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.     

The 14‐3‐3 protein GF14f negatively affects grain filling of inferior spikelets of rice (Oryza sativa L.)

In rice (Oryza sativa L.), later flowering inferior spikelets (IS), which are located on proximal secondary branches, fill slowly and produce smaller and lighter grains than earlier flowering superior spikelets (SS). Many genes have been reported to be involved in poor grain filling of IS, however the underlying molecular mechanisms remain unclear. The present study determined that GF14f, a member of the 14‐3‐3 protein family, showed temporal and spatial differences in expression patterns between SS and IS. Using GF14f–RNAi plants, we observed that a reduction in GF14f expression in the endosperm resulted in a significant increase in both grain length and weight, which in turn improved grain yield. Furthermore, pull‐down assays indicated that GF14f interacts with enzymes that are involved in sucrose breakdown, starch synthesis, tricarboxylic acid (TCA) cycle and glycolysis. At the same time, an increase in the activity of sucrose synthase (SuSase), adenosine diphosphate‐glucose pyrophosphorylase (AGPase), and starch synthase (StSase) was observed in the GF14f–RNAi grains. Comprehensive analysis of the proteome and metabolite profiling revealed that the abundance of proteins related to the TCA cycle, and glycolysis increased in the GF14f–RNAi grains together with several carbohydrate intermediates. These results suggested that GF14f negatively affected grain development and filling, and the observed higher abundance of the GF14f protein in IS compared with SS may be responsible for poor IS grain filling. The study provides insights into the molecular mechanisms underlying poor grain filling of IS and suggests that GF14f could serve as a potential tool for improving rice grain filling.     

Short-term effects of experimental warming and enhanced ultraviolet-B radiation on photosynthesis and antioxidant defense of <Emphasis Type="Italic">Picea asperata</Emphasis> seedlings

It is generally accepted that sucrose synthase (SuSy), ADP-glucose pyrophosphorylase (AGPase), soluble starch synthase (SSS), granule-bound starch synthases (GBSS) and starch branching enzyme (SBE) play a key role in starch synthesis in wheat grains. Starch synthesis in wheat grains is influenced by genotype and environment. However, what is not known is the degree of variation in enzyme activity during starch accumulation of wheat cultivars differing in kernel types. The present study was carried out to characterize the changing activities of key enzymes during grain filling in two kernel type winter wheat cultivars. Results showed that starch accumulation rate (SAR) and activities of SuSy, AGPase, SSS, GBSS and SBE in large kernel types were significantly higher than those in small kernel types. The soil water deficit experienced during the course of the experiment led to an increase at early grain-filling period and decrease during late grain-filling, respectively, in SAR and activities of key enzymes involved in starch synthesis, especially SuSy, AGPase, SSS, and SBE. Water deficit enhanced grain starch accumulation in small kernel types. It suggests that rainfed treatment increase physiological activities during early grain-filling and promote starch accumulation in small kernel types. The simulation with Richards’ equation showed that it was accumulation duration and SAR that determined the starch accumulation in large kernel types. Compared with small kernel types, plants of large kernel types maintained longer filling duration, higher SAR and greater activities of related enzymes during mid and late grain-filling. These observations suggest stronger sink activities in large kernel types at a later stage of development. Consequently, large kernel types have advantages over the small kernel types in terms of the amount of starch accumulation at mid and late stage, but are sensitive to water deficit.     

Post-anthesis development of inferior and superior spikelets in rice in relation to abscisic acid and ethylene

The purpose of this study was to test the hypothesis that the interaction between abscisic acid (ABA) and ethylene may be involved in mediating the post-anthesis development of spikelets in rice (Oryza sativa L.). Two rice genotypes were field-grown, and the changes of ABA, ethylene, and 1-aminocylopropane-1-carboxylic acid (ACC) levels in spikelets during grain filling and their relationships with endosperm-division and grain-filling rates were investigated. The results showed that earlier-flowering superior spikelets exerted dominance over later-flowering inferior spikelets in endosperm cell-division and grain-filling rates. The two genotypes behaved the same. Later-flowering spikelets had higher levels of ethylene and ACC than earlier-flowering spikelets. The ethylene evolution rate was significantly and negatively correlated with the cell division and grain filling rates. By contrast to ethylene, later-flowering spikelets contained a lower ABA content/concentration and showed a low content ratio of ABA to ACC than earlier-flowering ones. The cell-division and grain-filling rates were significantly and positively correlated with both ABA contents and the ratio of ABA to ACC. Application of cobalt ion (inhibitor of ethylene synthesis) or ABA at an early grain-filling stage significantly increased endosperm cell division rate and cell number, grain-filling rate, and grain weight of inferior spikelets. Application of ethephon (an ethylene-releasing agent) or fluridone (an inhibitor of carotenoid synthesis) had the opposite effect. The results suggest that antagonistic interactions between ABA and ethylene mediate endosperm cell-division and grain-filling in rice. A higher ratio of ABA to ethylene in rice spikelets is required to maintain a faster grain-filling rate.     

Increasing the abscisic acid level in maize grains induces precocious maturation by accelerating grain filling and dehydration

Elevated levels of abscisic acid (ABA) are closely associated with cereal grain filling under water deficit. However, grain dehydration during grain filling has received little attention. In this paper, three experiments with drought stress and exogenous ABA treatments were conducted to investigate the relationship between ABA and grain dehydration in maize (Zea mays L.) during the grain-filling period. The results indicated that exogenous ABA application and drought stress led to the same tendency of the grain ABA concentration, carbohydrate concentration and dehydration rate to increase but the moisture content to decrease. Moreover, the time to reach the maximum grain-filling rate was advanced, and the grain-filling period was shortened. In in vitro culture experiments, the sucrose-to-starch conversion was promoted, mainly influenced by sucrose synthase, ADP-glucose pyrophosphorylase (AGPase), and soluble starch synthase during the middle grain-filling stage, and the improvement in starch synthesis was possibly induced by AGPase. Correlation analysis showed that the ABA level was significantly negatively correlated with the moisture content and positively correlated with the starch level. A close and notably negative correlation was observed between the grain moisture content and starch level. In summary, adequate grain ABA promoted sucrose-to-starch conversion, shortened the duration of grain filling and accelerated grain dehydration, resulting in precocious grain maturation.     

Effects of the activities of key enzymes involved in starch biosynthesis on the fine structure of amylopectin in developing rice (Oryza sativa L.) endosperms

The dynamic changes of the activities of enzymes involving in starch biosynthesis, including ADP-glucose pyrophosphorylase (AGPase), soluble starch synthases (SSS), starch branching enzyme (SBE) and starch debranching enzymes (DBE) were studied, and changes of fine structure of amy- lopectin were characterized by isoamylase treatment during rice grain development, using trans anti-waxy gene rice plants. The relationships between the activities of those key enzymes were also analyzed. The amylose synthesis was significantly inhibited in transgenic Wanjing 9522, but the total starch content and final grain weight were less affected as compared with those of non-transgenic Wanjing 9522 rice cultivar. Analyses on the changes of activities of enzymes involving in starch bio- synthesis showed that different enzyme activities were expressed differently during rice endosperm development. Soluble starch synthase is relatively highly expressed in earlier stage of endosperm de- velopment, whilst maximal expression of granule-bound starch synthase (GBSS) occurred in mid-stage of endosperm development. No obvious differences in changes of the activities of AGPase and SBE between two rice cultivars investigated, except the DBEs. Distribution patterns of branches of amy- lopectin changed continually during the development of rice grains and varied between two rice culti- vars. It was suggested that amylopectin synthesis be prior to the synthesis of amylose and different enzymes have different roles in controlling syntheses of branches of amylopectin.     

Hormones in the grains and roots in relation to post-anthesis development of inferior and superior spikelets in japonica/indica hybrid rice

Grain filling is usually not adequate in later-flowering inferior spikelets in japonica/indica (J/I) hybrid rice (Oryza sativa) although it shows stronger hybrid vigor than indica/indica (I/I) hybrid. This study investigated the potential causes by examining changes in zeatin (Z) + zeatin riboside (ZR), indole-3-acetic acid (IAA), gibberellins (GAs, GA₁ + GA₄), and abscisic acid (ABA) in spikelets and roots during the grain filling period. The inferior spikelets of J/I hybrid exhibited low rate of endosperm cell division and slow grain filling. During the early grain filing period, they had less Z + ZR, IAA, and ABA, but more GAs, than the earlier-flowering superior spikelets. If compared to the inferior spikelets of the I/I hybrid, the J/I inferior spikelets also had less Z + ZR, IAA, and ABA. Rates of endosperm cell division and grain filling were positively and significantly correlated with Z + ZR and ABA contents in both grains and roots or IAA in grains, whereas not significantly correlated with GAs either in grains or roots or IAA in roots. Applications of kinetin, IAA, or ABA to spikelets, or kinetin and ABA to roots, enhanced cell division and grain filling in the inferior spikelets. Results suggest that low contents of cytokinins and ABA in both grains and roots and low contents of IAA in grains may result in the poor filling of inferior spikelets in the J/I hybrid.     

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.     

Gene expression of the biosynthetic enzymes and biosynthesis of starch during Rice Grain development

Amylose and amylopectin are determinants of the physicochemical properties for starch and grain quality in rice. Their biosynthesis is catalyzed by the interplay of ADP-glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), soluble starch synthase (SSS), a starch branching enzyme (SBE), and a starch debranching enzyme (SDE). In this study, the genes for these enzymes were highly expressed 7 to 28 days after flowering during grain development, and their expression closely matched increases in both starch content and grain weight Among all the tested cultivars, amylose contents in the rice grains remained essentially constant throughout their development The AGPase gene was highly expressed in the high-yield cultivars of both glutinous and non-glutinous rice. The SSS gene was actively expressed when mature GBSS mRNA decreased. Genes responsible for amylopectin biosynthesis were simultaneously expressed in the late stage of grain development. We have now demonstrated that the expression patterns of starch biosynthetic genes differ between glutinous and non-glutinous rice, and between Tongil (a Japonica/ Indica hybrid) and Japonica types.     

不同类型玉米发育籽粒中淀粉合成及相关酶活性比较

以普通玉米、爆裂玉米、甜玉米和糯玉米为试材,分析和比较不同类型的玉米品种之间籽粒发育过程中淀粉合成及相关酶活性的变化。结果表明,淀粉合成速率和蔗糖合成酶(SS)、可溶性淀粉合成酶(SSS)、束缚态淀粉合成酶(GBSS)、淀粉分支酶(SBE)、去分支酶(DBE)活性都呈单峰曲线变化。30～40 DAP,普通玉米的SS活性显著高于其他3种类型;类型间平均和最大SSS活性水平的顺序为普通玉米>糯玉米>爆裂玉米>甜玉米;30～40 DAP,普通玉米GBSS活性最高,糯玉米GBSS活性最低;20～40 DAP,糯玉米SBE活性最高;甜玉米的DBE活性很低,并且在40 DAP完全丧失。淀粉合成速率与SS、SSS、GBSS和SBE活性相关程度比较高,与腺苷二磷酸葡萄糖焦磷酸化酶(AGP酶)和DBE活性相关不显著。推测AGP酶虽然为淀粉合成提供直接前体ADPG,但可能SS活性过低致使其限速作用比AGP酶的还强,AGP酶潜在的限速作用无法表现,SS成为玉米籽粒淀粉合成的限速因子。GBSS对直链淀粉积累起重要的促进作用;SSS和SBE对支链淀粉积累起重要的促进作用。     

Involvement of polyamines in the post-anthesis development of inferior and superior spikelets in rice

Early-flowered superior spikelets usually exhibit a faster grain filling rate and heavier grain weight than late-flowered inferior spikelets in rice (Oryza sativa L.). But the intrinsic factors responsible for the variations between the two types of spikelets are unclear. This study investigated whether and how polyamines (PAs) are involved in regulating post-anthesis development of rice spikelets. Six rice genotypes differing in grain filling rate were field grown, and PA levels and activities of the enzymes involved in PA biosynthesis were measured in both superior and inferior spikelets. The results showed that superior spikelets exhibited higher levels of free spermidine (Spd) and free spermine (Spm) and higher activities of arginine decarboxylase (ADC, EC 4.1.1.19), S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) and Spd synthase (EC 2.5.1.16) than inferior spikelets at the early endosperm cell division and grain filling stage. The maximum concentrations of free Spd and free Spm and the maximum activities of ADC, SAMDC and Spd synthase were significantly correlated with the maximum cell division and grain filling rates, maximum cell number and grain weight. Application of Spd and Spm to panicles resulted in significantly higher rates of endosperm cell division and grain filling in inferior spikelets along with the activities of sucrose synthase (EC 2.4.1.13), ADP glucose pyrophosphorylase (EC 2.7.7.27) and soluble starch synthase (EC 2.4.1.21), suggesting that these PAs are involved in the sucrose-starch metabolic pathway. The results indicate that the poor development of inferior spikelets is attributed, at least partly, to the low PA level and its low biosynthetic activity.     

秸秆还田对旱作马铃薯块茎淀粉合成关键酶活性及基因表达的影响

为探讨秸秆还田下旱作马铃薯块茎形成过程中淀粉合成关键酶活性及基因表达特性,以马铃薯栽培品种"青薯9号"为材料,以露地栽培为对照,设置秸秆还田处理,研究了马铃薯块茎形成过程中淀粉合成关键酶活性、基因表达、淀粉糊化及累积指标。结果表明:秸秆还田显著提高了旱作马铃薯SSS酶活性,降低了AGPP、GBSS酶活性,而对SBE酶活性没有显著影响;显著提高了SSⅡ、SSⅢ基因表达量,降低了AGPase、GBSSⅠ、SBEⅠ、SBEⅡ基因表达量;显著增加了淀粉崩解值,减少了淀粉各阶段粘度、回生值,而对淀粉糊化温度没有显著影响;显著增加了直链淀粉含量及直/支链淀粉比,减少了总淀粉含量; GBSS酶活性与AGPase、SBEⅠ基因表达量呈显著正相关,与直链淀粉含量、直/支链淀粉比呈显著负相关; SBE酶活性与SSⅡ基因表达量、峰值粘度、低谷粘度、最终粘度、总淀粉含量呈显著正相关,与崩解值、糊化温度呈显著负相关; AGPase基因表达量与直链淀粉含量呈显著负相关;GBSSⅠ基因表达量与最终粘度、回生值呈显著正相关,与糊化温度呈显著负相关;淀粉糊化与累积无显著相关性。     

Regulation of leaf senescence, grain-filling and yield of rice by kinetin and abscisic acid

Senescence of rice ( Oryza sativa L. cv. Jaya) leaves was regulated with kinetin and abscisic acid (ABA) sprays at the reproductive stage. The effect of such sprays on grain-filling and yield was analyzed. Spraying 100-day-old plants with kinetin solution (100 μg ml^-1) significantly delayed senescence as indicated by higher total chlorophyll and protein levels in the three uppermost leaves compared with the controls. In contrast, spraying with ABA (15 μg ml^-1) significantly promoted foliar senescence. The number of spikelets per panicle, number of panicles, percentage filled grains, panicle weight and grain yield per plant and the mobilization and harvest indices were significantly increased by kinetin treatment, while ABA decreased most of them. The possibility of increased grain-filling and thus, yield due to delayed foliar senescence by kinetin treatment and decreased grain-filling due to hastening of senescence by ABA is discussed.     

Hormones in the grains in relation to sink strength and postanthesis development of spikelets in rice

Inferior spikelets usually exhibit a slower grain filling rate and lower grain weight than superior spikelets in a rice (Oryza sativa L.) panicle. This study investigated whether the variations in grain filling between the two kinds of spikelets were attributed to their sink strength and whether the sink strength was regulated by the hormonal levels in the grains. Using two field-grown rice genotypes, the division rate of endosperm cells, hormonal levels in the grains, and grain weight of both superior and inferior spikelets were determined during the grain filling period. The results showed that superior spikelets had dominance over inferior spikelets in endosperm cell division rate and cell number, grain filling and grain weight. Changes in zeatin (Z) and zeatin riboside (ZR) contents paralleled and were very significantly correlated with the cell division rate and cell number. Cell division rate and the content of indole-3-acetic acid (IAA) in the grains were also significantly correlated. Gibberellin (GAs; GA₁+ GA₄) content of the grains was high but ABA levels were low at the early grain filling stage. ABA increased substantially during the linear phase of grain growth and was very significantly correlated with grain dry weight during this period. Application of kinetin at 2 through 6 days post anthesis (DPA) significantly increased cell number, while spraying ABA at 11 through 15 DPA significantly increased the grain filling rate. The results suggest that differences in sink strength are responsible for variations in grain filling between superior and inferior spikelets. Both cytokinins and IAA in the grains may mediate cell division in rice endosperm at early grain filling stages, and therefore regulate the sink size of the grain, whereas ABA content correlates with sink activity during the linear period of grain growth.     

氮素水平对冬小麦籽粒灌浆过程中淀粉合成关键酶活性的影响

小麦籽粒灌浆过程中,淀粉合成关键酶腺苷二磷酸葡萄糖焦磷酸化酶(ADPG-PPase)、可溶性淀粉合成酶(SSS)、淀粉分支酶(SBE)和束缚态淀粉合成酶(GBSS)均随着灌浆进程呈单峰曲线变化,峰值出现在花后25d;不同氮肥施用量对灌浆前期酶活性的影响较小,而在花后20d之后影响较大;随着氮肥施用量的增加,4种酶活性均呈增加趋势,但氮肥过量时酶活性下降,表明适当增加施氮量有利于淀粉合成关键酶活性的提高。     

重穗型杂交水稻籽粒灌浆过程中强势和弱势颖花内源IAA、ABA和GA水平的动态状况

研究了重穗型杂交水稻培矮 6 4s/E3 2的灌浆过程和强、弱势颖花中内源IAA、ABA和GA1 GA3水平的动态状况。籽粒发育过程中不同内源激素水平高低依次为 :IAA >GA1 GA3>ABA。IAA和ABA水平在强势颖花中较高而GA1 GA3水平在弱势颖花中较高。 3种激素水平的变化与谷粒增重速率之间均存在正相关 ,两个最高的相关系数值分别存在于单位鲜重样本的IAA含量(ng/gFW ) 与籽粒鲜重的增重速率之间 (r =0 .82 1 8 )和单个籽粒IAA含量 (ng/grain)与籽粒干重的增重速率之间 (r =0 .8485 )。推测启动和维持籽粒灌浆过程可能需要较高的IAA水平 ;ABA可能具有促进籽粒中同化物的累积和种子成熟的作用 ;GA1 GA3可能具有保持弱势颖花活性的特殊作用     

重穗型杂交水稻籽粒灌浆过程中强势和强势颖花内源IAA、ABA和GA水平的动态状况

研究了重穗型杂交水稻培矮 6 4s/E3 2的灌浆过程和强、弱势颖花中内源IAA、ABA和GA1 GA3水平的动态状况。籽粒发育过程中不同内源激素水平高低依次为 :IAA >GA1 GA3>ABA。IAA和ABA水平在强势颖花中较高而GA1 GA3水平在弱势颖花中较高。 3种激素水平的变化与谷粒增重速率之间均存在正相关 ,两个最高的相关系数值分别存在于单位鲜重样本的IAA含量(ng/gFW ) 与籽粒鲜重的增重速率之间 (r =0 .82 1 8 )和单个籽粒IAA含量 (ng/grain)与籽粒干重的增重速率之间 (r =0 .8485 )。推测启动和维持籽粒灌浆过程可能需要较高的IAA水平 ;ABA可能具有促进籽粒中同化物的累积和种子成熟的作用 ;GA1 GA3可能具有保持弱势颖花活性的特殊作用     

Effects of exogenous ABA and cytokinin on leaf photosynthesis and grain protein accumulation in wheat ears cultured <Emphasis Type="Italic">in vitro</Emphasis>

A detached culture system and steady-state ¹⁵N labeling technique were used to study the effects of exogenous ABA and ZR on photosynthetic characteristics, nitrogen remobilization and the activities of key enzymes for nitrogen metabolism in detached wheat parts during grain protein accumulation. The differences in net photosynthetic rate, chlorophyll content (SPAD value) and soluble protein content in the flag leaves of detached culture system between the treatments of ABA and ZR showed that ABA facilitates the post-anthesis senescence course compared to the ZR treatment. The differences in the changes of ¹⁵N amount in different organs in the detached culture system between the ABA and ZR treatments showed that nitrogen remobilization from vegetative organs to the grain is accelerated by the ABA treatment but is delayed by ZR. The activities of GS and GPT in grains treated with ABA were significantly higher than those with the control treatment at 5 DAC, but reduced significantly compared with control at 11 DAC. The two enzyme activities in grains were reduced significantly by ZR at 5 DAC and increased significantly at 11 DAC, compared with those treated with ABA. The above changes of enzyme activity showed that the ABA treatment hastens amino acid conversion into grains and protein accumulation in grains, whereas the ZR treatment delays these processes. A significant reduction in grain weight with ABA treatment is associated with the reduction of net photosynthesis, chlorophyll content, and soluble protein content in flag leaves. Compared with the control and ZR treatments, a significant increase in grain protein content with the ABA treatment may result from the accelerating effects of ABA on N remobilization, amino acid conversion into grains and protein accumulation in grains.