Constitutive expression of apple endo-POLYGALACTURONASE1 in fruit induces early maturation, alters skin structure and accelerates softening.

During fruit ripening, polygalacturonases (PGs) are key contributors to the softening process in many species. Apple is a crisp fruit that normally exhibits only minor changes to cell walls and limited fruit softening. Here, we explore the effects of PG overexpression during fruit development using transgenic apple lines overexpressing the ripening-related endo-POLYGALACTURONASE1 gene. MdPG1-overexpressing (PGox) fruit displayed early maturation/ripening with black seeds, conversion of starch to sugars and ethylene production occurring by 80 days after pollination (DAP). PGox fruit exhibited a striking, white-skinned phenotype that was evident from 60 DAP and most likely resulted from increased air spaces and separation of cells in the hypodermis due to degradation of the middle lamellae. Irregularities in the integrity of the epidermis and cuticle were also observed. By 120 DAP, PGox fruit cracked and showed lenticel-associated russeting. Increased cuticular permeability was associated with microcracks in the cuticle around lenticels and was correlated with reduced cortical firmness at all time points and extensive post-harvest water loss from the fruit, resulting in premature shrivelling. Transcriptomic analysis suggested that early maturation was associated with upregulation of genes involved in stress responses, and overexpression of MdPG1 also altered the expression of genes involved in cell wall metabolism (e.g. ß-galactosidase, MD15G1221000) and ethylene biosynthesis (e.g. ACC synthase, MD14G1111500). The results show that upregulation of PG not only has dramatic effects on the structure of the fruit outer cell layers, indirectly affecting water status and turgor, but also has unexpected consequences for fruit development.

Transposon insertions regulate genome-wide allele-specific expression and underpin flower colour variations in apple (Malus spp.).

Allele-specific expression (ASE) can lead to phenotypic diversity and evolution. However, the mechanisms regulating ASE are not well understood, particularly in woody perennial plants. In this study, we investigated ASE genes in the apple cultivar 'Royal Gala' (RG). A high quality chromosome-level genome was assembled using a homozygous tetra-haploid RG plant, derived from anther cultures. Using RNA-sequencing (RNA-seq) data from RG flower and fruit tissues, we identified 2091 ASE genes. Compared with the haploid genome of 'Golden Delicious' (GD), a parent of RG, we distinguished the genomic sequences between the two alleles of 817 ASE genes, and further identified allele-specific presence of a transposable element (TE) in the upstream region of 354 ASE genes. These included MYB110a that encodes a transcription factor regulating anthocyanin biosynthesis. Interestingly, another ASE gene, MYB10 also showed an allele-specific TE insertion and was identified using genome data of other apple cultivars. The presence of the TE insertion in both MYB genes was positively associated with ASE and anthocyanin accumulation in apple petals through analysis of 231 apple accessions, and thus underpins apple flower colour evolution. Our study demonstrated the importance of TEs in regulating ASE on a genome-wide scale and presents a novel method for rapid identification of ASE genes and their regulatory elements in plants.

Regulation of wound ethylene biosynthesis by NAC transcription factors in kiwifruit.

BACKGROUND: The phytohormone ethylene controls many processes in plant development and acts as a key signaling molecule in response to biotic and abiotic stresses: it is rapidly induced by flooding, wounding, drought, and pathogen attack as well as during abscission and fruit ripening. In kiwifruit (Actinidia spp.), fruit ripening is characterized by two distinct phases: an early phase of system-1 ethylene biosynthesis characterized by absence of autocatalytic ethylene, followed by a late burst of autocatalytic (system-2) ethylene accompanied by aroma production and further ripening. Progress has been made in understanding the transcriptional regulation of kiwifruit fruit ripening but the regulation of system-1 ethylene biosynthesis remains largely unknown. The aim of this work is to better understand the transcriptional regulation of both systems of ethylene biosynthesis in contrasting kiwifruit organs: fruit and leaves. RESULTS: A detailed molecular study in kiwifruit (A. chinensis) revealed that ethylene biosynthesis was regulated differently between leaf and fruit after mechanical wounding. In fruit, wound ethylene biosynthesis was accompanied by transcriptional increases in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), ACC oxidase (ACO) and members of the NAC class of transcription factors (TFs). However, in kiwifruit leaves, wound-specific transcriptional increases were largely absent, despite a more rapid induction of ethylene production compared to fruit, suggesting that post-transcriptional control mechanisms in kiwifruit leaves are more important. One ACS member, AcACS1, appears to fulfil a dominant double role; controlling both fruit wound (system-1) and autocatalytic ripening (system-2) ethylene biosynthesis. In kiwifruit, transcriptional regulation of both system-1 and -2 ethylene in fruit appears to be controlled by temporal up-regulation of four NAC (NAM, ATAF1/2, CUC2) TFs (AcNAC1-4) that induce AcACS1 expression by directly binding to the AcACS1 promoter as shown using gel-shift (EMSA) and by activation of the AcACS1 promoter in planta as shown by gene activation assays combined with promoter deletion analysis. CONCLUSIONS: Our results indicate that in kiwifruit the NAC TFs AcNAC2-4 regulate both system-1 and -2 ethylene biosynthesis in fruit during wounding and ripening through control of AcACS1 expression levels but not in leaves where post-transcriptional/translational regulatory mechanisms may prevail.

Peridermal fruit skin formation in Actinidia sp. (kiwifruit) is associated with genetic loci controlling russeting and cuticle formation.

BACKGROUND: The skin (exocarp) of fleshy fruit is hugely diverse across species. Most fruit types have a live epidermal skin covered by a layer of cuticle made up of cutin while a few create an outermost layer of dead cells (peridermal layer). RESULTS: In this study we undertook crosses between epidermal and peridermal skinned kiwifruit, and showed that epidermal skin is a semi-dominant trait. Furthermore, backcrossing these epidermal skinned hybrids to a peridermal skinned fruit created a diverse range of phenotypes ranging from epidermal skinned fruit, through fruit with varying degrees of patches of periderm (russeting), to fruit with a complete periderm. Quantitative trait locus (QTL) analysis of this population suggested that periderm formation was associated with four loci. These QTLs were aligned either to ones associated with russet formation on chromosome 19 and 24, or cuticle integrity and coverage located on chromosomes 3, 11 and 24. CONCLUSION: From the segregation of skin type and QTL analysis, it appears that skin development in kiwifruit is controlled by two competing factors, cuticle strength and propensity to russet. A strong cuticle will inhibit russeting while a strong propensity to russet can create a continuous dead skinned periderm.

A gene expression atlas for kiwifruit (Actinidia chinensis) and network analysis of transcription factors.

BACKGROUND: Transcriptomic studies combined with a well annotated genome have laid the foundations for new understanding of molecular processes. Tools which visualise gene expression patterns have further added to these resources. The manual annotation of the Actinidia chinensis (kiwifruit) genome has resulted in a high quality set of 33,044 genes. Here we investigate gene expression patterns in diverse tissues, visualised in an Electronic Fluorescent Pictograph (eFP) browser, to study the relationship of transcription factor (TF) expression using network analysis. RESULTS: Sixty-one samples covering diverse tissues at different developmental time points were selected for RNA-seq analysis and an eFP browser was generated to visualise this dataset. 2839 TFs representing 57 different classes were identified and named. Network analysis of the TF expression patterns separated TFs into 14 different modules. Two modules consisting of 237 TFs were correlated with floral bud and flower development, a further two modules containing 160 TFs were associated with fruit development and maturation. A single module of 480 TFs was associated with ethylene-induced fruit ripening. Three "hub" genes correlated with flower and fruit development consisted of a HAF-like gene central to gynoecium development, an ERF and a DOF gene. Maturing and ripening hub genes included a KNOX gene that was associated with seed maturation, and a GRAS-like TF. CONCLUSIONS: This study provides an insight into the complexity of the transcriptional control of flower and fruit development, as well as providing a new resource to the plant community. The Actinidia eFP browser is provided in an accessible format that allows researchers to download and work internally.

Cell type-specific gene expression underpins remodelling of cell wall pectin in exocarp and cortex during apple fruit development.

In apple (Malus×domestica) fruit, the different layers of the exocarp (cuticle, epidermis, and hypodermis) protect and maintain fruit integrity, and resist the turgor-driven expansion of the underlying thin-walled cortical cells during growth. Using in situ immunolocalization and size exclusion epitope detection chromatography, distinct cell type differences in cell wall composition in the exocarp were revealed during apple fruit development. Epidermal cell walls lacked pectic (1→4)-ß-d-galactan (associated with rigidity), whereas linear (1→5)-α-l-arabinan (associated with flexibility) was exclusively present in the epidermal cell walls in expanding fruit and then appeared in all cell types during ripening. Branched (1→5)-α-l-arabinan was uniformly distributed between cell types. Laser capture microdissection and RNA sequencing (RNA-seq) were used to explore transcriptomic differences controlling cell type-specific wall modification. The RNA-seq data indicate that the control of cell wall composition is achieved through cell-specific gene expression of hydrolases. In epidermal cells, this results in the degradation of galactan side chains by possibly five ß-galactosidases (BGAL2, BGAL7, BGAL10, BGAL11, and BGAL103) and debranching of arabinans by α-arabinofuranosidases AF1 and AF2. Together, these results demonstrate that flexibility and rigidity of the different cell layers in apple fruit during development and ripening are determined, at least in part, by the control of cell wall pectin remodelling.

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.

The hybrid non-ethylene and ethylene ripening response in kiwifruit (Actinidia chinensis) is associated with differential regulation of MADS-box transcription factors.

BACKGROUND: Ripening in tomato is predominantly controlled by ethylene, whilst in fruit such as grape, it is predominantly controlled by other hormones. The ripening response of many kiwifruit (Actinidia) species is atypical. The majority of ripening-associated fruit starch hydrolysis, colour change and softening occurs in the apparent absence of ethylene production (Phase 1 ripening) whilst Phase 2 ripening requires autocatalytic ethylene production and is associated with further softening and an increase in aroma volatiles. RESULTS: To dissect the ripening response in the yellow-fleshed kiwifruit A. chinensis ('Hort16A'), a two dimensional developmental stage X ethylene response time study was undertaken. As fruit progressed through maturation and Phase 1 ripening, fruit were treated with different concentrations of propylene and ethylene. At the start of Phase 1 ripening, treated fruit responded to ethylene, and were capable of producing endogenous ethylene. As the fruit progressed through Phase 1 ripening, the fruit became less responsive to ethylene and endogeneous ethylene production was partially repressed. Towards the end of Phase 1 ripening the fruit were again able to produce high levels of ethylene. Progression through Phase 1 ripening coincided with a developmental increase in the expression of the ethylene-unresponsive MADS-box FRUITFUL-like gene (FUL1). The ability to respond to ethylene however coincided with a change in expression of another MADS-box gene SEPALLATA4/RIPENING INHIBITOR-like (SEP4/RIN). The promoter of SEP4/RIN was shown to be transactivated by EIN3-like transcription factors, but unlike tomato, not by SEP4/RIN itself. Transient over-expression of SEP4/RIN in kiwifruit caused an increase in ethylene production. CONCLUSIONS: These results suggest that the non-ethylene/ethylene ripening response observed in kiwifruit is a hybrid of both the tomato and grape ripening progression, with Phase 1 being akin to the RIN/ethylene inhibitory response observed in grape and Phase 2 akin to the RIN-associated autocatalytic ethylene response observed in tomato.

Chilling-related cell damage of apple (Malus × domestica Borkh.) fruit cortical tissue impacts antioxidant, lipid and phenolic metabolism.

'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) ß-d-glucoside and sitosteryl (6'-O-stearate) ß-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, ß-carotene, catechin, epicatechin, α-tocopherol, violaxanthin and sitosteryl ß-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.

Apple SEPALLATA1/2-like genes control fruit flesh development and ripening.

Flowering plants utilize different floral structures to develop flesh tissue in fruits. Here we show that suppression of the homeologous SEPALLATA1/2-like genes MADS8 and MADS9 in the fleshy fruit apple (Malus x domestica) leads to sepaloid petals and greatly reduced fruit flesh. Immunolabelling of cell-wall epitopes and differential staining showed that the developing hypanthium (from which the apple flesh develops) of MADS8/9-suppressed apple flowers lacks a tissue layer, and the remaining flesh tissue of fully developed apples has considerably smaller cells. From these observations, it is proposed that MADS8 and MADS9 control the development of discrete zones within the hypanthium tissue, and therefore fruit flesh, and also act as foundations for development of different floral organs. At fruit maturity, the MADS8/9-suppressed apples do not ripen in terms of both developmentally controlled ripening characters, such as starch degradation, and ethylene-modulated ripening traits. Transient assays suggest that, like the RIN gene in tomato, the MADS9 gene acts as a transcriptional activator of the ethylene biosynthesis enzyme, 1-aminocyclopropane-1-carboxylate (ACC) synthase 1. The existence of a single class of genes that regulate both flesh formation and ripening provides an evolutionary tool for controlling two critical aspects of fleshy fruit development.

Ethylene regulates Apple (Malus x domestica) fruit softening through a dose x time-dependent mechanism and through differential sensitivities and dependencies of cell wall-modifying genes.

In fleshy fruit species that have a strong requirement for ethylene to ripen, ethylene is synthesized autocatalytically, producing increasing concentrations as the fruits ripen. Apple fruit with the ACC OXIDASE 1 (ACO1) gene suppressed cannot produce ethylene autocatalytically at ripening. Using these apple lines, an ethylene sensitivity dependency model was previously proposed, with traits such as softening showing a high dependency for ethylene as well as low sensitivity. In this study, it is shown that the molecular control of fruit softening is a complex process, with different cell wall-related genes being independently regulated and exhibiting differential sensitivities to and dependencies on ethylene at the transcriptional level. This regulation is controlled through a dose × time mechanism, which results in a temporal transcriptional response that would allow for progressive cell wall disassembly and thus softening. This research builds on the sensitivity dependency model and shows that ethylene-dependent traits can progress over time to the same degree with lower levels of ethylene. This suggests that a developmental clock measuring cumulative ethylene controls the fruit ripening process.

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.

The genetic control of herkogamy.

Herkogamy is the spatial separation of anthers and stigmas within complete flowers, and is a key floral trait that promotes outcrossing in many angiosperms. The degree of separation between pollen-producing anthers and receptive stigmas has been shown to influence rates of self-pollination amongst plants, with a reduction in herkogamy increasing rates of successful selfing in self-compatible species. Self-pollination is becoming a critical issue in horticultural crops grown in environments where biotic pollinators are limited, absent, or difficult to utilise. In these cases, poor pollination results in reduced yield and misshapen fruit. Whilst there is a growing body of work elucidating the genetic basis of floral organ development, the genetic and environmental control points regulating herkogamy are poorly understood. A better understanding of the developmental and regulatory pathways involved in establishing varying degrees of herkogamy is needed to provide insights into the production of flowers more adept at selfing to produce consistent, high-quality fruit. This review presents our current understanding of herkogamy from a genetics and hormonal perspective.

A MADS-box gene-induced early flowering pear (Pyrus communis L.) for accelerated pear breeding.

There have been a considerable number of studies that have successfully sped up the flowering cycle in woody perennial horticultural species. One particularly successful study in apple (Malus domestica) accelerated flowering using a silver birch (Betula pendula) APETALA1/FRUITFULL MADS-box gene BpMADS4, which yielded a good balance of vegetative growth to support subsequent flower and fruit development. In this study, BpMADS4 was constitutively expressed in European pear (Pyrus communis) to establish whether this could be used as a tool in a rapid pear breeding program. Transformed pear lines flowered within 6-18 months after grafting onto a quince (Cydonia oblonga) rootstock. Unlike the spindly habit of early flowering apples, the early flowering pear lines displayed a normal tree-like habit. Like apple, the flower appearance was normal, and the flowers were fertile, producing fruit and seed upon pollination. Seed from these transformed lines were germinated and 50% of the progeny flowered within 3 months of sowing, demonstrating a use for these in a fast breeding program.

Down-regulation of POLYGALACTURONASE1 alters firmness, tensile strength and water loss in apple (Malus x domestica) fruit.

BACKGROUND: While there is now a significant body of research correlating apple (Malus x domestica) fruit softening with the cell wall hydrolase ENDO-POLYGALACTURONASE1 (PG1), there is currently little knowledge of its physiological effects in planta. This study examined the effect of down regulation of PG1 expression in 'Royal Gala' apples, a cultivar that typically has high levels of PG1, and softens during fruit ripening. RESULTS: PG1-suppressed 'Royal Gala' apples harvested from multiple seasons were firmer than controls after ripening, and intercellular adhesion was higher. Cell wall analyses indicated changes in yield and composition of pectin, and a higher molecular weight distribution of CDTA-soluble pectin. Structural analyses revealed more ruptured cells and free juice in pulled apart sections, suggesting improved integrity of intercellular connections and consequent cell rupture due to failure of the primary cell walls under stress. PG1-suppressed lines also had reduced expansion of cells in the hypodermis of ripe apples, resulting in more densely packed cells in this layer. This change in morphology appears to be linked with reduced transpirational water loss in the fruit. CONCLUSIONS: These findings confirm PG1's role in apple fruit softening and suggests that this is achieved in part by reducing cellular adhesion. This is consistent with previous studies carried out in strawberry but not with those performed in tomato. In apple PG1 also appears to influence other fruit texture characters such as juiciness and water loss.

Differential expression of ethylene biosynthetic and receptor genes in pollination-induced senescence of Dendrobium flowers.

Following successful pollination, Dendrobium orchid flowers rapidly undergo senescence. In Dendrobium cv. Khao Chaimongkol, compatible pollination resulted in faster ethylene production and more rapid development of senescence symptoms, such as drooping, epinasty, venation and yellowing, compared with non-pollinated controls or pollination with incompatible pollinia. The DenACS1 and DenACO1 genes in the perianth of florets that had been pollinated with compatible pollinia were expressed more highly than those in non-pollinated open florets. Incompatible pollinia reduced the expression of DenACS1 and DenACO1 genes in the perianth. Transcript levels of the ethylene receptor gene DenERS1 and signaling genes DenEIL1 and DenERF1 showed differential spatial regulation with greater expression in the perianth than in the column plus ovary following compatible pollination. Compatible pollinia increased ethylene production concomitant with premature senescence and the increased expression of the DenACS1 and DenACO1 genes, and suppressed the ethylene receptor gene DenERS1, whereas incompatible pollinia did not stimulate ethylene production nor induce premature senescence but induced higher expression of DenERS1 both in the perianth and in the column plus ovary. These results suggest that the increased ethylene production in open florets pollinated with compatible pollen was partially due to an increase in the expression of DenACS1 and DenACO1 genes. The compatible pollinia induced a negative regulation of DenERS1 which may play an important role in ethylene perception and in modulating ethylene signaling transduction during pollinia-induced flower senescence.

Fruit development of the diploid kiwifruit, Actinidia chinensis 'Hort16A'.

BACKGROUND: With the advent of high throughput genomic tools, it is now possible to undertake detailed molecular studies of individual species outside traditional model organisms. Combined with a good understanding of physiological processes, these tools allow researchers to explore natural diversity, giving a better understanding of biological mechanisms. Here a detailed study of fruit development from anthesis through to fruit senescence is presented for a non-model organism, kiwifruit, Actinidia chinensis ('Hort16A'). RESULTS: Consistent with previous studies, it was found that many aspects of fruit morphology, growth and development are similar to those of the model fruit tomato, except for a striking difference in fruit ripening progression. The early stages of fruit ripening occur as the fruit is still growing, and many ripening events are not associated with autocatalytic ethylene production (historically associated with respiratory climacteric). Autocatalytic ethylene is produced late in the ripening process as the fruit begins to senesce. CONCLUSION: By aligning A. chinensis fruit development to a phenological scale, this study provides a reference framework for subsequent physiological and genomic studies, and will allow cross comparison across fruit species, leading to a greater understanding of the diversity of fruits found across the plant kingdom.

The role of ethylene and cold temperature in the regulation of the apple POLYGALACTURONASE1 gene and fruit softening.

Fruit softening in apple (Malus x domestica) is associated with an increase in the ripening hormone ethylene. Here, we show that in cv Royal Gala apples that have the ethylene biosynthetic gene ACC OXIDASE1 suppressed, a cold treatment preconditions the apples to soften independently of added ethylene. When a cold treatment is followed by an ethylene treatment, a more rapid softening occurs than in apples that have not had a cold treatment. Apple fruit softening has been associated with the increase in the expression of cell wall hydrolase genes. One such gene, POLYGALACTURONASE1 (PG1), increases in expression both with ethylene and following a cold treatment. Transcriptional regulation of PG1 through the ethylene pathway is likely to be through an ETHYLENE-INSENSITIVE3-like transcription factor, which increases in expression during apple fruit development and transactivates the PG1 promoter in transient assays in the presence of ethylene. A cold-related gene that resembles a COLD BINDING FACTOR (CBF) class of gene also transactivates the PG1 promoter. The transactivation by the CBF-like gene is greatly enhanced by the addition of exogenous ethylene. These observations give a possible molecular mechanism for the cold- and ethylene-regulated control of fruit softening and suggest that either these two pathways act independently and synergistically with each other or cold enhances the ethylene response such that background levels of ethylene in the ethylene-suppressed apples is sufficient to induce fruit softening in apples.

Dissecting the role of climacteric ethylene in kiwifruit (Actinidia chinensis) ripening using a 1-aminocyclopropane-1-carboxylic acid oxidase knockdown line.

During climacteric fruit ripening, autocatalytic (Type II) ethylene production initiates a transcriptional cascade that controls the production of many important fruit quality traits including flavour production and softening. The last step in ethylene biosynthesis is the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by the enzyme ACC oxidase (ACO). Ten independent kiwifruit (Actinidia chinensis) lines were generated targeting suppression of fruit ripening-related ACO genes and the fruit from one of these lines (TK2) did not produce detectable levels of climacteric ethylene. Ripening behaviour in a population of kiwifruit at harvest is asynchronous, so a short burst of exogenous ethylene was used to synchronize ripening in TK2 and control fruit. Following such a treatment, TK2 and control fruit softened to an 'eating-ripe' firmness. Control fruit produced climacteric ethylene and softened beyond eating-ripe by 5 d. In contrast, TK2 fruit maintained an eating-ripe firmness for >25 d and total volatile production was dramatically reduced. Application of continuous exogenous ethylene to the ripening-arrested TK2 fruit re-initiated fruit softening and typical ripe fruit volatiles were detected. A 17 500 gene microarray identified 401 genes that changed after ethylene treatment, including a polygalacturonase and a pectate lyase involved in cell wall breakdown, and a quinone oxidoreductase potentially involved in volatile production. Many of the gene changes were consistent with the softening and flavour changes observed after ethylene treatment. However, a surprisingly large number of genes of unknown function were also observed, which could account for the unique flavour and textural properties of ripe kiwifruit.