Transcription factor LcNAC002 coregulates chlorophyll degradation and anthocyanin biosynthesis in litchi.

Chlorophyll degradation and anthocyanin biosynthesis, which often occur almost synchronously during fruit ripening, are crucial for vibrant coloration of fruits. However, the interlink point between their regulatory pathways remains largely unknown. Here, 2 litchi (Litchi chinensis Sonn.) cultivars with distinctively different coloration patterns during ripening, i.e. slow-reddening/stay-green "Feizixiao" (FZX) vs rapid-reddening/degreening "Nuomici" (NMC), were selected as the materials to study the key factors determining coloration. Litchi chinensis STAY-GREEN (LcSGR) was confirmed as the critical gene in pericarp chlorophyll loss and chloroplast breakdown during fruit ripening, as LcSGR directly interacted with pheophorbide a oxygenase (PAO), a key enzyme in chlorophyll degradation via the PAO pathway. Litchi chinensis no apical meristem (NAM), Arabidopsis transcription activation factor 1/2, and cup-shaped cotyledon 2 (LcNAC002) was identified as a positive regulator in the coloration of litchi pericarp. The expression of LcNAC002 was significantly higher in NMC than in FZX. Virus-induced gene silencing of LcNAC002 significantly decreased the expression of LcSGR as well as L. chinensis MYELOBLASTOSIS1 (LcMYB1), and inhibited chlorophyll loss and anthocyanin accumulation. A dual-luciferase reporter assay revealed that LcNAC002 significantly activates the expression of both LcSGR and LcMYB1. Furthermore, yeast-one-hybrid and electrophoretic mobility shift assay results showed that LcNAC002 directly binds to the promoters of LcSGR and LcMYB1. These findings suggest that LcNAC002 is an important ripening-related transcription factor that interlinks chlorophyll degradation and anthocyanin biosynthesis by coactivating the expression of both LcSGR and LcMYB1.

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.

Single-nucleus RNA sequencing and mRNA hybridization indicate key bud events and LcFT1 and LcTFL1-2 mRNA transportability during floral transition in litchi.

In flowering plants, floral induction signals intersect at the shoot apex to modulate meristem determinacy and growth form. Here, we report a single-nucleus RNA sequence analysis of litchi apical buds at different developmental stages. A total of 41 641 nuclei expressing 21 402 genes were analyzed, revealing 35 cell clusters corresponding to 12 broad populations. We identify genes associated with floral transition and propose a model that profiles the key events associated with litchi floral meristem identity by analyzing 567 identified floral meristem cells at single cell resolution. Interestingly, single-nucleus RNA-sequencing data indicated that all putative FT and TFL1 genes were not expressed in bud nuclei, but significant expression was detected in bud samples by RT-PCR. Based on the expression patterns and gene silencing results, we highlight the critical role of LcTFL1-2 in inhibiting flowering and propose that the LcFT1/LcTFL1-2 expression ratio may determine the success of floral transition. In addition, the transport of LcFT1 and LcTFL1-2 mRNA from the leaf to the shoot apical meristem is proposed based on in situ and dot-blot hybridization results. These findings allow a more comprehensive understanding of the molecular events during the litchi floral transition, as well as the identification of new regulators.

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.

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.

LcERF2 modulates cell wall metabolism by directly targeting a UDP-glucose-4-epimerase gene to regulate pedicel development and fruit abscission of litchi.

Elucidating the biochemical and molecular basis of premature abscission in fruit crops should help develop strategies to enhance fruit set and yield. Here, we report that LcERF2 contributes to differential abscission rates and responses to ethylene in Litchi chinensis (litchi). Reduced LcERF2 expression in litchi was observed to reduce fruit abscission, concurrent with enhanced pedicel growth and increased levels of hexoses, particularly galactose, as well as pectin abundance in the cell wall. Ecoptic expression of LcERF2 in Arabidopsis thaliana caused enhanced petal abscission, together with retarded plant growth and reduced pedicel galactose and pectin contents. Transcriptome analysis indicated that LcERF2 modulates the expression of genes involved in cell wall modification. Yeast one-hybrid, dual-luciferase reporter and electrophoretic mobility shift assays all demonstrated that a UDP-glucose-4-epimerase gene (LcUGE) was the direct downstream target of LcERF2. This result was further supported by a significant reduction in the expression of the A. thaliana homolog AtUGE2-4 in response to LcERF2 overexpression. Significantly reduced pedicel diameter and enhanced litchi fruit abscission were observed in response to LcUGE silencing. We conclude that LcERF2 mediates fruit abscission by orchestrating cell wall metabolism, and thus pedicel growth, in part by repressing the expression of LcUGE.

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.

Two Birds with One Stone: Surface Functionalization and Delamination of Multilayered Ti3C2Tx MXene by Grafting a Ruthenium(II) Complex to Achieve Conductivity-Enhanced Electrochemiluminescence.

Two-dimensional (2D) nanosheets have captured significant attention in constructing highly efficient electrochemiluminescent (ECL) materials because their high surface area and fully exposed postmodification sites could greatly increase the loading amount of luminophores. However, traditional 2D nanosheets as carriers exhibited natively poor electrical conductivity that restricted the electrochemical activation and the utilization ratio of ECL luminophores. Herein, to overcome this drawback, we utilized conductive 2D Ti3C2Tx MXene nanosheets as carriers to graft Ru(bpy)2(mcpbpy)2+ (bpy = 2,2'-bipyridine, mcpbpy = 4-(4'-methyl-[2,2'-bipyridin]-4-yl) butanoic acid) via a dehydrative condensation reaction and electrostatic interaction. Interestingly, Ru(bpy)2(mcpbpy)2+ played the role of "two birds with one stone", where Ru(bpy)2(mcpbpy)2+ acted as both an ECL luminophore and an intercalation molecule to achieve surface functionalization and delamination of multilayered Ti3C2Tx successfully, obtaining 2D ultrathin Ru-complex-grafted MXene nanosheets (Ru@MXene). Owing to the high load capacity and superior electrical conductivity of an ultrathin 2D MXene nanosheet, the obtained Ru@MXene exhibited a superb ECL emission. As expected, compared with the nonconductive 2D ultrathin metal-organic layers (MOLs) as carriers to graft Ru(bpy)2(mcpbpy)2+, the ECL intensity and ECL efficiency of Ru@MXene presented about 5-fold and 1.7-fold enhancement, respectively. Considering these advantages, Ru@MXene was applied to construct an ECL sensor for ultrasensitive determination of mucin 1 (MUC1), which displayed superb sensitivity (100 ag/mL to 10 ng/mL) with a low detection limit of 26.9 ag/mL. Overall, the conductivity-enhanced ECL based on Ru@MXene opened a fire-new chapter to develop splendent performance ECL emitters and shed new light on the application potential of conductive materials in the bioanalysis field.

Ruthenium(II) Complex-Grafted Hollow Hierarchical Metal-Organic Frameworks with Superior Electrochemiluminescence Performance for Sensitive Assay of Thrombin.

Metal-organic frameworks (MOFs) with porous structures exhibit favorable promise in synthesizing high-performance electrochemiluminescence (ECL) materials, yet their micropores and narrow channels not only restrict the loading capacity of ECL luminophores but also constrain the diffusion of coreactants, ions, and electrons. Hence, we developed a new and simple hydrothermal etching strategy for the fabrication of a hollow hierarchical MOF (HH-UiO-66-NH2) with a hierarchical-pore shell, which was employed as a carrier to graft Ru(bpy)2(mcpbpy)2+ (bpy = 2,2'-bipyridine, mcpbpy = 4-(4'-methyl-[2,2'-bipyridin]-4-yl) butanoic acid) onto the coordinatively unsaturated Zr6 nodes of HH-UiO-66-NH2, creating the Ru-complex-grafted HH-UiO-66-NH2 (abbreviated as HH-Ru-UiO-66-NH2). Impressively, the HH-Ru-UiO-66-NH2 presented brilliant ECL emission. On the one hand, the HH-UiO-66-NH2 with a hierarchical-pore shell and hollow cavity was conducive to immobilize the Ru(bpy)2(mcpbpy)2+ of large steric hindrance into the interior of the MOF, markedly improving the load number of luminophores. On the other hand, the hierarchical-pore shell of HH-UiO-66-NH2 permitted fast diffusion of coreactants, ions, and electrons that facilitated the excitation of more grafted luminophores and greatly enhanced the utilization ratio of ECL luminophores. Inspired by the superior ECL performance of HH-Ru-UiO-66-NH2, an ECL sensing platform was constructed on the basis of HH-Ru-UiO-66-NH2 as an ECL beacon combining catalytic hairpin assembly as a signal amplification strategy, showing excellent selectivity and high sensitivity for thrombin determination. This proof-of-concept work proposed a simple and feasible hydrothermal etching strategy to construct hollow hierarchical MOFs that served as carrier materials to immobilize ECL luminophores, providing significant inspiration to develop highly efficient ECL materials and endowing hollow hierarchical MOFs with ECL sensing applications for the first time.

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.

Matrix Coordination-Induced Electrochemiluminescence Enhancement of Tetraphenylethylene-Based Hafnium Metal-Organic Framework: An Electrochemiluminescence Chromophore for Ultrasensitive Electrochemiluminescence Sensor Construction.

Here, we discovered that rigidifying the tetraphenylethylene (TPE)-based ligand H4TCBPE (H4TCBPE = 1,1,2,2-tetra(4-carboxylbiphenyl)ethylene) into Hf-based metal-organic framework (Hf-TCBPE) could lead to a stronger electrochemiluminescence (ECL) emission in comparison to H4TCBPE aggregates and H4TCBPE monomers. Due to the lack of close-packed TCBPE chromophores in Hf-TCBPE, which was required for aggregation-induced ECL (AI-ECL) enhancement, we defined this unprecedented phenomenon as matrix coordination-induced ECL (MCI-ECL) enhancement. The strong ECL intensity of Hf-TCBPE not only originated from the fixation of the TCBPE ligand between Hf6 clusters that restricted the intramolecular free motions of TCBPE and suppressed the nonradiative relaxation but also stemmed from the high porosity of Hf-TCBPE that rendered both internal and external TCBPE chromophores able to be excited. Considering the unique ECL characteristic of Hf-TCBPE, we combined the new ECL indicator of Hf-TCBPE as well as the phosphate-terminal ferrocene (Fc)-labeled hairpin DNA (Fc-HP3) aptamer together as a signal probe (Hf-TCBPE/Fc-HP3), which was employed to construct a novel "off-on" ECL sensor for ultrasensitive mucin 1 (MUC1) detection with the assistance of the exonuclease III (Exo III)-assisted recycling amplification strategy. As expected, the ECL sensor displayed a desirable linear response range from 1 fg/mL to 1 ng/mL and the detection limit down to 0.49 fg/mL. The MCI-ECL enhancement demonstrated by the Hf-TCBPE developed a new and promising strategy to design and synthesize high-performance metal-organic framework (MOF)-based ECL materials for constructing ultrasensitive ECL sensors.

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.

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.

Characterization of a novel litchi R2R3-MYB transcription factor that involves in anthocyanin biosynthesis and tissue acidification.

BACKGROUND: Maturation of litchi (Litchi chinensis) fruit is characterized by dramatic changes in pigments in the pericarp and flavor compounds in the aril. Among them, the biosynthesis of anthocyanins is most noticeable. Previous studies showed that LcMYB1 and LcbHLH transcription factors participated in regulating the anthocyanin biosynthesis in litchi. However, the roles of other MYB factors remain unclear. RESULTS: In this study, we cloned and characterized the function of LcMYB5, a novel R2R3-MYB identified from litchi transcriptome. Although LcMYB5 was constitutively expressed in litchi tissues and its expressions was not correlated with tissue coloration, overexpression of LcMYB5 resulted in enhanced biosynthesis of anthocyanins in tobacco and petunia concurrent with the up-regulation of their endogenous bHLHs and key structural genes in anthocyanin precursor biosynthesis. These results indicate that LcMYB5 is an R2R3 transcriptional factor regulates anthocyanin biosynthesis either by directly activating the expression of key structural genes such as DFR or by indirectly up regulating the expressions of endogenous bHLH regulators. More interestingly, the pH values in petals and leaves from transgenic lines were significant lower than those in both untransformed tobacco and petunia, indicating LcMYB5 is also associated with pH regulation. The expressions of LcMYB5 and its bHLH partner LcbHLH1 were consistent with the expression of putative tissue acidification gene LcPH1, and the changes in malic acid provided further evidence for the close relationship between LcMYB5 and tissue acidification. CONCLUSIONS: Taking together, our study indicated that LcMYB5 is involved in not only anthocyanin biosynthesis but also tissue acidification.