Physio-Biochemical Insights into the Cold Resistance Variations among Nectarine (Prunus persica (L.) Batsch var. nectarina) Cultivars.

Cold stress occurs in late winter and early spring threatens greatly the nectarine industry. In this study, the semi-lethal low temperature (LT50) and thirteen cold resistance related parameters of five nectarine cultivars, including 'Nonglehong little princess' (LP), 'Luyou No. 5' (LY), 'Nonglehong No. 6' (NL), 'Zhongyou No. 20' (ZY) and 'Qiuhongzhu' (QH), were determined. Based on these parameters, they were categorized into high-(HR, including NL and LP), moderate-(MR, including QH) and low-cold resistant (LR, including ZY and LY) groups. The relative water (RW), proline (PRO), soluble sucrose (SS) and soluble protein (SP) contents, and superoxide dismutase (SOD) and peroxidase (POD) activities of HR cultivars were higher while their relative electronic conductivity (RE), malondialdehyde (MDA) and gibberellin acid (GA3) contents and catalase (CAT) activity were lower than other cultivars during natural overwintering. Redundancy analysis revealed that the lowest temperature in a day (LT) and LT50 significantly explains 69.8% and 10.9% of these physiological variables, respectively. Moreover, GA3 and indoleacetic acid (IAA) contents and CAT activity were positively correlated, while PRO, SS, ABA and RW contents were negatively correlated with both LT and LT50. Our study will be helpful in understanding the cold resistance variations of nectarine germplasm resources.

Functional and Transcriptome Analysis Reveal Specific Roles of Dimocarpus longan DlRan3A and DlRan3B in Root Hair Development, Reproductive Growth, and Stress Tolerance.

Ran GTPases play essential roles in plant growth and development. Our previous studies revealed the nuclear localization of DlRan3A and DlRan3B proteins and proposed their functional redundancy and distinction in Dimocarpus longan somatic embryogenesis, hormone, and abiotic stress responses. To further explore the possible roles of DlRan3A and DlRan3B, gene expression analysis by qPCR showed that their transcripts were both more abundant in the early embryo and pulp in longan. Heterologous expression of DlRan3A driven by its own previously cloned promoter led to stunted growth, increased root hair density, abnormal fruits, bigger seeds, and enhanced abiotic stress tolerance. Conversely, constitutive promoter CaMV 35S (35S)-driven expression of DlRan3A, 35S, or DlRan3B promoter-controlled expression of DlRan3B did not induce the alterations in growth phenotype, while they rendered different hypersensitivities to abiotic stresses. Based on the transcriptome profiling of longan Ran overexpression in tobacco plants, we propose new mechanisms of the Ran-mediated regulation of genes associated with cell wall biosynthesis and expansion. Also, the transgenic plants expressing DlRan3A or DlRan3B genes controlled by 35S or by their own promoter all exhibited altered mRNA levels of stress-related and transcription factor genes. Moreover, DlRan3A overexpressors were more tolerant to salinity, osmotic, and heat stresses, accompanied by upregulation of oxidation-related genes, possibly involving the Ran-RBOH-CIPK network. Analysis of a subset of selected genes from the Ran transcriptome identified possible cold stress-related roles of brassinosteroid (BR)-responsive genes. The marked presence of genes related to cell wall biosynthesis and expansion, hormone, and defense responses highlighted their close regulatory association with Ran.

Comparison of Bioactive Compounds and Antioxidant Activities in Differentially Pigmented Cerasus humilis Fruits.

Chinese dwarf cherry (Cerasus humilis) is a wild fruit tree and medicinal plant endemic to China. Its fruits are rich in various bioactive compounds, such as flavonoids and carotenoids, which contribute greatly to their high antioxidant capacity. In this study, the contents of bioactive substances (chlorophyll, carotenoids, ascorbic acid, anthocyanin, total flavonoids, and total phenols), antioxidant capacities, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonicacid) (ABTS+) scavenging ability, and ferric-reducing antioxidant power (FRAP)) in differentially pigmented C. humilis fruits of four varieties were determined and compared. The results revealed that anthocyanin, total flavonoids and total phenols were the three main components responsible for the antioxidant activity of C. humilis fruits. 'Jinou No.1' fruits with dark red peel and red flesh had the highest contents of anthocyanin, total flavonoids, and total phenols, as well as the highest antioxidant capacities; 'Nongda No.5' fruits with yellow-green peel and yellow flesh had the highest contents of carotenoids and chlorophyll, while 'Nongda No.6' fruit had the highest ascorbic acid content. To further reveal the molecular mechanism underlying differences in the accumulation of carotenoids and flavonoids among differentially pigmented C. humilis fruits, the expression patterns of structural genes involved in the biosynthesis of the two compounds were investigated. Correlation analysis results revealed that the content of carotenoids in C. humilis fruits was very significantly positively correlated with the expression of the ChCHYB, ChZEP, ChVDE, ChNSY, ChCCD1, ChCCD4, ChNCED1, and ChNCED5 genes (p < 0.01) and significantly negatively correlated with the expression of ChZDS (p < 0.05). The anthocyanin content was very significantly positively correlated with ChCHS, ChFLS, and ChUFGT expression (p < 0.01). The total flavonoid content was very significantly positively correlated with the expression of ChCHS, ChUFGT, and ChC4H (p < 0.01) and significantly positively correlated with ChFLS expression (p < 0.05). This study can provide a basis for understanding the differences in the accumulation of bioactive substances, and is helpful for clarifying the mechanisms underlying the accumulation of various carotenoids and flavonoids among differentially pigmented C. humilis fruits.

Characterization and Functional Analysis of Chalcone Synthase Genes in Highbush Blueberry (Vaccinium corymbosum).

Chalcone synthase (CHS) is the first key enzyme-catalyzing plant flavonoid biosynthesis. Until now, however, the blueberry CHS gene family has not been systematically characterized and studied. In this study, we identified 22 CHS genes that could be further classified into four subfamilies from the highbush blueberry (Vaccinium corymbosum) genome. This classification was well supported by the high nucleotide and protein sequence similarities and similar gene structure and conserved motifs among VcCHS members from the same subfamily. Gene duplication analysis revealed that the expansion of the blueberry CHS gene family was mainly caused by segmental duplications. Promoter analysis revealed that the promoter regions of VcCHSs contained numerous cis-acting elements responsive to light, phytohormone and stress, along with binding sites for 36 different types of transcription factors. Gene expression analysis revealed that Subfamily I VcCHSs highly expressed in fruits at late ripening stages. Through transient overexpression, we found that three VcCHSs (VcCHS13 from subfamily II; VcCHS8 and VcCHS21 from subfamily I) could significantly enhance the anthocyanin accumulation and up-regulate the expression of flavonoid biosynthetic structural genes in blueberry leaves and apple fruits. Notably, the promoting effect of the Subfamily I member VcCHS21 was the best. The promoter of VcCHS21 contains a G-box (CACGTG) and an E-box sequence, as well as a bHLH binding site. A yeast one hybridization (Y1H) assay revealed that three anthocyanin biosynthesis regulatory bHLHs (VcAN1, VcbHLH1-1 and VcbHLH1-2) could specifically bind to the G-box sequence (CACGTG) in the VcCHS21 promoter, indicating that the expression of VcCHS21 was regulated by bHLHs. Our study will be helpful for understanding the characteristics and functions of blueberry CHSs.

Influences of Jujube Witches' Broom (JWB) Phytoplasma Infection and Oxytetracycline Hydrochloride Treatment on the Gene Expression Profiling in Jujube.

Jujube witches' broom disease (JWB), caused by Candidatus Phytoplasma ziziphi, is the most destructive phytoplasma disease threatening the jujube industry. Tetracycline derivatives treatments have been validated to be capable of recovering jujube trees from phytoplasma infection. In this study, we reported that oxytetracycline hydrochloride (OTC-HCl) trunk injection treatment could recover more than 86% of mild JWB-diseased trees. In order to explore the underlying molecular mechanism, comparative transcriptomic analysis of healthy control (C group), JWB-diseased (D group) and OTC-HCl treated JWB-diseased (T group) jujube leaves was performed. In total, 755 differentially expressed genes (DEGs), including 488 in 'C vs. D', 345 in 'D vs. T' and 94 in 'C vs. T', were identified. Gene enrichment analysis revealed that these DEGs were mainly involved in DNA and RNA metabolisms, signaling, photosynthesis, plant hormone metabolism and transduction, primary and secondary metabolisms, their transportations, etc. Notably, most of the DEGs identified in 'C vs. D' displayed adverse change patterns in 'D vs. T', suggesting that the expression of these genes was restored after OTC-HCl treatment. Our study revealed the influences of JWB phytoplasma infection and OTC-HCl treatment on gene expression profiling in jujube and would be helpful for understanding the chemotherapy effects of OTC-HCl on JWB-diseased jujube.

Characterization of Carotenoid Cleavage Oxygenase Genes in Cerasus humilis and Functional Analysis of ChCCD1.

Carotenoid cleavage oxygenases (CCOs) are key enzymes that function in degrading carotenoids into a variety of apocarotenoids and some other compounds. In this study, we performed genome-wide identification and characterization analysis of CCO genes in Cerasus humilis. Totally, nine CCO genes could be classified into six subfamilies, including carotenoid cleavage dioxygenase 1 (CCD1), CCD4, CCD7, CCD8, CCD-like and nine-cis-epoxycarotenoid dioxygenase (NCED), were identified. Results of gene expression analysis showed that ChCCOs exhibited diverse expression patterns in different organs and in fruits at different ripening stages. To investigate the roles of ChCCOs in carotenoids degradation, enzyme assays of the ChCCD1 and ChCCD4 were performed in Escerichia coli BL21(DE3) that can accumulate lycopene, ß-carotene and zeaxanthin. The prokaryotic expressed ChCCD1 resulted in obvious degradation of lycopene, ß-carotene and zeaxanthin, but ChCCD4 did not show similar functions. To further determine the cleaved volatile apocarotenoids of these two proteins, headspace gas chromatography/mass spectrometer analysis was performed. Results showed that ChCCD1 could cleave lycopene at 5, 6 and 5', 6' positions to produce 6-methy-5-hepten-2-one and could catalyze ß-carotene at 9, 10 and 9', 10' positions to generate ß-ionone. Our study will be helpful for clarifying the roles of CCO genes especially ChCCD1 in regulating carotenoid degradation and apocarotenoid production in C. humilis.

Characterization of Banana SNARE Genes and Their Expression Analysis under Temperature Stress and Mutualistic and Pathogenic Fungal Colonization.

SNAREs (soluble N-ethylmaleimide-sensitive-factor attachment protein receptors) are engines for almost all of the membrane fusion and exocytosis events in organism cells. In this study, we identified 84 SNARE genes from banana (Musa acuminata). Gene expression analysis revealed that the expression of MaSNAREs varied a lot in different banana organs. By analyzing their expression patterns under low temperature (4 °C), high temperature (45 °C), mutualistic fungus (Serendipita indica, Si) and fungal pathogen (Fusarium oxysporum f. sp. Cubense Tropical Race 4, FocTR4) treatments, many MaSNAREs were found to be stress responsive. For example, MaBET1d was up-regulate by both low and high temperature stresses; MaNPSN11a was up-regulated by low temperature but down-regulated by high temperature; and FocTR4 treatment up-regulated the expression of MaSYP121 but down-regulated MaVAMP72a and MaSNAP33a. Notably, the upregulation or downregulation effects of FocTR4 on the expression of some MaSNAREs could be alleviated by priorly colonized Si, suggesting that they play roles in the Si-enhanced banana wilt resistance. Foc resistance assays were performed in tobacco leaves transiently overexpressing MaSYP121, MaVAMP72a and MaSNAP33a. Results showed that transient overexpression of MaSYP121 and MaSNPA33a suppressed the penetration and spread of both Foc1 (Foc Race 1) and FocTR4 in tobacco leaves, suggesting that they play positive roles in resisting Foc infection. However, the transient overexpression of MaVAMP72a facilitated Foc infection. Our study can provide a basis for understanding the roles of MaSNAREs in the banana responses to temperature stress and mutualistic and pathogenic fungal colonization.

Identification and Characterization of Long Non-Coding RNAs: Implicating Insights into Their Regulatory Role in Kiwifruit Ripening and Softening during Low-Temperature Storage.

Long non-coding RNAs (lncRNAs) are crucial players regulating many biological processes in plants. However, limited knowledge is available regarding their roles in kiwifruit ripening and softening. In this study, using lncRNA-seq technology, 591 differentially expressed (DE) lncRNAs (DELs) and 3107 DE genes (DEGs) were identified from kiwifruit stored at 4 °C for 1, 2, and 3 weeks in comparison with non-treated control fruits. Of note, 645 DEGs were predicted to be targets of DELs (DEGTLs), including some DE protein-coding genes (such as ß-amylase and pectinesterase). DEGTL-based GO enrichment analysis revealed that these genes were significantly enriched in cell wall modification and pectinesterase activity in 1 W vs. CK and 3 W vs. CK, which might be closely related to the fruit softening during low-temperature storage. Moreover, KEGG enrichment analysis revealed that DEGTLs were significantly associated with starch and sucrose metabolism. Our study revealed that lncRNAs play critical regulatory roles in kiwifruit ripening and softening under low-temperature storage, mainly by mediating the expression of starch and sucrose metabolism and cell wall modification related genes.

MicroRNA miR171b Positively Regulates Resistance to Huanglongbing of Citrus.

Huanglongbing (HLB) is one of the most severe citrus diseases in the world, causing huge economic losses. However, efficient methods of protecting citrus from HLB have not yet been developed. microRNA (miRNA)-mediated regulation of gene expression is a useful tool to control plant diseases, but the miRNAs involved in regulating resistance to HLB have not yet been identified. In this study, we found that miR171b positively regulated resistance to HLB in citrus. Upon infection with HLB bacteria, the bacteria were detected in the second month in the control plants. However, in the miR171b-overexpressing transgenic citrus plants, the bacteria could not be detected until the 24th month. RNA-seq data indicated that multiple pathways, such as photosynthesis, plant-pathogen interaction, the MAPK signaling pathway, etc., might be involved in improving the resistance to HLB in miR171b-overexpressing plants compared with the control. Finally, we determined that miR171b could target SCARECROW-like (SCL) genes to downregulate their expression, which then led to promoted resistance to HLB stress. Collectively, our results demonstrate that miR171b plays a positive regulatory role in resistance to citrus HLB, and provides a new insight into the role of miRNAs in the adaptation of citrus to HLB stress.

Characterization of Highbush Blueberry (Vaccinium corymbosum L.) Anthocyanin Biosynthesis Related MYBs and Functional Analysis of VcMYB Gene.

As one of the most important transcription factors regulating plant anthocyanin biosynthesis, MYB has attracted great attentions. In this study, we identified fifteen candidate anthocyanin biosynthesis related MYB (ABRM) proteins, including twelve R2R3-MYBs and three 1R-MYBs, from highbush blueberry. The subcellular localization prediction results showed that, with the exception of VcRVE8 (localized in chloroplast and nucleus), all of the blueberry ABRMs were nucleus-localized. The gene structure analysis revealed that the exon numbers of the blueberry ABRM genes varied greatly, ranging between one and eight. There are many light-responsive, phytohormone-responsive, abiotic stress-responsive and plant growth and development related cis-acting elements in the promoters of the blueberry ABRM genes. It is noteworthy that almost all of their promoters contain light-, ABA- and MeJA-responsive elements, which is consistent with the well-established results that anthocyanin accumulation and the expression of MYBs are influenced significantly by many factors, such as light, ABA and JA. The gene expression analysis revealed that VcMYB, VcMYB6, VcMYB23, VcMYBL2 and VcPH4 are expressed abundantly in blueberry fruits, and VcMYB is expressed the highest in the red, purple and blue fruits among all blueberry ABRMs. VcMYB shared high similarity with functionally proven ABRMs from many other plant species. The gene cloning results showed that VcMYB had three variable transcripts, but only the transient overexpression of VcMYB-1 promoted anthocyanin accumulation in the green fruits. Our study can provide a basis for future research on the anthocyanin biosynthesis related MYBs in blueberry.

Characterization of three tandem-duplicated calcium binding protein (CaBP) genes and promoters reveals their roles in the phytohormone and wounding responses in citrus.

Accumulated evidences have revealed the critical roles of calcium binding protein (CaBP) in growth and stress responses of plants. However, its function in woody plants is poorly understood. In this study, we cloned the CDS, gDNA and promoter sequences of three tandem-duplicated CaBPs (CsCaBP1, CsCaBP2 and CsCaBP3) from Citrus sinensis, analyzed their sequence characteristics, and investigated their gene expression patterns and promoter activities under treatments of CaCl2, several phytohormones and wounding. Results showed that the three CsCaBPs have high sequence similarity. Their expression was strongly induced by CaCl2, ethylene, jasmonic acid, salicylic acid and wounding, and the promoting effect of wounding on their expression was found to be partially ethylene-dependent. Consistently, we identified many phytohormone-related cis-acting elements in their promoters, and their promoter activity could be induced significantly by ethylene, jasmonic acid, salicylic acid and wounding. All the three CsCaBPs can interact with WRKY40, whose encoding gene showed a similar expression pattern to CsCaBPs under phytohormone and wounding treatments. In addition, CsERF14, CsERF21, CsERF3 and CsERF2 could bind to their promoters. The results obtained in this study indicated that the three duplicated CsCaBPs were functionally redundant and played similar roles in the phytohormone and wounding responses of C. sinensis.

Integrated metabolic, transcriptomic and chromatin accessibility analyses provide novel insights into the competition for anthocyanins and flavonols biosynthesis during fruit ripening in red apple.

Fruit ripening is accompanied by a wide range of metabolites and global changes in gene expression that are regulated by various factors. In this study, we investigated the molecular differences in red apple 'Hongmantang' fruits at three ripening stages (PS1, PS5 and PS9) through a comprehensive analysis of metabolome, transcriptome and chromatin accessibility. Totally, we identified 341 and 195 differentially accumulated metabolites (DAMs) in comparison I (PS5_vs_PS1) and comparison II (PS9_vs_PS5), including 57 and 23 differentially accumulated flavonoids (DAFs), respectively. Intriguingly, among these DAFs, anthocyanins and flavonols showed opposite patterns of variation, suggesting a possible competition between their biosynthesis. To unveil the underlying mechanisms, RNA-Seq and ATAC-Seq analyses were performed. A total of 852 DEGs significantly enriched in anthocyanin metabolism and 128 differential accessible regions (DARs) significantly enriched by MYB-related motifs were identified as up-regulated in Comparison I but down-regulated in Comparison II. Meanwhile, the 843 DEGs significantly enriched in phenylalanine metabolism and the 364 DARs significantly enriched by bZIP-related motifs showed opposite trends. In addition, four bZIPs and 14 MYBs were identified as possible hub genes regulating the biosynthesis of flavonols and anthocyanins. Our study will contribute to the understanding of anthocyanins and flavonols biosynthesis competition in red apple fruits during ripening.

Genome-Wide Identification and Characterization of Banana Ca2+-ATPase Genes and Expression Analysis under Different Concentrations of Ca2+ Treatments.

Ca2+-ATPases have been confirmed to play very important roles in plant growth and development and in stress responses. However, studies on banana (Musa acuminata) Ca2+-ATPases are very limited. In this study, we identified 18 Ca2+-ATPase genes from banana, including 6 P-IIA or ER (Endoplasmic Reticulum) type Ca2+-ATPases (MaEACs) and 12 P-IIB or Auto-Inhibited Ca2+-ATPases (MaACAs). The MaEACs and MaACAs could be further classified into two and three subfamilies, respectively. This classification is well supported by their gene structures, which are encoded by protein motif distributions. The banana Ca2+-ATPases were all predicted to be plasma membrane-located. The promoter regions of banana Ca2+-ATPases contain many cis-acting elements and transcription factor binding sites (TFBS). A gene expression analysis showed that banana Ca2+-ATPases were differentially expressed in different organs. By investigating their expression patterns in banana roots under different concentrations of Ca2+ treatments, we found that most banana Ca2+-ATPase members were highly expressed under 4 mM and 2 mM Ca2+ treatments, but their expression decreased under 1 mM and 0 mM Ca2+ treatments, suggesting that their downregulation might be closely related to reduced Ca accumulation and retarded growth under low Ca2+ and Ca2+ deficiency conditions. Our study will contribute to the understanding of the roles of Ca2+-ATPases in banana growth and Ca management.

Integrated Metabolomic and Transcriptomic Analysis Reveals Differential Flavonoid Accumulation and Its Underlying Mechanism in Fruits of Distinct Canarium album Cultivars.

Canarium album fruit has great potential to be consumed as a raw material not only for food but also medicine. The diverse active metabolites composition and content of C. album fruits greatly affect their pharmacological effects. However, up to now, there has been no report on the global metabolome differences among fruits from distinct C. album cultivars. In our present study, by using non-targeted metabolomics techniques, we identified 87 DAMs (differentially accumulated metabolites) including 17 types of flavonoids from fruits of four different C. album cultivars. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis revealed that the flavone and flavonol biosynthesis- and flavonoid biosynthesis-related DAMs were major factors determining their metabolome differences. Comparative transcriptomic analysis revealed that 15 KEGG pathways were significantly enriched by genes of the identified 3655 DEGs (differentially expressed genes) among different C. album cultivars. Consistent with the metabolome data, flavonoid biosynthesis-related DEGs, including eight key structural genes (such as FLS, CCoAOMT, CHI, C4H, DFR, LAR, and C3'H, etc.) and several regulatory transcription factor (TF) genes (including 32 MYBs and 34 bHLHs, etc.), were found to be significantly enriched (p < 0.01). Our study indicated that the differential expression of flavonoid biosynthesis-related genes and accumulation of flavonoids played dominant roles in the various metabolome compositions of fruits from different C. album cultivars.

The Upregulated Expression of the Citrus RIN4 Gene in HLB Diseased Citrus Aids Candidatus Liberibacter Asiaticus Infection.

The citrus industry has been threatened by Huanglongbing (HLB) for over a century. Here, an HLB-induced Arabidopsis RPM1-interacting protein 4 (RIN4) homologous gene was cloned from Citrus clementina, and its characteristics and function were analyzed to determine its role during citrus-Candidatus Liberibacter asiaticus (CLas) interactions. Quantitative real-time PCR showed that RIN4 was expressed in roots, stems, leaves and flowers, with the greatest expression level in leaves. Its expression was suppressed by gibberellic acid, indole-3-acetic acid, salicylic acid and jasmonic acid treatments, but was induced by abscisic acid and salt treatments, as well as wounding. The transient expression of a RIN4-GFP showed that RIN4 was localized in the cell membrane. RIN4-overexpressing transgenic C. maxima cv. 'Shatianyou' plants were obtained, and some transgenic plants showed greater sensitivity to CLas infection and earlier HLB symptoms appearance than non-transgenic controls. Results obtained in this study indicated that the upregulated expression of RIN4 in HLB diseased citrus may aid CLas infection.

Influences of Serendipita indica and Dictyophorae echinovolvata on the Growth and Fusarium Wilt Disease Resistance of Banana.

Recently, many control methods have been tried and applied in the Fusarium wilt disease control of banana and have achieved definite progresses. In this study, by using 'Zhongjiao No.3' and 'Zhongjiao No.4' banana seedlings as materials, the effects of Serendipita indica and bamboo fungus (Dictyophorae echinovolvata) culture substrates on the growth and Fusarium wilt disease resistance of banana were investigated. Results showed that the plant height, leaf length, leaf width, root length and root thickness, aboveground part fresh weight, root fresh weight, and relative chlorophyll content and nitrogen content in leaves of banana seedlings colonized with S. indica were all greater than those of non-colonized controls, while these parameters of banana seedlings grown in nutrient soil containing D. echinovolvata culture substrates were significantly suppressed. Both S. indica non-colonized and colonized seedlings cultivated in nutrient containing 1/4 D. echinovolvata culture substrates showed much milder symptoms compared with those cultivated in normal nutrient soil, indicating that the addition of bamboo fungus substrates to the soil can enhance the Fusarium wilt resistance of banana. The results obtained in this study can provide a basis for the application of S. indica and bamboo fungus in the prevention and control of banana Fusarium wilt disease.

Genome-wide identification of FAD gene family and their contributions to the temperature stresses and mutualistic and parasitic fungi colonization responses in banana.

Fatty acid desaturase (FAD) plays important roles in plant growth and development and plant defense processes. In this study, we identified 27 MaFAD genes from the banana genome. According to the amino acid sequence similarities, their encoded proteins could be classified into five subfamilies. This classification is consistently supported by their gene and protein structures, conserved motifs and subcellular localizations. Segmental duplication events were found to play predominant roles in the MaFAD gene family expansion. Thirty miRNAs targeting MaFADs were identified and many hormone- and stress-responsive cis-acting elements and transcription factor binding sites (TFBSs) were identified in their promoters, indicating that the MaFADs expression regulation was very complicated. Gene expression analysis showed that some MaFADs showed significant differential expression in response to high and low temperature. FocTR4 influenced greatly the expression of several MaFADs and greatly induced the fatty acid (FA) accumulations in roots. Although S. indica showed no significant influence on the expression of most MaFADs, it could greatly alleviate the influence of FocTR4 on several MaFADs and FA biosynthesis. Our study revealed that MaFADs contributed greatly to the responses of high and low temperature stresses and mutualistic and parasitic fungi colonization in banana.

Identification, Characterization and Expression Analysis of Anthocyanin Biosynthesis-related bHLH Genes in Blueberry (Vaccinium corymbosum L.).

Basic helix-loop-helix proteins (bHLHs) play very important roles in the anthocyanin biosynthesis of many plant species. However, the reports on blueberry anthocyanin biosynthesis-related bHLHs were very limited. In this study, six anthocyanin biosynthesis-related bHLHs were identified from blueberry genome data through homologous protein sequence alignment. Among these blueberry bHLHs, VcAN1, VcbHLH42-1, VcbHLH42-2 and VcbHLH42-3 were clustered into one group, while VcbHLH1-1 and VcbHLH1-2 were clustered into the other group. All these bHLHs were of the bHLH-MYC_N domain, had DNA binding sites and reported conserved amino acids in the bHLH domain, indicating that they were all G-box binding proteins. Protein subcellular location prediction result revealed that all these bHLHs were nucleus-located. Gene structure analysis showed that VcAN1 gDNA contained eight introns, while all the others contained seven introns. Many light-, phytohormone-, stress- and plant growth and development-related cis-acting elements and transcription factor binding sites (TFBSs) were identified in their promoters, but the types and numbers of cis-elements and TFBSs varied greatly between the two bHLH groups. Quantitative real-time PCR results showed that VcAN1 expressed highly in old leaf, stem and blue fruit, and its expression increased as the blueberry fruit ripened. Its expression in purple podetium and old leaf was respectively significantly higher than in green podetium and young leaf, indicating that VcAN1 plays roles in anthocyanin biosynthesis regulation not only in fruit but also in podetium and leaf. VcbHLH1-1 expressed the highest in young leaf and stem, and the lowest in green fruit. The expression of VcbHLH1-1 also increased as the fruit ripened, and its expression in blue fruit was significantly higher than in green fruit. VcbHLH1-2 showed high expression in stem but low expression in fruit, especially in red fruit. Our study indicated that the anthocyanin biosynthesis regulatory functions of these bHLHs showed certain spatiotemporal specificity. Additionally, VcAN1 might be a key gene controlling the anthocyanin biosynthesis in blueberry, whose function is worth exploring further for its potential applications in plant high anthocyanin breeding.

Establishment of a Novel and Efficient Agrobacterium-Mediated in Planta Transformation System for Passion Fruit (Passiflora edulis).

Passion fruit (Passiflora edulis) is an important fruit crop with high economic value. Genetic engineering plays an important role in crop improvement with desired traits and gene functional studies. The lack of a simple, efficient, and stable transformation system for passion fruit has greatly limited gene functional studies. In this study, a simple and efficient Agrobacterium-mediated in planta transformation system for passion fruit was established, using Agrobacterium virulent strain EHA105 harboring the binary vectors pCAMBIA1301 and pCAMBIA1302 with GUS and GFP reporter genes. The system requires less time and labor costs than conventional transformation systems, and no additional phytohormones and sterile conditions are required. Regeneration efficiency of 86% and transformation efficiency of 29% were achieved, when the wounds were wrapped with Parafilm and the plants were kept in darkness for 15 days. Approximately 75% of the regenerated plants had a single shoot and 26% multiple shoots. The transformation was confirmed at the DNA and RNA levels as well as by GUS staining and GFP fluorescent measurements. The developed protocol will contribute to the genetic improvement of passion fruit breeding.