Overexpression of a Bcl-2-associated athanogene SlBAG9 negatively regulates high-temperature response in tomato.

The Bcl-2-associated athanogene (BAG) gene is a multi-functional family of co-chaperones regulator, modulating plant stress response. Our previous study revealed that the SlBAG9 of tomato (Solanum lycopersicum) had the higher expression level induced by high-temperature (HT) at the transcriptional and protein levels, but its biological function was still unclear. Here, we conducted an in-depth analysis of SlBAG9. SlBAG9 protein was not located in the mitochondria but in the cytoplasm and nucleus. Many cis-acting elements involved in plant stress and hormone responses were located in the promoter regions of SlBAG9 including heat-shock element (HSE1). The ß-glucuronidase (GUS) histochemical analysis showed that SlBAG9 promoter could drive GUS gene expression in transiently transformed Nicotiana tabacum leaves under non-inducing condition and HSE1 is critical for HT-induced GUS activity under HT. The transcription of SlBAG9 was expressed in different organs and was regulated by HT, cold, drought, and salt stresses as well as exogenous abscisic acid (ABA) and H2O2. To further elucidate SlBAG9 function in response to HT, the transgenic tomato plants overexpressing SlBAG9 were developed. Compared to the wild-type plants, SlBAG9-overexpressing plants exhibited more sensitivity to HT stress, reflected by the burning symptoms, the degradation of chlorophyll, and the reduction of photosynthetic rates. Additionally, SlBAG9-overexpressing lines showed higher accumulation of lipid peroxidation production (MDA) and H2O2, but lower activities of superoxide dismutase, catalase, and peroxidase. Therefore, it is speculated that SlBAG9 plays a negative role in thermotolerance probably by inhibition of antioxidant enzyme system leading to the oxidative damage, consequently aggravating the HT-caused injury phenotype.

Characteristics of SlCML39, a Tomato Calmodulin-like Gene, and Its Negative Role in High Temperature Tolerance of Arabidopsis thaliana during Germination and Seedling Growth.

Calmodulin-like (CML) proteins are primary calcium sensors and function in plant growth and response to stress stimuli. However, so far, the function of plant CML proteins, including tomato, is still unclear. Previously, it was found that a tomato (Solanum lycopersicum) CML, here named SlCML39, was significantly induced by high temperature (HT) at transcription level, but its biological function is scarce. In this study, the characteristics of SlCML39 and its role in HT tolerance were studied. SlCML39 encodes a protein of 201 amino acids containing four EF hand motifs. Many cis-acting elements related to plant stress and hormone response appear in the promoter regions of SlCML39. SlCML39 is mainly expressed in the root, stem, and leaf and can be regulated by HT, cold, drought, and salt stresses as well as ABA and H2O2. Furthermore, heterologous overexpression of SlCML39 reduces HT tolerance in Arabidopsis thaliana at the germination and seedling growth stages. To better understand the molecular mechanism of SlCML39, the downstream gene network regulated by SlCML39 under HT was analyzed by RNA-Seq. Interestingly, we found that many genes involved in stress responses as well as ABA signal pathway are down-regulated in the transgenic seedlings under HT stress, such as KIN1, RD29B, RD26, and MAP3K18. Collectively, these data indicate that SlCML39 acts as an important negative regulator in response to HT stress, which might be mediated by the ABA signal pathway.

The role of plant-specific VQ motif-containing proteins: An ever-thickening plot.

VQ proteins are a class of plant-specific proteins containing the conserved motif FxxhVQxhTG（h denotes hydrophobic residues and x represents any amino acid）and are named VQ for the V and Q residues. By analyzing the structure of VQ members it was found that most VQ genes do not contain introns and the number of encoded amino acids is less than 300 aa. A majority of VQ proteins are located in the nucleus. Accumulated evidence has highlighted the importance of VQ proteins mainly participating in signal pathways through interacting with partners (eg. WRKYs and MAPKs) to regulate plant growth and development and respond to biotic and abiotic stresses. This review primarily focuses on the structure of VQ members in plant kingdom and the biological function and the mechanism of VQ protein action, and discusses recent advances in understanding the pivotal role of VQ-motif, which provides a solid foundation for further exploration on VQ proteins.

In-depth proteome analysis reveals multiple pathways involved in tomato SlMPK1-mediated high-temperature responses.

High temperature (HT) is one of the major environmental factors which limits plant growth and yield. The mitogen-activated protein kinase (MAPK) plays vital roles in environmental stress responses. However, the mechanisms triggered by MAPKs in plants in response to HT are still extremely limited. In this study, the proteomic data of differences between SlMPK1 RNA-interference mutant (SlMPK1i) and wild type and of tomato (Solanum lycopersicum) plants under HT stress using isobaric tags for relative and absolute quantitation (iTRAQ) was re-analyzed in depth. In total, 168 differently expressed proteins (DEPs) were identified in response to HT stress, including 38 DEPs only found in wild type, and 84 DEPs specifically observed in SlMPK1i after HT treatment. The majority of higher expression of 84 DEPs were annotated into photosynthesis, oxidation-reduction process, protein folding, translation, proteolysis, stress response, and amino acid biosynthetic process. More importantly, SlMPK1-mediated photosynthesis was confirmed by the physiological characterization of SlMPK1i with a higher level of photosynthetic capacity under HT stress. Overall, the results reveal a set of potential candidate proteins helping to further understand the intricate regulatory network regulated by SlMPK1 in response to HT.

Genome-wide analysis of the plant-specific VQ motif-containing proteins in tomato (Solanum lycopersicum) and characterization of SlVQ6 in thermotolerance.

The VQ motif-containing (VQ) proteins are plant-specific proteins with a conserved "FxxhVQxhTG" amino acid sequence, which regulate plant growth and development. Little is known, however, about the function of VQ proteins in tomato (Solanum lycopersicum). Here, a total of 26 SlVQ proteins were confirmed and characterized using a comprehensive genome-wide analysis. The SlVQ proteins all contain the conserved motif with seven variations, which are classified into eight groups (I, II, IV-VI, VIII-X). Most of them were predicted to be localized in the nucleus. Besides, a network including SlVQ proteins interaction with WRKY transcription factors (SlWRKYs) and mitogen-activated protein kinases (SlMPKs) is proposed. In addition, among the SlVQ genes, SlVQ6 was expressed in the range of organs and tissues with the highest levels and could response to different stresses. Ectopically overexpression of SlVQ6 in Arabidopsis plants decreased high temperature tolerance. RNA sequencing analysis revealed that several stress-related genes, such as HSP70-4, RD20, GolS1 and AT4g36010 were down-regulated in SlVQ6 overexpressing plants compared to these in wild-type under normal growth conditions. This study provides critical information about SlVQ genes and their encoded proteins, as well as further research on SlVQ functions in tomato growth and development.

Iron and callose homeostatic regulation in rice roots under low phosphorus.

BACKGROUND: Phosphorus (Pi) deficiency induces root morphological remodeling in plants. The primary root length of rice increased under Pi deficiency stress; however, the underlying mechanism is not well understood. In this study, transcriptome analysis (RNA-seq) and Real-time quantitative PCR (qRT-PCR) techniques were combined with the determination of physiological and biochemical indexes to research the regulation mechanisms of iron (Fe) accumulation and callose deposition in rice roots, to illuminate the relationship between Fe accumulation and primary root growth under Pi deficient conditions. RESULTS: Induced expression of LPR1 genes was observed under low Pi, which also caused Fe accumulation, resulting in iron plaque formation on the root surface in rice; however, in contrast to Arabidopsis, low Pi promoted primary root lengthening in rice. This might be due to Fe accumulation and callose deposition being still appropriately regulated under low Pi. The down-regulated expression of Fe-uptake-related key genes (including IRT, NAS, NAAT, YSLs, OsNRAMP1, ZIPs, ARF, and Rabs) inhibited iron uptake pathways I, II, and III in rice roots under low Pi conditions. In contrast, due to the up-regulated expression of the VITs gene, Fe was increasingly stored in both root vacuoles and cell walls. Furthermore, due to induced expression and increased activity of ß-1-3 glucanase, callose deposition was more controlled in low Pi treated rice roots. In addition, low Pi and low Fe treatment still caused primary root lengthening. CONCLUSIONS: The obtained results indicate that Low phosphorus induces iron and callose homeostatic regulation in rice roots. Because of the Fe homeostatic regulation, Fe plays a small role in rice root morphological remodeling under low Pi.

The Tomato Mitogen-Activated Protein Kinase SlMPK1 Is as a Negative Regulator of the High-Temperature Stress Response.

High-temperature (HT) stress is a major environmental stress that limits plant growth and development. MAPK cascades play key roles in plant growth and stress signaling, but their involvement in the HT stress response is poorly understood. Here, we describe a 47-kD MBP-phosphorylated protein (p47-MBPK) activated in tomato (Solanum lycopersicum) leaves under HT and identify it as SlMPK1 by tandem mass spectrometry analysis. Silencing of SlMPK1 in transgenic tomato plants resulted in enhanced tolerance to HT, while overexpression resulted in reduced tolerance. Proteomic analysis identified a set of proteins involved in antioxidant defense that are significantly more abundant in RNA interference-SlMPK1 plants than nontransgenic plants under HT stress. RNA interference-SlMPK1 plants also showed changes in membrane lipid peroxidation and antioxidant enzyme activities. Furthermore, using yeast two-hybrid screening, we identified a serine-proline-rich protein homolog, SlSPRH1, which interacts with SlMPK1 in yeast, in plant cells, and in vitro. We demonstrate that SlMPK1 can directly phosphorylate SlSPRH1. Furthermore, the serine residue serine-44 of SlSPRH1 is a crucial phosphorylation site in the SlMPK1-mediated antioxidant defense mechanism activated during HT stress. We also demonstrate that heterologous expression of SlSPRH1 in Arabidopsis (Arabidopsis thaliana) led to a decrease in thermotolerance and lower antioxidant capacity. Taken together, our results suggest that SlMPK1 is a negative regulator of thermotolerance in tomato plants. SlMPK1 acts by regulating antioxidant defense, and its substrate SlSPRH1 is involved in this pathway.

Revealing new insights into different phosphorus-starving responses between two maize (Zea mays) inbred lines by transcriptomic and proteomic studies.

Phosphorus (P) is an essential plant nutrient, and deficiency of P is one of the most important factors restricting maize yield. Therefore, it is necessary to develop a more efficient program of P fertilization and breeding crop varieties with enhanced Pi uptake and use efficiency, which required understanding how plants respond to Pi starvation. To understand how maize plants adapt to P-deficiency stress, we screened 116 inbred lines in the field and identified two lines, DSY2 and DSY79 that were extreme low-P resistant and sensitive, respectively. We further conducted physiological, transcriptomic, and proteomic studies using the roots of DSY2 and DSY79 under normal or low-P conditions. The results showed that the low-P resistant line, DSY2 had larger root length, surface area and volume, higher root vitality, as well as acid phosphatase activity as compared with the low-P sensitive line, DSY79 under the low-P condition. The transcriptomic and proteomic results suggest that dramatic more genes were induced in DSY2, including the plant hormone signaling, acid phosphatase, and metabolite genes, as compared with DSY79 after being challenged by low-P stress. The new insights generated in this study will be useful toward the improvement of P-utilize efficiency in maize.

Expression of sulfur uptake assimilation-related genes in response to cadmium, bensulfuron-methyl and their co-contamination in rice roots.

The responses of sulfur (S) uptake assimilation-related genes' expression in roots of two rice cultivars to cadmium (Cd), bensulfuron-methyl (BSM) and their co-contamination (Cd+BSM) were investigated by gene-chip microarray analysis and quantitative real-time PCR (QRT-PCR) technology. Treatments of Cd and Cd+BSM induced expression of sulfate transporter and permease genes, and promoted sulfate uptake in rice roots. Cd+BSM could alleviate Cd toxicity to cv. Fengmeizhan seedlings, probably due to Cd+BSM promoting greater S absorption by seedlings. Cd and Cd+BSM induced expression of sulfate assimilation-related genes, and thus activated the sulfur assimilation pathway. Cd and Cd+BSM induced expression of phytochelatin synthase and metallothionein genes, and induced expression of glutathione S-transferases (GSTs), glutathione synthase (GS) and S-containing antioxidation enzyme genes, which detoxified Cd(2+). It is suggested that (to cope with the toxicity of Cd, BSM and their co-contamination) the S uptake and assimilation pathway was activated in rice roots by increased expression of related genes, thus enhancing the supply of organic S for synthesis of Cd or BSM resistance-related substances.

Characterization of seedling proteomes and development of markers to distinguish the Brassica A and C genomes.

The diploid species Brassica rapa (genome AA) and B. oleracea (genome CC) were compared by full-scale proteome analyses of seedling. A total of 28.2% of the proteins was common to both species, indicating the existence of a basal or ubiquitous proteome. However, a number of discriminating proteins (32.0%) and specific proteins (39.8%) of the Brassica A and C genomes, respectively, were identified, which could represent potentially species-specific functions. Based on these A or C genome-specific proteins, a number of PCR-based markers to distinguish B. rapa and B. oleracea species were also developed.

Responses of wheat seedlings to cadmium, mercury and trichlorobenzene stresses.

The molecular response of wheat (Triticum aestivum L., cv. Yangmai 13) seedlings to heavy metal (Cd, Hg) and 1,2,4-trichlorobenzene (TCB) stresses were examined by two-dimensional gel electrophoresis, image analysis, and peptide mass fingerprinting. The results showed inhibitions of root and shoot growth by Cd, Hg, and TCB. These stresses led to water deficit and lipid phosphorylation in the seedling which also promoted protein phophorylation in the leaves. Hg stress inhibited protein synthesis while Cd and TCB stresses induced or up-regulated more proteins in the leaves. Most of these induced proteins played important roles in the biochemical reactions involved in tolerance of wheat to Cd and TCB stresses. The primary functions of Cd- and TCB-induced proteins included methionine metabolism, Rubisco modification, protein phosphorylation regulation, protein configuration protection, H+ transmembrane transportation and also the synthesis of ethylene, defense substances and cell wall compounds.

A proteomic analysis of rice seedlings responding to 1,2,4-trichlorobenzene stress.

The proteomic analysis of rice (Oryza sativa L.) roots and leaves responding to 1,2,4-trichlorobenzene (TCB) stress was carried out by two dimensional gel electrophoresis, mass spectrometric (MS), and protein database analysis. The results showed that 5 mg/L TCB stress had a significant effect on global proteome in rice roots and leaves. The analysis of the category and function of TCB stress inducible proteins showed that different kinds of responses were produced in rice roots and leaves, when rice seedlings were exposed to 5 mg/L TCB stress. Most responses are essential for rice defending the damage of TCB stress. These responses include detoxication of toxic substances, expression of pathogenesis-related proteins, synthesis of cell wall substances and secondary compounds, regulation of protein and amino acid metabolism, activation of methionine salvage pathway, and also include osmotic regulation and phytohormone metabolism. Comparing the TCB stress inducible proteins between the two cultivars, the beta-glucosidase and pathogenesis-related protein family 10 proteins were particularly induced by TCB stress in the roots of rice cultivar (Oryza sativa L.) Aizaizhan, and the glutathione S-transferase and aci-reductone dioxygenase 4 were induced in the roots of rice cultivar Shanyou 63. This may be one of the important mechanisms for Shanyou 63 having higher tolerance to TCB stress than Aizaizhan.

[Optimization of SRAP & ISSR technology and its application in the identification of seeds of Brassica oleracea L].

In this study, the molecular marker technology of SRAP and ISSR were applied in rapid identification of seeds from eight species of Brassica oleracea L. Firstly, using the genomic DNA of cabbage as template, SRAP and ISSR reaction systems were optimized through testing every factor, respectively, that affects PCR amplification. Then, using the optimized reaction systems, 30 SRAP primer pairs and 15 ISSR primers were applied to amplify genomic DNA of cabbage, savoy, purple cabbage, borecole, cauliflower, broccoli, Brussels sprouts, and kohlrabi The results showed that high polymorphisms were exhibited among the eight species of Brassica oleracea L. by SRAP primer pairs of M3-E5 and M4-E5, as well as ISSR primers of 844 and 888, especially primer 844 which can identify all eight materials efficiently.