Regulatory mechanism of GA3 application on grape (Vitis vinifera L.) berry size.

Gibberellin A3 (GA3) is often used as a principal growth regulator to increase plant size. Here, we applied Tween-20 (2%)-formulated GA3 (T1:40 mg/L; T2:70 mg/L) by dipping the clusters at the initial expansion phase of 'Red Globe' grape (Vitis vinifera L.) in 2018 and 2019. Tween-20 (2%) was used as a control. The results showed that GA3 significantly increased fruit cell length, cell size, diameter, and volume. The hormone levels of auxin (IAA) and zeatin (ZT) were significantly increased at 2 h (0 d) -1 d after application (DAA0-1) and remained significantly higher at DAA1 until maturity. Conversely, ABA exhibited an opposite trend. The mRNA and non-coding sequencing results yielded 436 differentially expressed mRNA (DE_mRNAs), 79 DE_lncRNAs and 17 DE_miRNAs. These genes are linked to hormone pathways like cysteine and methionine metabolism (ko00270), glutathione metabolism (ko00480) and plant hormone signal transduction (ko04075). GA3 application reduced expression of insensitive dwarf 2 (GID2, VIT_07s0129g01000), small auxin-upregulated RNA (SAUR, VIT_08s0007g03120) and 1-aminocyclopropane-1-carboxylate synthase (ACS, VIT_18s0001g08520), but increased SAUR (VIT_04s0023g00560) expression. These four genes were predicted to be negatively regulated by vvi-miR156, vvi-miR172, vvi-miR396, and vvi-miR159, corresponding to specific lncRNAs. Therefore, miRNAs could affect grape size by regulating key genes GID2, ACS and SAUR. The R2R3 MYB family member VvRAX2 (VIT_08s0007g05030) was upregulated in response to GA3 application. Overexpression of VvRAX2 in tomato transgenic lines increased fruit size in contrast to the wild type. This study provides a basis and genetic resources for elucidating the novel role of ncRNAs in fruit development.

Insights on the stem elongation of spur-type bud sport mutant of 'Red Delicious' apple.

MAIN CONCLUSION: The decreased capacity of auxin-, CTK-, and BR-mediated cell division and cell enlargement pathways, combined with the enhanced capacity of GA and ETH-, JA-, ABA-, SA-mediated stress-resistant pathways were presumed to be the crucial reasons for the formation of spur-type 'Red Delicious' mutants. Vallee Spur', which exhibit short internodes and compact tree shape, is the fourth generation of the spur-type bud sport mutant of 'Red Delicious'. However, the underlying molecular mechanism of these properties remains unclear. Here, comparative phenotypic, full-length transcriptome and phytohormone analyses were performed between 'Red Delicious' (NSP) and 'Vallee Spur' (SP). The new shoot internode length of NSP was ˃ 1.53-fold higher than that of the SP mutant. Cytological analysis showed that the stem cells of the SP mutant were smaller and more tightly arranged relative to the NSP. By Iso-Seq, a total of 1426 differentially expressed genes (DEGs) were detected, including 808 upregulated and 618 downregulated genes in new shoot apex with 2 leaves of the SP mutant. Gene expressions involved in auxin, cytokinin (CTK), and brassinosteroid (BR) signal transduction were mostly downregulated in the SP mutant, whereas those involved in gibberellin (GA), ethylene (ETH), jasmonate (JA), ABA, and salicylic acid (SA) signal transduction were mostly upregulated. The overall thermogram analysis of hormone levels in the shoot apex carrying two leaves detected by LC-MS/MS absolute quantification showed that the levels of IAA-Asp, IAA, iP7G, OPDA, and 6-deoxyCS were significantly upregulated in the SP mutant, while the remaining 28 hormones were significantly downregulated. It is speculated that the decreased capacity of auxin, CTK, and BR-mediated cell division and cell enlargement pathways is crucial for the formation of the SP mutant. GA and stress-resistant pathways of ETH, JA, ABA, and SA also play vital roles in stem elongation. These results highlight the involvement of phytohormones in the formation of stem elongation occurring in 'Red Delicious' spur-type bud sport mutants and provide information for exploring its biological mechanism.

MYB_SH[AL]QKY[RF] transcription factors MdLUX and MdPCL-like promote anthocyanin accumulation through DNA hypomethylation and MdF3H activation in apple.

Heritable DNA methylation is a highly conserved epigenetic mark that is important for many biological processes. In a previous transcriptomic study on the fruit skin pigmentation of apple (Malus domestica Borkh.) cv. 'Red Delicious' (G0) and its four continuous-generation bud sport mutants including 'Starking Red' (G1), 'Starkrimson' (G2), 'Campbell Redchief' (G3) and 'Vallee spur' (G4), we identified MYB transcription factors (TFs) MdLUX and MdPCL-like involved in regulating anthocyanin synthesis. However, how these TFs ultimately determine the fruit skin color traits remains elusive. Here, bioinformatics analysis revealed that MdLUX and MdPCL-like contained a well-conserved motif SH[AL]QKY[RF] in their C-terminal region and were located in the nucleus of onion epidermal cells. Overexpression of MdLUX and MdPCL-like in 'Golden Delicious' fruits, 'Gala' calli and Arabidopsis thaliana promoted the accumulation of anthocyanin, whereas MdLUX and MdPCL-like suppression inhibited anthocyanin accumulation in 'Red Fuji' apple fruit skin. Yeast one-hybrid assays revealed that MdLUX and MdPCL-like may bind to the promoter region of the anthocyanin biosynthesis gene MdF3H. Dual-luciferase assays indicated that MdLUX and MdPCL-like activated MdF3H. The whole-genome DNA methylation study revealed that the methylation levels of the mCG context at the upstream (i.e., promoter region) of MdLUX and MdPCL-like were inversely correlated with their mRNA levels and anthocyanin accumulation. Hence, the data suggest that MYB_SH[AL]QKY[RF] TFs MdLUX and MdPCL-like promote anthocyanin biosynthesis in apple fruit skins through the DNA hypomethylation of their promoter regions and the activation of the structural flavonoid gene MdF3H.

Synthesis of light-inducible and light-independent anthocyanins regulated by specific genes in grape 'Marselan' (V. vinifera L.).

Anthocyanin is an important parameter for evaluating the quality of wine grapes. However, the effects of different light intensities on anthocyanin synthesis in grape berry skin and its regulation mechanisms are still unclear. In this experiment, clusters of wine grape cv. 'Marselan' were bagged using fruit bags with different light transmittance of 50%, 15%, 5%, and 0, designated as treatment A, B, C and D, respectively. Fruits that were not bagged were used as the control, designated as CK. The anthocyanin composition and concentration, as well as gene expression profiles in the berry skin were determined. The results showed that the degree of coloration of the berry skin reduced with the decrease of the light transmittance, and the veraison was postponed for 10 days in D when compared with the CK. Total anthocyanin concentration in the berry skin treated with D decreased by 51.50% compared with CK at the harvest stage. A total of 24 and 21 anthocyanins were detected in CK and D, respectively. Among them, Malvidin-3-O-coumaroylglucoside (trans), which showed a significant positive correlation with the total concentration of anthocyanins at the harvest stage (r = 0.775) and was not detected in D, was presumed to be light-induced anthocyanin. Other anthocyanins which were both synthesized in CK and D were considered to be light-independent anthocyanins. Among them, Malvidin-3-O-coumaroylglucoside (cis) and Malvidin-3-O-acetylglucoside were typical representatives. Remarkably, the synthesis of light-inducible anthocyanins and light-independent anthocyanins were regulated by different candidate structural genes involved in flavonoid biosynthesis pathway and members of MYB and bHLH transcription factors.

Anthocyanin accumulation correlates with hormones in the fruit skin of 'Red Delicious' and its four generation bud sport mutants.

BACKGROUND: Bud sport mutants of apple (Malus domestica Borkh.) trees with a highly blushed colouring pattern are mainly caused by the accumulation of anthocyanins in the fruit skin. Hormones are important factors modulating anthocyanin accumulation. However, a good understanding of the interplay between hormones and anthocyanin synthesis in apples, especially in mutants at the molecular level, remains elusive. Here, physiological and comparative transcriptome approaches were used to reveal the molecular basis of color pigmentation in the skin of 'Red Delicious' (G0) and its mutants, including 'Starking Red' (G1), 'Starkrimson' (G2), 'Campbell Redchief' (G3) and 'Vallee spur' (G4). RESULTS: Pigmentation in the skin gradually proliferated from G0 to G4. The anthocyanin content was higher in the mutants than in 'Red Delicious'. The activation of early phenylpropanoid biosynthesis genes, including ASP3, PAL, 4CL, PER, CHS, CYP98A and F3'H, was more responsible for anthocyanin accumulation in mutants at the color break stage. In addition, IAA and ABA had a positive regulatory effect on the synthesis of anthocyanins, while GA had the reverse effect. The down-regulation of AACT1, HMGS, HMGR, MVK, MVD2, IDI1 and FPPS2 involved in terpenoid biosynthesis influences anthocyanin accumulation by positively regulating transcripts of AUX1 and SAUR that contribute to the synthesis of IAA, GID2 to GA, PP2C and SnRK2 to ABA. Furthermore, MYB and bHLH members, which are highly correlated (r=0.882-0.980) with anthocyanin content, modulated anthocyanin accumulation by regulating the transcription of structural genes, including CHS and F3'H, involved in the flavonoid biosynthesis pathway. CONCLUSIONS: The present comprehensive transcriptome analyses contribute to the understanding of the the relationship between hormones and anthocyanin synthesis as well as the molecular mechanism involved in apple skin pigmentation.

Proteomic analysis of conidia germination in Fusarium oxysporum f. sp. cubense tropical race 4 reveals new targets in ergosterol biosynthesis pathway for controlling Fusarium wilt of banana.

Conidial germination is a crucial step of the soilborne fungus Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), a most important lethal disease of banana. In this study, a total of 3659 proteins were identified by isobaric tags for relative and absolute quantitation (iTRAQ)-based comparative proteomic approach, of which 1009 were differentially expressed during conidial germination of the fungus at 0, 3, 7, and 11 h. Functional classification and bioinformatics analysis revealed that the majority of the differentially expressed proteins are involved in six metabolic pathways. Particularly, all differential proteins involved in the ergosterol biosynthesis pathway were significantly upregulated, indicating the importance of the ergosterol biosynthesis pathway to the conidial germination of Foc TR4. Quantitative RT-PCR, western blotting, and in vitro growth inhibition assay by several categories of fungicides on the Foc TR4 were used to validate the proteomics results. Four enzymes, C-24 sterol methyltransferase (ERG6), cytochrome P450 lanosterol C-14α-demethylase (EGR11), hydroxymethylglutaryl-CoA synthase (ERG13), and C-4 sterol methyl oxidase (ERG25), in the ergosterol biosynthesis pathway were identified and verified, and they hold great promise as new targets for effective inhibition of Foc TR4 early growth in controlling Fusarium wilt of banana. To the best of our knowledge, this report represents the first comprehensive study on proteomics profiling of conidia germination in Foc TR4. It provides new insights into a better understanding of the developmental processes of Foc TR4 spores. More importantly, by host plant-induced gene silencing (HIGS) technology, the new targets reported in this work allow us to develop novel transgenic banana leading to high protection from Fusarium wilt and to explore more effective antifungal drugs against either individual or multiple target proteins of Foc TR4.

Transcriptome profiling of resistant and susceptible Cavendish banana roots following inoculation with Fusarium oxysporum f. sp. cubense tropical race 4.

BACKGROUND: Fusarium wilt, caused by the fungal pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is considered the most lethal disease of Cavendish bananas in the world. The disease can be managed in the field by planting resistant Cavendish plants generated by somaclonal variation. However, little information is available on the genetic basis of plant resistance to Foc TR4. To a better understand the defense response of resistant banana plants to the Fusarium wilt pathogen, the transcriptome profiles in roots of resistant and susceptible Cavendish banana challenged with Foc TR4 were compared. RESULTS: RNA-seq analysis generated more than 103 million 90-bp clean pair end (PE) reads, which were assembled into 88,161 unigenes (mean size = 554 bp). Based on sequence similarity searches, 61,706 (69.99%) genes were identified, among which 21,273 and 50,410 unigenes were assigned to gene ontology (GO) categories and clusters of orthologous groups (COG), respectively. Searches in the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) mapped 33,243 (37.71%) unigenes to 119 KEGG pathways. A total of 5,008 genes were assigned to plant-pathogen interactions, including disease defense and signal transduction. Digital gene expression (DGE) analysis revealed large differences in the transcriptome profiles of the Foc TR4-resistant somaclonal variant and its susceptible wild-type. Expression patterns of genes involved in pathogen-associated molecular pattern (PAMP) recognition, activation of effector-triggered immunity (ETI), ion influx, and biosynthesis of hormones as well as pathogenesis-related (PR) genes, transcription factors, signaling/regulatory genes, cell wall modification genes and genes with other functions were analyzed and compared. The results indicated that basal defense mechanisms are involved in the recognition of PAMPs, and that high levels of defense-related transcripts may contribute to Foc TR4 resistance in banana. CONCLUSIONS: This study generated a substantial amount of banana transcript sequences and compared the defense responses against Foc TR4 between resistant and susceptible Cavendish bananas. The results contribute to the identification of candidate genes related to plant resistance in a non-model organism, banana, and help to improve the current understanding of host-pathogen interactions.