Overexpression of transcription factor FaMYB63 enhances salt tolerance by directly binding to the SOS1 promoter in Arabidopsis thaliana.

Salinity is a pivotal abiotic stress factor with far-reaching consequences on global crop growth, yield, and quality and which includes strawberries. R2R3-MYB transcription factors encompass a range of roles in plant development and responses to abiotic stress. In this study, we identified that strawberry transcription factor FaMYB63 exhibited a significant upregulation in its expression under salt stress conditions. An analysis using yeast assay demonstrated that FaMYB63 exhibited the ability to activate transcriptional activity. Compared with those in the wild-type (WT) plants, the seed germination rate, root length, contents of chlorophyll and proline, and antioxidant activities (SOD, CAT, and POD) were significantly higher in FaMYB63-overexpressing Arabidopsis plants exposed to salt stress. Conversely, the levels of malondialdehyde (MDA) were considerably lower. Additionally, the FaMYB63-overexpressed Arabidopsis plants displayed a substantially improved capacity to scavenge active oxygen. Furthermore, the activation of stress-related genes by FaMYB63 bolstered the tolerance of transgenic Arabidopsis to salt stress. It was also established that FaMYB63 binds directly to the promoter of the salt overly sensitive gene SOS1, thereby activating its expression. These findings identified FaMYB63 as a possible and important regulator of salt stress tolerance in strawberries.

ATP-binding cassette protein ABCC8 promotes anthocyanin accumulation in strawberry fruits.

Anthocyanins are important health-promoting flavonoid compounds that substantially contribute to fruit quality. Anthocyanin biosynthesis and most regulatory mechanisms are relatively well understood. However, the functions of anthocyanin transport genes in strawberry fruit remain unclear. In this study, a gene encoding an ATP-binding cassette (ABC) protein of type C, ABCC8, was isolated from strawberry fruits. qRT-PCR analysis demonstrated that the transcript levels of FvABCC8 were the highest and were strongly correlated with anthocyanin accumulation during strawberry fruit ripening. Transient overexpression and RNAi of FvABCC8 led to an increase and decrease in anthocyanin content in strawberry fruits, respectively. Moreover, the ABCC8 promoter was activated by MYB and bHLH transcription factors MYB10, bHLH33, and MYC1. Sucrose enhanced anthocyanin accumulation in FvABCC8-overexpressing Arabidopsis, particularly at higher concentrations. FvABCC8-overexpressing lines were less sensitive to ABA during seed germination and seedling development. These results suggest that strawberry vacuolar anthocyanin transport may be mediated by the ABCC transporter ABCC8, the expression of which may be regulated by transcription factors MYB10, bHLH33, and MYC1.

R2R3-MYB transcription factor FaMYB5 is involved in citric acid metabolism in strawberry fruits.

The citrate content of strawberry fruits affects their organoleptic quality. However, little is known about the transcriptional regulatory mechanisms of citric acid metabolism in strawberry fruits. In this study, the R2R3-MYB transcription factor FaMYB5 was identified and placed in the R2R3-MYB subfamily. FaMYB5 is found in the nucleus and shows tissue- and stage-specific expression levels. Citric acid content was positively correlated with FaMYB5 transcript levels. Upregulated FaMYB5 increased citric acid accumulation in transient FaMYB5-overexpressing strawberry fruits, whereas transient RNA silencing of FaMYB5 in strawberry fruits resulted in a reduction of citric acid content. The role of FaMYB5 was verified using stable transgenic NC89 tobacco. Furthermore, a yeast one-hybrid assay revealed that FaMYB5 influences citric acid accumulation by binding to the FaACO (aconitase), FaGAD (glutamate decarboxylase), and FaCS2 (citrate synthase) promoters. Dual-luciferase assays were used to demonstrate that FaMYB5 could activate FaCS2 expression and repress the transcription levels of FaACO and FaGAD. This study identified important roles of FaMYB5 in the regulation of citric acid metabolism and provided a potential target for improving strawberry fruit taste in horticultural crops.

The R2R3-MYB transcription factor FaMYB63 participates in regulation of eugenol production in strawberry.

The biosynthetic pathway of volatile phenylpropanoids, including 4-allyl-2-methoxyphenol (eugenol), has been investigated in petunia (Petunia hybrida). However, the regulatory network for eugenol accumulation in strawberry (Fragaria × ananassa Duch.) fruit remains unclear. Here, an R2R3-type MYB transcription factor (TF; FaMYB63) was isolated from strawberry by yeast one-hybrid (Y1H) screening using the promoter of the FaEGS1 (eugenol synthase 1 [EGS 1]) gene, which encodes the enzyme responsible for the last step in eugenol biosynthesis. FaMYB63 is phylogenetically distinct from other R2R3-MYB TFs, including FaEOBІІ (EMISSION OF BENZENOID II [EOBII]), which also participates in regulating eugenol biosynthesis in strawberry receptacles. Reverse transcription quantitative PCR (RT-qPCR) assays showed that the expression of FaMYB63 was tissue-specific and consistent with eugenol content through strawberry fruit development, was repressed by abscisic acid, and was activated by auxins (indole-3-acetic acid). Overexpression and RNA interference-mediated silencing of FaMYB63 resulted in marked changes in the transcript levels of the biosynthetic genes FaEGS1, FaEGS2, and FaCAD1 (cinnamyl alcohol dehydrogenase 1 [CAD1]) and, thereby, the accumulation of eugenol. Electrophoretic mobility shift, Y1H, GUS activity, and dual-luciferase activity assays demonstrated that the transcript levels of FaEOBІІ and FaMYB10 were regulated by FaMYB63, but not the other way around. Together, these results demonstrate that FaMYB63 directly activates FaEGS1, FaEGS2, FaCAD1, FaEOBІІ, and FaMYB10 to induce eugenol biosynthesis during strawberry fruit development. These findings deepen the understanding of the regulatory network that influences eugenol metabolism in an edible fruit crop.

FvMYB24, a strawberry R2R3-MYB transcription factor, improved salt stress tolerance in transgenic Arabidopsis.

Salinity is one of the major environmental stresses that limit crop growth and productivity. In this study, the FvMYB24 gene that encodes an R2R3-type MYB transcription factor was cloned and characterized. An expression analysis showed that FvMYB24 had a tissue- and stage-specific profile and was induced by salt treatment. Arabidopsis plants that overexpressed transgenic FvMYB24 exhibited a higher germination rate, fresh weight, chlorophyll content, and longer root length than the wild type (WT) under salt stress. The transgenic plants had higher activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) and the accumulation of proline, while these plants accumulated lower amounts of malondialdehyde (MDA) compared with the WT. Furthermore, our results also revealed that the overexpression of FvMYB24 up-regulated the expression of several stress-related genes (AtSOS1, AtSOS2, AtSOS3, AtSOD, AtPOD, AtCAT1, AtNHX1, and AtLEA3) in response to salt stress, thus, enhancing the tolerance of transgenic Arabidopsis. An analysis of the cis-acting elements in the SOS1, SOS2, and SOS3 promoters revealed MYB-binding sites. However, FvMYB24 could only bind to the SOS1 promoter to mediate salt tolerance but not to the SOS2 and SOS3 promoters. These findings suggest that FvMYB24 could potentially be used as a positive regulator in transgenic plant breeding to improve the tolerance of strawberry plants to salt.

Volatile constituents and ellagic acid formation in strawberry fruits of selected cultivars.

Strawberries (Fragaria × ananassa Duch.) are considered a functional food and pleasing fruit in China, mainly because of their high concentration of ellagic acid (EA) and their aroma. A total of 127 volatile compounds were identified by HS-SPME-GC-MS. Changes in volatile constituents and EA were investigated in 50 strawberry cultivars in the red-ripening stage and in 6 cultivars, including 'Benihoppe', 'Snow White', 'Yanli', 'Kaorino', 'Tokun', and 'Xiaobai', at four developmental stages. The results indicated that the components and amounts of volatile compounds and EA markedly varied among and within cultivars. Through multivariate statistical analysis of the volatile compounds, 50 cultivars were divided into 4 clusters. Aromatic components that affected the cluster formation of cultivars were detected. Volatile compounds varied quantitatively among the 6 varieties during the developmental stages, and distinct changes were observed in both red-turning fruits and red-ripening fruits compared with white fruits. Except for 'Xiaobai', which showed the highest EA content at the red-ripening stage, the other 5 cultivars exhibited the highest EA level at the large green fruit stage. Partial least squares-discriminant analysis (PLS-DA) of the profiles of volatile compounds indicated that large green fruits were characterized by EA and aldehydes; white fruits were characterized by ketones and alkanes; and red-ripening fruits were characterized by esters, acids, furans, and alcohols. The results contribute new and important information to breeding programs and the desirable cultivation of strawberry production.

The regulatory module MdPUB29-MdbHLH3 connects ethylene biosynthesis with fruit quality in apple.

The plant hormone ethylene is critical for climacteric fruit ripening, while glucose and anthocyanins determine the fruit quality of climacteric fruits such as apple. Understanding the exact molecular mechanism for this process is important for elucidating the interconnection of ethylene and fruit quality. Overexpression of apple MdbHLH3 gene, an anthocyanin-related basic helix-loop-helix transcription factor (bHLH TF) gene, promotes ethylene production, and transgenic apple plantlets and trees exhibit ethylene-related root developmental abnormalities, premature leaf senescence, and fruit ripening. Biochemical analyses demonstrate that MdbHLH3 binds to the promoters of three genes that are involved in ethylene biosynthesis, including MdACO1, MdACS1, and MdACS5A, activating their transcriptional expression, thereby promoting ethylene biosynthesis. High glucose-inhibited U-box-type E3 ubiquitin ligase MdPUB29, the ortholog of Arabidopsis AtPUB29 in apple, influences the expression of ethylene biosynthetic genes and ethylene production by direct ubiquitination of the MdbHLH3 protein. Our findings provide new insights into the ubiquitination of MdbHLH3 by glucose-inhibited ubiquitin E3 ligase MdPUB29 in the regulation of ethylene biosynthesis as well as indicate that the regulatory module MdPUB29-MdbHLH3 connects ethylene biosynthesis with fruit quality in apple.

The small ubiquitin-like modifier E3 ligase MdSIZ1 promotes anthocyanin accumulation by sumoylating MdMYB1 under low-temperature conditions in apple.

MdMYB1 acts as a crucial component of the MYB-bHLH-WD40 complex to regulate anthocyanin biosynthesis in red-skinned apples (Malus domestica), but little is known about its post-translational regulation. Here, a small ubiquitin-like modifier E3 ligase MdSIZ1 was screened out as an MdMYB1-interacting protein with a yeast two-hybridization approach. The interaction between MdSIZ1 and MdMYB1 was further verified with pull-down and CoIP assays. Furthermore, it was found that MdSIZ1 directly sumoylated MdMYB1 proteins in vivo and in vitro, especially under moderately low temperature (17 °C) conditions, and that this sumoylation was required for MdMYB1 protein stability. Moreover, the transcription level of MdSIZ1 gene was remarkably induced by low temperature and phosphorus deficiency, and MdSIZ1 overexpression exerted a large positive influence on anthocyanin accumulation and red fruit coloration, suggesting its important role in the regulation of anthocyanin biosynthesis under stress conditions. Our findings reveal an important role for a small ubiquitin-like modifier modification of MYB transcription factors in regulation of anthocyanin biosynthesis in plants.

Effect of sunlight-exposure on antioxidants and antioxidant enzyme activities in 'd'Anjou' pear in relation to superficial scald development.

Influence of preharvest sunlight exposure on superficial scald development in 'd'Anjou' pears during cold storage was investigated. The biochemical changes related to scald including α-farnesene, conjugated trienols (CTols), antioxidants, antioxidant enzyme activities were monitored among separated blushed and shaded peels of unbagged fruit as well as the whole peel of bagged fruit. In unbagged fruit, scald symptom was restricted to shaded peel; while there was no difference in α-farnesene between blushed and shaded peels, CTols increased significantly in shaded peel along with scald development after 3months storage. Bagging treatment increased both α-farnesene and CTols significantly and enhanced scald. Preharvest sunlight exposure significantly increased certain antioxidant contents and antioxidant enzyme activities in blushed peel at harvest and during storage. These results reveal a direct role of CTols during development of scald, however, antioxidant systems may play an important role in α-farnesene oxidation to CTols and scald susceptibility in 'd'Anjou pears.

MdMYB9 and MdMYB11 are involved in the regulation of the JA-induced biosynthesis of anthocyanin and proanthocyanidin in apples.

Anthocyanin and proanthocyanidin (PA) are important secondary metabolites and beneficial to human health. Their biosynthesis is induced by jasmonate (JA) treatment and regulated by MYB transcription factors (TFs). However, which and how MYB TFs regulate this process is largely unknown in apple. In this study, MdMYB9 and MdMYB11 which were induced by methyl jasmonate (MeJA) were functionally characterized. Overexpression of MdMYB9 or MdMYB11 promoted not only anthocyanin but also PA accumulation in apple calluses, and the accumulation was further enhanced by MeJA. Subsequently, yeast two-hybrid, pull-down and bimolecular fluorescence complementation assays showed that both MYB proteins interact with MdbHLH3. Moreover, Jasmonate ZIM-domain (MdJAZ) proteins interact with MdbHLH3. Furthermore, chromatin immunoprecipitation-quantitative PCR and yeast one-hybrid assays demonstrated that both MdMYB9 and MdMYB11 bind to the promoters of ANS, ANR and LAR, whereas MdbHLH3 is recruited to the promoters of MdMYB9 and MdMYB11 and regulates their transcription. In addition, transient expression assays indicated that overexpression of MdJAZ2 inhibits the recruitment of MdbHLH3 to the promoters of MdMYB9 and MdMYB11. Our findings provide new insight into the mechanism of how MeJA regulates anthocyanin and PA accumulation in apple.

The effect of postharvest calcium application in hydro-cooling water on tissue calcium content, biochemical changes, and quality attributes of sweet cherry fruit.

To improve storage/shipping quality of sweet cherry (Prunus avium L.), the effect of calcium chloride (CaCl2) added to hydro-cooling water on physiological and biochemical processes related to fruit and pedicel quality was investigated on two major cultivars. The fruit tissue Ca content increased up to 29-85% logarithmically for 'Sweetheart' and 39-188% linearly for 'Lapins' as CaCl2 rate increased from 0.2% to 2.0% at 0 °C for 5 min. The increase of fruit tissue Ca content was accompanied by reductions in respiration rate, ascorbic acid degradation, and membrane lipid peroxidation, which enhanced total phenolics content and total antioxidant capacity, and resulted in increases in fruit firmness and pitting resistance and decreases in titratable acidity loss and decay of both cultivars. Pedicel browning was inhibited by CaCl2 at 0.2% and 0.5%, but increased by higher rates at 1.0% and 2.0%, possibly via modifying membrane lipid peroxidation.

A dsRNA-binding protein MdDRB1 associated with miRNA biogenesis modifies adventitious rooting and tree architecture in apple.

Although numerous miRNAs have been already isolated from fruit trees, knowledge about miRNA biogenesis is largely unknown in fruit trees. Double-strand RNA-binding (DRB) protein plays an important role in miRNA processing and maturation; however, its role in the regulation of economically important traits is not clear yet in fruit trees. EST blast and RACE amplification were performed to isolate apple MdDRB1 gene. Following expression analysis, RNA binding and protein interaction assays, MdDRB1 was transformed into apple callus and in vitro tissue cultures to characterize the functions of MdDRB1 in miRNA biogenesis, adventitious rooting, leaf development and tree growth habit. MdDRB1 contained two highly conserved DRB domains. Its transcripts existed in all tissues tested and are induced by hormones. It bound to double-strand RNAs and interacted with AtDCL1 (Dicer-Like 1) and MdDCL1. Chip assay indicated its role in miRNA biogenesis. Transgenic analysis showed that MdDRB1 controls adventitious rooting, leaf curvature and tree architecture by modulating the accumulation of miRNAs and the transcript levels of miRNA target genes. Our results demonstrated that MdDRB1 functions in the miRNA biogenesis in a conserved way and that it is a master regulator in the formation of economically important traits in fruit trees.

The bHLH transcription factor MdbHLH3 promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples.

Low environmental temperatures promote anthocyanin accumulation and fruit colouration by up-regulating the expression of genes involved in anthocyanin biosynthesis and regulation in many fruit trees. However, the molecular mechanism by which fruit trees regulate this process in response to low temperature (LT) remains largely unknown. In this study, the cold-induced bHLH transcription factor gene MdbHLH3 was isolated from an apple tree and was found to interact physically and specifically through two regions (amino acids 1-23 and 186-228) at the N terminus with the MYB partner MdMYB1 (allelic to MdMYB10). Subsequently, MdbHLH3 bound to the promoters of the anthocyanin biosynthesis genes MdDFR and MdUFGT and the regulatory gene MdMYB1 to activate their expression. Furthermore, the MdbHLH3 protein was post-translationally modified, possibly involving phosphorylation following exposure to LTs, which enhanced its promoter-binding capacity and transcription activity. Our results demonstrate the molecular mechanism by which MdbHLH3 regulates LT-induced anthocyanin accumulation and fruit colouration in apple.

The cold-induced basic helix-loop-helix transcription factor gene MdCIbHLH1 encodes an ICE-like protein in apple.

BACKGROUND: Plant growth is greatly affected by low temperatures, and the expression of a number of genes is induced by cold stress. Although many genes in the cold signaling pathway have been identified in Arabidopsis, little is known about the transcription factors involved in the cold stress response in apple. RESULTS: Here, we show that the apple bHLH (basic helix-loop-helix) gene MdCIbHLH1 (Cold-Induced bHLH1), which encodes an ICE-like protein, was noticeably induced in response to cold stress. The MdCIbHLH1 protein specifically bound to the MYC recognition sequences in the AtCBF3 promoter, and MdCIbHLH1 overexpression enhanced cold tolerance in transgenic Arabidopsis. In addition, the MdCIbHLH1 protein bound to the promoters of MdCBF2 and favorably contributed to cold tolerance in transgenic apple plants by upregulating the expression of MdCBF2 through the CBF (C-repeat-binding factor) pathway. Our findings indicate that MdCIbHLH1 functions in stress tolerance in different species. For example, ectopic MdCIbHLH1 expression conferred enhanced chilling tolerance in transgenic tobacco. Finally, we observed that cold induces the degradation of the MdCIbHLH1 protein in apple and that this degradation was potentially mediated by ubiquitination and sumoylation. CONCLUSIONS: Based on these findings, MdCIbHLH1 encodes a transcription factor that is important for the cold tolerance response in apple.

Electronic interactions in a branched chromophore investigated by nonlinear optical and time-resolved spectroscopy.

The third order nonlinear optical properties of a trimer branched chromophore system and its linear molecule analog are investigated. Two-photon absorption and degenerate four wave mixing measurements were carried out on both systems. An enhancement in the nonlinear optical effect is observed for the branched trimer molecule in comparison to the linear chromophore system. Ultrafast time-resolved measurements were carried out to probe the excited state dynamics in the branched structures. The time-resolved measurements suggest that the two important processes affecting the nonlinear optical properties in the trimer system, charge transfer stabilization and initial electronic delocalization, occur on two different time scales.