Expression and regulation of pear 1-aminocyclopropane-1-carboxylic acid synthase gene (PpACS1a) during fruit ripening,under salicylic acid and indole-3-acetic acid treatment,and in diseased fruit

In plants, the level of ethylene is determined by the activity of the key enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). A gene encoding an ACC synthase protein was isolated from pear (Pyrus pyrifolia). This gene designated PpACS1a (GenBank accession no. KC632526) was 1488 bp in length with an open reading frame (ORF) encoding a protein of 495 amino acids that shared high similarity with other pear ACC synthase proteins. The PpACS1a was grouped into type-1 subfamily of plant ACS based on its conserved domain and phylogenetic status. Real-time quantitative PCR indicated that PpACS1a was differentially expressed in pear tissues and predominantly expressed in anthers. The expression signal of PpACS1a was also detected in fruit and leaves, but no signal was detected in shoots and petals. Furthermore, the PpACS1a expression was regulated during fruit ripening. In addition, the PpACS1a gene expression was regulated by salicylic acid (SA) and indole-3-acetic acid (IAA) in fruit. Moreover, the expression of the PpACS1a was up-regulated in diseased pear fruit. These results indicated that PpACS1a might be involved in fruit ripening and response to SA, IAA and disease.     

Expression and regulation of the ethylene receptor PpERS gene during pear fruit development and following salicylic acid treatment

A gene encoding an ethylene receptor protein was isolated from pear (Pyrus pyrifolia). This gene, designated PpERS (GenBank accession No. KC517482), was 1,918 bp in length with an open reading frame encoding a protein of 638 amino acids that shared high similarity with another pear ethylene receptor protein PpERS1, and two apple ethylene receptor proteins MdERS and MdERS1. The PpERS was grouped into the ETR1 subfamily of ethylene receptor based on its conserved domain and phylogenetic status. The PpERS gene contained five exons interrupted by four introns. Quantitative RT-PCR indicated that PpERS was differentially expressed in pear tissues and predominantly expressed in petals, shoots, anthers, and 160 days after full bloom fruit. The PpERS expression was regulated during fruit development. In addition, the PpERS gene expression was regulated by salicylic acid (SA) and ethylene in fruit. The results indicated that PpERS might participate in ethylene and SA signaling transduction during pear fruit development.     

Identification of 1-aminocyclopropane-1-carboxylic acid synthase genes controlling the ethylene level of ripening fruit in Japanese pear (Pyrus pyrifolia Nakai)

The shelf life of Japanese pear fruit is determined by its level of ethylene production. Relatively high levels of ethylene reduce storage potential and fruit quality. We have identified RFLP markers tightly linked to the locus that determines the rate of ethylene evolution in ripening fruit of the Japanese pear. The study was carried out using sequences of two types of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase genes (PPACS1 and pPPACS2) and a ACC oxidase gene (PPAOX1) as probes on 35 Japanese pear cultivars expressing different levels of ethylene (0.0∼300 μl/kg fresh weight/h) in ripening fruit. When total DNA was digested with HindIII and probed with pPPACS1, we identified a band of 2.8 kb which was specific to cultivars having very high ethylene levels (≧10 μ1/kg f.w./h) during fruit ripening. The probe pPPACS2 identified a band of 0.8 kb specific to cultivars with moderate ethylene levels (0.5 μl/kg f.w./h–10 μl/kg f.w./h) during fruit ripening. The cultivars that produce high levels of ethylene possess at least one additional copy of pPPACS1 and those producing moderate levels of ethylene have at least one additional copy of pPPACS2. These results suggest that RFLP analysis with different ACC synthase genes could be useful for predicting the maximum ethylene level during fruit ripening in Japanese pear. Received: 1 July 1998 / Accepted: 6 October 1998     

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.     

Induction of defense responses against Alternaria rot by different elicitors in harvested pear fruit

Pear fruit (Pyrus pyrifolia L. cv. Yali) treated by different elicitors, such as salicylic acid (SA), oxalic acid, calcium chloride, and antagonistic yeast Cryptococcus laurentii, were investigated to determine the induction of defense responses. The possible mechanism by which elicitors induced the resistance of pear fruit against postharvest disease was also evaluated. The results indicated that all the elicitors could significantly enhance defense-related enzyme activities, such as β-1,3-glucanase, phenylalanine ammonia lyase, peroxidase, and polyphenol oxidase activity, and reduce the disease incidence caused by Alternaria alternata in pear fruit (P=0.05). Among these different elicitors, SA treatment showed the best result in inducing the defense responses and reducing the decay in pear fruit.     

砂梨脂氧合酶cDNA片段克隆与RNAi载体构建

以砂梨‘若光’的成熟果实为材料,根据脂氧合酶氨基酸保守区设计1对简并引物,采用RT-PCR克隆到1段长827 bp的序列,经Blast比对和DNAstar软件聚类分析,结果表明由该序列推导出的氨基酸序列含有脂氧合酶氨基酸保守结构域,与马铃薯脂氧合酶基因的氨基酸序列相似性达到77.5%,判断其为砂梨脂氧合酶基因片段,命名为LOX1,并将序列登录到GenBank,登录号为EF215448。根据RNAi载体的构建原则,选择LOX1一开放阅读框设计携带酶切位点的特异引物,通过PCR扩增正、反向基因片段,再与YYT间隔区串连,插入植物表达载体pYF7713中的相应位置,成功地构建了干扰LOX基因表达的RNAi的植物双元表达载体pYL028,为深入研究该基因在砂梨果实成熟过程中的功能及耐贮藏基因工程育种奠定了基础。     

Rapid identification of 1-aminocyclopropane-1-carboxylate (ACC) synthase genotypes in cultivars of Japanese pear (Pyrus pyrifolia Nakai) using CAPS markers

In Japanese pear (Pyrus pyrifolia Nakai), fruit storage potential is closely related to the amount of ethylene produced. We have developed a rapid and accurate method for analyzing genes involved in high ethylene production during fruit ripening in Japanese pear. This involves cleaved-amplified polymorphic sequences (CAPS) of two 1-aminocyclopropane-1-carboxylate (ACC) synthase genes (PPACS1 and PPACS2). Two CAPS markers (A for PPACS1 and B for PPACS2), associated with the amount of ethylene produced, were identified. Marker A was associated with high ethylene producers and marker B with moderate ethylene producers. The absence of these two markers enabled the identification of low ethylene producers. Using these markers, we have identified ethylene genotypes for 40 Japanese pear cultivars and two Chinese pear (P. bretschneideri) cultivars that are commercially important and used in breeding programs. Furthermore, we performed linkage analysis of these two genes in the F(2) population, which revealed that the recombination frequency between the two markers was 20.8 +/- 3.6%. This information is critical to the selection of parents and in breeding strategies to improve storage ability of Japanese pears.     

A tomato HD-Zip homeobox protein, LeHB-1, plays an important role in floral organogenesis and ripening

Ethylene is required for climacteric fruit ripening. Inhibition of ethylene biosynthesis genes, 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase, prevents or delays ripening, but it is not known how these genes are modulated during normal development. LeHB-1, a previously uncharacterized tomato homeobox protein, was shown by gel retardation assay to interact with the promoter of LeACO1 , an ACC oxidase gene expressed during ripening. Inhibition of LeHB-1 mRNA accumulation in tomato fruit, using virus-induced gene silencing, greatly reduced LeACO1 mRNA levels, and inhibited ripening. Conversely, ectopic overexpression of LeHB-1 by viral delivery to developing flowers elsewhere on injected plants triggered altered floral organ morphology, including production of multiple flowers within one sepal whorl, fusion of sepals and petals, and conversion of sepals into carpel-like structures that grew into fruits and ripened. Our findings suggest that LeHB-1 is not only involved in the control of ripening but also plays a critical role in floral organogenesis.