Response of linear and cyclic electron flux to moderate high temperature and high light stress in tomato.

OBJECTIVE: To evaluate the possible photoprotection mechanisms of cyclic and linear electron flux (CEF and LEF) under specific high temperature and high light (HH) stress. METHODS: Six-leaf-stage tomato seedlings ("Liaoyuanduoli", n=160) were divided into four parts: Part 1, served as control under 25 °C, 500 µmol/(m2·s); Part 2, spayed with distilled water (H2O) under 35 °C, 1000 µmol/(m2·s) (HH); Part 3, spayed with 100 µmol/L diuron (DCMU, CEF inhibitor) under HH; Part 4, spayed with 60 µmol/L methyl viologen (MV, LEF inhibitor) under HH. Energy conversion, photosystem I (PSI), and PSII activity, and trans-thylakoid membrane proton motive force were monitored during the treatment of 5 d and of the recovering 10 d. RESULTS: HH decreased photochemical reaction dissipation (P) and the maximal photochemical efficiency of PSII (Fv/Fm), and increased the excitation energy distribution coefficient of PSII (ß); DCMU and MV aggravated the partition imbalance of the excitation energy (γ) and the photoinhibition degree. With prolonged DCMU treatment time, electron transport rate and quantum efficiency of PSI (ETRI and YI) significantly decreased whereas acceptor and donor side limitation of PSI (YNA and YND) increased. MV led to a significant decline and accession of yield of regulated and non-regulated energy YNPQ and YNO, respectively. Membrane integrity and ATPase activity were reduced by HH stress, and DCMU and MV enhanced inhibitory actions. CONCLUSIONS: The protective effects of CEF and LEF were mediated to a certain degree by meliorations in energy absorption and distribution as well as by maintenance of thylakoid membrane integrity and ATPase activity.

Proteomics profiling of ethylene-induced tomato flower pedicel abscission.

The control of abscission is an important agricultural concern because of its substantial effect on crop yield and quality. Changes in gene expression are correlated with the ethylene-mediated execution of abscission. However, only few large-scale proteomic studies focused on tomato pedicel abscission. Isobaric tag for relative and absolute quantification labeling was used to examine the protein and phosphoprotein changes in the tomato pedicel AZ (AZ) treated with ethylene or 1-methylcyclopropene. Among the 1429 quantified proteins, 383 unique peptides corresponding to 166 proteins showed higher than 1.5-fold change in abundance. A total of 450 phosphopeptides were detected, among which 85 phosphopeptides corresponding to 73 phosphoproteins were significantly regulated (>1.5-fold abundance change) in response to ethylene. Protein and phosphoprotein sets showed 26 similar proteins. Six phosphorylation motifs were extracted from the 138 phosphorylation sites. By analyzing translational and modification levels, we found that the modification level was not due to the translational changes. Comparison between the protein and phosphoprotein functions revealed that the proteins acted mainly in the metabolic process and showed catalytic activity, whereas most of the phosphoproteins showed signaling and transporting activities. Data revealed the unique features of the AZ phosphoproteomics, thereby suggesting the involvement of a complex network of kinase-substrate and phosphatase-substrate interactions in response to ethylene. Some phosphorylation sites from calcium-dependent protein kinase (CDPK5(S523)), CDPK5(S527), and SRL3(S329) were also found to perform protective functions for AZ and to be helpful in ethylene signal transduction. BIOLOGICAL SIGNIFICANCE: Organ abscission has both positive and negative roles. Abscission is conducive for the fall of ripe fruits and the release and dispersion of seeds, but abscission has been a major limiting factor for crop productivity. Hence, more details about the process may aid in the regulation of organ abscission. However, at present, the detailed mechanism of abscission is still unclear. In tomato, several transcriptome analyses were performed using pedicels as materials. Yet, no large-scale proteomics and phosphoproteomic studies of abscission zone have been reported so far. Hence, in this present study, we determined the ethylene-induced changes in the proteomics and phosphoproteomics of tomato flower AZ tissue using the isobaric tag for relative and absolute quantification (iTRAQ). Proteomics data from both data sets revealed the differentially expressed proteins that are associated with the translational and modification levels relevant to abscission mechanism. Two key proteins (CDPK (CDPK5(S523) and CDPK5(S527)) and SRL3(S329)) among ethylene signal transduction and defense-related proteins were obtained from the phosphoproteins. The set of tomato phosphorylation sites presented in this work is useful in at least two ways. First, as a database resource, the data would facilitate research on the identified phosphoproteins. Second, the identified sites of the related proteins could provide enough knowledge for further experiments. Hence, our results contribute to the understanding of the mechanism of abscission in plants.

Ultrastructural localization of polygalacturonase in ethylene-stimulated abscission of tomato pedicel explants.

Polygalacturonase (PG) is crucial in plant organ abscission process. This paper investigated the cellular and subcellular localization of PG in ethylene-stimulated abscission of tomato pedicel explants. Confocal laser scanning microscopy of abscission zone sections with the fluorescent probe Cy3 revealed that PG was initially accumulated in parenchyma cells in cortical and vascular tissues after 8 h of ethylene treatment and then extended throughout the abscission zone when the abscission zone separated at 24 h after ethylene treatment. At the subcellular level, transmission electron microscopy with immunogold staining showed that PG showed abundant accumulation in the cortical and vascular tissues at 8 h after ethylene treatment, and the distribution area extended to the central parenchyma cells at 16 h after ethylene treatment. In addition, PGs were observed in the distal and proximal parts of the tomato pedicel explants throughout the abscission process. The results provided a visualized distribution of PG in the pedicel abscission zone and proved that PG was closely related to abscission.

Functional expression of FIP-fve, a fungal immunomodulatory protein from the edible mushroom Flammulina velutipes in Pichia pastoris GS115.

FIP-fve is a bioactive protein isolated from the mushroom Flammulina velutipes, which belongs to the fungal immunomodulatory protein (FIP) family and demonstrates several kinds of biological activities including anti-allergy, anti-tumor and immunomodulation. In the current study, the FIP-fve gene was cloned and expressed in the yeast Pichia pastoris GS115, and its correctness was confirmed by SDS-PAGE and Western blot. Optimal expression of rFIP-fve was observed when the P. pastoris cells were cultured in 1% methanol for 9 6h, which resulted in a yield of 258.2 mg l(-1). The rFIP-fve protein was subsequently purified via ammonium sulfate precipitation and Sephadex G-100 gel chromatography. In vitro bioactivity examination showed that rFIP-fve could agglutinate human red blood cells and stimulate the cell viability of murine splenocytes. The immunomodulatory capacity and anti-tumor activity of rFIP-fve were demonstrated by enhanced interleukin-2 secretion and interferon-γ release from the murine lymphocytes, similar to the biological FIP-fve. In conclusion, the FIP-fve gene was functionally and effectively expressed in P. pastoris, and rFIP-fve displayed biological activities similar to those of native FIP-fve. These results indicated the potential use of rFIP-fve from P. pastoris as an effective and feasible source for therapeutic studies and medical applications.