Unveiling Interactions among Mitochondria,Caspase-Like Proteases,and the Actin Cytoskeleton during Plant Programmed Cell Death (PCD)

Aponogeton madagascariensis produces perforations over its leaf surface via programmed cell death (PCD). PCD begins between longitudinal and transverse veins at the center of spaces regarded as areoles, and continues outward, stopping several cells from these veins. The gradient of PCD that exists within a single areole of leaves in an early stage of development was used as a model to investigate cellular dynamics during PCD. Mitochondria have interactions with a family of proteases known as caspases, and the actin cytoskeleton during metazoan PCD; less is known regarding these interactions during plant PCD. This study employed the actin stain Alexa Fluor 488 phalloidin, the actin depolymerizer Latrunculin B (Lat B), a synthetic caspase peptide substrate and corresponding specific inhibitors, as well as the mitochondrial pore inhibitor cyclosporine A (CsA) to analyze the role of these cellular constituents during PCD. Results depicted that YVADase (caspase-1) activity is higher during the very early stages of perforation formation, followed by the bundling and subsequent breakdown of actin. Actin depolymerization using Lat B caused no change in YVADase activity. In vivo inhibition of YVADase activity prevented PCD and actin breakdown, therefore substantiating actin as a likely substrate for caspase-like proteases (CLPs). The mitochondrial pore inhibitor CsA significantly decreased YVADase activity, and prevented both PCD and actin breakdown; therefore suggesting the mitochondria as a possible trigger for CLPs during PCD in the lace plant. To our knowledge, this is the first in vivo study using either caspase-1 inhibitor (Ac-YVAD-CMK) or CsA, following which the actin cytoskeleton was examined. Overall, our findings suggest the mitochondria as a possible upstream activator of YVADase activity and implicate these proteases as potential initiators of actin breakdown during perforation formation via PCD in the lace plant.     

Leaf‐shape remodeling: programmed cell death in fistular leaves of Allium fistulosum

Some species of Allium in Liliaceae have fistular leaves. The fistular lamina of Allium fistulosum undergoes a process from solid to hollow during development. The aims were to reveal the process of fistular leaf formation involved in programmed cell death (PCD) and to compare the cytological events in the execution of cell death to those in the unusual leaf perforations or plant aerenchyma formation. In this study, light and transmission electron microscopy were used to characterize the development of fistular leaves and cytological events. Terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL) assays and gel electrophoresis were used to determine nuclear DNA cleavage during the PCD. The cavity arises in the leaf blade by degradation of specialized cells, the designated pre‐cavity cells, in the center of the leaves. Nuclei of cells within the pre‐cavity site become TUNEL‐positive, indicating that DNA cleavage is an early event. Gel electrophoresis revealed that DNA internucleosomal cleavage occurred resulting in a characteristic DNA ladder. Ultrastructural analysis of cells at the different stages showed disrupted vacuoles, misshapen nuclei with condensed chromatin, degraded cytoplasm and organelles and emergence of secondary vacuoles. The cell walls degraded last, and residue of degraded cell walls aggregated together. These results revealed that PCD plays a critical role in the development of A. fistulosum fistular leaves. The continuous cavity in A. fistulosum leaves resemble the aerenchyma in the pith of some gramineous plants to improve gas exchange.     

益智胚珠的珠心冠原与承珠盘细胞壁的组织化学研究

用组织化学方法研究了益智胚珠中珠心冠原与承珠盘细胞壁的组成。珠心冠原细胞壁含有纤维素、胼胝质、果胶质,但不含栓质。承珠盘细胞壁含有纤维素、木质素、果胶质,也不含栓质。讨论了珠心冠原与承珠盘细胞壁的组成及承珠盘的可能功能。     

Programmed cell death and leaf morphogenesis in <Emphasis Type="Italic">Monstera obliqua</Emphasis> (Araceae)

The unusual perforations in the leaf blades of Monstera obliqua (Araceae) arise through programmed cell death early in leaf development. At each perforation site, a discrete subpopulation of cells undergoes programmed cell death simultaneously, while neighboring protoderm and ground meristem cells are unaffected. Nuclei of cells within the perforation site become terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive, indicating that DNA cleavage is an early event. Gel electrophoresis indicates that DNA cleavage is random and does not result in bands that represent multiples of internucleosomal units. Ultrastructural analysis of cells at the same stage reveals misshapen, densely stained nuclei with condensed chromatin, disrupted vacuoles, and condensed cytoplasm. Cell walls within the perforation site remain intact, although a small disk of dying tissue becomes detached from neighboring healthy tissues as the leaf expands and stretches the minute perforation. Exposed ground meristem cells at the rim of the perforation differentiate as epidermal cells. The cell biology of perforation formation in Monstera resembles that in the aquatic plant Aponogeton madagascariensis (Aponogetonaceae; Gunawardena et al. 2004), but the absence of cell wall degradation and the simultaneous execution of programmed cell death throughout the perforation site reflect the convergent evolution of this distinct mode of leaf morphogenesis in these distantly related plants.     

The lace plant: a novel model system to study plant proteases during developmental programmed cell death in vivo

Programmed cell death (PCD) plays a major role in plant development and defense throughout the plant kingdom. Within animal systems, it is well accepted that caspases play a major role in the PCD process, although no true caspases have yet to be identified in plants. Despite this, vast amounts of evidence suggest the existence of caspase-like proteases in plants. The lace plant (Aponogeton madagascariensis) forms perforations in a predictable pattern between longitudinal and transverse veins over its entire leaf surface via PCD. Due to the thin nature of the leaf, allowing for long-term live cell imaging, a perfected method for sterile culture, as well as the feasibility of pharmacological experiments, the lace plant provides an excellent model to study developmental PCD. In this review, we report the suitability of the lace plant as a novel organism to study proteases in vivo during developmentally regulated cell death.     

Apoplastic polyesters in Arabidopsis surface tissues--a typical suberin and a particular cutin

Cutinized and suberized cell walls form physiological important plant-environment interfaces as they act as barriers limiting water and nutrient loss and protect from radiation and invasion by pathogens. Due to the lack of protocols for the isolation and analysis of cutin and suberin in Arabidopsis, the model plant for molecular biology, mutants and transgenic plants with a defined altered cutin or suberin composition are unavailable, causing that structure and function of these apoplastic barriers are still poorly understood. Transmission electron microscopy (TEM) revealed that Arabidopsis leaf cuticle thickness ranges from only 22 nm in leaf blades to 45 nm on petioles, causing the difficulty in cuticular membrane isolation. We report the use of polysaccharide hydrolases to isolate Arabidopsis cuticular membranes, suitable for depolymerization and subsequent compositional analysis. Although cutin characteristic omega-hydroxy acids (7%) and mid-chain hydroxylated fatty acids (8%) were detected, the discovery of alpha,omega-diacids (40%) and 2-hydroxy acids (14%) as major depolymerization products reveals a so far novel monomer composition in Arabidopsis cutin, but with chemical analogy to root suberin. Histochemical and TEM analysis revealed that suberin depositions were localized to the cell walls in the endodermis of primary roots and the periderm of mature roots of Arabidopsis. Enzyme digested and solvent extracted root cell walls when subjected to suberin depolymerization conditions released omega-hydroxy acids (43%) and alpha,omega-diacids (24%) as major components together with carboxylic acids (9%), alcohols (6%) and 2-hydroxyacids (0.1%). This similarity to suberin of other species indicates that Arabidopsis roots can serve as a model for suberized tissue in general.     

Apoplasmic barriers and oxygen transport properties of hypodermal cell walls in roots from four amazonian tree species

The formation of suberized and lignified barriers in the exodermis is suggested to be part of a suite of adaptations to flooded or waterlogged conditions, adjusting transport of solutes and gases in and out of roots. In this study, the composition of apoplasmic barriers in hypodermal cell walls and oxygen profiles in roots and the surrounding medium of four Amazon tree species that are subjected to long-term flooding at their habitat was analyzed. In hypodermal cell walls of the deciduous tree Crateva benthami, suberization is very weak and dominated by monoacids, 2-hydroxy acids, and omega-hydroxycarboxylic acids. This species does not show any morphological adaptations to flooding and overcomes the aquatic period in a dormant state. Hypodermal cells of Tabernaemontana juruana, a tree which is able to maintain its leaf system during the aquatic phase, are characterized by extensively suberized walls, incrusted mainly by the unsaturated C(18) omega-hydroxycarboxylic acid and the alpha,omega-dicarboxylic acid analogon, known as typical suberin markers. Two other evergreen species, Laetia corymbulosa and Salix martiana, contained 3- to 4-fold less aliphatic suberin in the exodermis, but more than 85% of the aromatic moiety of suberin are composed of para-hydroxybenzoic acid, suggesting a function of suberin in pathogen defense. No major differences in the lignin content among the species were observed. Determination of oxygen distribution in the roots and rhizosphere of the four species revealed that radial loss of oxygen can be effectively restricted by the formation of suberized barriers but not by lignification of exodermal cell walls.     

Programmed cell death and tissue remodelling in plants

The use of programmed cell death (PCD) to remodel plants at the cellular, tissue, and organ levels is particularly fascinating and occurs in such processes as tracheary element differentiation, lysigenous aerenchyma formation, development of functionally unisexual flowers from bisexual floral primordia, and leaf morphogenesis. The formation of complex leaf shape through the use of PCD is a rare event across vascular plants and occurs only in a few species of Monstera and related genera, and in the lace plant (Aponogeton madagascariensis). During early development, the lace plant leaf forms a pattern of equidistantly positioned perforations across the surface of the leaf, giving it a lattice-like appearance. Due to the accessibility and predictability of this process, the lace plant provides highly suitable material for the study of developmentally regulated PCD in plants. A sterile lace plant culture system has been successfully established, providing material free of micro-organisms for experimental study. The potential role of ethylene and caspase-like activity in developmentally regulated PCD in the lace plant is currently under investigation, with preliminary results indicating that both may play a role in the cell death pathway.     

Chemical composition and ultrastructure of broad bean (<Emphasis Type="Italic">Vicia faba</Emphasis> L.) nodule endodermis in comparison to the root endodermis

Ultrastructure and development of apoplastic barriers within indeterminate root nodules formed by Vicia faba L. were examined by light and electron microscopy. The nodule outer cortex is separated from the inner cortex by a heavily suberized nodule endodermis, which matures in submeristematic regions and possesses suberin lamellae. Unsuberized passage cells are present near vascular strands, which are surrounded by a vascular endodermis attached on the inner side of the nodule endodermal cell walls. The vascular endodermis appears immediately below the meristematic apex in developmental state I (Casparian bands), gradually develops suberin lamellae, and attains developmental state II at the base of the nodule. For chemical analysis apoplastic barrier tissues were dissected after enzymatic digestion of non-impregnated tissues. Root epidermal and endodermal cell walls as well as nodule outer cortex could be isolated as pure fractions; nodule endodermal cell walls could not be separated from vascular endodermal cell walls and enclosed xylem vessels. Gas chromatography-flame ionization detection and gas chromatography-mass spectrometry were applied for quantitative and qualitative analysis of suberin and lignin in isolated cell walls of these tissues. The suberin content of isolated endodermal cell walls of nodules was approximately twice that of the root endodermal cell walls. The suberin content of the nodule outer cortex and root epidermal cell walls was less than one-tenth of that of the nodule endodermal cell wall. Substantial amounts of lignin could only be found in the nodule endodermal cell wall fraction. Organic solvent extracts of the isolated tissues revealed long-chain aliphatic acids, steroids, and triterpenoid structures of the lupeol type. Surprisingly, extract from the outer cortex consisted of 89% triterpenoids whereas extracts from all other cell wall isolates contained not more than 16% total triterpenoids. The results of ultrastructural and chemical composition are in good correspondence and underline the important role of the examined tissues as apoplastic barriers.     

Accumulation of aluminium in the cell wall pectin in cultured tobacco (Nicotiana tabacum L.) cells treated with a combination of aluminium and iron

In nutrient medium, aluminium (Al) accumulation in tobacco cells occurs only in the presence of ferrous ion [Fe(II)]. The localization of Al was examined to elucidate a mechanism of Al accumulation. After the digestion of Al-treated cells with cellulase and pectolyase together, the resulting spheroplasts contained as much Al as the intact cells. However, the cell walls isolated from Al-treated cells also contained as much Al as the intact cells. Comparison of sugar and Al contents in polysaccharide components extracted chemically from cell walls isolated from intact cells and spheroplasts revealed that the enzymes digested most of the cellulose and hemicellulose, but only half of the pectin, and that Al mainly existed in the pectin remaining in the spheroplasts. Gel-permeation chromatography of the pectin fraction (NH₄-oxalate extract) from the cell walls of the intact cells indicated that Al was associated with small polysaccharides of approximately 3–7 kDa. These results suggest that a minor part of pectin is a major site of Al accumulation. The content of cell wall pectin increased during Al treatment in nutrient medium. Taken together, we hypothesize that Al may bind to the pectin newly produced during Al treatment.     

Alternative modes of leaf dissection in monocotyledons

Although a majority of monocotyledons have simple leaves, pinnately or palmately dissected blades are found in four orders, the Alismatales, Pandanales, Dioscoreales and Arecales. Independent evolutionary origins of leaf dissection are indicated by phylogenetic analyses and are reflected in the diversity of mechanisms employed during leaf development. The mechanism of blastozone fractionation through localized enhancement and suppression of growth of the free margin of the leaf primordium occurs in the Araceae and Dioscoreaceae. By contrast, the corrugated, dissected leaves of palms (Arecaceae) develop through a two-step process: first, plications are formed through intercalary growth in a submarginal position and, second, the initially simple leaf blade is dissected through an abscission-like process of leaflet separation. A third mechanism, perforation formation, is employed in Monstera and five related genera of the Araceae. In this mode, discrete patches of cells undergo programmed cell death during lamina development, resulting in formation of open perforations. When perforations are positioned near the leaf margin, mechanical disruption of the thin bridges of marginal tissue results in a deeply pinnatisect blade. Whereas blastozone fractionation defines the early primary morphogenesis phase of leaf development, the other two modes occur later, during the secondary morphogenesis/histogenesis phase. Evolution of these mechanisms presumably has involved recruitment of other developmental programmes into the development of dissected leaves.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 150 , 25–44.     

Tissue Specific Localization of Pectin–Ca2+ Cross-Linkages and Pectin Methyl-Esterification during Fruit Ripening in Tomato (Solanum lycopersicum)

Fruit ripening is one of the developmental processes accompanying seed development. The tomato is a well-known model for studying fruit ripening and development, and the disassembly of primary cell walls and the middle lamella, such as through pectin de-methylesterified by pectin methylesterase (PE) and depolymerization by polygalacturonase (PG), is generally accepted to be one of the major changes that occur during ripening. Although many reports of the changes in pectin during tomato fruit ripening are focused on the relation to softening of the pericarp or the Blossom-end rot by calcium (Ca²⁺) deficiency disorder, the changes in pectin structure and localization in each tissues during tomato fruit ripening is not well known. In this study, to elucidate the tissue-specific role of pectin during fruit development and ripening, we examined gene expression, the enzymatic activities involved in pectin synthesis and depolymerisation in fruit using biochemical and immunohistochemical analyses, and uronic acids and calcium (Ca)-bound pectin were determined by secondary ion-microprobe mass spectrometry. These results show that changes in pectin properties during fruit development and ripening have tissue-specific patterns. In particular, differential control of pectin methyl-esterification occurs in each tissue. Variations in the cell walls of the pericarp are quite different from that of locular tissues. The Ca-binding pectin and hairy pectin in skin cell layers are important for intercellular and tissue–tissue adhesion. Maintenance of the globular form and softening of tomato fruit may be regulated by the arrangement of pectin structures in each tissue.     

Overexpression of polygalacturonase in transgenic apple trees leads to a range of novel phenotypes involving changes in cell adhesion

Polygalacturonases (PGs) cleave runs of unesterified GalUA that form homogalacturonan regions along the backbone of pectin. Homogalacturonan-rich pectin is commonly found in the middle lamella region of the wall where two adjacent cells abut and its integrity is important for cell adhesion. Transgenic apple (Malus domestica Borkh. cv Royal Gala) trees were produced that contained additional copies of a fruit-specific apple PG gene under a constitutive promoter. In contrast to previous studies in transgenic tobacco (Nicotiana tabacum) where PG overexpression had no effect on the plant (K.W. Osteryoung, K. Toenjes, B. Hall, V. Winkler, A.B. Bennett [1990] Plant Cell 2: 1239-1248), PG overexpression in transgenic apple led to a range of novel phenotypes. These phenotypes included silvery colored leaves and premature leaf shedding due to reduced cell adhesion in leaf abscission zones. Mature leaves had malformed and malfunctioning stomata that perturbed water relations and contributed to a brittle leaf phenotype. Chemical and ultrastructural analyses were used to relate the phenotypic changes to pectin changes in the leaf cell walls. The modification of apple trees by a single PG gene has offered a new and unexpected perspective on the role of pectin and cell wall adhesion in leaf morphology and stomatal development.     

Suberin--a biopolyester forming apoplastic plant interfaces

Suberized cell walls form physiologically important plant-environment interfaces because they act as barriers that limit water and nutrient transport and protect plants from invasion by pathogens. Plants respond to environmental stimuli by modifying the degree of suberization in root cell walls. Salt stress or drought-induced suberization leads to a decrease in radial water transport in roots. Although reinforced, suberized cell walls never act as absolutely impermeable barriers. Deeper insights into the structure and biosynthesis of suberin are required to elucidate what determines the barrier properties. Progress has been obtained from analytical methods that enabled the structural characterization of oligomeric building blocks in suberin, and from the opening of suberin research to molecular genetic approaches by the elucidation of the chemical composition and tissue distribution of suberin in the model species Arabidopsis.     

ANATOMY OF SOME LEAF GALLS OF ROSA WOODSII (ROSACEAE)

Infection of Rosa woodsii by some members of the order Hymenoptera results in neoplasmic outgrowths on the leaves. One type of outgrowth produces a spherical swelling (leaf gall) while the other has extensive hair-like proliferations (hairy gall). The anatomy and ultrastructure of these galls were examined by light microscopy and transmission electron microscopy. The leaf gall cells were considerably larger than normal cells, lacked well-developed chloroplasts and were loosely arranged with prominent intercellular spaces. Vascular bundles were scattered throughout the gall tissue. The upper three cell layers of the leaf gall tissue resembles a periderm, having many suberin lamellae. The suberin lamellae were often traversed by pores which may represent incomplete plasmodesmata. Phenolic compounds were commonly seen both in the normal and gall cells. A layer of internal cells of the hairy galls have remarkably thickened cell walls, presumably due to the deposition of cellulosic substances. Unlike leaf galls, the epidermal cells of the hairy galls were not heavily cuticularized and no periderm was found. The hair-like outgrowths present on the outer surface of these galls had a central vascular bundle. The epidermis of the outgrowths also had thickened cell walls, and trichomes occurred on the outer surface. The structural modifications brought about by the insect invasion in these two galls are compared and their roles in gall formation are discussed.     

Programmed cell death remodels lace plant leaf shape during development

Programmed cell death (PCD) functions in the developmental remodeling of leaf shape in higher plants, a process analogous to digit formation in the vertebrate limb. In this study, we provide a cytological characterization of the time course of events as PCD remodels young expanding leaves of the lace plant. Tonoplast rupture is the first PCD event in this system, indicated by alterations in cytoplasmic streaming, loss of anthocyanin color, and ultrastructural appearance. Nuclei become terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling positive soon afterward but do not become morphologically altered until late stages of PCD. Genomic DNA is fragmented, but not into internucleosomal units. Other cytoplasmic changes, such as shrinkage and degradation of organelles, occur later. This form of PCD resembles tracheary element differentiation in cytological execution but requires unique developmental regulation so that discrete panels of tissue located equidistantly between veins undergo PCD while surrounding cells do not.     

Studies onArtemisia afra Jacq.: The phloem in stem and leaf

Summary The structure of the phloem was studied in stem and leaf ofArtemisia afra Jacq., with particular attention being given to the sieve element walls. Both primary and secondary sieve elements of stem and midvein have nacreous walls, which persist in mature cells. Histochemical tests indicated that the sieve element wall layers contained some pectin. Sieve element wall layers lack lignin. Sieve elements of the minor veins (secondary and tertiary veins) lack nacreous thickening, although their walls may be relatively thick. These walls and those of contiguous transfer cells are rich in pectic substances. Transfer cell wall ingrowths are more highly developed in tertiary than in secondary veins.     

The role of the parenchyma sheath and PCD during the development of oil cavities in Pterodon pubescens (Leguminosae-Papilionoideae)

Pterodon pubescens cavities are constituted by lumen and uniseriated epithelium surrounded by multiseriate parenchyma sheath. We studied the development of secretory cavities, including the role of parenchyma sheath, using light and transmission electron microscopy. A Tunel assay was performed to verify whether programmed cell death (PCD) occurs during the process. The lumen is formed by schizogeny and lysigeny occur in later developmental stages of the secretory cavities. Ultrastructurally, epithelial cells in later developmental stages become dark and with sinuous walls; the protoplast becomes retracted and the cytoplasm shows low organelle definition. Degenerated cells are released toward the lumen. Our results showed that PCD occurs during later developmental stages of cavities and plays a critical role in functioning of these glands. New cells originated from the parenchyma sheath differentiate into secretory cells and replace those degenerated ones. This fact associated to PCD guarantees epithelium renovation during the secretory cycle and the maintenance of secretory activity of cavities.     

The primary walls of cotton fibers contain an ensheathing pectin layer

Summary Cotton fiber walls (1–2 days post anthesis) are distinctly bilayered compared to those of nonfiber epidermal cells, with a more electron-opaque outer layer and a less electron-opaque, more finely fibrillar inner layer. When probed with antibodies and affinity probes to various saccharides, xyloglucans and cellulose are found exclusively in the inner layer and de-esterified pectins and extensin exclusively in the outer layer. Ovular epidermal cells that do not differentiate into fibers have no pectin sheath, but are labelled throughout with antixyloglucan and cellulase-gold probes. Middle lamellae between adjacent cells were clearly labelled with the antibodies to de-esterified pectins, however. Similarly, cell walls of leaf trichomes have a bilayered wall strongly enriched in pectin, whereas other epidermal cells are not bilayered and are pectin poor. These data indicate that one of the early markers of fiber and trichome cells from other epidermal cells involves the production of a pectin layer. The de-esterified pectins present in the ensheathing layer may allow for expansion and elongation of the fiber cells that does not occur in the other epidermal cells without such a sheath or may even be a consequence of the elongation process.     

Immunocytochemical detection of rhamnogalacturonan II on forming cell plates in cultured tobacco cells

Rhamnogalacturonan II (RG-II) is a region of pectin macromolecules that is present in plant primary cell walls. The RG-II region serves as the site of borate cross-linking within pectin, via which pectin macromolecules link together to form a gel. In this study, we examined whether RG-II is present in the cell plate, the precursor of primary cell walls that forms during cytokinesis. A structure inside dividing cells was labeled with a rabbit polyclonal anti-RG-II antibody and detected by immunofluorescence microscopy. An antibody against callose, a marker polysaccharide for the cell plate, also labeled the structure. In immunoelectron microscopy analyses using the anti-RG-II antibody, gold particles were distributed in electron-lucent vesicular structures that appeared to correspond to the forming cell plates in late anaphase cells. Together, these results suggest that RG-II is present in cell plates from the early phase of their assembly.