Comparative Transcriptome Analysis Reveals the Transcriptional Alterations in Growth- and Development-Related Genes in Sweet Potato Plants Infected and Non-Infected by SPFMV, SPV2, and SPVG.

Field co-infection of multiple viruses results in considerable losses in the yield and quality of storage roots in sweet potato. However, little is known about the molecular mechanisms underlying developmental disorders of sweet potato subjected to co-infection by multiple viruses. Here, a comparative transcriptomic analysis was performed to reveal the transcriptional alterations in sweet potato plants infected (VCSP) and non-infected (VFSP) by Sweet potato mild mottle virus (SPFMV), Sweet potato virus Y (SPV2) and Sweet potato virus G (SPVG). A total of 1580 and 12,566 differentially expressed genes (DEGs) were identified in leaves and storage roots of VFSP and VCSP plants, respectively. In leaves, 707 upregulated and 773 downregulated genes were identified, whereas 5653 upregulated and 6913 downregulated genes were identified in storage roots. Gene Ontology (GO) classification and pathway enrichment analysis showed that the expression of genes involved in chloroplast and photosynthesis and brassinosteroid (BR) biosynthesis in leaves and the vitamin biosynthetic process in storage roots was inhibited by co-infection of three viruses: SPFMV, SPV2, and SPVG. This was likely closely related to better photosynthesis and higher contents of Vitamin C (Vc) in storage roots of VFSP than that of VCSP. While some genes involved in ribosome and secondary metabolite-related pathways in leaves and alanine, aspartate, and glutamate metabolism in storage roots displayed higher expression in VCSP than in VFSP. Quantitative real-time PCR analysis demonstrated that the expression patterns of 26 DEGs, including 16 upregulated genes and 10 downregulated genes were consistent with the RNA-seq data from VFSP and VCSP. Taken together, this study integrates the results of morphology, physiology, and comparative transcriptome analyses in leaves and storage roots of VCSP and VFSP to reveal transcriptional alterations in growth- and development-related genes, providing new insight into the molecular mechanisms underlying developmental disorders of sweet potato subjected to co-infection by multiple viruses.

Comparative transcriptome analysis reveals the transcriptional alterations in heat-resistant and heat-sensitive sweet maize (Zea mays L.) varieties under heat stress.

BACKGROUND: Despite the heat-related physiology and heat-shock proteins in maize have been extensively studied, little is known about the transcriptome profiling of how the maize varieties with different genotypes responding to high temperatures. Seedling mortality of Xiantian 5 (XT) is significantly lower than that of Zhefengtian (ZF) when exposed to high temperature (42 °C for 6 h) and followed by a recovery growth (25 °C for one week). Therefore, we performed a transcriptome analysis using the total RNA extracted from the leaves of XT and ZF that were previously subjected to heat stress at 42 °C for 0 h, 0.5 h, and 3 h, respectively. RESULTS: A total of 516 commonly up-regulated and 1,261 commonly down-regulated genes were identified among XT/ZF, XT0.5/ZF0.5 and XT3/ZF3 using transcriptome analysis. Gene Ontology classification of the 516 up-regulated genes showed that their encoded proteins were significantly assigned to 18 cellular components, and were classified into 9 functional categories, and were involved in 9 biological processes. Most of proteins encoded by up-regulated genes were localized in chloroplast and its structural components, and involved in multiple biological processes associated with photosynthesis, indicating that these chloroplast proteins play an important role in increasing heat tolerance in sweet maize. While the proteins encoded by 1,261 down-regulated genes were significantly assigned to 31 cellular components, and were classified into 3 functional categories, and were involved in 9 biological processes. Interestingly, these proteins were involved in a series of biological processes from gene expression to translation, suggesting that lowering these processes may contribute to improved heat resistance in sweet maize. The up-regulated genes were identified to be involved in 36 distinct metabolic pathways, of which the most significant ones was secondary metabolite biosynthetic pathway. While the down-regulated genes were identified to be involved in 23 distinct metabolic pathways, of which the most significant ones were found in ribosome. Quantitative real-time PCR analysis demonstrated that 5 genes involved in the biosynthesis of secondary metabolites and photosynthesis in XT have higher abundance than those in ZF, whereas 5 ribosome genes in XT showed lower abundance than those in ZF. In addition, heat-tolerant sweet maize may keep at lower growth level than heat-sensitive one through dowregulating expression of genes related to zeatin and brassinosteroid biosynthesis to better regulate heat stress responses. CONCLUSIONS: Comparative transcriptomic profiling reveals transcriptional alterations in heat-resistant and heat-sensitive sweet maize varieties under heat stress, which provides a new insight into underlying molecular mechanism of maize in response to heat stress.

A zinc finger protein, interacted with cyclophilin, affects root development via IAA pathway in rice.

The plant hormone auxin plays a crucial role in lateral root development. To better understand the molecular mechanisms underlying lateral root formation, an auxin-responsive gene OsCYP2 (Os02g0121300) was characterized from rice. Compared to the wild type, OsCYP2-RNAi (RNA interference) lines exhibited distinctive defects in lateral root development. Yeast two-hybrid and glutathione S-transferase pull-down results confirmed that OsCYP2 interacted with a C2HC-type zinc finger protein (OsZFP, Os01g0252900) which is located in the rice nucleus. T2 OsZFP-RNAi lines had significantly fewer lateral roots than did wild-type plants, which suggests a role for OsCYP2 and OsZFP in regulating lateral root development. Quantitative real-time polymerase chain reaction showed that the expression of certain Aux/IAA (auxin/indole-3-acetic acid) genes was altered in OsCYP2- and OsZFP-RNAi lines in response to IAA. These findings imply that OsCYP2 and OsZFP participate in IAA signal pathways controlling lateral root development. More importantly, OsIAA11 showed functional redundancy not only in OsCYP2-RNAi lines but also in OsZFP-RNAi lines, which provides important clues for the elucidation of mechanisms controlling lateral root development in response to auxin.

Comparative proteomic analysis reveals alterations in development and photosynthesis-related proteins in diploid and triploid rice.

BACKGROUND: Polyploidy has pivotal influences on rice (Oryza sativa L.) morphology and physiology, and is very important for understanding rice domestication and improving agricultural traits. Diploid (DP) and triploid (TP) rice shows differences in morphological parameters, such as plant height, leaf length, leaf width and the physiological index of chlorophyll content. However, the underlying mechanisms determining these morphological differences are remain to be defined. To better understand the proteomic changes between DP and TP, tandem mass tags (TMT) mass spectrometry (MS)/MS was used to detect the significant changes to protein expression between DP and TP. RESULTS: Results indicated that both photosynthesis and metabolic pathways were highly significantly associated with proteomic alteration between DP and TP based on biological process and pathway enrichment analysis, and 13 higher abundance chloroplast proteins involving in these two pathways were identified in TP. Quantitative real-time PCR analysis demonstrated that 5 of the 13 chloroplast proteins ATPF, PSAA, PSAB, PSBB and RBL in TP were higher abundance compared with those in DP. CONCLUSIONS: This study integrates morphology, physiology and proteomic profiling alteration of DP and TP to address their underlying different molecular mechanisms. Our finding revealed that ATPF, PSAA, PSAB, PSBB and RBL can induce considerable expression changes in TP and may affect the development and growth of rice through photosynthesis and metabolic pathways.

Cell wall proteomics contributes to explore the functional proteins of Brachypodium distachyon grains.

The plant cell wall is the first barrier in response to external stimuli and cell wall proteins (CWPs) can play an important role in the modulation of plant growth and development. In the past 10 years, the plant cell wall proteomics has increasingly become a very active research filed, which provides a broader understanding of CWPs for people. The cell wall proteome of Arabidopsis, rice, and other model plants has begun to take shape, and proteomic technology has become an effective way to identify the candidate functional CWPs in large scale. The challenging work of Francin-Allami et al. (Proteomics 2015, 15, 2296-2306) is a vital step toward building the most extensive cell wall proteome of a monocot species. They identified 299 cell wall proteins in Brachypodium distachyon grains, and also compared the grain cell wall proteome with those of B. distachyon culms and leaves, which provides a new perspective for further explaining the plant cell wall structures and remodeling mechanism.

Proteomic dissection of plant responses to various pathogens.

During their growth and development, plants are vulnerable to the effects of a variety of pathogens. Proteomics technology plays an important role in research studies of plant defense mechanisms by mining the expression changes of proteins in response to various biotic stresses. This review article provides a comprehensive overview of the latest developments in international proteomic research on plant biotic stress. It summarizes the methods commonly used in plant proteomic research to investigate biotic stress, analyze the protein responses of plants in adverse conditions, and reviews the applications of proteomics combined with transgenic technology in plant protection.

Proteomic identification of OsCYP2, a rice cyclophilin that confers salt tolerance in rice (Oryza sativa L.) seedlings when overexpressed.

BACKGROUND: High Salinity is a major environmental stress influencing growth and development of rice. Comparative proteomic analysis of hybrid rice shoot proteins from Shanyou 10 seedlings, a salt-tolerant hybrid variety, and Liangyoupeijiu seedlings, a salt-sensitive hybrid variety, was performed to identify new components involved in salt-stress signaling. RESULTS: Phenotypic analysis of one protein that was upregulated during salt-induced stress, cyclophilin 2 (OsCYP2), indicated that OsCYP2 transgenic rice seedlings had better tolerance to salt stress than did wild-type seedlings. Interestingly, wild-type seedlings exhibited a marked reduction in maximal photochemical efficiency under salt stress, whereas no such change was observed for OsCYP2-transgenic seedlings. OsCYP2-transgenic seedlings had lower levels of lipid peroxidation products and higher activities of antioxidant enzymes than wild-type seedlings. Spatiotemporal expression analysis of OsCYP2 showed that it could be induced by salt stress in both Shanyou 10 and Liangyoupeijiu seedlings, but Shanyou 10 seedlings showed higher OsCYP2 expression levels. Moreover, circadian rhythm expression of OsCYP2 in Shanyou 10 seedlings occurred earlier than in Liangyoupeijiu seedlings. Treatment with PEG, heat, or ABA induced OsCYP2 expression in Shanyou 10 seedlings but inhibited its expression in Liangyoupeijiu seedlings. Cold stress inhibited OsCYP2 expression in Shanyou 10 and Liangyoupeijiu seedlings. In addition, OsCYP2 was strongly expressed in shoots but rarely in roots in two rice hybrid varieties. CONCLUSIONS: Together, these data suggest that OsCYP2 may act as a key regulator that controls ROS level by modulating activities of antioxidant enzymes at translation level. OsCYP2 expression is not only induced by salt stress, but also regulated by circadian rhythm. Moreover, OsCYP2 is also likely to act as a key component that is involved in signal pathways of other types of stresses-PEG, heat, cold, or ABA.

Comparative proteomics analysis of proteins expressed in the I-1 and I-2 internodes of strawberry stolons.

BACKGROUND: Strawberries (Fragaria ananassa) reproduce asexually through stolons, which have strong tendencies to form adventitious roots at their second node. Understanding how the development of the proximal (I-1) and distal (I-2) internodes of stolons differ should facilitate nursery cultivation of strawberries. RESULTS: Herein, we compared the proteomic profiles of the strawberry stolon I-1 and I-2 internodes. Proteins extracted from the internodes were separated by two-dimensional gel electrophoresis, and 164 I-1 protein spots and 200 I-2 protein spots were examined further. Using mass spectrometry and database searches, 38 I-1 and 52 I-2 proteins were identified and categorized (8 and 10 groups, respectively) according to their cellular compartmentalization and functionality. Many of the identified proteins are enzymes necessary for carbohydrate metabolism and photosynthesis. Furthermore, identification of proteins that interact revealed that many of the I-2 proteins form a dynamic network during development. Finally, given our results, we present a mechanistic scheme for adventitious root formation of new clonal plants at the second node. CONCLUSIONS: Comparative proteomic analysis of I-1 and I-2 proteins revealed that the ubiquitin-proteasome pathway and sugar-hormone pathways might be important during adventitious root formation at the second node of new clonal plants.

Proteomic analysis of 2-chloroethanol extracts of rice (Oryza sativa L.) seeds.

Ethanol-soluble proteins, including prolamins, are one of the most important seed proteins in rice (Oryza sativa L.). However, little is known about the proteomic profile of ethanol-soluble protein fraction extracted from rice grain. In this work, the differential profile of ethanol-soluble proteins extracted by 2-chloroethanol and ethanol has been documented. Proteome analysis utilizing LC-MS/MS identified a total of 64 unique proteins in the 2-chloroethanol extract of rice seeds. The majority of these proteins had low molecular weight ranging from 10 to 25 kD and isoelectric point (pI) in mid-acidic (pH 5-pH 7) and mid-basic (pH 7-pH 9) ranges. Database searches combined with transmembrane domain (TMD) analysis revealed that >70% of identified proteins were hydrophobic, i.e., had at least one TMD. Gene ontology classification and enrichment analysis showed that the identified proteins were involved in13 types of biological processes, 5 types of cell components, and 17 types of molecular functions. These results were significant based on the hyper p-value of <0.05. The most frequent categories of biological processes, cell components, and molecular functions were, respectively, type I hypersensitivity, extracellular space and extracellular region, and serine-type endopeptidase inhibitor activity. Interestingly, in addition to seed storage proteins such as prolamins and glutelins, certain allergen proteins, protease inhibitors, and lipid transfer proteins were identified in the extracts. Together, the collected data provide novel insights into the protein profile of 2-chloroethanol extract of rice seeds.

Nystose regulates the response of rice roots to cold stress via multiple signaling pathways: A comparative proteomics analysis.

Small fructans improve plant tolerance for cold stress. However, the underlying molecular mechanisms are poorly understood. Here, we have demonstrated that the small fructan tetrasaccharide nystose improves the cold stress tolerance of primary rice roots. Roots developed from seeds soaked in nystose showed lower browning rate, higher root activity, and faster growth compared to seeds soaked in water under chilling stress. Comparative proteomics analysis of nystose-treated and control roots identified a total of 497 differentially expressed proteins. GO classification and KEGG pathway analysis documented that some of the upregulated differentially expressed proteins were implicated in the regulation of serine/threonine protein phosphatase activity, abscisic acid-activated signaling, removal of superoxide radicals, and the response to oxidative stress and defense responses. Western blot analysis indicated that nystose promotes the growth of primary rice roots by increasing the level of RSOsPR10, and the cold stress-induced change in RSOsPR10levelis regulated by jasmonate, salicylic acid, and abscisic acid signaling pathways in rice roots. Furthermore, OsMKK4-dependentmitogen-activated protein kinase signaling cascades may be involved in the nystose-induced cold tolerance of primary rice roots. Together, these results indicate that nystose acts as an immunostimulator of the response to cold stress by multiple signaling pathways.

[Advances in plant proteomics--I. Key techniques of proteome].

With the completion of genome sequences of the model plants, such as rice (Oryza sativa L.) and Arabidopsis thaliana, it has come into plant functional genomics era, which becomes a hard base of the appearance and development of plant proteomics. This review focuses on the background of proteomics, concept of proteomics and key techniques of proteomics. The key techniques of proteomics include separation, such as 2-DE (Two-Dimensional Electrophoresis), RP-HPLC (Reverse Phase High Performance Liquid Chromatography) and SELDI (Surface Enhanced Laser Desorption/ Ionization) protein chip, mass spectrometry, such as MALDI-TOF-MS (Matrix Assisted Laser Desorption/Ionization-Time Of Flight-Mass Spectrometry) and ESI-MS/MS (Electrospray Ionization Mass Spectrometry/Mass Spectrometry), databases related to proteomics, quantitative proteome, TAP (Tandem Affinity Purification) and yeast two-hybrid system. Challenges and prospects of proteomic techniques are discussed.

[Advances in plant proteomics. II. Application of proteome techniques to plant biology research].

Proteome techniques have widely been applied to the fields of plant genetics, plant development, and plant physiology and ecology to investigate plant genetic diversity, plant development such as seed maturation and germination processes, differentiation of plant tissue and organ, separation and functional identification of novel component of various organells, mechanisms of plant adapted to abiotic or biotic stresses including high temperature, low temperature, high salt, drought, and pathogens and insects, and interaction of plant with microbe. The prospects of plant proteomics are discussed.

Global analysis of lysine acetylation in strawberry leaves.

Protein lysine acetylation is a reversible and dynamic post-translational modification. It plays an important role in regulating diverse cellular processes including chromatin dynamic, metabolic pathways, and transcription in both prokaryotes and eukaryotes. Although studies of lysine acetylome in plants have been reported, the throughput was not high enough, hindering the deep understanding of lysine acetylation in plant physiology and pathology. In this study, taking advantages of anti-acetyllysine-based enrichment and high-sensitive-mass spectrometer, we applied an integrated proteomic approach to comprehensively investigate lysine acetylome in strawberry. In total, we identified 1392 acetylation sites in 684 proteins, representing the largest dataset of acetylome in plants to date. To reveal the functional impacts of lysine acetylation in strawberry, intensive bioinformatic analysis was performed. The results significantly expanded our current understanding of plant acetylome and demonstrated that lysine acetylation is involved in multiple cellular metabolism and cellular processes. More interestingly, nearly 50% of all acetylated proteins identified in this work were localized in chloroplast and the vital role of lysine acetylation in photosynthesis was also revealed. Taken together, this study not only established the most extensive lysine acetylome in plants to date, but also systematically suggests the significant and unique roles of lysine acetylation in plants.

Differential Proteomic Analysis Using iTRAQ Reveals Alterations in Hull Development in Rice (Oryza sativa L.).

Rice hull, the outer cover of the rice grain, determines grain shape and size. Changes in the rice hull proteome in different growth stages may reflect the underlying mechanisms involved in grain development. To better understand these changes, isobaric tags for relative and absolute quantitative (iTRAQ) MS/MS was used to detect statistically significant changes in the rice hull proteome in the booting, flowering, and milk-ripe growth stages. Differentially expressed proteins were analyzed to predict their potential functions during development. Gene ontology (GO) terms and pathways were used to evaluate the biological mechanisms involved in rice hull at the three growth stages. In total, 5,268 proteins were detected and characterized, of which 563 were differentially expressed across the development stages. The results showed that the flowering and milk-ripe stage proteomes were more similar to each other (r=0.61) than either was to the booting stage proteome. A GO enrichment analysis of the differentially expressed proteins was used to predict their roles during rice hull development. The potential functions of 25 significantly differentially expressed proteins were used to evaluate their possible roles at various growth stages. Among these proteins, an unannotated protein (Q7X8A1) was found to be overexpressed especially in the flowering stage, while a putative uncharacterized protein (B8BF94) and an aldehyde dehydrogenase (Q9FPK6) were overexpressed only in the milk-ripe stage. Pathways regulated by differentially expressed proteins were also analyzed. Magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclase (Q9SDJ2), and two magnesium-chelatase subunits, ChlD (Q6ATS0), and ChlI (Q53RM0), were associated with chlorophyll biosynthesis at different developmental stages. The expression of Q9SDJ2 in the flowering and milk-ripe stages was validated by qRT-PCR. The 25 candidate proteins may be pivotal markers for controlling rice hull development at various growth stages and chlorophyll biosynthesis pathway related proteins, especially magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclase (Q9SDJ2), may provide new insights into the molecular mechanisms of rice hull development and chlorophyll associated regulation.

Proteomic analysis of strawberry leaves infected with Colletotrichum fragariae.

Understanding the defense mechanisms used by anthracnose-resistant strawberries against Colletotrichum infection is important for breeding purposes. To characterize cell responses to Colletotrichum infection, proteomes from strawberry seedling leaves that had or had not been infected with Colletotrichum fragariae were characterized at different time points post infection by 2-DE and by MALDI-TOF/TOF MS/MS and database-searching protein identification. Mass spectrometry identified 49 differentially expressed proteins with significant intensity differences (>1.5-fold, p<0.05) in mock- and C. fragariae-infected leaves at least at one time point. Notably, 2-DE analysis revealed that C. fragariae infection increased the expression of well-known and novel pathogen-responsive proteins whose expression patterns tended to correlate with physiological changes in the leaves. Quantitative real-time PCR was used to examine the transcriptional profiles of infected and uninfected strawberry leaves, and western blotting confirmed the induction of ß-1,3-glucanase and a low-molecular-weight heat shock protein in response to C. fragariae infection. During the late phase of infection, proteins involved in the Calvin cycle and glycolysis pathway had suppressed expression. The abundance changes, putative functions, and participation in physiological reactions for the identified proteins produce a pathogen-responsive protein network in C. fragariae-infected strawberry leaves. Together, these findings increase our knowledge of pathogen resistance mechanisms, especially those found in non-model plant species.