The involvement of 1-aminocyclopropane-1-carboxylic acid synthase isogene, Pp-ACS1, in peach fruit softening

Ethylene promotes fruit ripening, including softening. The fruit of melting-flesh peach (Prunus persica (L). Batsch) cultivar 'Akatsuki' produces increasing levels of ethylene, and the flesh firmness softens rapidly during the ripening stage. On the other hand, the fruit of stony hard peach cultivars 'Yumyeong', 'Odoroki', and 'Manami' does not soften and produces little ethylene during fruit ripening and storage. To clarify the mechanism of suppression of ethylene production in stony hard peaches, the expression patterns of four ethylene biosynthesis enzymes were examined: ACC synthases (Pp-ACS1, Pp-ACS2, and Pp-ACS3) and ACC oxidase (Pp-ACO1). In the melting-flesh cultivar 'Akatsuki', Pp-ACS1 mRNA was dramatically induced after harvesting, and a large amount of ethylene was produced. On the other hand, in stony hard peaches, Pp-ACS1 mRNA was not induced during the ripening stage, and ethylene production was inhibited. Since Pp-ACS1 mRNA was induced normally in senescing flowers, wounded leaves, and wounded immature fruit of 'Yumyeong', Pp-ACS1 was suppressed only at the ripening stage, and was not a defect in Pp-ACS1. These results indicate that the suppression of fruit softening in stony hard peach cultivars was caused by a low level of ethylene production, which depends on the suppressed expression of Pp-ACS1.     

桃ACC合酶基因的分离及其差异表达

利用5′/3′RACE PCR技术,从桃(Prunus persica (L.) Batsch)果实中克隆了植物乙烯生物合成的关键酶--ACC合酶的全长cDNA pacs,对pacs基因进行全序列测定表明,该基因全长1 848个碱基,编码区为1 449个碱基,5′端有177个碱基的非编码区序列,3′端有219个碱基的非编码区序列(不包括终止密码子TAA).pacs基因编码区共编码483个氨基酸,蛋白质大小为54 kD,等电点为6.43.pacs与番茄(S19677)、梅(AB031026)、番木瓜(U68216)、苹果(AB034993)等其他植物ACC合酶cDNA氨基酸序列同源性分别为65%、70%、75%、90%,并存在与这些ACC合酶氨基酸的活性位点保守序列SLSKDMGFPGFR.RT-PCR结合杂交分析表明,pacs和我们以前克隆的桃ACC合酶cDNA pacs12(AF467782)在叶片和花中基因表达模式基本一致,伤处理和IAA均能诱导叶片pacs 和pacs12基因的表达,但pacs在伤处理叶片的表达水平比pacs12高;pacs 和pacs12基因在果实表达有所不同,pacs在绿熟和成熟果实中均有表达,而pacs12在绿熟果实中基本检测不到,在成熟果实中才有表达,两者在果实中的表达水平比伤处理和IAA处理叶片和花中要低.     

Identification of an ETR1-homologue from mango fruit expressing during fruit ripening and wounding

We have isolated a mango (Mangifera indica L.) cDNA homologue of the ethylene receptor gene ETR-1, referred to as METR1, which codes for a polypeptide of 802 amino acids with a predicted molecular weight of 89 kDa. The amino acid sequence is highly homologous (over 80 percnt;) to ETRs from other fruits. Genomic Southern blot analysis indicates that two or more ETR homologues exist in mango. RNA blot analysis revealed that the level of METR1 mRNA in the mesocarp increased during fruit ripening. In addition, it was found that the METR1 mRNA increases transiently during wounding of the tissue. This is the first report of an ETR homologue showing an induction during fruit ripening and wounding.     

Ethylene receptor expression is regulated during fruit ripening, flower senescence and abscission

Using theArabidopsis ethylene receptorETR1 as a probe, we have isolated a tomato homologue (tETR) from a ripening cDNA library. The predicted amino acid sequence is 70% identical toETR1 and homologous to a variety of bacterial two component response regulators over the histidine kinase domain. Sequencing of four separate cDNAs indicates that tETR lacks the carboxyl terminal response domain and is identical to that encoded by the tomatoNever ripe gene. Ribonuclease protection showed tETR mRNA was undetectable in unripe fruit or pre-senescent flowers, increased in abundance during the early stages of ripening, flower senescence, and in abscission zones, and was greatly reduced in fruit of ripening mutants deficient in ethylene synthesis or response. These results suggest that changes in ethylene sensitivity are mediated by modulation of receptor levels during development.     

Characterization of ripening-regulated cDNAs and their expression in ethylene-suppressed charentais melon fruit

Charentais melons (Cucumis melo cv Reticulatus) are climacteric and undergo extremely rapid ripening. Sixteen cDNAs corresponding to mRNAs whose abundance is ripening regulated were isolated to characterize the changes in gene expression that accompany this very rapid ripening process. Sequence comparisons indicated that eight of these cDNA clones encoded proteins that have been previously characterized, with one corresponding to ACC (1-aminocyclopropane-1-carboxylic acid) oxidase, three to proteins associated with pathogen responses, two to proteins involved in sulfur amino acid biosynthesis, and two having significant homology to a seed storage protein or a yeast secretory protein. The remaining eight cDNA sequences did not reveal significant sequence similarities to previously characterized proteins. The majority of the 16 ripening-regulated cDNAs corresponded to mRNAs that were fruit specific, although three were expressed at low levels in vegetative tissues. When examined in transgenic antisense ACC oxidase melon fruit, three distinct patterns of mRNA accumulation were observed. One group of cDNAs corresponded to mRNAs whose abundance was reduced in transgenic fruit but inducible by ethylene treatment, indicating that these genes are directly regulated by ethylene. A second group of mRNAs was not significantly altered in the transgenic fruit and was unaffected by treatment with ethylene, indicating that these genes are regulated by ethylene-independent developmental cues. The third and largest group of cDNAs showed an unexpected pattern of expression, with levels of mRNA reduced in transgenic fruit and remaining low after exposure to ethylene. Regulation of this third group of genes thus appears to ethylene independent, but may be regulated by developmental cues that require ethylene at a certain stage in fruit development. The results confirm that both ethylene-dependent and ethylene-independent pathways of gene regulation coexist in climacteric fruit.     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Structure and expression of an ethylene-related mRNA from tomato.

Messenger RNAs homologous to a cDNA clone (pTOM 13) derived from a ripe-tomato-specific cDNA library are expressed during tomato fruit ripening and after the wounding of leaf and green fruit material. Both responses involve the synthesis of the hormone ethylene. Accumulation of the pTOM 13--homologous RNA during ripening is rapid and sustained, and reaches its maximum level in orange fruit. Following mechanical wounding of tomato leaves a pTOM 13--homologous RNA shows rapid induction within 30 minutes, which occurs before maximal ethylene evolution (2-3 h). This RNA also accumulates following the wounding of green tomato fruit. Northern blot analysis of poly(A)+ RNA indicates that the length of the mRNA is about 1400 nucleotides. Nucleotide sequence analysis showed the cDNA insert to contain the complete coding region of the pTOM 13 protein (33.5 kD) and an unusual 5' structure of ten dT-nucleotides. Hybridisation of the pTOM 13 cDNA insert to Southern blots of tomato DNA indicates the presence of only a small number of homologous sequences in the tomato genome.     

Expression of ACC synthase is enhanced earlier than that of ACC oxidase during fruit ripening of mume (Prunus mume)

Mume (Japanese apricot: Prunus mume Sieb. et Zucc.) is a climacteric fruit that produces large amounts of ethylene as it ripens. Ripening is accompanied by marked increases in the activities of two ethylene-biosynthetic enzymes, namely, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. To study the molecular aspects of ripening of mume, we isolated cDNA clones for proteins that we considered likely to be involved in the biosynthesis and perception of ethylene during ripening, namely, ACC synthase, ACC oxidase and the ethylene receptor. Northern blotting analysis revealed the markedly increased expression of ACC synthase prior to that of ACC oxidase and the increase in ethylene production during ripening. Overall, the levels of the mRNAs for the genes corresponded closely to the levels of activity of the ethylene-biosynthetic enzymes. Exposure of mature green mume fruit to ethylene for 12 h induced strong expression of ACC synthase, as well as of ACC oxidase. Wounding of the pericarp of mume fruit induced the expression of ACC synthase but not of ACC oxidase. The rate of ethylene production increased only slightly after wounding. These results suggest that expression of the genes for ACC synthase and ACC oxidase must be activated sequentially for maximum production of ethylene during ripening of mume fruit and that several mechanisms regulate the expression of ethylene-biosynthetic genes during ripening.     

猕猴桃果实乙烯受体基因Ad-ETR1的克隆及其表达

以"布鲁诺"美味猕猴桃(Actinidia deliciosa cv.Bruno)果实为材料,根据其它植物乙烯受体氨基酸保守区序列,设计简并引物,通过RT-PCR扩增出1个657bp大小的cDNA片段(Ad-ETR1)该片段编码219个氨基酸,与其它植物乙烯受体及其基因的氨基酸及核苷酸同源性在72%～90%之间.Northern杂交结果表明,猕猴桃果实成熟衰老进程中Ad-ETR1 mRNA的积累趋于增加.这种积累的最大值出现在乙烯进入跃变之后;乙烯处理可以促使Ad-ETR1 mRNA最大值提前出现,乙酰水杨酸(ASA)处理则显著抑制Ad-ETR1表达.     

Differential regulation of the tomato ETR gene family throughout plant development

Ethylene perception in plants is co-ordinated by multiple hormone receptor candidates sharing sequence commonalties with prokaryotic environmental sensor proteins known as two-component regulators. Two tomato homologs of the Arabidopsis ethylene receptor ETR1 were cloned from a root cDNA library. Both cDNAs, termed LeETR1 and LeETR2, were highly homologous to ETR1, exhibiting ～ 90% deduced amino acid sequence similarity and 80% deduced amino acid sequence identity. LeETR1 and LeETR2 contained all the major structural elements of two-component regulators, including the response regulator motif absent in LeETR3, the gene encoding tomato NEVER RIPE (NR). Using RNase protection analysis, the mRNAs of LeETR1, LeETR2 and NR were quantified in tissues engaged in key processes of the plant life cycle, including seed germination, shoot elongation, leaf and flower senescence, floral abscission, fruit set and fruit ripening. LeETR1 was expressed constitutively in all plant tissues examined. LeETR2 mRNA was expressed at low levels throughout the plant but was induced in imbibing tomato seeds prior to germination and was down-regulated in elongating seedlings and senescing leaf petioles. NR expression was developmentally regulated in floral ovaries and ripening fruit. Notably, hormonal regulation of NR was highly tissue-specific. Ethylene biosynthesis induced NR mRNA accumulation in ripening fruit but not in elongating seedlings or in senescing leaves or flowers. Furthermore, the abundance of mRNAs for all three LeETR genes remained uniform in multiple plant tissues experiencing marked changes in ethylene sensitivity, including the cell separation layer throughout tomato flower abscission.     

Novel, Starch-Like Polysaccharides Are Synthesized by an Unbound Form of Granule-Bound Starch Synthase in Glycogen-Accumulating Mutants of Chlamydomonas reinhardtii

We isolated two muskmelon (Cucumis melo) cDNA homologs of the Arabidopsis ethylene receptor genes ETR1 and ERS1 and designated them Cm-ETR1 (C. melo ETR1; accession no. AF054806) and Cm-ERS1 (C. melo ERS1; accession no. AF037368), respectively. Northern analysis revealed that the level of Cm-ERS1 mRNA in the pericarp increased in parallel with the increase in fruit size and then markedly decreased at the end of enlargement. In fully enlarged fruit the level of Cm-ERS1 mRNA was low in all tissues, whereas that of Cm-ETR1 mRNA was very high in the seeds and placenta. During ripening Cm-ERS1 mRNA increased slightly in the pericarp of fruit before the marked increase of Cm-ETR1 mRNA paralleled climacteric ethylene production. These results indicate that both Cm-ETR1 and Cm-ERS1 play specific roles not only in ripening but also in the early development of melon fruit and that they have distinct roles in particular fruit tissues at particular developmental stages.     

Characterization of expression, and cloning, of beta-D-xylosidase and alpha-L-arabinofuranosidase in developing and ripening tomato (Lycopersicon esculentum Mill.) fruit

Modifications to the cell wall of developing and ripening tomato fruit are mediated by cell wall-degrading enzymes, including a beta-d-xylosidase or alpha-l-arabinofuranosidase, which participate in the breakdown of xylans and/or arabinoxylans. The activity of both enzymes was highest during early fruit growth, before decreasing during later development and ripening. Two beta-d-xylosidase cDNAs, designated LeXYL1 and LeXYL2, and an alpha-l-arabinofuranosidase cDNA, designated LeARF1, were obtained. Accumulation of mRNAs for beta-d-xylosidase and alpha-l-arabinofuranosidase was examined during fruit development and ripening. LeARF1 and LeXYL2 genes were relatively highly expressed during fruit development and decreased after the onset of ripening. By contrast, LeXYL1 was not expressed during fruit development, but was expressed later, particularly during over-ripening. The expression of all three genes was also followed in ripening-impaired mutants, Nr, Nr2, nor, and rin of cv. Ailsa Craig fruit. LeXYL2 mRNA was detected in the ripe fruits of all the mutants and its abundance was similar to that in mature green wild-type fruit. By contrast, LEXYL1 mRNA was expressed only in the ripe fruits of the Nr mutant, suggesting that the two beta-d-xylosidase genes are subject to distinct regulatory control during fruit development and ripening. LeARF1 mRNA was detected in ripe fruits of Nr2, nor and rin, and not in ripe fruit of the Nr mutant. The accumulation of LeARF1 in ripe fruit was restored by 1-methylcyclopropene (1-MCP), an inhibitor of ethylene action, while 1-MCP had no effect on the expression of LeXYL1 or LeXYL2. This suggests that LeARF1 expression is subject to negative regulation by ethylene and that the two beta-d-xylosidase genes are independent of ethylene action.