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.     

Programmed cell death in cereal aleurone

Progress in understanding programmed cell death (PCD) in the cereal aleurone is described. Cereal aleurone cells are specialized endosperm cells that function to synthesize and secrete hydrolytic enzymes that break down reserves in the starchy endosperm. Unlike the cells of the starchy endosperm, aleurone cells are viable in mature grain but undergo PCD when germination is triggered or when isolated aleurone layers or protoplasts are incubated in gibberellic acid (GA). Abscisic acid (ABA) slows down the process of aleurone cell death and isolated aleurone protoplasts can be kept alive in media containing ABA for up to 6 months. Cell death in barley aleurone occurs only after cells become highly vacuolated and is manifested in an abrupt loss of plasma membrane integrity. Aleurone cell death does not follow the apoptotic pathway found in many animal cells. The hallmarks of apoptosis, including internucleosomal DNA cleavage, plasma membrane and nuclear blebbing and formation of apoptotic bodies, are not observed in dying aleurone cells. PCD in barley aleurone cells is accompanied by the accumulation of a spectrum of nuclease and protease activities and the loss of organelles as a result of cellular autolysis.     

Spatial and temporal regulation of DNA fragmentation in the aleurone of germinating barley

During germination of barley grains, the appearance of DNA fragmentation started in aleurone cells near the embryo and extended to the distal end in a time-dependent manner. DNA fragmentation was demonstrated to occur only after the expression of -amylase mRNA in the aleurone layer. In addition, cell wall degradation started in cells near the embryo on the sides facing the endosperm. Subsequently cell wall degradation extended to the lateral cell walls and to cells more to the distal end of the grain. A typical alteration of the nucleus was observed by electron microscopy and an almost complete degradation of DNA was found in the nucleus while the nuclear envelope remained intact. The results indicate that programmed cell death occurred in aleurone cells during germination. A model is proposed for the regulation of programmed cell death in aleurone cells during germination involving ABA levels and cell wall degradation.     

Apoptosis in barley aleurone during germination and its inhibition by abscisic acid

During germination of barley grains, DNA fragmentation was observed in the aleurone. The appearance of DNA fragmentation in the aleurone layer, observed by TUNEL staining in aleurone sections, started near the embryo and extended to the aleurone cells far from the embryo in a time dependent manner. The same spatial temporal activities of hydrolytic enzymes such as -amylase were observed in aleurone. DNA fragmentation could also be seen in vitro under osmotic stress, in isolated aleurone. During aleurone protoplast isolation, a very enhanced and strong DNA fragmentation occurred which was not seen in protoplast preparations of tobacco leaves. ABA was found to inhibit DNA fragmentation occurring in barley aleurone under osmotic stress condition and during protoplast isolation, while the plant growth regulator gibberellic acid counteracted the effect of ABA. Addition of auxin or cytokinin had no significant effect on DNA fragmentation in these cells. To study the role of phosphorylation in ABA signal transduction leading to control of DNA fragmentation (apoptosis), the effects of the phosphatase inhibitor okadaic acid and of phenylarisine oxide on apoptosis were studied. We hypothesize that the regulation of DNA fragmentation in aleurone plays a very important role in spatial and temporal control of aleurone activities during germination. The possible signal transduction pathway of ABA leading to the regulation of DNA fragmentation is discussed.     

Positional cues specify and maintain aleurone cell fate in maize endosperm development

A genetic analysis of maize aleurone development was conducted. Cell lineage was examined by simultaneously marking cells with C1 for anthocyanin pigmentation in the aleurone and wx1 for amylose synthesis in the starchy endosperm. The aleurone and starchy endosperm share a common lineage throughout development indicating that positional cues specify aleurone fate. Mutants in dek1 block aleurone formation at an early stage and cause peripheral endosperm cells to develop as starchy endosperm. Revertant sectors of a transposon-induced dek1 allele showed that peripheral endosperm cells remain competent to differentiate as aleurone cells until late in development. Ds-induced chromosome breakage was used to generate Dek1 loss-of-function sectors. Events occurring until late development caused aleurone cells to switch fate to starchy endosperm indicating that cell fate is not fixed. Thus, positional cues are required to specify and maintain aleurone fate and Dek1 function is required to respond to these cues. An analysis of additional mutants that disrupt aleurone differentiation suggests a hierarchy of gene functions to specify aleurone cell fate and then control aleurone differentiation. These mutants disrupt aleurone differentiation in reproducible patterns suggesting a relationship to endosperm pattern formation.     

大麦胚和胚乳发育的相关性及贮藏营养物质的积累

大麦(Hordeum vulgare L.)开花后1d,见合子及退化助细胞,游离核胚乳尚未形成;开花后2～3d,胚为5及10个细胞,胚乳为游离核期;开花后4及5、6d,胚为梨形及长梨形,胚乳达细胞化期;开花后8d,胚为胚芽鞘期,糊粉层原始细胞产生;开花后10d,胚具1叶,糊粉层1～2层;开花后13d胚为2叶胚,亚糊粉层发生;开花后17d,3叶胚形成,糊粉层多为3层并停止分裂,菱柱形及不规则胚乳细胞分化;开花后21～29d,胚为4叶胚,胚乳进一步分化;开花后33d,胚为5叶成熟胚,胚乳亦成熟。淀粉、蛋白质在胚中积累始于开花后13d。在盾片中由基向顶发生,在胚芽鞘及叶原基中,首先在顶端出现。成熟盾片顶端的淀粉消失。开花后6d,胚乳开始积累淀粉;开花后10d,糊粉层及胚乳细胞积累蛋白质。开花17d后胚乳的蛋白质体多聚集,29d后蛋白质体显著减少。开花后17d,在盾片及糊粉层细胞中检测到油脂。果长或果长与稃片长之比和盾片长可作为不同发育期胚和胚乳的形态指标。     

Change in wall composition of transfer and aleurone cells during wheat grain development

In addition to the starchy endosperm, a specialized tissue accumulating storage material, the endosperm of wheat grain, comprises the aleurone layer and the transfer cells next to the crease. The transfer cells, located at the ventral region of the grain, are involved in nutrient transfer from the maternal tissues to the developing endosperm. Immunolabeling techniques, Raman spectroscopy, and synchrotron infrared micro-spectroscopy were used to study the chemistry of the transfer cell walls during wheat grain development. The kinetic depositions of the main cell wall polysaccharides of wheat grain endosperm, arabinoxylan, and (1–3)(1–4)-β-glucan in transfer cell walls were different from kinetics previously observed in the aleurone cell walls. While (1–3)(1–4)-β-glucan appeared first in the aleurone cell walls at 90°D, arabinoxylan predominated in the transfer cell walls from 90 to 445°D. Both aleurone and transfer cell walls were enriched in (1–3)(1–4)-β-glucan at the mature stage of wheat grain development. Arabinoxylan was more substituted in the transfer cell walls than in the aleurone walls. However, arabinoxylan was more feruloylated in the aleurone than in the transfer cell walls, whatever the stage of grain development. In the transfer cells, the ferulic acid was less abundant in the outer periclinal walls while para-coumarate was absent. Possible implications of such differences are discussed.     

Gibberellic Acid enhancement of DNA turnover in barley aleurone cells

When imbibed, deembryonated halfseeds from barley (Hordeum vulgare L., var. Himalaya) are incubated in buffer, the DNA content of the aleurone layer increases 25 to 40% over a 24-hour period. In contrast, the DNA of isolated aleurone layers declines by 20% over the same time period. Gibberellic acid (GA) causes a reduction in DNA levels in both halfseed aleurone layers and isolated aleurone layers. GA also increases the specific radioactivity of [³H]thymidine-labeled halfseed aleurone layer DNA during the first 12 hours of treatment. Pulse-chase studies demonstrated that the newly synthesized DNA is metabolically labile.     

Cytological characterization of the embryo-lethal mutantdek-1 of maize

Summary The recessive embryo-lethal mutantdek-1 of maize, showing arrest of embryo development at the proembryo stage, lack of carotenoids and anthocyanins and absence in the endosperm of the aleurone layer, was characterized at a cytological level. Cytofluorimetric analysis excluded endoreduplication or polyploidization events in mutant embryonic cells, in spite of an evident increase in nucleolus and nucleus diameters.The data seem to point to an involvement ofDek-1 in the progression of the embryo toward specific developmental steps and in the differentiation of the aleurone layer in the endosperm. Cellular proliferation is not affected by the mutation, as is shown by DNA replication even after the arrest in development and by the possibility of inducing callus from mutant embryos.Abbreviation DAP days after pollination     

A gibberellin-induced nuclease is localized in the nucleus of wheat aleurone cells undergoing programmed cell death

The aleurone layer of cereal grains undergoes a gibberellin-regulated process of programmed cell death (PCD) following germination. We have applied a combination of ultrastructural and biochemical approaches to analyze aleurone PCD in intact wheat grains. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay revealed that PCD was initiated in aleurone cells proximal to the embryo and then extended to distal cells. DNA fragmentation and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis revealed PCD of aleurone cells in maize grains, although the process was delayed as compared with wheat. Aleurone cells undergoing PCD showed a rapid vacuolation with high lytic activity in the cytoplasm, whereas the nucleus, which adopted an irregular shape, appeared essentially intact and showed symptoms of degradation at the end of the process. A nuclease activity was identified localized in the nucleus of aleurone cells undergoing PCD, just prior to the appearance of DNA laddering. This nuclease was induced by gibberellic acid treatment and was not detected when gibberellin synthesis was inhibited or in gibberellic acid-insensitive mutants. This nuclease was activated by Ca(2+) and Mg(2+), strongly inhibited by Zn(2+), and showed optimum activity at neutral pH, resembling nucleases involved in apoptosis of animal cells.     

Studies on Endosperm Development and Deposition of Storage Reserves in Coix lacryma-jobi

The transition from free nuclear to cellular endosperm of Coix lacryma-jobi was eompleted 2 days after pollination. By 3 days after pollination the central cell was filled with endosperm cells. At first all cells of endosperm underwent division, later cell division was limited mainly in the peripheral region. 10 days after pollination the epidermal layer ceased its periclinal division and became the aleurone layer. Cell division persisted in the subepidermal 'cambium-like layers until the caryopsis nearly matured. Ceils of the inner region of endosperm became enlarged. Several layers of transfer cells were formed at the basal part of the endosperm. Starch grains appeared in endosperm cells on the 9th day after pollination. 10 days after pollination, lipid bodies occurred in the aleurone layer and the underlying layers. 13 and 15 days after pollination, the small vacuoles of aleurone cells contained protein and 20 days after pollenation they became aleurone grains. By 15 days after pollination pro tein bodies were formed in starch endosperm. Storage reserve deposition continued until the grain ripened. A correlation between endosperm and emoryo development was also observed.     

Endoplasmic Reticulum Formation during Germination of Wheat Seeds : A QUANTITATIVE ELECTRON MICROSCOPE STUDY

This study demonstrates germination-induced ultrastructural changes in wheat (Triticum aestivum L. cv Arthur) aleurone cells. Seeds imbided for 4 hours in water contained endoplasmic reticulum (ER) or ER-like membranes as vesicles or as short segments of membrane associated with the spherosomes on the periphery of aleurone grains. Aleurone cells incubated between 8 and 10 hours contained abundant ER membranes mainly associated with the nuclear envelope and, to a lesser extent, with the spherosomes surrounding the aleurone grain. The membranes located on the periphery of the nucleus occurred as regions of stacked cisternae. When aleurone cells were analyzed by morphometry, the increase in ER during incubation was found to be greater than 2-fold. During the same incubation period, other organelles did not change significantly. The early increase in ER was not affected by gibberellin incubation. Thus, the rapid proliferation of ER observed during the early stages of germination in aleurone cells of wheat is not likely to be controlled directly by gibberellin.     

外源CO及NO对干旱胁迫下水稻糊粉层细胞死亡的影响

采用荧光显微技术结合药理学方法,以水稻(Oryza sativa L.)种子及其糊粉层为实验材料,研究外源CO、NO对干旱胁迫下水稻种子萌发过程中糊粉层细胞DNA降解及死亡的影响。结果表明:(1)干旱胁迫促进糊粉层细胞的死亡,且近胚端糊粉层细胞的死亡进程早于远胚端的细胞。(2)外源CO及NO供体处理能缓解干旱胁迫下水稻糊粉层细胞DNA的降解,延迟细胞死亡进程;CO专一性抑制剂及NO清除剂能逆转CO及NO的效应,缩短细胞死亡进程。(3)外源CO及NO供体促进干旱胁迫下水稻种子的萌发,CO专一性抑制剂及NO清除剂能抑制干旱胁迫下水稻种子的萌发。(4)CO合成酶抑制剂并不能抑制外源NO对干旱胁迫伤害的缓解效应,即CO能通过NO介导调节干旱胁迫下水稻种子糊粉层细胞的死亡及种子萌发。     

A study of DNA depolyploidization and depolytenization of the heterochromatized gonosomal chromatin bodies in the secondary giant trophoblast cells of the field vole Microtus rossiaemeridionalis using cytophotometry

A study was made of the distribution of the heterochromatized gonosomal chromatin bodies (GCB) material in the course of nuclear fragmentation of secondary giant trophoblast cells resulting in polykaryocyte formation at the late stage of their differentiation. A simultaneous DNA cytophotometry in GCBs and nuclear fragments showed a progressive GCB DNA content decrease proportional to that of DNA content in nuclear fragments. DNA contents in the nuclear fragments corresponded to 2c, 4c and 8c. In most cases 1-2 GCBs were found in the nuclear fragments of different ploidy levels. Both the total DNA content in GCBs and the DNA content in separate GCBs well correlated with the ploidy levels of fragments. The data obtained demonstrate a regular, whole-genome distribution of chromosomal materials into the nuclear fragments exemplified by sex chromosome distribution in compliance with the ploidy of nuclear fragments. We discuss a possible mechanism of nuclear fragmentation that may ensure substantially a balanced genome of nuclear fragments without leading to mitotic cycle renewal in the giant trophoblast cell population.     

薏苡胚乳发育及营养物质积累的研究

薏苡 ( Coix lacryma- jobi)授粉后 2 d,游离核胚乳已转变为细胞胚乳。授粉后 3d,中央细胞被胚乳细胞充满。起初 ,全部胚乳细胞均进行分裂 ,一定时期后 ,细胞分裂主要发生在胚乳周边区。授粉后 1 0 d,表皮停止平周分裂变为糊粉层 ,内方的数层形成层状细胞行平周分裂直到颖果接近成熟。胚乳内部生长则依赖于细胞体积扩大。胚乳基部 (颖果基部的胚乳 )形成了数层传递细胞。授粉后 9d,淀粉积累。授粉后 1 0 d,糊粉层及其内方数层细胞产生了脂体 ,后者的脂体以后又消失。授粉后 1 3、1 5 d,糊粉层细胞的液泡积累蛋白质。授粉后 2 0 d,液泡变为糊粉粒。授粉后 1 5 d淀粉胚乳细胞产生蛋白质体 ,营养物质积累持续到颖果成熟。还观察了胚和胚乳发育的对应关系。