Programmed cell death in Ricinus and Arabidopsis: the function of KDEL cysteine peptidases in development.

Programmed cell death (PCD) in plants is a prerequisite for development as well as seed and fruit production. It also plays a significant role in pathogen defense. A unique group of papain-type cysteine endopeptidases, characterized by a C-terminal endoplasmic reticulum (ER) retention signal (KDEL CysEP), is involved in plant PCD. Genes for these endopeptidases have been sequenced and analyzed from 25 angiosperms and gymnosperms. They have no structural relationship to caspases involved in mammalian PCD and homologs to this group of plant cysteine endopeptidases have not been found in mammals or yeast. In castor beans (Ricinus communis), the CysEP is synthesized as pre-pro-enzyme. The pro-enzyme is transported to the cytosol of cells undergoing PCD in ER-derived vesicles called ricinosomes. These vesicles release the mature CysEP in the final stages of organelle disintegration triggered by acidification of the cytoplasm resulting from the disruption of the vacuole. Mature CysEP digests the hydroxyproline (Hyp)-rich proteins (extensins) that form the basic scaffold of the plant cell wall. The KDEL CysEPs accept a wide variety of amino acids at the active site, including the glycosylated Hyp residues of the extensins. In Arabidopsis, three KDEL CysEPs (AtCEP1, AtCEP2 and AtCEP3) are expressed in tissues undergoing PCD. In transgenic Arabidopsis plants expressing ß-glucuronidase under the control of the promoters for these three genes, cell- and tissue-specific activities were mapped during seedling, flower and seed development. KDEL CysEPs participate in the collapse of tissues in the final stage of PCD and in tissue re-modeling such as lateral root formation.

Probing the floral developmental stages, bisexuality and sex reversions in castor (Ricinus communis L.).

Castor (Ricinus communis L) is an ideal model species for sex mechanism studies in monoecious angiosperms, due to wide variations in sex expression. Sex reversion to monoecy in pistillate lines, along with labile sex expression, negatively influences hybrid seed purity. The study focuses on understanding the mechanisms of unisexual flower development, sex reversions and sex variations in castor, using various genotypes with distinct sex expression pattern. Male and female flowers had 8 and 12 developmental stages respectively, were morphologically similar till stage 4, with an intermediate bisexual state and were intermediate between type 1 and type 2 flowers. Pistil abortion was earlier than stamen inhibition. Sex alterations occurred at floral and inflorescence level. While sex-reversion was unidirectional towards maleness via bisexual stage, at high day temperatures (Tmax > 38 °C), femaleness was restored with subsequent drop in temperatures. Temperature existing for 2-3 weeks during floral meristem development, influences sexuality of the flower. We report for first time that unisexuality is preceded by bisexuality in castor flowers which alters with genotype and temperature, and sex reversions as well as high sexual polymorphisms in castor are due to alterations in floral developmental pathways. Differentially expressed (male-abundant or male-specific) genes Short chain dehydrogenase reductase 2a (SDR) and WUSCHEL are possibly involved in sex determination of castor.

Developmental studies on glyoxysomes in Ricinus endosperm.

The development of glyoxysomes and their associated enzymes, isocitrate lyase and malate synthetase, was studied in the endosperm of castor bean seeds during germination and early growth in darkness. The protein content of the glyoxysome fraction, separated by sucrose density centrifugation, increased linearly from day 2 to day 4 and declined subsequently, while maximum enzyme activities were reached at day 5. The specific activities of the enzymes in the glyoxysomes increased until day 5 and remained constant thereafter. At all stages of germination the only organelle with isocitrate lyase activity was the glyoxysome, but at the earlier stages a greater portion of the total activity was recovered in the soluble form. Malate synthetase was found primarily in the glyoxysomes after day 4, but at earlier stages part of the activity appeared at regions of lower density on the sucrose gradient. It was shown that this particulate malate synthetase activity was due to glyoxysomes broken during preparation, and that, as a result of this breakage, isocitrate lyase was solubilized. We conclude that both enzymes are housed in the glyoxysome in vivo throughout the germination period, and that the rise and fall in enzyme activities in phase with fat breakdown correspond to the net production and destruction of this organelle.

Endoplasmic reticulum as the site of lecithin formation in castor bean endosperm.

The properties of a discrete membranous fraction isolated on sucrose gradients from castor bean endosperm have been examined. This fraction was previously shown to be the exclusive site of phosphorylcholine-glyceride transferase. The distribution of NADPH-cytochrome c reductase and antimycin insensitive NADH-cytochrome c reductase across the gradient followed closely that of the phosphorylcholine-glyceride transferase. This fraction also had NADH diaphorase activity and contained cytochromes b(5) and P 450. On sucrose gradients containing 1 mM EDTA this fraction had a mean isopycnic density of 1.12 g/cm(3) and sedimented separately from the ribosomes; electron micrographs showed that it was comprised of smooth membranes. When magnesium was included in the gradients to prevent the dissociation of membrane-bound ribosomes, the isopycnic density of the membrane fraction with its associated enzymes was increased to 1.16 g/cm(3) and under these conditions the electron micrographs showed that the membranes had the typical appearance of rough endoplasmic reticulum. Together these data show that the endoplasmic reticulum is the exclusive site of lecithin formation in the castor bean endosperm and establish a central role for this cytoplasmic component in the biogenesis of cell membranes.

Cytochemical and developmental changes in microbodies (glyoxysomes) and related organelles of castor bean endosperm.

Structural changes in endosperm cells of germinating castor beans were examined and complemented with a cytochemical analysis of staining with diaminobenzidine (DAB). Deposition of oxidized DAB occurred only in microbodies due to the presence of catalase, and in cell walls associated with peroxidase activity. Seedling development paralleled the disappearance of spherosomes (lipid bodies) and matrix of aleurone grains in endosperm cells. 6 to 7 days after germination, a cross-section through the endosperm contained cells in all stages of development and senescence beginning at the seed coat and progressing inward to the cotyledons. Part of this aging process involved vacuole formation by fusion of aleurone grain membranes. This coincided with an increase in microbodies (glyoxsomes), mitochondria, plastids with an elaborate tubular network, and the formation of a new protein body referred to as a dilated cisterna, which is structurally and biochemically distinct from microbodies although both apparently develop from rough endoplasmic reticulum (ER). In vacuolate cells microbodies are the most numerous organelle and are intimately associated with spherosomes and dilated cisternae. This phenomenon is discussed in relation to the biochemical activities of these organelles. Turnover of microbodies involves sequestration into autophagic vacuoles as intact organelles which still retain catalase activity. Crystalloids present in microbodies develop by condensation of matrix protein and are the principal site of catalase formerly in the matrix.

23Na and (1)H NMR microimaging of intact plants.

(23)Na NMR microimaging is described to map, for the first time, the sodium distribution in living plants. As an example, the response of 6-day-old seedlings of Ricinus communis to exposure to sodium chloride concentrations from 5 to 300 mM was observed in vivo using (23)Na as well as (1)H NMR microimaging. Experiments were performed at 11.75 T with a double resonant (23)Na-(1)H probehead. The probehead was homebuilt and equipped with a climate chamber. T(1) and T(2) of (23)Na were measured in the cross section of the hypocotyl. Within 85 min (23)Na images with an in-plane resolution of 156 x 156 micrometer were acquired. With this spatial information, the different types of tissue in the hypocotyl can be discerned. The measurement time appears to be short compared to the time scale of sodium uptake and accumulation in the plant so that the kinetics of salt stress can be followed. In conclusion, (23)Na NMR microimaging promises great potential for physiological studies of the consequences of salt stress on the macroscopic level and thus may become a unique tool for characterizing plants with respect to salt tolerance and salt sensitivity.

Transformation of nuclear DNA in meristematic and embryogenic tissues.

Particle bombardment/biolistic delivery is a very popular method of genetic transformation of diverse targets including cells and intact tissues. Delivery of DNA through particle bombardment is genotype and species independent, nevertheless, an efficient protocol for large-scale generation of transgenic plants through embryogenic tissues with a high (≥80%) shoot regeneration efficiency is a prerequisite. Young embryogenic tissues or multiple shoot buds in early stages of induction are the most suited target tissues for recovery of transgenic plants. We describe the protocol for delivery of foreign genes using particle delivery system (Biorad gene gun, PDS-1000/He) in to the meristematic tissues of embryonic axes derived from mature seeds of castor. With the optimized physical and biological parameters, putative transformants were obtained at a frequency of 1.4% through particle gun bombardment of castor embryo axes. Also, transformation of embryogenic calli of sorghum using particle inflow gun (PIG) is described.

Sugar concentrations along and across the Ricinus communis L. hypocotyl measured by single cell sampling analysis.

Single cell sap sampling and analysis were used to measure the longitudinal and radial distribution of sucrose, glucose and fructose in the apical cell division zone and in the basal, elongated zone of the Ricinus hypocotyl. Sucrose and hexose increased in concentration from the apex to the base of the seedling axis. In the cell division zone low hexose and sucrose concentrations prevailed in cortex and pith, with a slightly higher hexose concentration in pith cells. The sucrose concentrations in sieve tubes and in phloem were much higher than in the cortex and pith cells. In the basal zone of the hypocotyl high levels of sucrose in phloem, cortex and pith were found, therefore radial, diffusional sucrose flow away from the phloem was considered unlikely. It is proposed that radial flow of growth-water to the hypocotyl periphery together with the down-regulation of a sucrose transporter at the phloem leads to a preferential sucrose flow to the expanding cortex. The pith cells, which do not experience flow of growth-water, are probably insufficiently supplied with sucrose from the phloem resulting eventually in cell death as the plant grows. Shortage of sucrose supply, experimentally achieved by removal of the endosperm, led to sucrose hydrolysis in the pith. The sucrose levels in the other tissues decreased less. It appears that the hydrolysis to hexose was initiated to maintain the osmotic value in the pith cell sap. It is speculated that high hexose levels in the cells are indicative of insufficient sucrose supply via the phloem and that the pith cells are confronted with that situation during early seedling development.

Cloning and characterization of a lupeol synthase involved in the synthesis of epicuticular wax crystals on stem and hypocotyl surfaces of Ricinus communis.

Pentacyclic triterpenoids are a large group of secondary metabolites found in many different plant species, either as glycoside conjugates or as aglycones. The latter in many cases accumulate to high amounts in the cuticular wax and hence at the surface of plant organs. In the present work, the cuticle-specific formation of triterpenoids was investigated in Ricinus communis stems, combining analytical and molecular genetic methods. Two phenotypes of castor bean could be distinguished based on the glaucous or glossy appearance of the surfaces of all stem portions including the hypocotyls, and were due to the presence or absence of thread-shaped epicuticular wax crystals, respectively. Comparative studies showed that these crystals are formed by the triperpenoid lupeol, present in high amounts on all stem surfaces. On the hypocotyl portion of stems, lupeol was found to accumulate rapidly during early development of the surface (10-15 days after emergence). Mature hypocotyls of glossy individuals were covered with 12.5 microg/cm2 of wax containing approximately 1% of lupeol, whereas the glaucous phenotype had a wax load of 51.9 microg/cm2 with 56% of lupeol. Two oxidosqualene cyclases from castor bean were cloned, functionally expressed in yeast, and characterized as a cycloartenol synthase (RcCAS) and a lupeol synthase (RcLUS). Phylogenetic analyses revealed that RcLUS is similar to two clades of known lupeol synthases, but also exhibits some similarities with beta-amyrin synthases. Both the organ-specific expression of RcLUS and the expression pattern during hypocotyl development exactly matched the accumulation of cuticular lupeol in castor bean. In contrast, RcCAS was constitutively expressed in all organs at various times. We conclude that the RcLUS enzyme is responsible for formation of the cuticular lupeol, and thus for the characteristic surface properties of R. communis stems.

Ricinosomes and endosperm transfer cell structure in programmed cell death of the nucellus during Ricinus seed development.

The ricinosome (precursor protease vesicle) is an organelle found exclusively in plant cells. Ricinosomes contain a 45-kDa pro-cysteine endopeptidase (CysEP) with a C-terminal KDEL endoplasmic reticulum retention signal. CysEP is a member of a unique group of papain-type cysteine peptidases found specifically in senescing and ricinosome-containing tissues. During seed development in the castor oil plant (Ricinus communis L.), the cells of the nucellus are killed as the major seed storage organ, the cellular endosperm, expands and begins to accumulate reserves. The destruction of the maternal seed tissues is a developmentally programmed cell death. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling revealed that nuclear DNA fragmentation occurs in the nucellar cells adjacent to the expanding endosperm. These cells exhibit ultrastructural features consistent with programmed cell death, including vesiculation of the cytosol, development of irregularly shaped nuclei, vacuolar collapse, and shrinkage of the cytoplasm. Ricinosomes containing the CysEP were identified in the nucellar cells by light and electron microscopy and immunocytochemistry. Both proCysEP and mature CysEP are present in protein extracts of the nucellar tissues during seed development. Upon collapse of the nucellar cells, the content of the ricinosomes is released into the cytoplasm, where the activated CysEP digests the remaining proteinaceous cellular debris. Digestion products of the nucellar cells are presumed taken up by the outermost cells of the endosperm, which have labyrinthine ingrowths of the outer walls typical of transfer cells.

Leaf development in Ricinus communis during drought stress: dynamics of growth processes, of cellular structure and of sink-source transition.

Dicot leaf growth is characterized by partly transient tip-to-base gradients of growth processes, structure and function. These gradients develop dynamically and interact with dynamically developing stress conditions like drought. In Ricinus communis plants growing under well-watered and drought conditions growth rates peaked during the late night and minimal values occurred in the late afternoon. During this diurnal course the leaf base always showed much higher rates than the leaf tip. The amplitude of this diurnal course decreased when leaves approached maturity and during drought stress without any significant alteration of the diurnal pattern and it increased during the first days after rewatering. Unique relationships between leaf size and cytological structure were observed. This provided the framework for the analysis of changes in assimilation, transpiration and dark respiration, chlorophyll, protein, carbohydrate, and amino acid concentrations, and of activities of sink-source-related enzymes at the leaf tip and base during leaf development in well-watered and drought-stressed plants. Gas exchange was dominated by physiological rather than by anatomical properties (stomatal density). Tip-to-base gradients in carbohydrate concentrations per dry weight and sink-source-related enzymes were absent, whereas significant gradients were found in amino acid concentrations per dry weight. During drought stress, growing leaves developed source function at smaller leaf size, before specific physiological adaptations to drought occurred. The relevance of the developmental status of individual leaves for the drought-stress response and of the structural changes for the biochemical composition changes is discussed.

Ricinosomes: an organelle for developmentally regulated programmed cell death in senescing plant tissues.

This review describes aspects of programmed cell death (PCD). Present research maps the enzymes involved and explores the signal transduction pathways involved in their synthesis. A special organelle (the ricinosome) has been discovered in the senescing endosperm of germinating castor beans (Ricinus communis) that develops at the beginning of PCD and delivers large amounts of a papain-type cysteine endopeptidase (CysEP) in the final stages of cellular disintegration. Castor beans store oil and proteins in a living endosperm surrounding the cotyledons. These stores are mobilized during germination and transferred into the cotyledons. PCD is initiated after this transfer is complete. The CysEP is synthesized in the lumen of the endoplasmic reticulum (ER) where it is retained by its C-terminal KDEL peptide as a rather inactive pro-enzyme. Large number of ricinosomes bud from the ER at the same time as the nuclear DNA is characteristically fragmented during PCD. The mitochondria, glyoxysomes and ribosomes are degraded in autophagic vacuoles, while the endopeptidase is activated by removal of the propeptide and the KDEL tail and enters the cytosol. The endosperm dries and detaches from the cotyledons. A homologous KDEL-tailed cysteine endopeptidase has been found in several senescing tissues; it has been localized in ricinosomes of withering day-lily petals and dying seed coats. Three genes for a KDEL-tailed cysteine endopeptidase have been identified in Arabidopsis. One is expressed in senescing ovules, the second in the vascular vessels and the third in maturing siliques. These genes open the way to exploring PCD in plants.

Intracellular distribution of enzymes of the cytidine diphosphate choline pathway in castor bean endosperm.

The occurrence and subcellular distribution of enzymes of the cytidine diphosphate choline pathway of lecithin synthesis have been examined. Choline kinase (EC 2.7.1.32) was completely soluble, while phosphorylcholine-cytidyl transferase (EC 2.7.7.15) and phosphorylcholine-glyceride transferase (EC 2.7.8.2) were associated with particulate fractions. Although components sedimenting at 10,000 to 100,000 x g contained both enzymes, phosphorylcholine-cytidyl transferase and particularly phosphorylcholine-glyceride transferase were present in the 10,000 x g pellet, which contained the major organelles, mitochondria, and glyoxysomes. When the crude homogenate was centrifuged on a sucrose density gradient, four major bands of particulate protein were recovered. A band at density 1.24 g/cm(3) contained the glyoxysomes and was devoid of phosphorylcholine-cytidyl transferase and phosphorylcholine-glyceride transferase activity. Enzyme activity was barely detectable in the mitochondria, at density 1.18 g/cm(2). Phosphorylcholine-glyceride transferase was found almost exclusively in a sharp band at density 1.12 g/cm(3), and phosphorylcholinecytidyl transferase was found in the uppermost band at density 1.08 g/cm(3). Thus, for the synthesis of lecithin in their membranes, the glyoxysomes and mitochondria depend on enzymes elsewhere in the cell; the final two steps in lecithin formation occur, apparently exclusively, in separate particulate cell components.

The ricinosomes of senescing plant tissue bud from the endoplasmic reticulum.

The ricinosome (synonym, precursor protease vesicle) is a novel organelle, found so far exclusively in plant cells. Electron microscopic studies suggest that it buds off from the endoplasmic reticulum in senescing tissues. Biochemical support for this unusual origin now comes from the composition of the purified organelle, which contains large amounts of a 45-kDa cysteine endoprotease precursor with a C-terminal KDEL motif and the endoplasmic reticulum lumen residents BiP (binding protein) and protein disulfide isomerase. Western blot analysis, peptide sequencing, and mass spectrometry demonstrate retention of KDEL in the protease proform. Acidification of isolated ricinosomes causes castor bean cysteine endopeptidase activation, with cleavage of the N-terminal propeptide and the C-terminal KDEL motif. We propose that ricinosomes accumulate during senescence by programmed cell death and are activated by release of protons from acidic vacuoles.