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.

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.

A Data Driven Approach to Assess Complex Colour Profiles in Plant Tissues.

The ability to quantify the colour of fruit is extremely important for a number of applied fields including plant breeding, postharvest assessment, and consumer quality assessment. Fruit and other plant organs display highly complex colour patterning. This complexity makes it challenging to compare and contrast colours in an accurate and time efficient manner. Multiple methodologies exist that attempt to digitally quantify colour in complex images but these either require a priori knowledge to assign colours to a particular bin, or fit the colours present within segment of the colour space into a single colour value using a thresholding approach. A major drawback of these methodologies is that, through the process of averaging, they tend to synthetically generate values that may not exist within the context of the original image. As such, to date there are no published methodologies that assess colour patterning using a data driven approach. In this study we present a methodology to acquire and process digital images of biological samples that contain complex colour gradients. The CIE (Commission Internationale de l'Eclairage/International Commission on Illumination) ΔE2000 formula was used to determine the perceptually unique colours (PUC) within images of fruit containing complex colour gradients. This process, on average, resulted in a 98% reduction in colour values from the number of unique colours (UC) in the original image. This data driven procedure summarised the colour data values while maintaining a linear relationship with the normalised colour complexity contained in the total image. A weighted ΔE2000 distance metric was used to generate a distance matrix and facilitated clustering of summarised colour data. Clustering showed that our data driven methodology has the ability to group these complex images into their respective binomial families while maintaining the ability to detect subtle colour differences. This methodology was also able to differentiate closely related images. We provide a high quality set of complex biological images that span the visual spectrum that can be used in future colorimetric research to benchmark colourimetric method development.

Genetic Analysis of Walnut (Juglans regia L.) Pellicle Pigment Variation Through a Novel, High-Throughput Phenotyping Platform.

Walnut pellicle color is a key quality attribute that drives consumer preference and walnut sales. For the first time a high-throughput, computer vision-based phenotyping platform using a custom algorithm to quantitatively score each walnut pellicle in L* a* b* color space was deployed at large-scale. This was compared to traditional qualitative scoring by eye and was used to dissect the genetics of pellicle pigmentation. Progeny from both a bi-parental population of 168 trees ('Chandler' × 'Idaho') and a genome-wide association (GWAS) with 528 trees of the UC Davis Walnut Improvement Program were analyzed. Color phenotypes were found to have overlapping regions in the 'Chandler' genetic map on Chr01 suggesting complex genetic control. In the GWAS population, multiple, small effect QTL across Chr01, Chr07, Chr08, Chr09, Chr10, Chr12 and Chr13 were discovered. Marker trait associations were co-localized with QTL mapping on Chr01, Chr10, Chr14, and Chr16. Putative candidate genes controlling walnut pellicle pigmentation were postulated.

Sensory-Directed Genetic and Biochemical Characterization of Volatile Terpene Production in Kiwifruit.

Terpene volatiles are found in many important fruit crops, but their relationship to flavor is poorly understood. Here, we demonstrate using sensory descriptive and discriminant analysis that 1,8-cineole contributes a key floral/eucalyptus note to the aroma of ripe 'Hort16A' kiwifruit (Actinidia chinensis). Two quantitative trait loci (QTLs) for 1,8-cineole production were identified on linkage groups 27 and 29a in a segregating A. chinensis population, with the QTL on LG29a colocating with a complex cluster of putative terpene synthase (TPS)-encoding genes. Transient expression in Nicotiana benthamiana and analysis of recombinant proteins expressed in Escherichia coli showed four genes in the cluster (AcTPS1a-AcTPS1d) encoded functional TPS enzymes, which produced predominantly sabinene, 1,8-cineole, geraniol, and springene, respectively. The terpene profile produced by AcTPS1b closely resembled the terpenes detected in red-fleshed A chinensis AcTPS1b expression correlated with 1,8-cineole content in developing/ripening fruit and also showed a positive correlation with 1,8-cineole content in the mapping population, indicating the basis for segregation is an expression QTL. Transient overexpression of AcTPS1b in Actinidia eriantha fruit confirmed this gene produced 1,8-cineole in Actinidia Structure-function analysis showed AcTPS1a and AcTPS1b are natural variants at key TPS catalytic site residues previously shown to change enzyme specificity in vitro. Together, our results indicate that AcTPS1b is a key gene for production of the signature flavor terpene 1,8-cineole in ripe kiwifruit. Using a sensory-directed strategy for compound identification provides a rational approach for applying marker-aided selection to improving flavor in kiwifruit as well as other fruits.

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.

Copy number variants in kiwifruit ETHYLENE RESPONSE FACTOR/APETALA2 (ERF/AP2)-like genes show divergence in fruit ripening associated cold and ethylene responses in C-REPEAT/DRE BINDING FACTOR-like genes.

The ETYHLENE RESPONSE FACTOR/APETALA2 (ERF/AP2) transcription factors have been shown to control a wide range of developmental and environmental responses in plants. These include hormonal responses to ethylene and Abscisic Acid (ABA) as well as to cold and drought. In Actinidia chinensis (kiwifruit), ripening is unusual: although it is sometimes classed as a climacteric fruit (ethylene-associated ripening), much of fruit ripening occurs independently from autocatalytic ethylene production. Initiation of ripening appears to be strongly developmentally controlled and modulated by low temperature. In this study, fruit treated with different temperatures showed an increase in soluble sugar accumulation, and a corresponding increase in ß-AMYLASE (BAM) genes (predominantly BAM3.2 and BAM9) with lower temperatures. To investigate the potential role of the AP2/ERF gene family in the control of fruit ripening in kiwifruit this family was investigated further. Using the new genome annotation and further genome sequence analysis we identified 226 ERF-like genes, 10 AP2L/RAV-like genes and 32 AP2-like genes. An RNA-seq screen from kiwifruit of different maturities, and following treatment with ethylene and temperatures between 0 and 16°C, revealed 4%, 26% and 18% of the ERF-like genes were upregulated by maturation, ethylene and cold temperatures, respectively. Focusing on the C-REPEAT/DRE BINDING FACTOR (CBF) cold master regulators, nine potential genes were identified based on sequence similarity. Five of these CBF-like genes were found in a copy number variant (CNV) cluster of six genes on chromosome 14. Expression analysis showed that two homeologous genes (ERF41 and ERF180) increased in abundance with cold and ethylene, while the cluster of CNV CBF-like genes had lost the ability to respond to cold and increased only with ethylene, suggesting an evolutionary progression of function of these genes.

Re-programming of Pseudomonas syringae pv. actinidiae gene expression during early stages of infection of kiwifruit.

BACKGROUND: Pseudomonas syringae is a widespread bacterial species complex that includes a number of significant plant pathogens. Amongst these, P. syringae pv. actinidiae (Psa) initiated a worldwide pandemic in 2008 on cultivars of Actinidia chinensis var. chinensis. To gain information about the expression of genes involved in pathogenicity we have carried out transcriptome analysis of Psa during the early stages of kiwifruit infection. RESULTS: Gene expression in Psa was investigated during the first five days after infection of kiwifruit plantlets, using RNA-seq. Principal component and heatmap analyses showed distinct phases of gene expression during the time course of infection. The first phase was an immediate transient peak of induction around three hours post inoculation (HPI) that included genes that code for a Type VI Secretion System and nutrient acquisition (particularly phosphate). This was followed by a significant commitment, between 3 and 24 HPI, to the induction of genes encoding the Type III Secretion System (T3SS) and Type III Secreted Effectors (T3SE). Expression of these genes collectively accounted for 6.3% of the bacterial transcriptome at this stage. There was considerable variation in the expression levels of individual T3SEs but all followed the same temporal expression pattern, with the exception of hopAS1, which peaked later in expression at 48 HPI. As infection progressed over the time course of five days, there was an increase in the expression of genes with roles in sugar, amino acid and sulfur transport and the production of alginate and colanic acid. These are both polymers that are major constituents of extracellular polysaccharide substances (EPS) and are involved in biofilm production. Reverse transcription-quantitative PCR (RT-qPCR) on an independent infection time course experiment showed that the expression profile of selected bacterial genes at each infection phase correlated well with the RNA-seq data. CONCLUSIONS: The results from this study indicate that there is a complex remodeling of the transcriptome during the early stages of infection, with at least three distinct phases of coordinated gene expression. These include genes induced during the immediate contact with the host, those involved in the initiation of infection, and finally those responsible for nutrient acquisition.

The effect of 1-methylcyclopropene (1-MCP) on expression of ethylene receptor genes in durian pulp during ripening.

Rapid fruit ripening is a significant problem that limits the shelf life of durian, with ethylene having a major impact on the regulation of this event. Durian treated with ethephon ripened 3 d after treatment with increased pulp total soluble solids, ethylene production of the whole fruit and decreased pulp firmness compared to the control fruit. 1-MCP treatment delayed ripening by up to 9 d with inhibited accumulation of total soluble solids, color change, softening and ethylene production. Genes related to ethylene perception (DzETR1 and DzETR2) and the signaling pathway (DzCTR1, DzEIL1 and DzEIL2) in the pulp were investigated during this process, using qPCR to quantify changes in gene transcription. All candidate genes were significantly up-regulated in ripening durian pulp. Ethephon treatment increased the expression of DzETR1 and DzETR2 genes, while expression of DzCTR1, DzEIL1 and DzEIL2 were slightly affected. 1-MCP treatment significantly inhibited the expression of the DzETR2 and DzEIL1 genes. The promoters of DzETR2 genes were isolated and their activation by fruit transcription factors studied using transient expression in tobacco leaves. It was found that members of the kiwifruit and apple EIL1, EIL2 and EIL3 genes strongly activated the DzETR2 promoter. These results suggest that ethylene-induced ripening of durian is via the regulation of DzETR2 by EIL transcription factors.

Cell separation in kiwifruit without development of a specialised detachment zone.

BACKGROUND: Unlike in abscission or dehiscence, fruit of kiwifruit Actinidia eriantha develop the ability for peel detachment when they are ripe and soft in the absence of a morphologically identifiable abscission zone. Two closely-related genotypes with contrasting detachment behaviour have been identified. The 'good-peeling' genotype has detachment with clean debonding of cells, and a peel tissue that does not tear. The 'poor-peeling' genotype has poor detachability, with cells that rupture upon debonding, and peel tissue that fragments easily. RESULTS: Structural studies indicated that peel detachability in both genotypes occurred in the outer pericarp beneath the hypodermis. Immunolabelling showed differences in methylesterification of pectin, where the interface of labelling coincided with the location of detachment in the good-peeling genotype, whereas in the poor-peeling genotype, no such interface existed. This zone of difference in methylesterification was enhanced by differential cell wall changes between the peel and outer pericarp tissue. Although both genotypes expressed two polygalacturonase genes, no enzyme activity was detected in the good-peeling genotype, suggesting limited pectin breakdown, keeping cell walls strong without tearing or fragmentation of the peel and flesh upon detachment. Differences in location and amounts of wall-stiffening galactan in the peel of the good-peeling genotype possibly contributed to this phenotype. Hemicellulose-acting transglycosylases were more active in the good-peeling genotype, suggesting an influence on peel flexibility by remodelling their substrates during development of detachability. High xyloglucanase activity in the peel of the good-peeling genotype may contribute by having a strengthening effect on the cellulose-xyloglucan network. CONCLUSIONS: In fruit of A. eriantha, peel detachability is due to the establishment of a zone of discontinuity created by differential cell wall changes in peel and outer pericarp tissues that lead to changes in mechanical properties of the peel. During ripening, the peel becomes flexible and the cells continue to adhere strongly to each other, preventing breakage, whereas the underlying outer pericarp loses cell wall strength as softening proceeds. Together these results reveal a novel and interesting mechanism for enabling cell separation.

Apple dwarfing rootstocks exhibit an imbalance in carbohydrate allocation and reduced cell growth and metabolism.

Apple dwarfing rootstocks cause earlier shoot termination and reduced root and shoot mass. To identify physiological factors responsible for rootstock-induced growth restriction, we compared vascular-enriched gene expression between two dwarfing rootstocks ('M27' and 'M9') and the vigorous rootstock 'M793' using RNA sequencing and quantitative reverse transcriptase PCR. Differentially expressed genes common to both dwarfing rootstocks belonged to five main biological processes: (1) primary metabolism, (2) cell wall synthesis and modification, (3) secondary metabolism, (4) hormone signalling and response and (5) redox homeostasis. Genes promoting the biosynthesis of amino acids, lipids and cell walls were downregulated in dwarfing rootstocks, whereas genes promoting the breakdown of these compounds were upregulated. The only exception to this trend was the upregulation of starch synthesis genes in dwarfing rootstocks. Non-structural carbohydrate analysis demonstrated that starch concentrations in 'M9' roots, stems and grafted 'Royal Gala' ('RG') scions were double that of equivalent tissues from 'RG' homo-grafted trees ('RG'/'RG'). Fructose and glucose concentrations were much lower in all three tissues of the 'RG'/'M9' trees. Together, these data indicate that dwarfing rootstocks are in a state of sugar depletion and reduced cellular activity despite having large starch reserves. Another significant finding was the over-accumulation of flavonoids and the downregulation of auxin influx transporters MdAUX1 and MdLAX2 in dwarfing rootstocks. We propose that both factors reduce polar auxin transport. The results of this study contribute novel information about the physiological state of dwarfing rootstocks.

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.

A dynamic interplay between phytohormones is required for fruit development, maturation, and ripening.

Plant species that bear fruit often utilize expansion of an ovary (carpel) or accessory tissue as a vehicle for seed dispersal. While the seed(s) develop, the tissue(s) of the fruit follow a common progression of cell division and cell expansion, promoting growth of the fruit. Once the seed is fully developed, the fruit matures and the surrounding tissue either dries or ripens promoting the dissemination of the seed. As with many developmental processes in plants, plant hormones play an important role in the synchronization of signals between the developing seed and its surrounding fruit tissue(s), regulating each phase of fruit development. Following pollination, fruit set is achieved through a de-repression of growth and an activation of cell division via the action of auxin and/or cytokinin and/or gibberellin. Following fruit set, growth of the fruit is facilitated through a relatively poorly studied period of cell expansion and endoreduplication that is likely regulated by similar hormones as in fruit set. Once the seeds reach maturity, fruit become ready to undergo ripening and during this period there is a major switch in relative hormone levels of the fruit, involving an overall decrease in auxin, gibberellin, and cytokinin and a simultaneous increase in abscisic acid and ethylene. While the role of hormones in fruit set and ripening is well documented, the knowledge of the roles of other hormones during growth, maturation, and some individual ripening components is sketchy.

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.

A rapid method of fruit cell isolation for cell size and shape measurements.

BACKGROUND: Cell size is a structural component of fleshy fruit, contributing to important traits such as fruit size and texture. There are currently a number of methods for measuring cell size; most rely either on tissue sectioning or digestion of the tissue with cell wall degrading enzymes or chemicals to release single cells. Neither of these approaches is ideal for assaying large fruit numbers as both require a considerable time to prepare the tissue, with current methods of cell wall digestions taking 24 to 48 hours. Additionally, sectioning can lead to a measurement of a plane that does not represent the widest point of the cell. RESULTS: To develop a more rapid way of measuring fruit cell size we have developed a protocol that solubilises pectin in the middle lamella of the plant cell wall releasing single cells into a buffered solution. Gently boiling small fruit samples in a 0.05 M Na2CO3 solution, osmotically balanced with 0.3 M mannitol, produced good cell separation with little cellular damage in less than 30 minutes. The advantage of combining a chemical treatment with boiling is that the cells are rapidly killed. This stopped cell shape changes that could potentially occur during separation. With this method both the rounded and angular cells of the apple cultivars SciRos 'Pacific Rose' and SciFresh 'Jazz' were observed in the separated cells. Using this technique, an in-depth analysis was performed measuring cell size from 5 different apple cultivars. Cell size was measured using the public domain ImageJ software. For each cultivar a minimum of 1000 cells were measured and it was found that each cultivar displayed a different distribution of cell size. Cell size within cultivars was similar and there was no correlation between flesh firmness and cell size. This protocol was tested on tissue from other fleshy fruit including tomato, rock melon and kiwifruit. It was found that good cell separation was achieved with flesh tissue from all these fruit types, showing a broad utility to this protocol. CONCLUSION: We have developed a method for isolating single cells from fleshy fruit that reduces the time needed for fruit cell separation. This method was used to demonstrate differences in cell size and shape for 5 different apple cultivars. While firmness between the different cultivars is independent of cell size, apples with more angular cells appear to be firmer.