Initital cellularization and differentiation of the aleurone cells in the ventral region of the developing wheat grain

Early cellularization of the free-nuclear endosperm and subsequent differentation of the aleurone cells in the ventral region of the developing wheatgrain (Triticumaestivum L. cv. Heron) were examined using both light and electron microscopy. In ovules harvested 1 d after anthesis, irregular wall ingroths typical of transfer cells protrude into the multinucleate cytoplasm. Initital cellularization occurs by a process of free wall formation in much the same fashion as in the dorsal region of the grain. In places, sheets of endoplasmic reticulum and dictyosomes appear to be closely associated with the growing wall. Like the wall ingrowths noted earlier, the freely growing walls are intensely fluorescent after staining with aniline blue. Initiatal cellularization is complete 2–3 days after anthesis. Unlike the first-formed cells in the dorsal region of the developing grain, those in the ventral region are not meristematic. These amitotic cells become the groove aleurone cells which at an early stage of development are set apart from the rest of the endosperm by their irregularly thickened walls and dense cytoplasm. Autofluorescence is first apparent in the walls of those cells next to the degenerating nucellus. In contrast to the aleurone cells in the dorsal region of the grain, at maturity only the inner wall layer of each of the groove aleurone cells remains autofluorescent. The aleurone grains are highly variable in appearance and contain no Type II inclusions.     

Effects of gibberellic acid and environmental factors on cytosolic calcium in wheat aleurone cells

Gibberellins (GAs) control a wide range of physiological functions in plants from germination to flowering. The cellular mechanisms by which gibberellic acid (GA₃) acts have been most extensively studied in the cereal aleurone. In this tissue, alterations in cellular calcium are known to be important for the primary response to GA, which is the production and secretion of hydrolytic enzymes. The extent to which cytosolic Ca²⁺ mediates the early events in GA action, however, is not known. In order to address this question, changes in cytosolic Ca²⁺ in wheat (Triticum aestivum L. cv. Inia) aleurone cells that occur rapidly after treatment with GA were characterized. In addition, GA-induced changes were compared with changes induced by three environmental stimuli that are known to modify the GA response: osmotic stress, salt (NaCl), and hypoxia. The Ca²⁺-sensitive dye fluo-3 was used to photometrically measure cytosolic Ca²⁺. It was found that GA₃ induced a steady-state increase in cytosolic Ca²⁺ of 100–500 nM. This increase was initiated within a few minutes of treatment with GA and was fully developed after 30–90 min. The changes in cytosolic Ca²⁺ that were induced by GA were distinct from those induced by mannitol, NaCl, or hypoxia. Mannitol caused a steady-state decrease whereas NaCl and hypoxia both increased cytosolic Ca²⁺. In the case of NaCl this increase was transient but for hypoxia the increase was prolonged as long as hypoxic conditions were maintained. Gibberellin-induced changes in cytosolic Ca²⁺ were not induced by the inactive GA, GA₈, nor did the GA-insensitive wheat mutant, D6899, respond to active GA₃ with altered cytosolic Ca²⁺. It is concluded that changes in cytosolic Ca²⁺ are an early and integral part of the GA response in aleurone cells. The data also indicate, however, that changes in Ca²⁺ are not sufficient, by themselves, to induce the GA response of aleurone cells.Abbreviations AM acetoxymethyl ester - GA gibberellin - GA₃ gibberellic acid - Mes 2-[N-morpholino]ethanesulfonic acid - PM plasma membrane The author is very grateful to Dr. T-h. D. Ho for his gift of D6899 grain and to Dr. R. Hooley for supplying the inactive GA₈. This work was supported by National Science Foundation Grant DCB-9206692.     

Gibberellin modulation of phosphatidyl-choline turnover in wheat aleurone tissue

Phosphatidyl choline (PC) is synthesised in wheat (Triticum aestivum L. cv. Flanders) aleurone tissue during early germination when new endomembranes are being formed. Although gibberellic acid does not ostensibly affect PC levels, it inhibits the incorporation of choline and differentially and specifically modulates the turnover of the N-methyl and methylene carbons of the choline headgroup of PC. Gibberellic acid has no effect on turnover of the phosphate moiety of either PC or the other major phosphatides. The possible biological importance of the findings is discussed.Abbreviations ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid - PA phosphatidic acid - PC phosphatidyl choline - PE phosphatidyl ethanolamine - PG phosphatidyl glycerol - PI phosphatidyl inositol - t_1/2 half-life     

Calcium localization in oat aleurone cells using chlorotetracycline and X-ray microanalysis

Calcium distribution was studied in oat caryopses. Using the chlorotetracycline method it was found that membrane-associated Ca²⁺ was present in the aleurone layer. X-ray microanalysis confirmed the presence of calcium in aleurone cells; it also demonstrated the presence of considerable amounts of calcium in the cell wall surrounding these cells.Abbreviation CTC chlorotetracycline     

Calmodulin stimulation of unidirectional calcium uptake by the endoplasmic reticulum of barley aleurone

The role of calmodulin (CaM) in gibberellic acid (GA₃)-stimulated Ca²⁺ uptake was investigated in endomembranes isolated from aleurone cells of barley (Hordeum vulgare L.). Unidirectional Ca²⁺ -uptake activity of endoplasmic reticulum (ER) was higher in membranes isolated from aleurone layers treated for 16 h with GA₃ and Ca²⁺ compared with those isolated from layers incubated in Ca²⁺ alone. However, the level of uptake from Ca²⁺-treated tissue could be stimulated to that of the GA₃-treated cells by applying exogenous CaM which increased the V _max of the Ca²⁺ transporter approximately threefold. Calcium uptake in ER from GA₃-treated tissue was inhibited by the CaM antagonist W7 in 50% of experiments, whereas the activity in membranes from non-GA₃-treated tissue was unaffected. Treatment with GA₃ also led to a twofold increase in CaM levels in aleurone layers within 4–6 h, paralleling the time course of the stimulation of Ca²⁺ uptake and preceding the stimulation of α-amylase secretion. We propose that the elevation of Ca²⁺ uptake into the ER induced by GA₃ may be coordinated and regulated by elevated levels of membrane-associated CaM and this may regulate Ca²⁺-dependent α-amylase synthesis in the lumen of the ER.     

The Golgi apparatus in developing endosperm of wheat (Triticum aestivum L.)

The ultrastructure and distribution of the Golgi apparatus in developing wheat endosperm was investigated using a zinc iodide-osmium tetroxide staining complex in conjunction with low and high voltage electron microscopy. Dictyosomes were numerous in starchy endosperm and aleurone at 15 days after anthesis, and during the period of rapid storage protein deposition 25 d after anthesis. Fewer dictyosomes were seen in maturing endosperm. Two types of vesicles were associated with the dictyosomes; small, heavily-stained vesicles were sited at the ends of fine tubules which extend from the cisternae, and larger less-stained vesicles were associated with the periphery of the cisternae. Stereo-pairs of micrographs up to 1 m thick were taken to demonstrate the interconnections between cisternal and tubular endoplasmic reticulum. Elements of tubular ER were closely associated with dictyosomes, but connections were not observed. These results are discussed in relation to the transport of endosperm storage proteins from their site of synthesis on the cisternal ER to their site of storage, the protein bodies.     

Development of aleurone and sub-aleurone layers in maize

Electron-microscope studies indicate that the aleurone tissue of maize (Zea mays L.) starts developing approximately 10–15 days after pollination in stocks that take ca. 40 days for the aleurone to mature completely. Development commences when specialized endosperm cells adjacent to the maternal nucellar layer start to differentiate. Differentiation is characterized by the formation of aleurone protein bodies and spherosomes. The protein bodies of the aleurone layer have a vacuolar origin whereas the protein bodies of the immediate underlying endosperm cells appear to develop from protrusions of the rough endoplasmic reticulum. Thus, two morphologically and developmentally distinct types of protein bodies are present in these adjacent tissues. The spherosomes of the aleurone layer form early in the development of this tissue and increase in number as the tissue matures. During the final stages of maturation, these spherosomes become closely apposed to the aleurone grains and the plasma membrane. No further changes are apparent in the structure of the aleurone cells after 40 days from pollination when the caryopsis begins to desiccate.     

Phospholipid metabolism of wheat grains: Enzymes of the CDP-diglyceride phospholipid biosynthetic pathway

Summary Enzymes of the CDP-diglyceride pathway of phospholipid synthesis, CDP-diacylglycerol synthetase, CDP-diacylglycerol: glycerol 3-phosphate phosphatidyl-transferase and enzymes of phosphatidylserine formation were initially of relatively high specific activities in aleurone cells of wheat and declined upon imbibition. Enzyme activity of phosphatidylinositol synthesis was not detected in dry grains but was present upon imbibition. CDP-diacylglycerol: glycerol 3-phosphate phosphatidyltransferase shifted during imbibition from 85% of the activity in the supernatant of aleurone layers from dry seeds to 98% associated with large particle fractions after 36 hours of imbibition. Phosphatidylserine formation shifted from a dominant location in the 1,500 x g fraction in the dry seed to a predominantly mitochondrial location after 36 hours of imbibition. The subcellular distribution of CDP-diacylglycerol synthetase did not change appreciably upon imbibition from that of the dry seed, 75 to 80% of the activity was found in the supernatant. Only CDP-diacylglycerol: glycerol 3-phosphate phosphatidyltransferase showed increased specific activity late in the imbibition period. GA₃ accelerated the decrease of already declining activities of the CDP-diglyceride enzymes and the changes in their patterns of distribution, augmented the activities of the phosphatidylinositol synthesizing enzyme, and both accelerated and augmented the increase in the activity of the enzyme of phosphatidylglycerol synthesis which occurred late in imbibition.Committee on Institutional Cooperation Travelling Scholar from the University of Chicago.     

The fine structure of aleurone cells in the soybean seed coat

Summary The morphology and fine structure of aleurone cells of soybean [Glycine max (L.) Merr.] seed coats were analyzed with transmission electron microscopy for the period of rapid seed fill up to physiological maturity. Thin sections and freeze-fracture replicas were prepared for each stage. The aleurone is a tissue lining the embryo sac and consists of a single layer of cells attached to the aerenchyma of the seed coat proper. During seed fill, aleurone cells contained numerous Golgi-derived vesicles in the basal region of the cytoplasm that were either free or attached to the plasma membrane along the lateral and basal regions of the cell wall. Correspondingly, the Golgi apparatus were well developed with individual dictyosomes having 5 to 8, highly fenestrated stacked cisternae. The degree of fenestration along the periphery of each cisterna increased from the cis to trans region. Rough endoplasmic reticulum (RER) was also abundant, often consisting of up to 30, stacked swollen cisternae which occupied large regions of cytoplasm. Plasmodesmata which connected adjacent aleurone cells was not observed along the dorsal walls of aleurone cells that faced aerenchyma. At physiological maturity, dictyosome cisternae were less fenestrated and had fewer associated secretory vesicles. Stacked lamellae of RER were absent, being replaced by short tubular cisternae and small vesicles. At physiological maturity, the aleurone cells had thick walls, and contained numerous lipid bodies in apposition to the plasma membrane. The cytoplasm appeared densely stained in thin-sections and contained protein bodies and amyloplasts with large starch grains. We conclude that during the period of rapid seed fill aleurone cells produce, package, transport and secrete vesicular contents toward the embryo, that is followed at physiological maturity by the storage of lipid, protein and starch in the same cells. The embryo is the most likely destination for secretory products during the period of rapid seed fill. The fate of the stored food reserves in aleurone cells at physiological maturity may be analogous to that of aleurone tissue of grasses, being utilized during imbibition for processes important to germination.     

Immunofluorescent visualization of arrays of transverse cortical actin microfilaments in wheat root-tip cells

Summary Actin microfilaments in isolated root-tip cells from wheat (Triticum aestivum L. cv. Kite) were visualized by immunofluorescence microscopy using two different antiactin monoclonal antibodies. Cells in interphase contain predominantly subcortical bundles of microfilaments, as described in many cell types, but in preprophase and prophase cells, immunodetectable actin is organized solely in ordered arrays of cortical microfilaments that cover the entire surface of the cell, transverse on lateral faces, random on end walls. Intermediate stages with random and transverse microfilaments are also seen on lateral faces. The cell cycle stage-dependent transverse cortical microfilaments described here are previously unreported in higher plant cells.Abbreviations Ig immunoglobulin - MF microfilament     

Total protein release from barley aleurone layers as a bioassay for gibberellins

The effect of gibberellic acid on the secretion of proteins from barley (Hordeum vulgare L.) aleurone layers has been investigated for its suitability as a gibberellin bioassay. Concentrations from 10^–4 g/ml to 100 g/ml of GA₃ resulted in the release of proportionally increasing amounts of total protein. The release of proteins is not affected by indoleacetic acid and kinetin. This method has been applied and compared with the -amylase assay for the estimation of gibberellin in extracts of tomato fruits and maize seedlings.Abbreviations GA₃ gibberellic acid - IAA indoleactic acid - K kinetin     

Diversity of salt tolerance in a germplasm collection of wheat (Triticum spp.)

Summary A collection (5,072 lines) of wheat germplasm was screened at the seedling stage for tolerance to salinity concentrations having electrical conductivities of 0.8 (control), 12.5, 18.75 and 25.0 dS/m. Surviving seedlings were expressed for each line as a percentage of the control value. The 442 lines with greater than 70% surviving seedlings were tested for whole-life cycle survival under each salinity condition. The data of the reactions to salinity at both the seedling stage and maturity were used to classify the collection according to: (1) country of origin (2) species and ploidy level. The data were then subjected to a diversity analysis using the Shannon-Weaver information index. Seedling stage tolerance to 12.5 dS/cm salinity was widely distributed in the collection (79% of lines), whereas only 9% were tolerant at 25.0 dS/m salinity. The seedling stage tolerance was indicative of maturity tolerance. At the seedling stage, entries from USA and Egypt showed the largest proportions of tolerant lines. In addition, USA, Mexico and Egypt entries exhibited the widest variability. Diversity among regions was greater than among countries within regions, while the diversity among species was greater than among ploidy levels. Tetraploids exceeded hexaploids and diploids in the proportion of tolerant lines and diversity. Wheat-rye derivatives showed a good potential for salt tolerance at early stages. Screening more germplasm from the arid and semi-arid regions especially from countries with salt affected soils was recommended.Contribution of Project (AR-1-73) SANCST     

Characterization of the calmodulin gene family in wheat: structure, chromosomal location, and evolutionary aspects

Calmodulin is a ubiquitous transducer of calcium signals in eukaryotes. In diploid plant species, several isoforms of calmodulin have been described. Here, we report on the isolation and characterization of calmodulin cDNAs corresponding to 10 genes from hexaploid (bread) wheat (Triticum aestivum). These genes encode three distinct calmodulin isoforms; one isoform is novel in that it lacks a conserved calcium binding site. Based on their nucleotide sequences, the 10 cDNAs were classified into four subfamilies. Using subfamily-specific DNA probes, calmodulin genes were identified and the chromosomal location of each subfamily was determined by Southern analysis of selected aneuploid lines. The data suggest that hexaploid wheat possesses at least 13 calmodulin-related genes. Subfamilies 1 and 2 were both localized to the short arms of homoeologous-group 3 chromosomes; subfamily 2 is located on all three homoeologous short arms (3AS, 3BS and 3DS), whereas subfamily 1 is located only on 3AS and 3BS but not on 3DS. Further analysis revealed thatAegilops tauschii, the presumed diploid donor of the D-genome of hexaploid wheat, lacks a subfamily-1 calmodulin gene homologue, whereas diploid species related to the progenitors of the A and B genomes do contain such genes. Subfamily 3 was localized to the short arm of homoeologous chromosomes 2A, 2B and 2D, and subfamily 4 was mapped to the proximal regions of 4AS, 4BL and 4DL. These findings suggest that the calmodulin genes within each subfamily in hexaploid wheat represent homoeoallelic loci. Furthermore, they also suggest that calmodulin genes diversified into subfamilies before speciation ofTriticum andAegilops diploid species.     

Localization of gibberellic acid-induced acid phosphatase activity in the endoplasmic reticulum of barley aleurone cells with the electron microscope

A metal-salt precipitation method with p-nitrophenyl phosphate as substrate has been used to localize in the electron microscope acid phosphatase activity in isolated aleurone layers of barley (Hordeum vulgare L.), treated for 16 h in the presence or absence of gibberellic acid (GA₃). The paper confirms results obtained earlier with an azo-dye precipitation method of enzyme localization. In addition the results show for the first time that in GA₃-treated tissue enzyme activity is associated with the endoplasmic reticulum (ER), there being reaction product deposited in the ER cisternae. It is suggested that this activity represents new enzyme synthesized on ER in response to GA₃ and probably destined for secretion.Abbreviation ER endoplasmic reticulum     

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     

The control of chloroplast number in wheat mesophyll cells

Chloroplast number per cell and mesophyll cell plan area were determined in populations of separated cells from the primary leaves of different wheat species representing three levels of ploidy. Mean chloroplast number per cell increases with ploidy level as mean cell size increases. But in addition the analysis of individual cells clearly shows that cells of a similar size but from species of different ploidies have similar numbers of chloroplasts. We conclude that the number of chloroplasts within a cell is closely correlated (P<0.001) with the size of the cell and this relationship is consistent for species of different ploidies over a wide range of cell sizes. These results are discussed in relation to the hypothesis that chloroplast number in leaf mesophyll cells is determined by the size of the cell.     

Gibberellic acid controls specific acid-phosphatase isozymes in aleurone cells and protoplasts of Avena fatua L.

In the presence of gibberellic acid (GA₃) aleurone layers and isolated aleurone protoplasts of Avena fatua accumulate specific isozymes of acid phosphatase (EC 3.1.3.2). Some of these may be involved in mobilizing aleurone-grain phosphate reserves during germination. The hormone also controls secretion of other specific molecular forms of the enzyme that probably assist in endosperm hydrolysis. The accumulation and secretion of putative cell-wall-associated isozymes are stimulated by the action of GA₃ in isolated protoplasts. This effect however, is apparently over-ridden in the intact tissue, possibly by a cell-wall-based feedback mechanism.Abbreviations GA₃ gibberellic acid - pI isoelectric point(s)     

Gibberellic-acid-induced synthesis and release of cell-wall-degrading endoxylanase by isolated aleurone layers of barley

When aleurone layers of barley (Hordeum vulgare L.) are incubated with gibberellic acid (GA₃) xylose and arabinose—both as free sugars and bound to larger molecules—are released into the medium. Release begins 10–12h after the start of incubation and continues for at least 60h. At the same time there is a GA₃-induced breakdown of the cell wall resulting in a loss of 2/3 of the cell-wall pentose during 60h of incubation. GA₃ causes the appearance in the medium of an enzyme (or enzymes) which hydrolyze larchwood xylan and aleurone-layer arabinoxylan. Release of the enzyme(s) into the medium begins 28–32h after the start of incubation. Enzyme activity does not accumulate to any large extent in the tissue prior to release into the medium, and is present in very low levels only in the absence of GA₃. Xylanase activity is associated with a protein (or proteins) with a molecular weight of 29,000. The hydrolysis of the xylans is largely caused by endoxylanase activity, indicating the importance of endoglycosidases in the GA₃-induced breakdown of the aleurone cell wall.