Biotechnological approaches for predicting and controlling apple storage disorders.

Fruit storage disorders are major causes of crop losses and downgrades. Cold storage, either in air or in controlled atmospheres high in CO2 and low in O2, can result in chilling injury or respiratory injury (due to high internal CO2 concentrations). Here, we review biotechnological approaches currently being used to better understand these processes, to predict to provide resistance/tolerance to them. Reducing postharvest crop losses through improved cultivars or inventory management will be a major contributor to food security.

Biotechnological approaches for controlling postharvest fruit softening.

Fruit softening is the major factor determining the postharvest life of fruit, affecting bruise and damage susceptibility, pathogen colonisation, and consumer satisfaction, all of which contribute to product losses in the supply chain and consumers' homes. Ripening-related changes to the cell wall, cuticle and soluble sugars largely determine softening, and some are amenable to biotechnological intervention, for example, by manipulation of the expression of genes encoding cell wall-modifying proteins or wax and cutin synthases. In this review, we discuss work exploring the role of genes involved in cell wall and cuticle properties, and recent developments in the silencing of multiple genes by targeting single transcription factors. Identification of transcription factors that control the expression of suites of genes encoding cell wall-modifying proteins provides exciting targets for biotechnology.

Biotechnological approaches for reducing fruit losses caused by pathogenic infection.

Fruit loss due to disease occurs in both the field and postharvest. Knowledge of host immune responses and pathogen virulence is enabling the formulation of increasingly sophisticated strategies for disease control. Traditional genetic modification, typically involving overexpression of genes involved in pathogen perception and defence responses, is beginning to be superseded by CRISPR-Cas9 manipulation of host susceptibility targets. Moreover, the refinement of RNA interference (RNAi) strategies, including spray-induced gene silencing (SIGS), is allowing more nuanced control options. These latter approaches have the advantage over earlier technologies in that either they do not result in the generation of genetically modified organisms (RNAi-based SIGS), or the genetic manipulation used leaves no trace of introduced genetic material (gene editing). Thus, these strategies may be more widely acceptable for deployment for future disease control.

Manipulation of ZDS in tomato exposes carotenoid- and ABA-specific effects on fruit development and ripening.

Spontaneous mutations in fruit-specific carotenoid biosynthetic genes of tomato (Solanum lycopersicum) have led to improved understanding of ripening-associated carotenogenesis. Here, we confirm that ZDS is encoded by a single gene in tomato transcriptionally regulated by ripening transcription factors RIN, NOR and ethylene. Manipulation of ZDS was achieved through transgenic repression and heterologous over-expression in tomato. CaMV 35S-driven RNAi repression inhibited carotenoid biosynthesis in all aerial tissues examined resulting in elevated levels of ζ-carotene isomers and upstream carotenoids, while downstream all trans-lycopene and subsequent photoprotective carotenes and xanthophylls were diminished. Consequently, immature fruit displayed photo-bleaching consistent with reduced levels of the photoprotective carotenes and developmental phenotypes related to a reduction in the carotenoid-derived phytohormone abscisic acid (ABA). ZDS-repressed ripe fruit was devoid of the characteristic red carotenoid, all trans-lycopene and displayed brilliant yellow pigmentation due to elevated 9,9' di-cis-ζ-carotene. Over-expression of the Arabidopsis thaliana ZDS (AtZDS) gene bypassed endogenous co-suppression and revealed ZDS as an additional bottleneck in ripening-associated carotenogenesis of tomato. Quantitation of carotenoids in addition to multiple ripening parameters in ZDS-altered lines and ABA-deficient fruit-specific carotenoid mutants was used to separate phenotypic consequences of ABA from other effects of ZDS manipulation and reveal a unique and dynamic ζ-carotene isomer profile in ripe fruit.

Delayed response to cold stress is characterized by successive metabolic shifts culminating in apple fruit peel necrosis.

BACKGROUND: Superficial scald is a physiological disorder of apple fruit characterized by sunken, necrotic lesions appearing after prolonged cold storage, although initial injury occurs much earlier in the storage period. To determine the degree to which the transition to cell death is an active process and specific metabolism involved, untargeted metabolic and transcriptomic profiling was used to follow metabolism of peel tissue over 180 d of cold storage. RESULTS: The metabolome and transcriptome of peel destined to develop scald began to diverge from peel where scald was controlled using antioxidant (diphenylamine; DPA) or rendered insensitive to ethylene using 1-methylcyclopropene (1-MCP) beginning between 30 and 60 days of storage. Overall metabolic and transcriptomic shifts, representing multiple pathways and processes, occurred alongside α-farnesene oxidation and, later, methanol production alongside symptom development. CONCLUSIONS: Results indicate this form of peel necrosis is a product of an active metabolic transition involving multiple pathways triggered by chilling temperatures at cold storage inception rather than physical injury. Among multiple other pathways, enhanced methanol and methyl ester levels alongside upregulated pectin methylesterases are unique to peel that is developing scald symptoms similar to injury resulting from mechanical stress and herbivory in other plants.

Gene expression and metabolism preceding soft scald, a chilling injury of 'Honeycrisp' apple fruit.

BACKGROUND: 'Honeycrisp' is an apple cultivar that is susceptible to soft scald, a chilling injury expressed as necrotic patches on the peel. Improved understanding of metabolism associated with the disorder would improve our understanding of soft scald and contribute to developing more effective management strategies for apple storage. It was expected that specific gene expression and specific metabolite levels in the peel would be linked with soft scald risk at harvest and/or specific time points during cold storage. RESULTS: Fruit from nine 'Honeycrisp' apple orchards that would eventually develop different incidences of soft scald between 4 and 8 weeks of cold air storage were used to contrast and determine differential transcriptomic and metabolomic changes during storage. Untargeted metabolic profiling revealed changes in a number of distinct pathways preceding and concurrent with soft scald symptom development, including elevated γ-aminobutryic acid (GABA), 1-hexanol, acylated steryl glycosides, and free p-coumaryl acyl esters. At harvest, levels of sesquiterpenoid and triterpenoid acyl esters were relatively higher in peel of fruit that did not later develop the disorder. RNA-seq driven gene expression profiling highlighted possible involvement of genes and associated metabolic processes with soft scald development. These included elevated expression of genes involved in lipid peroxidation and phenolic metabolism in fruit with soft scald, and isoprenoid/brassinosteroid metabolism in fruit that did not develop soft scald. Expression of other stress-related genes in fruit that developed soft scald included chlorophyll catabolism, cell wall loosening, and lipid transport while superoxide dismutases were up-regulated in fruit that did not develop the disorder. CONCLUSIONS: This study delineates the sequential transcriptomic and metabolomic changes preceding soft scald symptom development. Changes were differential depending on susceptibility of fruit to the disorder and could be attributed to key stress related and mediating pathways.

Transcriptomic events associated with internal browning of apple during postharvest storage.

BACKGROUND: Postharvest ripening of apple (Malus x domestica) can be slowed down by low temperatures, and a combination of low O2 and high CO2 levels. While this maintains the quality of most fruit, occasionally storage disorders such as flesh browning can occur. This study aimed to explore changes in the apple transcriptome associated with a flesh browning disorder related to controlled atmosphere storage using RNA-sequencing techniques. Samples from a browning-susceptible cultivar ('Braeburn') were stored for four months under controlled atmosphere. Based on a visual browning index, the inner and outer cortex of the stored apples was classified as healthy or affected tissue. RESULTS: Over 600 million short single-end reads were mapped onto the Malus consensus coding sequence set, and differences in the expression profiles between healthy and affected tissues were assessed to identify candidate genes associated with internal browning in a tissue-specific manner. Genes involved in lipid metabolism, secondary metabolism, and cell wall modifications were highly modified in the affected inner cortex, while energy-related and stress-related genes were mostly altered in the outer cortex. The expression levels of several of them were confirmed using qRT-PCR. Additionally, a set of novel browning-specific differentially expressed genes, including pyruvate dehydrogenase and 1-aminocyclopropane-1-carboxylate oxidase, was validated in apples stored for various periods at different controlled atmosphere conditions, giving rise to potential biomarkers associated with high risk of browning development. CONCLUSIONS: The gene expression data presented in this study will help elucidate the molecular mechanism of browning development in apples at controlled atmosphere storage. A conceptual model, including energy-related (linked to the tricarboxylic acid cycle and the electron transport chain) and lipid-related genes (related to membrane alterations, and fatty acid oxidation), for browning development in apple is proposed, which may be relevant for future studies towards improving the postharvest life of apple.

Tomato GOLDEN2-LIKE transcription factors reveal molecular gradients that function during fruit development and ripening.

Fruit ripening is the summation of changes rendering fleshy fruit tissues attractive and palatable to seed dispersing organisms. For example, sugar content is influenced by plastid numbers and photosynthetic activity in unripe fruit and later by starch and sugar catabolism during ripening. Tomato fruit are sinks of photosynthate, yet unripe green fruit contribute significantly to the sugars that ultimately accumulate in the ripe fruit. Plastid numbers and chlorophyll content are influenced by numerous environmental and genetic factors and are positively correlated with photosynthesis and photosynthate accumulation. GOLDEN2-LIKE (GLK) transcription factors regulate plastid and chlorophyll levels. Tomato (Solanum lycopersicum), like most plants, contains two GLKs (i.e., GLK1 and GLK2/UNIFORM). Mutant and transgene analysis demonstrated that these genes encode functionally similar peptides, though differential expression renders GLK1 more important in leaves, while GLK2 is predominant in fruit. A latitudinal gradient of GLK2 expression influences the typical uneven coloration of green and ripe wild-type fruit. Transcriptome profiling revealed a broader fruit gene expression gradient throughout development. The gradient influenced general ripening activities beyond plastid development and was consistent with the easily observed yet poorly studied ripening gradient present in tomato and many fleshy fruits.

Understanding development and ripening of fruit crops in an 'omics' era.

Next generation sequencing has revolutionized plant biology. Not only has our understanding of plant metabolism advanced using model systems and modern chromatography, but application of 'omics'-based technology has been widely extended to non-model systems as costs have plummeted and efficiency increased. As a result, important fundamental questions relating to important horticultural crops are being answered, and novel approaches with application to industry are in progress. Here we review recent research advances on development and ripening of fruit crops, how next generation sequencing approaches are driving this advance and the emerging future landscape.

Biomarker development for external CO2 injury prediction in apples through exploration of both transcriptome and DNA methylation changes.

Several apple cultivars are susceptible to CO2 injury, a physiological disorder that can be expressed either externally or internally, and which can cause major losses of fruit during controlled atmosphere (CA) storage. Disorder development can also be enhanced using SmartFresh™ technology, based on the inhibition of ethylene perception by 1-methylcyclopropene (1-MCP). Injury development is associated with less mature fruit with lower ethylene production, but the aetiology of the disorder is poorly understood. Here we report on the progress made using mRNAseq approaches to explore the transcriptome during the development of external CO2 injury. Next-generation sequencing was used to mine the apple transcriptome for gene expression changes that are associated with the development of external CO2 injury. 'Empire' apples from a single orchard were treated with either 1 µL L(-1) 1-MCP or 1 g L(-1) diphenylamine or left untreated, and then stored in a CA of 5 kPa CO2 and 2 kPa O2. In addition, susceptibility to the disorder in the 'Empire' apples from five different orchards was investigated and the methylation state of the ACS1 promoter investigated using McrBC endonuclease digestion and real-time quantitative polymerase chain reaction. Expression of over 30 000 genes, aligned to the apple genome, was monitored, with clear divergence of expression among treatments after 1 day of CA storage. Symptom development, internal ethylene concentrations (IECs) and methylation state of the ACS1 promoter were different for each of five orchards. With transcriptomic changes affected by treatment, this dataset will be useful in discovering biomarkers that assess disorder susceptibility. An inverse correlation between the frequency of this disorder and the IEC was detected in a multiple orchard trial. Differential methylation state of the ACS1 promoter correlated with both IEC and injury occurrence, indicating epigenetic regulation of ethylene biosynthesis and possibly events leading to disorder development.

Molecular and genetic regulation of fruit ripening.

Fleshy fruit undergo a novel developmental program that ends in the irreversible process of ripening and eventual tissue senescence. During this maturation process, fruit undergo numerous physiological, biochemical and structural alterations, making them more attractive to seed dispersal organisms. In addition, advanced or over-ripening and senescence, especially through tissue softening and eventual decay, render fruit susceptible to invasion by opportunistic pathogens. While ripening and senescence are often used interchangeably, the specific metabolic activities of each would suggest that ripening is a distinct process of fleshy fruits that precedes and may predispose the fruit to subsequent senescence.

Biology and genetic engineering of fruit maturation for enhanced quality and shelf-life.

Commercial regulation of ripening is currently achieved through early harvest, by controlling the postharvest storage atmosphere and genetic selection for slow or late ripening varieties. Although these approaches are often effective, they are not universally applicable and often result in acceptable, but poor quality, products. With increased understanding of the molecular biology underlying ripening and the advent of genetic engineering technologies, researchers have pursued new strategies to address problems in fruit shelf-life and quality. These have been guided by recent insights into mechanisms by which ethylene and a complex network of transcription factors regulate ripening, and by an increased appreciation of factors that contribute to shelf-life, such as the fruit cuticle.

Ripening-specific stigmasterol increase in tomato fruit is associated with increased sterol C-22 desaturase (CYP710A11) gene expression.

Phytosterol content and composition and sterol C-22 desaturase (LeSD1; CYP710A11) transcript levels in pericarp tissue of 'Rutgers' tomato fruit were compared in the wild-type (wt) and isogenic lines of the nonripening mutants nor and rin at four stages of ripening/aging. Wild-type fruit were harvested at the mature-green (MG), breaker (BK), breaker plus 3 days (B + 3), and breaker plus 6 days (B + 6) stages, whereas nor and rin fruits were harvested at comparable chronological ages (days after pollination). At the MG stage, wt and mutant fruits had closely similar sterol contents, compositions, and conjugations, with >91% of the total sterols in the acylated steryl glycoside plus steryl glycoside (ASG + SG) fraction. During ripening/aging, there were substantial increases in total sterols and the percentage of sterols in the free plus esterified (FS + SE) fraction. Both changes were greater in wt than in nor or rin. In fruit of wt, rin, and nor, respectively, the increases in total sterols between MG and B + 6 were 2.1-, 1.9-, and 1.5-fold, and at B + 6 the percentages of total sterols in FS + SE were 42, 21, and 24. Among all sterol lipids (ASG, SG, FS, and SE), the ratio of stigmasterol (stigmasta-5,22-dien-3beta-ol) to beta-sitosterol (stigmast-5-en-3beta-ol), the two major sterols in tomato, increased 2.3-fold during ripening of wt fruit but declined slightly during comparable aging of nor and rin fruits. In accord with these changes, the abundance of LeSD1 transcript increased 4-fold in pericarp of ripening wt fruit, peaking at B + 3, whereas transcript levels in nor and rin fruits fluctuated but never exceeded the abundance in wt fruit at the MG stage. These findings indicate that the ripening-specific increase in stigmasterol in wt fruit results from a marked increase in LeSD1 transcription and translation, which accelerates C-22 desaturation of the precursor sterol, beta-sitosterol.

Ethylene and alpha-farnesene metabolism in green and red skin of three apple cultivars in response to 1-methylcyclopropene (1-MCP) treatment.

Relationships among alpha-farnesene synthesis and oxidation, ethylene production and perception, antioxidative enzyme activities, and superficial scald development in fruit of three commercial apple cultivars were investigated at the biochemical and gene transcriptional levels. Scald-susceptible Cortland and Law Rome and scald-resistant Idared apples were untreated or treated with the ethylene action inhibitor 1-methylcyclopropene (1-MCP) and stored for up to 25 weeks at 0.5 degrees C. Separate blushed (red) and unblushed (green) peel tissue samples were taken at harvest and after 2, 4, 6, 10, 15, 20, and 25 weeks of storage. Large increases in peel tissue concentrations of alpha-farnesene and its conjugated trienol (CTol) oxidation products occurred in untreated Cortland and Law Rome and were about 4-9-fold greater than those in Idared. In both Cortland and Law Rome, accumulation of CTols in green peel was nearly twice that in red peel. 1-MCP treatment delayed and attenuated alpha-farnesene and CTol accumulation in each cultivar. Activities of peroxidase (POX) and catalase (CAT) were lower in red peel than in green peel, with the exception of CAT in Law Rome, whereas no effects of 1-MCP on enzyme activities were detected except for Cortland. In control fruit, internal ethylene concentrations (IECs) increased during the first 4-6 weeks to reach highest levels in Cortland, intermediate levels in Law Rome, and low levels in Idared. In 1-MCP-treated fruit, IECs increased gradually to modest levels by 25 weeks in Cortland and Law Rome but were almost nil in Idared. Expression patterns of the alpha-farnesene synthase gene MdAFS1, the ethylene receptor gene MdERS1, and the ethylene biosynthetic genes MdACS1 and MdACO1 were generally in accord with the patterns of alpha-farnesene and ethylene production. In particular, MdAFS1 and MdACS1 showed similar patterns of expression in each cultivar. Among the controls, transcript levels increased more rapidly in Cortland and Law Rome than in Idared during the first few weeks of storage. In 1-MCP-treated fruit, transcript abundance in Cortland and Law Rome rose to untreated control levels after 10-15 weeks but remained low in Idared. Scald symptoms were restricted to unblushed skin, and the incidence in controls after 25 weeks was nearly 100% in Cortland and Law Rome compared with 1% in Idared. 1-MCP treatment reduced scald incidence to 14, 3, and 0% in Cortland, Law Rome, and Idared, respectively. Overall, the results support the proposed role of CTols in scald induction and indicate that alpha-farnesene synthesis is tightly regulated by ethylene. However, gene transcription alone does not account for the big differences in ethylene and alpha-farnesene production in Cortland, Law Rome, and Idared apples.

Senescence-associated down-regulation of 1-aminocyclopropane-1-carboxylate (ACC) oxidase delays harvest-induced senescence in broccoli.

To gain an in-depth understanding of the role of ethylene in post harvest senescence, we used broccoli (Brassica oleracea var. italica) as our model species. The senescence-associated asparagine synthetase (AS) promoter from asparagus was used to drive the expression of an antisense 1-aminocyclopropane-1-carboxylate oxidase (ACO) cDNA from broccoli, BoACO2, to reduce ethylene production following harvest. Physiological analyses revealed that transgenic broccoli lines harbouring the antisense BoACO2 gene construct (designated as AS-asACO) displayed delayed senescence in both detached leaves and detached heads as measured by hue angle. Harvested floret tissue from these plants also showed a delayed loss of chlorophyll, lower protease activity and higher total protein content, and changes in transcript levels of senescence marker genes when compared with wild type and transgenic lines transformed with an empty T-DNA. Genes that were down-regulated included those coding for cysteine protease (BoCP5), metallothionein-like protein (BoMT1), hexokinase (BoHK1), invertase (BoINV1) and sucrose transporters (BoSUC1 and BoSUC2). Northern analysis for BoACO1 and BoACO2, ACO assays and western analysis, revealed reduced ACO transcript, enzyme activity and protein accumulation, as well as reduced ethylene production in the transgenic AS-asACO lines when compared with controls, confirming that a key enzyme regulating ethylene biosynthesis was reduced in these plants. This, together with the changes observed in gene expression, confirm a significant role for ethylene in regulating the events leading to senescence in broccoli following harvest.

Novel jasmonate amino acid conjugates in Asparagus officinalis during harvest-induced and natural foliar senescence.

Five jasmonates, including novel tryptophan conjugates of jasmonic acid and dihydrojasmonic acid, were identified in extracts from spears of Asparagus officinalis L. by electrospray tandem mass spectrometry. Spears were harvested and were held dry or with bases immersed in water. The concentrations of jasmonic acid, dihydrojasmonic acid, their tryptophan conjugates, cucurbic acid and methyl jasmonate, were measured by ELISA in spears in the 10 d following harvest. A transient increase that occurred in all spear tips immediately following harvest in the concentration of jasmonates can be attributed to a wounding response. A second increase in the concentration of jasmonates occurred from 7 d after harvest but only in dry-treated spear tips indicating that jasmonates may have accumulated in response to water stress. Jasmonate levels were also monitored during natural foliar senescence. Increased levels of jasmonates occurred after the onset of senescence, implicating them as a consequence rather than a cause of senescence.