Nitrogen storage and remobilization in Brassica napus L. during the growth cycle: identification, characterization and immunolocalization of a putative taproot storage glycoprotein.

In taproot of oilseed rape (Brassica napus L.), a 23 kDa polypeptide has been recently identified as a putative vegetative storage protein (VSP) because of its accumulation during flowering and its specific mobilization to sustain grain filling when N uptake is strongly reduced. The objectives were to characterize this protein more precisely and to study the effect of environmental factors (N availability, daylength, temperature, water deficit, wounding) or endogenous signals (methyl jasmonate, abscisic acid) that might change the N source/sink relationships within the plant, and may therefore trigger its accumulation. The 23 kDa putative VSP has two isoforms, is glycosylated and both isoforms share the same N-terminal sequence which had been used to produce specific polyclonal antibodies. Low levels of an immunoreactive protein of 24 kDa were found in leaves and flowers. In taproot, the 23 kDa putative VSP seems to accumulate only in the vacuoles of peripheral cortical parenchyma cells, around the phloem vessels. Among all treatments tested, the accumulation of this protein could only be induced by abscisic acid and methyl jasmonate. When compared to control plants, application of methyl jasmonate reduced N uptake by 89% after 15 d, induced a strong remobilization of N from senescing leaves and a concomitant accumulation of the 23 kDa putative VSP. These results suggested that, in rape, the 23 kDa protein is used as a storage buffer between N losses from senescing leaves promoted by methyl jasmonate and grain filling.     

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.     

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.     

Subtilisin-like serine proteases involved in N remobilization during grain filling in wheat

The induction of two subtilisin-like proteases (P1 and P2) associated with stress-induced senescence in young plants was investigated in adult wheat plants during the grain-filling period. Western blot analysis of flag leaf extracts showed that P1 was induced very late in the life cycle of the plants (9 days post-anthesis) and that 7 days later it reached a 2.5-fold increase with respect to the initial value at anthesis. On the other hand, the P2 signal was already detected previous to anthesis and increased soon after anthesis, reaching a fourfold increase by the end of the grain-filling period. The induction of P1 and P2 temporally correlates with the degradation of the Rubisco small and large subunits in the flag leaf, as well as with nitrogen (N) accumulation in the ears. At the same time, a decrease in the endogenous concentration of the cytokinins isopentenyladenine and isopentenyladenosine (iP + iPA) in the leaves was observed. In detached leaves senescing in the dark, the levels of both proteases were affected by 6-benzylaminopurine application: the induction of P1 was completely prevented, whereas the induction of P2 was reduced. Our findings demonstrate that both P1 and P2 are expressed in leaves of adult plants and are induced during natural senescence. These results enable us to postulate their participation in N remobilization to developing grains during monocarpic senescence and their regulation by a cytokinin-mediated mechanism.     

A comparative study of the effects of methyl jasmonate and abscisic acid on some rice physiological processes

The effects of methyl jasmonate (MJ) and abscisic acid (ABA) on some physiological processes of rice were compared. MJ exhibited ABA-like effects by promoting senescence of detached leaves, by inducing acid phosphatase activity of detached leaves, by inhibiting ethylene production and shoot growth of seedlings, as well as inhibiting callus formation from anthers. However, MJ and ABA had opposite effects on 1-aminocyclopropane-1-carboxylic acid-dependent ethylene production in detached leaves. The regeneration ability of anther-derived callus was inhibited by MJ but not by ABA. MJ but not ABA markedly induced peroxidase activity in senescing detached leaves. It is concluded that not all physiological processes of rice affected by MJ are similar to those by ABA.Abbreviations ABA abscisic acid - MJ methyl jasmonate - ACC 1-aminocyclopropane-l-carboxylic acid - Apase acid phosphatase     

Nitrogen availability impacts oilseed rape (Brassica napus L.) plant water status and proline production efficiency under water-limited conditions

Large amounts of nitrogen (N) fertilizers are used in the production of oilseed rape. However, as low-input methods of crop management are introduced crops will need to withstand temporary N deficiency. In temperate areas, oilseed rape will also be affected by frequent drought periods. Here we evaluated the physiological and metabolic impact of nitrate limitation on the oilseed rape response to water deprivation. Different amounts of N fertilizer were applied to plants at the vegetative stage, which were then deprived of water and rehydrated. Both water and N depletion accelerated leaf senescence and reduced leaf development. N-deprived plants exhibited less pronounced symptoms of wilting during drought, probably because leaves were smaller and stomata were partially closed. Efficiency of proline production, a major stress-induced diversion of nitrogen metabolism, was assessed at different positions along the whole plant axis and related to leaf developmental stage and water status indices. Proline accumulation, preferentially in younger leaves, accounted for 25-85% of the free amino acid pool. This was mainly due to a better capacity for proline synthesis in fully N-supplied plants whether they were subjected to drought or not, as deduced from the expression patterns of the proline metabolism BnP5CS and BnPDH genes. Although less proline accumulated in the oldest leaves, a significant amount was transported from senescing to emerging leaves. Moreover, during rehydration proline was readily recycled. Our results therefore suggest that proline plays a significant role in leaf N remobilization and in N use efficiency in oilseed rape.     

Methyl jasmonate induces the formation of secondary abscission zone in stem of Bryophyllum calycinum Salisb

Methyl jasmonate (JA-Me) at a concentration of 0.5 % induced the formation of secondary abscission zone and senescence in several types of stem explants (only internode segment, internode segment with nodes and without leaves, internode segment with nodes and debladed petioles) of Bryophyllum calycinum when it was applied in various places of the stem or the debladed petiole as lanolin paste. In the presence of small leaves in stem explants methyl jasmonate also induced the formation of secondary abscission zone and senescence but the presence of larger leaves completely inhibited methyl jasmonate-induced processes. Auxin, (indole-3-acetic acid, IAA), at a concentration of 0.1 % extremely prevented the formation of secondary abscission zones and senescence in the stem tissues induced by methyl jasmonate. Similar relationship between auxin and methyl jasmonate to induce the formation of secondary abscission zone and senescence was found in decapitated shoot of the intact plant. Mechanisms of the formation of secondary abscission zone are also discussed in terms of the interaction of methyl jasmonate with auxin.     

Increased cytokinin levels in transgenic PSAG12–IPT tobacco plants have large direct and indirect effects on leaf senescence, photosynthesis and N partitioning

We studied the impact of delayed leaf senescence on the functioning of plants growing under conditions of nitrogen remobilization. Interactions between cytokinin metabolism, Rubisco and protein levels, photosynthesis and plant nitrogen partitioning were studied in transgenic tobacco (Nicotiana tabacum L.) plants showing delayed leaf senescence through a novel type of enhanced cytokinin syn‐thesis, i.e. targeted to senescing leaves and negatively auto‐regulated (P_SAG12–IPT), thus preventing developmental abnormalities. Plants were grown with growth‐limiting nitrogen supply. Compared to the wild‐type, endogenous levels of free zeatin (Z)‐ and Z riboside (ZR)‐type cytokinins were increased up to 15‐fold (total ZR up to 100‐fold) in senescing leaves, and twofold in younger leaves of P_SAG12–IPT. In these plants, the senescence‐associated declines in N, protein and Rubisco levels and photosynthesis rates were delayed. Senescing leaves accumulated more (¹⁵N‐labelled) N than younger leaves, associated with reduced shoot N accumulation (–60%) and a partially inverted canopy N profile in P_SAG12–IPT plants. While root N accumulation was not affected, N translocation to non‐senescing leaves was progressively reduced. We discuss potential consequences of these modified sink–source relations, associated with delayed leaf senescence, for plant productivity and the efficiency of utilization of light and minerals.     

Light Restriction Delays Leaf Senescence in Winter Oilseed Rape (Brassica napus L.)

Oilseed rape (Brassica napus L.) is a crop with a complex aerial architecture that can cause self-shading leading to a vertical light gradient over the foliage. Mutual shading between neighboring plants at a high sowing density also results in an alteration of photosynthetically active radiation (PAR) absorption by lower leaves. The aim of this study was to analyze the impact that light restriction on lower leaves has on shoot architecture, biomass production and allocation, nitrogen (N) fluxes, and progression of sequential senescence. Field-grown plants were collected at the end of the vegetative rest period and grown in hydroponic conditions until pod maturity. A shading treatment corresponding to a 43.4 % reduction of PAR was applied at the early flowering stage. N uptake and fluxes of N allocation and remobilization were determined by supplying K¹⁵NO₃ in the nutrient solution. Photosynthesis and expression of SAG12 and Cab genes (indicators of leaf senescence progression) were also analyzed on different leaf ranks. The results showed that shading enhanced leaf development on the main stem and ramifications to optimize light capture. The expression pattern of the SAG12/Cab molecular indicator suggested a delay in leaf senescence that allowed leaf life span to be extended resulting in a more efficient leaf compound remobilization, with lower N residual contents in fallen leaves under shading. N uptake increased and N remobilization fluxes were enhanced from source organs (leaves and stem) toward sink organs (flowers). Profuse branching and late senescing varieties would be of interest for further selection programs under high sowing densities.     

Expression of an amino acid biosynthesis gene in tomato flowers: developmental upregulation and MeJa response are parenchyma-specific and mutually compatible

The gene coding for threonine deaminase (TD), the enzyme which catalyzes the first committed step in the biosynthesis of isoleucine, was isolated from tomato as a consequence of its unusual 500-fold upregulation in floral organs. It was subsequently shown that TD is induced in potato leaves in response to wounding, abscisic acid and methyl jasmonate (MeJa). Detailed analysis presented here, reveals an intricate developmental regulation pattern of gene expression in flowers that is operating solely in parenchyma territories. Yet, despite its high pre-existing expression level, TD in flowers can be further induced by MeJa. Induction of TD in flowers as well as in leaves is effective only in the parenchyma domains, irrespective of the prior expression levels. TD is neither expressed nor induced in epidermal, vascular or sporogenous tissues. Promoter analysis in transgenic tomato plants indicates that induction of TD follows identical kinetics in flowers and leaves. Furthermore, the 'conditioning' of developmental upregulation in flowers, the response to MeJa in flowers and leaves, and the parenchyma-specific expression are all mediated by the cis -elements within the proximal 192 bp of the promoter. Promoter elements regulating the correct organ-specific expression are located, however, further upstream. The promoter constructs used in this study can serve as useful tools for expressing extremely high levels of transgenes in specific cells. A scheme explaining tissue-specific response to MeJa, in conjunction with developmental control, is discussed.     

Natural variation in the regulation of leaf senescence and relation to N and root traits in wheat

Objectives

To identify parameters that can be used for the analysis of natural variation in leaf senescence of wheat; and to understand the association between the onset and progression of leaf senescence with N uptake and root traits.

Methods

Chlorophyll content and the proportion of yellow leaves were used as senescence indicators and their relation with other morphological and physiological traits were measured in contrasting early senescing (ES) and late senescing (LS) wheat lines.

Results

There were significant genotype effects on the onset and progress of senescence. The ES lines in which leaf senescence commenced early had significantly lower root biomass and N uptake than LS lines. The strong negative association between the extent of leaf senescence with root biomass and N uptake indicated that the poor root growth induced N limitation caused the early senescence of ES lines.

Conclusions

The leaf senescence development in ES lines was precocious and constitutive as the trait expressed even under optimal growth conditions suggesting they could be useful in understanding the genetic regulation of senescence under different abiotic stress situations. Accelerated leaf senescence in wheat could be a mechanism to compensate for limitations in the root system that tend to restrict nutrient uptake.     

A leaf-specific 27 kDa protein of potato Kunitz-type proteinase inhibitor is induced in response to abscisic acid, ethylene, methyl jasmonate, and water deficit

The 22 kDa Kunitz-type potato proteinase inhibitor (22 kDa KPPI) was induced in tubers. However, the 27 kDa protein, which is immunologically related to the 22 kDa KPPI, was induced in leaves by wounding, hormones, and environmental stresses. The leaf-specific 27 kDa protein was induced in leaves that were treated with exogenous abscisic acid (ABA), ethephon, methyl jasmonate (MeJA), and water deficit. These results indicate that the 27 kDa protein in leaves could function as a defense protein against mechanical damages by herbivorous animals and abiotic environmental stresses that could induce plant hormones.     

Methyl jasmonate, calcium, and leaf senescence in rice

The possible involvement of calcium in the regulation of methyl jasmonate-promoted senescence of detached rice (Oryza sativa) leaves was investigated. Calcium effectively reduced methyl jasmonate-promoted senescence of detached rice leaves. The effect of methyl jasmonate on the senescence was also significantly reduced by calcium ionophore A23187. Methyl jasmonate-promoted senescence of detached rice leaves may be mediated through blocking the entrance of calcium ions into the cytosol.     

Arabidopsis thaliana Atvsp is homologous to soybean VspA and VspB,genes encoding vegetative storage protein acid phosphatases,and is regulated similarly by methyl jasmonate,wounding, sugars,light and phosphate

The soybean vegetative storage proteins, VSP and VSP, are acid phosphatases that accumulate to very high levels in hypocotyls, young leaves and flowers and pods. The genes encoding the soybean VSP are activated by jasmonate, wounding, sugars and light and down regulated by phosphate and auxin. In this study, expression of an Arabidopsis thaliana gene (Atvsp) encoding a protein homologous to soybean Vsp and Vsp, was examined and compared to expression of the soybean Vsp genes. Atvsp mRNA was present at high levels in flowers and buds and at low levels in roots, stems, leaves and siliques. Expression of Atvsp in leaves could be induced by wounding or by treatment of illuminated plants with methyl jasmonate and sucrose. Roots of plants with wounded leaves also accumulated Atvsp mRNA indicating that this gene can be regulated by a transmissible wound signal. Phosphate partially inhibited expression of Atvsp. Arabidopsis proteins of 29 and 30 kDa crossreacted with antibodies against soybean VSP. These proteins were very abundant in flowers and the proteins accumulated in leaves and roots of plants treated with methyl jasmonate. The level of these proteins in flowers was similar to the levels of soybean VSP in young soybean leaves. Overall, these data indicate that Arabidopsis Atvsp and soybean VspA/B genes are regulated similarly and that in both plants, the gene products can accumulate to high levels. This suggests that genes homologous to VspA/B may be of greater general significance than previously recognized.     

Effect of two contrasted N fertilisations on rapeseed growth and nitrate metabolism

Winter oilseed rape was grown under two nitrogen fertilisation conditions. The N1-plants and N5-plants were respectively supplied with 4.5 g N per plant (N-limiting condition) and 22.5 g N per plant (non-N-limiting condition). Growth parameters and nitrate reducing capacity were monitored at five sampling stages interspersed with ammonium nitrate applications. N5-plants showed a higher growth rate producing more leaves and stems, early flower and silique formation and delayed leaf senescence. They also contained more nitrate and a higher nitrate reductase activity (NRA) especially in leaves which represented the main site of nitrate reduction before flowering. However, stems and siliques contributed to NRA especially in nitrogen-limited plants that lost their leaves early. This present study outlines the importance of siliques as individual sinks reducing nitrate essentially in the pod walls. The soluble protein content decreased in senescing leaves which was indicative of the reallocation of proteinic nitrogen towards stems and siliques. In non-limiting conditions, other nitrogen compounds of leaves may account for such a reallocation. Hence, the timing of leaf fall could contribute to the low nitrogen recovery in rapeseed.     

Leaf senescence in a non-yellowing mutant of Festuca pratensis: implications of the stay-green mutation for photosynthesis, growth and nitrogen nutrition

Mutation of the nuclear gene sid disables chlorophyll degradation during leaf senescence in the pasture grass Festuca pratensis. This study investigated the effect of the mutation on photosynthesis and on leaf and whole plant growth under a range of nitrogen regimes. When plants were cultivated in a static hydroponic system, the chlorophyll content of fourth leaves of the stay-green mutant Bf993 remained virtually unchanged from full expansion to complete senescence, while tissue of the wild-type (cv. Rossa) became completely yellow. The retention of chlorophyll in Bf993 was not associated with maintenance of photosynthetic activity as shown by rates of light-saturated CO₂ fixation and apparent quantum efficiency. Higher levels of total N in senescing leaves of Bf993 than in Rossa indicated reduced nitrogen remobilization in the mutant. When using a range of [NH₄NO₃], dry matter production and tillering Mere lower for Bf993 at all but the highest [NH₄NO³, which was supra-optimal for the wild type. In contrast to the static system, where fluctuations in N supply occurred, growth and [NO₃^?] uptake were similar in mutant and wild type when [NO₃^?] was continuously maintained by a flowing solution culture system. The results are discussed in relation to the role of N supply and the effect of the stay-green mutation on N recycling.