A High-Quality Assembly and Comparative Analysis of the Mitogenome of Actinidia macrosperma.

The mitochondrial genome (mitogenome) of Actinidia macrosperma, a traditional medicinal plant within the Actinidia genus, remains relatively understudied. This study aimed to sequence the mitogenome of A. macrosperma, determining its assembly, informational content, and developmental expression. The results revealed that the mitogenome of A. macrosperma is circular, spanning 752,501 bp with a GC content of 46.16%. It comprises 63 unique genes, including 39 protein-coding genes (PCGs), 23 tRNA genes, and three rRNA genes. Moreover, the mitogenome was found to contain 63 SSRs, predominantly mono-nucleotides, as well as 25 tandem repeats and 650 pairs of dispersed repeats, each with lengths equal to or greater than 60, mainly comprising forward repeats and palindromic repeats. Moreover, 53 homologous fragments were identified between the mitogenome and chloroplast genome (cp-genome), with the longest segment measuring 4296 bp. This study represents the initial report on the mitogenome of the A. macrosperma, providing crucial genetic materials for phylogenetic research within the Actinidia genus and promoting the exploitation of species genetic resources.

Transcriptome Analysis Reveals Candidate Lignin-Related Genes and Transcription Factors during Fruit Development in Pomelo (Citrus maxima).

Juice sac granulation (a physiological disorder) leads to large postharvest losses of pomelo (Citrus maxima). Previous studies have shown that juice sac granulation is closely related to lignin accumulation, while the molecular mechanisms underlying this disorder remain elusive in pomelo. Our results showed that the lignin content in NC (near the core) and FC (far away from the core) juice sacs overall increased from 157 DPA (days post anthesis) to 212 DPA and reached a maximum at 212 DPA. Additionally, the lignin content of NC juice sacs was higher than that of FC juice sacs. In this study, we used transcriptome-based weighted gene co-expression network analysis (WGCNA) to address how lignin formation in NC and FC juice sacs is generated during the development of pomelo. After data assembly and bioinformatic analysis, we found a most correlated module (black module) to the lignin content, then we used the 11 DEGs in this module as hub genes for lignin biosynthesis. Among these DEGs, PAL (phenylalanine ammonia lyase), HCT (hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase), 4CL2 (4-coumarate: CoA ligase), C4H (cinnamate 4-hydroxylase), C3'H (p-coumarate 3-hydroxylase), and CCoAOMT1 (caffeoyl CoA 3-Omethyltransferase) were the most distinct DEGs in granulated juice sacs. Co-expression analysis revealed that the expression patterns of several transcription factors such as MYB, NAC, OFP6, and bHLH130 are highly correlated with lignin formation. In addition, the expression patterns of the DEGs related to lignin biosynthesis and transcription factors were validated by qRT-PCR, and the results were highly concordant with the RNA-seq results. These results would be beneficial for further studies on the molecular mechanism of lignin accumulation in pomelo juice sacs and would help with citrus breeding.

Identification and Functional Analysis of the CgNAC043 Gene Involved in Lignin Synthesis from Citrusgrandis "San Hong".

Overaccumulation of lignin (a physiological disorder known as granulation) often occurs during fruit ripening and postharvest storage in pomelo (Citrus grandis). It causes an unpleasant fruit texture and taste. Previous studies have shown that lignin metabolism is closely associated with the process of juice sacs granulation. At present, the underlying transcriptional regulatory mechanisms remain unclear. In this study, we identified and isolated a candidate NAC transcription factor, CgNAC043, that is involved in the regulation of lignin biosynthesis in Citrus grandis, which has homologs in Arabidopsis and other plants. We used the fruit juice sacs of 'San hong' as the material, the staining for lignin with HCl-phloroglucinol of fruit juice sacs became dark red from the various developmental stages at 172 to 212 days post anthesis (DPA). The RT-qPCR was used to analyze the gene expression of CgNAC043 and its target gene CgMYB46 in fruit sacs, it was found that the expression trend of CgNAC043 was basically same as CgMYB46, which increased gradually and peaked at 212 DPA. The expression level of CgNAC043 in juice sacs obtained away from the core was the lowest, while those near the core and granulated area were highly expressed. The transcriptional activation activity of CgNAC043 and CgMYB46 was analyzed by a yeast two-hybrid system, with only CgNAC043 showing transcriptional activation activity in Y2H Gold yeast. A transformation vector, p1301- CgNAC043, was transformed into the mesocarp of 'San hong' by Agrobacterium-mediated transformation. Results showed that the expression of transcription factors CgMYB58 and CgMYB46 are all upregulated. Further experiments proved that CgNAC043 not only can directly trans-activate the promoter of CgMYB46 but also trans-activate the promoters for the lignin biosynthesis-related genes CgCCoAOMT and CgC3H by dual luciferase assay. We isolated the CgNAC043 gene in pomelo and found CgNAC043 regulates target genes conferring the regulation of juice sacs granulation.

Fruit quality and volatile constituents of a new very early-ripening pummelo (Citrus maxima) cultivar 'Liuyuezao'.

'Liuyuezao' (LYZ) pummelo (Citrus maxima) originated from a spontaneous bud sport on a 'Guanxi' (GXB) pummelo tree and was released as a new very early-season cultivar. The objective of this study was to present the sensory and nutritional profiles of LYZ fruits, and compare it with other major commercialized pummelo cultivars including GXB, 'Sanhong' (SH) and 'Hongrou' (HR). LYZ had higher contents of organic acids (12.01 mg/g), phenols (669.01 mg/L), vitamin C (75.73 mg/100 mL) and stronger antioxidant capacity (77.65 mg/100 mL) but lower levels of soluble sugars (62.85 mg/g), carotenoids (0.25 mg/L) and flavonoids (46.3 mg/L) when compared to the other pummelos. Moreover, a smaller number (49) and much less content (7.63) of fruit volatiles were detected in LYZ than them in GXB, SH and HR. The relatively high levels of fructose (20.6 mg/g) and organic acids and low levels of volatile compounds in LYZ mainly contributed to its sweet and mildly sour taste and moderate aroma of pummelo note. LYZ is presented as an alternative pummelo cultivar with the potential for commercialization.

Molecular Analysis of 14-3-3 Genes in Citrus sinensis and Their Responses to Different Stresses.

14-3-3 proteins (14-3-3s) are among the most important phosphorylated molecules playing crucial roles in regulating plant development and defense responses to environmental constraints. No report thus far has documented the gene family of 14-3-3s in Citrus sinensis and their roles in response to stresses. In this study, nine 14-3-3 genes, designated as CitGF14s (CitGF14a through CitGF14i) were identified from the latest C. sinensis genome. Phylogenetic analysis classified them into ε-like and non-ε groups, which were supported by gene structure analysis. The nine CitGF14s were located on five chromosomes, and none had duplication. Publicly available RNA-Seq raw data and microarray databases were mined for 14-3-3 expression profiles in different organs of citrus and in response to biotic and abiotic stresses. RT-qPCR was used for further examining spatial expression patterns of CitGF14s in citrus and their temporal expressions in one-year-old C. sinensis "Xuegan" plants after being exposed to different biotic and abiotic stresses. The nine CitGF14s were expressed in eight different organs with some isoforms displayed tissue-specific expression patterns. Six of the CitGF14s positively responded to citrus canker infection (Xanthomonas axonopodis pv. citri). The CitGF14s showed expressional divergence after phytohormone application and abiotic stress treatments, suggesting that 14-3-3 proteins are ubiquitous regulators in C. sinensis. Using the yeast two-hybrid assay, CitGF14a, b, c, d, g, and h were found to interact with CitGF14i proteins to form a heterodimer, while CitGF14i interacted with itself to form a homodimer. Further analysis of CitGF14s co-expression and potential interactors established a 14-3-3s protein interaction network. The established network identified 14-3-3 genes and several candidate clients which may play an important role in developmental regulation and stress responses in this important fruit crop. This is the first study of 14-3-3s in citrus, and the established network may help further investigation of the roles of 14-3-3s in response to abiotic and biotic constraints.

Identification of Appropriate Reference Genes for Normalizing miRNA Expression in Citrus Infected by Xanthomonas citri subsp. citri.

MicroRNAs (miRNAs) are short noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Reverse transcription-quantitative PCR (RT-qPCR) is one of the most common methods used for quantification of miRNA expression, and the levels of expression are normalized by comparing with reference genes. Thus, the selection of reference genes is critically important for accurate quantification. The present study was intended to identify appropriate miRNA reference genes for normalizing the level of miRNA expression in Citrus sinensis L. Osbeck and Citrus reticulata Blanco infected by Xanthomonas citri subsp. citri, which caused citrus canker disease. Five algorithms (Delta Ct, geNorm, NormFinder, BestKeeper and RefFinder) were used for screening reference genes, and two quantification approaches, poly(A) extension RT-qPCR and stem-loop RT-qPCR, were used to determine the most appropriate method for detecting expression patterns of miRNA. An overall comprehensive ranking output derived from the multi-algorithms showed that poly(A)-tailed miR162-3p/miR472 were the best reference gene combination for miRNA RT-qPCR normalization in citrus canker research. Candidate reference gene expression profiles determined by poly(A) RT-qPCR were more consistent in the two citrus species. To the best of our knowledge, this is the first systematic comparison of two miRNA quantification methods for evaluating reference genes. These results highlight the importance of rigorously assessing candidate reference genes and clarify some contradictory results in miRNA research on citrus.