Decorrelating source and sink determinism of nitrogen remobilization during grain filling in wheat

Background and Aims

Nitrogen (N) remobilization is the major source of N for grain filling in wheat, the other being N uptake after anthesis (N_up); however, variations in remobilization efficiency are not fully understood. It is hard to tell whether the source or the sink effects predominate, because N in the culm at anthesis (N_ant) correlates strongly with both N remobilization (N_rem) and grain number (G_n), respectively the main source and the main sink.

Methods

A pot experiment was thus designed to assess the relative contributions of the source and sink to N_rem regulation. Using two cultivars of winter wheat (Triticum aestivum, ‘Apache’ and ‘Autan’), three pre-anthesis and two post-anthesis N fertilization levels were applied in order to vary the N sources, while ear trimming at anthesis reduced sink size.

Key Results

Unlike results observed at a scale of m², the equation binding N_ant to N_rem exhibited a negative intercept, challenging the concept of nitrogen remobilization efficiency. Before ear trimming, G_n fitted well to N_ant, with a slope dependent on genotype. To obtain a sink variable that was less correlated with N_ant, the difference δG_n was calculated between actual grain number and that which could be predicted from culm N before trimming. A multiple regression then predicted N_rem (r² = 0·95) from N_ant, N_up and δG_n, with fitting unbiased by fertilization treatment, trimming or genotype.

Conclusions

In untrimmed culms, δG_n had a negligible effect, so that N_rem could be fitted to N_ant and N_up only: grain N filling appeared to be determined by sources only (N_ant and N_up), not by sink, and the reduction of N_rem by N_up was quantified. In these ‘normal’ cases, the regulation of N_rem should thus be located within the N sources themselves. In contrast, ear-trimming needs to be considered with caution as it introduced a sink limitation on N_rem; moreover one with an important genotype effect.Key words: Triticum aestivum, winter wheat, source/sink, grain filling, nitrogen uptake, grain number, nitrogen harvest index, nitrogen remobilization efficiency, genotype × environment     

Distribution and remobilization of Zn and Mn during grain development in wheat

Wheat (Triticum aestivum cv. Aroona) was grown in siliceoussand with essential nutrients for unlimited growth except forthe following treatments: controls (sufficient Zn and Mn), lowMn (sufficient Zn) and low Zn (sufficient Mn) until anthesis.Replicate plants were harvested at anthesis; the remaining plantswere transferred to a chelate-buffered nutrient solution containingall essential nutrients except Zn and Mn to allow monitoringof the remobilization of existing Zn and Mn reserves withinthe plant. These plants were harvested 14 d post-anthesis andat grain maturity. At each harvest plants were separated intoindividual components. There were no growth differences between any of the treatmentsat the three harvests. Large amounts of Zn and Mn found in theroots and stems at anthesis were rapidly depleted during graindevelopment. The Zn content of the leaves increased from anthesisto 14 d post-anthesis, but then declined. The Mn content ofthe leaves increased throughout grain development in the controlswhilst remaining constant in the plants pre-grown at low Mn.The Zn and Mn content of the glumes, palea and lemma rose inthe controls from anthesis to 14 d post-anthesis; thereafterZn content declined but Mn content continued to increase. TheZn and Mn content of the grain rose sharply toward grain maturity.We conclude that Mn was not remobil-ized from the leaves ofwheat during grain development. Zinc was remobilized from theleaves, especially the flag leaf and from the leaves of thelow Zn plants. The post-anthesis accumulation of Zn and Mn withinthe glumes will be discussed in relation to the transport pathwaythat Zn and Mn use to enter the developing seed. Key words: Zinc, manganese, wheat, distribution, remobilization     

Effect of N source during soybean pod filling on nitrogen and sulfur assimilation and remobilization

During pod filling, a grain legume remobilizes vegetative nitrogen and sulfur to its developing fruit. This study was conducted to determine whether different nitrogen sources affected N and S assimilation and remobilization during pod filling. Well-nodulated plants fed 1.0 mM KNO₃, 0.5 mM urea, or 2.5 mM urea assimilated 0%, 37%, or 114% more N, respectively, and 25%, 46%, or 56% more S, respectively, than did the average non-nodulated control plant fed 5.0 mM KNO₃. Thus, N source during pod filling greatly affected both N and S assimilation. Depending upon N source, plant N concentration during pod filling decreased from 2.96% to between 1.36% and 1.82%. Non-nodulated control plants fed 5.0 mM KNO₃ had the highest residual N at harvest. During the same treatments, plant S concentration decreased from 0.246% to a relatively uniform 0.215%. Thus, during pod filling, vegetative N was seemingly remobilized more efficiently (38–54%) than was S (13%). N source also affected seed yield and seed quality. Non-nodulated control plants fed 5.0 mM KNO₃ produced the lowest yield (21.1 g seeds plant^-1), whereas well nodulated plants fed 1.0 mM KNO₃, 0.5 mM urea, or 2.5 mM urea produced yields of 26.2 g, 31.8 g, and 36.7 g seeds plant^-1, respectively. Non-nodulated plants fed 2.5 mM urea yielded 28.6 g of seeds plant^-1. Seed N concentrations of non-nodulated plants and nodulated plants fed 2.5 mM urea were high, 6.30% and 6.11% N, respectively, whereas their seed S concentrations were low, 0.348% and 0.330% S, respectively. N sources that produced both a relatively high seed yield and seed N concentration (i.e., a relatively high total seed N plant^-1) produced a proportionately smaller increase in total seed sulfur. Consequently, seed quality, as judged solely by seed S concentration, was lowered.     

Dynamics of light and nitrogen distribution during grain filling within wheat canopy

In monocarpic species, during the reproductive stage the growing grains represent a strong sink for nitrogen (N) and trigger N remobilization from the vegetative organs, which decreases canopy photosynthesis and accelerates leaf senescence. The spatiotemporal distribution of N in a reproductive canopy has not been described in detail. Here, we investigated the role of the local light environment on the spatiotemporal distribution of leaf lamina N mass per unit leaf area (SLN) during grain filling of field-grown wheat (Triticum aestivum). In addition, in order to provide some insight into the coordination of N depletion between the different vegetative organs, N dynamics were studied for individual leaf laminae, leaf sheaths, internodes, and chaff of the top fertile culms. At the canopy scale, SLN distribution paralleled the light gradient below the flag leaf collar until almost the end of grain filling. On the contrary, the significant light gradient along the flag leaf lamina was not associated with a SLN gradient. Within the top fertile culms, the time course of total (alive + necrotic tissues) N concentration of the different laminae and sheaths displayed a similar pattern. Another common pattern was observed for internodes and chaff. During the period of no root N uptake, N depletion of individual laminae and sheaths followed a first-order kinetics independent of leaf age, genotype, or N nutrition. The results presented here show that during grain filling, N dynamics are integrated at the culm scale and strongly depend on the local light conditions determined by the canopy structure.     

A germin-like protein of wheat leaf apoplast inhibits serine proteases

A protein resistant to heat and proteolysis that inhibits serine proteases was isolated from wheat leaf apoplasts. Based on trypsin inhibition, its more active form was a 66-69 kDa oligomer. It was dissociated in an 18-21 kDa monomer having an amino terminal sequence identical to the Box A of germins and germin-like proteins. Like these proteins, it was glycosylated and showed manganese superoxide dismutase activity. The monomer displayed three forms when examined by 2D western blot: two of 19 kDa, pI 5.8 and 6.2; and one of 21 kDa, pI 5.8. It was found that the protein controls serine protease activity in the apoplast of plants challenged with the fungus Septoria tritici.     

Abscisic acid and ethylene interact in wheat grains in response to soil drying during grain filling

Grain filling is an intensive transportation process regulated by soil drying and plant hormones. This study investigated how the interaction between abscisic acid (ABA) and ethylene is involved in mediating the effects of soil drying on grain filling in wheat (Triticum aestivum). Two wheat cultivars, cv. Yangmai 6 and cv. Yangmai 11, were field-grown, and three irrigation treatments, well-watered, moderately soil-dried (MD) and severely soil-dried (SD), were imposed from 9 d post anthesis until maturity. A higher ABA concentration and lower concentrations of ethylene and 1-aminocylopropane-1-carboxylic acid (ACC) were found in superior grains (within a spike, those grains that were filled earlier and reached a greater size) than in inferior grains (within a spike, those grains that were filled later and were smaller), and were associated with a higher filling rate in the superior grains. An increase in ABA concentration and reductions in ethylene and ACC concentrations in grains under MD conditions increased the grain-filling rate, whereas much higher ethylene, ACC and ABA concentrations under SD conditions reduced the grain-filling rate. Application of chemical regulators gave similar results. The results did not differ between the two cultivars. The grain-filling rate in wheat is mediated by the balance between ABA and ethylene in the grains, and an increase in the ratio of ABA to ethylene increases the grain-filling rate.     

QTLs for enzyme activities and soluble carbohydrates involved in starch accumulation during grain filling in maize

ADPglucose, the essential substrate for starch synthesis, is synthesized in maize by a pathway involving at least invertases, sucrose synthase, and ADPglucose pyrophosphorylase, as shown by the starch-deficient mutants, mn1, sh1, and bt2 or sh2, respectively. To improve understanding of the relationship between early grain-filling traits and carbohydrate composition in mature grain, QTLs linked to soluble invertase, sucrose synthase, and ADPglucose pyrophosphorylase activities and to starch, sucrose, fructose, and glucose concentrations were investigated. In order to take into account the specific time-course of each enzyme activity during grain filling, sampling was carried out at three periods (15, 25, and 35 d after pollination) on 100 lines from a recombinant inbred family, grown in the field. The MQTL method associated with QTL interaction analysis revealed numerous QTLs for all traits, but only one QTL was consistently observed at the three sampling periods. Some chromosome zones were heavily labelled, forming clusters of QTLs. Numerous possible candidate genes of the starch synthetic pathway co-located with QTLs. Four QTLs were found close to the locus Sh1 (bin 9.01) coding for the sucrose synthase. In order to confirm the importance of this locus, the CAPS polymorphism of the Sh1 gene was analysed in 45 genetically unrelated maize lines from various geographical origins. The DNA polymorphism was significantly associated with phenotypic traits related to grain filling (starch and amylose content, grain matter, and ADPglucose pyrophosphorylase activity at 35 DAP). Thus, the Sh1 locus could provide a physiologically pertinent marker for maize selection.     

Activities of fructan- and sucrose-metabolizing enzymes in wheat stems subjected to water stress during grain filling

This study investigated if a controlled water deficit during grain filling of wheat (Triticum aestivum L.) could accelerate grain filling by facilitating the remobilization of carbon reserves in the stem through regulating the enzymes involved in fructan and sucrose metabolism. Two high lodging-resistant wheat cultivars were grown in pots and treated with either a normal (NN) or high amount of nitrogen (HN) at heading time. Plants were either well-watered (WW) or water-stressed (WS) from 9 days post anthesis until maturity. Leaf water potentials markedly decreased at midday as a result of water stress but completely recovered by early morning. Photosynthetic rate and zeatin + zeatin riboside concentrations in the flag leaves declined faster in WS plants than in WW plants, and they decreased more slowly with HN than with NN when soil water potential was the same, indicating that the water deficit enhanced, whereas HN delayed, senescence. Water stress, both at NN and HN, facilitated the reduction in concentration of total nonstructural carbohydrates (NSC) and fructans in the stems but increased the sucrose level there, promoted the re-allocation of pre-fixed ¹⁴C from the stems to grains, shortened the grain-filling period, and accelerated the grain-filling rate. Grain weight and grain yield were increased under the controlled water deficit when HN was applied. Fructan exohydrolase (FEH; EC 3.2.1.80) and sucrose phosphate synthase (SPS; EC 2.4.1.14) activities were substantially enhanced by water stress and positively correlated with the total NSC and fructan remobilization from the stems. Acid invertase (EC 3.2.1.26) activity was also enhanced by the water stress and associated with the change in fructan concentration, but not correlated with the total NSC remobilization and ¹⁴C increase in the grains. Sucrose:sucrose fructosyltransferase (EC 2.4.1.99) activity was inhibited by the water stress and negatively correlated with the remobilization of carbon reserves. Sucrose synthase (EC 2.4.1.13) activity in the stems decreased sharply during grain filling and showed no significant difference between WW and WS treatments. Abscisic acid (ABA) concentration in the stem was remarkably enhanced by water stress and significantly correlated with SPS and FEH activities. Application of ABA to WW plants yielded similar results to those for WS plants. The results suggest that the increased remobilization of carbon reserves by water stress is attributable to the enhanced FEH and SPS activities in wheat stems, and that ABA plays a vital role in the regulation of the key enzymes involved in fructan and sucrose metabolism.Abbreviations ABA Abscisic acid - DAS Days after sowing - DPA Days post anthesis - ESC Ethanol-soluble carbohydrate - FEH Fructan exohydrolase - HN High amount of nitrogen - INV Invertase - NN Normal amount of nitrogen - NSC Nonstructural carbohydrate - _leaf Leaf water potential - _soil Soil water potential - Pr Photosynthetic rate - SPS Sucrose phosphate synthase - SS Sucrose synthase - SST Sucrose:sucrose fructosyltransferase - V_limit Limiting substrate - V_max Saturated substrate - WS Water stressed - WSC Water-soluble carbohydrate - WW Well watered - Z Zeatin - ZR Zeatin riboside     

Planting density effects on assimilation and partitioning of photosynthates during grain filling in the late-sown wheat

Leaf blades of the late-sown winter wheat produced the major portion, i.e., more than 60 %, of the total ¹⁴C-photosynthates at grain filling, but ear (rachis and glumes) only about 15 %, sheaths about 11 %, and stem internodes about 11 %. The change of plant density in this experiment had little influence on the ¹⁴CO2-photoassimilation of the ear (rachis and glumes), flag leaf lamina, sheaths and stem internodes, but markedly affected photosynthesis of the second, the third and lower leaves. The photosynthetic rate [expressed as specific radioactivity, s-¹ kg-¹(d.m.)] and the amount of ¹⁴CO₂ photosynthates decreased significantly in the second, the third and other lower leaves at a high plant density. Upon grain-filling of the late-sown wheat, the grain was the major importer of photosynthates. Yet partitioning to the stem internodes depended on the plant density. Stem was the importer of photosynthates at a low plant density, but the exporter at a high plant density. In plants at a low plant density a fairly large proportion of photosynthates was distributed into the roots. The middle and lower above-ground parts of the late-sown wheat at a high plant density decreased or lost their function early. As a result, the plant senesced earlier. However, the grain setting, filling and yielding were restricted. An appropriately low plant density was suitable for prolonging the function of the middle and lower organs, delaying the senescence of plant, increasing the source supply for grain filling, and improving the grain yield. This revised version was published online in August 2006 with corrections to the Cover Date.     

Activities of key enzymes in sucrose-to-starch conversion in wheat grains subjected to water deficit during grain filling

This study tested the hypothesis that a controlled water deficit during grain filling of wheat (Triticum aestivum) could accelerate grain-filling rate through regulating the key enzymes involved in Suc-to-starch pathway in the grains. Two high lodging-resistant wheat cultivars were field grown. Well-watered and water-deficit (WD) treatments were imposed from 9 DPA until maturity. The WD promoted the reallocation of prefixed 14C from the stems to grains, shortened the grain-filling period, and increased grain-filling rate or starch accumulation rate (SAR) in the grains. Activities of Suc synthase (SuSase), soluble starch synthase (SSS), and starch branching enzyme (SBE) in the grains were substantially enhanced by WD and positively correlated with the SAR. ADP Glc pyrophosphorylase activity was also enhanced in WD grains initially and correlated with SAR with a smaller coefficient. Activities of granule-bound starch synthase and soluble and insoluble acid invertase in the grains were less affected by WD. Abscisic acid (ABA) content in the grains was remarkably enhanced by WD and very significantly correlated with activities of SuSase, SSS, and SBE. Application of ABA on well-watered plants showed similar results as those by WD. Spraying with fluridone, an ABA synthesis inhibitor, had the opposite effect. The results suggest that increased grain-filling rate is mainly attributed to the enhanced sink activity by regulating key enzymes involved in Suc-to-starch conversion, especially SuSase, SSS, and SBE, in wheat grains when subjected to a mild water deficit during grain filling, and ABA plays a vital role in the regulation of this process.     

Enhanced expression of serine proteases during floral senescence in Gladiolus

Programmed cell death during senescence in plants is associated with proteolysis that helps in remobilization of nitrogen to other growing tissues. In this paper, we provide one of the few reports for the expression of specific serine proteases during senescence associated proteolysis in Gladiolus grandiflorus flowers. Senescence in tepals, stamens and carpels results in an increase in total protease activity and a decrease in total protein content. Of the total protease activity, serine proteases account for about 67-70% while cysteine proteases account for only 23-25%. In-gel assays using gelatin as a substrate and specific protease inhibitors reveal the enhanced activity of two trypsin-type serine proteases of sizes 75 kDa and 125 kDa during the course of senescence. The activity of the 125 kDa protease increases not only during tepal senescence but also during stamen and carpel senescence indicating that it is responsive to general senescence signals.     

Relation of source and sink during grain filling period in wheat and some aspects of its regulation

The effect of Mg²⁺, Na⁺, K⁺, ouabain and pH on ATPase activity of purified membrane fractions enriched in plasmalemma fragments from Hordeum vulgare L. (glycophyte) and Halocnemum strobilaceum L. (halophyte) was studied. Membrane ATPases from both plants were synergistically activated by K⁺ and Na⁺ in the presence of Mg²⁺. The maximum activity of the enzymes were observed at the ratio Na/K = 2–3. Ouabain (10^-4 M) almost completely eliminated the (Na⁺+ K⁺)-stimulated component of the ATPase activity. The Na, K, Mg-ATPase of Hordeum had a single pH optimum (pH 8), but that of the Halocnemum had two optima(pH 6 and 8). It appears that similar enzymes operate in the cells of both plants studied. The higher Na, K, Mg-ATPase activity of the halophyte compared to that of the glycophyte suggests the involvement of the enzyme in the extrusion of Na⁺ from the cytoplasm of cells of both plants.     

Ontogenetic changes in photosynthetic capacity and dry matter production of flag wheat leaves during the grain filling period

The relationships between photosynthetic capacity and dry matter accumulation during the grain filling period have been studied in flag leaves of Triticum aestivum L., cv. Kolibri grown in Mediterranean field conditions. Particular importance has been given to assimilate accumulation in relation to the onset of senescence. During grain filling, the time course of specific dry weight (SDW) was similar in the blade and in the sheath. Variations in SDW were about six times larger in the sheath than in the blade. Minimum blade SDW values occurred during heading and at anthesis. Maximum blade SDW values were observed two weeks after anthesis. After this, SDW values decreased sharply. The dry matter increase per grain in the period from two weeks after anthesis to the end, was only about 25% of final grain dry weight. The importance of environmental constraints on maximum SDW values are discussed. Maximum SDW values occurred at the beginning of the period of rapid decline in blade net CO₂ assimilation rate and leaf nitrogen content, that is, at the beginning of senescence. On the other hand, the stomatal resistance to CO₂ and the development of senescence are not apparently related. The maximum blade dry weight increase (considering a value of zero at heading) was about 60 mg dry weight per g fresh weight. The possible relationships between dry matter accumulation and senescence onset are discussed.     

Induction of cysteine and serine proteases during xylogenesis in Zinnia elegans

The terminal process of xylogenesis, autolysis, is essential for the formation of a tubular system for conduction of water and solutes throughout the whole plant. Several hydrolase types are implicated in autolysis responsible for the breakdown of cytoplasm. Here, we characterize p48h-17 cDNA from in vitro tracheary elements (TEs) of Zinnia elegans which encodes a preproprotein similar to papain. The putative mature protein, a cysteine protease, has a molecular mass of 22,699 Da with a pI of 5.7. DNA gel blot analysis indicated that p48h-17 is likely encoded by one or two genes. The p48h-17 mRNA accumulated markedly in in vitro differentiating TEs, whereas it appeared not to be induced in response to senescence and wounding in the leaves or H₂O₂ challenge in the cultured mesophyll cells. In stems, the expression of the p48h-17 gene was preferentially associated with differentiating xylem. Activity gel assays demonstrated that a cysteine and a serine protease, which had apparent molecular masses of 20 kDa and 60 kDa, respectively, were markedly induced during in vitro TE differentiation. The cysteine protease activity was also preferentially present in the xylem of Zinnia stems. Transient expression of the p48h-17 cDNA in tobacco protoplasts resulted in the production of a 20 kDa cysteine protease. Taken together, the results indicate that the p48h-17 gene appears to be preferentially associated with xylogenesis, and both the cysteine and serine proteases might be involved in autolysis during xylogenesis.     

Late foliar diseases in wheat crops decrease nitrogen yield through N uptake rather than through variations in N remobilization

Background and Aims

French wheat grains may be of little value on world markets because they have low and highly variable grain protein concentrations (GPC). This nitrogen-yield to yield ratio depends on crop nitrogen (N) fertilization as well as on crop capacity to use N, which is known to vary with climate and disease severity. Here an examination is made of the respective roles that N remobilization and post-anthesis N uptake play in N yield variations; in particular, when wheat crops (Triticum aestivum) are affected by leaf rust (Puccinia triticina) and Septoria tritici blotch (teleomorph Mycosphaerella graminicola).

Methods

Data from a 4-year field experiment was used to analyse N yield variations in wheat crops grown either with a third or no late N fertilization. Natural aerial epidemics ensured a range of disease severity, and fungicide ensured disease-free control plots. The data set of Gooding et al. (2005, Journal of Agricultural Science 143: 503–518) was incorporated in order to enlarge the range of conditions.

Key Results

Post-anthesis N uptake accounted for a third of N yield whilst N remobilization accounted for two-thirds in all crops whether affected by diseases or not. However, variations in N yield were highly correlated with post-anthesis N uptake, more than with N remobilization, in diseased and also healthy crops. Furthermore, N remobilization did not significantly correlate with N yield in healthy crops. These findings matched data from studies using various wheat genotypes under various management and climatic conditions. Leaf area duration (LAD) accurately predicted N remobilization whether or not crops were diseased; in diseased crops, LAD also accurately predicted N uptake.

Conclusions

Under the experimental conditions, N yield variations were closely associated with post-anthesis N uptake in diseased but also in healthy crops. Understanding the respective roles of N uptake and N remobilization in the case of diseased and healthy crops holds the promise of better modelling of variations in N yield, and thus in GPC.Key words: Triticum aestivum, Puccinia triticina, leaf rust, Mycosphaerella graminicola, Septoria tritici blotch, N uptake, N remobilization, N yield, Leaf area duration     

Relationship between activities of key enzymes involved in starch synthesis and accumulation in maize inbred lines during grain filling

Time course of starch production and the key enzyme activities in the grains of four maize inbred lines (two high-starch and two low-starch lines) were studied. Accumulation of grain starch and its components in four maize inbred lines rose continuously after pollination and increased as a sigmoid curve during grain filling. The accumulation rates showed single-peak curves. The accumulation rates of starch and its components reached their peaks on 25–32 days after pollination (DAP), respectively. Activities of adenosine diphosphoglucose pyrophosphorylase (AGPPase) and starch synthase in the grains of four lines followed single-peak curves with the peaks on 24–31 DAP. The highest activity of the starch-branching enzyme (Q-enzyme) in the grains of both high-starch lines appeared on 23 DAP, but that of both low-starch inbred lines showed double-peak curves, the peaks being at 15–20 DAP and 30–35 DAP. There was significant positive correlation between AGPPase, soluble starch synthase (SSS), and starch granule-bound synthase (GBSS) activities. The results indicated the Q-enzyme had different expression patterns in the high-and the low-starch maize inbred lines, and that AGPPase, SSS, and GBSS activities were significantly and positively correlated with amylose, amylopectin, and starch accumulation rates in all lines. This text was submitted by the authors in English