Differential effect of ammonium on the induction of nitrate and nitrite reductase activities in roots of barley (Hordeum vulgare) seedlings

The effect of exogenous NH₄⁺ on the induction of nitrate reductase activity (NRA; EC 1.6.6.1) and nitrite reductase activity (NiRA; EC 1.7.7.1) in roots of 8-day-old intact barley (Hordeum vulgare L.) seedlings was studied. Enzyme activities were induced with 0.1, 1 or 10 mM NO₃⁺ in the presence of 0, 1 or 10 mM NH₄⁺, Exogenous NH₄⁺ partially inhibited the induction of NRA when roots were exposed to 0.1 mM, but not to 1 or 10 mM NO₃⁺, In contrast, the induction of NiRA was inhibited by NH₄⁺ at all NO₃⁺ levels. Maximum inhibition of the enzyme activities occurred at 1.0 mM NH₄⁺ Pre-treatment with NH₄⁺ had no effect on the subsequent induction of NRA in the absence of additional NH₄⁺ whereas the induction of NiRA in NH₄⁺-pretreated roots was inhibited in the absence of NH₄⁺ At 10 mM NO₃⁺ L-methionine sulfoximine stimulated the induction of NRA whether or not exogenous NH₄⁺ was present. In contrast, the induction of NiRA was inhibited by L-methionine sulfoximine irrespective of NH₄⁺ supply. During the postinduction phase, exogenous NH₄⁺ decreased NRA in roots supplied with 0.1 mM but not with 1mM NH₃⁺ whereas, NiRA was unaffected by NH₄⁺ at either substrate concentration. The results indicate that exogenous NH₄⁺ regulates the induction of NRA in roots by limiting the availability of NO₃⁺. Conversely, it has a direct effect, independent of the availability of NO₃⁺, on the induction of NiRA. The lack of an NH₄⁺ effect on NiRA during the postinduction phase is apparently due to a slower turnover rate of that enzyme.     

Differential localization of two acid proteinases in germinating barley (Hordeum vulgare) seed

A cathepsin D-like aspartic proteinase (EC 3.4.23) is abundant in ungerminated barley ( Hordeum vulgare ) seed while a 30 kDa cysteine endoproteinase (EC 3.4.22) is one of the proteinases synthesized de novo in the germinating seed. In this work, the localization of these two acid proteinases was studied at both the tissue and subcellular levels by immunomicroscopy. The results confirm that they have completely different functions. The aspartic proteinase was present in the ungerminated seed and, during germination, it appeared in all the living tissues of the grain, including the shoot and root. Contrary to previous suggestions, it was not observed in the starchy endosperm. By immunoblotting, the high molecular mass form of the enzyme (32 + 16 kDa) was found in all the living tissues, whereas the low molecular mass form (29 + 11 kDa) was not present in the shoot or root, indicating that the two enzyme forms have different physiological roles. The aspartic proteinase was localized first in the scutellar protein bodies of germinating seed, and later in the vacuoles which are formed by fusion of the protein bodies. In contrast to the aspartic proteinase, the expression of the 30 kDa cysteine proteinase began during the first germination day, and it was secreted into the starchy endosperm; first from the scutellum and later from the aleurone layer. It was not found in either shoots or roots. The 30 kDa cysteine proteinase was detected in the Golgi apparatus and in the putative secretory vesicles of the scutellar epithelium. These results suggest that the aspartic proteinase functions only in the living tissues of the grain, as opposed to the 30 kDa cysteine proteinase which is apparently one of the proteases initiating the hydrolysis of storage proteins in the starchy endosperm.     

西藏青稞4个B组醇溶蛋白基因的克隆和特征

从两份西藏青稞材料中分离克隆出4个B组醇溶蛋白基因（BH1—BH4）,DNA测序结果表明：它们均包含完整的开放阅读框。推断的氨基酸序列与先前报道的大麦B组醇溶蛋白具有相同的蛋白质基本结构。系统分析表明：它们推断的氨基酸序列与栽培大麦中的B组醇溶蛋白具有较高的相关性,与野生大麦和山羊草属的醇溶谷蛋白相似性较低。并且,在4个基因BH1—BH4中,BH1与先前报道的B组醇溶蛋白基因有较低的序列相似性,因此我们对BH1基因进行了原核表达,含该基因的表达载体在大肠杆菌中表达出相对分子质量为28．15kDa并以包涵体形式存在的蛋白,进一步对其在青稞谷粒品质改良中的潜在价值进行了探讨。     

Influence of nitrate supply on concentrations and translocation of abscisic acid in barley (Hordeum vulgare)

Spring barley ( Hordeum vulgare L. cv. Golf) was grown at different nitrate supply rates, controlled by using the relative addition rate technique, in order to elucidate the relationship between nitrate-N supply and root and shoot levels of abscisic acid (ABA). The plants were maintained as (1) standard cultures where nitrate was supplied at relative addition rates (RAs) of 0.03, 0.09 and 0.18 day⁻¹, and (2) split-root cultures at RA 0.09 day⁻¹ but with the nitrate distributed between the two root parts in ratios of 100:0, 80:20 and 60:40. Time-dependent changes in root and shoot concentrations of ABA (determined by radioimmunoassay using a monoclonal antibody) were observed in both standard and split-root cultures during 12 days of acclimation to the different nitrate regimes. However, the ABA responses were similar at all nitrate supply rates. Further experiments were performed with split-root cultures where the distribution of nitrate between the two root parts was reversed from 80:20 to 20:80 so that short-term effects to local perturbations of nitrate supply could be studied without altering whole-plant N absorption. Transient increases in ABA concentrations (maximum of 25 to 40% after 3 to 4 h) were observed in both subroot parts, as well as in xylem sap and shoot tissue. By pruning the root system it was demonstrated that the change in ABA had its origin in the subroot part receiving the increased nitrate supply (i.e. switched from 20 to 80% of the total nitrate supply). The data indicate that ABA responses are easily transmitted between different organs, including transmission from one set of seminal roots to another via the shoot. The data do not provide any indication that long-term nitrate supplies or general nitrogen status of barley plants affect, or are otherwise related to, the average tissue ABA concentrations of roots and shoots.     

Control by phytochrome of the synthesis of protein related to senescence in chloroplasts of barley (Hordeum vulgare)

Protein synthesis has been measured in chloroplast isolated from detached leaves of barley ( Hordeum vulgare L. cv. Hassan). The effects of hormone and light treatments of the leaves on chloroplast protein synthesis have been compared with effects on other senescence symptoms. Interruption of the dark with red light retards senescence and increases chloroplast protein synthesis. The effect of red light was reversed by far-red light. Red light did not act additively with kinetin, and it competed with ethylene and abscisic acid, accelerators of senescence, which decreased protein synthesis. In contrast to the interruption of the dark with red light, continuous light decreased chloroplast protein synthesis. It may be concluded effects on chloroplast protein synthesis. The retardation of senescence by continuous light is not necessarily related to P_u Instead, energy provided by photosynthesis may be an important factor.     

Epi- and intracuticular lipids and cuticular transpiration rates of primary leaves of eight barley (Hordeum vulgare) cultivars

The major constituents of the epi- and intracuticular lipids of primary leaves of 8 cultivars of barley ( Hordeum vulgare L.) have been studied together with cuticular transpiration rates. The total amount of analysed cuticular lipids ranged from 9.6 to 13.4 μg cm⁻² and was dominated by the epicuticular fraction, which made up 73–84% of the total. There were variations in the percentages of the analysed lipid classes, alkanes, esters, aldehydes, β-diketones and alcohols, between epi- and intracuticular lipids among individual cultivars, but no clear tendency in these variations, except for the aldehydes, was found. The epicuticular lipids were richer in aldehydes than the intracuticular lipids. The cuticular transpiration rates were poorly correlated with the levels or composition of epi-, intra- or total cuticular lipids. The cuticular transpiration rates were considerably altered as a response to a water stress treatment, but these changes could not be correlated with any changes in amount or composition of the cuticular lipids. From these results it is concluded that some property other than amount or composition of cuticular lipids is the most important in regulation of water diffusion through the cuticle.     

Jasmonate-induced gene expression of barley (Hordeum vulgare) leaves -- the link between jasmonate and abscisic acid

In barley leaves a group of genes is expressed in response to treatment with jasmonates and abscisic acid (ABA) [21]. One of these genes coding for a jasmonate-induced protein of 23 kDa (JIP-23) was analyzed to find out the link between ABA and jasmonates by recording its expression upon modulating independently, the endogenous level of both of them. By use of inhibitors of JA synthesis and ABA degradation, and the ABA-deficient mutant Az34, as well as of cultivar-specific differences, it was shown that endogenous jasmonate increases are necessary and sufficient for expression of this gene. The endogenous rise of ABA did not induce synthesis of JIP-23, whereas exogenous ABA did not act via jasmonates. Different signalling pathways are suggested and discussed.     

Stimulation of ATPase activity in barley (Hordeum vulgare) root plasma membranes after treatment with triacontanol and calmodulin

Triacontanol (TRIA) treatment of plasma membrane-enriched vesicles from barley ( Hordeum vulgare L., cv. Conquest) roots resulted in stimulation of membrane-associated, divalent cation-dependent ATPase activity (EC 3.6.1.3). The stimulation at physiologically active concentrations of TRIA (10⁻¹¹–10⁻⁹ M ) occurred only when the vesicles were treated with TRIA in the presence of calmodulin. Octacosanol, the C₂₈-analogue of TRIA, had no effect on divalent cation-dependent ATPase activity. Consistent with in vivo studies, simultaneous treatment of vesicles with weight equivalents of TRIA and octacosanol reduced the stimulation of ATPase activity. The effect of calmodulin on the stimulation of ATPase activity was diminished by calmidazolium, a specific inhibitor of calmodulin. Circular dichroism studies did not show a change in the α-helix content of calmodulin in the presence of TRIA. TRIA also had no apparent effect on soluble calcium-calmodulin 3',5'-cyclic nucleotide phosphodiesterase activity. Removal of excess TRIA from the medium after treatment still resulted in stimulation of divalent cation-dependent ATPase activity in the presence of calmodulin was comparable to treated vesicles from which excess TRIA had not been removed. These data further support the contention that TRIA affects membrane structure and function.     

Chlorophyll organization in dark-grown and light-grown pine (Pinus brutia) and barley (Hordeum vulgare)

A study was conducted comparing the organization of chlorophyll during development of the photosynthetic apparatus in dark-grown and light-grown pine and barley. The rationale was that gymnosperms, but not angiosperms, have a capacity to synthesize chlorophyll in darkness. Seedlings of Pinus brutia were germinated and grown in darkness or under photoperiodic (day/night) conditions. The low-temperature (77 K) fluorescence spectra of newly-emerging dark-grown seedlings exhibited a single fluorescence band peaking at 678–679 nm, which decayed primarily with a ∼5.5 ns lifetime. Over the first few days of growth, the emission shifted to longer wavelengths and a subnanosecond lifetime component became prevalent. After several days of dark growth the emission spectrum and lifetime profile of the low temperature fluorescence came to resemble those of light-grown pine and barley. At room temperature, dark-grown pine showed little variable fluorescence, though addition of DCMU caused a substantial fluorescence rise. Illumination with moderate light for a few hours was sufficient to 'photoinduce' the appearance of normal variable fluorescence. At 77 K, DCMU-treated samples clearly showed a very long-lived (∼40 ns) fluorescence lifetime component in light-grown pine and barley. This component was undetectable in dark-grown pine. If, however, dark-grown samples were illuminated either before or after DCMU addition and then frozen to 77 K, the ∼40 ns lifetime component appeared at a fluorescence intensity similar to that in light-grown samples. These results are explained primarily in terms of photoactivation of the photosystem II (PSII) donor side. The temporary maintenance of PSII in an inactive, highly-quenched state is suggested to provide an available, yet protected precursor for active PSII.     

The cytological effects of the gametocides Ethrel and RH-531 on microsporogenesis in barley (Hordeum vulgare L)

Abstract. Barley plants ( Hordeum vulgare L. cv. Midas) raised under controlled environmental conditions were sprayed with either of the gametocides Ethrel and RH-531. At various times after spraying the anthers were fixed for light and electron microscopy. Abortion of sporogenous cells occurred in plants sprayed at both pre- and post-meiotic stages of microsporogenesis. In contrast, cells of the tapetum were insensitive to the immediate effects of gametocides. The cytological effects of the gametocides are similar to those induced by male sterile genes in a variety of plants. These range from the induction of additional mitotic divisions in the pollen mother cells to exine malformations on developing microspores. These observations are discussed in terms of the control mechanisms operating during microsporogenesis.     

Novel approaches for examining the effects of differential soil compaction on xylem sap abscisic acid concentration, stomatal conductance and growth in barley (Hordeum vulgare L.)

Novel techniques were devised to explore the mechanisms mediating the adverse effects of compacted soil on plants. These included growing plants in: (i) profiles containing horizons differing in their degree of compaction and; (ii) split-pots in which the roots were divided between compartments containing moderately (1·4 g cm ^{? 3}) and severely compacted (1·7 g cm ^{? 3}) soil. Wild-type and ABA-deficient genotypes of barley were used to examine the role of abscisic acid (ABA) as a root-to-shoot signal. Shoot dry weight and leaf area were reduced and root : shoot ratio was increased relative to 1·4 g cm ^{? 3} control plants whenever plants of both genotypes encountered severely compacted horizons. In bartey cultivar Steptoe, stomatal conductance decreased within 4 d of the first roots encountering 1·7 g cm ^{? 3} soil and increased over a similar period when roots penetrated from 1·7 g cm ^{? 3} into 1·4 g cm ^{? 3} soil. Conductance was again reduced by a second 1·7 g cm ^{? 3} horizon. These responses were inversely correlated with xylem sap ABA concentration. No equivalent stomatal responses occurred in Az34 (ABA deficient genotype), in which the changes in xylem sap ABA were much smaller. When plants were grown in 1·7 : 1·4 g cm ^{? 3} split-pots, shoot growth was unaffected relative to 1·4 g cm ^{? 3} control plants in Steptoe, but was significantly reduced in Az34. Excision of the roots in compacted soil restored growth to the 1·4 g cm ^{? 3} control level in Az34. Stomatal conductance was reduced in the split-pot treatment of Steptoe, but returned to the 1·4 g cm ^{? 3} control level when the roots in compacted soil were excised. Xylem sap ABA concentration was initially higher than in 1·4 g cm ^{? 3} control plants but subsequently returned to the control level; no recovery occurred if the roots in compacted soil were left intact. Xylem sap ABA concentration in the split-pot treatment of Az34 was initially similar to plants grown in uniform 1·7 g cm ^{? 3} soil, but returned to the 1·4 g cm ^{? 3} control level when the roots in the compacted compartment were excised. These results clearly demonstrate the involvement of a root-sourced signal in mediating responses to compacted soil; the role of ABA in providing this signal and future applications of the compaction procedures reported here are discussed.     

Conversion of D-tryptophan to indole-3-acetic acid in coleoptiles of a normal and a semi-dwarf barley (Hordeum vulgare) strain

Exogenously applied D-tryptophan (D-Trp) was more effective than L-Trp in inducing elongation of coleoptile segments of a normal barley ( Hordeum vulgare L. cv. Akashinriki) strain and a semi-dwarf strain with lower endogenous indole-3-acetic acid (IAA) level. D-cycloserine, an inhibitor of D-aminotransferase, completely inhibited both the D- and L-Trp-induced elongation of both strains. Addition of D-Trp increased IAA levels in both strains 4-fold over endogenous levels. The increase in IAA level was completely inhibited by D-cycloserine. The endogenous L-Trp level of semi-dwarf coleoptiles was similar to that of normal ones. These results suggested that IAA is synthesized by the conversion of L-Trp to indole-3-pyruvic acid via D-Trp in both strains, and that the lower IAA level of the semi-dwarf strain probably is a result of the impeded IAA biosynthesis involved in D-Trp.