Comparative Proteomic Analyses Provide New Insights into Low Phosphorus Stress Responses in Maize Leaves

Phosphorus deficiency limits plant growth and development. To better understand the mechanisms behind how maize responds to phosphate stress, we compared the proteome analysis results of two groups of maize leaves that were treated separately with 1,000 µM (control, +P) and 5 µM of KH₂PO₄ (intervention group, −P) for 25 days. In total, 1,342 protein spots were detected on 2-DE maps and 15.43% had changed (P<0.05; ≥1.5-fold) significantly in quantity between the +P and −P groups. These proteins are involved in several major metabolic pathways, including photosynthesis, carbohydrate metabolism, energy metabolism, secondary metabolism, signal transduction, protein synthesis, cell rescue and cell defense and virulence. The results showed that the reduction in photosynthesis under low phosphorus treatment was due to the down-regulation of the proteins involved in CO₂ enrichment, the Calvin cycle and the electron transport system. Electron transport and photosynthesis restrictions resulted in a large accumulation of peroxides. Maize has developed many different reactive oxygen species (ROS) scavenging mechanisms to cope with low phosphorus stress, including up-regulating its antioxidant content and antioxidase activity. After being subjected to phosphorus stress over a long period, maize may increase its internal phosphorus utilization efficiency by altering photorespiration, starch synthesis and lipid composition. These results provide important information about how maize responds to low phosphorus stress.     

Comparative proteomic analysis of <Emphasis Type="Italic">Phalaenopsis</Emphasis> leaves in the vegetative and flowering phase

Phalaenopsis, an epiphytic crassulacean acid metabolism (CAM) plant, requires moderate variations of day/night temperatures for flowering. In this study, changes in chlorophyll content, chlorophyll fluorescence, sugar components, titratable acidity and soluble protein content in Phalaenopsis leaves during flowering were observed. Comparative proteomic analysis of Phalaenopsis leaves in the vegetative and flowering phase was performed for the first time using iTRAQ (isobaric tags for relative and absolute quantification). A total of 126 proteins were differentially expressed in Phalaenopsis leaves. Analysis of potential functions revealed that the major categories of predicted function of the up-regulated proteins were protein destination (27 %), photosynthesis (15.9 %), primary metabolism (14.3 %) and defense (12.7 %) in the flowering phase, while the major categories of predicted function of the down-regulated proteins were protein destination (33.3 %), primary metabolism (20.6 %), transportation (14.3 %) and signal transduction (11.1 %). Proteome profile analysis indicated that the proteome changes were consistent with changes in sugar and protein metabolites. Some novel proteins were differentially expressed, most of which were identified as signaling proteins, including 14-3-3 proteins, fibrillin, rapid alkalinization factors (RALF), the Ras-related protein RABB1c, calreticulin and calmodulin. Histone, importin alpha, multidrug resistance proteins and the ABC transporters were also differentially expressed. These results provide insights into the mechanisms that regulate flowering in complex flowering plants.     

Identification of changes in Triticum aestivum L. leaf proteome in response to drought stress by 2D-PAGE and MALDI-TOF/TOF mass spectrometry

Drought is an abiotic stress that strongly influences plant growth, development and productivity. To gain a better understanding of the drought-stress responses at physiological and molecular level in wheat plants (Triticum aestivum cv. KTC86211), we performed a comparative physiological and proteomics analysis. Eight-day-old wheat seedlings were treated with polyethylene glycol-simulated drought stress for 0, 24, 48 and 72 h. Drought treatment resulted in alterations of morphology, increased relative electrolyte leakage and reduced length and weight on leaf and root. Stress-induced proteome changes were analyzed by two-dimensional gel electrophoresis in conjunction with MALDI-TOF/TOF. Twenty-three spots differed significantly between control and treated plants following 48 h of drought stress, with 19 upregulated, and 4 downregulated, in leaf tissues. All of the differentially expressed protein spots were identified, revealing that the majority of proteins altered by drought treatment were involved in reactive oxygen species scavenging enzymes and photosynthesis. Other proteins identified were involved in protein metabolism, cytoskeleton structure, defense response, acid metabolism and signal transduction. All proteins might contribute cooperatively to reestablish cellular homeostasis under drought stress. The present study not only provides new insights into the mechanisms of acclimation and tolerance to drought stress in wheat plants, but also provides clues for improving wheat’s drought tolerance through breeding or genetic engineering.     

Differential proteomic analysis of Trichoplusia ni cells after continuous selection with activated Cry1Ac toxin

Development of insect resistance to Bacillus thuringiensis (Bt) toxins threatens the sustained successful application of Bt-based biological control tactics. Multi-mechanisms of resistance have been proposed, such as alteration of toxin-binding proteins, changes of proteases in midgut and so on. The other responses of the Cry1Ac-selected insects might also contribute to the evolution of resistance. Here, the Cry1Ac-selected Trichoplusia ni TnH5 cells with high resistance were subjected to analysis of proteome and the differentially expressed proteins were identified using mass spectrometry. The differential proteins included transporter, molecular chaperon, structural molecules and many other molecules involved in protein metabolism, signal transduction, nucleotide binding, lipid biosynthesis, carbohydrates metabolism and energy production, suggesting that a complex mechanisms involved in the development of insect resistance to Bt Cry1Ac toxins at cellular levels. The decrease of protein synthesis, changes of signal transduction, more rapid energy production, the enhanced lipid synthesis and the decline of possible Cry1Ac-binding proteins in cytoplasm and other events might contribute to the development of resistance in the selected cells. Our results provide some new cues for understanding the mechanism of Bt resistance.     

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.     

杜仲叶片干旱胁迫响应相关差异蛋白的筛选与鉴定

为研究干旱胁迫下杜仲幼苗生理生化及分子响应机制,利用盆栽试验,通过持续（3、6、9、12、15 d）干旱胁迫处理和复水处理,研究杜仲幼苗的生理响应特性。同时,通过研究对照与处理15 d后的杜仲幼苗差异蛋白质组,分析杜仲幼苗对干旱胁迫的分子响应机制。结果表明,随着干旱处理时间的延长,杜仲叶片的水分饱和亏逐渐增加;光合速率、蒸腾速率、胞间二氧化碳浓度、气孔导度均逐渐减小;SOD、POD、CAT活性呈先上升后降低的趋势;丙二醛含量则呈现先上升,然后下降,最后又上升的变化特点;脯氨酸和可溶性糖含量的变化趋势与SOD等活性变化一致,前期上升,后期下降。在复水后,杜仲叶片的所有指标均有所恢复,但未达到干旱处理之前的水平。表明干旱胁迫影响了杜仲叶片的正常生长代谢。通过对干旱处理15 d后杜仲叶片总蛋白进行双向电泳分离和MALDI-TOF-TOF生物质谱鉴定,成功鉴定出36个差异表达蛋白,其中22个上调表达,14个下调表达。对36个差异蛋白进行功能分析发现,这些差异蛋白主要涉及信号传导、光合作用、碳代谢、能量代谢、次级代谢物合成、抗氧化保护酶、氨基酸代谢和蛋白质代谢。推测杜仲为适应干旱胁迫,首先是感应干旱胁迫信号,并传导至细胞内,影响杜仲叶片中光合作用、次级代谢物合成和蛋白质的生物合成;同时,通过过氧化物保护酶的作用,将过多活性氧加以清除;另一方面,则是通过增强糖酵解,磷酸戊糖途径,产生能量供杜仲正常生长所需。从生理机制来看,杜仲叶片同过增加胞内脯氨酸、可溶性糖含量,降低胞内渗透势,减少叶片中水分损失,与氨基酸合成和糖代谢相关蛋白的表达量上升的结果一致。     

A comprehensive differential proteomic study of nitrate deprivation in Arabidopsis reveals complex regulatory networks of plant nitrogen responses

Nitrogen (N) is an important nutrient and signal for plant growth and development. However, to date, our knowledge of how plants sense and transduce the N signals is very limited. To better understand the molecular mechanisms of plant N responses, we took two-dimensional gel-based proteomic and phosphoproteomic approaches to profile the proteins with abundance and phosphorylation state changes during nitrate deprivation and recovery in the model plant Arabidopsis thaliana. After 7-day-old seedlings were N-deprived for up to 48 h followed by 24 h recovery, a total of 170 and 38 proteins were identified with significant changes in abundance and phosphorylation state, respectively. Bioinformatic analyses implicate these proteins in diverse cellular processes including N and protein metabolisms, photosynthesis, cytoskeleton, redox homeostasis, and signal transduction. Functional studies of the selected nitrate-responsive proteins indicate that the proteasome regulatory subunit RPT5a and the cytoskeleton protein Tubulin alpha-6 (TUA6) play important roles in plant nitrate responses by regulating plant N use efficiency (NUE) and low nitrate-induced anthocyanin biosynthesis, respectively. In conclusion, our study provides novel insights into plant responses to nitrate at the proteome level, which are expected to be highly useful for dissecting the N response pathways in higher plants and for improving plant NUE.     

The Comparatively Proteomic Analysis in Response to Cold Stress in Cassava Plantlets

Cassava (Manihot esculenta Crantz) is a tropical root crop and sensitive to low temperature. However, it is poorly to know how cassava can modify its metabolism and growth to adapt to cold stress. An investigation aimed at a better understanding of cold-tolerant mechanism of cassava plantlets was carried out with the approaches of physiology and proteomics in the present study. The principal component analysis of seven physiological characteristics showed that electrolyte leakage (EL), chlorophyll content, and malondialdehyde (MDA) may be the most important physiological indexes for determining cold-resistant abilities of cassava. The genome-wide proteomic analysis showed that 20 differential proteins had the same patterns in the apical expanded leaves of cassava SC8 and Col1046. They were mainly related to photosynthesis, carbon metabolism and energy metabolism, defense, protein synthesis, amino acid metabolism, signal transduction, structure, detoxifying and antioxidant, chaperones, and DNA-binding proteins, in which 40 % were related with photosynthesis. The remarkable variation in photosynthetic activity and expression level of peroxiredoxin is closely linked with expression levels of proteomic profiles. Moreover, analysis of differentially expressed proteins under cold stress is an important step toward further elucidation of mechanisms of cold stress resistance.     

A proteomic approach to study pea (Pisum sativum) responses to powdery mildew (Erysiphe pisi)

As a global approach to gain a better understanding of the mechanisms involved in pea resistance to Erysiphe pisi, changes in the leaf proteome of two pea genotypes differing in their resistance phenotype were analyzed by a combination of 2-DE and MALDI-TOF/TOF MS. Leaf proteins from control non-inoculated and inoculated susceptible (Messire) and resistant (JI2480) plants were resolved by 2-DE, with IEF in the 5-8 pH range and SDS-PAGE on 12% gels. CBB-stained gels revealed the existence of quantitative and qualitative differences between extracts from: (i) non-inoculated leaves of both genotypes (77 spots); (ii) inoculated and non-inoculated Messire leaves (19 spots); and (iii) inoculated and non-inoculated JI2480 leaves (12 spots). Some of the differential spots have been identified, after MALDI-TOF/TOF analysis and database searching, as proteins belonging to several functional categories, including photosynthesis and carbon metabolism, energy production, stress and defense, protein synthesis and degradation and signal transduction. Results are discussed in terms of constitutive and induced elements involved in pea resistance against Erysiphe pisi.     

Comparative protein profiles of <Emphasis Type="Italic">Butea superba</Emphasis> tubers under seasonal changes

Seasonal changes are major factors affecting environmental conditions which induce multiple stresses in plants, leading to changes in protein relative abundance in the complex cellular plant metabolic pathways. Proteomics was applied to study variations in proteome composition of Butea. superba tubers during winter, summer and rainy season throughout the year using two-dimensional polyacrylamide gel electrophoresis coupled with a nanoflow liquid chromatography coupled to electrospray ionization quadrupole-time-of-flight tandem mass spectrometry. A total of 191 protein spots were identified and also classified into 12 functional groups. The majority of these were mainly involved in carbohydrate and energy metabolism (30.37 %) and defense and stress (18.32 %). The results exhibited the highest numbers of identified proteins in winter-harvested samples. Forty-five differential proteins were found in different seasons, involving important metabolic pathways. Further analysis indicated that changes in the protein levels were due mainly to temperature stress during summer and to water stress during winter, which affected cellular structure, photosynthesis, signal transduction and homeostasis, amino-acid biosynthesis, protein destination and storage, protein biosynthesis and stimulated defense and stress mechanisms involving glycolytic enzymes and relative oxygen species catabolizing enzymes. The proteins with differential relative abundances might induce an altered physiological status within plant tubers for survival. The work provided new insights into the better understanding of the molecular basis of plant proteomes and stress tolerance mechanisms, especially during seasonal changes. The finding suggested proteins that might potentially be used as protein markers in differing seasons in other plants and aid in selecting B. superba tubers with the most suitable medicinal properties in the future.