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.

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.

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.

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.

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.

Biosynthesis of quebrachitol, a transportable photosynthate, in Litchi chinensis.

Although methylated cyclitols constitute a major proportion of the carbohydrates in many plant species, their physiological roles and biosynthetic pathway are largely unknown. Quebrachitol (2-O-methyl-chiro-inositol) is one of the major methylated cyclitols in some plant species. In litchi, quebrachitol represents approximately 50% of soluble sugars in mature leaves and 40% of the total sugars in phloem exudate. In the present study, we identified bornesitol as a transient methylated intermediate of quebrachitol and measured the concentrations of methyl-inositols in different tissues and in tissues subjected to different treatments. 14CO2 feeding and phloem exudate experiments demonstrated that quebrachitol is one of the transportable photosynthates. In contrast to other plant species, the biosynthesis of quebrachitol in litchi is not associated with osmotic stress. High quebrachitol concentrations in tissues of the woody plant litchi might represent a unique carbon metabolic strategy that maintains osmolality under reduced-sucrose conditions. The presence of bornesitol but not ononitol in the leaves indicates a different biosynthetic pathway with pinitol. The biosynthesis of quebrachitol involves the methylation of myo-inositol and the subsequent epimerization of bornesitol. An inositol methyltransferase gene (LcIMT1) responsible for bornesitol biosynthesis was isolated and characterized for the first time, and the biosynthesis pathways of methyl-inositols are discussed.

LcGST4 is an anthocyanin-related glutathione S-transferase gene in Litchi chinensis Sonn.

KEY MESSAGE: A novel LcGST4 was identified and characterized from Litchi chinensis . Expression and functional analysis demonstrated that it might function in anthocyanin accumulation in litchi. Glutathione S-transferases (GSTs) have been defined as detoxification enzymes for their ability to recognize reactive electrophilic xenobiotic molecules as well as endogenous secondary metabolites. Anthocyanins are among the few endogenous substrates of GSTs for vacuolar accumulation. The gene encoding a GST protein that is involved in anthocyanin sequestration from Litchi chinensis Sonn. has not been reported. Here, LcGST4, an anthocyanin-related GST, was identified and characterized. Phylogenetic analysis showed that LcGST4 was clustered with other known anthocyanin-related GSTs in the same clade. Expression analysis revealed that the expression pattern of LcGST4 was strongly correlated with anthocyanin accumulation in litchi. ABA- and light-responsive elements were found in the LcGST4 promoter, which is in agreement with the result that the expression of LcGST4 was induced by both ABA and debagging treatment. A GST activity assay in vitro verified that the LcGST4 protein shared universal activity with the GST family. Functional complementation of an Arabidopsis mutant tt19 demonstrated that LcGST4 might function in anthocyanin accumulation in litchi. Dual luciferase assay revealed that the expression of LcGST4 was activated by LcMYB1, a key R2R3-MYB transcription factor that regulates anthocyanin biosynthesis in litchi.

Proteomic Analysis of Hylocereus polyrhizus Reveals Metabolic Pathway Changes.

Red dragon fruit or red pitaya (Hylocereus polyrhizus) is the only edible fruit that contains betalains. The color of betalains ranges from red and violet to yellow in plants. Betalains may also serve as an important component of health-promoting and disease-preventing functional food. Currently, the biosynthetic and regulatory pathways for betalain production remain to be fully deciphered. In this study, isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analyses were used to reveal the molecular mechanism of betalain biosynthesis in H. polyrhizus fruits at white and red pulp stages, respectively. A total of 1946 proteins were identified as the differentially expressed between the two samples, and 936 of them were significantly highly expressed at the red pulp stage of H. polyrhizus. RNA-seq and iTRAQ analyses showed that some transcripts and proteins were positively correlated; they belonged to "phenylpropanoid biosynthesis", "tyrosine metabolism", "flavonoid biosynthesis", "ascorbate and aldarate metabolism", "betalains biosynthesis" and "anthocyanin biosynthesis". In betalains biosynthesis pathway, several proteins/enzymes such as polyphenol oxidase, CYP76AD3 and 4,5-dihydroxy-phenylalanine (DOPA) dioxygenase extradiol-like protein were identified. The present study provides a new insight into the molecular mechanism of the betalain biosynthesis at the posttranscriptional level.

Transcriptomic analysis of Litchi chinensis pericarp during maturation with a focus on chlorophyll degradation and flavonoid biosynthesis.

BACKGROUND: The fruit of litchi (Litchi chinensis) comprises a white translucent edible aril surrounded by a pericarp. The pericarp of litchi has been the focus of studies associated with fruit size, coloration, cracking and shelf life. However, research at the molecular level has been limited by the lack of genomic and transcriptomic information. In this study, an analysis of the transcriptome of litchi pericarp was performed to obtain information regarding the molecular mechanisms underlying the physiological changes in the pericarp, including those leading to fruit surface coloration. RESULTS: Coincident with the rapid break down of chlorophyll, but substantial increase of anthocyanins in litchi pericarp as fruit developed, two major physiological changes, degreening and pigmentation were visually apparent. In this study, a cDNA library of litchi pericarp with three different coloration stages was constructed. A total of 4.7 Gb of raw RNA-Seq data was generated and this was then de novo assembled into 51,089 unigenes with a mean length of 737 bp. Approximately 70% of the unigenes (34,705) could be annotated based on public protein databases and, of these, 3,649 genes were significantly differentially expressed between any two coloration stages, while 156 genes were differentially expressed among all three stages. Genes encoding enzymes involved in chlorophyll degradation and flavonoid biosynthesis were identified in the transcriptome dataset. The transcript expression patterns of the Stay Green (SGR) protein suggested a key role in chlorophyll degradation in the litchi pericarp, and this conclusion was supported by the result of an assay over-expressing LcSGR protein in tobacco leaves. We also found that the expression levels of most genes especially late anthocyanin biosynthesis genes were co-ordinated up-regulated coincident with the accumulation of anthocyanins, and that candidate MYB transcription factors that likely regulate flavonoid biosynthesis were identified. CONCLUSIONS: This study provides a large collection of transcripts and expression profiles associated with litchi fruit maturation processes, including coloration. Since most of the unigenes were annotated, they provide a platform for litchi functional genomic research within this species.

CrWSKP1, an SKP1-like Gene, Is Involved in the Self-Incompatibility Reaction of "Wuzishatangju" (Citrus reticulata Blanco).

Plant S-phase kinase-associated protein 1 (SKP1) genes play crucial roles in plant development and differentiation. However, the role of SKP1 in citrus is unclear. Herein, we described a novel SKP1-like gene, designated as CrWSKP1, from "Wuzishatangju" (Citrus reticulata Blanco). The cDNA sequence of CrWSKP1 is 779 base pairs (bp) and contains an open reading frame (ORF) of 477 bp. The genomic sequence of the CrWSKP1 gene is 1296 bp with two exons and one intron. CrWSKP1 has high identity with SKP1-like genes from other plant species within two conserved regions. Approximately 85% of pollen tubes of self-pollinated CrWSKP1 transgenic tobaccos became twisted at four days after self-pollination. Pollen tube numbers of self-pollinated CrWSKP1 transformants entering into ovules were significantly fewer than that of the control. Seed number of self-pollinated CrWSKP1 transformants was significantly reduced. These results suggested that the CrWSKP1 is involved in the self-incompatibility (SI) reaction of "Wuzishatangju".

LcINH1 as an inhibitor of cell wall invertase LcCWIN5 regulates early seed development in Litchi chinensis Sonn.

Sugar signal mediated by Cell wall invertase (CWIN) plays a central role in seed development. In higher plants, invertase inhibitors (INHs) suppress CWIN activities at a post-translational level. In Litchi chinensis cultivar 'Nuomici', impaired CWIN expression is associated with seed abortion. Here, the expression of LcINH1 was significantly higher in the funicle of seed-aborting cultivar 'Nuomici' than big-seeded cultivar 'Heiye'. Promoter analyses found LcINH1 contained a 404 bp repeat fragment with an endosperm regulatory element of Skn-1_motif. LcINH1 and LcCWIN2/5 were located in plasma membrane. LcINH1 was able to interact with LcCWIN5, but not with LcCWIN2. In vitro enzyme activity assay demonstrated that LcINH1 could inhibit CWIN activity. Silencing LcINH1 in 'Nuomici' resulted in normal seed development, paralleled increased CWIN activities and glucose levels. Transcriptome analysis identified 1079 differentially expressed genes (DEGs) in LcINH1-silenced fruits. KEGG analysis showed significant enrichment of DEGs in pathways related to transporters and plant hormone signal transduction. Weighted gene co-expression network analysis indicated that the turquoise module was highly correlated with fructose content, and LcSWEET3b was closely associated with early seed development. These findings suggest that LcINH1 regulate LcCWIN5 activity at the post-translational level to alter sucrose metabolism, thereby affecting early seed development in litchi.

Genetic Analyses of Flower Main Traits from Two Pitayas and Their Progenies: A Cactus Plant.

Elucidation of the genetic foundation governing crucial traits in pitaya flowers is imperative for enhancing both the ornamental and economic values. In this study, the dynamic variation in flower genetics, segregation variation patterns, and a mixed inheritance model of the major and multigene flower traits of 'Dahong' and 'Honghuaqinglong' pitayas and their progenies were explored. The results showed that the main traits of flowers exhibited varying degrees of variation among the reciprocal F1 hybrids, with the data exhibiting the characteristics of quantitative traits. The betalain content, petal number, and stigma number exhibited values below the median values of the parents, suggesting a genetic inclination towards lower values. Perianth width, calyx tube width, petal number, and stigma number had the same genetic effects and significant correlation. Stigma-related traits had a clear maternal inheritance tendency. The heritability of flower length, stigma relative to anther distance, and petal betalain content was governed by two pairs of additive-dominant major genes. Perianth width, calyx tube width, petal number, and stigma number all conformed to the model of two pairs of equal-additive-dominant major genes. This study provides valuable information for parental selection, cross-breeding, and the enhancement of pitaya varieties to meet market preferences and environmental conditions.