The Rest Period of Rhododendron Flower Buds: III. CYTOLOGICAL STUDIES ON THE ACCUMULATION AND BREAKDOWN OF PROTEIN BODIES AND AMYLOPLASTS DURING FLOWER DEVELOPMENT

Rhododendron flower development occurs in three easily definedstages: a pre-rest stage, during which petal growth is mainlyby cell elongation; an indeterminate rest period; and an after-reststage, that begins when the flowers resume growth and ends atanthesis. Early in the pre-rest stage of development, protein bodies andamyloplasts accumulate in the petals. The epidermal cells accumulateonly protein bodies and the mesophyll cells accumulate amyloplaststhat have a few small protein bodies around the periphery. Thesubepidermal cells and the cells around the vascular bundlesaccumulate both large protein bodies and amyloplasts. Duringthe rest period there is a cessation of cell elongation andthe reserve protein bodies and amyloplasts remain intact. The protein bodies in all of the cells including those aroundthe amyloplasts are proteolized early in the after-rest stageof development. Digestion of the starch granules occurs whenthe petals are about one-half their final size. Epidermal-cell expansion during after-rest is relatively uniform;the walls between adjacent epidermal cells remain attached toeach other. The mesophyll cells elongate irregularly and thewalls of adjacent cells separate giving rise to large intercellularspaces. At anthesis the petal cells consist of a cell wall, a parietalcytoplasm, and a large central vacuole.     

The ADAXIALIZED LEAF1 gene functions in leaf and embryonic pattern formation in rice

The adaxial-abaxial axis in leaf primordia is thought to be established first and is necessary for the expansion of the leaf lamina along the mediolateral axis. To understand axis information in leaf development, we isolated the adaxialized leaf1 (adl1) mutant in rice, which forms abaxially rolled leaves. adl1 leaves are covered with bulliform-like cells, which are normally distributed only on the adaxial surface. An adl1 double mutant with the adaxially snowy leaf mutant, which has albino cells that specifically appear in the abaxial mesophyll tissue, indicated that adl1 leaves show adaxialization in both epidermal and mesophyll tissues. The expression of HD-ZIPIII genes in adl1 mutant increased in mature leaves, but not in the young primordia or the SAM. This indicated that ADL1 may not be directly involved in determining initial leaf polarity, but rather is associated with the maintenance of axis information. ADL1 encodes a plant-specific calpain-like cysteine proteinase orthologous to maize DEFECTIVE KERNEL1. Furthermore, we identified intermediate and strong alleles of the adl1 mutant that generate shootless embryos and globular-arrested embryos with aleurone layer loss, respectively. We propose that ADL1 plays an important role in pattern formation of the leaf and embryo by promoting proper epidermal development.     

Ethylene and senescence in petals of tradescantia

Flowers of Tradescantia (clone O2) which are ephemeral, produce ethylene during senescence with the maximum rates occurring during the initial period of fading. Senescing isolated petals produce ethylene in a similar manner, exhibit a loss of membrane semipermeability, and exogenous ethylene hastens the onset as well as the subsequent rate of this loss. The aminoethoxy analog of 0.1 millimolar rhizobitoxine completely inhibits ethylene production by isolated petals but only partially the loss of membrane semipermeability. Isolated petals acquire a sensitivity to ethylene as they mature, becoming fully sensitive on the day of anthesis.     

Intracellular energy depletion triggers programmed cell death during petal senescence in tulip

Programmed cell death (PCD) in petals provides a model system to study the molecular aspects of organ senescence. In this study, the very early triggering signal for PCD during the senescence process from young green buds to 14-d-old petals of Tulipa gesneriana was determined. The opening and closing movement of petals of intact plants increased for the first 3 d and then gradually decreased. DNA degradation and cytochrome c (Cyt c) release were clearly observed in 6-d-old flowers. Oxidative stress or ethylene production can be excluded as the early signal for petal PCD. In contrast, ATP was dramatically depleted after the first day of flower opening. Sucrose supplementation to cut flowers maintained their ATP levels and the movement ability for a longer time than in those kept in water. The onset of DNA degradation, Cyt c release, and petal senescence was also delayed by sucrose supplementation to cut flowers. These results suggest that intracellular energy depletion, rather than oxidative stress or ethylene production, may be the very early signal to trigger PCD in tulip petals.     

Spatial and temporal progress of programmed cell death in the developing starchy endosperm of rice

Programmed cell death (PCD) is the genetically regulated disassembly of cells, and occurs in the endosperm of cereals during seed maturation. Since PCD determines the lifetime of cells, it can affect endosperm growth and, therefore, cereal yield. However, the features and mechanisms of PCD in the developing starchy endosperm in the Poaceae remain unclear. In the present study, we investigated the characteristics of PCD in developing starchy endosperm of rice (Oryza sativa L.) by fluorescence microscopy, focusing on the spatial and temporal progress of PCD-associated responses. Cell death commenced in the central region of starchy endosperm, and then spread to the peripheral region. PCD-associated responses, such as mitochondrial membrane permeabilization and activation of the protease that cleaves the amino acid sequence VEID, showed similar spatial patterns to that of cell death, but preceded cell death. Degradation of nuclear DNA could not be detected in developing starchy endosperm by the TUNEL assay. These results indicated that PCD in developing starchy endosperm of rice proceeds via a highly organized pattern. In addition, these results suggested that PCD in developing starchy endosperm of rice is characterized by the involvement of mitochondrial signaling and the activity of a caspase-like protease that cleaves the VEID sequence.     

Is a cysteine proteinase inhibitor involved in the regulation of petal wilting in senescing carnation (Dianthus caryophyllus L.) flowers?

Senescence of carnation petals is accompanied by autocatalytic ethylene production and wilting of the petals; the former is caused by the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase genes and the latter is related to the expression of a cysteine proteinase (CPase) gene. CPase is probably responsible for the degradation of proteins, leading to the decomposition of cell components and resultant cell death during the senescence of petals. The carnation plant also has a gene for the CPase inhibitor (DC-CPIn) that is expressed abundantly in petals at the full opening stage of flowers. In the present study, DC-CPIn cDNA was cloned and expressed in E. coli. The recombinant DC-CPIn protein completely inhibited the activities of a proteinase (CPase) extracted from carnation petals and papain. Northern blot analysis showed that the mRNA for CPase (DC-CP1) accumulated in large amounts, whereas that for DC-CPIn disappeared, corresponding to the onset of petal wilting in flowers undergoing natural senescence and exogenous ethylene-induced senescence. Based on these findings, a role of DC-CPIn in the regulation of petal wilting is suggested; DC-CPIn acts as a suppressor of petal wilting, which probably functions to fine-tune petal wilting in contrast to coarse tuning, the up-regulation of CPase activity by gene expression.     

Expression of defender against apoptotic death (DAD-1) in Iris and Dianthus petals

The gene defender against apoptotic death ( DAD-1 ) prevents programmed cell death in animal cells. We investigated the expression pattern of DAD-1 in petals of iris ( Iris  ×  hollandica cv. Blue Magic) and carnation ( Dianthus caryophyllus cv. Etarro). DAD-1 expression in Iris petals was strongly reduced by the time of visible senescence, which occurs 4 days after flower opening. Microscopic analysis showed that most mesophyll cells had died prior to a clear decrease in DAD-1 expression and that epidermis cells started to die by that time. In carnation petals DAD-1 expression also decreased by the time of massive cell death. After ethylene treatment, DAD-1 expression in carnation again decreased concomitant with the advance in massive cell death. In conclusion, DAD-1 is not an early regulator of petal cell death. Its expression may be required for the programmed dismantling of cells, as it ceases only just prior to, or concomitant with, cell death.     

A homolog of the defender against apoptotic death gene (DAD1) in senescing gladiolus petals is down-regulated prior to the onset of programmed cell death

We isolated a homolog of the potential anti-apoptotic gene, defender against apoptotic death (DAD1) from gladiolus petals as full-length cDNA (GlDAD1), and investigated the relationship between its expression and the execution processes of programmed cell death (PCD) in senescing petals. RNA gel blotting showed that GlDAD1 expression in petals was drastically reduced, considerably before the first visible senescence symptom (petal wilting). A few days after down-regulation GlDAD1 expression, DNA and nuclear fragmentation were observed, both specific for the execution phase of PCD.     

Time-course of programmed cell death during leaf senescence in <Emphasis Type="Italic">Eucommia ulmoides</Emphasis>

Leaves of Eucommia ulmoides Oliv. harvested between April to November were examined for programmed cell death (PCD) during growth and senescence. Leaves developed in April, becoming fully expanded in late May, remaining unchanged until November when they started to dehisce. Falling leaves retained a green color. Our results showed that (1) mesophyll cells gradually reduced their nuclei from September to November, (2) positive TUNEL signals appeared on the nuclei from August, (3) ladder-like DNA fragmentation occurred in September and October, and (4) a 20-kDa Ca²⁺-dependent DNase appeared in these same months. In fallen leaves, intact mesophyll cell nuclei could not be detected, but a few cells around the vascular bundle had nuclei. Therefore, (1) programmed cell death (PCD) of leaf cells occurred in the leaves of E. ulmoides, (2) the progress of mesophyll cell PCD lasted for more than 2 months, and (3) PCD of leaf cells was asynchronous in natural senescing leaves. Electronic Publication     

Activity of Ageing Carnation Flower Parts and the Effects of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid-Induced Ethylene

Peak levels of 1-aminocyclopropane-l-carboxylic acid (ACC) in flower parts of ageing carnations (Dianthus caryophyllus L. cv Scanea 3C) were detected 6 to 9 days after flower opening. The ethylene climacteric and the first visible sign of wilting was observed 7 days after opening. The concentration of conjugated ACC in these same tissues peaked at day three with reduction of 70% by day 4. From day 5 to day 9 all parts followed a diurnal pattern of increasing in conjugate levels 1 day and decreasing the next. Concentrations of conjugated ACC were significantly higher than those of ACC in all ageing parts. Preclimacteric petals treated with ACC or 1-(malonylamino)-cycloprane-1-carboxylic acid (MACC), started to senesce 30 to 36 hours after treatment. When petals were treated with MACC plus by 0.1 millimolar aminoethyoxyvinylglycine, premature senescence was induced, while ethylene production was suppressed relative to MACC-treated petals. Petals treated with MACC and silver complex produced ethylene, but did not senesce. The MACC-induced ethylene was inhibited by the addition of 1.0 millimolar CoC1₂. These results demonstrate MACC-induced senescence in preclimacteric petals. The patterns of ACC and MACC detected in the flower parts support the view that an individual part probably does not export an ethylene precursor to the remainder of the flower inducing senescence.     

Expression of cysteine proteinase during developmental events associated with programmed cell death in brinjal

Here we show that the expression of a cysteine proteinase coincides with several developmental events associated with programmed cell death (PCD) in Solanum melongena (brinjal), i.e. during leaf senescence, fruit senescence, xylogenesis, nucellar cell degeneration and anther senescence. We have isolated a cDNA encoding brinjal cysteine proteinase (SmCP) that shares high (90-92%) amino acid identity to cysteine proteinases of tobacco (CYP-8) and tomato (LCYP-2) that have not been previously reported to be senescence-associated. In contrast, SmCP shows lower (39-41%) amino acid identity to other senescence-related cysteine proteinases and, unlike most of them, it is not preferentially expressed in certain organs or cell types. Northern analysis of leaves, fruits and flowers at different stages of development showed that SmCP expression increased significantly at senescence in leaf and fruit, but was highly expressed throughout flower development. In situ hybridization studies on flower sections using an antisense RNA probe localized the SmCP mRNA to the xylem, the epidermis and the endothecium of the anther and the nucellar cells, suggesting its involvement in PCD during xylogenesis, anther senescence and ovule development, respectively. Its expression during nucellar cell degeneration suggests that protein reserves of the nucellus are released to the developing embryo. Polarity in its pattern of expression in the nucellus of the developing seed (40DAP) further implies a directional flow of these nutrients.     

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 μm 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.     

The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants

Programmed cell death (PCD) is a process by which cells in many organisms die. The basic morphological and biochemical features of PCD are conserved between the animal and plant kingdoms. Cysteine proteases have emerged as key enzymes in the regulation of animal PCD. Here, we show that in soybean cells, PCD-activating oxidative stress induced a set of cysteine proteases. The activation of one or more of the cysteine proteases was instrumental in the PCD of soybean cells. Inhibition of the cysteine proteases by ectopic expression of cystatin, an endogenous cysteine protease inhibitor gene, inhibited induced cysteine protease activity and blocked PCD triggered either by an avirulent strain of Pseudomonas syringae pv glycinea or directly by oxidative stress. Similar expression of serine protease inhibitors was ineffective. A glutathione S-transferase-cystatin fusion protein was used to purify and characterize the induced proteases. Taken together, our results suggest that plant PCD can be regulated by activity poised between the cysteine proteases and the cysteine protease inhibitors. We also propose a new role for proteinase inhibitor genes as modulators of PCD in plants.