers when making decisions on fertilizer application rates (Schepers et al., 1992). Measurements of leaf tissue nitrate primarily determine nitrate stored in the vacuole. However, vacuolar nitrate accumulation within cereal leaf cells differs; the highest concentrations accumulate in epidermal cells (Fricke et al., 1994). Barley root epidermal and cortical cell vacuolar nitrate can be remobilized during times of N deficiency and this source can maintain cytosolic nitrate concentrations in the short term (van der Leij et al., 1998). Some recent papers have suggested that there is a close link between cytosolic nitrate activity and nitrate reductase activity (NRA). For example, in leaf cells of Arabidopsis (Cookson et al., 2005) and barley root cells (Fan et al., 2006), changes in cytosolic nitrate activity could be measured under conditions when cellular NRA was altered. As cytosolic nitrate activity is important for determining the thermodynamic gradients for transport to and from the vacuole (Miller and Smith, 1992; De Angeli et al., 2006) how NRA and mRNA expression changed during the remobilization of stored nitrate has also been examined. In order to study the relationship between NUE and key steps determining N distribution within the plant such as vacuolar storage and cytosolic nitrate activity these parameters were compared in two crop cultivars. Two rice cultivars were used for these measurements because this species shows large variation in NUE (Koutroubas and Ntanos, 2003; Peng et al., 2006). Rice cultivation is particularly wasteful as large amounts of applied N fertilizer are lost into the surrounding environment (Vlek and Byrnes, 1986). The rice cultivars used in this study have differing levels of N accumulation efficiency when grown in soil or hydroponics with either nitrate or ammonium. In an earlier study, two Chinese rice cultivars were shown to have differing nitrate uptake rates when supplied with 1 mM nitrate: Nong Ken (NK, japonica) taking up less than Yang Dao (YD, indica) (Fan et al., 2005). Furthermore, the pattern of nitrate transporter expression (OsNRT1.1 and OsNRT2.1) was different in the two cultivars (Fan et al., 2005). These cultivars were chosen for this further study, first to compare their NUE and then to measure how their physiological properties differ. Mater (...truncated)