Aleurone Layer Cells Research Articles

Evidence for “gibberellin-like” activity of coumarin

The existing body of information defines some regulatory functions to secondary metabolites like coumarin (COU). Experience and some experimental results in our laboratory tempted us to test a GA-like activity of COU. Experiments were conducted with endospermic (de-embryonated) and embryonic wheat half grains treated with different concentrations of COU, for different time courses, alone or in combination with the GA-biosynthesis inhibitor paclobutrazol (P). Results showed that treatment with COU caused stimulation of amylase synthesis and secretion from aleurone layer cells. P did not affect synthesis but slightly reduced excretion of amylase. However, germination percentage of intact wheat grains and seedling growth were inhibited by COU in a concentration-dependent manner. This indicates that COU has some GA-like effect, and also affects other aspects of germination and seedling establishment. This GA-like effect was also confirmed by the observed increase in elongation of wheat seedlings second leaf sheath and pea stem when treated with COU. These potential regulatory roles of COU may indicate that secondary metabolites have more important roles during different plant developmental processes.

Protein and gene expression characteristics of a rice phosphoenolpyruvate carboxylase Osppc3; its unique role for seed cell maturation

Phosphoenolpyruvate carboxylase (PEPCase) has been considered a probable key enzyme for seed development. We previously isolated cDNA of a PEPCase isogene Osppc3 from developing rice seeds. However, its physiological role has been unclear. Here, we report an expression analysis of Osppc3 in developing seeds at the protein and mRNA levels. Osppc3 protein was preferentially expressed in developing seeds at an early stage, from 3 to 14 DAF, and was localized in the embryo and in the aleurone layer, the cells of which accumulate storage lipids. Co-expression analysis of Osppc3 revealed a gene expression pattern similar to those of fatty acid biosynthetic pathway genes as well as ribosomal protein genes and DNA replication genes, implying that Osppc3 cooperates with those gene products in seed development. We validated similarity of gene expression patterns of these genes by semi-quantitative RT-PCR in developing seeds. The gene expression pattern of Osppc3 was related to triacylglycerol accumulation in embryo and aleurone layer cells. Microscopic observations of embryo and aleurone layer tissues also revealed differences in the timepoint at which triacylglycerol drastically accumulates during cell maturation. These results indicate that Osppc3 should play roles in both the arrangements of cell components and storage lipid accumulation in rice seeds.

Two maize END-1 orthologs, BETL9 and BETL9like, are transcribed in a non-overlapping spatial pattern on the outer surface of the developing endosperm.

In the course of a project aimed to isolate transfer cells-specific genes in maize endosperm we have identified the BETL9 gene. BETL9 encodes for a small protein very similar in sequence to the product of the barley transfer cell-specific gene END-1. Both BETL9 and END-1 proteins are lipid transfer proteins, but their function is currently unknown. In situ hybridization analysis confirms that the BETL9 gene is exclusively transcribed in the basal endosperm transfer cell layer during seed development since 10 days after pollination. However, immunolocalization data indicates that the BETL9 protein accumulates in the maternal placento-chalaza cells located just beside the transfer cell layer. This suggests that the BETL9 protein should be transported to the maternal side to exert its, still unknown, function. In addition, we have identified a second maize gene very similar in sequence to BETL9 and we have named it BETL9like. In situ hybridization shows that BETL9like is also specifically transcribed in the developing maize endosperm within the same time frame that BETL9, but in this case it is exclusively expressed in the aleurone cell layer. Consequently, the BETL9 and BETL9like genes are transcribed in a non-overlapping pattern on the outer surface of the maize endosperm. The BETL9 and BETL9like promoter sequences, fused to the GUS reporter gene, accurately reflected the expression pattern observed for the genes in maize. Finally, we have identified in the Arabidopsis genome a set of four genes orthologous to BETL9 and BETL9like and analyzed the activity of their promoters in Arabidopsis transgenic plants carrying fusions of their promoter sequences to the GUS reporter. As in the case of the maize genes, the Arabidopsis orthologs showed highly complementary expression patterns.

Localisation of Storage Reserves in Developing Seeds of Pongamia pinnata (L.) Pierre, a Potential Agroforestry Tree

AbstractPongamia pinnata is an important oil yielding perennial tree species. The aim of the present study was to document the histological and ultrastructural change that is occurring during pongamia seed development. The seeds were sampled at five stages of development at intervals of 3 weeks starting from 30 weeks after flowering up to 42 weeks. The seed development was followed microscopically using toluidine blue staining. The seed coat was made up of an external layer of palisade cells, an internal layer of hourglass cells followed by a parenchymatous cell layer and aleurone cell layer. The seed reserve compounds such as polysaccharides, proteins and starch showed distinct histochemical characterisation. Lignin was mainly found in the seed coat cell layers, while polysaccharides, proteins and starch granules in the cotyledon cells. The ultrastructural studies showed marked cellular changes during the seed development. The cell size varied from 9.4 to 78 μm during the seed development. The number of oil bodies per cell ranged from 200 to 300 at 42 weeks after flowering. Protein storage vacuoles were observed during the later stages of seed development. The plastids contained electron‐dense starch granules. The seeds harvested after 42 weeks after flowering had maximum physiological maturity with high oil content and other seed reserve materials. This basic knowledge on pongamia seed development could invariably be used for further understanding of biochemical changes that might be involved in the biosynthetic pathway of oil.

Soil-Weed Seed Communication Systems

The nature of weeds is a complex adaptive, soil-seed communication system. The nature of weedy Setaria life history is an adaptable, changeable system in which complex behaviors emerge when self-similar plant components self-organize into functional traits possessing biological information about spatial structure and temporal behavior. Setaria life history behavior is a Markov chain of irreversible and reversible processes regulated by morpho-physiological traits acting through environment-plant communication systems (environment-plant-seed, soil-seed). Heritable functional traits are the physical reservoirs of information guiding life history development, emergent behavior. The consequence of structural self-similarity and behavioral self-organization has been the evolution of a complex adaptive seed-soil communication system. Weedy Setaria life history is represented in algorithmic form as FoxPatch, a model to forecast seed behavior. Weedy Setaria seed life history behaviors are controlled by environmental information (signals) flowing from the soil to the seed embryo. The specific signal to which Setaria is tuned affecting seed behavior in the soil is the amount of oxygen and heat (T, thermal) in soil water over time, oxy-hydro-thermal time (O2-H2O-T-Time). The Shannon environmental-biological communication system between the soil and the Setaria seed contains the five elements (E) and components:E1, information source, soil; E2, transmitter, soil particle contact with seed surface water films; E3, channel, continuous soil particle-seed surface water films; E4, receiver, living seed interior from the transfer aleurone cell layer (TACL) membrane to aleurone layer to embryo; E5, destination, embryo. The signal is soil O2-H2O-T-Time; the message is O2-H2O-T stimulating embryo respiration. The Setaria soil-seed communication system seed behavior can be also expressed as operations (processes) computed by seed algorithms. Information is physical: memory resides in several locations in the Setaria seed. Memory is expressed in the long-term by responsiveness to O2-H2O-heat messages as determined by the morpho-physiological soil-seed communication system (hull, TACL membrane, scavenger protein). The message is remembered: plants pass on a range of heteromorphic seeds appropriate to continuing, successful local adaptation.

Differences in phosphatidic acid signalling and metabolism between ABA and GA treatments of barley aleurone cells

Phosphatidic acid (PA) is the common lipid product in abscisic acid (ABA) and gibberellic acid (GA) response. In this work we investigated the lipid metabolism in response to both hormones. We could detect an invivo phospholipase D activity (PLD, EC 3.1.4.4). This PLD produced [(32)P]PA (phosphatidic acid) rapidly (minutes) in the presence of ABA, confirming PA involvement in signal transduction, and transiently, indicating rapid PA removal after generation. The presence of PA removal by phosphatidate phosphatase 1 and 2 isoforms (E.C. 3.1.3.4) was verified in isolated aleurone membranes invitro, the former but not the latter being specifically responsive to the presence of GA or ABA. The invitro DGPP phosphatase activity was not modified by short time incubation with GA or ABA while the invitro PA kinase - that allows the production of 18:2-DGPP from 18:2-PA - is stimulated by ABA. The long term effects (24h) of ABA or GA on lipid and fatty acid composition of aleurone layer cells were then investigated. An increase in PC and, to a lesser extent, in PE levels is the consequence of both hormone treatments. ABA, in aleurone layer cells, specifically activates a PLD whose product, PA, could be the substrate of PAP1 and/or PAK activities. Neither PLD nor PAK activation can be monitored by GA treatment. The increase in PAP1 activity monitored after ABA or GA treatment might participate in the increase in PC level observed after 24h hormone incubation.

Dynamic Changes in the Distribution of Minerals in Relation to Phytic Acid Accumulation during Rice Seed Development

Phytic acid (inositol hexakisphosphate [InsP(6)]) is the storage compound of phosphorus in seeds. As phytic acid binds strongly to metallic cations, it also acts as a storage compound of metals. To understand the mechanisms underlying metal accumulation and localization in relation to phytic acid storage, we applied synchrotron-based x-ray microfluorescence imaging analysis to characterize the simultaneous subcellular distribution of some mineral elements (phosphorus, calcium, potassium, iron, zinc, and copper) in immature and mature rice (Oryza sativa) seeds. This fine-imaging method can reveal whether these elements colocalize. We also determined their accumulation patterns and the changes in phosphate and InsP(6) contents during seed development. While the InsP(6) content in the outer parts of seeds rapidly increased during seed development, the phosphate contents of both the outer and inner parts of seeds remained low. Phosphorus, calcium, potassium, and iron were most abundant in the aleurone layer, and they colocalized throughout seed development. Zinc was broadly distributed from the aleurone layer to the inner endosperm. Copper localized outside the aleurone layer and did not colocalize with phosphorus. From these results, we suggest that phosphorus translocated from source organs was immediately converted to InsP(6) and accumulated in aleurone layer cells and that calcium, potassium, and iron accumulated as phytic acid salt (phytate) in the aleurone layer, whereas zinc bound loosely to InsP(6) and accumulated not only in phytate but also in another storage form. Copper accumulated in the endosperm and may exhibit a storage form other than phytate.

Presence of base excision repair enzymes in the wheat aleurone and their activation in cells undergoing programmed cell death

Cereal aleurone cells are specialized endosperm cells that produce enzymes to hydrolyze the starchy endosperm during germination. Aleurone cells can undergo programmed cell death (PCD) when incubated in the presence of gibberellic acid (GA) in contrast to abscisic acid (ABA) which inhibits the process. The progression of PCD in aleurone layer cells of wheat grain is accompanied by an increase in deoxyribonuclease (DNase) activities and the internucleosomal degradation of nuclear DNA. Reactive oxygen species (ROS) are increased during PCD in the aleurone cells owing to the β-oxidation of triglycerides and inhibition of the antioxidant enzymes possibly leading to extensive oxidative damage to DNA. ROS generate mainly non-bulky DNA base lesions which are removed in the base excision repair (BER) pathway, initiated by the DNA glycosylases. At present, very little is known about oxidative DNA damage repair in cereals. Here, we study DNA repair in the cell-free extracts of wheat aleurone layer incubated or not with phytohormones. We show, for the first time, the presence of 8-oxoguanine-DNA and ethenoadenine-DNA glycosylase activities in wheat aleurone cells. Interestingly, the DNA glycosylase and AP endonuclease activities are strongly induced in the presence of GA. Based on these data we propose that GA in addition to activation of nuclear DNases also induces the DNA repair activities which remove oxidized DNA bases in the BER pathway. Potential roles of the wheat DNA glycosylases in GA-induced oligonucleosomal fragmentation of DNA and metabolic activation of aleurone layer cells via repair of transcribed regions are discussed.

New insights into globoids of protein storage vacuoles in wheat aleurone using synchrotron soft X-ray microscopy.

Mature developed seeds are physiologically and biochemically committed to store nutrients, principally as starch, protein, oils, and minerals. The composition and distribution of elements inside the aleurone cell layer reflect their biogenesis, structural characteristics, and physiological functions. It is therefore of primary importance to understand the mechanisms underlying metal ion accumulation, distribution, storage, and bioavailability in aleurone subcellular organelles for seed fortification purposes. Synchrotron radiation soft X-ray full-field imaging mode (FFIM) and low-energy X-ray fluorescence (LEXRF) spectromicroscopy were applied to characterize major structural features and the subcellular distribution of physiologically important elements (Zn, Fe, Na, Mg, Al, Si, and P). These direct imaging methods reveal the accumulation patterns between the apoplast and symplast, and highlight the importance of globoids with phytic acid mineral salts and walls as preferential storage structures. C, N, and O chemical topographies are directly linked to the structural backbone of plant substructures. Zn, Fe, Na, Mg, Al, and P were linked to globoid structures within protein storage vacuoles with variable levels of co-localization. Si distribution was atypical, being contained in the aleurone apoplast and symplast, supporting a physiological role for Si in addition to its structural function. These results reveal that the immobilization of metals within the observed endomembrane structures presents a structural and functional barrier and affects bioavailability. The combination of high spatial and chemical X-ray microscopy techniques highlights how in situ analysis can yield new insights into the complexity of the wheat aleurone layer, whose precise biochemical composition, morphology, and structural characteristics are still not unequivocally resolved.

Transcription factor profiling identifies an aleurone-preferred NAC family member involved in maize seed development

Transcription factors (TFs) preferentially expressed in developing endosperm were identified by searching a maize transcriptome database. One thousand and two hundred TFs were annotated, 414 of which were shown to express in the seeds. Of these, 113 presented preferential expression in developing endosperm. Among the endosperm-preferred TFs 13% were represented by members of the zinc-finger family, 17% by members of the bZIP family and 11% by members of the NAC family. Among the NAC family TFs, we identified APN-1, whose expression was detected 5 days after pollination (DAP) and peaked at 20 DAP. Analysis of the promoter sequence of gene encoding APN-1 revealed the presence of cis-elements related to endosperm-specificity, abscisic acid (ABA) and gibberellic acid (GA) signalling. Transient expression analysis of a construct containing the β-glucuronidase gene driven by the Apn-1 gene promoter showed that expression was restricted to the aleurone cell layer. Analysis of mRNA levels in developing maize endosperm treated with hormones revealed that the expression of the gene encoding APN-1 was stimulated by ABA and inhibited by GA.

The defective seed5 (des5) mutant: effects on barley seed development and HvDek1, HvCr4, and HvSal1 gene regulation

Barley, one of the major small grain crops, is especially important in climatically demanding agricultural areas of the world, with multiple uses within food, feed, and beverage. The barley endosperm is further of special scientific interest due to its three aleurone cell layers, with the potential of bringing forward the molecular understanding of seed development and cell specification from Arabidopsis and maize. Work done in Arabidopsis and maize indicate the presence of conserved seed developmental pathways where Crinkly4 (Cr4), Defective kernel1 (Dek1), and Supernumerary aleurone layer1 (Sal1) are key players. With the use of microscopy, a comprehensive phenotypic characterization of the barley defective seed5 (des5) mutant is presented here. The analysis further extends to molecular quantification of gene expression changes in the des5 mutant by qRT-PCR. Moreover, full-length genomic sequences of the barley orthologues were generated and these were annotated as HvDek1, HvCr4, and HvSal1. The most striking results in this study are the patchy reduction in number of aleurone cells, rudimentary anticlinal aleurone cell walls, and the specific change of HvCr4 expression compared to HvDek1 and HvSal1. The data presented support the involvement of Hvdes5 in establishing aleurone cells. Finally, how these results might affect the current model of aleurone and epidermal cell identity and development is discussed with a speculation regarding a possible role of Des5 in regulating cell division/ secondary cell wall building.

Detailed Analysis of the Expression of an Alpha-gliadin Promoter and the Deposition of Alpha-gliadin Protein During Wheat Grain Development

Alpha-gliadin proteins are important for the industrial quality of bread wheat flour, but they also contain many epitopes that can trigger celiac (coeliac) disease (CD). The B-genome-encoded alpha-gliadin genes, however, contain very few epitopes. Controlling alpha-gliadin gene expression in wheat requires knowledge on the processes of expression and deposition of alpha-gliadin protein during wheat grain development. A 592-bp fragment of the promotor of a B-genome-encoded alpha-gliadin gene driving the expression of a GUS reporter gene was transformed into wheat. A large number of transgenic lines were used for data collection. GUS staining was used to determine GUS expression during wheat kernel development, and immunogold labelling and tissue printing followed by staining with an alpha-gliadin-specific antibody was used to detect alpha-gliadin protein deposited in developing wheat kernels. The promoter sequence was screened for regulatory motifs and compared to other available alpha-gliadin promoter sequences. GUS expression was detected primarily in the cells of the starchy endosperm, notably in the subaleurone layer but also in the aleurone layer. The alpha-gliadin promoter was active from 11 days after anthesis (DAA) until maturity, with an expression similar to that of a 326-bp low molecular weight (LMW) subunit gene promoter reported previously. An alpha-gliadin-specific antibody detected alpha-gliadin protein in protein bodies in the starchy endosperm and in the subaleurone layer but, in contrast to the promoter activity, no alpha-gliadin was detected in the aleurone cell layer. Sequence comparison showed differences in regulatory elements between the promoters of alpha-gliadin genes originating from different genomes (A and B) of bread wheat both in the region used here and upstream. The results suggest that additional regulator elements upstream of the promoter region used may specifically repress expression in the aleurone cell layer. Observed differences in expression regulator motifs between the alpha-gliadin genes on the different genomes (A and B) of bread wheat leads to a better understanding how alpha-gliadin expression can be controlled.

Dimerization properties of the transmembrane domains of Arabidopsis CRINKLY4 receptor-like kinase and homologs

CRINKLY4 (CR4) is a plant serine–threonine receptor kinase. In Zea mays, CR4 functions in the differentiation of the leaf epidermis and the aleurone cell layer and, in Arabidopsis thaliana, the ortholog ACR4 is involved in the development of the integument and seed coat. The Arabidopsis genome also encodes four CR4-related proteins (CRR) whose functions are not known. Based on studies of animal receptor kinase proteins it is likely that the molecular basis of function of CR4 and related proteins is mediated by receptor dimerization. The importance of the transmembrane (TM) domain in the dimerization of several receptor kinases has been demonstrated by the TOXCAT system, a genetic assay that measures helix interactions in a natural membrane environment. In this study, we have used the TOXCAT assay to investigate the potential of the CR4 and CR4-related TM domains to homo-dimerize. Our investigation indicates that the CR4 TM domain and the CRR TM domains have higher propensities for homo-dimerization than the ACR4 TM domain. Interestingly, the dimerization potential of the ACR4 TM domain is significantly weaker even though 13 of 24 amino acids are identical to that of the CR4 TM domain. In order to determine the contributions of specific amino acids to the higher dimerization potential of CR4 compared to ACR4, mutations were made at specific sites in ACR4 TM domain and the strength of the dimer assessed by the TOXCAT assay. One mutation restored the activity to the CR4 level, while other mutations produced either no change or significantly increased the dimerization potential of the ACR4 TM domain. Our results indicate that the TM domains of CR4, ACR4 and the CRR receptor family of proteins have the intrinsic capacity to homo-dimerize, albeit with varying degrees of affinity.

Differences in the dimerization properties mediated by the transmembrane domains of Zea mays and Arabidopsis CRINKLY4 AND CRINKLY4‐like receptor kinases

CRINKLY4 is a transmembrane (TM) containing serine/threonine receptor kinase that functions in the differentiation of the leaf epidermis and the aleurone cell layer in Z. mays (ZmCR4) and development of the integument and seed coat in Arabidopsis (AtCR4). In addition, the Arabidopsis genome also encodes four CRINKLY4 homologs whose functions are not known. Based on studies of animal receptor kinase proteins, it is likely that CRINKLY4, and its homologs, also function as dimers. The importance of the TM domain in the dimerization of several receptor kinases has been demonstrated by the TOXCAT system, a genetic assay that measures helix interactions in a natural membrane environment. In this study, we have used the TOXCAT assay to investigate the potential of the CRINKLY4 and CRINKLY4‐like TM domains to homodimerize. Our investigation indicates that the ZmCR4 TM domain and the Arabidopsis homologs have similar homodimerization potentials. In contrast, the dimerization potential of the AtCR4 TM domain is significantly weaker, which suggests that the functional mechanisms of ZmCR4 and AtCR4 may be different. Interestingly, 13 of 24 amino acids are identical between the ZmCR4 and AtCR4 TM domains. We also report on studies aimed at delineating the contribution of specific amino acids to differences in the dimerization potential of CRINKLY4 TM domains of the two organisms.

Inheritance of the number and thickness of cell layers in barley aleurone tissue (Hordeum vulgare L.): an approach using F2–F3 progeny

The aleurone tissue of cereal grains, nutritionally rich in minerals and vitamins, is an important target for the improvement of cereals. Inheritance of the thickness and the number of cell layers in barley aleurone was studied on the F2-F3 progeny of an Erhard Frederichen x Criolla Negra cross in which the parental lines have three or two aleurone layers, respectively. F3 grain was sampled from each F2 plant and 96.8% of the entire F3 grain population was classified as being either the 2- or 3-layer type. Using microsatellite, single nucleotide polymorphism (SNP) and morphological markers on 190 F2 plants, a linkage map was built. Three quantitative trait loci (QTLs) affecting aleurone traits were revealed on chromosome 5H (max. LOD = 5.83) and chromosome 7H (max. LOD = 4.45) by interval mapping, and on chromosome 2H by marker analysis with an unmapped marker. These QTLs were consistent with genetic sub-models involving either 2-cell type dominance for 7H and 2H, or putative partial dominance for 5H where 2-cell-layer dominance and additivity gave similar LODs. The number of aleurone cell layers and aleurone thickness were strongly correlated and QTL results for these traits were alike. An SNP marker of sal1, an orthologue of the maize multilayer aleurone gene was mapped to the 7HL chromosome arm. However, the 7H QTL did not co-locate with the barley sal1 SNP, suggesting that an additional gene is involved in determining aleurone traits. These new mapping data allow comparisons to be made with related studies.

Hordoindolines are Predominantly Expressed in the Aleurone Layer in Late Kernel Development in Barley

In order to isolate grain-specific genes during kernel development, two differential screening methods—suppression subtractive hybridization (SSH) and differential hybridization (DH)—were applied. Two cDNAs encoding hordoindolines known to be related to grain hardness were isolated and designated as Hordeum vulgare indoline a and b (HvIDa and HvIDb). The cDNAs encoding HvIDa and HvIDb contained a 450 bp and a 444 bp open reading frame (ORF) that encoded the putative hordoindoline-a and -b precursors consisting of 150 and 148 amino acids, respectively. The deduced amino acid sequences of both HvIDa and HvIDb contained one tryptophan-rich domain and ten highly conserved cysteine residues. The expression of the HvIDa gene was high at 5 DAF, reached a peak at 8 DAF and decreased slightly until 20 DAF, while the expression of HvIDb began to be detectable at 8 DAF when it was higher than that of other developmental stages and decreased slightly until 20 DAF. The HvIDa and HvIDb genes were predominantly detected in the aleurone cell layers in the late part of kernel development, e.g. at 20 DAF. These findings may provide clues to the molecular mechanisms of kernel development and contribute to the determination of the grain texture in barley.

Expressions of rice sucrose non-fermenting-1 related protein kinase 1 genes are differently regulated during the caryopsis development

The rice sucrose non-fermenting-1 related protein kinase 1 ( SnRK1) family consists of three genes, which were named OSK1, OSK24 and OSK35. In order to elucidate the distinct functions of OSK genes, we identified precise regions for their expression by the promoter: GUS expression analyses as well as in situ mRNA localization experiments. At first, we isolated genomic clones corresponding to each member of OSKs in order to obtain the promoter sequences. All OSK genes house 11 exons and 10 introns and the positions of introns within the coding regions are fully conserved in all these genes. Histochemical analyses using OSK promoter: β-glucronidase ( OSKP:GUS) reporter genes showed that expression patterns of OSK1P:GUS and OSK24P:GUS were quite different in the developing caryopsis. The expression of OSK1P:GUS was nearly restricted in the vascular tissues during the caryopsis development. In contrast, the OSK24P:GUS expression was detected in the pericarp at the early stage with a shift to the endosperm as the endosperm cells were formed, and GUS staining was confined to both aleurone layer and endosperm cells around 15 days after flowering, when cell division of cellular endosperm were almost finished. The shifting pattern of the OSK24 expression was correlated with the appearance of starch granules in each tissue. Similar correlation between OSK24 expression and emergence of starch granules was also observed at another temporal sink organ, the basal part of leaf sheath. These results suggest that OSK24 (rice SnRK1b) most probably have a special role in carbohydrate metabolism of the sink organs.

The concentrations and distribution of phytic acid-phosphorus and other mineral nutrients in wild-type andlow phytic acid1-1 (lpa1-1) corn (Zea maysL.) grains and grain parts

A comparison of mineral nutrient and phytic acid-phosphorus (PA-P) distribution in the grains of wild-type (WT) and low phytic acid1-1 (lpa1-1) corn (Zea mays L.) was conducted to determine how the lpa1-1 mutation influences mineral element concentrations in different grain parts and impacts the structure of phosphorus-rich inclusions (globoids) in the grain cells. This is the first report regarding total phosphorus (P) and PA-P concentrations in scutellum and root-shoot axis portions of cereal embryos of WT in comparison to its matching lpa1-1 genotype. In WT, 95% of the grain PA-P was located in the embryo, mostly in the scutellum. The lpa1-1 mutation reduced whole-grain PA-P by 62% but influenced the scutella more than the root-shoot axes and rest-of-grain fractions. In spite of the lpa1-1 mutants containing greatly reduced PA-P, whole-grain amounts of Mg, Fe, and Mn were higher in lpa1-1 than in WT, K and Zn were similar, and Ca was lower. Iron was 1/3 higher in lpa1-1 grains than WT while Ca was 18% lower. Decreased phytic acid in lpa1-1 grains resulted in reduction in globoid size in both scutellum and aleurone layer cells. Most lpa1-1 aleurone globoids were non-spherical and scutellum globoids were clusters of small spheres while WT globoids were large discrete spheres. X-ray analyses of globoids in both grain types revealed major amounts of P, K, and Mg and traces of Ca, Fe, and Zn. Both grain types contained almost no mineral nutrient stores in the starchy endosperm.Key words: corn (Zea mays L.), phytic acid-phosphorus, low phytic acid1-1 (lpa1-1) grains, mineral nutrients, globoids, electron microscopy.

Metabolism of hydrogen peroxide during reserve mobilization and programmed cell death of barley (Hordeum vulgare L.) aleurone layer cells.

During germination, aleurone layer cells of barley (Hordeum vulgare) grains synthesize and secrete hydrolytic enzymes (principally alpha-amylase) in response to gibberellic acid (GA); shortly thereafter, the aleurone layer cells undergo programmed death. Gluconeogenesis of lipid reserves within aleurone cells, which supports this hydrolytic enzyme synthesis, results in the generation of H(2)O(2), which is catabolized by glyoxysomal catalase. Lowered amounts of catalase may contribute to aleurone cell death because of a compromised capacity to cope with reactive oxygen species generated by glyoxysomes and mitochondria. In the presence of GA, cells of intact aleurone layers underwent programmed death between 18 and 48 h; in the presence of ABA, no cell death was evident over 60 h. The capacity of GA-treated layers to metabolize exogenous H(2)O(2) increased steadily over the first 24 h, during the stage of lipid mobilization and the major synthesis and secretion of alpha-amylase; thereafter, this capacity declined markedly. In contrast, cells of ABA-treated aleurone layers exhibited little change in their capacity for H(2)O(2)-metabolism. Glyoxysomal catalase increased in activity over the first 12-24 h of GA treatment, which was accompanied by an increase in catalase-1 transcripts between 12 and 18 h. Catalase protein and activity declined after 24 h in GA-treated layers, prior to the onset of rapid programmed death at 30 h. These data suggest that a decline in glyoxysomal catalase precedes death of aleurone cells and may indeed contribute to an increase in cellular oxidative stress.

Molecular cloning and functional analysis of a novel type of Bowman-Birk inhibitor gene family in rice.

Bowman-Birk inhibitor (BBI) genes encode serine protease inhibitors that have repetitive cysteine-rich domains with reactive sites for the trypsin or chymotrypsin family. We have identified seven BBI genes from japonica rice (Oryza sativa subsp. japonica var Teqing). All of the genes identified were found in a single cluster on the southern end of the long arm of rice chromosome 1. Four of the seven BBI genes have two repetitive cysteine-rich domains, whereas one has a truncated domain with only one reactive site. We have also identified three novel BBI genes, each of which possesses three repetitive domains instead of two. In situ hybridization analyses indicated that the accumulation of rice BBI transcripts was differentially regulated in germinating embryos and also in the leaves, roots, and flower organs at later developmental stages. Different members of the rice BBI gene family displayed different expression patterns during rice seed germination, and wounding induced the expression of rice BBI transcripts. The three-domain BBIs had higher expression levels than the two-domain BBIs. It was also found that the mRNA of rice BBI genes was present in abundant amounts in scutellar epithelium and aleurone layer cells. RBBI3-1, one of the three-domain RBBI, exhibited in vitro trypsin-inhibiting activity but no chymotrypsin-inhibiting activity. Overexpression of RBBI2-3 in transgenic rice plants resulted in resistance to the fungal pathogen Pyricularia oryzae, indicating that proteinase inhibitors confer resistance against the fungal pathogen in vivo and that they might play a role in the defense system of the rice plant.