Diamine oxidase is involved in H(2)O(2) production in the chalazal cells during barley grain filling

The localization and activities of diamine oxidase (DAO, EC 1.4.3.6) and polyamine oxidase (PAO, EC 1.4.3.4) together with polyamine levels have been investigated in developing grains of barley (Hordeum vulgare L.). DAO (pH 7.5) is present mainly in vascular tissue and its neighbouring cells, namely chalazal cells and nucellar projection, while PAO (pH 6.0) is mainly localized in the chlorenchymatous cells of the crease and at the base of the vascular tissue. Activities of both these enzymes appear to be independently-regulated, as DAO activity increased steadily throughout grain development while PAO activity was higher during the early stages of grain filling, declined thereafter and again increased towards maturity. The maximum activities of DAO coincided with the maximum content of putrescine while the levels of PAO did not seem to be directly correlated with spermidine or spermine contents. Isoelectric focusing (IEF) of DAO and PAO activities revealed the presence of bands at 30 and 45 DPA. The possible involvement of DAO and PAO in the supply of H(2)O(2) to peroxidase-catalysed reactions in the chalazal cells during grain filling is discussed.     

Spatial and temporal dynamics of peroxidase and amine oxidase activity is linked to polyamines and lignin in wheat grains

The regulation of contents and activities of peroxidase (POX), diamine oxidase (DAO) and polyamine oxidase (PAO) were determined in relation to polyamines and lignin content in wheat (Triticum aestivum L.) grains. Two cultivars WH 542 (heat susceptible) and PBW 343 (heat tolerant) were used. Activities of POX, DAO and PAO were substantially higher in PBW 343 as compared with WH 542 and appeared to be independently regulated. POX and PAO showed peak activities at mid-milky stage (15 d post anthesis) while the activity of DAO showed continuous decline. Histochemical localization of POX and PAO in situ revealed their presence in the chalazal cell walls, crease and seed coat. Substantially higher activities of enzymes in PBW 343 correlated well with a higher degree of lignification in the chalazal cells as compared to WH 542.     

The location of (1-3)-β-glucan in the nucellar projection and in the vascular tissue of the crease in developing barley grain using a (1-3)-β-glucan-specific monoclonal antibody

Immunocytochemical localization of the (1-3)-beta-glucan, callose, in developing barley (Hordeum vulgare L.) grain was investigated using a specific monoclonal antibody and observed by means of confocal laser-scanning microscopy. The nucellar projection (NP) and vascular tissue (VT) of the crease cells were specifically labelled by this antibody at all stages of grain development. Maximum intensity of label was found in the NP at 12-15 days post anthesis; thereafter, label was localized in the VT of the crease. The location of (1-3)-beta-glucan in the NP and VT of the crease was also monitored by means of aniline blue-induced fluorescence of callose. The results obtained using both methods were found to be similar. The possible significance of the presence of callose in these tissues is discussed in relation to the uptake of assimilates into the developing grain.     

Peroxidase isozyme patterns in the skin of maturing tomato fruit

The cessation of tomato fruit growth is thought to be induced by an increase in the activity of enzymes which rigidify cell walls in the fruit skin. Peroxidase could catalyse such wall‐stiffening reactions, and marked rises in peroxidase activity were recently reported in skin cell walls towards fruit maturity. Peroxidase isoforms in the fruit are here analysed using native gel electrophoresis. New isoforms of apparent M_r 44, 48 and 53 kDa are shown to appear in cell walls of the fruit skin at around the time of cessation of growth. It is inferred that these isozymes are present in the cell wall in vivo. Fruit from a range of non‐ripening mutants were also examined. Some of these do not soften or ripen for many weeks after achieving their final size. The new isozymes were found in skin cell walls of mature fruit in each of these mutants, as in the wild‐type and commercial varieties. It is concluded that the late‐appearing isozymes are not associated with fruit ripening or softening, and are probably not ethylene‐induced. They may act to control fruit growth by cross‐linking wall polymers within the fruit skin, thus mechanically stiffening the walls and terminating growth.     

Soluble peroxidase from winter wheat seedlings with phenoloxidase-like activity

Peroxidase isozymes from winter wheat (Triticum aestivum L. cv Orso) seedlings extracts showed phenoloxidase-like activity, becoming visible on polyacrylamide gels also in the absence of hydrogen peroxide. The results obtained after a characterization of the two activities, based on their substrate specificity, on their selective inhibition, and on the possible occurrence of artifacts, suggested the existence of polyfunctional peroxidase isozymes. Different isozymes possessing only phenol oxidase activity were not found in the same plant material. This appears to be the first evidence of phenoloxidase-acting isoperoxidases in winter wheat.     

Partial purification of tomato fruit peroxidase and its effect on the mechanical properties of tomato fruit skin

Peroxidase (EC 1.11.1.7)-mediated stiffening of cell walls within the fruit skin of tomato is hypothesized to regulate fruit growth. However, to date, there is no experimental evidence demonstrating that peroxidase affects the mechanical properties of skin tissue. Here, the mechanical properties of skin strips excised from a range of fruits at different ages were determined using an 'Instron' universal material testing instrument. The stiffness of tomato fruit skin strips increases 3-fold with increasing fruit age. Application of partially-purified peroxidase from the cell walls of mature tomato fruit skin significantly increased the stiffness of fruit skin irrespective of the age of fruit. Furthermore, the application of hydrogen peroxide significantly increased the stiffness of skin strips excised from fruit of an age when endogenous peroxidase isozymes associated with the termination of growth are first detected. The results support the hypothesis that the tomato fruit skin plays an integral role in the regulation of tomato fruit growth, and that changes in its mechanical properties may be mediated by peroxidase. As far as is known, this is the first demonstration that peroxidases alter the mechanical properties of the plant cell wall.     

Effects of the barley mutants Risø 1508 and 527 high lysine genes on the cellular development of the endosperm

The dry grain weight of the Risø barley ( Hordeum vulgare L. var. disticum ) high lysine mutants 1508 and 527 at maturity was 32 and 37% lower, respectively, than the grains of the cultivar Bomi. Dry grain weight of the double mutant 527/1508 was reduced by 57%. Total number of endosperm nuclei from cv. Bomi, mutants 1508, 527 and 527/1508 at 24 days after anthesis was 173 000,156 000,121 000 and 111 000, respectively. Transverse mid-grain sections from mutants 1508 and 527 contained fewer aleurone cells and approximately the same number of starchy endosperm cells as cv. Bomi. The cellular organization of the endosperm of the double mutant deviated substantially from the normal. Cell volume in the central starchy endosperm of cv. Bomi, mutants 1508 and 527 at 33 days after anthesis averaged 390 000, 270 000 and 180 000 um5, respectively. Cell volume in the double mutant was smaller than in 527, but could not be accurately estimated due to the irregular shape of the cells. The mean section area of individual large starch granules in the central endosperm of mutants 1508, 527 and 527/1508 at 33 days after anthesis was 30, 48 and 72% smaller, respectively, than those of cv. Bomi. The average aleurone cell volume in cv. Bomi at 33 days after anthesis was 6 200 μm³.     

Changes in chloroplast peroxidase activities in relation to chlorophyll loss in barley leaf segments

Total peroxidase activity increased during senescence of excised barley ( Hordeum vulgare L. cv. Kashimamugi) leaves. Kinetin treatment furter increased total peroxidase activity but repressed chlorophyll degradation in excised barley leaves. When isoperoxidases were extracted from barley leaf segments. 4 cationic and 4 anionic isozymes were found in polyacrylamide gel electrophorests during leaf senescence. The chloroplasts contained only two cationic isoperoxidase activities. One (designated C4) was repressed by kinetin. and the other (C3) was increased by kinetin. Glucosamine, which also repressed the degradation of chlorophyll, completely repressed C4 activity but did not affect C3 activity. The induction with senescence, and the repression with kinetin and glucosamine, suggest chat chloroplast isoperoxidase C4 may function as a chlorophyll-degrading enzyme during barley leaf senescence.     

Subcellular localization of peroxidase in tomato fruit skin and the possible implications for the regulation of fruit growth

The cessation of tomato fruit growth has been associated with the appearance of three 'wall-bound' peroxidase isozymes in the skin of tomato fruit. However, the presence of these isozymes in the ionically eluted 'wall-bound' fraction may be an artefact of either non-specific binding of symplastic peroxidase to the cell wall, or isozymes bound to membranes included in the 'wall-bound' fraction. Therefore, subcellular localization of peroxidase in both immature and mature tomato fruit skins was studied. Immature fruits showed intense peroxidase activity associated with the tonoplast and pro-vacuolar membranes, but little or no activity associated with the cell wall. However, the presence of peroxidase activity within the cell wall of mature green fruits was confirmed. Furthermore, peroxidase activity was also observed associated with the plasma membrane and large vesicles allied to the plasma membrane. While cross-linking in cell wall components was previously assumed to be the mechanism by which peroxidase might control fruit growth, the incorporation of 'lignin-like' phenolics may also play a part. Isoelectric focusing (IEF) of both symplastic and apoplastic peroxidase extracted from immature and mature tomato fruit skin showed that all peroxidase isozymes present were highly anionic. In this current study, histochemical techniques are used to demonstrate a developmental increase in 'lignin-like' phenolics within the sub-cuticular cell walls of the fruit skin. The localization of peroxidase within tomato fruit skin is discussed in relation to its potential role in the regulation of tomato fruit growth.     

Changes in Activities of Antioxidant Enzymes in Sunflower Leaves of Different Ages

Role of superoxide dismutase isozymes and other antioxidant enzymes was studied in relation to leaf age in sunflower (Helianthus annuus L. cv. ACC 1508) at pre-flowering and grain filling stages. Relative water content (RWC) did not change much in leaves of different age and at the two stages. Protein content declined continuously from the youngest to the oldest leaf, while chlorophyll (Chl) and carotenoids (Car) contents increased down to 7^th/9^th leaf and declined in subsequent older leaves. Protein, Chl and Car contents were higher at pre-flowering than at seed filling stage. Superoxide dismutase (SOD), its isozymes, and ascorbate peroxidase (APO) and catalase (CAT) activities were highest in the 9^th leaf and declined in subsequent older leaves. SOD and APO activities were higher at seed filling, except in oldest senescent (13^th, 15^th) leaves. Among SOD isozymes, Cu/Zn-SOD and Mn-SOD activities accounted for most of the total SOD, and only marginal activity was observed for Fe-SOD. Peroxidase activity increased from youngest to the oldest leaf at pre-flowering stage and down to 13^th leaf at seed filling stage. This revised version was published online in July 2006 with corrections to the Cover Date.     

粳稻细胞质雄性不育系、保持系POD同工酶的比较研究

粳稻３４１Ａ、３４１Ｂ的不同器官和花药及雌蕊的不同发育时期共检测出９种ＰＯＤ同工酶谱带,其中ＰＯＤ＿５、ＰＯＤ＿６在所有１０个器官中存在,因此可以认为是粳稻３４１Ａ、３４１Ｂ的基本谱带。ＰＯＤ＿８、ＰＯＤ＿９谱带仅在少数器官中存在,具有一定的器官专一性。花粉粒发育的两个关键时刻,ＰＯＤ同工酶均多于保持系,这可能是雄性不育细胞质基因对核基因表达的调控作用所致,细胞质基因与细胞核基因存在着一定的相互关系,雄性不育可能是这两种基因共同作用的结果。     

Specific aspects of detection of peroxidase isozymes and their genetic control in barley

Identical specimens were separated by electrophoresis in two gels to detect and fix peroxidase isozymes. Both gels were stained by Coomassie brilliant blue for detecting proteins. One gel was previously incubated for detecting peroxidase activity. The differences in electrophoretic patterns between the gels indicate the zones of peroxidase activity. It has been shown that locus Prx 6H, controlling a low-mobility grain peroxidase (PRX 6H), is localized to barley chromosome 6. Two loci, Alb 4H and Alb 7H, controlling the biosyntheses of water-soluble proteins of barley endosperm, were localized to chromosomes 4 and 7. It has been demonstrated that barley species is polymorphic at multiple molecular forms of peroxidase.     

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 foliar applications of fungicides during plant growth on grain development and grain germinability in spring barley

In 1990 and 1991 grain samples of spring barley were obtained from plots in which fungal diseases were controlled until ear emergence using fungicide sprays, and from comparable plots which had received no fungicide applications. The grain was stored at 10^oC, and tested for germination at intervals during storage. Grain from plots treated with fungicide consistently exhibited more dormancy than grain from untreated plots. In 1992, grain development was monitored from anthesis to harvest-ripeness in treated and untreated plots of cvs Blenheim, Camargue and Tyne. In cvs Blenheim and Camargue, grains in fungicide-treated plots had a greater dry weight at harvest-ripeness, and dehydrated later in development than grains in untreated plots. Neither of these differences was observed in cv. Tyne. Possible interpretations of the effects of fungicide treatment of the parent plant on the physiology of the grain are discussed.     

The chemical composition of suberin in apoplastic barriers affects radial hydraulic conductivity differently in the roots of rice (Oryza sativa L. cv. IR64) and corn (Zea mays L. cv. Helix)

Apoplastic transport barriers in the roots of rice (Oryza sativa L. cv. IR64) and corn (Zea mays L. cv. Helix) were isolated enzymatically. Following chemical degradation (monomerization, derivatization), the amounts of aliphatic and aromatic suberin monomers were analysed quantitatively by gas chromatography and mass spectrometry. In corn, suberin was determined for isolated endodermal (ECW) and rhizo-hypodermal (RHCW) cell walls. In rice, the strong lignification of the central cylinder (CC), did not allow the isolation of endodermal cell walls. Similarly, exodermal walls could not be separated from the rhizodermal and sclerenchyma cell layers. Suberin analyses of ECW and RHCW of rice, thus, refer to either the entire CC or to the entire outer part of the root (OPR), the latter lacking the inner cortical cell layer. In both species, aromatic suberin was mainly composed of coumaric and ferulic acids. Aliphatic suberin monomers released from rice and corn belonged to five substance classes: primary fatty acids, primary alcohols, diacids, omega-hydroxy fatty acids, and 2-hydroxy fatty acids, with omega-hydroxy fatty acids being the most prominent substance class. Qualitative composition of aliphatic suberin of rice was different from that of corn; (i) it was much less diverse, and (ii) besides monomers with chain lengths of C(16), a second maximum of C(28) was evident. In corn, C(24) monomers represented the most prominent class of chain lengths. When suberin quantities were related to surface areas of the respective tissues of interest (hypodermis and/or exodermis and endodermis), exodermal cell walls of rice contained, on average, six-times more aliphatic suberin than those of corn. In endodermal cell walls, amounts were 34 times greater in rice than in corn. Significantly higher amounts of suberin detected in the apoplastic barriers of rice corresponded with a substantially lower root hydraulic conductivity (Lp(r)) compared with corn, when water flow was driven by hydrostatic pressure gradients across the apoplast. As the OPR of rice is highly porous and permeable to water, it is argued that this holds true only for the endodermis. The results imply that some caution is required when discussing the role of suberin in terms of an efficient transport barrier for water. The simple view that only the quantity of suberin present is important, may not hold. A more detailed consideration of both the chemical nature of suberins and of the microstructure of deposits is required, i.e. how suberins impregnate wall pores.     

Iron Deficiency Decreases Suberization in Bean Roots through a Decrease in Suberin-Specific Peroxidase Activity

The suberin content of young root parts of iron-deficient and iron-sufficient Phaseolus vulgaris L. cv Prélude was determined. The aliphatic components that could be released from suberin-enriched fractions by LiAID₄ depolymerization were identified by gas chromatography-mass spectrometry. In the normal roots, the major aliphatic components were ω-hydroxy acids and dicarboxylic acids in which saturated C₁₆ and monounsaturated C₁₈ were the dominant homologues. Iron-deficient bean roots contained only 11% of the aliphatic components of suberin found in control roots although the relative composition of the constituents was not significantly affected by iron deficiency. Analysis of the aromatic components of the suberin polymer that could be released by alkaline nitrobenzene oxidation of bean root samples showed a 95% decrease in p-hydroxybenzaldehyde, vanillin, and syringaldehyde under iron-deficient conditions. The inhibition of suberin synthesis in bean roots was not due to a decrease in Fe-dependent ω-hydroxylase activity since normal ω-hydroxylation could be demonstrated, both in vitro with microsomal preparations and in situ by labeling of ω-hydroxy and dicarboxylic acids with [¹⁴C]acetate. The level of the isozyme of peroxidase that is specifically associated with suberization was suppressed by iron deficiency to 25% of that found in control roots. None of the other extracted isozymes of peroxidase was affected by the iron nutritional status. The activity of the suberin-associated peroxidase was restored within 3 to 4 days after application of iron to the growth medium. The results suggest that, in bean roots, iron deficiency causes inhibition of suberization by causing a decrease in the level of isoperoxidase activity which is required for polymerization of the aromatic domains of suberin, while the ability to synthesize the aliphatic components of the suberin polymer is not impaired.     

Histological analysis of the maturation of native and wound periderm in potato (Solanum tuberosum L.) Tuber

Maturation of potato (Solanum tuberosum L.) tuber native and wound periderm and development of resistance to periderm abrasion were investigated utilizing cytological and histochemical techniques. Both native and wound periderm consist of three different tissues: phellem, phellogen and phelloderm. It was previously determined that the phellogen walls of immature native periderm are thin and prone to fracture during harvest, leading to periderm abrasion (excoriation). Phellogen walls thicken and become less susceptible to fracture upon maturation of the periderm, leading to resistance to excoriation. We now demonstrate that phellogen cells of immature wound periderm also have thin radial walls and that wound periderm abrasion is due to fracture of these walls. Maturation of the wound periderm is also associated with an increase in the thickness of the phellogen radial walls. Histological analysis with ruthenium red and hydroxylamine-FeCI2, which stain unesterified and highly methyl-esterified pectins, respectively, indicates that the phellogen cell walls of native and wound periderm differ significantly regardless of the stage of maturity. Results obtained by staining with ruthenium red and hydroxylamine-FeCI2 imply that phellogen cell walls of immature native periderm contain methyl-esterified pectin, but are lacking in unesterified (acidic) pectins. Maturation of native periderm is accompanied by an apparent increase in unesterified pectins in the walls of phellogen cells, which may allow for the strengthening of phellogen cell walls via calcium pectate formation. Histological staining of the phellogen walls of wound periderm, on the other hand, implies that these walls are deficient in pectins. Moreover, maturation of wound periderm is not accompanied by an increase in unesterified pectins in these walls. Since peroxidase is known to catalyse the cross-linking of cell wall polymers, we stained native and wound periderm for the presence of peroxidase utilizing guaiacol as a substrate. Peroxidase staining was strong in the phellogen walls of both immature and mature native periderm and we could not detect any differences in staining between them. Peroxidase staining was weak in the phellogen walls of immature wound periderm and was not detectably different in mature wound periderm. Peroxidase data imply that there are distinct differences between native and wound periderm, though our data do not indicate that changes in peroxidase activity are involved in the development of resistance to periderm abrasion that occurs upon maturation of the periderm. However, we cannot rule out the involvement in this process of peroxidase isozymes that have low affinity for the substrates utilized here.     

Development of Embryo and Endosperm and Nutrient Accumulation in Vigna sesquipedalis (L.) Fruwirth

The structure of embryo sac, fertilization and development of embryo and endosperm in Vigina sesquipedalis (L.) Fruwirth were investigated. Pollization occures 7–10h before anthesis, and fertilization is completed 10 h after anthesis. After fertilization, wall ingrowths are formed at the micropylar and chalazal ends of the embryo sac. Embryo development conforms to the Onagrad type, and passes through 2 or more celled proembryo, long stick-shaped, globular, heart shaped, torpedo, young embryo, growing and enlarging embryo and mature embryo. Wall ingrowths are formed on the walls of basal cells and outer walls of the cells at basal region of suspenser. The suspensor remains as the seed reaches maturity. The starch grains accumulate in the cells of cotyledons by 9–16 days after anthesis, and proteins accumulate by 12–18 days after. The endosperm development follows the nuclear type. The endosperm ceils form at the micropylar end, and remain free nuclear phase at chalazal end. The outer cells are transfer cells. Those cells at the micropylar end form folded cells with wall ingrowths. At heartembryo stage, the endosperm begins to degenerate and disintegrates before the embryo matures.     

Effects of barley mildew on green leaf area and grain yield in field and greenhouse experiments

In 1980 the relationships between mildew severity, green leaf area (GLA) and grain yield of spring barley were examined using greenhouse-grown plants and plants grown in micro-plots in the field. Mildew, by causing premature senescence, reduced GLA and grain yield was strongly correlated with GLA integrated from growth stages 2–10.5 on the Feekes scale. Early mildew attack reduced all yield components (including grain size) even when fungicidal control had eliminated mildew by anthesis. Analyses of culms at anthesis and harvest supported the view that the smaller grain size associated with early mildew attack resulted from a deficiency in carbohydrate stored in culms before anthesis and available for retranslocation to the developing grain. Amounts of total soluble carbohydrate at anthesis and the amounts lost beween anthesis and harvest were both strongly correlated with GLA up to anthesis.     

The cellular pathway of photosynthate transfer in the developing wheat grain. II. A structural analysis and histochemical studies of the pathway from the crease phloem to the endosperm cavity

In the developing wheat grain, photosynthate is transferred longitudinally along the crease phloem and then laterally into the endosperm cavity through the crease vascular parenchyma, pigment strand and nucellar projection. In order to clarify this cellular pathway of photosynthate unloading, and hence the controlling mechanism of grain filling, the potential for symplastic and apoplastic transfer was examined through structural and histochemical studies on these tissue types. It was found that cells in the crease region from the phloem to the nucellar projection are interconnected by numerous plasmodesmata and have dense cytoplasm with abundant mitochondria. Histochemical studies confirmed that, at the stage of grain development studied, an apoplastic barrier exists in the cell walls of the pigment strand. This barrier is composed of lignin, phenolics and suberin. The potential capacity for symplastic transfer, determined by measuring plasmodesmatal frequencies and computing potential sucrose fluxes through these plasmodesmata, indicated that there is sufficient plasmodesmatal cross-sectional area to support symplastic unloading of photosynthate at the rate required for normal grain growth. The potential capacity for membrane transport of sucrose to the apoplast was assessed by measuring plasma membrane surface areas of the various cell types and computing potential plasma membrane fluxes of sucrose. These fluxes indicated that the combined plasma membrane surface areas of the sieve element–companion cell (se–cc) complexes, vascular parenchyma and pigment strand are not sufficient to allow sucrose transfer to the apoplast at the observed rates. In contrast, the wall ingrowths of the transfer cells in the nucellar projection amplify the membrane surface area up to 22-fold, supporting the observed rates of sucrose transfer into the endosperm cavity. We conclude that photosynthate moves via the symplast from the se–cc complexes to the nucellar projection transfer cells, from where it is transferred across the plasma membrane into the endosperm cavity. The apoplastic barrier in the pigment strand is considered to restrict solute movement to the symplast and block apoplastic solute exchange between maternal and embryonic tissues. The implications of this cellular pathway in relation to the control of photosynthate transfer in the developing grain are discussed.