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.

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.

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.

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.