The embryo sac of Cynara cardunculus: ultrastructure of the development and localisation of the aspartic proteinase cardosin B

Cynara cardunculus is a native plant with flowers that are used traditionally in the manufacture of ewe’s cheese in the Iberian Peninsula. Milk clotting ability of the plant is attributed to the high concentrations of aspartic proteinases (APs), named cardosins, found in the flowers. Although these enzymes are well characterised on a molecular and biochemical basis, the biological role of the majority of plant APs is yet unassigned. We suspected APs play an important role in ovule function, and we characterised the maturation of the ovules of C. cardunculus and its Polygonum-type embryo sacs. The internal layer of the integument differentiates into an endothelium as described for other Asteraceae, with differentiation of two nucellar layers, a podium and a hypostase coinciding with the onset of pollen receptivity. In flowering plants, programmed cell death (PCD) events are essential for the success of nucellar maturation and consequent differentiation of a fully functional embryo sac. In C. cardunculus, nucellar PCD is integral to the maturation of the embryo sac, which in turn is closely correlated with the accumulation of the AP cardosin B specifically in the hypostase. The onset of cardosin B expression temporally coincides with the degeneration of nucellar cells. In fully mature embryo sacs, cardosin B is localised in both the hypostase and epistase, two regions that differentiate through PCD. Thus, cardosin B localisations closely correlate with events of PCD in the nucellus of C. cardunculus suggesting involvement in ovule and embryo sac development and further suggest the biological significance of APs like cardosin B, in this particular process. This work contributes new data to the plant AP research field and indicates an involvement of cardosin B in the PCD-dependent degeneration of the nucellus.     

Programmed Cell Death Progresses Differentially in Epidermal and Mesophyll Cells of Lily Petals

In the petals of some species of flowers, programmed cell death (PCD) begins earlier in mesophyll cells than in epidermal cells. However, PCD progression in each cell type has not been characterized in detail. We separately constructed a time course of biochemical signs and expression patterns of PCD-associated genes in epidermal and mesophyll cells in Lilium cv. Yelloween petals. Before visible signs of senescence could be observed, we found signs of PCD, including DNA degradation and decreased protein content in mesophyll cells only. In these cells, the total proteinase activity increased on the day after anthesis. Within 3 days after anthesis, the protein content decreased by 61.8%, and 22.8% of mesophyll cells was lost. A second peak of proteinase activity was observed on day 6, and the number of mesophyll cells decreased again from days 4 to 7. These biochemical and morphological results suggest that PCD progressed in steps during flower life in the mesophyll cells. PCD began in epidermal cells on day 5, in temporal synchrony with the time course of visible senescence. In the mesophyll cells, the KDEL-tailed cysteine proteinase (LoCYP) and S1/P1 nuclease (LoNUC) genes were upregulated before petal wilting, earlier than in epidermal cells. In contrast, relative to that in the mesophyll cells, the expression of the SAG12 cysteine proteinase homolog (LoSAG12) drastically increased in epidermal cells in the final stage of senescence. These results suggest that multiple PCD-associated genes differentially contribute to the time lag of PCD progression between epidermal and mesophyll cells of lily petals.     

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.     

Photosynthesis nuclear genes generally lack TATA-boxes: a tobacco photosystem I gene responds to light through an initiator

A maternal plant exquisitely promotes the success of its offspring by orchestrating embryo development and endowing protection even after the embryos mature. It uses ovule integuments for physical and physiological contact with the developing embryo and for subsequently equipping the seed with a seed coat (testa). The testa is developmentally and metabolically dynamic, but its molecular biology is not well understood. We show here that the inner integument in Brassica napus undergoes organized development and then programmed cell death (PCD), as evident from vacuolation, starch mobilization, DNA fragmentation and eventual compression. We have identified a cysteine proteinase gene (BnCysP1) that is expressed only in the inner integument as it undergoes PCD, well before the embryo begins storage protein synthesis. Two paralogous Cys proteinases have been recruited in rapeseed for the PCD of testa and for leaf senescence, and these differ 25% in their primary structure and post-translational modifications. Despite Arabidopsis being closely related to rapeseed, and an indication of developmental compression of its inner integument, the Arabidopsis genome is suggestive of only one Cys proteinase that shows approximately 72% identity to BnCysP1. It is, however, leaf senescence-associated, and the other Cys proteinases are <52% identical. BnCysP1 also differs from ricinosome-deployed PCD Cys endopeptidases in lacking the hallmark KDEL tail and being glycosylated. BnCysP1, one of the very few plant genes known to function only in the seed coat, will be useful in dissecting post-fertilization development of this important organ in rapeseed.     

Cysteine proteinases from Schistosoma haematobium adult worms.

To identify and characterize cysteine proteinases from Schistosoma haematobium, lyophilized adult worms were homogenized, and enzymes were isolated and purified. From extracts prepared in acidic buffer, 3 putative cysteine proteinases were identified either directly or indirectly. The first proteinase (ShCP1) was identified by labeling with a radioiodinated inhibitor, Z-Tyr-Ala-CHN2, as a 35-kDa protein. However, it could not be detected by silver staining, amino acid sequencing, or by a monoclonal antibody specific for a similar molecule from Schistosoma mansoni. A second cysteine proteinase, ShCP2, was purified by gel filtration and dialysis. This 32-kDa molecule was thiol-dependent and was labeled with Z-Tyr-Ala-CHN2. The amino terminal amino acid sequence of ShCP2 showed remarkable similarity (up to 77%) to that of S. mansoni cathepsin B (SmCP2) as well as to mammalian cysteine proteinases. Both ShCP1 and ShCP2 reacted with polyclonal antibodies against S. mansoni, suggesting the existence of shared antigenic epitopes. A third activity, ShCP3, was identified as possibly a distinct proteinase based on its similarities to a 28-kDa cysteine proteinase from S. mansoni. This preliminary investigation demonstrates that the overall profile of cysteine proteinases in S. haematobium is very similar to that of S. mansoni.     

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.     

Pollen tube reuses intracellular components of nucellar cells undergoing programmed cell death in Pinus densiflora

Through the process known as programmed cell death (PCD), nucelli of Pinus densiflora serve as the transmitting tissue for growth of the pollen tube. We sought to clarify the processes of degradation of nucellar cell components and their transport to the pollen tube during PCD in response to pollen tube penetration of such nucelli. Stimulated by pollination, synthesis of large amounts of starch grains occurred in cells in a wide region of the nucellus, but as the pollen tube penetrated the nucellus, starch grains were degraded in amyloplasts of nucellar cells. In cells undergoing PCD, electron-dense vacuoles with high membrane contrast appeared, assumed a variety of autophagic structures, expanded, and ultimately collapsed and disappeared. Vesicles and electron-dense amorphous materials were released inside the thickened walls of cells undergoing PCD, and those vesicles and materials reaching the pollen tube after passing through the extracellular matrix were taken into the tube by endocytosis. These results show that in PCD of nucellar cells, intracellular materials are degraded in amyloplasts and vacuoles, and some of the degraded material is supplied to the pollen tube by vesicular transport to support tube growth.     

Nucellar Senescence and Ethylene Production as they Relate to Avocado Fruitlet Abscission

Events preliminary to avocado (Persea americana Mill) fruitletabscission include senescence of the nucellus and seed coat.The dynamics of nucellar deterioration and ethylene productionleading to seed abortion and abscission in avocado was examined.Excised branches bearing clusters of fruit from 1.0–2.5cm diameter were placed in humid chambers to reduce transpirationalwater loss. Fruitlets synchronously began nucellar and seedcoat deterioration 27–33 h after excision and rapidlyprogressed through stages of increasing degradation culminatingin abscission approximately 2 days later. The nucellus-seedcoat produced a temporary burst of ethylene at the first visiblesign of nucellar senescence followed by less ethylene productionin the mesocarp approximately 12 h later. All fruit underwentnucellar degradation prior to abscission. Exogenously appliedethylene accelerated fruitlet abscission with concentrationsas low as 1.0µ 1^–1 and with maximum response at100µl^–1 or greater. Maximal response took 2 days.Aminoethoxyvinyl-glycine (AVG) at 30 µ M inhibited ethyleneproduction and fruitlet abscission. The senescence process,however, was not af fected in any way by ethylene or AVG treatments.Observations of attached fruit suggest that nucellar-seed coatsenescence, concomitant ethylene production, and resulting abscissiontake place in a manner and within a time period similar to thatobserved on detached branches. It is concluded that nucellarand seed coat senescence is prerequisite to avocado fruitletabscission, and the time required from the first indicationof nucellar breakdown to abscission of that fruitlet appearsto be approximately 2 days. The senescence process is responsiblefor a large, transient rate increase in ethylene productionby the nucellus and perhaps seed coat. Ethylene is consideredto be the result rather than the cause of nucellar-seed coatsenescence. The ethylene thus produced induces fruit abscission.     

银杏贮粉室发生部位的珠心细胞程序性死亡的形态学观察

银杏（Ginkgo bilobaL)贮粉室的发生涉及位于珠孔端的珠心细胞的程序性死亡（PCD）,本研究观察了贮粉室发生过程中发生PCD的珠心细胞的形态学变化。这些珠心细胞在PCD过程中形态变化显著,细胞组分有序地降解,液泡在此起关键作用。在液泡化过程中,细胞质基质和一些细胞器被液泡所吞噬,此时的细胞器结构完整。当液泡膜破裂,细胞质基质消失之后。细胞器才逐步解体。最终,这些珠心细胞仅具有残留的细胞壁,随着胚珠的生长,细胞壁也被破坏,在整个PCD过程中,内膜系统发生明显改变;细胞质膜出泡,产生多泡体;形成多环膜结构;出现由膜包围的小体,其中含有细胞质基质和一些细胞器;液泡膜破裂;细胞器解体;细胞中出现大量的小膜泡。珠孔端的珠心表皮开裂形成贮粉室的开口有两种方式：一种为专一细胞的自溶,而另一种是在两个邻接细胞的中胶层处分离,没有发生细胞的自溶破裂。贮粉室开口位置的特定表皮细胞在开裂发生前就死亡,从而提前标示出表皮开裂的发生位置。这些细胞形态的变化反映出银杏珠心细胞的死亡是受发育调控的PCD过程。     

银杏贮粉室发生部位的珠心细胞程序性死亡的形态学观察

银杏(Ginkgo biloba L.)贮粉室的发生涉及位于珠孔端的珠心细胞的程序性死亡(PCD).本研究观察了贮粉室发生过程中发生PCD的珠心细胞的形态学变化.这些珠心细胞在PCD过程中形态变化显著,细胞组分有序地降解,液泡在此起关键作用.在液泡化过程中,细胞质基质和一些细胞器被液泡所吞噬,此时的细胞器结构完整.当液泡膜破裂、细胞质基质消失之后,细胞器才逐步解体.最终,这些珠心细胞仅具有残留的细胞壁.随着胚珠的生长,细胞壁也被破坏.在整个PCD过程中,内膜系统发生明显改变:细胞质膜出泡,产生多泡体;形成多环膜结构;出现由膜包围的小体,其中含有细胞质基质和一些细胞器;液泡膜破裂;细胞器解体;细胞中出现大量的小膜泡.珠孔端的珠心表皮开裂形成贮粉室的开口有两种方式:一种为专一细胞的自溶,而另一种是在两个邻接细胞的中胶层处分离,没有发生细胞的自溶破裂.贮粉室开口位置的特定表皮细胞在开裂发生前就死亡,从而提前标示出表皮开裂的发生位置.这些细胞形态的变化反映出银杏珠心细胞的死亡是受发育调控的PCD过程.     

The cysteine proteinases of the pineapple plant.

The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct.     

Senescence and programmed cell death: substance or semantics?

The terms senescence and programmed cell death (PCD) have led to some confusion. Senescence as visibly observed in, for example, leaf yellowing and petal wilting, has often been taken to be synonymous with the programmed death of the constituent cells. PCD also obviously refers to cells, which show a programme leading to their death. Some scientists noted that leaf yellowing, if it has not gone too far, can be reversed. They suggested calling leaf yellowing, before the point of no return, 'senescence' and the process after it 'PCD'. However, this runs into several problems. It is counter to the historical definitions of senescence, both in animal and plant science, which stipulate that senescence is programmed and directly ends in death. It would also mean that only leaves and shoots show senescence, whereas several other plant parts, where reversal has not (yet) been shown, have no senescence, but only PCD. This conflicts with ordinary usage (as in root and flower senescence). Moreover, a programme can be reversible and therefore it is not counter to logic to regard the cell death programme as potentially reversible. In green leaf cells a decision to die, in a programmed way, has been taken, in principle, before the cells start to remobilize their contents (that is, before visible yellowing) and only rarely is this decision reversed. According to the arguments developed here there are no good reasons to separate a senescence phase and a subsequent PCD phase. Rather, it is asserted, senescence in cells is the same as PCD and the two are fully synchronous.     

Programmed cell death of the nucellus during Sechium edule Sw. seed development is associated with activation of caspase-like proteases

The nucellus is a maternal tissue that embeds and feeds the developing embryo and secondary endosperm. During seed development, the cells of the nucellus suffer a degenerative process soon after fertilization as the cellular endosperm expands and accumulates reserves. Nucellar cell degeneration has been considered to be a form of developmentally programmed cell death (PCD). It was investigated whether or not this degenerative process is characterized by apoptotic hallmarks. Evidence showed that cell death is mostly localized in the border region of the tissue adjacent to the expanding endosperm. Cell death is accompanied by profound changes in the morphology of the nuclei and by a huge degradation of nuclear DNA. Moreover, an increase of activity of different classes of proteinases is reported, and the induction of caspase-like proteases sensitive to specific inhibitors was detected. Nucellar caspase-like proteases are characterized by an acid pH optimum suggesting a possible localization in the vacuole.     

Degradation of ribulose-bisphosphate carboxylase by vacuolar enzymes of senescing French bean leaves: Immunocytochemical and ultrastructural observations

Summary The possible involvement of vacuolar cysteine proteinases in degradation of ribulose-bisphosphate carboxylase (Rubisco) in senescing French bean leaves was studied by ultrastructural and immunocytochemical analyses with antibodies raised against the large subunit (LSU) of Rubisco and SH-EP, a cysteine proteinase fromVigna mungo that is immunologically identical to one of the major proteinases of French bean plants. Primary leaves of 10-day-old plants were detached and placed at 25 °C in darkness for 0, 4, and 8 days to allow their senescence to proceed. The leaves at each senescence stage were subjected to the conventional electron microscopic and immunocytochemical studies. The results indicated that the chloroplasts of senescing French bean leaves were separated from the cytoplasm of the cell periphery and taken into the central vacuole and that the Rubisco LSU in the chloroplasts was degraded by vacuolar enzymes such as an SH-EP-related cysteine proteinase that developed in senescing leaves. The present results together with the results of previous biochemical studies using vacuolar lysates support the view that Rubisco is degraded through the association of chloroplasts with the central vacuole during the senescence of leaves that were detached and placed in darkness.     

Redox regulation of peroxiredoxin and proteinases by ascorbate and thiols during pea root nodule senescence

Redox factors contributing to nodule senescence were studied in pea. The abundance of the nodule cytosolic peroxiredoxin but not the mitochondrial peroxiredoxin protein was modulated by ascorbate. In contrast to redox-active antioxidants such as ascorbate and cytosolic peroxiredoxin that decreased during nodule development, maximal extractable nodule proteinase activity increased progressively as the nodules aged. Cathepsin-like activities were constant throughout development but serine and cysteine proteinase activities increased during senescence. Senescence-induced cysteine proteinase activity was inhibited by cysteine, dithiotreitol, or E-64. Senescence-dependent decreases in redox-active factors, particularly ascorbate and peroxiredoxin favour decreased redox-mediated inactivation of cysteine proteinases.