Ethylene-Mediated Programmed Cell Death during Maize Endosperm Development of Wild-Type and shrunken2 Genotypes

We characterized the progression of programmed cell death during maize (Zea mays L.) endosperm development of starchy (Su; wild-type) and shrunken2 (sh2) genotypes and tested the involve ment of ethylene in mediating this process. Histological and viability staining demonstrated that endosperm cell death was initiated earlier and progressed more rapidly in sh2 endosperm compared with Su endosperm. Internucleosomal DNA fragmentation accompanied endosperm cell death and occurred more extensively in sh2 endosperm. 1-Aminocyclopropane-1-carboxylic acid levels peaked approximately 16 d after pollination (dap) in Su endosperm and gradually decreased during subsequent development, whereas two large 1-aminocyclopropane-1-carboxylic acid peaks were observed in sh2 endosperm, the first between 16 and 20 dap and the second at 36 dap. Ethylene levels were elevated in sh2 kernels compared with Su kernels, with an initial peak 20 dap approximately 3-fold higher than in Su kernels and a second peak 36 dap approximately 5-fold higher than that in Su kernels. Ethylene treatment of Su kernels resulted in earlier and more extensive endosperm cell death and DNA fragmentation. Aminoethoxyvinylglycine treatment of sh2 kernels reduced the extent of DNA fragmentation. We conclude that ethylene is involved in triggering programmed cell death in developing maize endosperm and is responsible for the aberrant phenotype of sh2 kernels.     

Sugar utilization by developing wild type and shrunken-2 maize kernels

To characterize the movement of sugars during kernel development in maize, a newly devised in vitro kernel development scheme was utilized. Viable seeds of wild type maize (Zea mays L.) as well as the mutant shrunken-2 (sh2) were found to mature when grown in culture with reducing sugars or sucrose as the carbon source. However, wild type and sh2 kernels had greater germination, starch content, and seed weight when sucrose, rather than reducing sugars, was the carbon source. By the use of labeled sucrose it was shown that sucrose can move into endosperm tissue without intervening degradation and resynthesis. These results show that when grown in vitro the maize seed can utilize reducing sugars for development, but it prefers sucrose.     

The maize disorganized aleurone layer 1 and 2 ( dil1, dil2) mutants lack control of the mitotic division plane in the aleurone layer of developing endosperm

The maize (Zea mays L.) endosperm consists of an epidermal like layer of isodiametric aleurone cells surrounding a central body of starchy endosperm cells. In disorgal1 (dil1) and disorgal2 (dil2) mutants the control of the mitotic division plane is relaxed or missing, resulting in mature grains with disorganized aleurone layers. In addition to orientation of the division plane, both the shape and size of the aleurone cells are affected, and often more than one layer of aleurone cells is present. Homozygous dil1 and dil2 grains are shrunken due to reduced accumulation of starchy endosperm and premature developmental arrest of the embryo, and mature mutant grains germinate at a very low rate and fail to develop into plants. However, homozygous mutant plants can be obtained through embryo rescue, revealing that both mutants have an irregular leaf epidermis as well as roots with a strongly reduced number of root hairs and aberrant root hair morphology. Our results suggest the presence of common regulatory mechanisms for the control of cell division orientation in the aleurone and plant epidermis.Abbreviations DAP days after pollination - dek defective kernel mutant - dil disorganized aleurone layer mutant - GUS -glucuronidase - LM light microscopy - PPB pre-prophase band - SEM scanning electron microscopy - TUSC Trait Utility System for Corn     

ULTRASTRUCTURE OF THE MATURE UNGERMINATED RICE (ORYZA SATIVA) CARYOPSIS. THE CARYOPSIS COAT AND THE ALEURONE CELLS

The results of a light and electron microscopic study of the caryopsis coat and aleurone cells in ungerminated, unimbibed rice (Oryza sativa) caryopses are presented. Surrounding the rice grain is the caryopsis coat composed of the pericarp, seed coat and nucellar layers. The outermost layer, the pericarp, consists of crushed cells and is about 10 μm thick. The seed coat, interior to the pericarp, is one cell thick and has a thick cuticle. Between the seed coat cuticle and endosperm are the remains of the nucellus. The nucellus is about 2.5 μm thick and has a thick cuticle adjacent to the seed coat cuticle. Interior to the caryopsis coat is the aleurone layer of the endosperm. The aleurone completely surrounds the rice grain and is composed of two cell types—aleurone cells that surround the starchy endosperm and modified aleurone cells that surround the germ. The aleurone cells of the starchy endosperm contain many aleurone grains and lipid bodies around a centrally located nucleus. The modified aleurone cells lack aleurone grains, have fewer lipid bodies than the other aleurone cells, and contain filament bundles (fibrils). Plastids of aleurone cells exhibit a unique morphology in which the outer membranes invaginate to form tubules and vesicles within the plastid. Transfer aleurone cells are not observed in the mature rice caryopsis.     

Endosperm differentiation in barley wild-type and sex mutants

The cereal endosperm is a storage organ consisting of the central starchy endosperm surrounded by the aleurone layer. In barley, endosperm development is subdivisible into four main stages, i.e. the syncytial (I), the cellularization (II), the differentiation (III) and the maturation stage (IV). During stage I, a multinucleate syncytium is formed, which in stage II develops into the undifferentiated cellular endosperm. During stage III the cells of the endosperm differentiate into two types of aleurone cells (peripheral and modified) and three different starchy endosperm cell types (irregular, prismatic and subaleurone). To elucidate the ontogenetic relationship between the endosperm tissues, the phenotypes of sex (shrunken endosperm mutants expressing xenia) mutant endosperms were studied. These mutants can be classified into two groups, i.e. those in which development is arrested at one of the four wild-type stages described above, and those with abnormal development with new organizational patterns in the endosperm or with novel cell types. Based on these studies, it is suggested that the two endosperm halves represent cell lines derived from the two daughter nuclei of the primary endosperm nucleus, and that the prismatic starchy endosperm cells arise from a peripheral endosperm meristematic activity during stage III. Finally, a model for the main molecular events underlying the morphogenetic processes is discussed.     

Changes in cell wall polysaccharides of germinating barley grains

Decorticated barley grains were germinated at 25° for 6 days, until the endosperm reserves were nearly exhausted. The neutral monosaccharide components of the hydrolysates of the cell walls and gums from the embryo, aleurone layer and starchy endosperm and the endospermic starch were determined at daily intervals. The amount of embryo cell wall polysaccharide increased 40 times and glucose became the major component, followed in abundance by xylose and arabinose. The cell wall and gum polysaccharides of the aleurone layer (plus testa) and the starchy endosperm declined during germination and their compositions altered. The endospermic starch also decreased. In the early stages of germination the apparent composition of the cell walls of the aleurone layer and starchy endosperm depended upon how they had been prepared. After 6 days the cell walls and gums had provided a significant carbohydrate supply to the living tissues, equivalent to 18.5% of the endospermic polysaccharide degraded during growth, starch having provided the remaining 81.5%.     

Brittle-1, an Adenylate Translocator, Facilitates Transfer of Extraplastidial Synthesized ADP-Glucose into Amyloplasts of Maize Endosperms

Amyloplasts of starchy tissues such as those of maize (Zea mays L.) function in the synthesis and accumulation of starch during kernel development. ADP-glucose pyrophosphorylase (AGPase) is known to be located in chloroplasts, and for many years it was generally accepted that AGPase was also localized in amyloplasts of starchy tissues. Recent aqueous fractionation of young maize endosperm led to the conclusion that 95% of the cellular AGPase was extraplastidial, but immunolocalization studies at the electron- and light-microscopic levels supported the conclusion that maize endosperm AGPase was localized in the amyloplasts. We report the results of two nonaqueous procedures that provide evidence that in maize endosperms in the linear phase of starch accumulation, 90% or more of the cellular AGPase is extraplastidial. We also provide evidence that the brittle-1 protein (BT1), an adenylate translocator with a KTGGL motif common to the ADP-glucose-binding site of starch synthases and bacterial glycogen synthases, functions in the transfer of ADP-glucose into the amyloplast stroma. The importance of the BT1 translocator in starch accumulation in maize endosperms is demonstrated by the severely reduced starch content in bt1 mutant kernels.     

Vacuolar H<Superscript>+</Superscript>-translocating inorganic pyrophosphatase (Vpp1) marks partial aleurone cell fate in cereal endosperm development

Cereal endosperm is a model system for cell fate determination in plants. In wild-type plants the outermost endosperm cells adopt aleurone cell fate, while all underlying cells display starchy endosperm cell fate. Mutant analysis showed that cell fate is determined by position rather than lineage. To further characterise the precise cell fate of the outermost cells, we performed a differential screen and isolated the novel marker gene Vpp1. It encodes a vacuolar H⁺-translocating inorganic pyrophosphatase (V-PPase) and is mainly expressed in kernels, leaves and tassels. In kernels, its expression is restricted to the aleurone layer with the maximum of expression shifting from the adaxial to the abaxial side during early stages. Together with three other marker genes Vpp1 was then used to analyse the cell fate of the outermost cells in Dap3, Dap7, cr4 and dek1 mutants, all of which have aberrant aleurone layers. In the Dap3 and Dap7 mutants the Vpp1 and Ltp2 markers but not the A1 and Zein markers were expressed in patches without aleurone indicating that the outermost cells had some but not all features of aleurone cells and did not simply adopt starchy endosperm cell fate. A similar result was obtained in the cr4 mutant, although Ltp2 expression was less generalised. In other Dap7 patches characterised by multiple aleurone-like cell layers the expression of Vpp1 and Ltp2 confirmed the aleurone cell fate of the cells in the additional cell layers. The analysis of dek1 mutants confirmed the starchy endosperm cell fate of the majority but not all outermost cells. Based on these data we propose a model suggesting a stepwise commitment to aleurone cell fate. Sequential steps are marked by the expression of Vpp1, the expression of Ltp2, the acquisition of a regular shape and thick walls and finally pigmentation coupled with A1 expression.     

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.     

Agrobacterium tumefaciens-Mediated Transformation of Maize Endosperm as a Tool to Study Endosperm Cell Biology

Developing maize (Zea mays) endosperms can be excised from the maternal tissues and undergo tissue/cell-type differentiation under in vitro conditions. We have developed a method to transform in vitro-grown endosperms using Agrobacterium tumefaciens and standard binary vectors. We show that both aleurone and starchy endosperm cells can be successfully transformed using a short cocultivation with A. tumefaciens cells. The highest transformation rates were obtained with the A. tumefaciens EHA101 strain and the pTF101.1 binary vector. The percentage of aleurone cells transformed following this method varied between 10% and 22% whereas up to the eighth layer of starchy endosperm cells underneath the aleurone layer showed transformed cells. Cultured endosperms undergo normal cell type (aleurone and starchy endosperm) differentiation and storage protein accumulation, making them suitable for cell biology and biochemical studies. In addition, transgenic cultured endosperms are able to express and accumulate epitope-tagged storage proteins that can be isolated for biochemical assays or used for immunolabeling techniques.The endosperm is a unique plant tissue that arises from a second fertilization event between a male gamete and the central cell. Its main function is to provide nutrients to the embryo either during seed development or during germination. In cereals, the endosperm consists of three main cell types: the starchy endosperm cells, which constitute the bulk of the endosperm and accumulate large quantities of storage proteins and starch; the epidermal aleurone cells; and the transfer cells, which are in contact with the maternal vascular tissues (Olsen, 2004). The cereal endosperm is important as a model system to study plant development, cell differentiation, programmed cell death, and synthesis, trafficking, and accumulation of storage compounds. In addition, it is a major source of carbohydrate and proteins for human and animal nutrition.In spite of its importance, cell biology studies on the cereal endosperm using modern imaging approaches such as expression of fluorescent subcellular markers are very scarce because: (1) the endosperm is deeply immersed in maternal tissues and therefore, not readily available for imaging analysis and (2) the long time required for transformation and regeneration of stable transgenic plants. Although several approaches for culturing maize (Zea mays) endosperm in vitro have been reported in the past years (Shimamoto et al., 1983), only recently a novel method developed by Odd-Arne Olsen and colleagues (Gruis et al., 2006) has proven to be successful in retaining endosperm tissue and cell type identity in in vitro conditions. Cultures derived from transgenic maize lines in which endosperm cell types are identified by the activity of specific promoters have shown that aleurone and starchy endosperm cell identity continues to be established in vitro (Gruis et al., 2006).Although Agrobacterium tumefaciens is not a natural pathogen of most monocots (Cleene, 1985; Binns and Thomashow, 1988), it has been successfully used to transform many cereals, including maize, wheat (Triticum aestivum), Sorghum, barley (Hordeum vulgare), and rice (Oryza sativa; Grimsley et al., 1989; Gould et al., 1991; Chan et al., 1993; Ishida et al., 1996, 2007; Gurel et al., 2009; Harwood et al., 2009; Hensel et al., 2009). In the case of maize, stable transgenic plants can be obtained by A. tumefaciens-mediated transformation using either super-binary or standard-binary vectors (Frame et al., 2002; Mohanty et al., 2009a, 2009b). However, transformation of isolated maize endosperms have been only possible using transient transformation approaches such as biolistic methods (Torrent et al., 1997; Gruis et al., 2006) and protoplast transfection (Gallie and Young, 1994). Unfortunately, these two methods are not always ideal for cell biology studies. On one hand, biolistic methods often result in high-copy number transgenic events and on the other, protoplasts are usually highly stressed cells not suitable for detailed protein localization studies. A. tumefaciens-mediated transformation methods circumvent these disadvantages by resulting in a low-copy number of transgenes in intact tissues.We have developed a method to transform in vitro-grown endosperms using a brief incubation time with A. tumefaciens cells carrying standard binary vectors. We present here a detailed explanation of the method and quantitative information on the transformation efficiency using different A. tumefaciens strains, culture density, and incubation time. We also provide evidence that the in vitro-differentiated aleurone and starchy endosperm cells are comparable to the corresponding cell types differentiated in planta and therefore, suitable for cell biology studies. In addition, we show that transgenic cultured endosperms are able to express and accumulate epitope-tagged storage proteins that can be isolated for biochemical assays or used for immunolabeling imaging techniques.     

Sugar Uptake and Metabolism in the Developing Endosperm of Tassel-seed Tunicate (Ts-5 Tu) Maize

Factors regulating assimilate transport into developing maize (Zea mays L.) kernels have been difficult to determine because of the structural complexity of basal kernel tissues and the damage that results from tissue dissection. The sensitivity of maize kernels to experimental manipulation is such that substantial maternal tissue is required to support kernel growth in vitro. Consequently, sugar transport experiments with isolated seed tissues or detached kernels have not unequivocally demonstrated how sugar transport occurs. In the present study, Tassel-seed Tunicate (Ts-5 Tu) maize kernels were investigated as a model system for introducing test solutions into the pedicel apoplast with minimal wounding. Transpiration in leafy glumes drew ¹⁴C-sugar solutions up the 8- to 10-millimeter-long pedicel stalks into the basal endosperm transfer cell region. ¹⁴C from fructose was incorporated into starch for 8 days. Sugar uptake into endosperm and embryo tissue showed specificity and inhibitor sensitivity. In particular, p-chloromercuribenzene sulfonate partially inhibited fructose uptake into the endosperm but had no effect on the metabolic conversion of that fructose that entered the endosperm. These results are consistent with active, carrier-mediated sugar transport, but a definitive determination would require more detailed tissue analysis. We propose that further refinement of the incubation solution may allow long-term kernel growth without cob tissue and thus provide a more precise determination of which maternal factors influence seed development.     

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.     

Proteomic Profiling of the Aleurone Layer of Mature Arabidopsis thaliana Seed

The aleurone layer of mature Arabidopsis thaliana seed plays important roles in seed germination and dormancy. However, the proteomic profile of this cell layer is unknown partly because it is difficult to separate this thin cell layer from the mature seeds. In this study, we have used a simple technique to separate the aleurone layer along with the seed coat following germination of seeds and determined for the first time the putative protein composition of this cell layer. By subjecting the total proteins extracted from the seed coat to 2D gel electrophoresis followed by liquid chromatography/tandem mass spectrometry, we identified four AGI loci, AT4G28520, AT5G44120, AT1G03880, and AT1G03890; all of which belong to the seed storage family of proteins. Because in Arabidopsis the diploid aleurone cells of the seed coat perform protein storage functions similar to that of triploid endosperm of other plant species, it is assumed that the above AGI loci are associated with the aleurone layer of the seed coat.     

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.     

Enhancement by gibberellic acid and asymmetric distribution of lysophospholipase in germinating barley

Germination of whole barley seeds for 4 and 6 days followed by measurement of lysophospholipase (lysolecithin acyl hydrolase, LAH) in the embryo-containing and embryo-free halves revealed a gradient of activity between the two halves of the seed. Most of the activity appeared in the embryo-containing half. This gradient decreased slightly in the aleurone and dramatically in the starchy endosperm during the 2 day germination interval. Embryo-containing and embryo-free half seeds of surface sterilized barley were placed separately on sterile agar plates. After 4 and 6 days LAH was observed in both the aleurone and starchy endosperm of the embryo-containing halves. In the embryo-free halves, LAH appeared at low levels in the aleurone and was virtually absent in the starchy endosperm. The scutellum of germinating seeds contains LAH activity. Exposure of embryo-free half seeds to GA₃ for 24 hr showed enhancement of acidic and alkaline LAH activities in the aleurone fraction and in the GA₃-medium in which the half seeds were treated. The LAH activity of the starchy endosperm of these half seeds was little changed by GA₃ treatment. Exposure of isolated aleurones to GA₃ for 24 hr resulted in substantial enhancement of acidic and alkaline LAH activities in the bathing medium and in fractions prepared from the aleurone. The physiological significance of the influence of GA₃ on LAH activity during barley germination is discussed.     

Proteolytic activities in the developing seeds of ×Haynaldoticum sardoum

Aminopeptidase, carboxypeptidase and proteinase activities were measured in endosperms from unripe and ripe seeds of ×Haynaldoticum sardoum. Aminopeptidase and proteinase activities were high during the early maturation stages and then decreased. In contrast, carboxypeptidase activity increased with maturation. Localization studies demonstrated that aminopeptidase and carboxypeptidase activities were present in the three tissues examined (pericarp, green layer plus aleurone, and starchy endosperm). Proteinase activity against gliadin was located in the pericarp and in the green layer plus aleurone, but was absent in the starchy endosperm. The presence of proteolytic activities in the outer kernel layers might be correlated to the hydrolysis of transitory protein reserves during the senescence of the seed coat?. Aminopeptidase and carboxypeptidase activities located in the starchy endosperm could participate in the breakdown of protein reserves during the early phases of seed germination.