Avocado (Persea americana Mill.) fruit hang on the tree in a physiologically mature but unripe state for many months. We hypothesised that this was due to an on-tree ripening block that prevents the development of ethylene sensitivity in the fruit. Physiologically mature fruit, determined by size and dry matter content, were categorised into ethylene sensitive or insensitive based on whether an ethylene treatment at harvest accelerated ripening. Using tissue biopsies taken from the fruit 2 d prior to their harvest, ethylene-insensitive mature fruit were found to have retained the transcriptome profile of very young immature fruit and were enriched in transcripts related to gibberellin biosynthesis and auxin signalling. Transcript abundance of GIBBERELLIN 20-OXIDASE, GIBBERELLIN METHYLTRANSFERASE and IAA13 in mesocarp biopsies taken 7 d before the fruit were harvested could predict the ethylene sensitivity status of the fruit. Upon harvest, the fruit with low transcript abundance of these genes responded to the ethylene challenge by producing ethylene and ripening earlier, whereas the fruit with high transcript abundance were unresponsive to ethylene and ripened with similar timing to fruit not exposed to ethylene. We conclude that avocado fruit on-tree have different strengths of ripening inhibition, which is caused by the differential retention of gibberellin and auxin-related biology that affects their sensitivity to ethylene.

This article comments on: Liu G-S, Huang H, Grierson D, Gao Y, Ji X, Peng Z-Z, Li H-L, Niu X-L, Jia W, He J-L, Xiang L-T, Gao H-Y, Qu G-Q, Zhu H-L, Zhu B-Z, Luo Y-B, Fu D-Q. 2024. NAC transcription factor SlNOR-like1 plays a dual regulatory role in tomato fruit cuticle formation. Journal of Experimental Botany 75, 1903–1918.

EDITORIAL article Front. Sustain. Food Syst., 04 January 2023Sec. Agro-Food Safety Volume 6 - 2022 | https://doi.org/10.3389/fsufs.2022.1061079

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.

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.

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.

BACKGROUND:MicroRNAs (miRNAs) are short non-coding RNAs that can influence gene expression via diverse mechanisms. Tomato is a fruit widely consumed for its flavor, culinary attributes, and high nutritional quality. Tomato fruit are climacteric and fleshy, and their ripening is regulated by endogenous and exogenous signals operating through a coordinated genetic network. Much research has been conducted on mechanisms of tomato fruit ripening, but the roles of miRNA-regulated repression/expression of specific regulatory genes are not well documented.RESULTS:In this study, we demonstrate that miR172 specifically targets four SlAP2 transcription factor genes in tomato. Among them, SlAP2a was repressed by the overexpression of SlmiR172, manifesting in altered flower morphology, development and accelerated ripening. miR172 over-expression lines specifically repressed SlAP2a, enhancing ethylene biosynthesis, fruit color and additional ripening characteristics. Most previously described ripening-regulatory genes, including RIN-MADS, NR, TAGL1 and LeHB-1 were not influenced by miR172 while CNR showed altered expression.CONCLUSIONS:Tomato fruit ripening is directly influenced by miR172 targeting of the APETALA2 transcription factor, SlAP2a, with minimal influence over additional known ripening-regulatory genes. miR172a-guided SlAP2a expression provides insight into another layer of genetic control of ripening and a target for modifying the quality and nutritional value of tomato and possibly other fleshy fruit crops.

SummarySpontaneous 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.

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.

The selection of reference genes for RT-qPCR requires the measurement of gene expression across experimental samples. A reference gene with a low variance in its expression for one set of samples may not be suitable for other experiments sampled across different developmental stages, tissue types and seasons. The latter are often typical of postharvest gene expression experiments. In this study we initially used 61 mRNA-seq data sets from different tissues, stages of development and stress conditions of apple (Malus x domestica) to select 10 potential reference genes. These were tested using RT-qPCR, reference gene selection software, and 156 mRNA-seq datasets from a second independent laboratory. From these results we have selected four potential reference genes for use in postharvest and other apple gene expression experiments. Use of this mRNA-seq data method may facilitate selection of reference genes in other crop species for which these data are available.

‘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.

The use of 1-methylcyclopropene (1-MCP) to maintain quality attributes of horticultural products has been investigated extensively. Commercial applications of 1-MCP (SmartFresh (TM)), are made to a range of horticultural crops, including avocados, bananas, melons, persimmons and tomatoes, but the majority of 1-MCP use is for apples. Apples are commonly kept in controlled atmosphere (CA) storage, and while 1-MCP can maintain the quality of fruit in air, the most consistent responses to 1-MCP are obtained when 1-MCP treated fruit are maintained in CA. An interesting feature of 1-MCP is the extent to which its use has revealed the interactions between ethylene production and the development of physiological storage disorders in a variety of fruits and vegetables. This has been especially true for apples because of their susceptibility to a wide range of disorders arising at least in part because of the extended storage periods routinely used. Here we review the effects of 1-MCP on quality and physiological disorders of fruits and vegetables, including studies on the interactions of 1-MCP with CA and modified atmosphere (MA) storage. We also present an overview of an international project on genomics and metabolomics concerning interactions between external carbon dioxide injury and flesh browning in CA stored apple fruit.

To accurately measure gene expression using PCR-based approaches, there is the need for reference genes that have low variance in expression (housekeeping genes) to normalise the data for RNA quantity and quality. For non-model species such as Malus x domestica (apples), previously, the selection of reference genes relied on using homology to reference genes in model species. In this study, a genomics approach was used to identify apple genes with low variance in expression in 217 messenger RNA (mRNA)-seq data sets covering different tissues, during fruit development, and treated with a range of different stress conditions. Ten potential reference genes were chosen for validation by quantitative PCR (qPCR) over 29 different tissue types and treatments. From the combined mRNA-seq and qPCR results, three potential reference genes are proposed that can be used as good controls for PCR based expression studies. The three genes show homology to lipid transfer proteins, phytochrome protein phosphatase and the ubiquitination pathway. With the progression of research away from non-model species, this approach provides a robust method for selecting candidate genes for use as reference genes in qPCR.

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.

'Soggy breakdown' (SB) is an internal flesh disorder of 'Honeycrisp' apple (Malus × domestica Borkh.) fruit that occurs during low temperature storage. The disorder is a chilling injury (CI) in which visible symptoms typically appear after several weeks of storage, but information about the underlying metabolism associated with its induction and development is lacking. The metabolic profile of flesh tissue from wholly healthy fruit and brown and healthy tissues from fruit with SB was characterized using gas chromatography-mass spectrometry (GC-MS) and liquid chromatograph-mass spectrometry (LC-MS). Partial least squares discriminant analysis (PLS-DA) and correlation networks revealed correlation among ester volatile compounds by composition and differences in phytosterol, phenolic and putative triacylglycerides (TAGs) metabolism among the tissues. anova-simultaneous component analysis (ASCA) was used to test the significance of metabolic changes linked with tissue health status. ASCA-significant components included antioxidant compounds, TAGs, and phytosterol conjugates. Relative to entirely healthy tissues, elevated metabolite levels in symptomatic tissue included γ-amino butyric acid, glycerol, sitosteryl (6'-O-palmitoyl) β-d-glucoside and sitosteryl (6'-O-stearate) β-d-glucoside, and TAGs containing combinations of 16:0, 18:3, 18:2 and 18:1 fatty acids. Reduced metabolite levels in SB tissue included 5-caffeoyl quinate, β-carotene, catechin, epicatechin, α-tocopherol, violaxanthin and sitosteryl β-d glucoside. Pathway analysis indicated aspects of primary metabolism differed according to tissue condition, although differences in metabolites involved were more subtle than those of some secondary metabolites. The results implicate oxidative stress and membrane disruption processes in SB development and constitute a diagnostic metabolic profile for the disorder.

Background: Postharvest ripening of apple (Malus x domestica) can be slowed down by low temperatures, and a combination of low O-2 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.

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.