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.

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.

Function of a non-enzymatic hexokinase LcHXK1 as glucose sensor in regulating litchi fruit abscission.

Fruit abscission is a severe hindrance to commercial crop production, and a lack of carbohydrates causes fruit abscission to intensify in a variety of plant species. However, the precise mechanism by which carbohydrates affect fruit setting potential has yet to be determined. In the current study, we noticed negative correlation between hexose level and fruit setting by comparing different cultivars, bearing shoots of varying diameters, and girdling and defoliation treatments. The cumulative fruit-dropping rate was significantly reduced in response to exogenous glucose dipping. These results suggested that hexose, especially glucose, is the key player in lowering litchi fruit abscission. Moreover, five putative litchi hexokinase genes (LcHXKs) were isolated and the subcellular localization as well as activity of their expressed proteins in catalyzing hexose phosphorylation were investigated. LcHXK2 was only found in mitochondria and expressed catalytic protein, whereas the other four HXKs were found in both mitochondria and nuclei and had no activity in catalyzing hexose phosphorylation. LcHXK1 and LcHXK4 were found in the same cluster as previously reported hexose sensors AtHXK1 and MdHXK1. Furthermore, VIGS-mediated silencing assay confirms that LcHXK1 suppression increases fruit abscission. These findings revealed that LcHXK1 functions as hexose sensor, negatively regulating litchi fruit abscission.

A chromosome-scale genome sequence of pitaya (Hylocereus undatus) provides novel insights into the genome evolution and regulation of betalain biosynthesis.

Pitaya (Hylocereus) is the most economically important fleshy-fruited tree of the Cactaceae family that is grown worldwide, and it has attracted significant attention because of its betalain-abundant fruits. Nonetheless, the lack of a pitaya reference genome significantly hinders studies focused on its evolution, as well as the potential for genetic improvement of this crop. Herein, we employed various sequencing approaches, namely, PacBio-SMRT, Illumina HiSeq paired-end, 10× Genomics, and Hi-C (high-throughput chromosome conformation capture) to provide a chromosome-level genomic assembly of 'GHB' pitaya (H. undatus, 2n = 2x = 22 chromosomes). The size of the assembled pitaya genome was 1.41 Gb, with a scaffold N50 of ~127.15 Mb. In total, 27,753 protein-coding genes and 896.31 Mb of repetitive sequences in the H. undatus genome were annotated. Pitaya has undergone a WGT (whole-genome triplication), and a recent WGD (whole-genome duplication) occurred after the gamma event, which is common to the other species in Cactaceae. A total of 29,328 intact LTR-RTs (~696.45 Mb) were obtained in H. undatus, of which two significantly expanded lineages, Ty1/copia and Ty3/gypsy, were the main drivers of the expanded genome. A high-density genetic map of F1 hybrid populations of 'GHB' × 'Dahong' pitayas (H. monacanthus) and their parents were constructed, and a total of 20,872 bin markers were identified (56,380 SNPs) for 11 linkage groups. More importantly, through transcriptomic and WGCNA (weighted gene coexpression network analysis), a global view of the gene regulatory network, including structural genes and the transcription factors involved in pitaya fruit betalain biosynthesis, was presented. Our data present a valuable resource for facilitating molecular breeding programs of pitaya and shed novel light on its genomic evolution, as well as the modulation of betalain biosynthesis in edible fruits.

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.

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.

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.

Restriction of intramolecular motions (RIM) by metal-organic frameworks for electrochemiluminescence enhancement:2D Zr12-adb nanoplate as a novel ECL tag for the construction of biosensing platform.

Herein, a new phenomenon of enhanced electrochemiluminescence (ECL) emission by restricting intramolecular motion in the 2D ultra-thin Zr12-adb (adb = 9,10-anthracene dibenzoate) metal-organic framework (MOF) nanoplate was discovered for the first time. The coordination immobilization of adb in porous ultra-thin Zr12-adb nanoplate endowed the Zr12-adb excellent ECL performance, including stronger ECL signal and higher ECL efficiency relative to those of H2adb monomers and H2adb aggregates. In the 2D Zr12-adb nanoplate, the bridging ligand adb was stretched and fixed between two Zr12 clusters, which restricted intramolecular rotations and suppressed unnecessary energy loss caused by self-rotation, thereby remarkably improved the ECL intensity and efficiency. More importantly, the porous ultra-thin structure of Zr12-adb MOF nanoplate not only allowed the coreactants to diffuse into the MOF interior, making both internal and external adb be excited, but also shortened the migration distance of electrons, ions, coreactants and coreactant intermediates, which further improved the ECL efficiency of Zr12-adb and overcame the shortcoming of H2adb aggregates in which the internal luminophores were not easily excited. Regarding the excellent ECL properties above, Zr12-adb nanoplate was selected as a new ECL emitter incorporated with the bipedal walking molecular machine together to fabricate a biosensor for sensitive detection of mucin 1. The enhanced ECL by restriction of intramolecular motions in MOFs provided a new pathway to improve ECL intensity and efficiency, which lighted up a lamp for the design and manufacture of high-performance ECL materials based on MOFs, thus offering new opportunities to develop ultrasensitive ECL biosensors.

An AIEgen-based 2D ultrathin metal-organic layer as an electrochemiluminescence platform for ultrasensitive biosensing of carcinoembryonic antigen.

In this work, a novel two-dimensional (2D) ultrathin metal-organic layer (MOL) based on the aggregation-induced emission (AIE) ligand H4ETTC (H4ETTC = 4',4''',4''''',4'''''''-(ethene-1,1,2,2-tetrayl)tetrakis(([1,1'-biphenyl]-4-carboxylic acid))) was developed and used to construct a novel electrochemiluminescence (ECL) aptasensor for ultrasensitive detection of carcinoembryonic antigen (CEA). The newly synthesized AIE luminogen (AIEgen)-based MOL (Hf-ETTC-MOL) yielded a higher ECL intensity and efficiency than did H4ETTC monomers, H4ETTC aggregates and 3D bulk Hf-ETTC-MOF. This improvement occurred not only because the ETTC ligands were coordinatively immobilized in a rigid MOL matrix, which restricted the intramolecular free rotation and vibration of these ligands and then reduced the non-radiative transition, but also because the porous ultrathin 2D MOL greatly shortened the transport distances of ions, electrons, coreactant (triethylamine, TEA) and coreactant intermediates (TEA˙ and TEA˙+), which made more ETTC luminophores able to be excited and yielded a high ECL efficiency. On the basis of using the Hf-ETTC-MOL as a novel ECL emitter and rolling circle amplification (RCA) as a signal amplification strategy, the constructed ECL aptasensor exhibited a linear range from 1 fg mL-1 to 1 ng mL-1 with a detection limit of 0.63 fg mL-1. This work has opened up new prospects for developing novel ECL materials and is expected to lead to increased interest in using AIEgen-based MOLs for ECL sensing.

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.

A highly sensitive self-enhanced aptasensor based on a stable ultrathin 2D metal-organic layer with outstanding electrochemiluminescence property.

3D bulk metal-organic frameworks (MOFs) have received growing interest in electrochemiluminescence (ECL) assays because they can provide a high specific surface for loading a large quantity of ECL luminophores, but the ECL efficiency of bulk MOFs is still low since some interior luminophores are difficult to be excited. Herein, an ultrathin 2D metal-organic layer (MOL) for grafting self-enhanced ruthenium complexes (Ru-l-Lys) was first synthesized to greatly increase the utilization ratio of luminophores. Compared with 3D bulk MOFs, ultrathin 2D MOL could provide more accessible postmodification sites for grafting the Ru-l-Lys complexes; the self-enhanced Ru-l-Lys complexes on MOL were easily excited by electrons due to the shortened ion/electron-transport distance and the removal of diffusion barriers. Furthermore, the electron transfer path between the Ru(ii) luminophore and coreactant (l-Lys) was shortened and the energy loss of the luminophores decreased, which significantly improved the ECL efficiency. As expected, our work manifested that the Zr-MOL's loading amount of Ru-l-Lys was about 1.23-fold higher than that of a 3D bulk Zr-MOF, and the ECL intensity and efficiency of Ru-l-Lys-Zr-MOL were around 93.45-fold and 1.64-fold higher than those of control Ru-l-Lys-Zr-MOF, respectively. Considering all of these merits, in this work, we utilized the prepared Ru-l-Lys-Zr-MOL as a highly efficient ECL indicator for the first time to fabricate a highly sensitive self-enhanced aptasensor for mucin 1 (MUC1) determination. The proposed aptasensor showed high sensitivity with a linear range from 1 fg mL-1 to 100 pg mL-1 with a detection limit of 0.72 fg mL-1; it also exhibited excellent specificity and stability. It is noteworthy that this work not only provides a new strategy to design and synthesize high-performance ECL materials, but also opens a new way to develop ultrasensitive ECL sensors for bioanalysis.

Highly stable Ru-complex-grafted 2D metal-organic layer with superior electrochemiluminescent efficiency as a sensing platform for simple and ultrasensitive detection of mucin 1.

This work utilized ultrathin metal-organic layer (MOL) to immobilize luminophores for effectively shortening the ion/electron-transport distance and relieving the diffusional constraints of ion/electron, which greatly enhanced the ECL efficiency and intensity. Moreover, the MOL's immobilization amount of luminophores should be higher than these of bulk MOFs because MOLs possess more accessible postmodification sites for the luminophores with minimal diffusion barriers. As expected, our proof-of-concept experiment indicated that the Hf-MOL's loading number of Ru(bpy)2(mcpbpy)2+ was about 1.74 times that of a 3D mesoporous MOF (PCN-777), and the ECL efficiency and intensity of PEI@Ru-Hf-MOL were around 1.27 times and 14.5 times those of PEI@Ru-PCN-777, respectively. In view of these merits, this work utilized the prepared PEI@Ru-Hf-MOL as a highly efficient sensing platform for simple, rapid and sensitive detection of mucin 1, which exhibited a broad linearity from 1 fg/mL to 10 ng/mL and a low detection limit of 0.48 fg/mL. This work provided a practicable strategy to develop high-performance ECL materials, and therefore opened up a new avenue to design ultrasensitive ECL biosensors, which expanded the application potential of MOLs in ECL assays.

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.

Highly Stable Mesoporous Luminescence-Functionalized MOF with Excellent Electrochemiluminescence Property for Ultrasensitive Immunosensor Construction.

In this work, a novel mesoporous luminescence-functionalized metal-organic framework (Ru-PCN-777) with high stability and excellent electrochemiluminescence (ECL) performance was synthesized by immobilizing Ru(bpy)2(mcpbpy)2+ on the Zr6 cluster of PCN-777 via a strong coordination bond between Zr4+ and -COO-. Consequently, the Ru(bpy)2(mcpbpy)2+ could not only cover the surface of PCN-777 but also graft into the interior of PCN-777, which greatly increased the loading amount of Ru(bpy)2(mcpbpy)2+ and effectively prevented the leaching of the Ru(bpy)2(mcpbpy)2+ resulting in a stable and high ECL response. Considering the above merits, we utilized the mesoporous Ru-PCN-777 to construct an ECL immunosensor to detect mucin 1 (MUC1) based on proximity-induced intramolecular DNA strand displacement (PiDSD). The ECL signal was further enhanced by the enzyme-assisted DNA recycling amplification strategy. As expected, the immunosensor had excellent sensitivity, specificity, and responded wide linearly to the concentration of MUC1 from 100 fg/mL to 100 ng/mL with a low detection limit of 33.3 fg/mL (S/N = 3). It is the first time that mesoporous Zr-MOF was introduced into ECL system to assay biomolecules, which might expand the application of mesoporous metal-organic frameworks (MOFs) in bioanalysis. This work indicates that the use of highly stable mesoporous luminescence-functionalized MOFs to enhance the ECL intensity and stability is a feasible strategy for designing and constructing high-performance ECL materials, and therefore may shed light on new ways to develop highly sensitive and selective ECL sensors.

Two LcbHLH Transcription Factors Interacting with LcMYB1 in Regulating Late Structural Genes of Anthocyanin Biosynthesis in Nicotiana and Litchi chinensis During Anthocyanin Accumulation.

Anthocyanin biosynthesis requires the MYB-bHLH-WD40 protein complex to activate the late biosynthetic genes. LcMYB1 was thought to act as key regulator in anthocyanin biosynthesis of litchi. However, basic helix-loop-helix proteins (bHLHs) as partners have not been identified yet. The present study describes the functional characterization of three litchi bHLH candidate anthocyanin regulators, LcbHLH1, LcbHLH2, and LcbHLH3. Although these three litchi bHLHs phylogenetically clustered with bHLH proteins involved in anthcoyanin biosynthesis in other plant, only LcbHLH1 and LcbHLH3 were found to localize in the nucleus and physically interact with LcMYB1. The transcription levels of all these bHLHs were not coordinated with anthocyanin accumulation in different tissues and during development. However, when co-infiltrated with LcMYB1, both LcbHLH1 and LcbHLH3 enhanced anthocyanin accumulation in tobacco leaves with LcbHLH3 being the best inducer. Significant accumulation of anthocyanins in leaves transformed with the combination of LcMYB1 and LcbHLH3 were noticed, and this was associated with the up-regulation of two tobacco endogenous bHLH regulators, NtAn1a and NtAn1b, and late structural genes, like NtDFR and NtANS. Significant activity of the ANS promoter was observed in transient expression assays either with LcMYB1-LcbHLH1 or LcMYB1-LcbHLH3, while only minute activity was detected after transformation with only LcMYB1. In contrast, no activity was measured after induction with the combination of LcbHLH2 and LcMYB1. Higher DFR expression was also oberseved in paralleling with higher anthocyanins in co-transformed lines. LcbHLH1 and LcbHLH3 are essential partner of LcMYB1 in regulating the anthocyanin production in tobacco and probably also in litchi. The LcMYB1-LcbHLH complex enhanced anthocyanin accumulation may associate with activating the transcription of DFR and ANS.

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.

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.

LcMYB1 is a key determinant of differential anthocyanin accumulation among genotypes, tissues, developmental phases and ABA and light stimuli in Litchi chinensis.

The red coloration of litchi fruit depends on the accumulation of anthocyanins. The anthocyanins level in litchi fruit varies widely among cultivars, developmental stages and environmental stimuli. Previous studies on various plant species demonstrate that anthocyanin biosynthesis is controlled at the transcriptional level. Here, we describe a litchi R2R3-MYB transcription factor gene, LcMYB1, which demonstrates a similar sequence as other known anthocyanin regulators. The transcription levels of the LcMYB1 and anthocyanin biosynthetic genes were investigated in samples with different anthocyanin levels. The expression of LcMYB1 was strongly associated with tissue anthocyanin content. LcMYB1 transcripts were only detected in anthocyanin-accumulating tissues and were positively correlated with anthocyanin accumulation in the pericarps of 12 genotypes. ABA and sunlight exposure promoted, whereas CPPU and bagging inhibited the expression of LcMYB1 and anthocyanin accumulation in the pericarp. Cis-elements associated with light responsiveness and abscisic acid responsiveness were identified in the promoter region of LcMYB1. Among the 6 structural genes tested, only LcUFGT was highly correlated with LcMYB1. These results suggest that LcMYB1 controls anthocyanin biosynthesis in litchi and LcUFGT might be the structural gene that is targeted and regulated by LcMYB1. Furthermore, the overexpression of LcMYB1 induced anthocyanin accumulation in all tissues in tobacco, confirming the function of LcMYB1 in the regulation of anthocyanin biosynthesis. The upregulation of NtAn1b in response to LcMYB1 overexpression seems to be essential for anthocyanin accumulation in the leaf and pedicel. In the reproductive tissues of transgenic tobacco, however, increased anthocyanin accumulation is independent of tobacco's endogenous MYB and bHLH transcriptional factors, but associated with the upregulation of specific structural genes.

Molecular characterization and expression analysis of S1 self-incompatibility locus-linked pollen 3.15 gene in Citrus reticulata.

Gametophytic self-incompatibility (GSI) is controlled by a highly polymorphic locus called the S-locus, which is an important factor that can result in seedless fruit in Citrus. The S1 self-incompatibility locus-linked pollen 3.15 gene (S1-3.15 ) belongs to a type of S locus gene. The role of S1-3.15 in the SI reaction of Citrus has not yet been reported. In this study, full-length sequences of cDNA and DNA encoding the S1-3.15 gene, referred to as CrS1-3.15 , were isolated from 'Wuzishatangju' (Self-incompatibility, SI) and 'Shatangju' (Self-compatibility, SC). The predicted amino acid sequences of CrS1-3.15 between 'Wuzishatangju' and 'Shatangju' differ by only three amino acids. Compared to 'Wuzishatangju', three bases were substituted in the genomic DNA of CrS1-3.15 from 'Shatangju'. Southern blot results showed that one copy of CrS1-3.15 existed in the genomic DNA of both 'Wuzishatangju' and 'Shatangju'. The expression level of the CrS1-3.15 gene in the ovaries of 'Shatangju' was approximately 60-fold higher than that in the ovaries of 'Wuzishatangju'. When 'Wuzishatangju' was cross-pollinated, the expression of CrS1-3.15 was upregulated in the ovaries at 3 d, and the highest expression levels were detected in the ovaries at 6 d after cross-pollination of 'Wuzishatangju' × 'Shatangju'. To obtain the CrS1-3.15 protein, the full-length cDNA of CrS1-3.15 genes from 'Wuzishatangju' and 'Shatangju' was successfully expressed in Pichia pastoris. Pollen germination frequency of 'Wuzishatangju' was inhibited significantly with increasing CrS1-3.15 protein concentrations from SI 'Wuzishatangju'.

Molecular characterization and expression analysis of ubiquitin-activating enzyme E1 gene in Citrus reticulata.

Ubiquitin-activating enzyme E1 (UBE1) catalyzes the first step in the ubiquitination reaction, which targets a protein for degradation via a proteasome pathway. UBE1 plays an important role in metabolic processes. In this study, full-length cDNA and DNA sequences of UBE1 gene, designated CrUBE1, were obtained from 'Wuzishatangju' (self-incompatible, SI) and 'Shatangju' (self-compatible, SC) mandarins. 5 amino acids and 8 bases were different in cDNA and DNA sequences of CrUBE1 between 'Wuzishatangju' and 'Shatangju', respectively. Southern blot analysis showed that there existed only one copy of the CrUBE1 gene in genome of 'Wuzishatangju' and 'Shatangju'. The temporal and spatial expression characteristics of the CrUBE1 gene were investigated using semi-quantitative RT-PCR (SqPCR) and quantitative real-time PCR (qPCR). The expression level of the CrUBE1 gene in anthers of 'Shatangju' was approximately 10-fold higher than in anthers of 'Wuzishatangju'. The highest expression level of CrUBE1 was detected in pistils at 7days after self-pollination of 'Wuzishatangju', which was approximately 5-fold higher than at 0 h. To obtain CrUBE1 protein, the full-length cDNA of CrUBE1 genes from 'Wuzishatangju' and 'Shatangju' were successfully expressed in Pichia pastoris. Pollen germination frequency of 'Wuzishatangju' was significantly inhibited with increasing of CrUBE1 protein concentrations from 'Wuzishatangju'.