BackgroundFrom field harvest to the consumer’s table, fresh citrus fruit spends a considerable amount of time in shipment and storage. During these processes, physiological disorders and pathological diseases are the main causes of fruit loss. Heat treatment (HT) has been widely used to maintain fruit quality during postharvest storage; however, limited molecular information related to this treatment is currently available at a systemic biological level.ResultsMature ‘Kamei’ Satsuma mandarin (Citrus unshiu Marc.) fruits were selected for exploring the disease resistance mechanisms induced by HT during postharvest storage. Proteomic analyses based on two-dimensional gel electrophoresis (2-DE), and metabolomic research based on gas chromatography coupled to mass spectrometry (GC-MS), and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) were conducted. The results show resistance associated proteins were up-regulated in heat treated pericarp, such as beta-1, 3-glucanase, Class III chitinase, 17.7 kDa heat shock protein and low molecular weight heat-shock protein. Also, redox metabolism enzymes were down-regulated in heat treated pericarp, including isoflavone reductase, oxidoreductase and superoxide dismutase. Primary metabolic profiling revealed organic acids and amino acids were down-regulated in heat treated pericarp; but significant accumulation of metabolites, including tetradecanoic acid, oleic acid, ornithine, 2-keto-d-gluconic acid, succinic acid, turanose, sucrose, galactose, myo-inositol, glucose and fructose were detected. Noticeably, H2O2 content decreased, while, lignin content increased in heat treated pericarp compared to the control, which might increase fruit resistibility in response to external stress. Also, flavonoids, substances which are well-known to be effective in reducing external stress, were up-regulated in heat treated pericarp.ConclusionsThis study provides a broad picture of differential accumulation of proteins and metabolites in postharvest citrus fruit, and gives new insights into HT improved fruit disease resistance during subsequent storage of ‘Kamei’ Satsuma mandarin. Interpretation of the data for the proteins and metabolites revealed reactive oxygen species (ROS) and lignin play important roles in heat treatment induced fruit resistance to pathogens and physiological disorders.

Although the functions of carotenogenic genes are well documented, little is known about the mechanisms that regulate their expression, especially those genes involved in α - and β-branch carotenoid metabolism. In this study, an R2R3-MYB transcriptional factor (CrMYB68) that directly regulates the transformation of α- and β-branch carotenoids was identified using Green Ougan (MT), a stay-green mutant of Citrus reticulata cv Suavissima. A comprehensive analysis of developing and harvested fruits indicated that reduced expression of β-carotene hydroxylases 2 (CrBCH2) and 9-cis-epoxycarotenoid dioxygenase 5 (CrNCED5) was responsible for the delay in the transformation of α- and β-carotene and the biosynthesis of ABA. Additionally, the expression of these genes was negatively correlated with the expression of CrMYB68 in MT. Further, electrophoretic mobility shift assays (EMSAs) and dual luciferase assays indicated that CrMYB68 can directly and negatively regulate CrBCH2 and CrNCED5. Moreover, transient overexpression experiments using leaves of Nicotiana benthamiana indicated that CrMYB68 can also negatively regulate NbBCH2 and NbNCED5. To overcome the difficulty of transgenic validation, we quantified the concentrations of carotenoids and ABA, and gene expression in a revertant of MT. The results of these experiments provide more evidence that CrMYB68 is an important regulator of carotenoid metabolism.

The chloroplast thioredoxins (TRXs) function as messengers of redox signals from ferredoxin to target enzymes. In this work, we studied the regulatory impact of pea (Pisum sativum) TRX-F on the magnesium (Mg) chelatase CHLI subunit and the enzymatic activation of Mg chelatase in vitro and in vivo. In vitro, reduced TRX-F activated the ATPase activity of pea CHLI and enhanced the activity of Mg chelatase reconstituted from the three recombinant subunits CHLI, CHLD, and CHLH in combination with the regulator protein GENOMES UNCOUPLED4 (GUN4). Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that TRX-F physically interacts with CHLI but not with either of the other two subunits or GUN4. In vivo, virus-induced TRX-F gene silencing (VIGS-TRX-F) in pea plants did not result in an altered redox state of CHLI. However, simultaneous silencing of the pea TRX-F and TRX-M genes (VIGS-TRX-F/TRX-M) resulted in partially and fully oxidized CHLI in vivo. VIGS-TRX-F/TRX-M plants demonstrated a significant reduction in Mg chelatase activity and 5-aminolevulinic acid synthesizing capacity as well as reduced pigment content and lower photosynthetic capacity. These results suggest that, in vivo, TRX-M can compensate for a lack of TRX-F and that both TRXs act as important redox regulators of Mg chelatase. Furthermore, the silencing of TRX-F and TRX-M expression also affects gene expression in the tetrapyrrole biosynthesis pathway and leads to the accumulation of reactive oxygen species, which may also serve as an additional signal for the transcriptional regulation of photosynthesis-associated nuclear genes.

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The first committed and highly regulated step of chlorophyll biosynthesis is the insertion of Mg(2+) into protoporphyrin IX, which is catalyzed by Mg chelatase that consists of CHLH, CHLD and CHLI subunits. In this study, CHLI and CHLD genes were suppressed by virus-induced gene silencing (VIGS-CHLI and VIGS-CHLD) in pea (Pisum sativum), respectively. VIGS-CHLI and VIGS-CHLD plants both showed yellow leaf phenotypes with the reduced Mg chelatase activity and the inactivated synthesis of 5-aminolevulinic acid. The lower chlorophyll accumulation correlated with undeveloped thylakoid membranes, altered chloroplast nucleoid structure, malformed antenna complexes and compromised photosynthesis capacity in the yellow leaf tissues of the VIGS-CHLI and VIGS-CHLD plants. Non-enzymatic antioxidant contents and the activities of antioxidant enzymes were altered in response to enhanced accumulation of reactive oxygen species (ROS) in the chlorophyll deficient leaves of VIGS-CHLI and VIGS-CHLD plants. Furthermore, the results of metabolite profiling indicate a tight correlation between primary metabolic pathways and Mg chelatase activity. We also found that CHLD induces a feedback-regulated change of the transcription of photosynthesis-associated nuclear genes. CHLD and CHLI silencing resulted in a rapid reduction of photosynthetic proteins. Taken together, Mg chelatase is not only a key regulator of tetrapyrrole biosynthesis but its activity also correlates with ROS homeostasis, primary interorganellar metabolism and retrograde signaling in plant cells.

Tomato fruit-weight 2.2 (FW2.2) was reported to control up to 30 % fruit weight. Recent studies demonstrated that FW2.2-like (FWL) genes also play important roles in plant growth and development. For instance, a maize homolog of FW2.2, named cell number regulator 1 (CNR1), negatively regulates plant and organ size. However, FWL genes in rice have not been characterized yet. In this study, eight FWL genes were identified in rice genome and designated as OsFWL1-8. The chromosome location, gene structure, protein motif, and phylogenetic relationship of OsFWL genes were analyzed. RT-PCR result and microarray data revealed that OsFWL genes exhibited diverse expression patterns and the detailed expression patterns of OsFWL5, 6, and 7 negatively correlated with leaf growth activity. Rice protoplast transient transformation experiment showed that most OsFWL proteins locate at cell membrane but OsFWL8 is present in the nucleus. In addition, the functions of OsFWL genes were investigated by analyzing two T-DNA insertion lines for OsFWL3 and 5. Compared with wild type, the grain weight of osfwl3 mutant and the plant height of osfwl5 mutant were increased by 5.3 and 12.5 %, respectively. We also found that the increase in grain length of osfwl3 mutant was due chiefly to incremental cell number, not cell size and the expression of OsFWL3 negatively correlated with glume growth activity. These results provide a comprehensive foundation for further study of OsFWL functions in rice.

Although abscisic acid (ABA) is a vital regulator of fruit ripening and several transcription factors have been reported to regulate ABA biosynthesis, reports of the effect of ABA on citrus ripening and the regulation of its biosynthesis by a multiple-transcription-factor complex are scarce. In the present study, a systematic metabolic, cytological, and transcriptome analysis of an ABA-deficient mutant (MT) of Citrus reticulata cv. Suavissima confirmed the positive effect of ABA on the citrus ripening process. The analysis of transcriptome profiles indicated that CrNAC036 played an important role in the ABA deficiency of the mutant, most likely due to an effect on the expression of 9-cis-epoxycarotenoid dioxygenase 5 (CrNCED5). Electrophoretic mobility shift assays and dual luciferase assays demonstrated that CrNAC036 can directly bind and negatively regulate CrNCED5 expression. Furthermore, yeast two-hybrid, bimolecular fluorescence complementation, and dual luciferase assays demonstrated that CrNAC036 interacted with CrMYB68, also down-regulating the expression of CrNCED5. Taken together, our results suggest that CrNAC036 and CrMYB68 synergistically inhibit ABA biosynthesis in citrus fruit by regulating the expression of CrNCED5.