The potential biofuel crop Jatropha curcas has very low tolerance to low temperatures, which is the bottleneck for its cultivation and commercialization. BTF3 (Basic transcription factor 3) function as a key regulator of plant growth and development as well as in the tolerance to biotic and abiotic stress. JcBTF3 was isolated from J. curcas by screening the cold stress responsive yeast hybrid library, which has five introns and six exons that contain a single ORF of 501 bp, encoding a protein of 166 amino acids. In addition to its NAC domain, phylogenetic analysis suggests that the entire sequence of the JcBTF3 protein and the gene structure are highly conserved in the plant kingdom. One hour of cold exposure caused a significant up-regulation of JcBTF3, while abscisic acid, methyl jasmonate, salt and drought stresses slightly induced its expression. Assessing expression in different tissues showed that JcBTF3 is expressed ubiquitously, with lower levels in the stem. Further experiments demonstrated that JcBTF3 is distributed throughout the cytoplasm and in the nucleus. Overexpression of JcBTF3 increased the tolerance to cold stresses in transgenic Arabidopsis. These results suggest that the conserved BTF3 gene structure could be an important hint for the functional identification of orthologous genes in additional plant species and that JcBTF3 might be used to improve the cold stress resistance of J. curcas.

•Eighty-three oil bodies related protein species were identified by LTQ-ES-MS/MS from Jatropha curcas seeds.•Twenty-eight differential protein species were found among three Jatropha curcas seeds with different lipid content.•The ratio of lipids to caleosin-like was related to the regulation of OB size.•Mutant induced by ethylmethylsulfone treatment was related to the caleosin protein species.•Glycine-rich RNA binding protein (GRP) was well targeted to OBs.To reveal the difference among three mature Jatropha curcas seeds (JcVH, variant with high lipid content; JcW, wild type and JcVL, variant with low lipid content) with different lipid content, comparative proteomics was employed to profile the changes of oil body (OB) associated protein species by using gels-based proteomic technique. Eighty-three protein species were successfully identified through LTQ-ES-MS/MS from mature JcW seeds purified OBs. Two-dimensional electrophoresis analysis of J. curcas OB associated protein species revealed they had essential interactions with other organelles and demonstrated that oleosin and caleosin were the most abundant OB structural protein species. Twenty-eight OB associated protein species showed significant difference among JcVH, JcW and JcVL according to statistical analysis. Complementary transient expression analysis revealed that calcium ion binding protein (CalBP) and glycine-rich RNA binding protein (GRP) were well targeted in OBs apart from the oleosins. This study demonstrated that ratio of lipid content to caleosins abundance was involved in the regulation of OB size, and the mutant induced by ethylmethylsulfone treatment might be related to the caleosin like protein species. These findings are important for biotechnological improvement with the aim to alter the lipid content in J. curcas seeds.Biological significanceThe economic value of Jatropha curcas largely depends on the lipid content in seeds which are mainly stored in the special organelle called oil bodies (OBs). In consideration of the biological importance and applications of J. curcas OB in seeds, it is necessary to further explore the components and functions of J. curcas OBs. Although a previous study concerning the J. curcas OB proteome revealed oleosins were the major OB protein component and additional protein species were similar to those in other oil seed plants, these identified OB associated protein species were corresponding to the protein bands instead of protein spots in the electrophoresis gels. Furthermore, the interaction of OB associated protein species and their contribution to OB formation and stabilization are still blank. In this study, with the overall object of profiling OB protein species from mature J. curcas seeds with different lipid content, we provided a setting of comparative OB proteomics with biochemical data and transient expression to explore the core of OB associated protein species involved in the regulation of OB size and lipid accumulation. The results were important for biotechnological improvement with the aim to a global modification of lipid storage in J. curcas seeds. Meanwhile, this study gave insight into possible associations between OBs and other organelles in mature J. curcas seeds. It may represent new aspects of the biological functions of the OBs during the oil mobilization. Combined the technique of transient transformation, a newly reported protein species, glycine-rich RNA binding protein (GRP) was successfully targeted in OBs. Therefore, further molecular analysis of these protein species is warranted to verify this association and what role they have in OBs.

Rice blast, caused by Magnaporthe grisea threatens rice production worldwide. It is important to develop novel and environment-safe strategies to control the fungus. Here we reported that Bacillus subtilis KB-1122 could strikingly inhibit the growth of M. grisea P131 in agar diffusion assays. To further understand the molecular mechanism on the suppressive role of B. subtilis on M. grisea, the antagonist–pathogen interaction of the two strains was studied by using comparative proteome analysis in this report. The cellular and culture supernatant (CSN) proteins were prepared from co-culture and subjected to two-dimensional polyacrylamide gel electrophoresis. Proteome analysis revealed 33 cellular and 18 CSN proteins showing changes upon co-culture respectively. Importantly, down-regulated cellular proteins came from M. grisea, whereas up-regulated proteins derived from B. subtilis. Results suggested that glyceraldehyde-3-phosphate dehydrogenase and serine protein kinase might contribute to antifungal activity of B. subtilis KB-1122. Of CSN proteins identified, the endo-1,4-beta-glucanase (involved in degradation of polysaccharides) was up-regulated consistently at different times of incubation. This suggests that this enzyme plays an important role in the interaction between B. subtilis KB-1122 with M. grisea P131.

The chloroplast-to-chromoplast difference is a highly regulated process. Tomato (Solanum lycopersicum) high-pigment mutant hp-1 shows an enhanced accumulation of carotenoids coupled with an increase in the volume and number of plastids. However, how chromoplasts of hp-1 acquire their specific metabolic characteristics is still unclear. A comparison of proteome profiles from plastids at the mature green, breaker, and red stages of tomato fruits showed 45 differentially expressed proteins. These identified proteins fell into six different functional categories. Our results showed that most of the proteins related to the Calvin cycle increased transitorily only at the early breaker stage, and remained unchanged at the early red stage. We found 18 proteins that were differentially expressed between the wild type and hp-1. The abundance of GCPE in chromoplasts of hp-1 was higher than that in the wild type. Meanwhile, we found that heat shock proteins were only present specifically in the mature green stage chromoplasts of hp-1 but not in the wild type. This suggested that GCPE and heat shock proteins might play important roles in the accumulation of high carotenoids in hp-1. Overall, our results could be helpful for understanding chloroplast-to-chromoplast differentiation and the function of hp-1.

•We have characterized the proteomic profiles of the seed development of J. curcas.•104 differential proteins have been identified by MALDI-TOF/TOF.•33 proteins and 19 mRNAs are involved in the metabolism of lipid accumulation.•G-6-P and triose-Ps are the major carbon sources for fatty acid synthesis.•C16:0 is the precursor for the elongation to C18:1 and C18:2.To characterize the metabolic signatures of lipid accumulation in Jatropha curcas seeds, comparative proteomic technique was employed to profile protein changes during the seed development. Temporal changes in comparative proteome were examined using gels-based proteomic technique at six developmental stages for lipid accumulation. And 104 differentially expressed proteins were identified by MALDI-TOF/TOF tandem mass spectrometry. These protein species were classified into 10 functional categories, and the results demonstrated that protein species related to energy and metabolism were notably accumulated and involved in the carbon flux to lipid accumulation that occurs primarily from early to late stage in seed development. Glycolysis and oxidative pentose phosphate pathways were the major pathways of producing carbon flux, and the glucose-6-phosphate and triose-phosphate are the major carbon source for fatty acid synthesis. Lipid analysis revealed that fatty acid accumulation initiated 25 days after flowering at the late stage of seed development of J. curcas. Furthermore, C16:0 was initially synthesized as the precursor for the elongation to C18:1 and C18:2 in the developing seeds of J. curcas. Together, the metabolic signatures on protein changes in seed development provide profound knowledge and perspective insights into understanding lipid network in J. curcas.Biological significanceDue to the abundant oil content in seeds, Jatropha curcas seeds are being considered as the ideal materials for biodiesel. Although several studies had carried out the transcriptomic project to study the genes expression profiles in seed development of J. curcas, these ESTs hadn't been confirmed by qRT-PCR. Yet, the seed development of J. curcas had been described for a pool of developing seeds instead of being characterized systematically. Moreover, cellular metabolic events are also controlled by protein–protein interactions, posttranslational protein modifications, and enzymatic activities which cannot be described by transcriptional profiling approaches alone. In this study, within the overall objective of profiling differential protein abundance in developing J. curcas seeds, we provide a setting of physiological data with dynamic proteomic and qRT-PCR analysis to characterize the metabolic pathways and the relationship between mRNA and protein patterns from early stage to seed filling during the seed development of J. curcas. The construction of J. curcas seed development proteome profiles will significantly increase our understanding of the process of seed development and provide a foundation to examine the dynamic changes of the metabolic network during seed development process and certainly suggest some clues to improve the lipid content of J. curcas seeds.

To gain a better understanding of the mechanism of rice (Oryza sativa L.) in response to salt stress, we performed a proteomics analysis of rice in response to 250 mM NaCl treatment using shoots of 3-day-old nascent seedlings. The changes of protein patterns were monitored with two-dimensional gel electrophoresis. Of 57 protein spots showing changes in abundance in response to salt stress, 52 were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The identified proteins were classified into eight functional categories. Several novel salt stress-responsive proteins, including protein synthesis inhibitor I, photosystem II stability/assembly factor HCF136, trigger factor-like protein and cycloartenol-C24-methyltransferase are upregulated upon salt stress. In order to figure out the different and similar molecular mechanism among salt and other stresses, regulation of some salt responsive proteins under other abiotic stress (cold and dehydration) and abscisic acid application was also analyzed. The possible molecular mechanism of rice seedlings in response to salinity and other stresses were discussed.

Physiological responses of two Jerusalem artichoke (Helianthus tuberosus L.) cultivars with different drought sensitivity to drought stress induced by polyethylene glycol (PEG) 6000 were investigated by characterizing water status, membrane lipid peroxidation, key antioxidant enzymes activity, and proline accumulation. It was observed that the drought-tolerant Jerusalem artichoke cv. Xiuyan maintained a relatively higher water status than the drought-sensitive cv. Yulin upon drought treatments. Meanwhile, lower levels of malondialdehyde (MDA) as well as higher levels of free proline occurred in cv. Xiuyan after 36 h drought treatments. Moreover, the activities of catalase (CAT) and superoxide dismutase (SOD) in cv. Xiuyan were higher than cv. Yulin after drought stress. These results indicated that drought sensitivities actually differ between Jerusalem artichoke cv. Xiuyan and cv. Yulin, and the cv. Xiuyan was more tolerant to drought stress caused by polyethylene glycol.

Two cultivars of Jerusalem artichoke (Helianthus tuberosus L.) differing in genotype, Red skin (cv. R., salt-tolerant but low-yield) and White skin (cv. W., salt-sensitive but high-yield), were used to investigate malondialdehyde (MDA) content and antioxidant enzyme activity changes in their roots under a hydroponic culture system with 250 mM NaCl. The results showed that MDA contents in roots of the two genotypes increased, but MDA content of cv. R. was higher than that of cv. W. Changes in all antioxidant enzymes in roots of both varieties exhibited a similar trend, namely increased initially and then decreased. However, there were still some differences existing between the two cultivars. In other words, activities of the other two antioxidant enzymes except catalase (CAT) and peroxidase (POD) in roots of cv. R. were less than controls at 48 h, while all others except ascorbate peroxidase (APX) in roots of cv. W. were greater than controls. The peak of superoxide dismutase (SOD) activity of cv. W. was observed to appear earlier than that of cv. R. CAT activity of cv. W. was significantly greater than the value of cv. R. and the latter showed a moderate trend. POD activity of cv. R. obtained the maximum at 6 h, whereas the peak of cv. W. displayed at 24 h. APX activity of cv. R. declined more than that of cv. W. These results suggested that there was a lower efficiency of scavenging reactive oxygen species (ROS) in cv. R. roots. Concomitantly, salt stress caused more severe damage to roots of cv. R. Antioxidant enzymes in roots were inadequate to elucidate salt-tolerance mechanisms of the whole plant.

Dehydration-responsive element-binding (DREB) proteins are important transcription factors in plant stress responses and signal transduction. Based on high-throughput sequencing results, a new cDNA sequence encoding an LcDREB3a transcription factor from the drought-resistant forage grass, Leymuschinensis, was isolated by RACE PCR. Sequence similarity analysis indicates that the gene product is active in the ABA-responsive pathway, and real-time PCR-based expression analysis shows the transcript accumulates in response to a variety of stress treatments. These results indicate that LcDREB3a is involved in both ABA-dependent and -independent signal transduction in the stress-responsive process of L.chinensis. The identity of the gene product as a DREB transcription factor is supported by observations of its nuclear localization when transiently expressed as a GFP fusion in onion epidermal cells. Furthermore, LcDREB3a is able to activate reporter gene expression, and the protein is shown to specifically bind to the conserved DRE element in a yeast one-hybrid assay. The transgenic expression of LcDREB3a in Arabidopsis causes no growth retardation and induces the increased expression of stress tolerance genes compared to control, resulting in improved drought and salt stress tolerance. Thus, LcDREB3a, encoding a stress-inducible DREB transcription factor, could enhance the abiotic stress tolerance of plants.

Enhanced salt tolerance of rice seedlings by abscisic acid (ABA) pretreatment was observed from phenotypic and physiological analyses. Total proteins from rice roots treated with ABA plus subsequent salt stress were analyzed by using proteomics method. Results showed that, 40 protein spots were uniquely upregulated in the seedlings under the condition of ABA pretreatment plus subsequent salt stress, whereas only 16 under the condition of salt treatment. About 78% (31 spots) of the 40 protein spots were only upregulated in the presence of the subsequent salt stress, indicating that plants might have an economical strategy to prevent energy loss under a false alarm. The results also showed that more enzymes involved in energy metabolism, defense, primary metabolism, etc. were upregulated uniquely in ABA-pretreated rice seedlings, suggesting more abundant energy supply, more active anabolism (nitrogen, nucleotide acid, carbohydrate, etc), and more comprehensive defense systems in ABA-pretreated seedlings than in salt stressed ones.

To understand oil mobilization in germinating seeds, we performed ultrastructural observation and proteomic analysis of endosperm in germinating Jatropha curcas seeds. Results showed that the oil mobilization was initiated during germination, and then the oil was consumed for early seedling development. The significant change in abundance of 50 protein spots during germination indicated that several pathways including β-oxidation, glyoxylate cycle, glycolysis, citric acid cycle, gluconeogenesis, and pentose phosphate pathway were involved in the oil mobilization.

To understand the responses of rice seedlings to different high-temperature stresses, seven-day-old rice seedlings were exposed to different high temperatures for 48 h, and the maximal quantum yield of PS II photochemistry measurements, ascorbate peroxidase activity assays and proteomic analyses in leaf tissue were performed. The results showed that when rice seedlings were exposed to high temperatures at 35 °C, 40 °C and 45 °C, the maximal quantum yield of photosystem II photochemistry, the activity of ascorbate peroxidase and the proteome changed greater at higher temperature. The proteomics analysis showed that proteins such as lignification-related proteins were regulated by high temperature and distinct proteins related to protection were up-regulated at different high temperatures. All the results indicated that different strategies were adopted at different levels of high temperature: the higher the temperature, the more protection machineries were involved. At 35 °C, some protective mechanisms were activated to maintain the photosynthetic capability. At 40 °C, antioxidative pathways were also active. When rice seedlings encountered high-temperature stress at 45 °C, in addition to those induced at 35 °C and 40 °C, heat shock proteins were effectively induced.

Physcomitrella patens is well known because of its importance in the study of plant systematics and evolution. The tolerance of P. patens for high-salinity environments also makes it an ideal candidate for studying the molecular mechanisms by which plants respond to salinity stresses. We measured changes in the proteome of P. patens gametophores that were exposed to high-salinity (250, 300, and 350 mM NaCl) using two-dimensional gel electrophoresis (2-DE) via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sixty-five protein spots were significantly altered by exposure to the high-salinity environment. Among them, 16 protein spots were down-regulated and 49 protein spots were up-regulated. These proteins were associated with a variety of functions, including energy and material metabolism, protein synthesis and degradation, cell defense, cell growth/division, transport, signal transduction, and transposons. Specifically, the up-regulated proteins were primarily involved in defense, protein folding, and ionic homeostasis. In summary, we outline several novel insights into the response of P. patens to high-salinity; (1) HSP70 is likely to play a significant role in protecting proteins from denaturation and degradation during salinity stress, (2) signaling proteins, such as 14-3-3 and phototropin, may work cooperatively to regulate plasma membrane H+-ATPase and maintain ion homeostasis, (3) an increase in photosynthetic activity may contribute to salinity tolerance, and (4) ROS scavengers were up-regulated suggesting that the antioxidative system may play a crucial role in protecting cells from oxidative damage following exposure to salinity stress in P. patens.

A cDNA clone, named JcERF, was isolated from Jatropha curcas seedlings (a woody oil plant). It was classified as an ERF subfamily member based on multiple sequence alignment and phylogenetic characterization. The deduced amino acid sequences of the JcERF clone showed no significant sequence similarity with other known ERF proteins except for the conserved AP2/EREBP DNA-binding domain. Expression of the JcERF gene was rapidly induced upon salinity, drought, ethylene and mechanical wounding treatments. No significant changes in the JcERF expression were observed under ABA stress. Gel retardation assay revealed that the JcERF protein could bind specifically to the GCC box as well as to the C/DRE motif. Also it can be inferred from the gel-shift that there is a possibility that the near sequence of the GCC box has an important effect on the DNA-binding activity. In yeast, the JcERF protein specifically bound to the DRE sequence and activated the transcription of two reporter genes His3 and LacZ driven by the DRE sequence. When fused to the LexA DNA-binding domain, the full-length JcERF functioned effectively as a trans-activator in the yeast one-hybrid assay. Overexpression of JcERF cDNA in transgenic Arabidopsis enhanced the salt and freezing tolerance. Meanwhile the seed germination of JcERF transgenic plants was not affected by various concentrations ABA in MS medium. Taken together, the results showed that JcERF functioned as a novel transcription factor and it exhibited a mechanism of plant response to environmental factors like the other AP2/EREBP regulons that also exist in tropical woody plants.

Comparison of protein expression in necrotic leaves and in normal leaves of wheat (Triticum aestivum L.) showed that the abundance of 39 proteins was changed significantly, and 26 of these proteins were identified. Analysis of the function of the differentially expressed proteins in the necrotic hybrid leaves showed that the cytoprotective heat shock proteins may be induced to maintain the integrity of other proteins, facilitating the intercellular transportation of vital cellular enzymes upon necrosis. The increased abundance of NADH dehydrogenase indicated that the chloroplasts of necrotic leaves were under photo-oxidative stress. In addition, the light and dark events of photosynthesis were impacted differently during necrosis. The increased abundance of the hormone-sensitive enzymes phospholipase and β-1,3-glucanase suggested that the level of plant hormones may be increased in necrotic leaves. Both DNA helicase and maturase K were down-regulated in necrotic leaves, indicating basic genetic processes, including replication, repair, recombination, transcription and translation, were impacted during necrosis. The results of this study give a comprehensive picture of the post-transcriptional response to necrosis in hybrid wheat leaves and serve as a platform for further characterization of gene function and regulation in wheat hybrid necrosis.

Jatropha curcas L. is an all-purpose biodiesel plant and is widely distributed in tropical and subtropical climates. It can grow well on poor quality soil which is not qualified for crop cultivation. This is very important for relieving land, food and energy crises. However, tropical and subtropical distribution limits the production of J. curcas seed. So it is valuable to know the molecular mechanism of J. curcas response to adverse abiotic environmental factors, especially freezing stress, in order to change the plant's characteristics. Until now there are just a few reports about J. curcas molecular biology. In this paper, we cloned and characterized a DNA binding protein from this plant, designated as JcDREB. Sequence analysis and yeast one-hybrid assays show that JcDREB can effectively function as a transcription factor of DREB protein family belonging to A-6 subgroup member. Expression patterns of JcDREB showed that it was induced by cold, salt and drought stresses, not by ABA. Over-expression of JcDREB in transgenic Arabidopsis exhibited enhanced salt and freezing stresses. Understanding the molecular mechanisms of J. curcas responses to environmental stresses, for example, high salinity, drought and low temperature, is crucial for improving their stress tolerance and productivity. This work provides more information about A-6 subgroup members of DREB subfamily.Highlights► A DNA binding protein JcDREB gene was isolated from biodiesel plant Jatropha curcas L. ► JcDREB was a transcription factor and induced by cold, salt and drought, but not by ABA. ► Transgenic Arabidopsis with over-expression JcDREB showed salt and freezing tolerance.

Plant-specific DNA-binding transcription factors with one finger (Dof) perform important roles in several biological processes. A yeast one-hybrid cDNA library of Jatropha curcas was used to identify Dof-type transcription factors. JcDof3, isolated from the library as a full-length cDNA, encoded a protein of 518 amino acids and contained a highly conserved Dof domain. Yeast one-hybrid systems and subcellular localization assays confirmed that JcDof3 was a typical transcription factor. In contrast to arrhythmic expression at basal level in etiolated cotyledons under continuous dark conditions, the circadian oscillations of JcDof3 transcripts were observed under long day, short day or continuous light regimes. A phylogenetic analysis showed that JcDof3 was clustered into the same clade with CYCLING DOF FACTOR (CDF), which interacts with F-box protein to regulate photoperiodic flowering. Moreover, a yeast two-hybrid assay showed that JcDof3 also interacted with F-box proteins. Our results suggest that JcDof3 is a circadian clock regulated gene, and might be involved in the flowering time regulation of J. curcas.Highlights► JcDof3 is a typical transcription factor. ► JcDof3 is a circadian clock regulated gene. ► JcDof3 transcripts exhibited daily oscillation in long day, short day or continuous light regimes. ► JcDof3 interacted with Arabidopsis F-box proteins.

Jatrpha curcas L., a non-model woody plant belonging to Euphorbiaceae family, is a promising economic plant due to the high oil content in seed and high tolerance to drought and salt stress. The embryo and endosperm of J. curcas seed differ in morphology, function and ploidy. To characterize the protein profiles of these two tissues, we have performed proteomic analysis with the dry mature J. curcas seeds. The data showed that the 2-DE profiles of endosperm and embryo were similar to each other. There are 66 differential proteins between the two seed tissues, in which 28 proteins distributed in 9 distinct functional classes, have been identified successfully in endosperm or embryo. The major groups of differential proteins are associated with metabolism (25%) and disease/defence (18%). Our results demonstrated that in the dry mature J. curcas seeds, the proteins involved in oil mobilization, signal transduction, transcription, protein synthesis, and cell cycle which are essential for the seed germination have occurred in endosperm and embryo, reflecting the fact that proteins required for germination are already present in the dry mature seed.Highlights► We compare the endosperm and embryo of dry mature J. curcas seeds basing on proteomics. ► The 2-DE profiles of endosperm and embryo from J. curcas dry mature seed are similar. ► 28 differential proteins between endosperm and embryo have been identified by LC–MS/MS. ► Some proteins required for germination have been present in the dry mature J. curcas seed.

Soil salinization has become a severe global problem and salinity is one of the most severe abiotic stresses inhibiting growth and survival of mycorrhizal fungi and their host plants. Salinity tolerance of ectomycorrhizal fungi and survival of ectomycorrhizal inocula is essential to reforestation and ecosystem restoration in saline areas. Proteomic changes of an ectomycorrhizal fungus, Boletus edulis, when exposed to salt stress conditions (4 % NaCl, w/v) were determined using two-dimensional electrophoresis (2DE) and mass spectrometry (MS) techniques. Twenty-two protein spots, 14 upregulated and 8 downregulated, were found changed under salt stress conditions. Sixteen changed protein spots were identified by nanospray ESI Q-TOF MS/MS and liquid chromatography MS/MS. These proteins were involved in biosynthesis of methionine and S-adenosylmethionine, glycolysis, DNA repair, cell cycle control, and general stress tolerance, and their possible functions in salinity adaptation of Boletus edulis were discussed.

Grazing is accompanied by a multitude of processes including wounding, saliva deposition, and defoliation. Previous studies have focused on the effects of the grazing or clipping intensity on plant regrowth, survival, and composition in the grassland. However, the impact of saliva deposition on plants is poorly understood. In this study, rice was used as a model plant to study the differentially expressed proteins after ovine saliva treatment. The shoots of 2-week-old seedlings were crosscut and the lower parts were daubed with ovine saliva at the cut surface. After 2, 6, 12 and 24 h, proteomics analysis was performed using proteins extracted from the saliva-treated shoots. The results showed that proteins involved in multiple pathways were differentially expressed in response to ovine saliva, including catalase (CAT), peroxiredoxin (Prx), ATP synthase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Moreover, real-time quantitative reverse-transcription-PCR (RT-PCR) data showed that most of the genes were also regulated at the transcript level. Our results indicate the ovine saliva induces an early response in the rice seedling by stress-related pathways. This study provides information about the response of rice seedlings to ovine saliva at the protein level.