Nitric oxide and hydrogen peroxide involvement during programmed cell death of Sechium edule nucellus

The nucellus is a maternal tissue that feeds the developing embryo and the secondary endosperm. During seed development the cells of the nucellus suffer a degenerative process early after fertilization as the cellular endosperm expands and accumulates reserves. Nucellar cell degeneration has been characterized as a form of developmentally programmed cell death (PCD). In this work we show that nucellus PCD is accompanied by a considerable production of both nitric oxide and hydrogen peroxide (NO and H₂O₂). Interestingly, each of the two molecules is able to induce the production of the other and to cause cell death when applied to a living nucellus. We show that the induced cell death has features of a PCD, accompanied by profound changes in the morphology of the nuclei and by a massive degradation of nuclear DNA. Moreover, we report that NO and H₂O₂ cause an induction of caspase‐like proteases previously characterized in physiological nucellar PCD.     

Caspase-Like Activities Accompany Programmed Cell Death Events in Developing Barley Grains

Programmed cell death is essential part of development and cell homeostasis of any multicellular organism. We have analyzed programmed cell death in developing barley caryopsis at histological, biochemical and molecular level. Caspase-1, -3, -4, -6 and -8-like activities increased with aging of pericarp coinciding with abundance of TUNEL positive nuclei and expression of HvVPE4 and HvPhS2 genes in the tissue. TUNEL-positive nuclei were also detected in nucellus and nucellar projection as well as in embryo surrounding region during early caryopsis development. Quantitative RT-PCR analysis of micro-dissected grain tissues revealed the expression of HvVPE2a, HvVPE2b, HvVPE2d, HvPhS2 and HvPhS3 genes exclusively in the nucellus/nucellar projection. The first increase in cascade of caspase-1, -3, -4, -6 and -8-like activities in the endosperm fraction may be related to programmed cell death in the nucellus and nucellar projection. The second increase of all above caspase-like activities including of caspase-9-like was detected in the maturating endosperm and coincided with expression of HvVPE1 and HvPhS1 genes as well as with degeneration of nuclei in starchy endosperm and transfer cells. The distribution of the TUNEL-positive nuclei, tissues-specific expression of genes encoding proteases with potential caspase activities and cascades of caspase-like activities suggest that each seed tissue follows individual pattern of development and disintegration, which however harmonizes with growth of the other tissues in order to achieve proper caryopsis development.     

Caspase-like activity in the seedlings of Pisum sativum eliminates weaker shoots during early vegetative development by induction of cell death

Activation of aspartate-specific cysteine proteases (caspases) plays a crucial role in programmed cell death (PCD) in animals. Although to date caspases have not been identified in plants, caspase-like activity was described in tobacco during a hypersensitive response to pathogens and in Arabidopsis and tomato cell cultures during chemical-induced PCD. Caspase-like activity was also detected in the course of plant development during petal senescence and endosperm PCD. It is shown here that caspase-like proteases play a crucial role in the developmental cell death of secondary shoots of pea seedlings that emerge after removal of the epicotyl. Caspase-like activity was induced in senescing secondary shoots, but not in dominant growing shoots, in contrast to the papain-like cysteine protease activity that was stronger in the dominant shoot. Revitalization of the senescing shoot by cutting of the dominant shoot reduced the caspase-like activity. Injection of caspase or cysteine protease inhibitors into the remaining epicotyl tissue suppressed the death of the secondary shoots, producing seedlings with two equal shoots. These results suggest that shoot selection in pea seedlings is controlled by PCD, through the activation of caspase-like proteases.     

Developmental regulation of a VEIDase caspase-like proteolytic activity in barley caryopsis

Caspases are essential in animal programmed cell death both as initiator and executioner proteases. Plants do not have close caspase homologues, but several instances of caspase-like proteolytic activity have been demonstrated in connection with programmed cell death in plants. It was asked if caspase-like proteases are involved during development of the barley caryopsis. The presence of a caspase-6-like proteolytic activity that preferentially cleaved the sequence VEID was demonstrated. A range of protease inhibitors was tested and only caspase-specific inhibitors showed major inhibitory effects. The profile of VEIDase activity in developing starchy endosperm, embryo, and whole caryopsis was measured and showed a general trend of higher activity in young, rapidly developing tissues. The VEIDase activity was localized in vivo to vesicles, shown to be autophagosomes, in randomly distributed cells of the starchy endosperm. The VEIDase activity detected in barley caryopsis is similar to activities described previously in mammals, spruce, yeast, and thale cress. In mammals, spruce, and yeast, VEIDase activity has been shown to be positively correlated with the occurrence of programmed cell death. Several manifestations of programmed cell death exist in developing barley caryopsis, indicating a connection between VEIDase activity and developmental programmed cell death in barley.     

Development of maternal seed tissue in barley is mediated by regulated cell expansion and cell disintegration and coordinated with endosperm growth

After fertilization, filial grain organs are surrounded by the maternal nucellus embedded within the integuments and pericarp. Rapid early endosperm growth must be coordinated with maternal tissue development. Parameters of maternal tissue growth and development were analysed during early endosperm formation. In the pericarp, cell proliferation is accomplished around the time of fertilization, followed by cell elongation predominantly in longitudinal directions. The rapid cell expansion coincides with endosperm cellularization. Distribution of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)-positive nuclei reveals distinct patterns starting in the nucellus at anthesis and followed later by the inner cell rows of the pericarp, then spreading to the whole pericarp. The pattern suggests timely and spatially regulated programmed cell death (PCD) processes in maternal seed tissues. When the endosperm is coenocytic, PCD events are only observed within the nucellus. Thereby, remobilization of nucellar storage compounds by PCD could nourish the early developing endosperm when functional interconnections are absent between maternal and filial seed organs. Specific proteases promote PCD events. Characterization of the barley vacuolar processing enzyme (VPE) gene family identified seven gene members specifically expressed in the developing grain. HvVPE2a (known as nucellain) together with closely similar HvVPE2b and HvVPE2d might be involved in nucellar PCD. HvVPE4 is strongly cell specific for pericarp parenchyma. Correlative evidence suggests that HvVPE4 plays a role in PCD events in the pericarp. Possible functions of PCD in the maternal tissues imply a potential nutritive role or the relief of a physical restraint for endosperm growth. PCD could also activate post-phloem transport functions.     

The nucellus degenerates by a process of programmed cell death during the early stages of wheat grain development

The nucellus, which is the maternal tissue of the wheat grain, degenerates during the early stages of development. We have investigated whether or not this degenerative process may be considered as programmed cell death (PCD). The analysis of DNA of tissues dissected from developing wheat (Triticum aestivum L. cv Chinese Spring) grains at 5-20 days post anthesis (dpa) showed the presence of DNA laddering, which is indicative of internucleosomal fragmentation of nuclear DNA, in maternal tissues but not in the endosperm. The TUNEL assay showed in-situ internucleosomal fragmentation of DNA in nuclei of parenchymal and epidermal cells of the nucellus, as well as in the pericarp, during the early stages of grain development (5 dpa). Furthermore, internucleosomal fragmentation of nuclear DNA was observed in nucellar projection cells in the middle stages of grain development (13-18 dpa), thus showing a process of PCD in these maternal tissues. Electron-transmission microscopy analysis allowed the morphology of PCD to be characterized in this plant tissue. Initially, fragmentation of the cytoplasm was observed, the nuclear envelope appeared dilated and to be forming vacuoles, and the content of heterochromatin increased. A progressive degradation of the cytosolic contents and organelles was observed, and the plasma membrane was disrupted. However, the Golgi apparatus remained intact and apparently functional even in the final stages of cell death.     

The nucellus: between cell elimination and sugar transport

The architecture of the seed is shaped by the processes of tissue partitioning, which determines the volume ratio of maternal and zygotic tissues, and nutrient partitioning, which regulates nutrient distribution among tissues. In angiosperms, early seed development is characterized by antagonistic development of the nucellus maternal tissue and the endosperm fertilization product to become the main sugar sink. This process marked the evolution of angiosperms and outlines the most ancient seed architectures. In Arabidopsis, the endosperm partially eliminates the nucellus and imports sugars from the seed coat. Here, we show that the nucellus is symplasmically connected to the chalaza, the seed nutrient unloading zone, and works as both a sugar sink and source alongside the seed coat. After fertilization, the transient nucellus accumulates starch early on and releases it in the apoplasmic space during its elimination. By contrast, the persistent nucellus exports sugars toward the endosperm through the SWEET4 hexose facilitator. Finally, we analyzed sugar metabolism and transport in the transparent testa 16 mutant, which fails to undergo nucellus cell elimination, which shed light on the coordination between tissue and nutrient partitioning. Overall, this study identifies a path of sugar transport in the Arabidopsis seed and describes a link between sugar redistribution and the nucellus cell-elimination program.

A path of sugar transport through the nucellus maternal tissue of the Arabidopsis seed is coordinated with the process of nucellus cell elimination.     

Caspase-like proteases and their role in programmed cell death in plants

The investigations performed over recent few years have proved the existence of caspase-like proteases in plants. Three groups of caspase-like proteases: metacaspases, legumain family proteases (VPEs) and saspases have been identified and characterized in plants so far. A considerable amount of evidence supports the role of these enzymes in programmed cell death (PCD) occurring during plant development, their organ senescence as well as hypersensitive response (HR) after pathogen attack. Current knowledge of these enzyme molecular and biochemical structures is summarized in the paper. The homology of caspase-like proteases to animal caspases has been also indicated. Some future perspectives of research concerning the signal pathway during PCD, the regulation of activity and mode of action of these proteases are presented in the article.     

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.     

Caspase-like protease involvement in the control of plant cell death

Cell death as a highly regulated process has now been recognized to be an important, if not essential, pathway that is ubiquitous in all multicellular eukaryotes. In addition to playing key roles in the morphogenesis and sculpting of the organs to give rise to highly specialized forms and shapes, cell death also participates in the programmed creation of specialized cell types for essential functions such as the selection of B cells in the immune system of mammals and the formation of tracheids in the xylem of vascular plants. Studies of apoptosis, the most well-characterized form of animal programmed cell death, have culminated in the identification of a central tripartite death switch the enzymatic component of which is a conserved family of cysteine proteases called caspases. Studies in invertebrates and other animal models suggest that caspases are conserved regulators of apoptotic cell death in all metazoans. In plant systems, the identities of the main executioners that orchestrate cell death remain elusive. Recent evidence from inhibitor studies and biochemical approaches suggests that caspase-like proteases may also be involved in cell death control in higher plants. Furthermore, the mitochondrion and reactive oxygen species may well constitute a common pathway for cell death activation in both animal and plant cells. Cloning of plant caspase-like proteases and elucidation of the mechanisms through which mitochondria may regulate cell death in both systems should shed light on the evolution of cell death control in eukaryotes and may help to identify essential components that are highly conserved in eukaryotes.     

A VPE-like protease NtTPE8 exclusively expresses in the integumentary tapetum and is involved in seed development

Programmed cell death(PCD) is an essential process for development, and shows conserved cytological features in both plants and animals. Caspases are well-known critical components of the PCD machinery in animals. However, currently few typical counterparts have been identified in plants and only several caspase-like proteases are known to be involved in plant PCD, indicating the existence of great challenge for confirming new caspase-like proteases and elucidating the mechanisms regulating plant PCD. Here, we report a novel cysteine protease, NtTPE8, which was extracted from tobacco seeds and confirmed as a new caspase-like protease.Recombinant NtTPE8 exhibited legumain and caspase-like proteolytic activities, both of which could be inhibited by the pan-caspase inhibitor(Z-VAD-FMK). Notably, NtTPE8 possessed several caspase activities and the capacity to cleave the cathepsin H substrate FVR, indicating a unique character of NtTPE8. NtTPE8 was exclusively expressed in the integumentary tapetum and thus, is the first specific molecular marker reported to date for this cell type. Downregulation of NtTPE8 caused seed abortion, via disturbing early embryogenesis, indicating its critical role in embryogenesis and seed development. In conclusion, we identified a novel caspase-like cysteine protease, NtTPE8,exclusively expressed in the integumentary tapetum that is involved in seed development.     

Plant caspase-like proteases in plant programmed cell death

Programmed cell death (PCD) is a genetically-controlled disassembly of the cell. In animal systems, the central core execution switch for apoptotic PCD is the activation of caspases (Cysteine-containing Aspartate-specific proteases). Accumulating evidence in recent years suggests the existence of caspase-like activity in plants and its functional involvement in various types of plant PCD, although no functional homologs of animal caspases were identified in plant genome. In this mini-review, we will cover the recent results on the existence of plant caspase-like proteases and introduce major technologies used in detecting the activation of caspase-like proteases during plant PCD.Key words: caspase-like proteases, fluorescence resonance energy transfer, programmed cell death     

植物类Caspase及其调控细胞程序性死亡的研究进展

植物细胞程序性死亡（PCD）在植物生长发育和逆境适应中发挥重要作用。半胱氨酸蛋白酶（caspase）调控动物PcD的启动、执行及信号转导。通过人工合成底物、动物caspase抑制剂等方法已证实在植物中存在类caspase,可分为metacas．pases、VPEs（vacuolar processing enzymes）和saspases等。本文综述了植物类caspase的种类、结构、定位、功能及其调控PCD的研究进展,提出植物PCD中类caspase作用的调控途径,为深入研究植物PCD提供参考。     

Programmed cell death in seeds of angiosperms

During the diversi fication of angiosperms, seeds have evolved structural, chemical, molecular and physiologically developing changes that specially affect the nucellus and endosperm. All through seed evolution, programmed cell death(PCD) has played a fundamental role. However,examples of PCD during seed development are limited. The present review examines PCD in integuments, nucellus,suspensor and endosperm in those representative examples of seeds studied to date.     

Caspase inhibitors affect the kinetics and dimensions of tracheary elements in xylogenic Zinnia (<Emphasis Type="Italic">Zinnia elegans</Emphasis>) cell cultures

Background  

The xylem vascular system is composed of fused dead, hollow cells called tracheary elements (TEs) that originate through trans-differentiation of root and shoot cambium cells. TEs undergo autolysis as they differentiate and mature. The final stage of the formation of TEs in plants is the death of the involved cells, a process showing some similarities to programmed cell death (PCD) in animal systems. Plant proteases with functional similarity to proteases involved in mammalian apoptotic cell death (caspases) are suggested as an integral part of the core mechanism of most PCD responses in plants, but participation of plant caspase-like proteases in TE PCD has not yet been documented.     

植物细胞程序性死亡中的类胱天蛋白酶研究进展

胱天蛋白酶(caspases)在动物细胞程序性死亡(programmed cell death,PCD)的起始、执行以及信号转导阶段起着关键作用,目前在植物中也发现有类胱天蛋白酶(caspase-like proteases,CLPs)的存在,并确认液泡加工酶(VPEs)、metacaspases和丝氨酸内肽酶(sapases)具有CLPs的作用,并证实CLPs参与植物的生长发育、抗病性及胁迫诱导的细胞程序性死亡等.本文对植物CLPs活性、生化结构及生理作用等方面的研究进展进行综述,并与动物caspases进行比较,为今后CLPs活性调节、作用方式及其在植物细胞程序性死亡中的作用等方面的研究提供参考.     

Death by proteases in plants: whodunit

Several studies have shown that protease inhibitors can suppress programmed cell death in various plant species and plant tissues. This is especially true of caspase inhibitors that can block programmed cell death and its marker DNA laddering. There are up to six different caspase-like activities that can be measured in plant extracts, the most prominent being caspase1-like and caspase3-like. These activities can be located in vacuoles and also in the nucleus or the cytoplasm. This represents a striking apparent similarity with animal programmed cell death. Because there are no caspase orthologue in plant genomes, a major challenge is to identify these proteases. Recently two proteases with caspase-like activities have been recognized as belonging to two different protease families that are not closely related to animal caspases. Various other protease families have been implicated and this suggests that complex protease networks have been recruited for the plant cell demise.     

VEIDase is a principal caspase-like activity involved in plant programmed cell death and essential for embryonic pattern formation

Plant embryogenesis is intimately associated with programmed cell death. The mechanisms of initiation and control of programmed cell death during plant embryo development are not known. Proteolytic activity associated with caspase-like proteins is paramount for control of programmed cell death in animals and yeasts. Caspase family of proteases has unique strong preference for cleavage of the target proteins next to asparagine residue. In this work, we have used synthetic peptide substrates containing caspase recognition sites and corresponding specific inhibitors to analyse the role of caspase-like activity in the regulation of programmed cell death during plant embryogenesis. We demonstrate that VEIDase is a principal caspase-like activity implicated in plant embryogenesis. This activity increases at the early stages of embryo development that coincide with massive cell death during shape remodeling. The VEIDase activity exhibits high sensitivity to pH, ionic strength and Zn(2+) concentration. Altogether, biochemical assays show that VEIDase plant caspase-like activity resembles that of both mammalian caspase-6 and yeast metacaspase, YCA1. In vivo, VEIDase activity is localised specifically in the embryonic cells during both the commitment and in the beginning of the execution phase of programmed cell death. Inhibition of VEIDase prevents normal embryo development via blocking the embryo-suspensor differentiation. Our data indicate that the VEIDase activity is an integral part in the control of plant developmental cell death programme, and that this activity is essential for the embryo pattern formation.     

Iatercellular Migration of Protoplasm and Its Relation to the Development of Embryo Sac in Nucellus of Wheat

During the development of the ovule before pollination, deterioration of successive layers of nucellar tissue, beginning from the nnermost, constantly takes place and consistently forms a zone of disorganization surrounding the periphery of the enlarging embryo sac. Nucellar tissue deteriorates much more profusely near the antipodal end of the sac. "Nuclear extrusion" taken as an indication of intercellular movement of the protoplasm which has undergone partial disassembly, can be seen among the nucellar tissues and between the nucellus and the embryo sac. The intruding nuclear fragments, some of which assume the form of nucleolus, can be found in the antipodal cells. The results interpreted according to our previous hypothesis, are as follows. The nucellar cell by means of intercellular movement of its own protoplasm in the state of partial disassembly, furnishes the embryo sac with composite units of various polymers and organelles. Consequently, the antipodal cells proliferate and flourish The interrelationship between nucellus and embryo sac has been discussed from the viewpoint of supply and utilization of nourishment, which is necessary for the rapid development of the embryo sac.