Proteomic analysis of rice seedlings during cold stress

Low temperature is one of the important environmental changes that affect plant growth and agricultural production. To investigate the responses of rice to cold stress, changes in protein expression were analyzed using a proteomic approach. Two-week-old rice seedlings were exposed to 5 degrees C for 48 h, then total crude proteins were extracted from leaf blades, leaf sheaths and roots, separated by 2-DE and stained with CBB. Of the 250-400 protein spots from each organ, 39 proteins changed in abundance after cold stress, with 19 proteins increasing, and 20 proteins decreasing. In leaf blades, it was difficult to detect the changes in stress-responsive proteins due to the presence of an abundant protein, ribulose bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU), which accounted for about 50% of the total proteins. To overcome this problem, an antibody-affinity column was prepared to trap RuBisCO LSU, and the remaining proteins in the flow through from the column were subsequently separated using 2-DE. As a result, slight changes in stress responsive proteins were clearly displayed, and four proteins were newly detected after cold stress. From identified proteins, it was concluded that proteins related to energy metabolism were up-regulated, and defense-related proteins were down-regulated in leaf blades, by cold stress. These results suggest that energy production is activated in the chilling environment; furthermore, stress-related proteins are rapidly up-regulated, while defense-related proteins disappear, under long-term cold stress.     

Priming effect of exogenous ABA on heat stress tolerance in rice seedlings is associated with the upregulation of antioxidative defense capability and heat shock-related genes

Plant Growth Regulation - Heat stress is a major restrictive factor that suppresses rice production. In this study, we investigated the potential priming effect of exogenous abscisic acid (ABA) on...     

A proteomic analysis of cold stress responses in rice seedlings

Using proteomic analysis, an investigation aimed at a better understanding of the molecular adaptation mechanisms of cold stress was carried out in rice (Oryza sativa). The seedlings were exposed to a progressively low temperature stress treatment from normal temperature to 15, 10, and 5 degrees C. Proteins were extracted from the leaves collected from both control and stressed seedlings. By fractionation, approximately 1700 protein spots were separated and visualized on CBB-stained 2-D gels. Sixty protein spots were found to be up-regulated in responding to the progressively low temperature stress and displayed different dynamic patterns. As an initial work, 41 of these proteins were identified using MALDI-TOF MS or ESI/MS/MS. These cold responsive proteins, besides two proteins of unknown function, include four factors of protein biosynthesis, four molecular chaperones, two proteases, and eight enzymes involved in biosynthesis of cell wall components, seven antioxidative/detoxifying enzymes, and proteins linked to energy pathway, as well as a protein involved in signal transduction. The functional proteomes illuminate the facts, at least in plant cell, that protein quality control mediated by chaperones and proteases and enhancement of cell wall components play important roles in tolerance to cold stress. Using TargetP program, the subcellular localization of the identified proteins was analyzed. Proteins (43.9%) were predicted to be located in the chloroplasts, implying that chloroplast proteome is virtually subjective to cold stress. The physiological implications, revealed from the experimental data, are discussed in context of a complex metabolic network in plant cells responsive to cold stress.     

铝胁迫下水稻幼苗根系的生理特性

以4叶1心期的水稻幼苗为材料,在水培条件下,研究了0、0.5和7.5 mmol·L^-1Al胁迫下水稻Al敏感品种IR24、耐Al品种金优725和两优培九幼苗根系的生理特性.结果表明：在7.5 mmol·L^-1Al胁迫下,耐Al品种根系活力下降幅度远小于Al敏感品种,Al敏感品种H₂O₂含量较Al耐性品种高.在Al胁迫下,各品种根系线粒体中CAT的增加幅度较小;IR24和金优725根系的线粒体POD活性随Al胁迫浓度的增加先升后降,两优培九POD活性则呈增加趋势;IR24根系线粒体APX活性随Al胁迫浓度的增加先升后降,金优725和两优培九的线粒体APX活性则呈上升趋势.随Al胁迫浓度的增加,各品种根系谷氨酸含量先增加后下降,柠檬酸含量下降,磷酸烯醇式丙酮酸含量增加.Al敏感品种抗氰呼吸速率占总呼吸的比率较耐Al品种明显降低.     

Identification and expression analysis of four heat shock protein genes associated with thermal stress in rice weevil, Sitophilus oryzae

In order to explore the function of heat shock proteins during thermal stress in rice weevil, Sitophilus oryzae, four heat shock protein genes were cloned and characterized. These heat shock protein genes (hsps) were named as Sohsp70–1, Sohsp70–2, Sohsc70, and Sohsp90, respectively. These hsps showed high sequence conservation with the maximum identity with hsps of Tribolium castaneum and other insects. All the four genes showed the highest mRNA expression in pupal stage and the lowest levels in larval stage. The induced expression of the two Sohsp70s (Sohsp70–1 and Sohsp70–2) were reached to the highest levels (15.59-fold and 12.66-fold) after 2?h of incubation at 37?°C, respectively. Expression of Sohsp90 not only was significantly elevated by heat stress but also by cold stress. Whereas, expression level of Sohsc70 was not induced either by heat or cold stress. Furthermore, for rapid heat hardening, the expression levels of Sohsp70–1, Sohsp70–2, Sohsc70 and Sohsp90 were observed as 2.57, 2.53, 3.33 and 2.33-fold higher than control, respectively; for rapid cold hardening, the expression levels of Sohsp70–1, Sohsp70–2, Sohsc70 and Sohsp90 were reported as 2.27, 3.02, 3.37 and 2.23-fold higher than control, respectively. Hence, our results revealed that the four Sohsps were associated with temperature adaption under rapid heat or cold hardening.     

Ethylene production by loblolly pine seedlings associated with water stress

Effects of water stress on production of ethylene and its precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), by loblolly pine ( Pinus taeda L.) seedlings from a Texas drought-hardy and a Virginia Coastal Plain source were investigated. Ethylene production rates in needles from the Virgnia source increased slightly with initial stress (-1.3 MPa), declined until water potential reached -1.6 MPa and then increased sharply at -2.5 MPa. The ethylene production rates in needles from the Texas also increased slightly with initial stress, then decreased with decreasing water potential. Ethylene production by root tissue was two to three times higher than needle tissue and decreased with decreasing water potential. ACC concentrations in needles of both seed sources decreased as water potential began decreasing. Below -1.4 MPa, ACC levels started increasing (Texas source) or remained constant until -2.8 MPa (Virginia source) at which time its level increased three-fold. Mean ACC levels in root tissue [122 nmol (g dry weight)⁻¹] were slightly higher than the mean levels in the needle tissue [92 nmol (g dry weight) ⁻¹]; roots apparently were more efficient in converting it to ethylene since ethylene production was two to three times higher than needle tissue. The modulation of ethylene synthesis by ACC synthase and ethyleneforming enzyme appeared to be influenced by stress level, organ and seed source.     

Comparative chloroplast proteome analysis of exogenously supplied trehalose to wheat seedlings under heat stress

The aim of our study was to investigate the underlying molecular mechanisms of exogenously supplied trehalose affecting wheat photosynthesis under heat stress. The amount of ATP synthase (ATPase), oxygen-evolving enhancer protein (OEE), PsbP, Rubisco, chloroplast fructose-bisphosphate aldolase (FBPA), and ferredoxin-NADP(H) oxidoreductase (FNR) were downregulated, while PSI reaction center subunits were upregulated under heat stress. However, in the trehalose-pretreated groups, the amount of FNR, cytochrome b₆f complex, PSI reaction center subunits, ATPase, FBPA, and Rubisco were upregulated under normal growth conditions and heat stress. Besides, during the recovery period, the upregulation in CAB, PsbP, OEE2, and ATPase suggested that trehalose pretreatment might help to the recovery of PSII and PSI. These results indicate that trehalose pretreatment effectively regulates the levels of the photosynthesis-related proteins and relieves the damage of heat stress to wheat chloroplast.     

Comparative analysis of physical stress responses in soybean seedlings using cloned heat shock cDNAs

Summary Soybean seedlings were subjected to a wide range of physical (abiotic) or environmental stresses. Cloned cDNAs to heat shock (hs)-induced mRNAs were used to assess whether these diverse stresses induced the accumulation of poly(A)RNAs in common with those induced by hs. Northern blot hybridization analyses indicated that a wide range of stress agents lead to the accumulation of detectable levels of several of the hs-induced poly(A)RNAs; the relative concentration of those RNAs induced by the wide range of stress agents (e.g. water stress, salt stress, anaerobiosis, high concentrations of hormones, etc.), was generally in the order of 100-fold lower than that induced by hs. There are two notable exceptions to that pattern of response to the stress agents. First, arsenite treatment resulted in accumulation of the hs poly(A)RNAs to levels similar to those induced by hs. Cadmium also induced a somewhat normal spectrum of the hs poly(A)RNAs, but generally lower levels accumulated than in hs- and arsenite0treated tissues. Second, one set of poly(A)RNAs which are present at low and variable levels in control (non-stressed tissue) tissue, and which are increased some 5- to 10-fold by hs, increased in relative concentration in response to a wide range of the stress agents similarly to the response to hs. The physiological significance of the accumulation of this set of poly(A)RNAs (which translate into four electrophoretically different 27 kd proteins) is not known, but they certainly seem to serve as a monitor (or barometer) of physiological stress conditions. Cadmium treatment results in the accumulation of those same poly(A)RNAs and an additional band of higher molecular weight poly(A)RNA homologous to the same hs cDNA clone (clone pCE 54). Ethylene seems to have no obvious causal relationship to the hs response, even though hs-treated seedlings display some symptoms similar to those exhibited by ethylene-treated seedlings.     

Novel insight into kinetin-inducible stress responses in rice seedlings

Kinetin (KN) action in rice self-defense mechanism was studied using our established 2-week-old rice (Oryza sativa L. japonica-type cv. Nipponbare) seedling in vitro model system. It was strikingly observed that KN caused formation of brownish necrotic microlesions in leaves, suggesting it triggers a stress response in rice. Subsequent northern analyses revealed differential regulation (both up-and down-regulations) of 10 prominent defense/stress-related marker genes, including the critical pathogenesis-related (PR) protein genes of class 1, 5 and 10. A systemic effect of KN in leaves was shown using OsPR1b (basic) and OsPOX (peroxidase) genes as representatives. KN also exclusively triggered potent accumulation of PR proteins (OsPR5 and OsPR10), and a phytoalexin, sakuranetin. Interestingly, as KN failed to induce jasmonic acid (JA) inducible genes (OsPR1a and JIOsPR10), and had almost no effect on accumulated endogenous JA level due to wounding by cut, KN might act through a yet unknown (and JA-independent) pathway. These results provide a new aspect on the role of KN as a potent activator of the stress responses in the rice plant.     

Proteomics of rice in response to heat stress and advances in genetic engineering for heat tolerance in rice

Rice is the most important food crop worldwide. Global warming inevitably affects the grain yields of rice. Recent proteomics studies in rice have provided evidence for better understanding the mechanisms of thermal adaptation. Heat stress response in rice is complicated, involving up- or down-regulation of numerous proteins related to different metabolic pathways. The heat-responsive proteins mainly include protection proteins, proteins involved in protein biosynthesis, protein degradation, energy and carbohydrate metabolism, and redox homeostasis. In addition, increased thermotolerance in transgenic rice was obtained by overexpression of rice genes and genes from other plants. On the other hand, heterologous expression of some rice proteins led to enhanced thermotolerance in bacteria and other easily transformed plants. In this paper, we review the proteomic characterization of rice in response to high temperature and achievements of genetic engineering for heat tolerance in rice.     

The decline in potassium concentration is associated with cadmium toxicity of rice seedlings

Cadmium (Cd) is one of the most dangerous environmental pollutants, among other things, affecting plant mineral composition. Thus, in this study, we investigated the changes in potassium (K) concentration in Cd-treated rice (Oryza sativa L.) seedlings of two cultivars. On treatment with 5 μM CdCl₂, the Cd concentration increased in the shoot and roots of Cd-sensitive cultivar (cv. Taichung Native 1, TN1) but not or slightly in the Cd-tolerant cultivar (cv. Tainung 67, TNG67). The decrease in K concentration in the shoot and roots of TN1 caused by Cd was more pronounced than that of TNG67. Exogenous addition of KCl decreased Cd concentration and reduced Cd toxicity of TN1 seedlings. Evidence presented in this study suggests that the improvement of K status is able to reduce toxicity of rice seedlings to CdCl₂.     

Effect of cadmium uptake and heat stress on root ultrastructure, membrane damage and antioxidative response in rice seedlings

Excess cadmium (Cd²⁺) in the soil environment is taken up by plants and can cause phytotoxicity. Elevated temperatures also lead to deleterious effects on plants. Plants are very often exposed to a combination of stresses rather than a single stress. The effect of Cd²⁺ and heat stress (HS) on the growth, root ultrastructure, lipid peroxidation (MDA), hydrogen peroxide accumulation and the activities of antioxidant enzymes peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) of rice roots from sensitive cv. DR-92 and tolerant cv. Bh-1 were investigated at 10 and 20 day of growth under controlled conditions. At day 10 under all Cd²⁺ treatments, the Cd²⁺ content between the two rice cultivars were almost similar. Application of 500 μM Cd²⁺ significantly increased metal concentrations at day 20 in the roots of rice seedlings resulting in a maximum accumulation of 44.25 μg Cd²⁺ g^-1 dry wt in cv. DR-92 and 30 μg Cd²⁺ g^-1 dry wt in cv. Bh-1 with a ~25 % decline in Relative Growth Index (RGI) in cv. DR-92. TEM studies revealed slight disorganization with cell wall ingrowths in root tissues from cv. DR-92 grown in 100 μM Cd²⁺ + HS. Uptake and accumulation of Cd²⁺ increased upon heat treatment in parenchyma, vacuoles and vascular cylinder of root tissues. Peroxidase primarily located in cell walls, the intensity being higher in sensitive cv. DR-92. Under Cd²⁺ stress alone, plants of sensitive cv. DR-92 significantly increased the H₂O₂ and MDA levels together with increased activities of the enzymes POD, CAT and APX at day 10 but remained almost stable at day 20. A strong increase in MDA levels was noted at day 20 in tolerant cv. Bh-1. Cd²⁺ + HS treatments in tolerant cv.Bh-1 led to a decreased H₂O₂ and MDA levels and decreased activities of the enzymes POD, CAT and APX. Results suggest stimulation of root antioxidant system under combination of two stresses and that heat stress seem to have a direct protective role by mitigating the effect of mild Cd²⁺ toxicity largely by enhanced Cd²⁺-MT formation contributing thereby towards the management of Cd²⁺ toxicity at cellular level that confers Cd²⁺ tolerance to rice cv. Bh-1.     

New QTLs identified for leaf correlative traits in rice seedlings under cadmium stress

Cadmium (Cd) is a non-essential toxic metal that is primarily released into the environment from artificial sources in recent decades. To investigate the genetics of Cd toxicity tolerance at the seedling stage in rice, a QTL analysis was carried out under cadmium stress conditions with two toxicity-linked traits—leaf rolling (LR) and the green leaf ratio (GLR). Using 127 rice lines of doubled haploid (DH) population derived from a cross between a japonica JX17 and indica ZYQ8, two QTLs for LR (qLR-1 and qLR-9) and one QTL for GLR (qGLR-3) were detected. Among them, the phenotypic variation of qLR-1 and qGLR-3 were 19.27 and 16.09, values which are useful for marker-assistant selection in breeding elite rice cultivars that have the capacity to tolerate Cd. The results further demonstrate that visual measurements of both LR and GLR in seedlings are effective methods for screening tolerant rice germplasm in cadmium stress scenarios.     

不同耐性水稻幼苗根系对镉胁迫的形态及生理响应

采用水培试验,以两个耐镉性不同的水稻品种为材料,研究了不同浓度镉胁迫对水稻幼苗根系形态、根系活力、游离脯氨酸含量及抗氧化酶活性的影响。结果表明:低于5 μmol/L Cd胁迫对2个水稻品种总根长、根表面积、根体积、根干重、根系活力无明显影响,在1 μmol/L Cd时,甚至起促进作用。随Cd浓度增加表现出一定的抑制效应,秀水63在10 μmol/L Cd胁迫下根系形态、根系活力明显受到抑制,而秀水09在25 μmol/L Cd胁迫下明显受抑。随Cd胁迫浓度的增加,游离脯氨酸含量、丙二醛(MDA)含量、超氧化物歧化酶(SOD)、过氧化物酶(POD)活性均呈上升趋势,两品种相比,秀水09的游离脯氨酸含量、SOD和POD增幅大于秀水63,而MDA含量增幅小于秀水63;过氧化氢酶(CAT)活性变化表现为先上升后下降,10-100 μmol/L Cd胁迫下秀水63根系中CAT活性明显低于秀水09。总之,水稻对Cd毒害响应存在明显的品种差异,且Cd胁迫下根系生理响应的差异是品种间耐性差异的重要原因之一。