Nitrogen recycling and remobilization are differentially controlled by leaf senescence and development stage in Arabidopsis under low nitrogen nutrition.

Five recombinant inbred lines (RILs) of Arabidopsis (Arabidopsis thaliana), previously selected from the Bay-0 x Shahdara RIL population on the basis of differential leaf senescence phenotypes (from early senescing to late senescing) when cultivated under nitrogen (N)-limiting conditions, were analyzed to monitor metabolic markers related to N assimilation and N remobilization pathways. In each RIL, a decrease of total N, free amino acid, and soluble protein contents with leaf aging was observed. In parallel, the expression of markers for N remobilization such as cytosolic glutamine synthetase, glutamate dehydrogenase, and CND41-like protease was increased. This increase occurred earlier and more rapidly in early-senescing lines than in late-senescing lines. We measured the partitioning of (15)N between sink and source leaves during the vegetative stage of development using (15)N tracing and showed that N remobilization from the source leaves to the sink leaves was more efficient in the early-senescing lines. The N remobilization rate was correlated with leaf senescence severity at the vegetative stage. Experiments of (15)N tracing at the reproductive stage showed, however, that the rate of N remobilization from the rosettes to the flowering organs and to the seeds was similar in early- and late-senescing lines. At the reproductive stage, N remobilization efficiency did not depend on senescence phenotypes but was related to the ratio between the biomasses of the sink and the source organs.

N-protein mobilisation associated with the leaf senescence process in oilseed rape is concomitant with the disappearance of trypsin inhibitor activity.

Brassica napus L. (oilseed rape) is an important crop plant characterised by low nitrogen (N) use efficiency. This is mainly due to a weak N recycling from leaves that is related to incomplete protein degradation. Assuming that protease inhibitors are involved throughout protein mobilisation, the goal of this study was to determine their role in the control of N mobilisation associated with leaf senescence. Results showed that a 19-kDa polypeptide exhibiting trypsin inhibitor (TI) activity presented an increased gradient from the older to the younger leaves. According to the SAG12/Cab gene expression profile, which is an indicator of leaf senescence, mature leaves of nitrate-deprived plants presented an earlier initiation of senescence and a decrease in protein concentration when compared with nitrate-replete plants. This coincided with disappearance of both TI activity and a reduction in the transcript level of the BnD22 gene (encoding a protein sharing homology with Künitz protease inhibitor). In young leaves of N-deprived plants, initiation of senescence was delayed; soluble protein concentration was maintained while both TI activity and BnD22 transcripts were high. This indicates that in oilseed rape growing under nitrate deprivation, the more efficient N recycling from mature leaves contributes to the maintenance of growth in young leaves. The data suggest a significant role for protease inhibitors in the regulation of proteolytic processes associated with N mobilisation during leaf senescence.

The expression patterns of SAG12/Cab genes reveal the spatial and temporal progression of leaf senescence in Brassica napus L. with sensitivity to the environment.

Despite a high nitrate uptake capacity, the nitrogen use efficiency (NUE) of oilseed rape is weak due to a relatively low N remobilization from vegetative (mostly leaves) to growing parts of the plant. Thus, this crop requires a high rate of N fertilization and leaves fall with a high N content. In order to reduce the rate of N fertilization and to improve the environmental impact of oilseed rape, new genotypes could be selected on their capacity to mobilize the foliar N. Various indicators of leaf senescence in oilseed rape were analysed during plant growth, as well as during senescence induced by N deprivation. Metabolic changes in leaves of increasing age were followed in N-supplied and N-deprived rosettes by measuring chlorophyll, total N, and soluble protein contents. Similarly, the expression of genes known to be up-regulated (SAG12) or down-regulated (Cab) during leaf senescence was monitored. The amount of soluble proteins per leaf was a better indicator of leaf senescence than chlorophyll or total N content, but was not evaluated as an accurate indicator under conditions of N deprivation. On the other hand, up-regulation of SAG12 concomitantly with down-regulation of Cab in the leaf revealed the spatial and temporal progression of leaf senescence in oilseed rape. This study shows, for the first time at the whole plant level, that the SAG12/Cab gene expressions match the sink/source transition for N during both developmental and nutrient stress-induced leaf senescence.

Effects of a cold treatment of the root system on white clover (Trifolium repens L.) morphogenesis and nitrogen reserve accumulation.

The ability of white clover (Trifolium repens L.) to undergo cold acclimation is an important determinant of its persistence in mixed swards since growth rate at low temperatures sustains higher clover contents at the start of spring. During a re-growth period following defoliation, a gradual exposure of the root system (cv. Grasslands Huia) led to some physiological and morphological changes of cold-adaptive significance, similar to those developed by clover ecotypes originating in northern areas of Europe. Thus, cold exposure of the root system resulted in small-leaved prostrate forms of white clover after one month of re-growth. Similarly, cold exposure increased the ability of plants to store nitrogen since the application of low temperatures to the root system enhanced soluble protein accumulation in roots and in stolons. More specifically, cold exposure of the roots induced gene expression of a vegetative storage protein (17.3 kDa VSP) in both organs. These results demonstrate that the root system of clover plants should be a site of perception of the low-temperature stimulus, and gave rise to the question of the transduction of the cold signal from the roots to the aerial parts. On the basis of this study and taking into account molecular aspects concerning the clover VSP, it is suggested that this protein could participate in cold acclimation in addition to its role in nitrogen storage.

Morphological pattern of development affects the contribution of nitrogen reserves to regrowth of defoliated white clover (Trifolium repens L.).

The contribution of nitrogen reserves to regrowth following defoliation was studied in white clover plants (Trifolium repens cv. Huia). This was found to be closely linked to the morphological pattern of development of the aerial parts during the same period. Low temperature (6 degrees C) and short day exposure (8 h photoperiod) were used to induce dwarf development, i.e. to increase branching rate and to enhance new sites of leaf production during a period of regrowth. Treated plants exhibited a large reduction in leaf area and a large increase in leaf pool size for the first 10 d of a subsequent regrowth under standard culture conditions (16 h daylight; 22/18 degrees C day/night). The contribution of nitrogen from storage compounds in organs remaining after defoliation (sources) to regrowing tissues (sinks) was assessed by 15N pulse-chase labelling during regrowth following shoot removal. The mobilization of nitrogen reserves from storage tissues of regrowing clover was closely linked to the pattern of differentiation of the newly developed organs. It appeared that regrowth was supported less by endogenous N for the first 10 d after defoliation in treated plants, compared with control plants grown continuously in standard conditions. It is assumed that dwarf plants exhibit a lower dependence upon the mobilization of soluble proteins previously accumulated in roots and uncut stolons. The relationship between leaf development rate and N-uptake recovery following defoliation is discussed.