Characterization of Starch Degradation Related Genes in Postharvest Kiwifruit.

Starch is one of the most important storage carbohydrates in plants. Kiwifruit typically accumulate large amounts of starch during development. The fruit retain starch until commercial maturity, and its postharvest degradation is essential for consumer acceptance. The activity of genes related to starch degradation has, however, rarely been investigated. Based on the kiwifruit genome sequence and previously reported starch degradation-related genes, 17 novel genes were isolated and the relationship between their expression and starch degradation was examined using two sets of materials: ethylene-treated (100 µL/L, 20 °C; ETH) vs. control (20 °C; CK) and controlled atmosphere stored (CA, 5% CO2 + 2% O2, 0 °C) vs. normal atmosphere in cold storage (NA, 0 °C). Physiological analysis indicated that ETH accelerated starch degradation and increased soluble solids content (SSC) and soluble sugars (glucose, fructose and sucrose), while CA inhibited starch reduction compared with NA. Using these materials, expression patterns of 24 genes that may contribute to starch degradation (seven previously reported and 17 newly isolated) were analyzed. Among the 24 genes, AdAMY1, AdAGL3 and AdBAM3.1/3L/9 were significantly induced by ETH and positively correlated with starch degradation. Furthermore, these five genes were also inhibited by CA, conforming the likely involvement of these genes in starch degradation. Thus, the present study has identified the genes with potential for involvement in starch degradation in postharvest kiwifruit, which will be useful for understanding the regulation of kiwifruit starch content and metabolism.

[Pollen viability, stigma receptivity and fruiting characteristics of botanical origin of Jinxianlian].

The viability and life span of pollen were evaluated by TTC (2,3,5-triphenyl tetrazlium chloride) and the peroxidase solution, the stigma receptivity were estimated by benzidine-H2O2 method and the fruiting characteristics were investigated. The results showed that (1) Anoectochilus roxburghii and A. formosanus appeared the same up-and-down trend of the pollen viability, increased and then decreased. The storage temperature and storage time had significant impact on the pollen viability. With the extension of storage time, the pollen activity decreased. 4 degrees C refrigerator storage may be extended the pollen vitality. (2) The stigma had receptivity in 1st day and reached the highest level in the 4th day after blooming. A. roxburghii lost receptivity in the 8th day while A. formosanus lost receptivity in the 10th day after blooming. (3) The different pollination had significant impact on seed setting rate. The seed setting rate of artificial cross-pollination was higher than that of the artificial self-pollination. Collecting pollen in the 3rd day and carrying out artificial cross-pollination in the 4th day after blooming can significantly improve seed setting rate. The results provided technical assurance for A. roxburghii and A. formosanus breeding of new varieties and seed breeding.

Coordinated regulation of anthocyanin biosynthesis in Chinese bayberry (Myrica rubra) fruit by a R2R3 MYB transcription factor.

Chinese bayberry (Myrica rubra) is a fruit crop with cultivars producing fruit ranging from white (Shuijing, SJ) to red (Dongkui, DK) and dark red-purple (Biqi, BQ), as a result of different levels of anthocyanin accumulation. Genes encoding the anthocyanin biosynthesis enzymes chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDPglucose: flavonoid 3-O-glucosyltransferase (UFGT), as well as MrMYB1, a R2R3 MYB transcription factor homologous to known activators of anthocyanin biosynthesis, were isolated from ripe fruit of BQ. Differences in mRNA abundance of MrF3H, MrF3'H, MrDFR1, MrANS and MrUFGT were highly correlated with differential accumulation of anthocyanins between cultivars, suggesting coordinated regulation by transcription factors. The transcript level of MrMYB1 was strongly associated with the anthocyanin content in ripe fruit of the three cultivars, as well as different anthocyanin containing tissues of BQ fruit. Fruit bagging strongly inhibited anthocyanin accumulation in fruit as well as the expression of all anthocyanin biosynthetic genes and MrMYB1. Overexpression of MrMYB1 stimulated both anthocyanin accumulation and activated an Arabidopsis-DFR promoter in tobacco (Nicotiana tabacum). MrMYB1d, an allele with a 1 bp deletion at nucleotide 30 of coding sequence, was observed in SJ and DK fruit, suggesting that a nonsense mutation of the MYB1 protein may be responsible for no or low expression of MYB1 in the white and red fruit. These results show that coordinated expression of multiple biosynthetic genes is involved in anthocyanin accumulation in Chinese bayberry fruit, and this is regulated by MrMYB1.

Characterization of cDNAs associated with lignification and their expression profiles in loquat fruit with different lignin accumulation.

The ripening fruit of two loquat (Eriobotrya japonica Lindl.) cultivars with different levels of lignin accumulation provide an intriguing example of lignification in flesh tissue. Increase in firmness as a result of lignification in ripening red-fleshed Luoyangqing (LYQ) fruit was confirmed, whereas white-fleshed Baisha (BS) fruit softened without lignification. Six cDNAs associated with the lignification pathway, i.e. EjPAL1, EjPAL2 (phenylalanine ammonia lyase, PAL, EC 4.3.1.5), Ej4CL (4-coumarate: coenzyme A ligase, 4CL, EC 6.2.1.12), EjCAD1, EjCAD2 (cinnamyl alcohol dehydrogenase, CAD, EC 1.1.1.195) and EjPOD (peroxidase, POD), were cloned from flesh tissue of LYQ fruit. Expression profiles of the six corresponding genes differed greatly in different tissues, and during fruit development and ripening in both LYQ and BS cultivars. Associated activities of PAL, 4CL, CAD, and POD enzymes were also measured. CAD and POD enzyme activities and the expression of EjCAD1 and EjPOD genes were most closely associated temporally with lignification of loquat flesh tissue. Levels of EjCAD1 transcripts were particularly aligned with changes in lignification during ripening as modified either by ethylene treatment or low temperature conditioning. The two PAL genes showed different expression patterns during fruit development, with EjPAL1 strongly expressed in mature fruit and EjPAL2 only expressed in early stages of development. In addition, EjCAD1 expression was stimulated by low temperature and may contribute to low temperature injury in the fruit. Our integrated data on lignin, monolignol precursors, and associated enzymes and genes, provide a consistent model of fruit lignification.

[Changes in respiratory rate, ethylene production rate and quality of postharvest Chinese bayberry fruits with different maturities].

Harvested fruits of three Chinese bayberry (Myrica rubra Sieb. & Zucc.) varieties, i.e. "Biqi", "Dongkui" and "Zaodamei" which were divided into three maturities (designated as "Immature", "Mature" and "Ripe") according to fruit colour, were investigated for the changes in climacteric pattern and quality at 20 degrees C. Respiration rate and ethylene production rate were underwent 3 h during 48 h storage. Our result showed that both Immature and Mature fruits underwent rises in respiration and ethylene production rate of a climacteric rise, but no such peak was observed in Ripe fruit (Fig.1 and 2). Total soluble solids (TSS) contents increased with maturity and decreased over the 48 h at 20 degrees C (Fig.3); titratable acidity (TA) decreased with the maturity and throughout 48 h storage period (Fig.4). In "Biqi" Chinese bayberry fruit, PAL activities increased in Immature and Mature fruit, but, it decreased in Ripe fruit during the storage period; the change in Cy-3-Glu with fruit ripening was consistent with PAL activities (Table 1); there was significant positive correlation between CIRG (Color Index for Red Grape) values and Cy-3-Glu content (r=0.96**). This study provides important information on the postharvest behaviour of Chinese bayberry fruit, and our result shows that it is climacteric fruit.

[Cloning and expression of ethylene receptor gene Ad-ETR1 from kiwifruit].

According to the conserved amino acid sequence from ethylene receptors in other plants, a pair of degenerate primers was designed and a 657-bp cDNA fragment encoding an ethylene receptor fruit was obtained by RT-PCR from ripening kiwifruit (Actinidia deliciosa cv. Bruno) (Fig. 1). The cDNA fragment encoding 219 amino acids was named Ad-ETR1, and its sequences shared high similarity at both nucleotide and polypeptide level with the sequences from the plants of Arabidopsis, tomato, persimmon, avocado, citrus and peach (Fig. 2, Table 1). Northern blot analysis indicated that the levels of Ad-ETR1 mRNA increased during kiwifruit ripening and reached peak at 144 h after treatment, then dropped immediately. The expression of Ad-ETR1 could be induced by ethylene treatment, while acetylsalicylic acid (ASA) treatment inhibited its expression (Fig. 4). In consistency with the changes of Ad-ETR1 mRNA, ethylene or ASA treatment had marked effects on the kiwifruit ethylene production and fruit softening (Fig. 3). The significance of these results was discussed.