Proteomic analysis of positive influence of alternate wetting and moderate soil drying on the process of rice grain filling

Rice (Oryza sativa L.) is one of the most important crops in the world to feed ever increasing world population. An increase in output of rice crop per unit is imminent. Alternate wetting and moderate soil drying (AWD) irrigation technology has been recommended as a good practice method to improve grain filling of rice crop at late growing stages. Physiological, molecular and agronomic parameters were adopted to elucidate the role of rice stem and sheath under AWD treatment as compared to the conventional irrigation during the grain filling stage. AWD treatment significantly increased stem and sheath dry weight, carbohydrate reserves and their remobilization to the grain, especially inferior spikelet grains. The results showed that the transport and conversion rate of the stem and sheath photoassimilates increased by 9.87 and 8.37%, respectively. Furthermore, protein expression profiles of the stem and sheath at 10, 20 and 30 days after flowering were analyzed. We examined 220 differentially expressed proteins, and successfully identified 166 proteins, including 71 proteins in the stem and 95 proteins in the sheath involved in thirteen important functional groups. Our results suggest that the AWD treatment at the rice grain filling stage is highly conducive to trigger the mobilization of the N assimilates from leave and root to the stem and sheath, and then promotes to remobilize the reserves to the grain through coordinately expressed proteins involved in photosynthesis, systematic senescence, oxidative stress defense, signal transduction and other metabolisms. This study reveals the metabolic mechanism of the stem and sheath in response to AWD at grain filling stage, and provides theoretical evidence for better quality control and scientific improvement of rice in practice.     

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.     

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。     

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.

     

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。     

Physiological mechanisms contributing to the increased water-use efficiency in winter wheat under deficit irrigation

Deficit irrigation in winter wheat has been practiced in the areas with limited irrigation water resources. The objectives of this study were to (i) understand the physiological basis for determinations of grain yield and water-use efficiency in grain yield (WUE) under deficit irrigation; and (ii) investigate the effect of deficit irrigation on dry matter accumulation and remobilization of pre-anthesis carbon reserves during grain filling. A field experiment was conducted in the Southern High Plains of the USA and winter wheat (cv. TAM 202) was grown on Pullman clay loam soil (fine mixed thermic Torretic Paleustoll). Treatments consisted of rain-fed, deficit irrigation from jointing to the middle of grain filling, and full irrigation. The physiological measurements included leaf water potential, net photosynthetic rate (Pn), stomatal conductance (Gs), and leaf area index. The rain-fed treatment had the lowest seasonal evapotranspiration (ET), biomass, grain yield, harvest index (HI) and WUE as a result of moderate to severe water stress from jointing to grain filling. Irrigation application increased seasonal ET, and ET increased as irrigation frequency increased. The seasonal ET increased 20% in one-irrigation treatments between jointing and anthesis, 32-46% in two-irrigation treatments, and 67% in three- and full irrigation treatments. Plant biomass, grain yield, HI and WUE increased as the result of increased ET. The increased yield under irrigation was mainly contributed by the increased number of spikes, and seeds per square meter and per spike. Among the irrigation treatments, grain yield increased significantly but the WUE increased slightly as irrigation frequency increased. The increased WUE under deficit irrigation was contributed by increased HI. Water stress during grain filling reduced Pn and Gs, and accelerated leaf senescence. However, the water stress during grain filling induced remobilization of pre-anthesis carbon reserves to grains, and the remobilization of pre-anthesis carbon reserves significantly contributed to the increased grain yield and HI. The results of this study showed that deficit irrigation between jointing and anthesis significantly increased wheat yield and WUE through increasing both current photosynthesis and the remobilization of pre-anthesis carbon reserves.     

Dynamic proteomic analysis reveals a switch between central carbon metabolism and alcoholic fermentation in rice filling grains

Accumulation of reserve materials in filling grains involves the coordination of different metabolic and cellular processes, and understanding the molecular mechanisms underlying the interconnections remains a major challenge for proteomics. Rice (Oryza sativa) is an excellent model for studying grain filling because of its importance as a staple food and the available genome sequence database. Our observations showed that embryo differentiation and endosperm cellularization in developing rice seeds were completed approximately 6 d after flowering (DAF); thereafter, the immature seeds mainly underwent cell enlargement and reached the size of mature seeds at 12 DAF. Grain filling began at 6 DAF and lasted until 20 DAF. Dynamic proteomic analyses revealed 396 protein spots differentially expressed throughout eight sequential developmental stages from 6 to 20 DAF and determined 345 identities. These proteins were involved in different cellular and metabolic processes with a prominently functional skew toward metabolism (45%) and protein synthesis/destination (20%). Expression analyses of protein groups associated with different functional categories/subcategories showed that substantially up-regulated proteins were involved in starch synthesis and alcoholic fermentation, whereas the down-regulated proteins in the process were involved in central carbon metabolism and most of the other functional categories/subcategories such as cell growth/division, protein synthesis, proteolysis, and signal transduction. The coordinated changes were consistent with the transition from cell growth and differentiation to starch synthesis and clearly indicated that a switch from central carbon metabolism to alcoholic fermentation may be important for starch synthesis and accumulation in the developmental process.     

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.     

Comparative proteomic analysis of early salt stress responsive proteins in roots and leaves of rice

Growth and productivity of rice (Oryza sativa L.) are severely affected by salinity. Understanding the mechanisms that protect rice and other important cereal crops from salt stress will help in the development of salt‐stress‐tolerant strains. In this study, rice seedlings of the same genetic species with various salt tolerances were studied. We first used 2DE to resolve the expressed proteome in rice roots and leaves and then used nanospray liquid chromatography/tandem mass spectrometry to identify the differentially expressed proteins in rice seedlings after salt treatment. The 2DE assays revealed that there were 104 differentially expressed protein spots in rice roots and 59 in leaves. Then, we identified 83 proteins in rice roots and 61 proteins in rice leaves by MS analysis. Functional classification analysis revealed that the differentially expressed proteins from roots could be classified into 18 functional categories while those from leaves could be classified into 11 functional categories. The proteins from rice seedlings that most significantly contributed to a protective effect against increased salinity were cysteine synthase, adenosine triphosphate synthase, quercetin 3‐O‐methyltransferase 1, and lipoxygenase 2. Further analysis demonstrated that the primary mechanisms underlying the ability of rice seedlings to tolerate salt stress were glycolysis, purine metabolism, and photosynthesis. Thus, we suggest that differentially expressed proteins may serve as marker group for the salt tolerance of rice.     

Landrace of japonica rice,Akamai exhibits enhanced root growth and efficient leaf phosphorus remobilization in response to limited phosphorus availability

Aims

Phosphorus (P) acquisition through extensive root growth and P allocation to different plant organs through efficient remobilization are important for acclimation of crop plants to P-limited environments. This study elucidated changes in rice root growth and leaf P-remobilization and their influence on grain yield under P deficiency.

Methods

Two pot experiments were conducted with (P100) and without (P0) inorganic P supply using two Japanese rice cultivars: Akamai (Yamagata) and Koshihikari. Multiple harvests were made until the panicle initiation stage. Root and shoot growth response, P acquisition, and temporal leaf P-remobilization efficiency were measured. A separate experiment ascertained the final yield and grain P status.

Results

The Akamai rice cultivar showed enhanced root growth and more acquired soil P. The Akamai root dry weight was 66% greater than that of Koshihikari under P0. Confronting P deficiency, Akamai remobilized some P from its lower mature leaves to upper younger leaves starting from early growth. The remobilized P fraction increased to 72% at panicle initiation under P0. Under P0, Akamai exhibited two-fold higher leaf P-remobilization efficiency than under P100.

Conclusions

Enhanced root growth that facilitates acquisition of more soil P through better soil exploration coupled with efficient leaf P remobilization from the early growth stage improves adaptation of Akamai rice cultivar to P-limited environments. Nevertheless, P-starvation responses did not facilitate higher grain yields in P-limited conditions.

     

Hormonal changes in the grains of rice subjected to water stress during grain filling

Lodging-resistant rice (Oryza sativa) cultivars usually show slow grain filling when nitrogen is applied in large amounts. This study investigated the possibility that a hormonal change may mediate the effect of water deficit that enhances whole plant senescence and speeds up grain filling. Two rice cultivars showing high lodging resistance and slow grain filling were field grown and applied with either normal or high amount nitrogen (HN) at heading. Well-watered and water-stressed (WS) treatments were imposed 9 days post anthesis to maturity. Results showed that WS increased partitioning of fixed (14)CO(2) into grains, accelerated the grain filling rate but shortened the grain filling period, whereas the HN did the opposite way. Cytokinin (zeatin + zeatin riboside) and indole-3-acetic acid contents in the grains transiently increased at early filling stage and WS treatments hastened their declines at the late grain filling stage. Gibberellins (GAs; GA(1) + GA(4)) in the grains were also high at early grain filling but HN enhanced, whereas WS substantially reduced, its accumulation. Opposite to GAs, abscisic acid (ABA) in the grains was low at early grain filling but WS remarkably enhanced its accumulation. The peak values of ABA were significantly correlated with the maximum grain filling rates (r = 0.92**, P < 0.01) and the partitioning of fixed (14)C into grains (r = 0.95**, P < 0.01). Exogenously applied ABA on pot-grown HN rice showed similar results as those by WS. Results suggest that an altered hormonal balance in rice grains by water stress during grain filling, especially a decrease in GAs and an increase in ABA, enhances the remobilization of prestored carbon to the grains and accelerates the grain filling rate.     

Proteomic analysis of rice leaf sheath during drought stress

Drought is one of the most severe limitations on the productivity of rainfed lowland and upland rice. To investigate the initial response of rice to drought stress, changes in protein expression were analyzed using a proteomic approach. Two-week-old rice seedlings were exposed to drought conditions from 2 to 6 days, and proteins were extracted from leaf sheaths, separated by two-dimensional polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. After drought stress for 2 to 6 days, 10 proteins increased in abundance and the level of 2 proteins decreased. The functional categories of these proteins were identified as defense, energy, metabolism, cell structure, and signal transduction. In addition to drought stress, accumulations of protein were analyzed under several different stress conditions. The levels of an actin depolymerizing factor, a light harvesting complex chain II, a superoxidase dismutase and a salt-induced protein were changed by drought and osmotic stresses, but not cold or salt stresses, or abscisic acid treatment. The effect of drought stress on protein in the leaf sheaths of drought-tolerant rice cultivar was also analyzed. The light harvesting complex chain II and the actin depolymerizing factor were present at high levels in a drought-tolerant rice cultivar before stress application. With drought stress, actin depolymerizing factor was expressed in leaf blades, leaf sheaths, and roots. These results suggest that actin depolymerizing factor is one of the target proteins induced by drought stress.     

籼稻桂华占、八桂香花后营养器官干物质流转与籽粒灌浆关系

以桂华占和八桂香2个籼稻品种为材料,研究籼稻花后不同部位器官物质积累、运转与籽粒生长的动态特征及相互关系。结果表明:(1)叶、叶鞘、节间干物质流转存在一定差异,倒2叶鞘对籽粒的贡献超过倒3叶鞘和倒1叶鞘,倒3节间对籽粒的贡献超过倒2节间和倒1节间;(2)不同部位籽粒的灌浆速率和拐点粒重呈现UPG(上部籽粒)MPG(中部籽粒)BPG(下部籽粒)变化趋势,拐点时间和活跃灌浆时间及持续灌浆时间均呈现BPGMPGUPG变化规律,UPG启动早,灌浆速率大,BPG的灌浆速率小,灌浆时间滞后,籽粒粒重呈现UPGMPGBPG;(3)叶片、叶鞘及节间干物质运转速度和运转率都与籽粒起始灌浆势呈正相关,其中器官间与起始灌浆势的相关系数大小表现为节间叶鞘叶片,不同叶位间与起始灌浆势的相关系数大小表现为倒2叶倒3叶倒1叶,其中节间干物质运转对籽粒生长的作用大于叶鞘,叶片干物质运转与籽粒生长的相关性最小。倒1节鞘物质输出与BPG生长时间上同步,倒2节鞘与MPG生长同步,倒3节鞘与UPG生长同步。     

土壤水分状况对花生和早稻叶片气体交换的影响

通过田间测坑试验研究了长期处于不同土壤水分状况下花生和早稻叶片气体交换的一些特点.结果表明,花生分枝期轻度和中度水分胁迫使气孔导度(Gs)和蒸腾速率(Tr)略有下降,净光合速率(Pn)和叶片水分利用效率(WUE)减小,轻度水分胁迫Gs/Tr略有上升而中度胁迫Gs/Tr变小.花生结荚期轻度和中度水分胁迫都使Gs、Tr、Gs/Tr和Pn显著降低,WUE大幅度上升.花生结荚期明显受土壤水分胁迫影响.早稻灌浆期轻度和中度水分胁迫Gs、Tr和Gs/Tr变化不显著,Pn和WUE增加,并且轻度水分胁迫下籽粒产量增加.Gs和Gs/Tr变化情况相结合可以作为作物水分胁迫程度的一个参考指标,即如果Gs和Gs/Tr同时下降则作物已经受到水分胁迫影响.     

Proteomics Analysis of Soybean Seedlings under Short-Term Water Deficit

Soybeans are one of the most important grain crops worldwide. Water deficit, which seriously affects the yield and quality of soybeans, is the main abiotic stress factor in soybean production. As a follow-up study, the droughttolerant soybean variant Heinong 44 was analyzed via proteome analysis. Soybean was exposed to water deficit for 0, 8, and 24 h, and protein samples were extracted for detection of differentially expressed proteins. Protein sequencing of leaf tissues under water stress yielded a total of 549 differentially expressed proteins: 75 and 320 upregulated proteins as well as 70 and 84 downregulated proteins were obtained after 8 and 24 h of water deficit, respectively. Gene Ontology analysis revealed that most of the differentially expressed proteins (DEPs) were involved in catalytic activity, molecular function, and metabolic processes, whereas some of them were involved in photosynthesis, carbon metabolism, and energy metabolism. We also identified some differentially expressed proteins that may be involved in the regulation of water deficit response. Our study provides a theoretical basis for the breeding of drought-resistant soybean varieties.     

Comparative proteomics analysis reveals an intimate protein network provoked by hydrogen peroxide stress in rice seedling leaves

Hydrogen peroxide (H2O2) plays a dual role in plants as the toxic by-product of normal cell metabolism and as a regulatory molecule in stress perception and signal transduction. However, a clear inventory as to how this dual function is regulated in plants is far from complete. In particular, how plants maintain survival under oxidative stress via adjustments of the intercellular metabolic network and antioxidative system is largely unknown. To investigate the responses of rice seedlings to H2O2 stress, changes in protein expression were analyzed using a comparative proteomics approach. Treatments with different concentrations of H2O2 for 6 h on 12-day-old rice seedlings resulted in several stressful phenotypes such as rolling leaves, decreased photosynthetic and photorespiratory rates, and elevated H2O2 accumulation. Analysis of approximately 2000 protein spots on each two-dimensional electrophoresis gel revealed 144 differentially expressed proteins. Of them, 65 protein spots were up-regulated, and 79 were down-regulated under at least one of the H2O2 treatment concentrations. Furthermore 129 differentially expressed protein spots were identified by mass spectrometry to match 89 diverse protein species. These identified proteins are involved in different cellular responses and metabolic processes with obvious functional tendencies toward cell defense, redox homeostasis, signal transduction, protein synthesis and degradation, photosynthesis and photorespiration, and carbohydrate/energy metabolism, indicating a good correlation between oxidative stress-responsive proteins and leaf physiological changes. The abundance changes of these proteins, together with their putative functions and participation in physiological reactions, produce an oxidative stress-responsive network at the protein level in H2O2-treated rice seedling leaves. Such a protein network allows us to further understand the possible management strategy of cellular activities occurring in the H2O2-treated rice seedling leaves and provides new insights into oxidative stress responses in plants.