Advancements in Reference Gene Selection for Fruit Trees: A Comprehensive Review.

Real-time quantitative polymerase chain reaction (qRT-PCR) has been widely used in gene expression analyses due to its advantages of sensitivity, accuracy and high throughput. The stability of internal reference genes has progressively emerged as a major factor affecting the precision of qRT-PCR results. However, the stability of the expression of the reference genes needs to be determined further in different cells or organs, physiological and experimental conditions. Methods for evaluating these candidate internal reference genes have also evolved from simple single software evaluation to more reliable and accurate internal reference gene evaluation by combining different software tools in a comprehensive analysis. This study intends to provide a definitive reference for upcoming research that will be conducted on fruit trees. The primary focus of this review is to summarize the research progress in recent years regarding the selection and stability analysis of candidate reference genes for different fruit trees.

Identification of genes associated with fatty acid biosynthesis based on 214 safflower core germplasm.

BACKGROUND: Safflower (Carthamus tinctorius L.) is an oilseed crop with substantial medicinal and economic value. However, the methods for constructing safflower core germplasm resources are limited, and the molecular mechanisms of lipid biosynthesis in safflower seeds are not well understood. RESULTS: In this study, 11 oil-related quantitative traits and 50 pairs of InDel markers were used to assess the diversity of a collection of 605 safflower germplasms. The original safflower germplasm exhibited rich phenotypic diversity, with high variation for most of the phenotypic traits under investigation. Similarly, high genetic diversity was evaluated in the original germplasm, in which the mean Shannon's information index (I), observed heterozygosity (H0), and expected heterozygosity (He) were 0.553, 0.182, and 0.374, respectively. Four subgroups with strong genetic structures were identified and a core germplasm of 214 cultivars was constructed, which is well represented in the original germplasm. Meanwhile, differential expression analysis of the transcriptomes of high and low linoleic acid safflower varieties at two stages of seed development identified a total of 47 genes associated with lipid biosynthesis. High expression of the genes KAS II and SAD enhanced the synthesis and accumulation of oleic acid, while FAD genes like FAD2 (Chr8G0104100), FAD3, FAD7 and FAD8 promoted the consumption of oleic acid conversion. The coordinated regulation of these multiple genes ensures the high accumulation of oleic acid in safflower seed oil. CONCLUSIONS: Based on these findings, a core germplasm of 214 cultivars was constructed and 47 candidate genes related to unsaturated fatty acid biosynthesis and lipid accumulation were identified. These results not only provide guidance for further studies to elucidate the molecular basis of oil lipid accumulation in safflower seeds, but also contribute to safflower cultivar improvements.

Betalain biosynthesis in red pulp pitaya is regulated via HuMYB132: a R-R type MYB transcription factor.

BACKGROUND: Multiple MYB transcription factors (TFs) are involved in the regulation of plant coloring. Betalain is a kind of natural plant pigment and its biosynthesis is regulated by a number of enzymes. Despite this, little is known about the molecular properties and roles of MYB TFs in pitaya betalain biosynthesis. RESULTS: In the present study, we identified a 1R-MYB gene, HuMYB132, which is preferentially expressed in red-pulp pitaya at the mature stage. It was clustered with Arabidopsis R-R-type genes and had two DNA-binding domains and a histidine-rich region. The expression assays in N. benthamiana and yeast indicated that HuMYB132 is a nucleus-localized protein with transcriptional activation activity. Dual luciferase reporter assay and electrophoretic mobility shift assays (EMSA) demonstrated that HuMYB132 could promote the transcriptional activities of HuADH1, HuCYP76AD1-1, and HuDODA1 by binding to their promoters. Silencing HuMYB132 reduced betalain accumulation and the expression levels of betalain biosynthetic genes in pitaya pulps. CONCLUSIONS: According to our findings, HuMYB132, a R-R type member of 1R-MYB TF subfamily, positively regulates pitaya betalain biosynthesis by regulating the expression of HuADH1, HuCYP76AD1-1, and HuDODA1. The present study provides a new theoretical reference for the management of pitaya betalain biosynthesis and also provides an essential basis for future regulation of betalain biosynthesis in Hylocereus.

Identification of HuSPL family and key role of HuSPL12 in regulation of betalain biosynthesis in pitaya.

The SQUAMOSA promoter binding protein-like (SPL) gene family is a unique family of plant-specific transcription factors (TFs), which plays vital roles in a variety of plant biological processes. Its role in betalain biosynthesis in Hylocereus undantus; however, is still unclear. Here, we report a total of 16 HuSPL genes from the pitaya genome, which were unevenly distributed among nine chromosomes. The HuSPL genes were clustered into seven groups, and most HuSPLs within the same group shared similar exon-intron structures and conserved motifs. Eight segment replication events in the HuSPL gene family were the main driving force behind the gene family expansion. Nine of the HuSPL genes had potential target sites for Hmo-miR156/157b. Hmo-miR156/157b-targeted HuSPLs exhibited differential expression patterns compared with constitutive expression patterns of most Hmo-miR156/157b-nontargeted HuSPLs. The expression of Hmo-miR156/157b gradually increased during fruit maturation, while the expression of Hmo-miR156/157b-targeted HuSPL5/11/14 gradually decreased. In addition, the lowest expression level of Hmo-miR156/157b-targeted HuSPL12 was detected 23rd day after flowering, when the middle pulps started to turn red. HuSPL5, HuSPL11, HuSPL12, and HuSPL14 were nucleus-localized proteins. HuSPL12 could inhibit the expression of HuWRKY40 by binding to its promoter. Results from yeast two-hybrid and bimolecular fluorescence complementation assays showed that HuSPL12 could interact with HuMYB1, HuMYB132, or HuWRKY42 TFs responsible for betalain biosynthesis. The results of the present study provide an essential basis for future regulation of betalain accumulation in pitaya.

Integrated metabolome and transcriptome analysis reveals the cause of anthocyanin biosynthesis deficiency in litchi aril.

Anthocyanins are health-promoting compounds with strong antioxidant properties that play important roles in disease prevention. Litchi chinensis Sonn. is a well-known and economically significant fruit due to its appealing appearance and nutritional value. The mature pericarp of litchi is rich in anthocyanins, whereas the aril (flesh) has an extremely low anthocyanin content. However, the mechanism of anthocyanin differential accumulation in litchi pericarp and aril remained unknown. Here, metabolome and transcriptome analysis were performed to unveil the cause of the deficiency of anthocyanin biosynthesis in litchi aril. Numerous anthocyanin biosynthesis-related metabolites and their derivatives were found in the aril, and the levels of rutin and (-)-epicatechin in the aril were comparable to those found in the pericarp, while anthocyanin levels were negligible. This suggests that the biosynthetic pathway from phenylalanine to cyanidin was present but that a block in cyanidin glycosylation could result in extremely low anthocyanin accumulation in the aril. Furthermore, 54 candidate genes were screened using weighted gene co-expression network analysis (WGCNA), and 9 genes (LcUFGT1, LcGST1, LcMYB1, LcSGR, LcCYP75B1, LcMATE, LcTPP, LcSWEET10, and LcERF61) might play a significant role in regulating anthocyanin biosynthesis. The dual-luciferase reporter (DLR) assay revealed that LcMYB1 strongly activated the promoters of LcUFGT1, LcGST4, and LcSWEET10. The results imply that LcMYB1 is the primary qualitative gene responsible for the deficiency of anthocyanin biosynthesis in litchi aril, which was confirmed by a transient transformation assay. Our findings shed light on the molecular mechanisms underlying tissue-specific anthocyanin accumulation and will help developing new red-fleshed litchi germplasm.

Pitaya Nutrition, Biology, and Biotechnology: A Review.

Pitaya (Hylocereus spp.) is a member of the cactus family that is native to Central and South America but is now cultivated throughout the sub-tropical and tropical regions of the world. It is of great importance due to its nutritional, ornamental, coloring, medicinal, industrial, and high consumption values. In order to effectively utilize and develop the available genetic resources, it is necessary to appreciate and understand studies pertaining to the usage, origin, nutrition, diversity, evaluation, characterization, conservation, taxonomy, and systematics of the genus Hylocereus. Additionally, to gain a basic understanding of the biology of the plant, this review has also discussed how biotechnological tools, such as cell and tissue culture, micropropagation (i.e., somatic embryogenesis, organogenesis, somaclonal variation, mutagenesis, androgenesis, gynogenesis, and altered ploidy), virus-induced gene silencing, and molecular marker technology, have been used to enhance pitaya germplasm.

Identification of HuSWEET Family in Pitaya (Hylocereus undatus) and Key Roles of HuSWEET12a and HuSWEET13d in Sugar Accumulation.

The sugar composition and content of fruit have a significant impact on their flavor and taste. In pitaya, or dragon fruit, sweetness is a crucial determinant of fruit taste and consumer preference. The sugars will eventually be exported transporters (SWEETs), a novel group of sugar transporters that have various physiological functions, including phloem loading, seed filling, nectar secretion, and fruit development. However, the role of SWEETs in sugar accumulation in pitaya fruit is not yet clear. Here, we identified 19 potential members (HuSWEET genes) of the SWEET family in pitaya and analyzed their conserved motifs, physiochemical characteristics, chromosomal distribution, gene structure, and phylogenetic relationship. Seven highly conserved α-helical transmembrane domains (7-TMs) were found, and the HuSWEET proteins can be divided into three clades based on the phylogenetic analysis. Interestingly, we found two HuSWEET genes, HuSWEET12a and HuSWEET13d, that showed strong preferential expressions in fruits and an upward trend during fruit maturation, suggesting they have key roles in sugar accumulation in pitaya. This can be further roughly demonstrated by the fact that transgenic tomato plants overexpressing HuSWEET12a/13d accumulated high levels of sugar in the mature fruit. Together, our result provides new insights into the regulation of sugar accumulation by SWEET family genes in pitaya fruit, which also set a crucial basis for the further functional study of the HuSWEETs.

Chloroplast Genomes and Phylogenetic Analysis of Three Carthamus (Asteraceae) Species.

The genus Carthamus Linnaeus, which belongs to the tribe Cardueae in the Asteraceae family, originated in the Mediterranean region and consists of approximately 20 species worldwide. Understanding the phylogeny of the Carthamus is crucial for the cultivation of C. tinctorius. Although chloroplast genomes are widely used for species identification and evolutionary studies, there have been limited investigations on the chloroplast genomes of Carthamus species. In this study, we assembled the chloroplast genomes of C. persicus, C. tinctorius × C. persicus, and C. lanatus and combined them with the five chloroplast genomes of C. tinctorius for comparative genomic analysis. The sizes of the chloroplast genomes of C. lanatus, C. persicus, and C. tinctorius × C. persicus were 152,602 bp, 153,177 bp, and 153,177 bp, respectively. Comparative analysis showed that the chloroplast genome structures of the four Carthamus species were highly conserved. Additionally, the phylogenomic analysis demonstrated that the plastid genome and angiosperms353 dataset significantly improved the phylogenetic support of Carthamus species. This analysis supported Carthamus as a monophyletic taxon and its internal division into the sect. Carthamus and sect. Atractylis. The Carthamus was closely related to Carduncellus, Femeniasia, Phonus, and Centaurea. In conclusion, this study not only expands our understanding of the cp genomes of Carthamus species but also provides support for more comprehensive phylogenetic studies of Carthamus.

HuNAC20 and HuNAC25, Two Novel NAC Genes from Pitaya, Confer Cold Tolerance in Transgenic Arabidopsis.

NAC transcription factors are one of the largest families of transcriptional regulators in plants, and members of the gene family play vital roles in regulating plant growth and development processes including biotic/abiotic stress responses. However, little information is available about the NAC family in pitaya. In this study, we conducted a genome-wide analysis and a total of 64 NACs (named HuNAC1-HuNAC64) were identified in pitaya (Hylocereus). These genes were grouped into fifteen subgroups with diversities in gene proportions, exon-intron structures, and conserved motifs. Genome mapping analysis revealed that HuNAC genes were unevenly scattered on all eleven chromosomes. Synteny analysis indicated that the segmental duplication events played key roles in the expansion of the pitaya NAC gene family. Expression levels of these HuNAC genes were analyzed under cold treatments using qRT-PCR. Four HuNAC genes, i.e., HuNAC7, HuNAC20, HuNAC25, and HuNAC30, were highly induced by cold stress. HuNAC7, HuNAC20, HuNAC25, and HuNAC30 were localized exclusively in the nucleus. HuNAC20, HuNAC25, and HuNAC30 were transcriptional activators while HuNAC7 was a transcriptional repressor. Overexpression of HuNAC20 and HuNAC25 in Arabidopsis thaliana significantly enhanced tolerance to cold stress through decreasing ion leakage, malondialdehyde (MDA), and H2O2 and O2- accumulation, accompanied by upregulating the expression of cold-responsive genes (AtRD29A, AtCOR15A, AtCOR47, and AtKIN1). This study presents comprehensive information on the understanding of the NAC gene family and provides candidate genes to breed new pitaya cultivars with tolerance to cold conditions through genetic transformation.

Genome-Wide Identification of WRKY Gene Family in Pitaya Reveals the Involvement of HmoWRKY42 in Betalain Biosynthesis.

The WRKY gene family is a plant-specific transcription factor (TF) that regulates many physiological processes and (a) biotic stress responses. Despite this, little is known about the molecular properties and roles of WRKY TFs in pitaya betalain biosynthesis. Here we report the identification of 70 WRKY in Hylocereus undatus, their gene structure, locations on each chromosome, systematic phylogenetic analysis, conserved motif analysis, and synteny of HuWRKY genes. HmoWRKY42 is a Group IIb WRKY protein and contains a coiled-coil motif, a WRKY domain and a C2H2 zinc-finger motif (CX5CX23HXH). Results from yeast one-hybrid and transient dual-luciferase assays showed that HmoWRKY42 was a transcriptional repressor and could repress HmocDOPA5GT1 expression by binding to its promoter. Yeast two-hybrid assays showed that HmoWRKY42 could interact with itself to form homodimers. Knocking out the coiled-coil motif of HmoWRKY42 prevented its self-interaction and prevented it from binding to the HmocDOPA5GT1 promoter. Knocking out the WRKY domain and C2H2 zinc-finger motif sequence of HmoWRKY42 also prevented it from binding to the HmocDOPA5GT1 promoter. The coiled-coil motif, the WRKY domain and the C2H2 zinc finger motif are key motifs for the binding of HmoWRKY42 to the HmocDOPA5GT1 promoter. HmoWRKY42 is localized in the nucleus and possesses trans-activation ability responsible for pitaya betalain biosynthesis by repressing the transcription of HmocDOPA5GT1. As far as we know, no reports are available on the role of HmoWRKY42 in pitaya betalain biosynthesis. The results provide an important foundation for future analyses of the regulation and functions of the HuWRKY gene family.

Sugar Transport, Metabolism and Signaling in Fruit Development of Litchi chinensis Sonn: A Review.

Litchi chinensis Sonn. is an important evergreen fruit crop cultivated in the tropical and subtropical regions. The edible portion of litchi fruit is the aril, which contains a high concentration of sucrose, glucose, and fructose. In this study, we review various aspects of sugar transport, metabolism, and signaling during fruit development in litchi. We begin by detailing the sugar transport and accumulation during aril development, and the biosynthesis of quebrachitol as a transportable photosynthate is discussed. We then document sugar metabolism in litchi fruit. We focus on the links between sugar signaling and seed development as well as fruit abscission. Finally, we outline future directions for research on sugar metabolism and signaling to improve fruit yield and quality.

Genome-Wide Identification of Aquaporin Gene Family in Pitaya Reveals an HuNIP6;1 Involved in Flowering Process.

Aquaporins (AQPs) are essential membrane proteins involved in seed maturation and germination, stomata movement, photosynthesis, and regulation of plant flowering processes. Pitaya flowers are open at night and wither at daybreak, which shows an obvious circadian rhythm. In this study, a comprehensive genome-wide analysis of AQPs in Hylocereus undantus was conducted to screen key genes associated with flowering processes. A total of 33 HuAQP genes were identified from the H. undantus genome. The 33 HuAQPs were grouped into four subfamilies: 10 PIPs, 13 TIPs, 8 NIPs, and 2 SIPs, which were distributed on 9 out of 11 pitaya chromosomes (Chr) (except for Chr7 and Chr10). Results from expression profiles showed that HuNIP6;1 may be involved in pitaya's floral opening. HuNIP6;1 was localized exclusively in the cell membrane. Overexpression of HuNIP6;1 in Arabidopsis thaliana significantly promoted early flowering through regulating negative flowering regulators of MJM30, COL9, and PRR5, suggesting that HuNIP6;1 plays key roles in regulating flowering time. The present study provides the first genome-wide analysis of the AQP gene family in pitaya and valuable information for utilization of HuAQPs.

A Novel WRKY Transcription Factor HmoWRKY40 Associated with Betalain Biosynthesis in Pitaya (Hylocereus monacanthus) through Regulating HmoCYP76AD1.

Betalains are water-soluble nitrogen-containing pigments with multiple bioactivities. Pitaya is the only large-scale commercially grown fruit containing abundant betalains for consumers. However, the upstream regulators in betalain biosynthesis are still not clear. In this study, HmoWRKY40, a novel WRKY transcription factor, was obtained from the transcriptome data of pitaya (Hylocereus monacanthus). HmoWRKY40 is a member of the Group IIa WRKY family, containing a conserved WRKY motif, and it is located in the nucleus. The betalain contents and expression levels of HmoWRKY40 increased rapidly during the coloration of pitaya and reached their maximums on the 23rd day after artificial pollination (DAAP). Yeast one-hybrid and transient expression assays showed that HmoWRKY40 could bind and activate the promoter of HmoCYP76AD1. Silencing the HmoWRKY40 gene resulted in a significant reduction of betacyanin contents. These results indicate that HmoWRKY40 transcriptionally activates HmoCYP76AD, which is involved in the regulation of pitaya betalain biosynthesis. The results of the present study provide new regulatory networks related to betalain biosynthesis in pitaya.

Integrated sRNAome and RNA-Seq analysis reveals miRNA effects on betalain biosynthesis in pitaya.

BACKGROUND: MicroRNAs (miRNAs) and their regulatory functions in anthocyanin, carotenoid, and chlorophyll accumulation have been extensively characterized in many plant species. However, the miRNA regulatory mechanism in betalain biosynthesis remains mostly unknown. RESULTS: In this study, 126 conserved miRNAs and 41 novel miRNAs were first isolated from Hylocereus monacanthus, among which 95 conserved miRNAs belonged to 53 miRNA families. Thirty-four candidate miRNAs related to betalain biosynthesis were differentially expressed. The expression patterns of those differential expressed miRNAs were analyzed in various pitaya tissues by RT-qPCR. A significantly negative correlation was detected between the expression levels of half those miRNAs and corresponding target genes. Target genes of miRNAs i.e. Hmo-miR157b-HmSPL6-like, Hmo-miR160a-Hpcyt P450-like3, Hmo-miR6020-HmCYP71A8-like, Hmo-novel-2-HmCYP83B1-like, Hmo-novel-15-HmTPST-like, Hmo-miR828a-HmTT2-like, Hmo-miR858-HmMYB12-like, Hmo-miR858-HmMYBC1-like and Hmo-miR858-HmMYB2-like were verified by 5'RACE and transient expression system in tobacco. CONCLUSIONS: Hmo-miR157b, Hmo-miR160a, Hmo-miR6020 Hmo-novel-2, Hmo-novel-15, Hmo-miR828a and Hmo-miR858 play important roles in pitaya fruit coloration and betalain accumulation. Our findings provide new insights into the roles of miRNAs and their target genes of regulatory functions involved in betalain biosynthesis of pitaya.

A lamin-like protein OsNMCP1 regulates drought resistance and root growth through chromatin accessibility modulation by interacting with a chromatin remodeller OsSWI3C in rice.

Lamin proteins in animals are implicated in important nuclear functions, including chromatin organization, signalling transduction, gene regulation and cell differentiation. Nuclear Matrix Constituent Proteins (NMCPs) are lamin analogues in plants, but their regulatory functions remain largely unknown. We report that OsNMCP1 is localized at the nuclear periphery in rice (Oryza sativa) and induced by drought stress. OsNMCP1 overexpression resulted in a deeper and thicker root system, and enhanced drought resistance compared to the wild-type control. An assay for transposase accessible chromatin with sequencing (ATAC-seq) analysis revealed that OsNMCP1-overexpression altered chromatin accessibility in hundreds of genes related to drought resistance and root growth, including OsNAC10, OsERF48, OsSGL, SNAC1 and OsbZIP23. OsNMCP1 can interact with SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin remodelling complex subunit OsSWI3C. The reported drought resistance or root growth-related genes that were positively regulated by OsNMCP1 were negatively regulated by OsSWI3C under drought stress conditions, and OsSWI3C overexpression led to decreased drought resistance. We propose that the interaction between OsNMCP1 and OsSWI3C under drought stress conditions may lead to the release of OsSWI3C from the SWI/SNF gene silencing complex, thus changing chromatin accessibility in the genes related to root growth and drought resistance.

Three LcABFs are Involved in the Regulation of Chlorophyll Degradation and Anthocyanin Biosynthesis During Fruit Ripening in Litchi chinensis.

During litchi (Litchi chinensis Sonn.) fruit ripening, two major physiological changes, degreening (Chl degradation) and pigmentation (anthocyanin biosynthesis), are visually apparent. However, the specific factor triggering this important transition is still unclear. In the present study, we found that endogenous ABA content increased sharply when Chl breakdown was initiated and the ABA level peaked just before the onset of anthocyanin accumulation, suggesting that ABA plays an important role during litchi fruit pigmentation. We characterized three ABSCISIC ACID RESPONSE ELEMENT-BINDING FACTORs (LcABF1/2/3) belonging to group A of the basic leucine zipper (bZIP) transcription factors previously shown to be involved in ABA signaling under abiotic stress. LcABF1 transcripts increased at the onset of Chl degradation, and the expression of LcABF3 accumulated in parallel with anthocyanin biosynthesis. In addition, dual luciferase and yeast one-hybrid assays indicated that LcABF1/2 recognized ABA-responsive elements in the promoter region of Chl degradation-related genes (PAO and SGR), while LcABF2/3 bound the promoter region of LcMYB1 and anthocyanin biosynthesis-related structural genes. Indeed, Nicotiana benthamiana leaves transiently expressing LcABF1/2 showed a senescence phenomenon with Chl degradation, and LcABF3 overexpression increased the accumulation of anthocyanin via activation of LcMYB1, which is the key determinant of anthocyanin biosynthesis. These data indicate that LcABF1/2/3 are important transcriptional regulators of ABA-dependent litchi fruit ripening involved in both Chl degradation and anthocyanin biosynthesis.

Genome-wide identification and expression analysis of SWEET gene family in Litchi chinensis reveal the involvement of LcSWEET2a/3b in early seed development.

BACKGROUND: SWEETs (Sugar Will Eventually be Exported transporters) function as sugar efflux transporters that perform diverse physiological functions, including phloem loading, nectar secretion, seed filling, and pathogen nutrition. The SWEET gene family has been identified and characterized in a number of plant species, but little is known about in Litchi chinensis, which is an important evergreen fruit crop. RESULTS: In this study, 16 LcSWEET genes were identified and nominated according to its homologous genes in Arabidopsis and grapevine. Multiple sequence alignment showed that the 7 alpha-helical transmembrane domains (7-TMs) were basically conserved in LcSWEETs. The LcSWEETs were divided into four clades (Clade I to Clade IV) by phylogenetic tree analysis. A total of 8 predicted motifs were detected in the litchi LcSWEET genes. The 16 LcSWEET genes were unevenly distributed in 9 chromosomes and there was one pairs of segmental duplicated events by synteny analysis. The expression patterns of the 16 LcSWEET genes showed higher expression levels in reproductive organs. The temporal and spatial expression patterns of LcSWEET2a and LcSWEET3b indicated they play central roles during early seed development. CONCLUSIONS: The litchi genome contained 16 SWEET genes, and most of the genes were expressed in different tissues. Gene expression suggested that LcSWEETs played important roles in the growth and development of litchi fruits. Genes that regulate early seed development were preliminarily identified. This work provides a comprehensive understanding of the SWEET gene family in litchi, laying a strong foundation for further functional studies of LcSWEET genes and improvement of litchi fruits.

Pitaya HpWRKY3 Is Associated with Fruit Sugar Accumulation by Transcriptionally Modulating Sucrose Metabolic Genes HpINV2 and HpSuSy1.

Sugar level is an important determinant of fruit taste and consumer preferences. However, upstream regulators that control sugar accumulation during fruit maturation are poorly understood. In the present work, we found that glucose is the main sugar in mature pitaya (Hylocereus) fruit, followed by fructose and sucrose. Expression levels of two sucrose-hydrolyzing enzyme genes HpINV2 and HpSuSy1 obviously increased during fruit maturation, which were correlated well with the elevated accumulation of glucose and fructose. A WRKY transcription factor HpWRKY3 was further identified as the putative binding protein of the HpINV2 and HpSuSy1 promoters by yeast one-hybrid and gel mobility shift assays. HpWRKY3 was localized exclusively in the nucleus and possessed trans-activation ability. HpWRKY3 exhibited the similar expression pattern with HpINV2 and HpSuSy1. Finally, transient expression assays in tobacco leaves showed that HpWRKY3 activated the expressions of HpINV2 and HpSuSy1. Taken together, we propose that HpWRKY3 is associated with pitaya fruit sugar accumulation by activating the transcriptions of sucrose metabolic genes. Our findings thus shed light on the transcriptional mechanism that regulates the sugar accumulation during pitaya fruit quality formation.

Functional characterization of a glucosyltransferase gene, LcUFGT1, involved in the formation of cyanidin glucoside in the pericarp of Litchi chinensis.

Anthocyanins generate the red color in the pericarp of Litchi chinensis. UDP-glucose: flavonoid 3-O-glycosyltransferase (UFGT, EC. 2.4.1.91) stabilizes anthocyanidin by attaching sugar moieties to the anthocyanin aglycone. In this study, the function of an UFGT gene involved in the biosynthesis of anthocyanin was verified through heterologous expression and virus-induced gene silencing assays. A strong positive correlation between UFGT activity and anthocyanin accumulation capacity was observed in the pericarp of 15 cultivars. Four putative flavonoid 3-O-glycosyltransferase-like genes, designated as LcUFGT1 to LcUFGT4, were identified in the pericarp of litchi. Among the four UFGT gene members, only LcUFGT1 can use cyanidin as its substrate. The expression of LcUFGT1 was parallel with developmental anthocyanin accumulation, and the heterologously expressed protein of LcUFGT1 displayed catalytic activities in the formation of anthocyanin. The LcUFGT1 over-expression tobacco had darker petals and pigmented filaments and calyxes resulting from higher anthocyanin accumulations compared with non-transformed tobacco. In the pericarp with LcUFGT1 suppressed by virus-induced gene silencing, pigmentation was retarded, which was well correlated with the reduced-LcUFGT1 transcriptional activity. These results suggested that the glycosylation-related gene LcUFGT1 plays a critical role in red color formation in the pericarp of litchi.