土壤水分亏缺对水稻茎秆贮藏碳水化合物向籽粒运转的调节

以两个茎秆贮藏物质利用效率不同的水稻(Oryza sativa)杂交组合(‘汕优63’和‘Pc311/早献党’)为材料,进行土壤水分亏缺处理(Water-deficit),以水层灌溉为对照(Well-watered),研究水分亏缺对水稻茎贮藏性碳水化合物运转及其关键酶活性的调节作用。结果表明,水分亏缺促进了水稻茎秆贮藏物质的运转和对籽粒产量的贡献,开花前茎秆贮藏的碳水化合物对产量贡献率分别提高了1.9~3.0倍(与水层灌溉相比)。土壤水分亏缺诱导了水稻茎节间α-淀粉酶、β-淀粉酶、α-葡萄糖苷酶、D-酶活性上升,但淀粉磷酸化酶受到了抑制,说明土壤水分亏缺加强水稻茎秆贮藏淀粉水解途径,而不是磷酸解途径。就蔗糖代谢而言,土壤水分亏缺提高了蔗糖磷酸合成酶的活性和活化状态,抑制蔗糖转化酶活性,促进蔗糖合成,加速贮藏物质快速降解和转移,从而调节稻株贮藏碳水化合物向籽粒的分配。     

Activities of starch hydrolytic enzymes and sucrose-phosphate synthase in the stems of rice subjected to water stress during grain filling

To understand the effect of water stress on the remobilization of prestored carbon reserves, the changes in the activities of starch hydrolytic enzymes and sucrose-phosphate synthase (SPS) in the stems of rice (Oryza sativa L.) during grain filling were investigated. Two rice cultivars, showing high lodging-resistance and slow remobilization, were grown in the field and subjected to well-watered (WW, psi(soil)=0) and water-stressed (WS, psi(soil)=-0.05 MPa) treatments 9 d after anthesis (DAA) till maturity. Leaf water potentials of both cultivars markedly decreased during the day as a result of WS treatment, but completely recovered by early morning. WS treatment accelerated the reduction of starch in the stems, promoted the reallocation of prefixed (14)C from the stems to grains, shortened the grain filling period, and increased the grain filling rate. More soluble sugars including sucrose were accumulated in the stems under WS than under WW treatments. Both alpha- and beta-amylase activities were enhanced by the WS, with the former enhanced more than the latter, and were significantly correlated with the concentrations of soluble sugars in the stems. The other two possible starch-breaking enzymes, alpha-glucosidase and starch phosphorylase, showed no significant differences in the activities between the WW and WS treatments. Water stress also increased the SPS activity that is responsible for sucrose production. Both V(limit) and V(max), the activities of the enzyme at limiting and saturating substrate concentrations, were enhanced and the activation state (V(limit)/V(max)) was also increased as a result of the more significant enhancement of V(limit). The enhanced SPS activity was closely correlated with an increase of sucrose accumulation in the stems. The results suggest that the fast hydrolysis of starch and increased carbon remobilization were attributed to the enhanced alpha-amylase activity and the high activation state of SPS when the rice was subjected to water stress.     

基于植株碳流的水稻籽粒淀粉积累模拟模型

通过解析水稻(Oryza sativa)植株碳素积累和转运的动态规律及其与环境因子和基因型之间的定量关系, 构建基于植株碳流动态的水稻籽粒淀粉积累模拟模型。水稻籽粒中的淀粉积累速率取决于库限制下的淀粉积累速率和源限制下的可获取碳源。库限制下的淀粉积累速率是潜在淀粉积累速率及温度、水分、氮素、淀粉合成能力等因子综合影响的结果; 源限制下的可获取碳源取决于花后光合器官生产的即时光合产物和营养器官向籽粒转运的储存光合产物。花后植株即时光合产物随花后生长度日呈对数递减。花后营养器官向籽粒转运的储存光合产物又分为叶片和茎中积累碳素的转运。利用不同栽培条件下的独立田间试验资料对籽粒淀粉积累的动态模型进行了检验, 结果显示籽粒淀粉积累量和含量的模拟值和观测值之间的根均方差均值分别为3.61%和4.51%, 决定系数分别为0.994和0.959, 表明该模型对不同栽培条件下的水稻单籽粒淀粉积累量和含量具有较好的预测性, 为水稻生产中籽粒淀粉指标的动态预测和管理调控提供了量化工具。     

A proteomics view on the role of drought-induced senescence and oxidative stress defense in enhanced stem reserves remobilization in wheat

Drought is one of the major factors limiting the yield of wheat (Triticum aestivum L.) particularly during grain filling. Under terminal drought condition, remobilization of pre-stored carbohydrates in wheat stem to grain has a major contribution in yield. To determine the molecular mechanism of stem reserve utilization under drought condition, we compared stem proteome patterns of two contrasting wheat landraces (N49 and N14) under a progressive post-anthesis drought stress, during which period N49 peduncle showed remarkably higher stem reserves remobilization efficiency compared to N14. Out of 830 protein spots reproducibly detected and analyzed on two-dimensional electrophoresis gels, 135 spots showed significant changes in at least one landrace. The highest number of differentially expressed proteins was observed in landrace N49 at 20days after anthesis when active remobilization of dry matter was observed, suggesting a possible involvement of these proteins in effective stem reserve remobilization of N49. The identification of 82 of differentially expressed proteins using mass spectrometry revealed a coordinated expression of proteins involved in leaf senescence, oxidative stress defense, signal transduction, metabolisms and photosynthesis which might enable N49 to efficiently remobilized its stem reserves compared to N14. The up-regulation of several senescence-associated proteins and breakdown of photosynthetic proteins in N49 might reflect the fact that N49 increased carbon remobilization from the stem to the grains by enhancing senescence. Furthermore, the up-regulation of several oxidative stress defense proteins in N49 might suggest a more effective protection against oxidative stress during senescence in order to protect stem cells from premature cell death. Our results suggest that wheat plant might response to soil drying by efficiently remobilize assimilates from stem to grain through coordinated gene expression.     

Promising Role of Moderate Soil Drying and Subsequent Recovery Through Moderate Wetting at Grain-Filling Stage for Rice Yield Enhancement

The study was conducted at the grain-filling stage to elucidate the physiological and molecular mechanisms of the root to enhance yield under alternate wetting and drying (AWD) compared with conventional irrigation. Measurements of root dry weight (RDW), seed setting rate, total kernel weight, and grain yield were determined along with 2D electrophoresis to detect altered protein expression in response to moderate soil drying (MD) and the subsequent recovery phase as moderate wetting (MW) under AWD compared with continuous wetting under CI. We found significant enhancement in RDW as well as 14.30 % increase in inferior spikelets^, seed setting and 10.32 g m⁻² increase in final yield. Among the total 55 differentially expressed proteins, 26 proteins were differentially expressed under both MD treatment and MW treatment, whereas 14 proteins under MD and 15 proteins under MW showed distinct expression. Differentially expressed proteins were involved in redox homeostasis, signaling, defense, energy, photoassimilate remobilization and included 14-3-3 proteins, cysteine-rich receptor-like protein kinase, monodehydroascorbate reductase, ascorbate peroxidase, glutathione S-transferases, translationally controlled tumor protein, remorin C-terminal domain containing protein, protein disulfide isomerase, DnaK family protein, cysteine synthase, aminotransferase, phosphoglycerate mutase, pyruvate phosphate dikinase, ATP synthase, and abscisic acid stress ripening (ASR1). The differential expression ratio of the signaling, redox, and defense group proteins was almost the same under MD and MW. ABA signaling, amino acid synthesis, and N remobilization were upregulated under MD, and the enzymes involved in carbohydrate, energy, and transportation metabolism were upregulated under MW. In conclusion, at the rice grain-filling stage, AWD is a potential technique to trigger signaling and the enzymatic protein network for systematic senescence initiation, root enlargement for maximum nutrient uptake, and maximize photoassimilate remobilization for yield enhancement.

     

Abscisic acid and cytokinins in the root exudates and leaves and their relationship to senescence and remobilization of carbon reserves in rice subjected to water stress during grain filling

The possible regulation of senescence-initiated remobilization of carbon reserves in rice (Oryza sativa L.) by abscisic acid (ABA) and cytokinins was studied using two rice cultivars with high lodging resistance and slow remobilization. The plants were grown in pots and either well-watered (WW, soil water potential = 0 MPa) or water-stressed (WS, soil water potential = -0.05 MPa) from 9 days after anthesis until they reached maturity. Leaf water potentials of both cultivars markedly decreased at midday as a result of water stress but completely recovered by early morning. Chlorophyll (Chl) and photosynthetic rate (Pr) of the flag leaves declined faster in WS plants than in WW plants, indicating that the water deficit enhanced senescence. Water stress accelerated starch remobilization in the stems, promoted the re-allocation of pre-fixed (14)C from the stems to grains, shortened the grain-filling period and increased the grain-filling rate. Sucrose phosphate synthase (SPS, EC 2.4.1.14) activity was enhanced by water stress and positively correlated with sucrose accumulation in both the stem and leaves. Water stress substantially increased ABA but reduced zeatin (Z) + zeatin riboside (ZR) concentrations in the root exudates and leaves. ABA significantly and negatively, while Z+ZR positively, correlated with Pr and Chl of the flag leaves. ABA, not Z+ZR, was positively and significantly correlated with SPS activity and remobilization of pre-stored carbon. Spraying ABA reduced Chl in the flag leaves, and enhanced SPS activity and remobilization of carbon reserves. Spraying kinetin had the opposite effect. The results suggest that both ABA and cytokinins are involved in controlling plant senescence, and an enhanced carbon remobilization is attributed to an elevated ABA level in rice plants subjected to water stress.     

Post‐flowering night respiration and altered sink activity account for high night temperature‐induced grain yield and quality loss in rice (Oryza sativa L.)

High night temperature (HNT) is a major constraint to sustaining global rice production under future climate. Physiological and biochemical mechanisms were elucidated for HNT‐induced grain yield and quality loss in rice. Contrasting rice cultivars (N22, tolerant; Gharib, susceptible; IR64, high yielding with superior grain quality) were tested under control (23°C) and HNT (29°C) using unique field‐based tents from panicle initiation till physiological maturity. HNT affected 1000 grain weight, grain yield, grain chalk and amylose content in Gharib and IR64. HNT increased night respiration (Rn) accounted for higher carbon losses during post‐flowering phase. Gharib and IR64 recorded 16 and 9% yield reduction with a 63 and 35% increase in average post‐flowering Rn under HNT, respectively. HNT altered sugar accumulation in the rachis and spikelets across the cultivars with Gharib and IR64 recording higher sugar accumulation in the rachis. HNT reduced panicle starch content in Gharib (22%) and IR64 (11%) at physiological maturity, but not in the tolerant N22. At the enzymatic level, HNT reduced sink strength with lower cell wall invertase and sucrose synthase activity in Gharib and IR64, which affected starch accumulation in the developing grain, thereby reducing grain weight and quality. Interestingly, N22 recorded lower Rn‐mediated carbon losses and minimum impact on sink strength under HNT. Mechanistic responses identified will facilitate crop models to precisely estimate HNT‐induced damage under future warming scenarios.     

Carbohydrate metabolism enzymes in CO2-enriched developing rice grains of cultivars varying in grain size

The increased supply of photosynthate from maternal tissue is known to promote grain growth in several crop species. However, the effect of increasing photosynthate supply on grain growth receives little attention in rice. This study was aimed at evaluating the effect of increasing photosynthate supply through CO₂ enrichment (650 μl I_-1) on grain growth in three rice cultivars differing in grain size. CO₂ enrichment was applied to the pot-grown plants between anthesis and final harvest. The results indicated that high CO₂ treatment enhanced the CO₂ exchange rate of leaf tissue, and subsequently increased the sucrose level of peduncle exudate, but it did not promote starch accumulation in the developing grains. This phenomenon was linked to the poor CO₂ responses for the grain activities of sucrose synthase, UDP-glucose pyrophosphorylase. ADP-glucose pyrophosphorylase, and starch synthases involved in the conversion of sucrose to starch. Significant cultivar differences also existed for the activities of sucrose to starch conversion enzymes with larger grain size cultivars tending to have higher enzymes activities (expressed on a grain basis), resulting in a greater carbohydrate accumulation.     

Involvement of abscisic acid and cytokinins in the senescence and remobilization of carbon reserves in wheat subjected to water stress during grain filling

This study investigated the possibility that abscisic acid (ABA) and cytokinins may mediate the effect of water deficit that enhances plant senescence and remobilization of pre‐stored carbon reserves. Two high lodging‐resistant wheat (Triticum aestivum L.) cultivars were field grown and treated with either a normal or high amount of nitrogen at heading. Well‐watered (WW) and water‐stressed (WS) treatments were imposed from 9 d post‐anthesis until maturity. Chlorophyll (Chl) and photosynthetic rate (Pr) of the flag leaves declined faster in WS plants than in WW plants, indicating that the water deficit enhanced senescence. Water stress facilitated the reduction of non‐structural carbohydrate in the stems and promoted the re‐allocation of prefixed ¹⁴C from the stems to grains, shortened the grain filling period and increased the grain filling rate. Water stress substantially increased ABA but reduced zeatin (Z) + zeatin riboside (ZR) concentrations in the stems and leaves. ABA correlated significantly and negatively, whereas Z + ZR correlated positively, with Pr and Chl of the flag leaves. ABA but not Z + ZR, was positively and significantly correlated with remobilization of pre‐stored carbon and grain filling rate. Exogenous ABA reduced Chl in the flag leaves, enhanced the remobilization, and increased grain filling rate. Spraying with kinetin had the opposite effect. The results suggest that both ABA and cytokinins are involved in controlling plant senescence, and an enhanced carbon remobilization and accelerated grain filling rate are attributed to an elevated ABA level in wheat plants when subjected to water stress.     

The use of comparative quantitative proteomics analysis in rice grain-filling in determining response to moderate soil drying stress

Moderate soil drying (MSD) stress at the grain filling stage can improve grain filling efficiently and thus increase grain yield. To elucidate the molecular response of grain filling to MSD stress, a labeling LC-based quantitative proteomics approach using tandem mass tags was applied to determine the changes in leaf and grain protein abundance level at 15 days after flowering. A total of 2109 leaf proteins and 3220 grain proteins were detected, and 251 leaf proteins and 220 grain proteins were differentially expressed under MSD stress. Based on MapMan ontology, differentially expressed proteins in leaf and grain were categorized within 22 and 18 functional categories, respectively. The patterns observed were interesting in that in some categories such as photosynthesis-related protein in leaf and cell division related proteins in grain showed higher expression abundant under MSD stress, which facilities increasing the source supply and sink size. In other categories, such as carbohydrate metabolism and mitochondrial electron transport, surprisingly showed a completely different expression pattern between leaf and grain under MSD stress, which led to faster and better remobilization of carbon from leaf to grain. Additionally, the complicated functional network including the small GTP-binding proteins, calmodulin, and 14-3-3 proteins play an important role in regulation carbon remobilization mediated by the stressful signals from soil after rice plants were treated with MSD at grain-filling stage. The findings provide theoretical evidence for better quality control and scientific improvement of rice in practice.     

不同水分管理下优质稻花后植株碳氮流转与籽粒生长及品质的相关性

以桂华占、八桂香为材料,在干湿交替灌溉、亏缺灌溉、淹水灌溉3种水分条件下,研究优质稻花后植株碳氮流转与籽粒生长及品质的相关性。结果表明:不同水分管理下,桂华占和八桂香花后碳氮流转与籽粒的生长间存在密切相关。主要表现在:(1)茎鞘和叶片干物质转运对籽粒干物质积累的贡献率为16.86%～25.68%,花后茎叶干物质运转速度和运转率与籽粒起始灌浆势呈显著甚至极显著正相关;籽粒最大灌浆速率、活跃灌浆期、持续灌浆时间与叶片干物质运转速度和运转率呈极显著正相关,与茎鞘干物质运转速度和运转率呈极显著负相关;(2)茎鞘碳同化物转运对籽粒的产量和淀粉产量的贡献率则为干湿交替灌溉>亏缺灌溉>淹水灌溉;但叶片碳同化物转运对籽粒的产量和淀粉产量的贡献率则为淹水灌溉>亏缺灌溉>干湿交替灌溉;茎叶可溶性糖积累量的减少和籽粒直链淀粉含量和积累量增加是同步的,且茎叶可溶性糖积累量快速递减期(花后3～12d)与直链淀粉含量和积累量快速递增期(花后6～12d)同步;(3)茎鞘和叶片氮素转运对籽粒氮素积累的贡献率为44.05%～117.66%,叶片总氮转运对籽粒氮素积累的贡献率大于茎鞘,茎鞘和叶片氮同化物对籽粒氮素的贡献率以淹水灌溉处理的最大,亏缺灌溉处理的次之,干湿交替灌溉处理的最小。     

Carbon footprint of rice production under biochar amendment – a case study in a Chinese rice cropping system

As a controversial strategy to mitigate global warming, biochar application into soil highlights the need for life cycle assessment before large‐scale practice. This study focused on the effect of biochar on carbon footprint of rice production. A field experiment was performed with three treatments: no residue amendment (Control), 6 t ha^?1 yr^?1 corn straw (CS) amendment, and 2.4 t ha^?1 yr^?1 corn straw‐derived biochar amendment (CBC). Carbon footprint was calculated by considering carbon source processes (pyrolysis energy cost, fertilizer and pesticide input, farmwork, and soil greenhouse gas emissions) and carbon sink processes (soil carbon increment and energy offset from pyrolytic gas). On average over three consecutive rice‐growing cycles from year 2011 to 2013, the CS treatment had a much higher carbon intensity of rice (0.68 kg CO₂‐C equivalent (CO₂‐C_e) kg^?1 grain) than that of Control (0.24 kg CO₂‐C_e kg^?1 grain), resulting from large soil CH₄ emissions. Biochar amendment significantly increased soil carbon pool and showed no significant effect on soil total N₂O and CH₄ emissions relative to Control; however, due to a variation in net electric energy input of biochar production based on different pyrolysis settings, carbon intensity of rice under CBC treatment ranged from 0.04 to 0.44 kg CO₂‐C_e kg^?1 grain. The results indicated that biochar strategy had the potential to significantly reduce the carbon footprint of crop production, but the energy‐efficient pyrolysis technique does matter.     

Involvement of abscisic acid and ethylene in the responses of rice grains to water stress during filling

This study was to test the hypothesis that the interaction between abscisic acid (ABA) and ethylene may be involved in mediating the effects of water stress on grain filling. Two high lodging‐resistant rice (Oryza sativa L.) cultivars were pot‐grown. Three treatments, well‐watered, moderate water‐stressed (MD), and severe water‐stressed (SD), were imposed from 9 d post‐anthesis until maturity. Grain filling rate and grain weight were significantly increased under MD but decreased under SD. The two cultivars behaved the same. ABA concentration in the grains was very low during the grain filling stage, reaching a maximum when the grain filling rate was highest. Both the grain filling rate and ABA concentration were substantially enhanced by water stress. In contrast to ABA, concentrations of ethylene and 1‐aminocylopropane ‐1‐carboxylic acid (ACC) in the grains were very high at early grain filling stage and sharply decreased during the linear period of grain growth. MD reduced, whereas SD remarkably increased, their accumulation. The ratio of ABA to ACC was increased in MD grains but decreased in SD grains, indicating that there was a greater enhancement of ABA concentration than ethylene production in the MD treatment only. Application of cobalt ion (inhibitor of ethylene synthesis) or ABA at the early grain filling stage significantly increased grain filling rate. Spraying with ethephon (ethylene‐releasing agent) or fluridone (inhibitor of ABA synthesis) had the opposite effect. The results suggest that antagonistic interactions between ABA and ethylene mediate the grain filling rate, and a high ratio of ABA to ethylene enhances grain filling rate.     

结实期土壤水分亏缺影响水稻籽粒灌浆的生理原因

通过分析结实期土壤水分亏缺对水稻(Oryza sativa)籽粒中蔗糖向淀粉合成的生理代谢中关键酶活性及籽粒灌浆的调节作用, 探讨土壤水分亏缺影响水稻籽粒灌浆的生理机制。结果表明, 适度土壤水分亏缺诱导了灌浆高峰期(花后15-20天)水稻籽粒中蔗糖合成酶、腺苷二磷酶葡萄糖焦磷酸化酶、可溶性淀粉合成酶及淀粉分支酶活性的增加, 提高了籽粒灌浆中前期(花后10-20天)籽粒中淀粉积累速率和籽粒灌浆速率。但在灌浆后期(花后20-30天)籽粒中, 上述关键酶活性下降较快, 籽粒活跃灌浆期明显缩短, 灌浆前中期灌浆速率的增加不能完全补偿灌浆期缩短带来的同化物积累损失, 导致水分亏缺处理水稻籽粒充实不良, 结实率、籽粒重和产量显著降低。研究认为, 灌浆期土壤水分亏缺引起的灌浆后期籽粒中蔗糖向淀粉合成代谢中一些关键酶活性快速下降和籽粒内容物的供应不足是籽粒淀粉积累总量减少、粒重降低的主要生理原因。     

Grain filling of cereals under soil drying

Monocarpic plants require the initiation of whole-plant senescence to remobilize and transfer assimilates pre-stored in vegetative tissues to grains. Delayed whole-plant senescence caused by either heavy use of nitrogen fertilizer or adoption of lodging-resistant cultivars/hybrids that remain green when the grains are due to ripen results in a low harvest index with much nonstructural carbohydrate (NSC) left in the straw. Usually, water stress during the grain-filling period induces early senescence, reduces photosynthesis, and shortens the grain-filling period; however, it increases the remobilization of NSC from the vegetative tissues to the grain. If mild soil drying is properly controlled during the later grain-filling period in rice (Oryza sativa) and wheat (Triticum aestivum), it can enhance whole-plant senescence, lead to faster and better remobilization of carbon from vegetative tissues to grains, and accelerate the grain-filling rate. In cases where plant senescence is unfavorably delayed, such as by heavy use of nitrogen and the introduction of hybrids with strong heterosis, the gain from the enhanced remobilization and accelerated grain-filling rate can outweigh the loss of reduced photosynthesis and the shortened grain-filling period, leading to an increased grain yield, better harvest index and higher water-use efficiency.     

Arbuscular mycorrhiza influences carbon‐use efficiency and grain yield of wheat grown under pre‐ and post‐anthesis salinity stress

• Soil salinity severely affects and constrains crop production worldwide. Salinity causes osmotic and ionic stress, inhibiting gas exchange and photosynthesis, ultimately impairing plant growth and development. Arbuscular mycorrhiza (AM) have been shown to maintain light and carbon use efficiency under stress, possibly providing a tool to improve salinity tolerance of the host plants. Thus, it was hypothesized that AM will contribute to improved growth and yield under stress conditions.
• Wheat plants (Triticum aestivum L.) were grown with (AMF+) or without (AMF?) arbuscular mycorrhizal fungi (AMF) inoculation. Plants were subjected to salinity stress (200 mm NaCl) either at pre‐ or post‐anthesis or at both stages. Growth and yield components, leaf chlorophyll content as well as gas exchange parameters and AMF colonization were analysed.
• AM plants exhibited a higher rate of net photosynthesis and stomatal conductance and lower intrinsic water use efficiency. Furthermore, AM wheat plants subjected to salinity stress at both pre‐anthesis and post‐anthesis maintained higher grain yield than non‐AM salinity‐stressed plants.
• These results suggest that AMF inoculation mitigates the negative effects of salinity stress by influencing carbon use efficiency and maintaining higher grain yield under stress.

     

‘Last In–First Out’: seasonal variations of non‐structural carbohydrates,glucose‐6‐phosphate and ATP in tubers of two Arum species

• Knowledge on the metabolism of polysaccharide reserves in wild species is still scarce. In natural sites we collected tubers of Arum italicum Mill. and A. maculatum L. – two geophytes with different apparent phenological timing, ecology and chorology – during five stages of the annual cycle in order to understand patterns of reserve accumulation and degradation.
• Both the entire tuber and its proximal and distal to shoot portion were utilised. Pools of non‐structural carbohydrates (glucose, sucrose and starch), glucose‐6‐phosphate and ATP were analysed as important markers of carbohydrate metabolism.
• In both species, starch and glucose content of the whole tuber significantly increased from sprouting to the maturation/senescence stages, whereas sucrose showed an opposite trend; ATP and glucose‐6‐phosphate were almost stable and dropped only at the end of the annual cycle. Considering the two different portions of the tuber, both ATP and glucose‐6‐phosphate concentrations were higher in proximity to the shoot in all seasonal stages, except the flowering stage.
• Our findings suggest that seasonal carbon partitioning in the underground organ is driven by phenology and occurs independently of seasonal climate conditions. Moreover, our results show that starch degradation, sustained by elevated ATP and glucose‐6‐phosphate pools, starts in the peripheral, proximal‐to‐shoot portion of the tuber, consuming starch accumulated in the previous season, as a ‘Last In–First Out’ mechanism of carbohydrate storage.