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.

Dendrobium orchids carrying antisense ACC oxidase: small changes in flower morphology and a delay of bud abortion, flower senescence, and abscission of flowers.

Four Dendrobium Sonia 'Earsakul' lines were generated by insertion of one, two or three antisense copies of a Carica papaya gene encoding 1-aminocyclopropane-1-carboxylic acid oxidase (CpACO). Whole vegetative plants of the transgenic lines showed about 50% of the basal ethylene production rate, while the increase in ethylene production in floral buds during opening and open flowers prior to visible senescence was delayed. Detailed analysis of more than 100 parameters in flowering plants showed no effect of antisense ACO on plant morphology and coloration, except for shorter length and width of some of the sepals and petals. In intact plants the water-soaking of floral buds as well as bud abscission were delayed by ACO antisense, as was the time to senescence of open flowers. Pollen viability and pollen tube growth were not affected in the transgenic lines. In cut inflorescences placed in water, bud yellowing, bud water soaking, and bud abscission were considerably delayed by the antisense construct, while the life span of open flowers were increased and abscission of open flowers were delayed. It is concluded that the reduction of ACO activity affected the shape of some petals/sepals and delayed the abortion in floral buds, and the senescence and abscission of open flowers.

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.

De novo transcriptome assembly and identification of the gene conferring a "pandan-like" aroma in coconut (Cocos nucifera L.).

Thailand's aromatic coconut (Cocos nucifera L.) is a special type of green dwarf coconut, the liquid endosperm of which is characterized by a pleasant "pandan-like" aroma due to the presence of 2-acetyl-1-pyrroline (2AP). The aim of this study was to perform a de novo assembly of transriptome from C. nucifera endosperm and to identify the gene responsible for 2AP biosynthesis. CnAMADH2 was identified as an ortholog of the rice aromatic gene and a G-to-C substitution found in exon 14 was associated with 2AP content in the aromatic green dwarf coconut accessions. The base substitution caused an amino-acid change, alanine-to-proline, at position 442 (P442A). The presence of P at this position might alter the steric conformation at the loop region and subsequently result in an unstabilized dimer conformation that could lower AMADH enzyme activity. Among AMADH/BADH protein sequences in different plant species, the P442A mutation was found exclusively in aromatic coconut. The PCR marker developed based on this sequence variation can perfectly detect the aromatic and non-aromatic alleles of the gene. This study confirms the hypothesis that plants may share a mechanism of 2AP biosynthesis. This is the first identification of the gene associated with 2AP biosynthesis in a tree plant.

Do mitochondria in Dendrobium petal mesophyll cells form vacuole-like vesicles?

Using transmission electron microscopy, we investigated the ultrastructure of mitochondria in petal mesophyll cells of the orchid Dendrobium cv. Lucky Duan, from the time of floral opening to visible petal senescence. Cells close to the vascular bundle contained many mitochondria, some of which showed internal degeneration. This inner mitochondrial breakdown was accompanied by an eightfold increase in mitochondrial volume. Small electron-dense granules (approximately 0.04 mum in diameter) at the periphery of the mitochondrial matrix remained. These granules were used as an indicator of still later stages of mitochondrial development in these cells. The apparent final stage of mitochondrial degeneration was a single-membrane-bound vesicle, resembling a vacuole. No evidence was found for the idea that mitochondria became transferred (intact or degenerated) into a lytic vacuole. Taken together, the data suggest the hypotheses that (a) mitochondria in cells close to the vascular bundle in petals of open Dendrobium cv. Lucky Duan flowers undergo large-scale internal degeneration and that (b) such degenerating mitochondria form vacuole-like vesicles.

Silencing polygalacturonase expression inhibits tomato petiole abscission.

Virus-induced gene silencing (VIGS) was used as a tool for functional analysis of cell wall-associated genes that have been suggested to be involved in leaf abscission. Tobacco rattle virus is an effective vector for VIGS in tomato (Lycopersicon esculentum). Silencing was more efficient when the plants were grown at 22 degrees C than when they were grown at 20 degrees C or 25 degrees C. The photobleaching phenotype resulting from silencing phytoene desaturase varied, depending on cultivar, from barely visible to photobleaching of entire leaves. To study the function of abscission-related genes, a purple transgenic tomato line constitutively expressing the maize anthocyanin regulatory gene, Leaf colour (Lc) was used. Silencing Lc expression in this line resulted in reduced anthocyanin production (reversing the colour from purple to green), thus providing a convenient silencing 'reporter'. Silencing tomato abscission-related polygalacturonases (TAPGs), using a TAPG1 fragment, delayed abscission and increased break strength of the abscission zone in explants treated with 1 mul l(-1) ethylene. The abundance of TAPG1 transcripts in the green (silenced) abscission zone tissues was <1% of that in the purple (non-silenced) controls. As a highly homologous region was used for all five polygalacturonases it is assumed that the effect of delayed abscission is the result of silencing all the genes in this family. By contrast, silencing abscission-related expansins (LeEXP11 and LeEXP12) and endoglucanases (LeCEL1 and LeCEL2) had no discernible effect on break strength, even when the two endoglucanase genes were silenced concurrently. Simultaneous silencing of TAPG and LeCEL1 was no more effective than silencing TAPG alone. The data demonstrate the importance of TAPGs in the abscission of leaf petioles.

The transcript abundance of an expansin gene in ripe sapodilla (Manilkara zapota) fruit is negatively regulated by ethylene.

After harvest, mature fruit of sapodilla (Manilkara zapota van Royen) exhibit rapid softening. The decrease in fruit firmness was hastened by ethylene and delayed by 1-methylcyclopropene (1-MCP). Two genes encoding expansins (called MzEXP1 and MzEXP2) were isolated. In both cultivars studied (Makok-Yai and Kra-Suay), MzEXP1 was transiently expressed early during fruit development on the plant. This suggests that it is involved in cell wall loosening during early fruit growth. In cv. Makok-Yai, MzEXP2 was expressed between 1 day before harvest and day 4 after harvest. In cv. Kra-Suay, the expression of MzEXP2 started 8 weeks before the normal harvest stage, and ended on day 3 after harvest. When the fruit of both cultivars was treated with ethylene (50 µL L-1 for 20 h at 25°C) just after harvest, the expression of MzEXP2 became undetectable. After treatment with 1-MCP MzEXP2 mRNA was highly abundant until day 5 after harvest, when in controls the transcript abundance had become undetectable. The onset of MzEXP2 expression seems not regulated by ethylene, as the concomitant ethylene levels are very low. The data strongly indicate that the decrease of MzEXP2 transcript abundance is due to ethylene production by the fruit, which is by then high. The expression of MzEXP2 ceased, both in controls and in ethylene-treated material, when the fruit had reached a rather low threshold firmness. The data suggest that the protein has a supporting and cooperative role in fruit softening.