Galactinol synthase 2 influences the metabolism of chlorophyll, carotenoids, and ethylene in tomato fruits.

Galactinol synthase (GolS), which catalyses the synthesis of galactinol, is the first critical enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs) and contributes to plant growth and development, and resistance mechanisms. However, its role in fruit development remains largely unknown. In this study, we used CRISPR/Cas9 gene-editing technology in tomato (Solanum lycopersicum) to create the gols2 mutant showing uniformly green fruits without dark-green shoulders, and promoting fruit ripening. Analysis indicated that galactinol was undetectable in the ovaries and fruits of the mutant, and the accumulation of chlorophyll and chloroplast development was suppressed in the fruits. RNA-sequencing analysis showed that genes related to chlorophyll accumulation and chloroplast development were down-regulated, including PROTOCHLOROPHYLLIDE OXIDOREDUCTASE, GOLDEN 2-LIKE 2, and CHLOROPHYLL A/B-BINDING PROTEINS. In addition, early color transformation and ethylene release was prompted in the gols2 lines by regulation of the expression of genes involved in carotenoid and ethylene metabolism (e.g. PHYTOENE SYNTHASE 1, CAROTENE CIS-TRANS ISOMERASE, and 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE2/4) and fruit ripening (e.g. RIPENING INHIBITOR, NON-RIPENING, and APETALA2a). Our results provide evidence for the involvement of GolS2 in pigment and ethylene metabolism of tomato fruits.

SlBEL11 regulates flavonoid biosynthesis, thus fine-tuning auxin efflux to prevent premature fruit drop in tomato.

Auxin regulates flower and fruit abscission, but how developmental signals mediate auxin transport in abscission remains unclear. Here, we reveal the role of the transcription factor BEL1-LIKE HOMEODOMAIN11 (SlBEL11) in regulating auxin transport during abscission in tomato (Solanum lycopersicum). SlBEL11 is highly expressed in the fruit abscission zone, and its expression increases during fruit development. Knockdown of SlBEL11 expression by RNA interference (RNAi) caused premature fruit drop at the breaker (Br) and 3 d post-breaker (Br+3) stages of fruit development. Transcriptome and metabolome analysis of SlBEL11-RNAi lines revealed impaired flavonoid biosynthesis and decreased levels of most flavonoids, especially quercetin, which functions as an auxin transport inhibitor. This suggested that SlBEL11 prevents premature fruit abscission by modulating auxin efflux from fruits, which is crucial for the formation of an auxin response gradient. Indeed, quercetin treatment suppressed premature fruit drop in SlBEL11-RNAi plants. DNA affinity purification sequencing (DAP-seq) analysis indicated that SlBEL11 induced expression of the transcription factor gene SlMYB111 by directly binding to its promoter. Chromatin immunoprecipitation-quantitative polymerase chain reaction and electrophoretic mobility shift assay showed that S. lycopersicum MYELOBLASTOSIS VIRAL ONCOGENE HOMOLOG111 (SlMYB111) induces the expression of the core flavonoid biosynthesis genes SlCHS1, SlCHI, SlF3H, and SlFLS by directly binding to their promoters. Our findings suggest that the SlBEL11-SlMYB111 module modulates flavonoid biosynthesis to fine-tune auxin efflux from fruits and thus maintain an auxin response gradient in the pedicel, thereby preventing premature fruit drop.

Galactinol Regulates JA Biosynthesis to Enhance Tomato Cold Tolerance.

Low temperatures can inhibit plant growth and development and reduce fruit yield. This study demonstrated that the expression of AnGolS1 from Ammopiptanthus nanus (A. nanus) encoding a galactinol synthase enhanced tomato cold tolerance. In AnGolS1-overexpressing plants, the jasmonic acid (JA) biosynthesis substrates 13-hydroperoxylinolenicacid and 12,13-epoxylinolenicacid were significantly accumulated, and the expression levels of the ethylene response factor (SlERF4-7) and serine protease inhibitor (SlSPI5) were increased. We speculated that there may be correlations among galactinol, ethylene signaling, the protease inhibitor, protease, and JA levels. The expression levels of SlERF4-7 and SlSPI5 as well as the JA content were significantly increased under exogenous galactinol treatment. Additionally, the expression of SlSPI5 was reduced in SlERF4-7-silenced plants, and SlERF4-7 was confirmed to bind to the dehydration-responsive element (DRE) of the SlSPI5 promoter. These results suggest that SlSPI5 is a target gene of the SlERF4-7 transcription factor. In addition, SlSPI5 interacted with cysteine protease (SlCPase), while SlCPase interacted with lipoxygenase (SlLOX5) and allene oxide synthase (SlAOS2). When SlCPase was silenced, JA levels increased and plant cold tolerance was enhanced. Therefore, galactinol regulates JA biosynthesis to enhance tomato cold tolerance through the SlERF4-7-SlSPI5-SlCPase-SlLOX5/SlAOS2 model. Overall, our study provides new perspectives on the role of galactinol in the JA regulatory network in plant adaptation to low-temperature stress.

A SlCLV3-SlWUS module regulates auxin and ethylene homeostasis in low light-induced tomato flower abscission.

Premature abscission of flowers and fruits triggered by low light stress can severely reduce crop yields. However, the underlying molecular mechanism of this organ abscission is not fully understood. Here, we show that a gene (SlCLV3) encoding CLAVATA3 (CLV3), a peptide hormone that regulates stem cell fate in meristems, is highly expressed in the pedicel abscission zone (AZ) in response to low light in tomato (Solanum lycopersicum). SlCLV3 knockdown and knockout lines exhibit delayed low light-induced flower drop. The receptor kinases SlCLV1 and BARELY ANY MERISTEM1 function in the SlCLV3 peptide-induced low light response in the AZ to decrease expression of the transcription factor gene WUSCHEL (SlWUS). DNA affinity purification sequencing identified the transcription factor genes KNOX-LIKE HOMEDOMAIN PROTEIN1 (SlKD1) and FRUITFULL2 (SlFUL2) as SlWUS target genes. Our data reveal that low light reduces SlWUS expression, resulting in higher SlKD1 and SlFUL2 expression in the AZ, thereby perturbing the auxin response gradient and causing increased ethylene production, eventually leading to the initiation of abscission. These results demonstrate that the SlCLV3-SlWUS signaling pathway plays a central role in low light-induced abscission by affecting auxin and ethylene homeostasis.

Photosynthetic Response Mechanism of Soil Salinity-Induced Cross-Tolerance to Subsequent Drought Stress in Tomato Plants.

Soil salinization and water shortage cause ion imbalance and hyperosmoticity in plant cells, adversely impairing photosynthesis efficiency. How soil salinity-induced photosynthetic acclimation influences the cross-tolerance to drought conditions represents a promising research topic. This study was to reveal the photosynthetic mechanism of soil salinity-induced resistance to the subsequent drought stress in tomato leaves through comprehensive photosynthesis-related spectroscopy analysis. We conducted soil salinity pretreatment and subsequent drought stress experiments, including irrigation with 100 mL water, 100 mL 100 mM NaCl solution (NaCl100), 50 mL water, and 50 mL 100 mM NaCl solution (NaCl50) for five days, followed by five-day drought stress. The results showed that soil salinity treatment by NaCl decreased the rate of photosynthetic gas exchange but enhanced CO2 assimilation, along with photosystem II [PS(II)] and photosystem I [PS(I)] activity and photochemical efficiency in tomato plants compared with water pretreatment after subsequent drought stress. NaCl100 and NaCl50 had the capacity to maintain non-photochemical quenching (NPQ) of chlorophyll fluorescence and the cyclic electron (CEF) flow around PSI in tomato leaves after being subjected to subsequent drought stress, thus avoiding the decrease of photosynthetic efficiency under drought conditions. NaCl100 and NaCl50 pretreatment induced a higher proton motive force (pmf) and also alleviated the damage to the thylakoid membrane and adenosine triphosphate (ATP) synthase of tomato leaves caused by subsequent drought stress. Overall, soil salinity treatment could enhance drought resistance in tomato plants by inducing NPQ, maintaining CEF and pmf that tradeoff between photoprotection and photochemistry reactions. This study also provides a photosynthetic perspective for salt and drought cross-tolerance.

MicroRNA1917 targets CTR4 splice variants to regulate ethylene responses in tomato.

Ethylene perception is regulated by receptors, and the downstream protein CONSTITUTIVE TRIPLE RESPONSE1 is a key suppressor of ethylene signalling. The non-conserved tomato (Solanum lycopersicum) microRNA1917 (Sly-miR1917) mediates degradation of SlCTR4 splice variants (SlCTR4sv) but the molecular details of this pathway remain unknown. Sly-miR1917 and the targeted SlCTR4sv are ubiquitously expressed in all tomato organs. Overexpression of Sly-miR1917 enhances ethylene responses, including the triple response in etiolated seedlings, in the absence of ethylene, as well as epinastic petiole growth, accelerated pedicel abscission, and fruit ripening. Enhanced ethylene signalling in Sly-miR1917-overexpressing plants (1917-OE) is accompanied by up-regulation of ethylene biosynthesis and signalling genes, and increased ethylene emission. These phenotypes were recovered by repressing the positive ethylene regulator EIN2. Moreover, the Sly-miR1917-targeted SlCTR4 splice variant SlCTR4sv3, expressed specifically in the abscission zone, exhibited the opposite expression pattern to Sly-miR1917. Complementation of the Arabidopsis thaliana ctr-1 mutant and yeast two-hybrid and bimolecular fluorescence complementation assays suggested that SlCTR4sv3 functions in ethylene signalling. Co-expression of Sly-miR1917 and SlCTR4sv3 in Nicotiana benthamiana further suggested that Sly-miR1917 cleaves SlCTR4sv3 in vivo. Database homology searching revealed a Solanum tuberosum CTR-like splice variant containing a Sly-miR1917 binding sequence, and a homologue of mature Sly-miR1917 in potato, indicating a conserved function for miR1917 and the regulatory miRNA-mediated ethylene network in solanaceous species.

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.

[The Feature of Skewing Distribution of TCR Vbeta T Cells from Peripheral Blood and Bone Marrow in Patients with AML-M(5)]

The TCR Vbeta 24 subfamily genes were amplified in peripheral blood and bone marrow mononuclear cells from 5 cases with acute monocytic leukemia (AML-M(5)) using RT-PCR, to observe the distribution of TCR Vbeta subfamilies. The results indicated that 1 - 19 Vbeta subfamily T cells could be identified in different samples from AML-M(5) cases. The variation of distribution of TCR Vbeta subfamily T cells could be found in different individual samples. The results provided the feature of cell immune function change in skewed distribution of TCR Vbeta subfamily T cells from peripheral blood and bone marrow of patients with AML-M(5).