Fruit softening: evidence for rhamnogalacturonan lyase action in vivo in ripe fruit cell walls.

BACKGROUND AND AIMS: The softening of ripening fruit involves partial depolymerization of cell-wall pectin by three types of reaction: enzymic hydrolysis, enzymic elimination (lyase-catalysed) and non-enzymic oxidative scission. Two known lyase activities are pectate lyase and rhamnogalacturonan lyase (RGL), potentially causing mid-chain cleavage of homogalacturonan and rhamnogalacturonan-I (RG-I) domains of pectin respectively. However, the important biological question of whether RGL exhibits action in vivo had not been tested. METHODS: We developed a method for specifically and sensitively detecting in-vivo RGL products, based on Driselase digestion of cell walls and detection of a characteristic unsaturated 'fingerprint' product (tetrasaccharide) of RGL action. KEY RESULTS: In model experiments, potato RG-I that had been partially cleaved in vitro by commercial RGL was digested by Driselase, releasing an unsaturated tetrasaccharide ('ΔUA-Rha-GalA-Rha'), taken as diagnostic of RGL action. This highly acidic fingerprint compound was separated from monosaccharides (galacturonate, galactose, rhamnose, etc.) by electrophoresis at pH 2, then separated from ΔUA-GalA (the fingerprint of pectate lyase action) by thin-layer chromatography. The 'ΔUA-Rha-GalA-Rha' was confirmed as 4-deoxy-ß-l-threo-hex-4-enopyranuronosyl-(1→2)-l-rhamnosyl-(1→4)-d-galacturonosyl-(1→2)-l-rhamnose by mass spectrometry and acid hydrolysis. Driselase digestion of cell walls from diverse ripe fruits [date, sea buckthorn, cranberry, yew (arils), mango, plum, blackberry, apple, pear and strawberry] yielded the same fingerprint compound, demonstrating that RGL had been acting in vivo in these fruits prior to harvest. The 'fingerprint' : (galacturonate + rhamnose) ratio in digests from ripe dates was approximately 1 : 72 (mol/mol), indicating that ~1.4 % of the backbone Rha→GalA bonds in endogenous RG-I had been cleaved by in-vivo RGL action. CONCLUSIONS: The results provide the first demonstration that RGL, previously known from studies of fruit gene expression, proteomic studies and in-vitro enzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening.

Comparison of the Effect of Hydrostatic and Dynamic High Pressure Processing on the Enzymatic Activity and Physicochemical Quality Attributes of 'Ataulfo' Mango Nectar.

The effects of hydrostatic (HHP) and dynamic (HPH) high-pressure treatments on the activity of pectin methylesterase (PME) and polyphenol oxidase (PPO) as well as the physicochemical quality attributes of 'Ataulfo' mango nectar were assessed. HHP reduced PME relative activity by 28% at 100 MPa for 5 min but increased PPO activity almost five-fold. Contrarily, HPH did not affect PME activity, but PPO was effectively reduced to 10% of residual activity at 300 MPa and at three passes. Color parameters (CIEL*a*b*), °hue, and chroma were differently affected by each type of high-pressure processing technology. The viscosity and fluid behavior were not affected by HHP, however, HPH changed the apparent viscosity at low dynamic pressure levels (100 MPa with one and three passes). The viscosity decreased at high shear rates in nectar samples, showing a shear-thinning effect. The results highlight how different effects can be achieved with each high-pressure technology; thus, selecting the most appropriate system for processing and preserving liquid foods like fruit beverages is recommended.

Prevention of Enzymatic Browning by Natural Extracts and Genome-Editing: A Review on Recent Progress.

Fresh fruits and vegetable products are easily perishable during postharvest handling due to enzymatic browning reactions. This phenomenon has contributed to a significant loss of food quality and appearance. Thus, a safe and effective alternative method from natural sources is needed to tackle enzymatic browning prevention. The capabilities of natural anti-browning agents derived from plant- and animal-based resources in inhibiting enzymatic activity have been demonstrated in the literature. Some also possess strong antioxidants properties. This review aims to summarize a recent investigation regarding the use of natural anti-browning extracts from different sources for controlling the browning. The potential applications of genome-editing in preventing browning activity and improving postharvest quality is also discussed. Moreover, the patents on the anti-browning extract from natural sources is also presented in this review. The information reviewed here could provide new insights, contributing to the development of natural anti-browning extracts and genome-editing techniques for the prevention of food browning.

Changes in the activity of flavanone 3ß-hydroxylase in blueberry fruit during storage in ozone-enriched atmosphere.

BACKGROUND: The present study aimed to determine whether the ozonation process affects the flavonoid biosynthesis in highbush blueberry (Vaccinum corymbosum L.) fruit. Flavanone 3ß-hydroxylase (F3H) was used as a marker of the flavonoid biosynthesis pathway. The activity of F3H, the expression of gene encoding F3H and the antioxidant status in blueberries treated with ozone at a concentration of 15 ppm for 30 min, every 12 h of storage, and maintained at 4 °C for 4 weeks were investigated. RESULTS: The results showed that ozonation process increases the expression of the F3H gene after 1 week of storage, which translates into a higher catalytic capacity of protein, as well as a higher content of flavonoids and total antioxidant potential of ozonated blueberries compared to non-ozonated fruits. CONCLUSION: The present study provides experimental evidence indicating that ozone treatment in proposed process conditions positively affects flavonoid metabolism in highbush blueberry fruit leading to the maintainance of the high quality of the fruit during storage. © 2021 Society of Chemical Industry.

CsAGA1 and CsAGA2 Mediate RFO Hydrolysis in Partially Distinct Manner in Cucumber Fruits.

A Raffinose family oligosaccharides (RFOs) is one of the major translocated sugars in the vascular bundle of cucumber, but little RFOs can be detected in fruits. Alpha-galactosidases (α-Gals) catalyze the first catabolism step of RFOs. Six α-Gal genes exist in a cucumber genome, but their spatial functions in fruits remain unclear. Here, we found that RFOs were highly accumulated in vascular tissues. In phloem sap, the stachyose and raffinose content was gradually decreased, whereas the content of sucrose, glucose and fructose was increased from pedicel to fruit top. Three alkaline forms instead of acid forms of α-Gals were preferentially expressed in fruit vascular tissues and alkaline forms have stronger RFO-hydrolysing activity than acid forms. By inducible gene silencing of three alkaline forms of α-Gals, stachyose was highly accumulated in RNAi-CsAGA2 plants, while raffinose and stachyose were highly accumulated in RNAi-CsAGA1 plants. The content of sucrose, glucose and fructose was decreased in both RNAi-CsAGA1 and RNAi-CsAGA2 plants after ß-estradiol treatment. In addition, the fresh- and dry-weight of fruits were significantly decreased in RNAi-CsAGA1 and RNAi-CsAGA2 plants. In cucurbitaceous plants, the non-sweet motif within the promoter of ClAGA2 is widely distributed in the promoter of its homologous genes. Taken together, we found RFOs hydrolysis occurred in the vascular tissues of fruits. CsAGA1 and CsAGA2 played key but partly distinct roles in the hydrolysis of RFOs.

Genome-Wide Identification of the Xyloglucan endotransglucosylase/Hydrolase (XTH) and Polygalacturonase (PG) Genes and Characterization of Their Role in Fruit Softening of Sweet Cherry.

Fruit firmness is an important economical trait in sweet cherry (Prunus avium L.) where the change of this trait is related to cell wall degradation. Xyloglucan endotransglycosylase/hydrolase (XTH) and polygalacturonases (PGs) are critical cell-wall-modifying enzymes that occupy a crucial position in fruit ripening and softening. Herein, we identified 18 XTHs and 45 PGs designated PavXTH1-18 and PavPG1-45 based on their locations in the genome of sweet cherry. We provided a systematical overview of PavXTHs and PavPGs, including phylogenetic relationships, conserved motifs, and expression profiling of these genes. The results showed that PavXTH14, PavXTH15 and PavPG38 were most likely to participated in fruit softening owing to the substantial increment in expression during fruit development and ripening. Furthermore, the phytohormone ABA, MeJA, and ethephon significantly elevated the expression of PavPG38 and PavXTH15, and thus promoted fruit softening. Importantly, transient expression PavXTH14, PavXTH15 and PavPG38 in cherry fruits significantly reduced the fruit firmness, and the content of various cell wall components including hemicellulose and pectin significantly changed correspondingly in the transgenic fruit. Taken together, these results present an extensive analysis of XTHs and PGs in sweet cherry and provide potential targets for breeding softening-resistant sweet cherry cultivars via manipulating cell wall-associated genes.

Genome-wide identification and expression profiling of durian CYPome related to fruit ripening.

Durian (Durio zibethinus L.) is a major economic crop native to Southeast Asian countries, including Thailand. Accordingly, understanding durian fruit ripening is an important factor in its market worldwide, owing to the fact that it is a climacteric fruit with a strikingly limited shelf life. However, knowledge regarding the molecular regulation of durian fruit ripening is still limited. Herein, we focused on cytochrome P450, a large enzyme family that regulates many biosynthetic pathways of plant metabolites and phytohormones. Deep mining of the durian genome and transcriptome libraries led to the identification of all P450s that are potentially involved in durian fruit ripening. Gene expression validation by RT-qPCR showed a high correlation with the transcriptome libraries at five fruit ripening stages. In addition to aril-specific and ripening-associated expression patterns, putative P450s that are potentially involved in phytohormone metabolism were selected for further study. Accordingly, the expression of CYP72, CYP83, CYP88, CYP94, CYP707, and CYP714 was significantly modulated by external treatment with ripening regulators, suggesting possible crosstalk between phytohormones during the regulation of fruit ripening. Interestingly, the expression levels of CYP88, CYP94, and CYP707, which are possibly involved in gibberellin, jasmonic acid, and abscisic acid biosynthesis, respectively, were significantly different between fast- and slow-post-harvest ripening cultivars, strongly implying important roles of these hormones in fruit ripening. Taken together, these phytohormone-associated P450s are potentially considered additional molecular regulators controlling ripening processes, besides ethylene and auxin, and are economically important biological traits.

Modification of 13-hydroperoxide lyase expression in olive affects plant growth and results in altered volatile profile.

The C6 aldehydes, alcohols, and the corresponding esters are the most important compounds of virgin olive oil aroma. These C6 volatile compounds are synthesized via the 13-hydroperoxide lyase (13-HPL) branch of the lipoxygenase pathway. In this investigation, a functional analysis of the olive (Olea europaea L.) 13-HPL gene by its overexpression and silencing in olive transgenic lines was carried out. With this aim, sense and RNAi constructs of the olive 13-HPL gene were generated and used for the transformation of embryogenic olive cultures. Leaves from overexpressing lines showed a slight increase in 13-HPL gene expression, whereas RNAi lines exhibited a strong decrease in their transcript levels. Quantification of 13-HPL activity in two overexpressing and two RNAi lines showed a positive correlation with levels of transcripts. Interestingly, RNAi lines showed a high decrease in the content of C6 volatiles linked to a strong increase of C5 volatile compounds, altering the volatile profile in the leaves. In addition, the silencing of the 13-HPL gene severely affected plant growth and development. This investigation demonstrates the role of the 13-HPL gene in the biogenesis of olive volatile compounds and constitutes a functional genomics study in olive related to virgin olive oil quality.

Milk-clotting and hydrolytic activities of an aspartic protease from Salpichroa origanifolia fruits on individual caseins.

In recent years, many attempts have been made to find new plant proteases to make artisan cheeses. The global increase in cheese consumption, together with a lower supply and increasing cost of calf rennet, religious factors (Islam and Judaism) and food choices (vegetarianism) have led to the search for suitable rennet substitutes for milk clotting. This study describes the milk-clotting and hydrolytic activities of an aspartic protease from Salpichroa origanifolia fruits (SoAP) on individual caseins to explore its potential use as an alternative to animal rennet. The milk-clotting index obtained for SoAP was 8.4 times lower than that obtained for chymosin. SoAP showed a higher degree of hydrolysis on α-casein than on the other fractions under the proposed conditions. RP-HPLC, mass spectrometry analyses and sequencing of the hydrolysates allowed identifying five peptides from α-casein, one peptide from ß-casein, and three peptides from k-casein. In silico analysis showed that the peptides identified may display a wide variety of potential biological activities. These results demonstrate the possibility of using SoAP for the manufacture of new types or artisan cheeses, with the simultaneous added value of the potential health-promoting benefits of the bioactive peptides generated during the hydrolysis.

Systems Biology Applied to the Study of Papaya Fruit Ripening: The Influence of Ethylene on Pulp Softening.

Papaya is a fleshy fruit that undergoes fast ethylene-induced modifications. The fruit becomes edible, but the fast pulp softening is the main factor that limits the post-harvest period. Papaya fast pulp softening occurs due to cell wall disassembling coordinated by ethylene triggering that massively expresses pectinases. In this work, RNA-seq analysis of ethylene-treated and non-treated papayas enabled a wide transcriptome overview that indicated the role of ethylene during ripening at the gene expression level. Several families of transcription factors (AP2/ERF, NAC, and MADS-box) were differentially expressed. ACO, ACS, and SAM-Mtase genes were upregulated, indicating a high rate of ethylene biosynthesis after ethylene treatment. The correlation among gene expression and physiological data demonstrated ethylene treatment can indeed simulate ripening, and regulation of changes in fruit color, aroma, and flavor could be attributed to the coordinated expression of several related genes. Especially about pulp firmness, the identification of 157 expressed genes related to cell wall metabolism demonstrated that pulp softening is accomplished by a coordinated action of several different cell wall-related enzymes. The mechanism is different from other commercially important fruits, such as strawberry, tomato, kiwifruit, and apple. The observed behavior of this new transcriptomic data confirms ethylene triggering is the main event that elicits fast pulp softening in papayas.

Chitosan Augments Tetramycin against Soft Rot in Kiwifruit and Enhances Its Improvement for Kiwifruit Growth, Quality and Aroma.

In this study, the co-application of chitosan and tetramycin against kiwifruit soft rot and its effects on the disease resistance, growth, quality and aroma of kiwifruit were investigated. The results show that chitosan could effectively enhance tetramycin against soft rot of kiwifruit with the field control efficacy of 85.33% for spraying chitosan 100 time + 0.3% tetramycin AS 5000-time dilution liquid, which was higher than 80.99% for 0.3% tetramycin AS 5000-time dilution liquid and significantly (p < 0.01) higher than 40.66% for chitosan 100-time dilution liquid. Chitosan could significantly (p < 0.05) improve the promoting effects of tetramycin on total phenolics, total flavonoids, SOD activity of kiwifruit compared to tetramycin during storage for 0-28 days and enhance the disease resistance of kiwifruit. Moreover, the co-application of chitosan and tetramycin was more effective than tetramycin or chitosan alone in enhancing fruit growth, improving fruit quality and increasing fruit aroma. This study highlights that chitosan can be used as an adjuvant to enhance tetramycin against soft rot of kiwifruit and promote tetramycin's improvement for the single fruit volume and weight, vitamin C, soluble sugar, soluble solid, dry matter, soluble protein, titratable acidity and aroma of kiwifruit.

Effect of gum Arabic, xanthan and carrageenan coatings containing antimicrobial agent on postharvest quality of strawberry: Assessing the physicochemical, enzyme activity and bioactive properties.

Effect of edible coatings of gum Arabic, carrageenan and xanthan gum containing lemon grass essential oil 1% w/v on postharvest quality of strawberry was studied under refrigeration for a period of 12 days. Results showed all the three coatings maintained fruit quality parameters during storage compared to control. Among all the coatings, carrageenan coated fruits showed delayed weight loss (10.1 to 8%), decay percentage (78.42 to 14.29%), retained ascorbic acid (0.15 to 0.27 g kg-1), antioxidant activity (18.17 to 25.85%), firmness (9.07 to 12.43 N), L* (32.38 to 40.42), a* (16.08 to 17.22) and b* (27.36 to 33.54). Carrageenan gum also showed lowest cellulase activity (0.03 units h-1 mg protein-1), pectin methylesterase activity (1.13 A620 min-1 mg protein-1) and ß-galactosidase activity (0.51 µmol min-1 mg protein-1), while showed maximum reduction in polygalacturonase activity (0.07 units h-1 mg protein-1) at the end of storage. Carrageenan gum was found effective in retention of anthocyanins and phenolic compounds during storage. Coatings loaded with antimicrobial agent inhibited psychrophilic bacteria, yeast and mold growth. It is concluded that carrageenan gum could better retain strawberry quality up to 12 days under refrigeration.

The NAC side of the fruit: tuning of fruit development and maturation.

Fruits and seeds resulting from fertilization of flowers, represent an incredible evolutionary advantage in angiosperms and have seen them become a critical element in our food supply.Many studies have been conducted to reveal how fruit matures while protecting growing seeds and ensuring their dispersal. As result, several transcription factors involved in fruit maturation and senescence have been isolated both in model and crop plants. These regulators modulate several cellular processes that occur during fruit ripening such as chlorophyll breakdown, tissue softening, carbohydrates and pigments accumulation.The NAC superfamily of transcription factors is known to be involved in almost all these aspects of fruit development and maturation. In this review, we summarise the current knowledge regarding NACs that modulate fruit ripening in model species (Arabidopsis thaliana and Solanum lycopersicum) and in crops of commercial interest (Oryza sativa, Malus domestica, Fragaria genus, Citrus sinensis and Musa acuminata).

Postharvest factors affect under-skin browning in 'Honey Gold' mango fruit.

BACKGROUND: Under-Skin Browning (USB) is a physiological skin disorder that significantly reduces quality of 'Honey Gold' mango (HG) fruit. Relationships between potential causative factors (vibration, holding temperature, sap) and expression factors (enzymes activities, phenolic concentration, anatomy) were investigated. RESULTS: USB incidence was 2.6-3.6-fold higher in ripe HG fruit vibrated for 3-18 h at 12 °C to simulate transport damage and held then at 12 °C for 8 days compared to control fruit held under the same conditions. USB severity of fruit lightly abraded with sand paper to simulate physical damage and artificially induce USB was higher in fruit held at 10 °C than at 6-8 °C or 12-13 °C for 6-8 days. Compared to non-affected skin, USB-affected tissue had a 7.4% increase in total phenolics concentration. However, polyphenol oxidase (PPO) and peroxidase (POD) activities decreased by 19%. Anatomical similarities were observed between USB symptoms and sapburn caused by spurt sap or terpinolene (a major sap component) to abraded skin areas. Incidence of sapburn was higher in abraded fruit held at 12 °C than at 20 °C. CONCLUSION: Holding HG mango fruit at 10 °C can intensify USB. Activities of PPO and POD appear not to be regulatory factors in USB expression in HG. Sap components may be involved in USB expression under conducive postharvest conditions. © 2021 Society of Chemical Industry.

Study of the olive ß-glucosidase gene family putatively involved in the synthesis of phenolic compounds of virgin olive oil.

BACKGROUND: Hydrolysis of the fruit phenolic glucosides occurring during the oil extraction process is the main biochemical reaction affecting the biosynthesis and accumulation of secoiridoid compounds in virgin olive oil. An integrated approach at the molecular, biochemical, and metabolic level was used to study the olive ß-glucosidase gene family in seven olive cultivars selected by their different phenolic profiles. RESULTS: Eight ß-glucosidase genes have been identified by in silico analysis of an olive transcriptome. Their expression levels were analyzed by reverse transcription quantitative polymerase chain reaction in olive fruits at different ripening stages: I, green fruits, 16-19 weeks after flowering (WAF); II, yellow-green fruits, 22-25 WAF; III, turning fruits, 28-31 WAF; and IV, fully ripe fruits, 35-40 WAF. Gene expression was compared with the level of ß-glucosidase activity in the fruit and with the phenolic composition of fruits and oils from different olive cultivars. Phylogenetic analysis of the encoded proteins and differences found among the ß-glucosidase genes based on Gene Ontology enrichment analysis data suggests maximum involvement of two genes, OeBGLU1A and OeBGLU1B, in the phenolic composition of virgin olive oil. Positive correlation coefficients were found within each olive cultivar between OeBGLU1A and OeBGLU1B gene expression data and the phenolic content of the oil. CONCLUSION: The results obtained suggest that the expression pattern of specific ß-glucosidase genes may be an accurate predictor for the phenolic content of virgin olive oil that could be used in olive breeding programs. © 2021 Society of Chemical Industry.

Study on browning mechanism of fresh-cut eggplant (Solanum melongena L.) based on metabolomics, enzymatic assays and gene expression.

Enzymatic browning is one of the crucial problems compromising the flavor and texture of fresh-cut fruit and vegetables. In this study, an untargeted metabolomics approach based on liquid chromatography-mass spectrometry (LC-MS) was used to explore the browning mechanism in fresh-cut eggplant. Metabolomics studies showed that with the increase of fresh-cut time, the contents of 946 metabolites changed dynamically. The metabolites having the same trend share common metabolic pathways. As an important browning substrate, the content of chlorogenic acid increased significantly, suggesting that may be more important to fresh-cut eggplant browning; all 119 common differential metabolites in 5 min/CK and 3 min/CK contrastive groups were mapped onto 31 KEGG pathways including phenylpropanol metabolism, glutathione metabolism pathway, et al. In physiological experiments, results showed that the Phenylpropanoid-Metabolism-Related enzymes (PAL, C4H, 4CL) were changed after fresh-cut treatment, the activities of three enzymes increased first and then decreased, and reached the maximum value at 5 min, indicating the accumulation of phenolic substances. At the same time, ROS were accumulated when plant tissue damaged by cutting, the activities of related antioxidant enzymes (SOD, APX and CAT) changed dynamically after oxidative damage. SOD and APX content increased significantly and reached the maximum value at 10 min after cutting, and then showed a downward trend. However, CAT activity increased sharply and reached the maximum value within 3 min after cutting, then maintained the same activity, and showed a downward trend after 30 min. These data fully demonstrated that the activities of browning related enzymes and gene expression increased with the prolonging of fresh cutting time. We explained the browning mechanism of fresh-cut eggplant by combining metabolomics and physiology, which may lay the foundation for better understanding the mechanism of browning during the fruits and vegetables during processing.

Fruit ripening-associated leucylaminopeptidase with cysteinylglycine dipeptidase activity from durian suggests its involvement in glutathione recycling.

BACKGROUND: Durian (Durio zibethinus L.) is a highly popular fruit in Thailand and several other Southeast Asian countries. It is abundant in essential nutrients and sulphur-containing compounds such as glutathione (GSH) and γ-glutamylcysteine (γ-EC). Cysteinylglycine (Cys-Gly) is produced by GSH catabolism and occurs in durian fruit pulp. Cysteine (Cys) is a precursor of sulphur-containing volatiles generated during fruit ripening. The aforementioned substances contribute to the strong odour and flavour of the ripe fruit. However, the genes encoding plant Cys-Gly dipeptidases are unknown. The aim of this study was to measure leucylaminopeptidase (LAP) activity in durian fruit pulp. RESULTS: We identified DzLAP1 and DzLAP2, which the former was highly expressed in the fruit pulp. DzLAP1 was expressed at various ripening stages and in response to ethephon/1-MCP treatment. Hence, DzLAP1 is active at the early stages of fruit ripening. DzLAP1 is a metalloenzyme ~ 63 kDa in size. It is activated by Mg2+ or Mn2+ and, like other LAPs, its optimal alkaline pH is 9.5. Kinetic studies revealed that DzLAP1 has Km = 1.62 mM for its preferred substrate Cys-Gly. DzLAP1-GFP was localised to the cytosol and targeted the plastids. In planta Cys-Gly hydrolysis was confirmed for Nicotiana benthamiana leaves co-infiltrated with Cys-Gly and expressing DzLAP1. CONCLUSIONS: DzLAP1 has Cys-Gly dipeptidase activity in the γ-glutamyl cycle. The present study revealed that the LAPs account for the high sulphur-containing compound levels identified in fully ripened durian fruit pulp.

Double NCED isozymes control ABA biosynthesis for ripening and senescent regulation in peach fruits.

During ripening, peach fruits (Prunus persica L. Batsch) rapidly progress to the senescent stage, resulting in a brief shelf life. Abscisic acid (ABA) plays an important role in regulating the ripening process, both in climacteric and non-climacteric fruits. A key enzyme for ABA biosynthesis in higher plants is 9-cis-epoxycarotenoid dioxygenase (NCED). In this study, two NCED isozymes, PpNCED1 and PpNCED5, were identified in peach fruits. While both NCED genes had similar transcriptional patterns (up-regulation) at the beginning of peach ripening, PpNCED5 showed a consistently lower expression level than PpNCED1. During the post-harvest stage, gene expression of PpNCED1 declined, while PpNCED5 expression increased relative to PpNCED1 expression. Considering the dynamic process of ABA accumulation during fruit ripening and senescence in peach, this study indicates that both NCED genes cooperatively control ABA biosynthesis in peach fruits. Moreover, spatio-temporal expression and transcriptional response to hormone and abiotic stress suggested that there is functional divergence between PpNCED1 and PpNCED5 genes in peach. A carotenoid-rich callus system was used to verify the function of PpNCED1 and PpNCED5. In the transgenic callus system, both PpNCED1 and PpNCED5 isozymes promoted ABA biosynthesis, which likely accelerated cell senescence through activating ROS signals. The results from this study provide evidence supporting an ABA biosynthetic regulation process via the two NCED genes in peach fruit, and suggest a mechanism of ABA-induced fruit ripening and senescence.

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.

CgPBA1 may be involved in nuclear degradation during secretory cavity formation by programmed cell death in Citrus grandis 'Tomentosa' fruits.

Caspase-3 is the crucial executor caspase of apoptosis in mammalian cells, which is essential for chromatin condensation and DNA fragmentation. Although plants have no caspase-3 homologs, PBA1 acts as a plant caspase-3-like enzyme in plant programmed cell death (PCD). PCD occurs during the formation of secretory cavities in Citrus fruits; hence, secretory cavities could be utilized as a new cell biology model for investigating the regulatory mechanisms of plant PCD. To further study the association between PBA1 and PCD during secretory cavity development in Citrus fruits, CgPBA1 was identified in the fruit of Citrus grandis 'Tomentosa'. The temporal and spatial expression of CgPBA1 during secretory cavity development were analyzed using quantitative real-time PCR and in situ hybridization, and the morphological changes in the apoptotic cell nuclei were observed using TUNEL assay and ultra-thin section technology. The results revealed that the full-length cDNA of CgPBA1 contains a 711 bp ORF that encodes a putative protein containing 236 amino acid with a proteasome-ß-6 functional domain that belongs to the Ntn hydrolase super family. CgPBA1 was predominantly expressed in the secretory cavities; its expression changes coincided with the morphological changes and DNA fragmentation in apoptotic cell nuclei. The green fluorescent fusion protein of CgPBA1 is also located in the nucleus of tobacco epidermal cells. Based on previous research and the findings of the present study, we speculate that CgPBA1 is a highly functional conserved protein in plants, and it might be involved in nuclear degradation during PCD for secretory cavity formation in C. grandis 'Tomentosa' fruits.