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.

Time course of programmed cell death, which included autophagic features, in hybrid tobacco cells expressing hybrid lethality.

KEY MESSAGE: PCD with features of vacuolar cell death including autophagy-related features were detected in hybrid tobacco cells, and detailed time course of features of vacuolar cell death were established. A type of interspecific Nicotiana hybrid, Nicotiana suaveolens × N. tabacum exhibits temperature-sensitive lethality. This lethality results from programmed cell death (PCD) in hybrid seedlings, but this PCD occurs only in seedlings and suspension-cultured cells grown at 28 °C, not those grown at 36 °C. Plant PCD can be classified as vacuolar cell death or necrotic cell death. Induction of autophagy, vacuolar membrane collapse and actin disorganization are each known features of vacuolar cell death, but observed cases of PCD showing all these features simultaneously are rare. In this study, these features of vacuolar cell death were evident in hybrid tobacco cells expressing hybrid lethality. Ion leakage, plasma membrane disruption, increased activity of vacuolar processing enzyme, vacuolar membrane collapse, and formation of punctate F-actin foci were each evident in these cells. Transmission electron microscopy revealed that macroautophagic structures formed and tonoplasts ruptured in these cells. The number of cells that contained monodansylcadaverine (MDC)-stained structures and the abundance of nine autophagy-related gene transcripts increased just before cell death at 28 °C; these features were not evident at 36 °C. We assessed whether an autophagic inhibitor, wortmannin (WM), influenced lethality in hybrid cells. After the hybrid cell began to die, WM suppressed increases in ion leakage and cell deaths, and it decreased the number of cells containing MDC-stained structures. These results showed that several features indicative of autophagy and vacuolar cell death were evident in the hybrid tobacco cells subject to lethality. In addition, we documented a detailed time course of these vacuolar cell death features.

Vesicles between plasma membrane and cell wall prior to visible senescence of Iris and Dendrobium flowers.

Cut Iris flowers (Iris x hollandica, cv. Blue Magic) show visible senescence about two days after full opening. Epidermal cells of the outer tepals collapse due to programmed cell death (PCD). Transmission electron microscopy (TEM) showed irregular swelling of the cell walls, starting prior to cell collapse. Compared to cells in flowers that had just opened, wall thickness increased up to tenfold prior to cell death. Fibrils were visible in the swollen walls. After cell death very little of the cell wall remained. Prior to and during visible wall swelling, vesicles (paramural bodies) were observed between the plasma membrane and the cell walls. The vesicles were also found in groups and were accompanied by amorphous substance. They usually showed a single membrane, and had a variety of diameters and electron densities. Cut Dendrobium hybrid cv. Lucky Duan flowers exhibited visible senescence about 14 days after full flower opening. Paramural bodies were also found in Dendrobium tepal epidermis and mesophyll cells, related to wall swelling and degradation. Although alternative explanations are well possible, it is hypothesized that paramural bodies carry enzymes involved in cell wall breakdown. The literature has not yet reported such bodies in association with senescence/PCD.

Expression of expansin genes in the pulp and the dehiscence zone of ripening durian (Durio zibethinus) fruit.

Durian (Durio zibethinus) fruit was harvested at the commercially mature stage and stored at 25°C. Durian fruit have 3-5 longitudinal dehiscence zones (DZs) in the peel, which are up to 40cm long and 2cm thick in large fruit. Dehiscence started a week after harvest, was hastened by exogenous ethylene, and delayed by 1-methylcyclopropene (1-MCP), showing that it is regulated by endogenous ethylene. Three genes encoding α-expansins (DzEXP1-3) were isolated. In the expression of these genes increased, prior to dehiscence. Pulp firmness decreased during storage. The decrease was hastened by ethylene and delayed by 1-methylcyclopropene (1-MCP). Exogenous ethylene promoted gene expression of DzEXP1 both in the DZs and in the pulp. It had a smaller effect on DzEXP2 in the zones and pulp, but did not affect DzEXP3 expression. 1-MCP inhibited the expression of DzEXP1 and, somewhat less, of DzEXP2, but did not affect DzEXP3 expression, both in DZs and pulp. It is concluded that the close relationship between expression of DzEXP1 and DzEXP2 and both dehiscence and fruit softening suggests that these genes are involved in both processes.

Two abscission zones proximal to Lansium domesticum fruit: one more sensitive to exogenous ethylene than the other.

Longkong (Lansium domesticum) fruit grows in bunches and is also sold as bunches. Individual fruit can separate from the bunch both before and after commercial harvest. The fruit has two separation sites. The first is located between bracts on the stem and the fused sepals (separation zone 1: SZ1) and the second between the fused sepals and the fruit (separation zone 2: SZ2). True abscission occurred at both zones. We investigated whether the two zones were active at different stages of development and if they were differentially sensitive to ethylene. Abscission occurred in the SZ1 in very young fruit (fruit still at the ovary stage), during early fruit development (5 weeks after full bloom; WAFB), and in ripe and overripe fruit (15-17 WAFB). Abscission did not spontaneously occur in the SZ2, but by the time the fruit was fully ripe, 15 WAFB, and later, a slight mechanical force was sufficient to break this zone. In fruit bunches severed from the tree at 5, 8, and 13 WAFB, break strength (BS) in SZ1 decreased much more after exogenous ethylene treatment than that in SZ2. Ethylene induced abscission in the SZ1, but not in SZ2. At 5, 8, and 13 WAFB, treatment with 1-methylcyclopropane (1-MCP; an inhibitor of ethylene perception) had a small effect on BS in the SZ1 and no effect in the SZ2. It is concluded that abscission in the SZ1 was much more sensitive to ethylene than that in the SZ2. In intact plants SZ1 reacts to endogenous ethylene, e.g., as a result of stress, while SZ2 apparently allows animals to remove the ripe fruit from the tree with minimal force.

Carotenoids in durian fruit pulp during growth and postharvest ripening.

Durian (Durio zibethinus) cvs. Chanee and Monthong fruit were severed from the tree during 14 day intervals, from 10 weeks after anthesis until commercial maturity. We determined the pulp (i.e. aril; fruit flesh) carotenoid composition, together with pulp firmness, color and total soluble solids (TSS) and postharvest quality. In ripe cv. Chanee fruit the main carotenoids were ß-carotene (about 80%), and α-carotene (20%), with minor levels of lutein and zeaxanthin. In ripe fruit total carotenoid concentration (expressed per gram FW) was about 9-fold higher in cv. Chanee than in cv. Monthong. Large differences between the cultivars were also found in ß-carotene levels (about 11 times more in cv. Chanee), and even larger ones in those of α-carotene. Differences in lutein and zeaxanthin concentrations were small. Pulp color was deeper yellow in cv. Chanee than in cv. Monthong, which was correlated with α-carotene and ß-carotene concentrations. Durian contains a high fat percentage, which is conducive to carotenoid uptake. It is concluded that it is advisable to consume cv. Chanee rather than cv. Monthong if intake of carotenoids is considered important.

Macroautophagy and microautophagy in relation to vacuole formation in mesophyll cells of Dendrobium tepals.

Prior to flower opening, mesophyll cells at the vascular bundles of Dendrobium tepals showed a large increase in vacuolar volume, partially at the expense of the cytoplasm. Electron micrographs indicated that this increase in vacuolar volume was mainly due to vacuole fusion. Macroautophagous structures typical of plant cells were observed. Only a small part of the decrease in cytoplasmic volume seemed due to macroautophagy. The vacuoles contained vesicles of various types, including multilamellar bodies. It was not clear if these vacuolar inclusions were due to macroautophagy or microautophagy. Only a single structure was observed of a protruding vacuole, indicating microautophagy. It is concluded that macroautophagy occurs in these cells but its role in vacuole formation seems small, while a possible role of microautophagy in vacuole formation might be hypothesized. Careful labeling of organelle membranes seems required to advance our insight in plant macro- and microautophagy and their roles in vacuole formation.

Ethylene and pollination decrease transcript abundance of an ethylene receptor gene in Dendrobium petals.

We studied the expression of a gene encoding an ethylene receptor, called Ethylene Response Sensor 1 (Den-ERS1), in the petals of Dendrobium orchid flowers. Transcripts accumulated during the young floral bud stage and declined by the time the flowers had been open for several days. Pollination or exposure to exogenous ethylene resulted in earlier flower senescence, an increase in ethylene production and a lower Den-ERS1 transcript abundance. Treatment with 1-methylcyclopropene (1-MCP), an inhibitor of the ethylene receptor, decreased ethylene production and resulted in high transcript abundance. The literature indicates two kinds of ethylene receptor genes with regard to the effects of ethylene. One group shows ethylene-induced down-regulated transcription, while the other has ethylene-induced up-regulation. The present gene is an example of the first group. The 5' flanking region showed binding sites for Myb and myb-like, homeodomain, MADS domain, NAC, TCP, bHLH and EIN3-like transcription factors. The binding site for the EIN3-like factor might explain the ethylene effect on transcription. A few other transcription factors (RAV1 and NAC) seem also related to ethylene effects.

Crystalloids in apparent autophagic plastids: remnants of plastids or peroxisomes?

Plant macroautophagy is carried out by autophagosome-type organelles. Recent evidence suggests that plastids also can carry out macroautophagy. The double membrane at the surface of plastids apparently invaginates, forming an intraplastidial space. This space contains a portion of cytoplasm that apparently becomes degraded. Here we report, in Tillandsia sp. and Aechmaea sp., the presence of almost square or diamond-shaped crystalloids inside what seems the intraplastidial space of autophagous plastids. The same type of crystalloids were observed in chloroplasts and other plastids, but were not found in the cytoplasm or the vacuole. Peroxisomes contained smaller and more irregularly shaped crystalloids compared to the ones observed in 'autophagous' plastids. It is hypothesized that plastids are able to sequester chloroplasts and other plastids.

Pollination increases ethylene production in Lilium hybrida cv. Brindisi flowers but does not affect the time to tepal senescence or tepal abscission.

In many species, pollination induces a rapid increase in ethylene production, which induces early petal senescence, petal abscission, or flower closure. Cross-pollination in Lilium hybrida cv. Brindisi resulted in a small increase in flower ethylene production. In intact plants and in isolated flowers, pollination had no effect on the time to tepal senescence or tepal abscission. When applied to closed buds of unpollinated flowers, exogenous ethylene slightly hastened the time to tepal senescence and abscission. However, exogenous ethylene had no effect when the flowers had just opened, i.e. at the time of pollination. Experiments with silver thiosulphate, which blocks the ethylene receptor, indicated that endogenous ethylene had a slight effect on the regulation of tepal senescence and tepal abscission, although only at the time the tepals were still inside buds and not in open flowers. Low ethylene-sensitivity after anthesis therefore explains why pollination had no effect on the processes studied.

Pollinia-borne chemicals that induce early postpollination effects in Dendrobium flowers move rapidly into agar blocks and include ACC and compounds with auxin activity.

The early visible effects of pollination in orchids are likely due to pollinia-borne chemicals. In Dendrobium we tested whether such compounds were water soluble and would diffuse in solid-aqueous phase, and determined both 1-aminocyclopropane-1-carboxylic acid (ACC) concentrations and auxin activity. Following pollination, the flower peduncle showed epinastic movement, followed by yellowing of the flower lip, flower senescence and ovary growth. Placing pollinia on agar blocks for 3, 6, 9 or 12h, prior to transferring them to the stigma, increased the time to these early postpollination effects or prevented them. Placing agar blocks that had been used for contact with the pollinia on the stigma also induced the early postpollination effects. The concentrations of ACC, the direct precursor of ethylene, in pollinia was lower the longer the pollinia had been in contact with the agar blocks, whilst the ACC content in the agar blocks increased with the period of contact. The auxin activity of the agar blocks also increased with the time of contact with pollinia. It is concluded that chemicals in the pollinia are responsible for the early visible postpollination effects, and that these (a) rapidly diffuse in aqueous media, and (b) comprise at least ACC and compounds with auxin activity. The idea is discussed that ACC plus auxin is adequate for the production of the early postpollination effects.

Lipid globules on the plastid surface in Iris tepal epidermis cells during tepal maturation and senescence.

Epidermis cells in the outer tepals of Iris flowers (Iris×hollandica, cv. Blue Magic) start programmed cell death (PCD) prior to floral opening. The tepals show visible senescence symptoms three days after full opening. Visible senescence coincides with collapse (death) of the upper epidermis cells. In these cells, electron-dense particles (plastoglobuli), membranes, and oil bodies were observed in the plastid interior. Electron-dense globules similar to plastoglobuli, thus apparently mainly consisting of lipids, were found on the plastid surface, from before flower opening until cell death. Such electron-dense globules were also present in the cytosol. The size of some of the globules on the plastid surface increased with time. The globules are likely involved in transfer of lipidic/proteinaceous material from the plastid to the cytosol. As the plastids contained ample oil bodies, up to the time of cell death, cell death was likely not due to lack of reserves. Mitochondrial ultrastructure also remained the same until cell death. The role of mitochondria in PCD is discussed.

Flower opening and closure: an update.

This review is an update of a 2003 review (Journal of Experimental Botany 54,1801-1812) by the same corresponding author. Many examples of flower opening have been recorded using time-lapse photography, showing its velocity and the required elongation growth. Ethylene regulates flower opening, together with at least gibberellins and auxin. Ethylene and gibberellic acid often promote and inhibit, respectively, the expression of DELLA genes and the stability of DELLA proteins. DELLA results in growth inhibition. Both hormones also inhibited and promoted, respectively, the expression of aquaporin genes required for cell elongation. Arabidopsis miRNA319a mutants exhibited narrow and short petals, whereby miRNA319a indirectly regulates auxin effects. Flower opening in roses was controlled by a NAC transcription factor, acting through miRNA164. The regulatory role of light and temperature, in interaction with the circadian clock, has been further elucidated. The end of the life span in many flowers is determined by floral closure. In some species pollination resulted in earlier closure of turgid flowers, compared with unpollinated flowers. It is hypothesized that this pollination-induced effect is only found in flowers in which closure is regulated by ethylene.

Expression of an AtNAP gene homolog in senescing morning glory (Ipomoea nil) petals of two cultivars with a different flower life span.

AtNAP, a NAC family transcription factor, has been shown to promote leaf senescence in Arabidopsis. We isolated an AtNAP homolog in morning glory (Ipomoea nil), designated InNAP, and investigated its expression during petal senescence. We used two cultivars, one showing a normal short flower life span (cv. Peking Tendan) and another a longer life span (cv. Violet). InNAP was highly expressed in both cultivars. Expression was high before that of the senescence marker gene InSAG12. InNAP and InSAG12 expression was high in cv. Peking Tendan before cv. Violet. The expression of both genes was therefore temporally related to the onset of the visible senescence symptoms. An inhibitor of ethylene action (silver thiosulphate, STS) delayed petal senescence in cv. Peking Tendan but had no effect in cv. Violet. STS treatment had no clear effect on the InNAP expression in petals of both cultivars, suggesting that endogenous ethylene may not be necessary for its induction. These data suggest the hypothesis that InNAP plays a role in petal senescence, independent of the role of endogenous ethylene.

Length of the dark period affects flower opening and the expression of circadian-clock associated genes as well as xyloglucan endotransglucosylase/hydrolase genes in petals of morning glory (Ipomoea nil).

KEY MESSAGE: We isolated differentially expressed and dark-responsive genes during flower development and opening in petals of morning glory. Flower opening usually depends on petal expansion and is regulated by both genetic and environmental factors. Flower opening in morning glory (Ipomoea nil) is controlled by the dark/light regime just prior to opening. Opening was normal after 8- or 12-h dark periods but progressed very slowly after a 4-h dark period or in continuous light. Four genes (InXTH1-InXTH4) encoding xyloglucan endotransglucosylase/hydrolases (XTHs) and three genes (InEXPA1-InEXPA3) encoding alpha-expansins (EXPAs) were isolated. The expression patterns of InXTH2, InXTH3, and InXTH4 in petals were closely correlated with the rate of flower opening controlled by the length of the dark period prior to opening, but those of the EXPA genes were not. The expression pattern of InXTH1 gene was closely correlated with petal elongation. Suppression subtractive hybridization was used to isolate dark-responsive genes accompanying flower opening. The expressions of ten isolated genes were associated with the length of the dark period prior to flower opening. One gene was highly homologous to Arabidopsis pseudo-response regulator7, which is associated with the circadian clock and phytochrome signaling; another to Arabidopsis REVEILLE1, which affects the output of the circadian clock. Other genes were related to light responses, plant hormone effects and signal transduction. The possible roles of these genes in regulation of flower opening are discussed.

Classical macroautophagy in Lobivia rauschii (Cactaceae) and possible plastidial autophagy in Tillandsia albida (Bromeliaceae) tapetum cells.

The tapetum in anthers is a tissue that undergoes programmed cell death (PCD) during the production of pollen. We observed two types of autophagy prior to cell death. In Lobivia rauschii (Cactaceae), tapetum cells showed plant-type autophagosomes-autolysosomes, which have been found previously exclusively in root meristem cells. The autophagic structures were formed by a network of tubules which apparently merged laterally, thereby sequestering a portion of the cytoplasm. The organelles observed in the sequestered material included multilamellar bodies, which have not been reported earlier in these organelles. By contrast, Tillandsia albida (Bromeliaceae) tapetum cells contained no such organelles but showed plastids that might possibly carry out autophagy, as they contained portions of the cytoplasm similar to the phenomenon reported earlier in Phaseolus and Dendrobium. However, the ultrastructure of the T. albida plastids was different from that in the previous reports. It is concluded that in L. rauschii classical plant macroautophagy was involved in degradation of the cytoplasm, while in T. albida such classical macroautophagy was not observed. Instead, the data in T. albida suggested the hypothesis that plastids are able to carry out degradation of the cytoplasm.

Ultrastructure of autophagy in plant cells: a review.

Just as with yeasts and animal cells, plant cells show several types of autophagy. Microautophagy is the uptake of cellular constituents by the vacuolar membrane. Although microautophagy seems frequent in plants it is not yet fully proven to occur. Macroautophagy occurs farther away from the vacuole. In plants it is performed by autolysosomes, which are considerably different from the autophagosomes found in yeasts and animal cells, as in plants these organelles contain hydrolases from the onset of their formation. Another type of autophagy in plant cells (called mega-autophagy or mega-autolysis) is the massive degradation of the cell at the end of one type of programmed cell death (PCD). Furthermore, evidence has been found for autophagy during degradation of specific proteins, and during the internal degeneration of chloroplasts. This paper gives a brief overview of the present knowledge on the ultrastructure of autophagic processes in plants.

Amorphous areas in the cytoplasm of Dendrobium tepal cells: production through organelle degradation and destruction through macroautophagy?

In Dendrobium flowers some tepal mesophyll cells showed cytoplasmic areas devoid of large organelles. Such amorphous areas comprised up to about 40% of the cross-section of a cell. The areas were not bound by a membrane. The origin of these areas is not known. We show data suggesting that they can be formed from vesicle-like organelles. The data imply that these organelles and other material become degraded inside the cytoplasm. This can be regarded as a form of autophagy. The amorphous areas became surrounded by small vacuoles, vesicles or double membranes. These seemed to merge and thereby sequester the areas. Degradation of the amorphous areas therefore seemed to involve macroautophagy.

Delay of iris flower senescence by cytokinins and jasmonates.

It is not known whether tepal senescence in Iris flowers is regulated by hormones. We applied hormones and hormone inhibitors to cut flowers and isolated tepals of Iris × hollandica cv. Blue Magic. Treatments with ethylene or ethylene antagonists indicated lack of ethylene involvement. Auxins or auxin inhibitors also did not change the time to senescence. Abscisic acid (ABA) hastened senescence, but an inhibitor of ABA synthesis (norflurazon) had no effect. Gibberellic acid (GA3 ) slightly delayed senescence in some experiments, but in other experiments it was without effect, and gibberellin inhibitors [ancymidol or 4-hydroxy-5-isopropyl-2-methylphenyltrimethyl ammonium chloride-1-piperidine carboxylate (AMO-1618)] were ineffective as well. Salicylic acid (SA) also had no effect. Ethylene, auxins, GA3 and SA affected flower opening, therefore did reach the flower cells. Jasmonates delayed senescence by about 2.0 days. Similarly, cytokinins delayed senescence by about 1.5-2.0 days. Antagonists of the phosphatidylinositol signal transduction pathway (lithium), calcium channels (niguldipine and verapamil), calmodulin action [fluphenazine, trifluoroperazine, phenoxybenzamide and N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide hydrochloride (W-7)] or protein kinase activity [1-(5-isoquinolinesulfonyl)-2-methylpiperazine hydrochloride (H-7), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-8) and N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride (H-9)] had no effect on senescence, indicating no role of a few common signal transduction pathways relating to hormone effects on senescence. The results indicate that tepal senescence in Iris cv. Blue Magic is not regulated by endogenous ethylene, auxin, gibberellins or SA. A role of ABA can at present not be excluded. The data suggest the hypothesis that cytokinins and jasmonates are among the natural regulators.

Opening of Iris flowers is regulated by endogenous auxins.

Flower opening in Iris (Iris×hollandica) requires elongation of the pedicel and ovary. This moves the floral bud upwards, thereby allowing the tepals to move laterally. Flower opening is requires with elongation of the pedicel and ovary. In cv. Blue Magic, we investigated the possible role of hormones other than ethylene in pedicel and ovary elongation and flower opening. Exogenous salicylic acid (SA) and the cytokinins benzyladenine (N6-benzyladenine, BA) and zeatin did not affect opening. Jasmonic acid (JA) and abscisic acid (ABA) were slightly inhibitory, but an inhibitor of ABA synthesis (norflurazon) was without effect. Flower opening was promoted by gibberellic acid (GA(3)), but two inhibitors of gibberellin synthesis (4-hydroxy-5-isopropyl-2-methylphenyltrimethyl ammonium chloride-1-piperidine carboxylate, AMO-1618; ancymidol) did not change opening. The auxins indoleacetic acid (IAA) and naphthaleneacetic acid (NAA) strongly promoted elongation and opening. An inhibitor of auxin transport (2,3,5-triodobenzoic acid, TIBA) and an inhibitor of auxin effects [α-(p-chlorophenoxy)-isobutyric acid; PCIB] inhibited elongation and opening. The data suggest that endogenous auxins are among the regulators of the pedicel and ovary elongation and thus of flower opening in Iris.