Dormancy regulator Prunus mume DAM6 promotes ethylene-mediated leaf senescence and abscission.

Leaf senescence and abscission in autumn are critical phenological events in deciduous woody perennials. After leaf fall, dormant buds remain on deciduous woody perennials, which then enter a winter dormancy phase. Thus, leaf fall is widely believed to be linked to the onset of dormancy. In Rosaceae fruit trees, DORMANCY-ASSOCIATED MADS-box (DAM) transcription factors control bud dormancy. However, apart from their regulatory effects on bud dormancy, the biological functions of DAMs have not been thoroughly characterized. In this study, we revealed a novel DAM function influencing leaf senescence and abscission in autumn. In Prunus mume, PmDAM6 expression was gradually up-regulated in leaves during autumn toward leaf fall. Our comparative transcriptome analysis using two RNA-seq datasets for the leaves of transgenic plants overexpressing PmDAM6 and peach (Prunus persica) DAM6 (PpeDAM6) indicated Prunus DAM6 may up-regulate the expression of genes involved in ethylene biosynthesis and signaling as well as leaf abscission. Significant increases in 1-aminocyclopropane-1-carboxylate accumulation and ethylene emission in DEX-treated 35S:PmDAM6-GR leaves reflect the inductive effect of PmDAM6 on ethylene biosynthesis. Additionally, ethephon treatments promoted autumn leaf senescence and abscission in apple and P. mume, mirroring the changes due to PmDAM6 overexpression. Collectively, these findings suggest that PmDAM6 may induce ethylene emission from leaves, thereby promoting leaf senescence and abscission. This study clarified the effects of Prunus DAM6 on autumn leaf fall, which is associated with bud dormancy onset. Accordingly, in Rosaceae, DAMs may play multiple important roles affecting whole plant growth during the tree dormancy induction phase.

Degenerate oligonucleotide primer MIG-seq: an effective PCR-based method for high-throughput genotyping.

Next-generation sequencing (NGS) library construction often involves using restriction enzymes to decrease genome complexity, enabling versatile polymorphism detection in plants. However, plant leaves frequently contain impurities, such as polyphenols, necessitating DNA purification before enzymatic reactions. To overcome this problem, we developed a PCR-based method for expeditious NGS library preparation, offering flexibility in number of detected polymorphisms. By substituting a segment of the simple sequence repeat sequence in the MIG-seq primer set (MIG-seq being a PCR method enabling library construction with low-quality DNA) with degenerate oligonucleotides, we introduced variability in detectable polymorphisms across various crops. This innovation, named degenerate oligonucleotide primer MIG-seq (dpMIG-seq), enabled a streamlined protocol for constructing dpMIG-seq libraries from unpurified DNA, which was implemented stably in several crop species, including fruit trees. Furthermore, dpMIG-seq facilitated efficient lineage selection in wheat and enabled linkage map construction and quantitative trait loci analysis in tomato, rice, and soybean without necessitating DNA concentration adjustments. These findings underscore the potential of the dpMIG-seq protocol for advancing genetic analyses across diverse plant species.

Retrograde sulfur flow from glucosinolates to cysteine in Arabidopsis thaliana.

Specialized (secondary) metabolic pathways in plants have long been considered one-way routes of leading primary metabolite precursors to bioactive end products. Conversely, endogenous degradation of such "end" products in plant tissues has been observed following environmental stimuli, including nutrition stress. Therefore, it is of general interest whether specialized metabolites can be reintegrated into primary metabolism to recover the invested resources, especially in the case of nitrogen- or sulfur-rich compounds. Here, we demonstrate that endogenous glucosinolates (GLs), a class of sulfur-rich plant metabolites, are exploited as a sulfur source by the reallocation of sulfur atoms to primary metabolites such as cysteine in Arabidopsis thaliana Tracer experiments using 34S- or deuterium-labeled GLs depicted the catabolic processing of GL breakdown products in which sulfur is mobilized from the thioglucoside group in GL molecules, potentially accompanied by the release of the sulfate group. Moreover, we reveal that beta-glucosidases BGLU28 and BGLU30 are the major myrosinases that initiate sulfur reallocation by hydrolyzing particular GL species, conferring sulfur deficiency tolerance in A. thaliana, especially during early development. The results delineate the physiological function of GL as a sulfur reservoir, in addition to their well-known functions as defense chemicals. Overall, our findings demonstrate the bidirectional interaction between primary and specialized metabolism, which enhances our understanding of the underlying metabolic mechanisms via which plants adapt to their environments.

Tobacco Root Endophytic Arthrobacter Harbors Genomic Features Enabling the Catabolism of Host-Specific Plant Specialized Metabolites.

Plant roots constitute the primary interface between plants and soilborne microorganisms and harbor microbial communities called the root microbiota. Recent studies have demonstrated a significant contribution of plant specialized metabolites (PSMs) to the assembly of root microbiota. However, the mechanistic and evolutionary details underlying the PSM-mediated microbiota assembly and its contribution to host specificity remain elusive. Here, we show that the bacterial genus Arthrobacter is predominant specifically in the tobacco endosphere and that its enrichment in the tobacco endosphere is partially mediated by a combination of two unrelated classes of tobacco-specific PSMs, santhopine and nicotine. We isolated and sequenced Arthrobacter strains from tobacco roots as well as soils treated with these PSMs and identified genomic features, including but not limited to genes for santhopine and nicotine catabolism, that are associated with the ability to colonize tobacco roots. Phylogenomic and comparative analyses suggest that these genes were gained in multiple independent acquisition events, each of which was possibly triggered by adaptation to particular soil environments. Taken together, our findings illustrate a cooperative role of a combination of PSMs in mediating plant species-specific root bacterial microbiota assembly and suggest that the observed interaction between tobacco and Arthrobacter may be a consequence of an ecological fitting process. IMPORTANCE Host secondary metabolites have a crucial effect on the taxonomic composition of its associated microbiota. It is estimated that a single plant species produces hundreds of secondary metabolites; however, whether different classes of metabolites have distinctive or common roles in the microbiota assembly remains unclear. Here, we show that two unrelated classes of secondary metabolites in tobacco play a cooperative role in the formation of tobacco-specific compositions of the root bacterial microbiota, which has been established as a consequence of independent evolutionary events in plants and bacteria triggered by different ecological effects. Our findings illustrate mechanistic and evolutionary aspects of the microbiota assembly that are mediated by an arsenal of plant secondary metabolites.

Discovery of a Family of Mixed Lineage Kinase Domain-like Proteins in Plants and Their Role in Innate Immune Signaling.

HeLo domain-containing mixed lineage kinase domain-like protein (MLKL), a pseudokinase, mediates necroptotic cell death in animals. Here, we report the discovery of a conserved protein family across seed plants that structurally resembles vertebrate MLKL. The Arabidopsis genome encodes three MLKLs (AtMLKLs) with overlapping functions in disease resistance mediated by Toll-interleukin 1-receptor domain intracellular immune receptors (TNLs). The HeLo domain of AtMLKLs confers cell death activity but is dispensable for immunity. Cryo-EM structures reveal a tetrameric configuration, in which the HeLo domain is buried, suggestive of an auto-repressed complex. The mobility of AtMLKL1 along microtubules is reduced by chitin, a fungal immunity-triggering molecule. An AtMLKL1 phosphomimetic variant exhibiting reduced mobility enhances immunity. Coupled with the predicted presence of HeLo domains in plant helper NLRs, our data reveal the importance of HeLo domain proteins for TNL-dependent immunity and argue for a cell death-independent immune mechanism mediated by MLKLs.

Low Temperature Storage Stimulates Fruit Softening and Sugar Accumulation Without Ethylene and Aroma Volatile Production in Kiwifruit.

Fruit ripening in response to propylene (an ethylene analog), 1-methylcyclopropene (1-MCP, an ethylene action inhibitor), and low temperature (5°C) treatments was characterized in "Kosui" kiwifruit (Actinidia rufa × A. chinensis). Propylene treatment induced ethylene production, with increased expression levels of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase 1 (AcACS1) and ACC oxidase 2 (AcACO2), and rapid fruit softening together with increased expression levels of polygalacturonase (AcPG) and expansin 1 (AcEXP1) within 5 days (d). Fruit soluble solids concentration (SSC) and contents of sucrose, glucose, and fructose together with the expression levels of ß-amylase 1 (Acß-AMY1), Acß-AMY2, and invertase (AcINV3-1) increased rapidly after 5 d exposure to propylene. Furthermore, propylene exposure for 5 d was sufficient to induce the production of key aroma volatile compounds, ethyl- and methyl butanoate, accompanied with increased expression levels of alcohol acyl transferase (AcAAT). Application of 1-MCP at the start of the experiment, followed by continuous exposure to propylene, significantly delayed fruit softening, changes in SSC and sugars, and strongly suppressed the production of ethylene, aroma volatiles, and expression of associated genes. During storage, fruit softening, SSC and sugar increase, and increased expression of genes associated with cell wall modification and carbohydrate metabolism were registered without detectable ethylene production; however, these changes occurred faster at 5°C compared to 22°C. Interestingly, ethyl and methyl butanoate as well as AcAAT expression were undetectable in kiwifruit during storage, while they were rescued by post-storage propylene exposure, indicating that the production of aroma volatile compounds is strongly ethylene-dependent. Transcript levels of a NAC-related transcription factor (TF), AcNAC3, increased in response to both propylene and low temperature treatments, while AcNAC5 was exclusively up-regulated by propylene. By contrast, transcript levels of a MADS-box TF, AcMADS2, exclusively increased in response to low temperature. The above findings indicate that kiwifruit ripening is inducible by either ethylene or low temperature signals. However, fruit ripened by low temperature were deficient in ethylene-dependent aroma volatiles, suggesting that ethylene signaling is non-functional during low temperature-modulated ripening in kiwifruit. These data provide further evidence that ethylene-dependent and low temperature-modulated ripening in kiwifruit involve different regulatory mechanisms.

Comparative transcriptome analysis reveals distinct ethylene-independent regulation of ripening in response to low temperature in kiwifruit.

BACKGROUND: Kiwifruit are classified as climacteric since exogenous ethylene (or its analogue propylene) induces rapid ripening accompanied by ethylene production under positive feedback regulation. However, most of the ripening-associated changes (Phase 1 ripening) in kiwifruit during storage and on-vine occur largely in the absence of any detectable ethylene. This ripening behavior is often attributed to basal levels of system I ethylene, although it is suggested to be modulated by low temperature. RESULTS: To elucidate the mechanisms regulating Phase 1 ripening in kiwifruit, a comparative transcriptome analysis using fruit continuously exposed to propylene (at 20 °C), and during storage at 5 °C and 20 °C was conducted. Propylene exposure induced kiwifruit softening, reduction of titratable acidity (TA), increase in soluble solids content (SSC) and ethylene production within 5 days. During storage, softening and reduction of TA occurred faster in fruit at 5 °C compared to 20 °C although no endogenous ethylene production was detected. Transcriptome analysis revealed 3761 ripening-related differentially expressed genes (DEGs), of which 2742 were up-regulated by propylene while 1058 were up-regulated by low temperature. Propylene exclusively up-regulated 2112 DEGs including those associated with ethylene biosynthesis and ripening such as AcACS1, AcACO2, AcPL1, AcXET1, Acß-GAL, AcAAT, AcERF6 and AcNAC7. Similarly, low temperature exclusively up-regulated 467 DEGS including AcACO3, AcPL2, AcPMEi, AcADH, Acß-AMY2, AcGA2ox2, AcNAC5 and AcbZIP2 among others. A considerable number of DEGs such as AcPG, AcEXP1, AcXET2, Acß-AMY1, AcGA2ox1, AcNAC6, AcMADS1 and AcbZIP1 were up-regulated by either propylene or low temperature. Frequent 1-MCP treatments failed to inhibit the accelerated ripening and up-regulation of associated DEGs by low temperature indicating that the changes were independent of ethylene. On-vine kiwifruit ripening proceeded in the absence of any detectable endogenous ethylene production, and coincided with increased expression of low temperature-responsive DEGs as well as the decrease in environmental temperature. CONCLUSIONS: These results indicate that kiwifruit possess both ethylene-dependent and low temperature-modulated ripening mechanisms that are distinct and independent of each other. The current work provides a foundation for elaborating the control of these two ripening mechanisms in kiwifruit.

Glutathione Transferase U13 Functions in Pathogen-Triggered Glucosinolate Metabolism.

Glutathione (GSH) and indole glucosinolates (IGs) exert key functions in the immune system of the model plant Arabidopsis (Arabidopsis thaliana). Appropriate GSH levels are important for execution of both pre- and postinvasive disease resistance mechanisms to invasive pathogens, whereas an intact PENETRATION2 (PEN2)-pathway for IG metabolism is essential for preinvasive resistance in this species. Earlier indirect evidence suggested that the latter pathway involves conjugation of GSH with unstable products of IG metabolism and further processing of the resulting adducts to biologically active molecules. Here we describe the identification of Glutathione-S-Transferase class-tau member 13 (GSTU13) as an indispensable component of the PEN2 immune pathway for IG metabolism. gstu13 mutant plants are defective in the pathogen-triggered biosynthesis of end products of the PEN2 pathway, including 4-O-ß-d-glucosyl-indol-3-yl formamide, indole-3-ylmethyl amine, and raphanusamic acid. In line with this metabolic defect, lack of functional GSTU13 results in enhanced disease susceptibility toward several fungal pathogens including Erysiphe pisi, Colletotrichum gloeosporioides, and Plectosphaerella cucumerina Seedlings of gstu13 plants fail also to deposit the (1,3)-ß-glucan cell wall polymer, callose, after recognition of the bacterial flg22 epitope. We show that GSTU13 mediates specifically the role of GSH in IG metabolism without noticeable impact on other immune functions of this tripeptide. We postulate that GSTU13 connects GSH with the pathogen-triggered PEN2 pathway for IG metabolism to deliver metabolites that may have numerous functions in the innate immune system of Arabidopsis.

Two Members of the Aluminum-Activated Malate Transporter Family, SlALMT4 and SlALMT5, are Expressed during Fruit Development, and the Overexpression of SlALMT5 Alters Organic Acid Contents in Seeds in Tomato (Solanum lycopersicum).

The aluminum-activated malate transporter (ALMT) family of proteins transports malate and/or inorganic anions across plant membranes. To demonstrate the possible role of ALMT genes in tomato fruit development, we focused on SlALMT4 and SlALMT5, the two major genes expressed during fruit development. Predicted proteins were classified into clade 2 of the family, many members of which localize to endomembranes. Tissue-specific gene expression was determined using transgenic tomato expressing the ß-glucuronidase reporter gene controlled by their own promoters. Both the genes were expressed in vascular bundles connecting to developing seeds in fruit and in the embryo of mature seeds. Further, SlALMT5 was expressed in embryo in developing seeds in fruit. Subcellular localization of both proteins to the endoplasmic reticulum (ER) was established by transiently expressing the green fluorescent protein fusions in plant protoplasts. SlALMT5 probably localized to other endomembranes as well. Localization of SlALMT5 to the ER was also confirmed by immunoblot analysis. The transport function of both SlALMT proteins was investigated electrophysiologically in Xenopus oocytes. SlALMT5 transported malate and inorganic anions such as nitrate and chloride, but not citrate. SlALMT4 also transported malate, but the results were less consistent perhaps because it did not localize strongly to the plasma membrane. To elucidate the physiological role of SlALMT5 further, we overexpressed SlALMT5 in tomato. Compared with the wild type, overexpressors exhibited higher malate and citrate contents in mature seeds, but not in fruit. We conclude that the malate transport function of SlALMT5 expressed in developing fruit influences the organic acid contents in mature seeds.

Low-temperature-modulated fruit ripening is independent of ethylene in 'Sanuki Gold' kiwifruit.

Fruit ripening in response to treatments with propylene, 1-methycyclopropene (1-MCP), and low temperature was characterized in 'Sanuki Gold' kiwifruit, Actinidia chinensis Planch. Propylene treatment immediately induced rapid fruit softening, increased AC-PG (polygalacturonase) and AC-EXP (expansin) mRNA accumulation, and stimulated an increase in the soluble solid concentration (SSC) and a decrease in titratable acidity (TA). After 3 d exposure to propylene, ethylene production and AC-PL (pectate lyase) mRNA accumulation were observed. 1-MCP treatment after 24 h exposure to propylene eliminated AC-PG mRNA accumulation and suppressed continued changes in SSC and TA. Application of 1-MCP at the start of the treatment, followed by continuous propylene exposure, markedly delayed fruit softening, and the expression of the cell wall-modifying genes, and changes in the SSC and TA, indicating that kiwifruit become insensitive to ethylene at least for 3 d following 1-MCP exposure. Surprisingly, significant fruit softening, mRNA accumulation of AC-PG, AC-PL, and AC-EXP, and decreased TA were observed without ethylene production in intact fruit stored at low temperature for 1 month, but not in fruit stored at room temperature. Repeated 1-MCP treatments (twice a week) failed to inhibit the changes that occurred in low temperature storage. These observations indicate that low temperature modulates the ripening of kiwifruit in an ethylene-independent manner, suggesting that kiwifruit ripening is inducible by either ethylene or low temperature signals.

Molecular characterization of plant acidic alpha-mannosidase, a member of glycosylhydrolase family 38, involved in the turnover of N-glycans during tomato fruit ripening.

It has been reported that acidic α-mannosidase activity increases during tomato fruit ripening, suggesting the turnover of N-glycoproteins is deeply associated with fruit ripening. As part of a study to reveal the relationship between the plant α-mannosidase activity and fruit maturation at the molecular level, we have already purified and characterized an α-mannosidase from tomato fruit (Hossain et al., Biosci. Biotechnol. Biochem. 2009;73:140-146). In this article, we describe the identification and expression of the tomato acidic α-mannosidase gene using the yeast-expression system. The α-mannosidase-gene located at chomosome 6 is a 10 kb spanned containing 30 exons. The gene-encoded-protein is single polypeptide chain of 1,028 amino acids containing glycosyl hydrolase domain-38 with predicted molecular mass of 116 kDa. The recombinant enzyme showed maximum activity at pH 5.5, and was almost completely inhibited by both of 1-deoxymannojirimycin and swainsonine. The recombinant α-mannosidase, like the native enzyme, could cleave α1-2, 1-3 and 1-6 mannosidic linkage from both high-mannose and truncated complex-type N-glycans. A molecular 3D modelling shows that catalytically important residues of animal lysosomal α-mannosidase could be superimposed on those of tomato α-mannosidase, suggesting that active site conformation is highly conserved between plant acidic α-mannosidase and animal lysosomal α-mannosidase.

Molecular identification and characterization of an acidic peptide:N-glycanase from tomato (Lycopersicum esculentum) fruits.

Plant acidic peptide:N-glycanase (PNGase) is one of the deglycosylation enzymes and has been considered to be involved in the catabolism of glycoproteins in plant cells. However, the tangible physiological significance involved in plant differentiation or growth is yet unclear. In this study, as a first step to elucidate the physiological role of free N-glycans and the de-N-glycosylation machinery working in developing plant cells, we have succeeded in expressing a cDNA from tomato fruits in Pichia pastoris and identified an acidic peptide:N-glycanase in the culture supernatant. The PNGase-gene-encoded protein is a single polypeptide chain of 588 amino acids with a predicted molecular mass of 65.8 kDa. The deduced amino acid sequence showed 57.9% similarity with almond PNGase A. The recombinant tomato PNGase showed optimum activity at pH 4.5 and 40 degrees C. It did not require any metal ions for full enzymatic activity and could release the complex-type N-glycan from glycopeptides. Our phylogenetic analysis reveals that the plant acidic PNGase is completely different from the ubiquitous cytosolic PNGase and is involved in a different de-N-glycosylation mechanism associated with plant growth and development.

Ripening-associated ethylene biosynthesis in tomato fruit is autocatalytically and developmentally regulated.

To investigate the regulatory mechanism(s) of ethylene biosynthesis in fruit, transgenic tomatoes with all known LeEIL genes suppressed were produced by RNA interference engineering. The transgenic tomato exhibited ethylene insensitivity phenotypes such as non-ripening and the lack of the triple response and petiole epinasty of seedlings even in the presence of exogenous ethylene. Transgenic fruit exhibited a low but consistent increase in ethylene production beyond 40 days after anthesis (DAA), with limited LeACS2 and LeACS4 expression. 1-Methylcyclopropene (1-MCP), a potent inhibitor of ethylene perception, failed to inhibit the limited increase in ethylene production and expression of the two 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) genes in the transgenic fruit. These results suggest that ripening-associated ethylene (system 2) in wild-type tomato fruit consists of two parts: a small part regulated by a developmental factor through the ethylene-independent expression of LeACS2 and LeACS4 and a large part regulated by an autocatalytic system due to the ethylene-dependent expression of the same genes. The results further suggest that basal ethylene (system 1) is less likely to be involved in the transition to system 2. Even if the effect of system 1 ethylene is eliminated, fruit can show a small increase in ethylene production due to unknown developmental factors. This increase would be enough for the stimulation of autocatalytic ethylene production, leading to fruit ripening.

Ethylene regulation of fruit softening and cell wall disassembly in Charentais melon.

Cell wall disassembly in ripening fruit is highly complex, involving the dismantling of multiple polysaccharide networks by diverse families of wall-modifying proteins. While it has been reported in several species that multiple members of each such family are expressed in the same fruit tissue, it is not clear whether this reflects functional redundancy, with protein isozymes from a single enzyme class performing similar roles and contributing equally to wall degradation, or whether they have discrete functions, with some isoforms playing a predominant role. Experiments reported here sought to distinguish between cell wall-related processes in ripening melon that were softening-associated and softening-independent. Cell wall polysaccharide depolymerization and the expression of wall metabolism-related genes were examined in transgenic melon (Cucumis melo var. cantalupensis Naud.) fruit with suppressed expression of the 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene and fruits treated with ethylene and 1-methylcyclopropene (1-MCP). Softening was completely inhibited in the transgenic fruit but was restored by treatment with exogenous ethylene. Moreover, post-harvest application of 1-MCP after the onset of ripening completely halted subsequent softening, suggesting that melon fruit softening is ethylene-dependent. Size exclusion chromatography of cell wall polysaccharides, from the transgenic fruits, with or without exogenous ethylene, indicated that the depolymerization of both pectins and xyloglucans was also ethylene dependent. However, northern analyses of a diverse range of cell wall-related genes, including those for polygalacturonases, xyloglucan endotransglucosylase/hydrolases, expansin, and beta-galactosidases, identified specific genes within single families that could be categorized as ethylene-dependent, ethylene-independent, or partially ethylene-dependent. These results support the hypothesis that while individual cell wall-modifying proteins from each family contribute to cell wall disassembly that accompanies fruit softening, other closely related family members are regulated in an ethylene-independent manner and apparently do not directly participate in fruit softening.

Differential feedback regulation of ethylene biosynthesis in pulp and peel tissues of banana fruit.

The feedback regulation of ethylene biosynthesis in banana [Musa sp. (AAA group, Cavendish subgroup) cv. Grand Nain] fruit was investigated in an attempt to clarify the opposite effect of 1-methylcyclopropene (1-MCP), an ethylene action inhibitor, before and after the onset of ripening. 1-MCP pre-treatment completely prevented the ripening-induced effect of propylene in pre-climacteric banana fruit, whereas treatment after the onset of ripening stimulated ethylene production. In pre-climacteric fruit, higher concentrations of propylene suppressed ethylene production more strongly, despite their earlier ethylene-inducing effect. Exposure of the fruit ripened by propylene to 1-MCP increased ethylene production concomitantly with an increase in 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and ACC content, and prevented a transient decrease in MA-ACS1 transcripts in the pulp tissues. In contrast, in the peel of ripening fruit, 1-MCP prevented the increase in ethylene production and subsequently the ripening process by reduction of the increase in MA-ACS1 and MA-ACO1 transcripts and of ACC synthase and ACC oxidase activities. These results suggest that ethylene biosynthesis in ripening banana fruit may be controlled negatively in the pulp tissue and positively in the peel tissue. This differential regulation by ethylene in pulp and peel tissues was also observed for MA-PL, MA-Exp, and MA-MADS genes.

Ethylene biosynthesis in detached young persimmon fruit is initiated in calyx and modulated by water loss from the fruit.

Persimmon (Diospyros kaki Thunb.) fruit are usually classified as climacteric fruit; however, unlike typical climacteric fruits, persimmon fruit exhibit a unique characteristic in that the younger the stage of fruit detached, the greater the level of ethylene produced. To investigate ethylene induction mechanisms in detached young persimmon fruit, we cloned three cDNAs encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (DK-ACS1, 2, and -3) and two encoding ACC oxidase (DK-ACO1 and -2) genes involved in ethylene biosynthesis, and we analyzed their expression in various fruit tissues. Ethylene production was induced within a few days of detachment in all fruit tissues tested, accompanied by temporally and spatially coordinated expression of all the DK-ACS and DK-ACO genes. In all tissues except the calyx, treatment with 1-methylcyclopropene, an inhibitor of ethylene action, suppressed ethylene production and ethylene biosynthesis-related gene expression. In the calyx, one ACC synthase gene (DK-ACS2) exhibited increased mRNA accumulation accompanied by a large quantity of ethylene production, and treatment of the fruit with 1-methylcyclopropene did not prevent either the accumulation of DK-ACS2 transcripts or ethylene induction. Furthermore, the alleviation of water loss from the fruit significantly delayed the onset of ethylene production and the expression of DK-ACS2 in the calyx. These results indicate that ethylene biosynthesis in detached young persimmon fruit is initially induced in calyx and is modulated by water loss through transcriptional activation of DK-ACS2. The ethylene produced in the calyx subsequently diffuses to other fruit tissues and acts as a secondary signal that stimulates autocatalytic ethylene biosynthesis in these tissues, leading to a burst of ethylene production.

Ethylene is required for both the initiation and progression of softening in pear (Pyrus communis L.) fruit.

In order to investigate the physiological role of ethylene in the initiation and subsequent progression of softening, pear fruit were treated with propylene, an analogue of ethylene or 1-methylcyclopropene (1-MCP), a gaseous inhibitor of ethylene action at the preclimacteric or ripening stages. The propylene treatment at the pre-ripe stage stimulated ethylene production and flesh softening while the 1-MCP treatment at the same stage markedly retarded the initiation of the ripening-related events. Moreover, 1-MCP treatment after the initiation of ripening markedly suppressed the subsequent flesh softening and ethylene production. These results clearly indicate that ethylene is not merely a by-product, but plays a crucial role in both the initiation and maintenance of regulating the softening process during ripening. The observations also suggest that ethylene in ripening is regulated entirely in an autocatalytic manner. The mRNA accumulation of pear polygalacturonases (PG) genes, PC-PG1 and PC-PG2, was in parallel with the pattern of fruit softening in both propylene and 1-MCP treatments. However, the expression pattern of pear endo-1,4-beta-D-glucanases (EGase) genes, PC-EG1 and PC-EG2, was not affected in both treatments. The results suggest that ethylene is required for PGs expression even in the late ripening stage, but not for EGases.