Barley aleurone cell death is not apoptotic: characterization of nuclease activities and DNA degradation

Barley aleurone cells undergo programmed cell death (PCD) when exposed to gibberellic acid (GA), but incubation in abscisic acid (ABA) prevent PCD. We tested the hypothesis that PCD in aleurone cells occurs by apoptosis, and show that the hallmark of apoptosis, namely DNA cleavage into 180 bp fragments, plasma membrane blebbing, and the formation of apoptotic bodies do not occur when aleurone cells die. We show that endogenous barley aleurone nucleases and nucleases present in enzymes used for protoplast preparation degrade aleurone DNA and that DNA degradation by these nucleases is rapid and can result in the formation of 180 bp DNA ladders. Methods are described that prevent DNA degradation during isolation from aleurone layers or protoplasts. Barley aleurone cells contain three nucleases whose activities are regulated by GA and ABA. CA induction and ABA repression of nuclease activities correlate with PCD in aleurone cells. Cells incubated in ABA remain alive and do not degrade their DNA, but living aleurone cells treated with GA accumulate nucleases and hydrolyze their nuclear DNA. We propose that barley nucleases play a role in DNA cleavage during aleurone PCD.     

Cell death of barley aleurone protoplasts is mediated by reactive oxygen species

The barley aleurone layer is a terminally differentiated secretory tissue whose activity is hormonally controlled. The plant hormone gibberellic acid (GA) stimulates the secretion of hydrolytic enzymes and triggers the onset of programmed cell death (PCD). Abscisic acid (ABA) antagonizes the effects of GA and inhibits enzyme secretion and PCD. Reactive oxygen species (ROS) are key players in many types of PCD, and data presented here implicate ROS in hormonally regulated death of barley aleurone cells. Incubation of aleurone layers or protoplasts in H(2)O(2)-containing media results in death of GA-treated but not ABA-treated aleurone cells. Cells that are programmed to die are therefore less able to withstand ROS than cells that are programmed to remain alive. Illumination of barley aleurone protoplasts with blue or UV-A light results in a rapid increase in intracellular H(2)O(2) production. GA-treated protoplasts die rapidly in response to this increase in intracellular H(2)O(2) production, but ABA-treated protoplasts do not die. The rate of light-induced death could be slowed by antioxidants, and incubating protoplasts in the dark with the antioxidant butylated hydroxy toluene reduces the rate of hormonally induced death. Taken together, these data demonstrate that GA-treated aleurone protoplasts are less able than ABA-treated protoplasts to tolerate internally generated or exogenously applied H(2)O(2), and strongly suggest that ROS are components of the hormonally regulated cell death pathway in barley aleurone cells.     

小麦淀粉胚乳发育期间的程序性细胞死亡

小麦淀粉胚乳在发育过程中经历程序性细胞死亡(PCD).小麦淀粉胚乳的DNA在发育的特定阶段呈现梯状电泳条带,用乙烯处理使DNA片段化发生的时间提前,而且ABA处理虽然不能推迟DNA片段化的发生时间,但能减弱DNA片段化的程度.小麦淀粉胚乳细胞在PCD过程中出现某些动植物细胞凋亡的共同的结构变化特征,但也有一些独特的结构变化.如染色质凝聚后仅少数染色质块发生趋边化;细胞核在PCD过程中最先开始衰退,细胞核解体时胞质中有丰富的细胞器,细胞核解体后细胞并未死亡,在胞质中仍在合成和积累淀粉和储藏蛋白,直到细胞被淀粉充满,细胞才死亡;不形成凋亡小体,死亡的淀粉胚乳细胞成为营养物质的储藏库.因此小麦淀粉胚乳细胞的PCD是一种特殊形式的PCD.     

Active oxygen and cell death in cereal aleurone cells

The cereal aleurone layer is a secretory tissue whose function is regulated by gibberellic acid (GA) and abscisic acid (ABA). Aleurone cells lack functional chloroplasts, thus excluding photosynthesis as a source of active oxygen species (AOS) in cell death. Incubation of barley aleurone layers or protoplasts in GA initiated the cell death programme, but incubation in ABA delays programmed cell death (PCD). Light, especially blue and UV-A light, and H(2)O(2) accelerate PCD of GA-treated aleurone cells, but ABA-treated aleurone cells are refractory to light and H(2)O(2) and are not killed. It was shown that light elevated intracellular H(2)O(2), and that the rise in H(2)O(2) was greater in GA-treated cells compared to cells in ABA. Experiments with antioxidants show that PCD in aleurone is probably regulated by AOS. The sensitivity of GA-treated aleurone to light and H(2)O(2) is a result of lowered amounts of enzymes that metabolize AOS. mRNAs encoding catalase, ascorbate peroxidase and superoxide dismutase are all reduced during 6-18 h of incubation in GA, but these mRNAs were present in higher amounts in cells incubated in ABA. The amounts of protein and enzyme activities encoded by these mRNAs were also dramatically reduced in GA-treated cells. Aleurone cells store and metabolize neutral lipids via the glyoxylate cycle in response to GA, and glyoxysomes are one potential source of AOS in the GA-treated cells. Mitochondria are another potential source of AOS in GA-treated cells. AOS generated by these organelles bring about membrane rupture and cell death.     

Hormonally regulated programmed cell death in barley aleurone cells

Cell death was studied in barley (cv Himalaya) aleurone cells treated with abscisic acid and gibberellin. Aleurone protoplasts incubated in abscisic acid remained viable in culture for at least 3 weeks, but exposure to gibberellin initiated a series of events that resulted in death. Between 4 and 8 days after incubation in gibberellin, >70% of all protoplasts died. Death, which occurred after cells became highly vacuolated, was manifest by an abrupt loss of plasma membrane integrity followed by rapid shrinkage of the cell corpse. Hydrolysis of DNA began before death and occurred as protoplasts ceased production of alpha-amylase. DNA degradation did not result in the accumulation of discrete low molecular weight fragments. DNA degradation and cell death were prevented by LY83583, an inhibitor of gibberellin signaling in barley aleurone. We conclude that cell death in aleurone cells is hormonally regulated and is the final step of a developmental program that promotes successful seedling establishment.     

Modulation of Calmodulin mRNA and Protein Levels in Barley Aleurone

Changes in calmodulin (CaM) mRNA and protein were investigated in aleurone layers of barley (Hordeum vulgare L. cv Himalaya) incubated in the presence and absence of calcium, gibberellic acid (GA3), and abscisic acid (ABA). CaM mRNA levels increased rapidly and transiently following incubation of aleurone layers in H2O, CaCl2, or GA3. The increase in CaM mRNA was prevented by ABA. This increase in CaM mRNA was brought about by physical stimulation during removal of the starchy endosperm from the aleurone layer. CaM protein levels did not increase in response to physical stimulation. Only incubation in GA3 plus CaCl2 brought about a rapid increase in CaM protein levels in the aleurone cell. ABA reduced the level of CaM protein below that found at the beginning of the incubation period. The rise in CaM protein preceded increases in the synthesis and secretion of [alpha]-amylase. Immunocytochemistry with monoclonal antibodies to carrot and mung bean CaM was used to localize CaM in aleurone protoplasts. Monoclonal antibodies to tubulin and polyclonal antibodies to tonoplast intrinsic protein and malate synthase were used as controls. CaM was localized to the nucleus, the vacuolar membrane, and the cytosol, but was not associated with microtubules.     

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.     

Perception of Gibberellin and Abscisic Acid at the External Face of the Plasma Membrane of Barley (Hordeum vulgare L.) Aleurone Protoplasts

The response of protoplasts isolated from aleurone layers of barley (Hordeum vulgare L. cv Himalaya) to internally and externally applied hormone was analyzed to localize the site of perception of the hormonal signal. Protoplasts responded to externally applied gibberellic acid (GA3) with increased synthesis and secretion of [alpha]-amylase, transient expression of the glucuronidase reporter gene fused to the hormone-responsive elements of the [alpha]-amylase promoter, and the vacuolation typical of GA3-treated aleurone cells. When up to 250 [mu]M GA3 was microinjected into the protoplast cytoplasm, none of these responses were observed. This did not reflect damage to the protoplasts during the microinjection procedure, since microinjected protoplasts remained responsive to externally applied hormone. Nor did it reflect loss of microinjected GA3 from the protoplast, since 50% of microinjected [3H]GA20 was retained by protoplasts for at least 24 h. Externally applied abscisic acid (ABA) could reverse the stimulation of [alpha]-amylase synthesis and secretion, whereas microinjecting up to 250 [mu]M ABA was ineffective at antagonizing the stimulatory effect of GA3. These results suggest that the site of perception of GA3 and ABA in the barley aleurone protoplast is on the external face of the plasma membrane.     

The cellular pathway of photosynthate transfer in the developing wheat grain. III. A structural analysis and physiological studies of the pathway from the endosperm cavity to the starchy endosperm

The cellular pathway of sucrose transfer from the endosperm cavity to the starchy endosperm of developing grains of wheat (Triticum turgidum) has been elucidated. The modified aleurone and sub-aleurone cells exhibit a dense cytoplasm enriched in mitochondria and endoplasmic relicilium. Significantly, the sub-aleurone cells are characterized by secondary wall ingrowths. Numerous plasmodesmata interconnect all cells between the modified aleurone and starchy endosperm. The pro-tonophore carbonylcyanide-m-chlorophenyl hydrazone (CCCP) slowed [¹⁴C]sucrose uptake by grain tissue slices enriched in modified aleurone and sub-aleurone cells but had no effect on uptake by the starchy endosperm. The fluorescent weak acid sulphorhodamine G (SRG) was preferentially accumulated by the modified aleurone and sub-aleurone cells, and this uptake was sensitive to CCCP. The combined plasma membrane surface areas of the modified aleurone and sub-aleurone cells appeared to be sufficient to support the in vivo rates of sucrose transfer to the starchy endosperm. Plasmolysis of intact excised grain inhibited [¹⁴C]sucrose transfer from the endosperm cavity to the starchy endosperm. The sulphydryl group modifier p-chloromercuribenzenesulphonie acid (PCMBS) decreased [¹⁴C]sucrose uptake by the modified aleurone and sub-aleurone cells but had little effect on uptake by the starchy endosperm. In contrast, when PCMBS and [¹⁴C]sucrose were supplied to the endosperm cavity of intact excised grain, PCMBS slowed accumulation by all tissues equally. Estimates of potential sucrose fluxes through the interconnecting plasmodesmata were found to be within the published range. It is concluded that the bulk of sucrose is accumulated from the endosperm cavity by the modified aleurone and sub-aleurone cells and subsequently transferred through the symplast to the starchy endosperm.     

Isolation and characterization of oat aleurone and starchy endosperm protein bodies

To compare oat (Avena sativa L. cv Froker) aleurone protein bodies with those of the starchy endosperm, methods were developed to isolate these tissues from mature seeds. Aleurone protoplasts were prepared by enzymic digestion and filtration of groat (caryopsis) slices, and starchy endosperm tissue was separated from the aleurone layer by squeezing slices of imbibed groats followed by filtration. Protein bodies were isolated from each tissue by sucrose density gradient centrifugation. Ultrastructure of the isolated protein bodies was not identical to that of the intact organelles, suggesting modification during isolation or fixation. Both aleurone and starchy endosperm protein bodies contained globulin and prolamin storage protein, but minor differences in the protein-banding pattern by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were evident. The amino acid compositions of the protein body fractions were similar and resembled that of oat globulin. The aleurone protein bodies contained phytic acid and protease activity, which were absent in starchy endosperm protein bodies.     

Rice caryopsis development Ⅱ: Dynamic changes in the endosperm

The rice endosperm plays crucial roles in nourishing the embryo during embryogenesis and seed germination. Although previous studies have provided the general information about rice endosperm, a systematic investigation throughout the entire endosperm developmental process is still lacking. In this study, we examined in detail rice endosperm development on a daily basis throughout the 30‐day period of post‐fertilization development. We observed that coenocytic nuclear division occurred in the first 2 days after pollination (DAP), cellularization occurred between 3 and 5 DAP, differentiation of the aleurone and starchy endosperm occurred between 6 and 9 DAP, and accumulation of storage products occurred concurrently with the aleurone/starchy endosperm differentiation from 6 DAP onwards and was accomplished by 21 DAP. Changes in cytoplasmic membrane permeability, possibly caused by programmed cell death, were observed in the central region of the starchy endosperm at 8 DAP, and expanded to the whole starchy endosperm at 21 DAP when the aleurone is the only living component in the endosperm. Further, we observed that a distinct multi‐layered dorsal aleurone formed near the dorsal vascular bundle, while the single‐ or occasionally two‐cell layered aleurone was located in the lateral and ventral positions of endosperm. Our results provide in detail the dynamic changes in mitotic divisions, cellularization, cell differentiation, storage product accumulation, and programmed cell death that occur during rice endosperm development.     

Programmed cell death during endosperm development

The endosperm of cereals functions as a storage tissue in which the majority of starch and seed storage proteins are synthesized. During its development, cereal endosperm initiates a cell death program that eventually affects the entire tissue with the exception of the outermost cells, which differentiate into the aleurone layer and remain living in the mature seed. To date, the cell death program has been described for maize and wheat endosperm, which exhibits common and unique elements for each species. The progression of endosperm programmed cell death (PCD) in both species is accompanied by an increase in nuclease activity and the internucleosomal degradation of nuclear DNA, hallmarks of apoptosis in animals. Moreover, ethylene and abscisic acid are key to mediating PCD in cereal endosperm. The progression of the cell death program in developing maize endosperm follows a highly organized pattern whereas in wheat endosperm, PCD initiates stochastically. Although the essential characteristics of cereal endosperm PCD are now known, the molecular mechanisms responsible for its execution remain to be identified.     

Defense-related signaling by interaction of arabinogalactan proteins and beta-glucosyl Yariv reagent inhibits gibberellin signaling in barley aleurone cells

Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins present at the plasma membrane and in extracellular spaces. A synthetic chemical, beta-glucosyl Yariv reagent (beta-GlcY), binds specifically to AGPs. We previously reported that gibberellin signaling is specifically inhibited by beta-GlcY treatment in barley aleurone protoplasts. In the present study, we found that beta-GlcY also inhibited gibberellin-induced programmed cell death (PCD) in aleurone cells. We examined the universality and specificity of the inhibitory effect of beta-GlcY on gibberellin signaling using microarray analysis and found that beta-GlcY was largely effective in repressing gibberellin-induced gene expression. In addition, >100 genes were up-regulated by beta-GlcY in a gibberellin-independent manner, and many of these were categorized as defense-related genes. Defense signaling triggered by several defense system inducers such as jasmonic acid and a chitin elicitor could inhibit gibberellin-inducible events such as alpha-amylase secretion, PCD and expression of some gibberellin-inducible genes in aleurone cells. Furthermore, beta-GlcY repressed the gibberellin-inducible Ca2+-ATPase gene which is important for gibberellin-dependent gene expression, and induced known repressors of gibberellin signaling, two WRKY genes and a NAK kinase gene. These effects of beta-GlcY were also phenocopied by the chitin elicitor and/or jasmonic acid. These results indicate that gibberellin signaling is under the regulation of defense-related signaling in aleurone cells. It is also probable that AGPs are involved in the perception of stimuli causing defense responses.     

Amylases in developing barley seeds

The amylases of developing barley seeds (Hordeum vulgare L. cv. Himalaya) were investigated by colorimetric and electrophoretic methods. Maxima of amylolytic activity appeared in the aleurone layers and starchy endosperm at 5 and 20 days after anthesis. Amylase from 5-day-old aleurone layers could be separated into four rapidly moving bands with α-amylase activity. By 20 days the four bands had been replaced by seven bands of medium mobility. These seven bands of amylase were electrophoretically identical to those observed when mature aleurone layers are treated with gibberellic acid. Immature aleurone layers failed to respond to exogenous gibberellic acid. In the starchy endosperm the seven bands of medium mobility were also present. Calcium-dependent alterations in the electrophoretic mobility and activity of particular bands occurred during the maturation of the starchy endosperm. Treatment of the immature starchy endosperm with papain yielded four forms of β-amylase.     

Kinetics of the Acidification Capacity of Aleurone Layer and Its Effect upon Solubilization of Reserve Substances from Starchy Endosperm of Wheat

The capacity of the isolated barley aleurone layer for endosperm acidification has been demonstrated (J Mikola, M Virtanen 1980 Plant Physiol 66: S-142). The kinetics of this acidification by isolated wheat aleurone layer and its effect on starchy endosperm solubilization are reported.     

Biotin-labeled abscisic acid as a probe for investigating abscisic acid binding sites on plasma membranes of barley aleurone protoplasts

Plant hormone abscisic acid (ABA) plays important roles in dormancy and stress responses, but its binding sites have not yet been fully elucidated. In this report, we suggest the utility of biotin-labeled abscisic acid (bioABA) as a probe to investigate ABA-binding sites on the plasma membrane of barley aleurone protoplasts. BioABA was approximately 100 times less effective than ABA in inhibiting expression of gibberellin-inducible alpha-amylase and in inducing expression of a reporter gene fused to the dehydrin promoter. To ascertain that bioABA could bind to ABA-binding sites on the plasma membrane, we used fluorescence flow cytometry to measure the fluorescence intensity of aleurone protoplasts treated with a combination of bioABA and fluorescence-labeled streptavidin. Addition of bioABA increased the fluorescence of aleurone protoplasts in a concentration-dependent manner, but addition of non-active bioABA derivatives did not. Furthermore, the increase in fluorescence intensity observed upon addition of bioABA was eliminated by co-treatment with excess ABA, but it was not eliminated by co-treatment with other plant hormones. These results suggest that bioABA binds to ABA-binding sites, and that bioABA should be a valuable probe for investigating ABA-binding sites on the plasma membrane.     

Proteomes of the barley aleurone layer: A model system for plant signalling and protein secretion

The cereal aleurone layer is of major importance due to its nutritional properties as well as its central role in seed germination and industrial malting. Cereal seed germination involves mobilisation of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellic acid produced by the embryo, the aleurone layer synthesises hydrolases that are secreted to the endosperm for the degradation of storage products. The barley aleurone layer can be separated from the other seed tissues and maintained in culture, allowing the study of the effect of added signalling molecules in an isolated system. These properties have led to its use as a model system for the study of plant signalling and germination. More recently, proteome analysis of the aleurone layer has provided new insight into this unique tissue including identification of plasma membrane proteins and targeted analysis of germination-related changes and the thioredoxin system. Here, analysis of intracellular and secreted proteomes reveals features of the aleurone layer system that makes it promising for investigations of plant protein secretion mechanisms.     

水稻淀粉胚乳程序性细胞死亡中的去核化

对水稻品种中籼8836淀粉胚乳细胞的去核化发育阶段的细胞超微结构变化和同期籽粒灌浆速率及相关酶活性的动态进行了观察和分析。开花受精后约在第3天胚乳完成细胞化,花后第5天少数淀粉胚乳细胞启动去核发育过程。核消亡是淀粉胚乳细胞程序性细胞死亡(PCD)的第一步。同一籽粒淀粉胚乳细胞的去核进程是不同步的。花后第13天所有淀粉胚乳细胞都已完成去核过程。在去核过程中,胚乳核的形态变化特征既有动植物PCD的共性又有其特殊性。伴随核降解过程,一部分线粒体解体,表明去核化与线粒体解体有一定联系。在去核化发育阶段,与PCD有关的酶类,如超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性非常高;与淀粉合成有关的酶类,如ADPG焦磷酸化酶、可溶性淀粉合成酶(SSS酶)、淀粉分支酶(或Q酶)也表现出很高的活性。去核化发育阶段籽粒灌浆速率最高,籽粒增重亦最快。淀粉胚乳细胞去核之后,细胞并未立即死亡,这些无核的细胞仍维持正常有序的代谢活动,继续进行淀粉和贮藏蛋白的合成与积累,但上述酶类的活性明显降低,灌浆速率也明显趋缓。淀粉胚乳细胞最终被贮藏物质充满时成为死细胞,完成其程序性死亡过程。Evan‘s blue染色鉴定表明淀粉胚乳细胞死亡不同步,细胞死亡在淀粉胚乳组织中是随机发生的。