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.

Sugar uptake in the Aril of litchi fruit depends on the apoplasmic post-phloem transport and the activity of proton pumps and the putative transporter LcSUT4.

The post-phloem unloading pathway and the mechanism of sugar accumulation remain unclear in litchi fruit. A combination of electron microscopy, transport of phloem-mobile symplasmic tracer (carboxyfluorescein, CF) and biochemical and molecular assays was used to explore the post-phloem transport pathway and the mechanism of aril sugar accumulation in litchi. In the funicle, where the aril originates, abundant plasmodesmata were observed, and CF introduced from the peduncle diffused to the parenchyma cells. In addition, abundant starch and pentasaccharide were detected and the sugar concentration was positively correlated with activities of sucrose hydrolysis enzymes. These results clearly showed that the phloem unloading and post-phloem transport in the funicle were symplastic. On the other hand, imaging of CF showed that it remained confined to the parenchyma cells in funicle tissues connecting the aril. Infiltration of both an ATPase inhibitor [eosin B (EB)] and a sucrose transporter inhibitor [p-chloromercuribenzene sulfonate (PCMBS)] inhibited sugar accumulation in the aril. These results indicated an apoplasmic post-phloem sugar transport from the funicle to the aril. Although facilitated diffusion might help sucrose uptake from the cytosol to the vacuole in cultivars with high soluble invertase, membrane ATPases in the aril, especially tonoplast ATPase, are crucial for aril sugar accumulation. The expression of a putative aril vacuolar membrane sucrose transporter gene (LcSUT4) was highly correlated with the sugar accumulation in the aril of litchi. These data suggest that apoplasmic transport is critical for sugar accumulation in litchi aril and that LcSUT4 is involved in this step.

Reference gene selection for quantitative real-time RT-PCR normalization in Iris. lactea var. chinensis roots under cadmium, lead, and salt stress conditions.

Quantitative real time PCR (RT-qPCR) has emerged as an accurate and sensitive method to measure the gene expression. However, obtaining reliable result depends on the selection of reference genes which normalize differences among samples. In this study, we assessed the expression stability of seven reference genes, namely, ubiquitin-protein ligase UBC9 (UBC), tubulin alpha-5 (TUBLIN), eukaryotic translation initiation factor (EIF-5A), translation elongation factor EF1A (EF1 α ), translation elongation factor EF1B (EF1b), actin11 (ACTIN), and histone H3 (HIS), in Iris. lactea var. chinensis (I. lactea var. chinensis) root when the plants were subjected to cadmium (Cd), lead (Pb), and salt stress conditions. All seven reference genes showed a relatively wide range of threshold cycles (C t ) values in different samples. GeNorm and NormFinder algorithms were used to assess the suitable reference genes. The results from the two software units showed that EIF-5A and UBC were the most stable reference genes across all of the tested samples, while TUBLIN was unsuitable as internal controls. I. lactea var. chinensis is tolerant to Cd, Pb, and salt. Our results will benefit future research on gene expression in response to the three abiotic stresses.

Differential modulation of nociceptive versus non-nociceptive synapses by endocannabinoids.

BACKGROUND: Although a number of clinical and preclinical studies have demonstrated analgesic effects of cannabinoid treatments, there are also instances when cannabinoids have had no effect or even exacerbated pain. The observed pro-nociceptive effects appear to be due to cannabinoid-induced disinhibition of afferent synaptic input to nociceptive circuits. To better understand how cannabinoid-mediated plasticity can have both pro- and anti-nociceptive effects, we examined the possibility that cannabinoids differentially modulate nociceptive vs. non-nociceptive synapses onto a shared postsynaptic target. These experiments were carried out in the central nervous system (CNS) of the medicinal leech, in which it is possible to intracellularly record from presynaptic nociceptive (N-cell) or pressure-sensitive (P-cell) neurons and their shared postsynaptic targets. RESULTS: The endocannabinoid 2-arachidonoyl glycerol (2AG) elicited significant long-lasting depression in nociceptive (N-cell) synapses. However, non-nociceptive (P-cell) synapses were potentiated following 2AG treatment. 2AG-induced potentiation of non-nociceptive synapses was blocked by the TRPV antagonist SB366791, suggesting involvement of the same TRPV-like receptor that has already been shown to mediate endocannabinoid-dependent depression in nociceptive inputs. Treatment with the GABA receptor antagonist bicuculline also blocked 2AG-induced potentiation, consistent with the idea that increased synaptic signaling was the result of endocannabinoid-mediated disinhibition. Interestingly, while bicuculline by itself increased non-nociceptive synaptic transmission, nociceptive synapses were depressed by this GABA receptor antagonist indicating that nociceptive synapses were actually excited by GABAergic input. Consistent with these observations, GABA application depolarized the nociceptive afferent and hyperpolarized the non-nociceptive afferent. CONCLUSIONS: These findings show that endocannabinoids can differentially modulate nociceptive vs. non-nociceptive synapses and that GABAergic regulation of these synapses plays an important role in determining whether endocannabinoids have a potentiating or depressing effect.

Combined application of ascorbic and oxalic acids delays postharvest browning of litchi fruits under controlled atmosphere conditions.

The effect of ascorbic acid [AA (40 mmol L-1)] and oxalic acid [OA (2 mmol L-1)] on browning of litchi fruit was investigated under 5% CO2 + 1% O2 controlled atmosphere (CA) and compared with air at 5 ± 1 °C for 28 days. The combined application of AA and OA suppressed browning index, soluble quinones, and activities of polyphenol oxidase and peroxidase under CA compared with control. The combination of CA along with AA + OA reduced weight loss and maintained higher anthocyanins, total phenolics, membrane integrity, ascorbate peroxidase, catalase, glutathione reductase and superoxide dismutase activities compared with control. In addition, AA + OA + CA combination showed markedly lower malondialdehyde, superoxide anion and hydrogen peroxide with substantially higher soluble solids content, ascorbic acid, titratable acidity and sensory quality compared with control. In conclusion, AA + OA combination could be considered appropriate to delay browning and to conserve litchi fruit visual appearance under CA storage conditions.

Inhibition of downy blight and enhancement of resistance in litchi fruit by postharvest application of melatonin.

Litchis are tasty fruit with economic importance. However, the extreme susceptibility of harvested litchis to litchi downy blight caused by Peronophythora litchii leads to compromised quality. This study aimed to study the effects of melatonin on postharvest resistance to P. litchii in 'Feizixiao' litchis. Results showed that melatonin restricted lesion expansion in litchis after P. litchi inoculation. Melatonin enhanced the activities of phenylalanine ammonia-lyase, cinnamate-4-hydroxylase and 4-hydroxycinnamate CoA ligase while promoting the accumulations of phenolics and flavonoids. Nicotinamide adenine dinucleotide phosphate content and glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase activities were higher in treated fruit than control fruit. Higher energy status along with elevated H+-ATPase, Ca2+-ATPase, succinate dehydrogenase and cytochrome C oxidase activities were observed in treated fruit. Ultrastructural observation showed reduced damage in mitochondria in treated fruit. The results suggest that melatonin induced resistance in litchis by modulating the phenylpropanoid and pentose phosphate pathways as well as energy metabolism. .

Effects of hydrogen water treatment on antioxidant system of litchi fruit during the pericarp browning.

Litchi fruit were exposed to 0.7 PPM hydrogen water (HW) before storage at 25 ± 1 â„ƒ. HW treatment delayed the pericarp browning and maintained the total soluble solids (TSS) of litchi fruit. Then, a total of 25 antioxidant system-related characters were determined to evaluate the effects of HW on antioxidant system during pericarp browning. Compared with control pericarp, the pericarp of HW-treated litchi fruit exhibited higher levels of superoxide radical (O2-·) scavenging activity, glutathione (GSH), monodehydroascorbate reductase (MDHAR), polyphenol oxidase (PPO) and total flavonoids during whole storage, higher levels of hydrogen peroxide (H2O2), catalase (CAT), glutathione disulfide (GSSG), ascorbate oxidase (AAO) and total phenols only on day 1, and higher levels of ascorbate peroxidase (APX), total anthocyanin, glutathione reductase (GR) and glutathione peroxidases (GPX) at later stage of storage. Those HW-induced antioxidant system-related characters might directly or indirectly enhanced the antioxidant capacity and delayed the pericarp browning of litchi.

Nullifying phosphatidic acid effect and controlling phospholipase D associated browning in litchi pericarp through combinatorial application of hexanal and inositol.

The role of phospholipid modification initiated by phospholipase D (PLD) in enzymatic browning has been revoked through this study. Various alcohols and aldehydes were tried to read out their PLD controlling behaviour. Based on in-vitro results, reagents like hexanal and inositol were used to regulate PLD activity of litchi fruit stored at ambient temperature and their effects on fruit quality and physiological characteristics were also investigated. The results showed that combinatorial chemical treatment was successful in maintaining freshness of fruit through delayed physiological loss in weight and hence maintaining firmness. Combinatorial treated fruit had lower browning index than control by day 7. This novel treatment also maintained comparable levels of total phenolics and lowered the level of malondialdehyde. Evaluation of antioxidative enzymatic profile also confirmed the alleviation of oxidative stress of litchi fruit at ambient temperature. Thus, this strategy of enzyme regulation could play a vital role in overall quality maintenance of litchi fruit.

Sodium para-aminosalicylate delays pericarp browning of litchi fruit by inhibiting ROS-mediated senescence during postharvest storage.

The effect of sodium para-aminosalicylate (PAS-Na) on litchi pericarp browning and the potential regulating mechanism was investigated in this study. Results showed that 0.3 g L-1 PAS-Na significantly inhibited the development of pericarp browning and reduced respiration rate of litchi fruit. PAS-Na inhibited the production of reactive oxygen species (ROS) and decreased the expression level of senescence-related genes. Additionally, PAS-Na treatment enhanced the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), which might contribute to the scavenging of ROS. Meanwhile, PAS-Na treatment maintained membrane integrity as indicated by reduced relative membrane leakage rate and malondialdehyde (MDA) content, as well as lower activities of membrane lipids-degrading enzymes: lipase and lipoxygenase (LOX). Amino acids, especially GABA, Glu, Met contents were also significantly affected by PAS-Na treatment. Taken together, we postulated that PAS-Na treatment might be a promising method for controlling postharvest browning and prolonging shelf-life of harvested litchi fruit.

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.

Postharvest application of antibrowning chemicals modulates oxidative stress and delays pericarp browning of controlled atmosphere stored litchi fruit.

Litchi fruit were treated with methionine [(0.25%) MN] and cysteine [(025%) CN] alone or in combination, and kept under 1% O2 + 5% CO2 controlled atmosphere (CA) at 5 ± 1ºC for 28 days. Among different treatments, CN was most effective to inhibit browning, than MN and CN + MN under CA conditions. Application of 0.25% CN significantly delayed browning index, reduced disease incidence, weight loss, malondialdehyde (MDA) contents, electrolyte leakage, hydrogen peroxide (H2 O2 ), superoxide anion (O2-• ) and polyphenol oxidase (PPO) and peroxidase (POD) activities with higher contents of total anthocyanins under CA-storage. In addition, 0.25% CN treatment showed higher contents of ascorbic acid, total phenolics (TPC), and 2,2-diphenyl-1-picrylhydrazyl-radical scavenging capacity and activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) enzymes having maintained quality attributes. Therefore, 0.25% CN pre-treatment could be considered a promising way for managing browning, and conserving litchi fruit quality under CA-storage. PRACTICAL APPLICATIONS: Litchi fruit are highly perishable due to rapid pericarp browning having limited postharvest market potential. The browning takes place due to enzymatic reactions and phenolic oxidation. However, it can be delayed by exogenous antibrowning treatments and suitable storage environment. The delayed incidence of pericarp browning may help to maintain its quality with extended storage potential suitable for domestic and international markets. So, the outcomes of the current work may help to maintain overall quality and to extend its storage potential that would be helpful in extending its market life with maintained visual quality at domestic and international destinations.

Degradation of tetracyclines in manure-amended soil and their uptake by litchi (Litchi chinensis Sonn.).

The environmental and human health risk posed by veterinary antibiotics is of global concern. Antibiotic uptake by herbal plants has been studied, but little is known about perennial woody fruit crops. Litchi (Litchi chinensis Sonn.), a longevial fruit tree, is routinely fertilized with animal manure and, therefore, may be at risk of antibiotic uptake into its fruits. This study investigated the degradation of chlortetracycline and doxycycline present in manure used to amend orchard soil, and their subsequent assimilation by litchi plant, as affected by manure application rate. The results show that half-lives of chlortetracycline and doxycycline in soil were decreased by increased manure rate, with an average of 27 and 59 days, respectively. Chlortetracycline was readily transported to litchi shoots and increased with the growth of litchi plants. Doxycycline predominantly remained in the roots, and underwent growth dilution in the plants. The two tetracyclines could not be detected in fruits from litchi trees when applied with manures, at various rates, over 2 years. For litchi, chlortetracycline may pose human health risk through manure application, but doxycycline is unlikely to do so. Long-term field experiments are required to monitor antibiotic accumulation in fruits of perennial fruit trees fertilized with animal manure.

Delay of Postharvest Browning in Litchi Fruit by Melatonin via the Enhancing of Antioxidative Processes and Oxidation Repair.

Melatonin acts as a crucial signaling and antioxidant molecule with multiple physiological functions in organisms. To explore effects of exogenous melatonin on postharvest browning and its possible mechanisms in litchi fruit, 'Ziniangxi' litchi fruits were treated with an aqueous solution of melatonin at 0.4 mM and then stored at 25 °C for 8 days. The results revealed that melatonin strongly suppressed pericarp browning and delayed discoloration during storage. Melatonin treatment reduced relative membrane-leakage rate and inhibited the generation of superoxide radicals (O2-·), hydrogen peroxide (H2O2), and malondialdehyde (MDA). Melatonin treatment markedly promoted the accumulation of endogenous melatonin; delayed loss of total phenolics, flavonoids, and anthocyanins; and enhanced the activities of antioxidant enzymes, including superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and glutathione reductase (GR, EC 1.6.4.2). By contrast, the activities of browning-related enzymes including polyphenoloxidase (PPO, EC 1.10.3.1) and peroxidase (POD, EC 1.11.1.7) were reduced. In addition, melatonin treatment up-regulated the expression of four genes encoding enzymes for repair of oxidized proteins, including LcMsrA1, LcMsrA2, LcMsrB1, and LcMsB2. These findings indicate that the delay of pericarp browning and senescence by melatonin in harvested litchi fruit could be attributed to the maintenance of redox homeostasis by the improvement of the antioxidant capacity and modulation of the repair of oxidatively damaged proteins.

Transcriptional changes in litchi (Litchi chinensis Sonn.) inflorescences treated with uniconazole.

In Arabidopsis, treating shoots with uniconazole can result in enhanced primary root elongation and bolting delay. Uniconazole spraying has become an important cultivation technique in controlling the flowering and improving the fruit-setting of litchi. However, the mechanism by which uniconazole regulates the complicated developmental processes in litchi remains unclear. This study aimed to determine which signal pathways and genes drive the responses of litchi inflorescences to uniconazole treatment. We monitored the transcriptional activity in inflorescences after uniconazole treatment by Illumina sequencing technology. The global expression profiles of uniconazole-treated litchi inflorescences were compared with those of the control, and 4051 differentially expressed genes were isolated. KEGG pathway enrichment analysis indicated that the plant hormone signal transduction pathway served key functions in the flower developmental stage under uniconazole treatment. Basing on the transcriptional analysis of genes involved in flower development, we hypothesized that uniconazole treatment increases the ratio of female flowers by activating the transcription of pistil-related genes. This phenomenon increases opportunities for pollination and fertilization, thereby enhancing the fruit-bearing rate. In addition, uniconazole treatment regulates the expression of unigenes involved in numerous transcription factor families, especially the bHLH and WRKY families. These findings suggest that the uniconazole-induced morphological changes in litchi inflorescences are related to the control of hormone signaling, the regulation of flowering genes, and the expression levels of various transcription factors. This study provides comprehensive inflorescence transcriptome data to elucidate the molecular mechanisms underlying the response of litchi flowers to uniconazole treatment and enumerates possible candidate genes that can be used to guide future research in controlling litchi flowering.

Maintaining quality of litchi fruit with acidified calcium sulfate.

The effect of acidified calcium sulfate (ACS) on the quality of litchi ( Litchi chinensis Sonn. cv. 'Brewster') fruit after harvest was evaluated. ACS at 1.25% or higher concentrations significantly inhibited the activities of polyphenol oxidase and peroxidase in the pericarp during storage at both 5 and 10 degrees C. These treatments also effectively prevented browning and retained the red color of the outer shell of the fruit. Total phenolic and total anthocyanin contents in pericarp were increased by ACS treatments in a dose-dependent manner. The radical scavenging activities for ROO(*), DPPH(*), (*)OH and O(2)(*-) were also enhanced by ACS, particularly by 2.5 and 5% concentrations. The activities of several antioxidant enzymes and enzymes of ascorbate-glutathione cycle including catalase, ascorbate peroxidase, glutathione peroxidase, glutathione reductase, dehydroascorbate reductase, and monodehydroascorbate reductase gradually declined during storage. However, ACS enhanced the activities of these enzymes, especially at the beginning of the storage. Samples treated with ACS generally had higher flavonoid levels than the control. The three major flavonoids, cyanidin-3-rutinoside, cyanidin-3-glucoside and quercetin-3-rutinoside, were found to be significantly increased by 2.5 and 5.0% ACS at both 5 and 10 degrees C. No differences were detected among various treatments in soluble solids content or sugar and organic acid levels in the pulp of litchi fruit, indicating that the internal quality of the fruit was not adversely affected by ACS treatment.