Genome-Scale Computational Identification and Characterization of UTR Introns in <i>Atalantia buxifolia</i>

Accumulated evidence has shown that CDS introns (CIs) play important roles in regulating gene expression. However, research on UTR introns (UIs) is limited. In this study, UIs (including 5′UTR and 3′UTR introns (5UIs and 3UIs)) were identified from the Atalantia buxifolia genome. The length and nucleotide distribution characteristics of both 5UIs and 3UIs and the distributions of cis-acting elements and transcription factor binding sites (TFBSs) in 5UIs were investigated. Moreover, PageMan enrichment analysis was applied to show the possible roles of transcripts containing UIs (UI-Ts). In total, 1077 5UIs and 866 3UIs were identified from 897 5UI-Ts and 670 3UI-Ts, respectively. Among them, 765 (85.28%) 5UI-Ts and 527 (78.66%) 3UI-Ts contained only one UI, and 94 (6.38%) UI-Ts contained both 5UI and 3UI. The UI density was lower than that of CDS introns, but their mean and median intron sizes were ~2 times those of the CDS introns. The A. buxifolia 5UIs were rich in gene-expression-enhancement-related elements and contained many TFBSs for BBR-BPC, MIKC_MADS, AP2 and Dof TFs, indicating that 5UIs play a role in regulating or enhancing the expression of downstream genes. Enrichment analysis revealed that UI-Ts involved in ‘not assigned’ and ‘RNA’ pathways were significantly enriched. Noteworthily, 119 (85.61%) of the 3UI-Ts were genes encoding pentatricopeptide (PPR) repeat-containing proteins. These results will be helpful for the future study of the regulatory roles of UIs in A. buxifolia

Exogenous Melatonin Enhances Cold Resistance by Improving Antioxidant Defense and Cold-Responsive Genes’ Expression in Banana

Accumulated evidence has revealed the mitigation effects of exogenous melatonin on cold stress in plants. In this study, to investigate the defensive roles of exogenous melatonin in banana under cold stress, we researched the influences of exogenous melatonin on the chlorophyll fluorescence parameters, antioxidant defense indexes and expression levels of cold-responsive genes in cold-stressed ‘Brazil’ banana seedlings. Results showed that 100 μM of exogenous melatonin achieved the best cold-resistance-promoting effect in banana. Exogenous melatonin treatment significantly increased the electron transfer rate, light harvesting efficiency, total antioxidant capacity, catalase and superoxidase activities and proline and soluble sugar contents and significantly reduced the accumulations of malondialdehyde, superoxide anion and hydrogen peroxide in the leaves of cold-stressed banana. In addition, under cold stress, melatonin significantly induced the expression of low-temperature-responsive genes, such as MaChiI1, MaCSD1C, MaWhy1, MaKIN10, MaADA1 and MaHOS1. It was concluded that the application of exogenous melatonin enhanced antioxidant defense and induced the expression of cold-responsive genes, thereby improving the cold resistance of banana. Our study will provide a basis for the application of exogenous melatonin in improving plant cold resistance

Comparison of micro RNA (miRNA) Profiles and Some miRNA Target Gene Expression levels in Roots of Non-infected and Huanglongbing-infected Tangerine (Citrus reticulata Blanco cv.‘Sanhu’ ) Trees

Solexa sequencing was used to reveal the changes in small RNAome profile in roots of mock-inoculated (CK) and Huanglongbing bacteria-inoculated (HLB) ‘Sanhu’ tangerine (Citrus reticulata Blanco) trees. Results showed that the number of reads of both unique and total sRNAs decreased apparently in roots following infection with HLB. Distribution in length of sRNAs changed also remarkably, showing an increase in 22 nt and 21 nt small RNAs (sRNAs) and a decrease in 24 nt sRNAs in HLB-infected samples. A total of 42 known micro RNAs (miRNAs) belonging to 27 highly conserved miRNA families were identified. Comparisons showed that 33 known miRNAs exhibited a significant expression difference between CK and HLB-inoculated samples. In addition, 34 novel miRNAs, among which 24 were differentially expressed, were also identified, and their expression levels were analyzed by qRT-PCR. Three hundred and eighty five potential target genes were predicted for most of the 57 differentially expressed miRNAs. GO and KEGG annotation analysis revealed that most miRNA-target genes were those implicated in developmental process, response to stress and stimulus, transcription and protein metabolism. The characterization of the miRNAomes between the healthy and HLB infected Sanhu tangerine roots provided new insight into the involvement of miRNAs in HLB infection of citrus

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

Identification, Characterization and Comparison of the Genome-Scale UTR Introns from Six Citrus Species

Ever since their discovery, introns within the coding sequence (CDS) of transcripts have been paid great attention. However, the introns located in the untranslated regions (UTRs) are often ignored. Here, we identified, characterized and compared the UTR introns (UIs) from six citrus species. Results showed that the average intron number of UTRs is greatly lower than that of CDSs. Among all six citrus species, the number and density of 5′UTR introns (5UIs) are higher than those of 3′UTR introns (3UIs). The UI densities varied greatly among different citrus species. There are 11 and 9 types of splice site (SS) pairs for the UIs of C. sinensis and C. medica, respectively. However, the UIs of the other four citrus species all own only three kinds of SS pairs. The ‘GT-AG’, accounting for more than 95% of both 5UIs and 3UIs SS pairs for all the six species, is the most popular type. Moreover, 81 5UIs and 26 3UIs were identified as common UIs among the six citrus species, and the transcripts containing these common UIs were mostly involved in gene expression or gene expression regulation. Our study revealed that the UIs’ length, abundance, density and SS pair types varied among different citrus species and that many UI-containing genes play important roles in gene expression regulation. Our findings have great implications for future citrus UI function research

The Upregulated Expression of the Citrus <i>RIN4</i> Gene in HLB Diseased Citrus Aids <i>Candidatus</i> 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

Exogenous Melatonin Enhances Cold Resistance by Improving Antioxidant Defense and Cold-Responsive Genes&rsquo; Expression in Banana

Accumulated evidence has revealed the mitigation effects of exogenous melatonin on cold stress in plants. In this study, to investigate the defensive roles of exogenous melatonin in banana under cold stress, we researched the influences of exogenous melatonin on the chlorophyll fluorescence parameters, antioxidant defense indexes and expression levels of cold-responsive genes in cold-stressed &lsquo;Brazil&rsquo; banana seedlings. Results showed that 100 &mu;M of exogenous melatonin achieved the best cold-resistance-promoting effect in banana. Exogenous melatonin treatment significantly increased the electron transfer rate, light harvesting efficiency, total antioxidant capacity, catalase and superoxidase activities and proline and soluble sugar contents and significantly reduced the accumulations of malondialdehyde, superoxide anion and hydrogen peroxide in the leaves of cold-stressed banana. In addition, under cold stress, melatonin significantly induced the expression of low-temperature-responsive genes, such as MaChiI1, MaCSD1C, MaWhy1, MaKIN10, MaADA1 and MaHOS1. It was concluded that the application of exogenous melatonin enhanced antioxidant defense and induced the expression of cold-responsive genes, thereby improving the cold resistance of banana. Our study will provide a basis for the application of exogenous melatonin in improving plant cold resistance

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&prime;H, etc.) and several regulatory transcription factor (TF) genes (including 32 MYBs and 34 bHLHs, etc.), were found to be significantly enriched (p &lt; 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

Characterization of Highbush Blueberry (<i>Vaccinium corymbosum</i> L.) Anthocyanin Biosynthesis Related MYBs and Functional Analysis of <i>VcMYB</i> 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