Proteomics approach to identify wound-response related proteins from rice leaf sheath

In order to avoid the complex conditions of the intact plant for simple analysis of proteins in wound-response stress, we used the detached rice leaf sheath which is a very active part of the rice seedling. Proteins were extracted from rice leaf sheath at 0, 12, 24, 48 h after cutting and separated by two-dimensional (2-D) polyacrylamide gel electrophoresis. Changes in differentially displayed proteins were found in leaf sheaths after cutting in the 0-48 h time course. Ten proteins were up-regulated, while 19 proteins were down-regulated compared with those on the four 2-D gels. Among them, 14 proteins were analyzed by N-terminal, or internal amino acid sequence. The clear functions of nine proteins could be identified. Six proteins did not yield amino acid sequence information due to their blocked N-termini. Furthermore, 11 proteins were determined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and identified protein database matching. It was shown that the down-regulated proteins were calreticulin (nos. 5, 6), histone H1 (no. 15) and hemoglobin (no. 17), putative peroxidase (no. 19); the up-regulated proteins were Bowman-Birk trypsin inhibitor (no. 23), putative receptor-like protein kinase (nos. 24, 25), calmodulin-related protein (no. 26), small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (no. 27), mannose-binding rice lectin (nos. 28, 29). Among all the above proteins, four (nos. 23, 24, 25, 26) have been confirmed to be wound-response proteins. The others cannot be excluded as also being related to wound-responses, such as the signal transduction-related proteins (nos. 5, 6), photosynthesis-related protein (no. 27), and stress-response proteins (nos. 19, 28, 29). This is the first time protein changes in response to wounding in rice leaf sheath have been shown.     

Differential expression of cell wall related genes in the elongation zone of rice roots under water deficit

Growth in the apical elongation zone of plant roots is central to the development of functional root systems. It has been known that rice seminal root elongation could be enhanced by water stress. In the present study, 17 cell-wall related genes were identified by cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. Five genes encoded cell-wall loosening enzymes and six genes were involved in the lignin biosynthesis. The six other genes were related to the metabolism of polysaccharide and protein matrices in cell wall. Northern blot analysis confirmed that they were differentially expressed in the elongation zone of rice seminal roots under water stress, and none of them was root-specific. The results indicated that the activity of cell-wall loosening enzymes was enhanced in the early stage (within 16 h), and some cell wall matrices were synthesized rapidly in the middle stages (from 16 to 48 h), while lignin biosynthesis was enhanced in the middle and late stages of water stress (from 48 to 72 h). Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 437–443. The text was submitted by the authors in English.     

An approach to identify cold-induced low-abundant proteins in rice leaf

A proteomic approach has been adopted to investigate the low-abundant proteins in rice leaf in response to cold stress. Rice seedlings were exposed to different temperatures, such as 5 or 10 degrees C, and samples were collected after different time course. To eliminate the high-abundant proteins in leaf tissues such as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), proteins were fractionated by polyethylene glycol (PEG). The elimination of Rubisco from the protein samples was confirmed by Western blot analysis. The PEG fractionated protein samples were separated by 2-DE and visualized by silver or CBB staining. A total 12 up-regulated protein spots were identified using the analysis of MALDI-TOF mass spectrometry or ESI MS/MS. We identified some novel proteins such as cysteine proteinase, thioredoxin peroxidase, a RING zinc finger protein-like, drought-inducible late embryogenesis abundant, and a fibrillin-like protein that had not yet been reported in the earlier reports on cold proteomic analysis. The identification of some novel low-abundant proteins in response to cold stress may provide a new homeostasis to develop enhanced cold tolerance transgenic plants. Thus, we propose that a PEG fractionation system can be used as an influential protein extraction method from the leaf samples, which can lead to knowledge of the expression pattern of low-abundant proteins in response to various biotic or abiotic stresses.     

Upland rice and lowland rice exhibited different PIP expression under water deficit and ABA treatment

Aquaporins play a significant role in plant water relations. To further understand the aquaporin function in plants under water stress, the expression of a subgroup of aquaporins, plasma membrane intrinsic proteins （PIPs）, was studied at both the protein and mRNA level in upland rice （Oryza sativa L. cv. Zhonghan 3） and lowland rice （Oryza sativa L. cv. Xiushui 63） when they were water stressed by treatment with 20% polyethylene glycol （PEG）. Plants responded differently to 20% PEG treatment. Leaf water content of upland rice leaves was reduced rapidly. PIP protein level increased markedly in roots of both types, but only in leaves of upland rice after 10 h of PEG treatment. At the mRNA level, OsPIP1,2, OsPIP1,3, OsPIP2;1 and OsPIP2;5 in roots as well as OsPIP1,2 and OsPIP1;3 in leaves were significantly up-regulated in upland rice, whereas the corresponding genes remained unchanged or down-regulated in lowland rice. Meanwhile, we observed a significant increase in the endogenous abscisic acid （ABA） level in upland rice but not in lowland rice under water deficit. Treatment with 60 μM ABA enhanced the expression of OsPIP1;2, OsPIP2;5 and OsPIP2;6 in roots and OsPIP1;2, OsPIP2;4 and OsPIP2;6 in leaves of upland rice. The responsiveness of PIP genes to water stress and ABA were different, implying that the regulation of PIP genes involves both ABA-dependent and ABA-independent signaling oathways during water deficit.     

A protein similar to PR (pathogenesis-related) proteins is elicited by metal toxicity in wheat roots

An acidic, low molecular weight protein called TA1-18 (T for Triticum. Al for aluminium and 18 for its approximate molecular weight) is induced in wheat roots that are exposed to growth-inhibiting concentrations of Al. Enhanced biosynthesis of TA1-18 began during the period 3 to 6 h after exposure to Al, and reached a maximum after 9 to 12 h of treatment. A protein with the same molecular weight and pl was also elicited during toxicity associated with Cu and Cd, with calcium deprivation, and low (3. 5) pH, but not by heat shock. TA1-18 was formed in small amounts in triticale, but was not detected in rye during exposure to growth-inhibiting levels of Al. Amino acid sequencing of trypsin fragments of TA1-18 revealed strong homology to pathogenesis-related protein PR2 from parsley cultures, with which TA1-18 also shares similar molecular weight and pl. Aluminium toxicity appears to have features in common with pathogenesis such that similar proteins are formed in response to both types of stress.     

Herb and conifer roots show similar high sensitivity to water deficit

Root systems play a major role in supplying the canopy with water, enabling photosynthesis and growth. Yet, much of the dynamic response of root hydraulics and its influence on gas exchange during soil drying and recovery remains uncertain. We examined the decline and recovery of the whole root hydraulic conductance (Kr) and canopy diffusive conductance (g_c) during exposure to moderate water stress in two species with contrasting root systems: Tanacetum cinerariifolium (herbaceous Asteraceae) and Callitris rhomboidea (woody conifer). Optical dendrometers were used to record stem water potential at high temporal resolution and enabled non-invasive measurements of Kr calculated from the rapid relaxation kinetics of water potential in hydrating roots. We observed parallel declines in Kr and g_c to <20% of unstressed levels during the early stages of water stress in both species. The recovery of Kr after rewatering differed between species. T. cinerariifolium recovered quickly, with 60% of Kr recovered within 2 h, while C. rhomboidea was much slower to return to its original Kr. Recovery of g_c followed a similar trend to Kr in both species, with C. rhomboidea slower to recover. Our findings suggest that the pronounced sensitivity of Kr to drought is a common feature among different plant species, but recovery may vary depending on root type and water stress severity. Kr dynamics are proposed to modulate g_c response during and following drought.

Root hydraulic conductance shows a similar high sensitivity to soil water deficit in herbaceous and woody roots and may control canopy diffusive conductance during drought and following recovery.     

Proteomics to identify resistance factors in corn-a review

The host resistance strategy for eliminating aflatoxins from corn has been advanced by the discovery of natural resistance traits such as proteins. This progress was aided by the development of a rapid laboratory-based kernel screening assay (KSA) used to separate resistant from susceptible seed, and for investigating kernel resistance.A. flavus GUS transformants have also been used, in conjunction with the KSA, to assess the amount of fungal growth in kernels and compare it with aflatoxin accumulation. Several proteins associated with resistance (RAPs) have been identified using 1 D PAGE. However, proteomics is now being used to further the discovery of RAPs. This methodology has led to the identification of stress-related RAPs as well as other antifungals. Characterization studies being conducted, including RNAi gene silencing experiments, may confirm roles for RAPs in host resistance.     

A proteomic approach to analyze nitrogen- and cytokinin-responsive proteins in rice roots

Nitrogen plays a central role in rice growth and development because it modulates a wide variety of processes, including cytokinin (CK) metabolism. CK-mediated signaling is also related to nitrogen metabolism. The functional relation between nitrogen and CK are extremely complex and unclear. In this study, a comparative proteomic analysis was carried out to analyze proteins regulated by nitrogen and CK in rice roots. Proteins extracted from rice roots are separated by two-dimensional polyacrylamide gel electrophoresis. Thirty-two protein spots that expressed similarly by nitrogen and CK treatments are selected for identification by mass spectrometry. Of these spots, 28 are successfully identified. These proteins were categorized into classes related to energy, metabolism, disease/defense, protein degradation, signal transduction, transposons, and unclear classification. Energy gives the largest functional category, suggesting that the glycolysis (two enzymes detected) and tricarboxylic acid cycle (six enzymes detected) are accurately regulated by nitrogen and CK, thus promoting the synthesis of amino acid. The identification of novel proteins provides new insights into the coordination of nitrogen and CK in rice. The possible role of these proteins is discussed.     

Polyamines induce adaptive responses in water deficit stressed cucumber roots

The aim of this study was to investigate the effect of exogenous polyamines (PAs) on the membrane status and proline level in roots of water stressed cucumber (Cucumis sativus cv. Dar) seedlings. It was found that water shortage resulted in an increase of membrane injury, lipoxygenase (LOX) activity, lipid peroxidation and proline concentration in cucumber roots during progressive dehydration. PA pretreatment resulted in a distinct reduction of the injury index, and this effect was reflected by a lower stress-evoked LOX activity increase and lipid peroxide levels at the end of the stress period. In contrast, PA-supplied stressed roots displayed a higher proline accumulation. The presented results suggest that exogenous PAs are able to alleviate water deficit-induced membrane permeability and diminish LOX activity. Observed changes were accompanied by an accumulation of proline, suggesting that the accumulation of this osmolyte might be another possible mode of action for PAs to attain higher membrane stability, and in this way mitigate water deficit effects in roots of cucumber seedlings.     

Water uptake by roots: effects of water deficit

The variable hydraulic conductivity of roots (Lp(r)) is explained in terms of a composite transport model. It is shown how the complex, composite anatomical structure of roots results in a composite transport of both water and solutes. In the model, the parallel apoplastic and cell-to-cell (symplastic and transcellular) pathways play an important role as well as the different tissues and structures arranged in series within the root cylinder (epidermis, exodermis, cortex, endodermis, stelar parenchyma). The roles of Casparian bands and suberin lamellae in the root's endo- and exodermis are discussed. Depending on the developmental state of these apoplastic barriers, the overall hydraulic resistance of roots is either more evenly distributed across the root cylinder (young unstressed roots) or is concentrated in certain layers (exo- and endodermis in older stressed roots). The reason for the variability of root Lp(r), is that hydraulic forces cause a dominating apoplastic flow of water around protoplasts, even in the endodermis and exodermis. In the absence of transpiration, water flow is osmotic in nature which causes a high resistance as water passes across many membranes on its passage across the root cylinder. The model allows for a high capability of roots to take up water in the presence of high rates of transpiration (high demands for water from the shoot). By contrast, the hydraulic conductance is low, when transpiration is switched off. Overall, this results in a non-linear relationship between water flow and forces (gradients of hydrostatic and osmotic pressure) which is otherwise hard to explain. The model allows for special root characteristics such as a high hydraulic conductivity (water permeability) in the presence of a low permeability of nutrient ions once taken up into the stele by active processes. Low root reflection coefficients are in line with the idea of some apoplastic bypasses for water within the root cylinder. According to the composite transport model, the switch from the hydraulic to the osmotic mode is purely physical. In the presence of heavily suberized roots, the apoplastic component of water flow may be too small. Under these conditions, a regulation of radial water flow by water channels dominates. Since water channels are under metabolic control, this component represents an 'active' element of regulation. Composite transport allows for an optimization of the water balance of the shoot in addition to the well-known phenomena involved in the regulation of water flow (gas exchange) across stomata. The model is employed to explain the responses of plants to water deficit and other stresses. During water deficit, the cohesion-tension mechanism of the ascent of sap in the xylem plays an important role. Results are summarized which prove the validity of the coehesion/tension theory. Effects of the stress hormone abscisic acid (ABA) are presented. They show that there is an apoplastic component of the flow of ABA in the root which contributes to the ABA signal in the xylem. On the other hand, (+)-cis-trans-ABA specifically affects both the cell level (water channel activity) and water flow driven by gradients in osmotic pressure at the root level which is in agreement with the composite transport model. Hydraulic water flow in the presence of gradients in hydrostatic pressure remains unchanged. The results agree with the composite transport model and resemble earlier findings of high salinity obtained for the cell (Lp) and root (Lp(r)) level. They are in line with known effects of nutrient deprivation on root Lp(r )and the diurnal rhythm of root Lp(r )recently found in roots of LOTUS.     

Upland rice and lowland rice exhibited different P/P expression under water deficit and ABA treatment

Aquaporins play a significant role in plant water relations.To further understand the aquaporin function in plants underwater stress,the expression of a subgroup of aquaporins,plasma membrane intrinsic proteins(PIPs),was studied at boththe protein and mRNA level in upland rice(Oryza sativa L.cv.Zhonghan 3)and lowland rice(Oryza sativa L.cv.Xiushui63)when they were water stressed by treatment with 20% polyethylene glycol(PEG).Plants responded differently to20% PEG treatment.Leaf water content of upland rice leaves was reduced rapidly.PIP protein level increased markedlyin roots of both types,but only in leaves of upland rice after 10h of PEG treatment.At the mRNA level,OsPIP1;2,Os-PIP1;3,OsPIP2;1 and OsPIP2;5 in roots as well as OsPIP1;2 and OsPIP1;3 in leaves were significantly up-regulatedin upland rice,whereas the corresponding genes remained unchanged or down-regulated in lowland rice.Meanwhile,weobserved a significant increase in the endogenous abscisic acid(ABA)level in upland rice but not in lowland rice underwater deficit.Treatment with 60μM ABA enhanced the expression of OsPIP1;2,OsPIP2;5 and OsPIP2;6 in roots andOsPIP1;2,OsPIP2;4 and OsPIP2;6 in leaves of upland rice.The responsiveness of PIP genes to water stress and ABAwere different,implying that the regulation of PIP genes involves both ABA-dependent and ABA-independent signalingpathways during water deficit.     

Comparison of two proteomics techniques used to identify proteins regulated by gibberellin in rice

Proteomics has become an essential methodology for large-scale analysis of proteins in various fields of plant biology. We compared two proteomics techniques, two-dimensional liquid chromatography (2D-LC) and fluorescence two-dimensional difference gel electrophoresis (2D-DIGE), for their ability to identify proteins regulated by gibberellin (GA) in rice. Two-week-old rice seedlings were treated with or without 5 microM GA3 for 48 h and proteins extracted from the basal region of the leaf sheath. After separation of the proteins by the two techniques, the amino acid sequences of GA3-responsive proteins were analyzed using a protein sequencer and mass spectrometry. 2D-LC and 2D-DIGE were able to resolve 1248 protein fractions and 1500 proteins, respectively. Out of these, 2D-LC identified 9 proteins that were up-regulated and 9 that were down-regulated by GA treatment; 2D-DIGE identified 4 up-regulated and 4 down-regulated proteins. The two techniques detected overlapping sets of proteins. For example, cytosolic glyceraldehyde-3-phosphate dehydrogenase and photosystem II oxygen-evolving complex protein were identified as GA3-regulated proteins by both methods. In addition, these two methods uncovered GA3-regulated unknown proteins which had not been reported previously, and novel proteins which are not detected in 2D-PAGE followed by Coomassie brilliant blue staining. These results suggest that these two methods are among some of the very useful tools for detecting proteins that may function in various physiological and developmental processes in plants.     

Impact of water deficit intensity on durum wheat seminal roots

Experiments were performed under controlled conditions to study seminal roots traits of durum wheat genotypes grown under four water conditions. Seminal root length, root-to-shoot dry matters ratio and piliferous layer cell size were measured. Root volume was assessed at three soil depths. Water stress has affected significantly root traits and piliferous layer cell size and this impact depends on its intensity. Severe water stress reduced markedly root traits. Water treatment by genotype interaction was observed. Middle-East genotypes responded differently from Algerian ones. Our results and those obtained elsewhere on the same genotypes for other physiological traits are discussed.     

水稻根系响应镉胁迫的蛋白质差异表达

为探讨水稻根系对镉胁迫的分子生理响应,以抗镉水稻PI312777和镉敏感水稻IR24为材料,设置Cd~(2+)浓度为0、50和100μmol/L的水培试验,处理7 d后分析了水稻根系的蛋白质差异表达。结果表明,在镉胁迫下水稻PI312777和IR24根系有18个蛋白质发生了差异表达,其中的12个得到MALDI-TOF/MS鉴定。这些鉴定的蛋白功能可分四类:(1)与活性氧(ROS)胁迫相关的过氧化物酶(POD)、蛋氨酸腺苷转移酶(MAT)、类萌发素蛋白前体;(2)与谷胱甘肽(GSH)合成相关的S-腺苷甲硫氨酸合成酶(SAMS)、谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(GDH);(3)与逆境胁迫相关的ABA胁迫诱导蛋白含HVA22域蛋白、ABA-胁迫-成熟诱导蛋白5(ASR5);(4)与细胞分裂调控相关的GTP结合核蛋白Ran-2。镉胁迫下SAMS和GTP结合核蛋白Ran-2在两种水稻根系均发生上调表达;MAT、POD、类萌发素蛋白前体和GS发生下调表达;依赖NADP-GDH、GDH和磷酸甘油酸变位酶在IR24根部均发生下调表达,在PI312777根部仅在100μmol/L Cd~(2+)处理发生下调表达;含HVA22域蛋白在PI312777根部上调表达,在IR24根部发生下调表达;ASR5在PI312777根部上调表达,在IR24根部的表达无显著差异;100μmol/L Cd~(2+)胁迫下60S酸性核糖体蛋白P0在水稻PI312777根部表达下调,在IR24根部表达上调。可见,镉胁迫使水稻根部ROS增加,形成氧化胁迫反应,造成毒害作用,而水稻根通过调节SAMS和GS提高GSH合成降低镉毒害。ASR5和HVA22蛋白等逆境胁迫蛋白的表达差异则是水稻品种间抗性差异的重要原因之一。     

Proteomics analysis reveals marker proteins for minor vein initiation in rice leaf

Leaf veins play a critical role in resource supplication and photosynthate translocation; thus, it is considered as an important agricultural trait for crop breeding. The rice minor veins are parallelly grown along all the parts of the leaf from base to tip. To understand the process of minor vein development, anatomy analysis was performed to reveal the initiation and development of minor veins in rice leaf. The frequency of minor vein initiation follows a decreased tendency from leaf base to tip. An iTRAQ-based proteomics analysis was performed in rice leaf sections. Photosynthesis- and carbon fixation-related proteins accumulated a high level in the middle part of leaves. Furthermore, marker proteins involved in sucrose degradation and starch synthesis were accumulated into initiation and mature parts of minor veins, respectively. It suggests a different source-sink activity in the initiation and mature parts of minor veins in terms of photosynthate translocation. The identified proteins are candidate markers for small vein initiation in rice leaves.     

Oxalic acid-induced resistance to Rhizoctonia solani in rice is associated with induction of phenolics,peroxidase and pathogenesis-related proteins

Abstract

Oxalic acid (1 mM) when applied as a foliar spray to rice plants induced resistance to challenge infection with Rhizoctonia solani, the rice sheath blight pathogen. Maximum reduction in sheath blight incidence was observed when the plants were sprayed with oxalic acid three days before inoculation with the fungus. The biochemical alterations in rice plants treated with oxalic acid was also investigated. When rice plants were treated with oxalic acid, a two-fold increase in phenolic content in leaf sheaths was recorded three days after treatment. Phenylalanine ammonia-lyase and peroxidase activities increased significantly starting from two days after treatment. Peroxidase (PO) isozyme analysis indicated that PO-3 and PO-4 were induced two days after treatment with oxalic acid. Western blot analysis revealed that two chitinases (28 and 35 kDa) and two β-1,3-glucanases (30 and 32 kDa) were strongly induced in rice sheaths four to six days after treatment with oxalic acid. Immunoblot analysis of protein extracts from oxalic acid-treated plants demonstrated the induction of a 23 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antibody. These results suggest that the enhanced activities of defense enzymes and defense-related compounds in oxalic acid-treated rice plants may contribute to resistance against R. solani.