Corrigendum: The LcKNAT1-LcEIL2/3 regulatory module is involved in fruitlet abscission in litchi.

[This corrects the article DOI: 10.3389/fpls.2021.802016.].

Increased DNA methylation of the splicing regulator SR45 suppresses seed abortion in litchi.

The gene regulatory networks that govern seed development are complex, yet very little is known about the genes and processes that are controlled by DNA methylation. Here, we performed single-base resolution DNA methylome analysis and found that CHH methylation increased significantly throughout seed development in litchi. Based on the association analysis of differentially methylated regions and weighted gene co-expression network analysis (WGCNA), 46 genes were identified as essential DNA methylation-regulated candidate genes involved in litchi seed development, including LcSR45, a homolog of the serine/arginine-rich (SR) splicing regulator SR45. LcSR45 is predominately expressed in the funicle, embryo, and seed integument, and displayed increased CHH methylation in the promoter during seed development. Notably, silencing of LcSR45 in a seed-aborted litchi cultivar significantly improved normal seed development, whereas the ectopic expression of LcSR45 in Arabidopsis caused seed abortion. Furthermore, LcSR45-dependent alternative splicing events were found to regulate genes involved in seed development. Together, our findings demonstrate that LcSR45 is hypermethylated, and plays a detrimental role in litchi seed development, indicating a global increase in DNA methylation at this stage.

LcERF10 functions as a positive regulator of litchi fruitlet abscission.

Phytohormone ethylene is well-known in positive modulation of plant organ abscission. However, the molecular mechanism underlying ethylene-induced abscission remains largely unknown. Here, we identified an ethylene-responsive factor, LcERF10, as a key regulatory gene in litchi fruitlet abscission. LcERF10 was strongly induced in the fruitlet abscission zone (FAZ) during the ethylene-activated abscission. Silencing of LcERF10 in litchi weakened the cytosolic alkalization of the FAZ and reduced fruitlet abscission. Moreover, LcERF10 directly bound the promoter and repressed the expression of LcNHX7, a Na+/H+ exchanger that was down-regulated in FAZ following the ethylene-activated abscission and up-regulated after LcERF10 silencing. Additionally, ectopic expression of LcERF10 in Arabidopsis promoted the cytosolic alkalization of the floral organ AZ and accelerated the floral organ abscission. Collectively, our results suggest that the transcription factor LcERF10 plays a positive role in litchi fruitlet abscission.

A LcDOF5.6-LcRbohD regulatory module controls the reactive oxygen species-mediated fruitlet abscission in litchi.

Reactive oxygen species (ROS) have been emerging as a key regulator in plant organ abscission. However, the mechanism underlying the regulation of ROS homeostasis in the abscission zone (AZ) is not completely established. Here, we report that a DOF (DNA binding with one finger) transcription factor LcDOF5.6 can suppress the litchi fruitlet abscission through repressing the ROS accumulation in fruitlet AZ (FAZ). The expression of LcRbohD, a homolog of the Arabidopsis RBOHs that are critical for ROS production, was significantly increased during the litchi fruitlet abscission, in parallel with an increased accumulation of ROS in FAZ. In contrast, silencing of LcRbohD reduced the ROS accumulation in FAZ and decreased the fruitlet abscission in litchi. Using in vitro and in vivo assays, we revealed that LcDOF5.6 was shown to inhibit the expression of LcRbohD via direct binding to its promoter. Consistently, silencing of LcDOF5.6 increased the expression of LcRbohD, concurrently with higher ROS accumulation in FAZ and increased fruitlet abscission. Furthermore, the expression of key genes (LcIDL1, LcHSL2, LcACO2, LcACS1, and LcEIL3) in INFLORESCENCE DEFICIENT IN ABSCISSION signaling and ethylene pathways were altered in LcRbohD-silenced and LcDOF5.6-silenced FAZ cells. Taken together, our results demonstrate an important role of the LcDOF5.6-LcRbohD module during litchi fruitlet abscission. Our findings provide new insights into the molecular regulatory network of organ abscission.

"Hong Long" Lychee (Litchi chinensis Sonn.) Is the Optimal Pollinizer for the Main Lychee Cultivars in Israel.

The lychee fruit is in high demand worldwide. However, the yields of many cultivars are low, including the high-quality cultivars "Nuomici" (NMC) and "Fei Zi Xiao" (FZX), which are very tasty and produce large fruit with a small seed, but tend to shed their fruitlets. In a previous work, we found that cross-hand pollination of "Mauritius" (MA) with pollen of another cultivar increased fruit set and reduced fruit-drop in comparison to self-hand pollination. In the current research, we aimed to identify the optimal pollen donor for three of the main cultivars grown in Israel: MA, FZX, and "Tamuz" (TA). We compared the effect of different pollinizers and found that the Vietnamese cultivar "Hong Long" (HL), which is becoming an important cultivar in Israel, was the optimal pollinizer for the three cultivars. In addition, we found that FZX and TA were not self-fertile under the Israeli environmental conditions since they tend to shed fruitlets that originated from self-fertilization. In contrast, MA is able to fertilize itself, although cross-pollination greatly increased its fruit number and size. We also identified a new PCR marker for lychee, M3, that enabled us to distinguish between self- and cross-fertilized FZX fruits pollinated by HL. Our results indicate that cross-pollination, particularly by HL, has beneficial effects on the production of lychee and it is especially important for cultivars that generate small seeds and tend to shed their fruitlets.

Characterizing the development of photosynthetic capacity in relation to chloroplast structure and mineral nutrition in leaves of three woody fruit species.

Plants have evolved different developmental patterns of photosynthetic capacity to better adapt to changing environmental conditions. Natural variation in photosynthetic development offers great potential for improving crop productivity. In this study, leaf developmental patterns were characterized in three woody fruit tree species with distinct photosynthetic capacity and growth habits. Changes in the photosynthetic rate, photosystem II (PSII) efficiency, chloroplast ultrastructure, activities of photosynthetic enzymes, and contents of carbohydrates and mineral nutrients were examined at five developmental stages to explore the interspecific variation in photosynthetic development. Rapid development of photosynthetic machinery and high photosynthetic capacity were found in Indian jujube (Ziziphus mauritiana) and apple (Malus domestica), whose net CO2 assimilation rate (A) peaked at full leaf expansion (FLE). Litchi (Litchi chinensis), a delayed-greening species, showed slow development of photosynthetic competence, with A peaked after FLE. The low photosynthetic capacity of litchi during early leaf expansion was associated with its delayed chloroplast development, low accumulation of starch, and low activities of ribulose-1,5-bisphosphate carboxylase/oxygenase and NADP-glyceraldehyde-3-phosphate dehydrogenase. Correlations between mineral contents and A across leaf stages and species identified manganese as the rate-limiting nutrients in photosynthetic development in new leaves. Foliar spray of MnSO4 solution (1 g l-1) induced a short-term increase in photosynthesis in young leaves of litchi. These findings suggest that a better understanding of interspecific variation in photosynthetic development facilitates the development of new strategies for improving the photosynthetic efficiency of woody fruit trees.

Metabolomics Analysis of Litchi Leaves during Floral Induction Reveals Metabolic Improvement by Stem Girdling.

Prolonged exposure to cold temperatures often results in a relatively low flowering rate in litchi (Litchi chinensis Sonn.) trees with younger leaves. This study aimed to verify the impact of stem girdling on litchi flowering by identifying and characterizing the induced metabolic changes. After a 60 day exposure to cold treatment at 15 °C/10 °C (12 h/12 h), the flowering rate of the girdled trees was 100%, while that of the non-girdled trees was 20%, indicating that girdling improved litchi flowering at its turning stage. The metabolic profiles of litchi leaves with and without stem girdling during floral induction were compared and 505 metabolites potentially associated with litchi flowering were detected. Most metabolites were involved in the metabolism of starch and sucrose, fatty acid, and phenylpyruvic acid. The metabolic pathways concerned with the biosynthesis of epinephrine, sucrose, and d-maltose were induced in leaves after girdling treatment. The level of galactitol, phenylpyruvic acid, acetyl-CoA, linoleic acid, alpha-linolenic acid, and 13-HPOT biosynthesis remained stable in the leaves from girdled trees but changed drastically in the leaves from non-girdled trees. In addition, 379 metabolites concerning flowering rate were characterized. Metabolism pathways of starch and sucrose, galactose, and linoleic acid are of great significance to the flowering of litchi. Linoleic acid exhibited the most significant variations between girdled trees and non-girdled trees with fold changes of up to 13.62. These results contribute to understanding the biological mechanism of litchi floral induction and the metabolic changes after stem girdling.

Pronounced anti-neuroinflammatory jasmonates and terpenes isolated from lychee seeds.

Lychee is a favorite fruit of the Cantonese and native to Southeast Asia. In this study, the anti-neuroinflammatory bioactive compounds of lychee seeds have been carried out. Five new jasmonates (1, 2, 6-8) and seventeen known compounds were isolated using a series of chemical and chromatographic methods. Their chemical structures were identified through comprehensive spectroscopic analysis. Anti-neuroinflammatory activities were assayed and evaluated for the purified compounds. Most of the compounds exhibited pronounced anti-neuroinflammatory activities on nitric oxide (NO) induced by lipopolysaccharide (LPS) in BV-2 microglia cells. Moreover, compounds 1, 2 and 20 could reduce the expression of LPS-induced pro-inflammatory factors (iNOS and COX-2), inhibit the expression of mRNA levels of iNOS, COX-2, IL-6 and block NF-κB nuclear translocation in dose-dependent manners. This study suggested that lychee phytochemicals could be benefit to some neuroinflammatory-associated diseases, such as Alzheimer's disease.

RNA-Seq Provides New Insights into the Molecular Events Involved in "Ball-Skin versus Bladder Effect" on Fruit Cracking in Litchi.

Fruit cracking is a disorder of fruit development in response to internal or external cues, which causes a loss in the economic value of fruit. Therefore, exploring the mechanism underlying fruit cracking is of great significance to increase the economic yield of fruit trees. However, the molecular mechanism underlying fruit cracking is still poorly understood. Litchi, as an important tropical and subtropical fruit crop, contributes significantly to the gross agricultural product in Southeast Asia. One important agricultural concern in the litchi industry is that some famous varieties with high economic value such as 'Nuomici' are susceptible to fruit cracking. Here, the cracking-susceptible cultivar 'Nuomici' and cracking-resistant cultivar 'Huaizhi' were selected, and the samples including pericarp and aril during fruit development and cracking were collected for RNA-Seq analysis. Based on weighted gene co-expression network analysis (WGCNA) and the "ball-skin versus bladder effect" theory (fruit cracking occurs upon the aril expanding pressure exceeds the pericarp strength), it was found that seven co-expression modules genes (1733 candidate genes) were closely associated with fruit cracking in 'Nuomici'. Importantly, we propose that the low expression level of genes related to plant hormones (Auxin, Gibberellins, Ethylene), transcription factors, calcium transport and signaling, and lipid synthesis might decrease the mechanical strength of pericarp in 'Nuomici', while high expression level of genes associated with plant hormones (Auxin and abscisic acid), transcription factors, starch/sucrose metabolism, and sugar/water transport might increase the aril expanding pressure, thereby resulting in fruit cracking in 'Nuomici'. In conclusion, our results provide comprehensive molecular events involved in the "ball-skin versus bladder effect" on fruit cracking in litchi.

The LcKNAT1-LcEIL2/3 Regulatory Module Is Involved in Fruitlet Abscission in Litchi.

Large and premature organ abscission may limit the industrial development of fruit crops by causing serious economic losses. It is well accepted that ethylene (ET) is a strong inducer of organ abscission in plants. However, the mechanisms underlying the control of organ abscission by ET are largely unknown. We previously revealed that LcKNAT1, a KNOTTED-LIKE FROM ARABIDOPSIS THALIANA1 (KNAT1)-like protein, acted as a negative regulator in control of fruitlet abscission through suppressing the expression of ET biosynthetic genes in litchi. In this study, we further reported that LcKNAT1 could also directly repress the transcription of LcEIL2 and LcEIL3, two ETHYLENE INSENSITIVE 3-like (EIL) homologs in litchi, which functioned as positive regulators in ET-activated fruitlet abscission by directly promoting the expression of genes responsible for ET biosynthesis and cell wall degradation. The expression level of LcKNAT1 was downregulated, while LcEIL2/3 was upregulated at the abscission zone (AZ) accompanying the fruitlet abscission in litchi. The results of electrophoretic mobility shift assays (EMSAs) and transient expression showed that LcKNAT1 could directly bind to the promoters of LcEIL2 and LcEIL3 and repress their expression. Furthermore, the genetic cross demonstrated that the ß-glucuronidase (GUS) expression driven by the promoters of LcEIL2 or LcEIL3 at the floral AZ was obviously suppressed by LcKNAT1 under stable transformation in Arabidopsis. Taken together, our findings suggest that the LcKNAT1-LcEIL2/3 regulatory module is likely involved in the fruitlet abscission in litchi, and we propose that LcKNAT1 could suppress both ET biosynthesis and signaling to regulate litchi fruit abscission.

LcEIL2/3 are involved in fruitlet abscission via activating genes related to ethylene biosynthesis and cell wall remodeling in litchi.

Fruit crops are subject to precocious fruit abscission, during which the phytohormone ethylene (ET) acts as a major positive regulator. However, the molecular basis of ET-induced fruit abscission remains poorly understood. Here, we show that two ETHYLENE INSENSITIVE 3-like (EIL) homologs in litchi, LcEIL2 and LcEIL3, play a role in ET-activated fruitlet abscission. LcEIL2/3 were significantly upregulated in the fruit abscission zone (AZ) during the ET-induced fruitlet abscission in litchi. The presence of LcEIL2/3 in wild-type Arabidopsis and ein3 eil1 mutants can accelerate the floral organ abscission. Moreover, the electrophoretic mobility shift assay and dual luciferase reporter analysis illustrated that LcEIL2/3 directly interacted with the gene promoters to activate the expression of cell wall remodeling genes LcCEL2/8 and LcPG1/2, and ET biosynthetic genes LcACS1/4/7 and LcACO2/3. Furthermore, we showed that LcPG1/2 were expressed in the floral abscission zone of Arabidopsis, and constitutive expression of LcPG2 in Arabidopsis promoted the floral organ abscission. In conclusion, we propose that LcEIL2/3 are involved in ET-induced fruitlet abscission via controlling expression of genes related to ET biosynthesis and cell wall remodeling in litchi.

KNOX protein KNAT1 regulates fruitlet abscission in litchi by repressing ethylene biosynthetic genes.

Abscission is triggered by multiple environmental and developmental cues, including endogenous plant hormones. KNOTTED-LIKE HOMEOBOX (KNOX) transcription factors (TFs) play an important role in controlling abscission in plants. However, the underlying molecular mechanism of KNOX TFs in abscission is largely unknown. Here, we identified LcKNAT1, a KNOTTED-LIKE FROM ARABIDOPSIS THALIANA1 (KNAT1)-like protein from litchi, which regulates abscission by modulating ethylene biosynthesis. LcKNAT1 is expressed in the fruit abscission zone and its expression decreases during fruitlet abscission. Furthermore, the expression of the ethylene biosynthetic genes LcACS1, LcACS7, and LcACO2 increases in the fruit abscission zone, in parallel with the emission of ethylene in fruitlets. In vitro and in vivo assays revealed that LcKNAT1 inhibits the expression of LcACS/ACO genes by directly binding to their promoters. Moreover, ectopic expression of LcKNAT1 represses flower abscission in tomatoes. Transgenic plants expressing LcKNAT1 also showed consistently decreased expression of ACS/ACO genes. Collectively, these results indicate that LcKNAT1 represses abscission via the negative regulation of ethylene biosynthesis.

Effects of three drying methods on polyphenol composition and antioxidant activities of Litchi chinensis Sonn.

The aim of this study was to investigate the effects of three different drying methods, freeze drying (FD), vacuum drying (VD) and oven drying (OD) on phenolic contents and antioxidant activities of litchi fruits. 20 polyphenols were exactly identified in the litchi fruits by UPLC-QqQ/MS. Significant losses were observed in the contents of total polyphenols and antioxidant activities in the dried litchi when compared with the fresh litchi. Principle component analysis indicated that there was significant difference of phenolic component between the use of thermal drying (VD and OD) and FD. Our results suggest that FD is the optimum drying method for litchi fruits considering the content of total polyphenols and antioxidant activities.

Molecular Events Involved in Fruitlet Abscission in Litchi.

Abscission in plants is an active and highly coordinated physiological process in which organs abscise from the plant body at the abscission zone (AZ) in responding to either developmental or environmental cues. Litchi (Litchi chinensis Sonn.) is an important economic fruit crop widely grown in Southeast Asia particularly in South China. However, the excessive fruit drop during fruit development is a major limiting factor for litchi production. Thus, it is an important agricultural concern to understand the mechanisms underlying the fruit abscission in litchi. Here, we present a review focusing on the molecular events involved in the fruitlet abscission. We also highlight the recent advances on genes specifically associated with fruit abscission and perspectives for future research.

Isolation and structural characterization of a non-competitive α-glucosidase inhibitory polysaccharide from the seeds of Litchi chinensis Sonn.

In this study, a novel homogeneous polysaccharide (LSP-W-4, MW = 6.70 kDa) was isolated and purified from the seeds of Litchi chinensis Sonn. Monosaccharide composition analysis indicated that LSP-W-4 is a heteropolysaccharide consisting of arabinose, mannose, glucose and galactose in a molar ratio of 6.33:3.88:10.35:1.00. A detailed structural analysis revealed that LSP-W-4 has a backbone consisting of 1,4-α-Glcp and 1,4-ß-Manp, as well as four branched chains including of T-α-Galp, T-α-Araf, α-Araf-(1 â†’ 5)-α-Araf-(1 â†’ and α-Araf-(1 â†’ 5)-α-Araf-(1 â†’ 5)-α-Araf-(1 â†’ attached to O6 of 1,4-ß-Manp and 1,4-α-Glcp. LSP-W-4 exhibited significant inhibitory activity both against yeast (Saccharomyces cerevisiae) and mammalian (rat-intestinal acetone powder) α-glucosidase, with IC50 values of 75.24 µM and 66.97 µM, respectively, with both such inhibitory activities being more powerful than those of acarbose. A kinetic analysis revealed that LSP-W-4 inhibited the activities of both yeast and mammalian α-glucosidase in a typical non-competitive manner, with KM values of 0.43 mmol/L and 0.53 mmol/L, respectively.

Involvement of HD-ZIP I transcription factors LcHB2 and LcHB3 in fruitlet abscission by promoting transcription of genes related to the biosynthesis of ethylene and ABA in litchi.

Abnormal fruitlet abscission is a limiting factor in the production of litchi, an economically important fruit in Southern Asia. Both ethylene and abscisic acid (ABA) induce organ abscission in plants. Although ACS/ACO and NCED genes are known to encode key enzymes required for ethylene and ABA biosynthesis, respectively, the transcriptional regulation of these genes is unclear in the process of plant organ shedding. Here, two polygalacturonase (PG) genes (LcPG1 and LcPG2) and two novel homeodomain-leucine zipper I transcription factors genes (LcHB2 and LcHB3) were identified as key genes associated with the fruitlet abscission in litchi. The expression of LcPG1 and LcPG2 was strongly associated with litchi fruitlet abscission, consistent with enhanced PG activity and reduced homogalacturonan content in fruitlet abscission zones (FAZs). The promoter activities of LcPG1/2 were enhanced by ethephon and ABA. In addition, the production of ethylene and ABA in fruitlets was significantly increased during fruit abscission. Consistently, expression of five genes (LcACO2, LcACO3, LcACS1, LcACS4 and LcACS7) related to ethylene biosynthesis and one gene (LcNCED3) related to ABA biosynthesis in FAZs were activated. Further, electrophoretic mobility shift assays and transient expression experiments demonstrated that both LcHB2 and LcHB3 could directly bind to the promoter of LcACO2/3, LcACS1/4/7 and LcNCED3 genes and activate their expression. Collectively, we propose that LcHB2/3 are involved in the litchi fruitlet abscission through positive regulation of ethylene and ABA biosynthesis.

The effect of desulfurization on the postharvest quality and sulfite metabolism in pulp of sulfitated "Feizixiao" Litchi (Litchi chinensis Sonn.) fruits.

The residual sulfite caused by sulfur fumigation (SF) is a hazard to health and influenced the export trade of litchi. Desulfurization (DS) is a valid chemical method to reduce the residual sulfite. However, the effect of DS on fumigated litchi has not been studied at physiological and molecular level. This study was aimed to evaluate the effect of DS (SF plus 3% desulfurizer) on the postharvest quality, sulfite residue, and the sulfite metabolism in sulfitated "Feizixiao" litchi during the 4°C storage. Results indicated that the DS promoted the color recovery of sulfitated litchi and achieved an effect similar to SF on controlling rot and browning. DS recovered the water content and respiration rate of sulfitated litchi pericarp. Thus, DS improves commodity properties of sulfitated litchi. Moreover, DS greatly reduced sulfite residue especially in pulp and ensured the edible safety of sulfitated litchi. The activities of sulfite oxidase, sulfite reductase, serine acetyltransferase, and O-acetylserine(thiol) lyase in pulp increased after SF but fell down after DS while the expressions of their encoding genes decreased after SF but then rallied after DS. These results indicated the key role of these enzymes in sulfite metabolism after SF and DS changed the sulfite metabolism at both enzymatic and transcriptional level. It could be concluded that DS used in this study was an effective method for improving the color recovery and ensuring the edible safety of sulfitated litchi by not only chemical reaction but also both of enzymatic and transcriptional regulation.

Cytokinin treatment modifies litchi fruit pericarp anatomy leading to reduced susceptibility to post-harvest pericarp browning.

Litchi (Litchi chinensis Sonn.) is a subtropical fruit known for its attractive red pericarp color, semi-translucent white aril and unique flavor and aroma. Rapid post-harvest pericarp browning strictly limits litchi fruit marketing. In the current research, we hypothesized that modification of litchi fruit pericarp anatomy by hormone application may reduce fruit susceptibility to post-harvest pericarp browning. In this context, we hypothesized that cytokinin treatment, known to induce cell division, may yield fruit with thicker pericarp and reduced susceptibility for fruit surface micro-crack formation, water loss and post-harvest pericarp browning. Exogenous cytokinin treatment was applied at different stages along the course of litchi fruit development and the effect on fruit pericarp anatomy, fruit maturation and postharvest pericarp browning was investigated. Interestingly, cytokinin treatment, applied 4 weeks after full female bloom (WFB), during the phase of pericarp cell division, led to mature fruit with thicker pericarp, reduced rate of post-harvest water loss and reduced susceptibility to post-harvest pericarp browning, as compared to non-treated control fruit. Histological sections ascribe the difference in pericarp anatomy to increased cell proliferation in the parenchymatic tissue and the highly-lignified brachysclereid cell layer. In contrast, exogenous cytokinin treatment applied 7 WFB, following the phase of pericarp cell division, significantly increased epidermal-cell proliferation but had no significant effect on overall fruit pericarp thickness and only minor affect on post-harvest water loss or pericarp browning. Interestingly, the late cytokinin treatment also significantly postponed fruit maturation-associated anthocyanin accumulation and chlorophyll degradation, as previously reported, but had no effect on other parameters of fruit maturation, like total soluble sugars and total titratable acids typically modified during aril maturation. In conclusion, exogenous cytokinin treatment at different stages in fruit development differentially modifies litchi fruit pericarp anatomy by induction of cell-type specific cell proliferation. Early cytokinin treatment during the phase of pericarp cell division may prolong litchi fruit storage by reducing fruit susceptibility to post-harvest water loss and pericarp browning.

Cloning and Characterization of a Flavonol Synthase Gene From Litchi chinensis and Its Variation Among Litchi Cultivars With Different Fruit Maturation Periods.

Litchi (Litchi chinensis) is an important subtropical fruit tree with high commercial value. However, the short and centralized fruit maturation period of litchi cultivars represents a bottleneck for litchi production. Therefore, the development of novel cultivars with extremely early fruit maturation period is critical. Previously, we showed that the genotypes of extremely early-maturing (EEM), early-maturing (EM), and middle-to-late-maturing (MLM) cultivars at a specific locus SNP51 (substitution type C/T) were consistent with their respective genetic background at the whole-genome level; a homozygous C/C genotype at SNP51 systematically differentiated EEM cultivars from others. The litchi gene on which SNP51 was located was annotated as flavonol synthase (FLS), which catalyzes the formation of flavonols. Here, we further elucidate the variation of the FLS gene from L. chinensis (LcFLS) among EEM, EM, and MLM cultivars. EEM cultivars with a homozygous C/C genotype at SNP51 all contained the same 2,199-bp sequence of the LcFLS gene. For MLM cultivars with a homozygous T/T genotype at SNP51, the sequence lengths of the LcFLS gene were 2,202-2,222 bp. EM cultivars with heterozygous C/T genotypes at SNP51 contained two different alleles of the LcFLS gene: a 2,199-bp sequence identical to that in EEM cultivars and a 2,205-bp sequence identical to that in MLM cultivar 'Heiye.' Moreover, the coding regions of LcFLS genes of other MLM cultivars were almost identical to that of 'Heiye.' Therefore, the LcFLS gene coding region may be used as a source of diagnostic SNP markers to discriminate or identify genotypes with the EEM trait. The expression pattern of the LcFLS gene and accumulation pattern of flavonol from EEM, EM, and MLM cultivars were analyzed and compared using quantitative real-time PCR (qRT-PCR) and high-performance liquid chromatography (HPLC) for mature leaves, flower buds, and fruits, 15, 30, 45, and 60 days after anthesis. Flavonol content and LcFLS gene expression levels were positively correlated in all three cultivars: both decreased from the EEM to MLM cultivars, with moderate levels in the EM cultivars. LcFLS gene function could be further analyzed to elucidate its correlation with phenotype variation among litchi cultivars with different fruit maturation periods.

Identification and expression profile analysis of the sucrose phosphate synthase gene family in Litchi chinensis Sonn.

Sucrose phosphate synthase (SPS, EC 2.4.1.14) is a key enzyme that regulates sucrose biosynthesis in plants. SPS is encoded by different gene families which display differential expression patterns and functional divergence. Genome-wide identification and expression analyses of SPS gene families have been performed in Arabidopsis, rice, and sugarcane, but a comprehensive analysis of the SPS gene family in Litchi chinensis Sonn. has not yet been reported. In the current study, four SPS gene (LcSPS1, LcSPS2, LcSPS3, and LcSPS4) were isolated from litchi. The genomic organization analysis indicated the four litchi SPS genes have very similar exon-intron structures. Phylogenetic tree showed LcSPS1-4 were grouped into different SPS families (LcSPS1 and LcSPS2 in A family, LcSPS3 in B family, and LcSPS4 in C family). LcSPS1 and LcSPS4 were strongly expressed in the flowers, while LcSPS3 most expressed in mature leaves. RT-qPCR results showed that LcSPS genes expressed differentially during aril development between cultivars with different hexose/sucrose ratios. A higher level of expression of LcSPS genes was detected in Wuheli, which accumulates higher sucrose in the aril at mature. The tissue- and developmental stage-specific expression of LcSPS1-4 genes uncovered in this study increase our understanding of the important roles played by these genes in litchi fruits.