Isolation and characterization of a urobilinogenoidic chlorophyll catabolite from Hordeum vulgare L

A new type of chlorophyll catabolite was isolated from extracts of de-greened primary leaves of barley (Hordeum vulgare cv. Lambic). Its constitution was elucidated by one-dimensional and two-dimensional [(1)H,(13)C]-homo- and heteronuclear NMR spectroscopic techniques and by high resolution mass spectroscopy. The isolated catabolite, a water-soluble, colorless, and nonfluorescent linear tetrapyrrole, resembles urobilinogen in which one of the propionic side chains forms a five membered isocylic ring system, indicating its origin from the chlorophylls.     

Organophosphate induced chlorophyll mutations in Hordeum vulgare

Summary Ten Organophosphorus (OP) insecticides are tested for their genetic toxicology in the Hordeum vulgare system. Of these, 8 OPs induced chlorophyll mutations of which 6 are new discoveries.     

Light-independent accumulation of chlorophyll a and b and protochlorophyllide in green barley (Hordeum vulgare)

Barley ( Hordeum vulgare L. cvs Clipper, Procter, Astrix) seedlings were transferred from daylight to darkness and changes in chlorophyll a , chlorophyll b , protochlorophyllide and chlorophyllide (μ leaf⁻¹) in either the first or second leaf determined spectrophotometrically after separating the esterified from unesterified pigments by partitioning between ammoniacal acetone and light petroleum ether. Chlorophyll a and b as well as protochlorophyllide accumulated in the dark. The ratio of chlorophyll to protochlorophyllide formed in the absence of light was 18:1. 5-aminolevulinic acid (10 m M ) promoted the synthesis of chlorophyll a and b and protochlorophyllide. Pigment synthesis and response to 5-aminolevulinic acid addition was related to tissue age. Mature tissue in the apical third of the leaf accumulated most chlorophyll, but per μg chlorophyll present at the time of transfer to darkness, was less efficient than immature tissue towards the base of the leaf. Immature tissue was also most responsive to added 5-aminolevulinic acid. Chlorophyll synthesis in the dark was accompanied by chloroplast development. Chloroplasts in immature leaf tissue increased in size and extent of thylakoid development when transferred from daylight to darkness. The results indicate that chlorophyll synthesis and chloroplast membrane development in light-grown barley continue into the dark phase of the diurnal cycle. A light-independent protochlorophyllide reductase in light-grown barley seedlings is postulated.     

Light induced transpiration in a chlorophyll deficient mutant of Hordeum vulgare

A chlorophyll deficient mutant of Hordeum vulgare L. was investigated with respect to its transpiration response to light pulses. Broad band blue light. 380–500 nm, caused a significant transpiration response, while broad band red light did not. The transpiration response to changes in the ambient CO₂-concentration was the same in the chlorophyll deficient mutant as in green plants. The absence of a transpiration response to red light in the mutant was therefore not the result of a defective CO₂-response. It is concluded that the specific blue light response is not mediated via photosynthetic CO₂-fixation. The nature of the blue light response is discussed.     

Chlorophyll Fluorescence as a Possible Tool for Salinity Tolerance Screening in Barley (Hordeum vulgare L.)

The application of chlorophyll fluorescence measurements to screening barley (Hordeum vulgare L.) genotypes for salinity tolerance has been investigated. Excised barley leaves were cut under water and incubated with the cut end immersed in water or in a 100-mM NaCl solution, either in the dark or in high light. Changes in rapid fluorescence kinetics occurred in excised barley leaves exposed to the saline solution only when the incubation was carried out in the presence of high light. Fluorescence changes consisted of decreases in the variable to maximum fluorescence ratio and in increases in the relative proportion of variable fluorescence leading to point I in the Kautsky fluorescence induction curve. These relative increases in fluorescence at point I appeared to arise from a delayed plastoquinone reoxidation in the dark, since they disappeared after short, far-red illumination, which is known to excite photosystem I preferentially. We show that a significant correlation existed between some fluorescence parameters, measured after a combined salt and high-light treatment, and other independent measurements of salinity tolerance. These results suggest that chlorophyll fluorescence, and especially the relative fluorescence at point I in the Kautsky fluorescence induction curve, could be used for the screening of barley genotypes for salinity tolerance.     

Reinvestigation of the chlorophyll distribution among the chlorophyll-proteins and chlorophyll-protein complexes of Hordeum vulgare L.

Solubilization of barley (Hordeum vulgare L.) thylakoid membranes with sodium dodecylsulphate plus sodium deoxycholate with or without Triton X-100 and subsequent fractionation in the polyacrylamide gel electrophoresis system described in this paper resulted: (1) in the resolution of the chlorophyll-proteins and chlorophyll-protein complexes commonly known as CP1a, CP1, LHCP¹, LHCP², CPa and LHCP³; (2) in the highly increased stability of CP1 and CP1a, as judged by their chlorophyll content, (3) at the expense of the free pigment concentration (4) which could be reduced to a negligible amount. Some 40% of the total chlorophyll contained in the mature higher plant thylakoid membrane is associated with CP1 and CP1a and as already suggested before [19] no significant amount of free chlorophyll occurs in vivo.     

Reinvestigation of the chlorophyll distribution among the chlorophyll-proteins and chlorophyll-protein complexes of Hordeum vulgare L.

Solubilization of barley (Hordeum vulgare L.) thylakoid membranes with sodium dodecylsulphate plus sodium deoxycholate with or without Triton X-100 and subsequent fractionation in the polyacrylamide gel electrophoresis system described in this paper resulted: (1) in the resolution of the chlorophyll-proteins and chlorophyll-protein complexes commonly known as CP1a, CP1, LHCP¹, LHCP², CPa and LHCP³; (2) in the highly increased stability of CP1 and CP1a, as judged by their chlorophyll content, (3) at the expense of the free pigment concentration (4) which could be reduced to a negligible amount. Some 40% of the total chlorophyll contained in the mature higher plant thylakoid membrane is associated with CP1 and CP1 a and as already suggested before [19] no significant amount of free chlorophyll occurs in vivo.Abbreviations chl chlorophyll - CP1 P₇₀₀-chla-protein - CPa P680-chla-protein - DOC sodium deoxychlolate - FC free chlorophyll - LHCP light-harvesting chlorophyll a/b-protein - PAGE(S) polyacrylamide gel electrophoresis (system) - SDS sodium dodecylsulphate - TX-100 Triton X-100     

Production of gynogenic haploids of Hordeum vulgare L.

Haploid plants were regenerated from cultured unfertilized ovaries of Hordeum vulgare L. (barley). Optimal response was obtained by the addition of 0.6 M 4-chloro-2-methylphenoxyacetic acid (MCPA), 2.8 M indole-3-acetic acid (IAA) and 4.4 M 6-benzyladenine (BA) in the N₆ medium. Further increase in the rate of callus formation and the number of green plants produced was possible with the addition of 90 g/l sucrose and 100 g/l coconut water. The stage of development of the ovaries at the time of culture was critical; the largest number of plants being produced by ovaries from flowers at the trinucleate stage of pollen.Abbreviations (BA) 6-benzyladenine - (MCPA) 4-chloro-2-methylphenoxyaceticacid - (2,4-D) 2,4-dichlorophenoxyaceticacid - (GA₃) gibberellic acid - (IAA) indole-3-acetic acid     

Chlorophyll a fluorescence--A useful tool for the early detection of temperature stress in spring barley (Hordeum vulgare L.)

The photosynthetic activity of two Syrian barley (Hordeum vulgare L.) landraces, Arabi Abiad (A. Abiad) and Arabi Aswad (A. Aswad), grown under low- and high- temperature stresses, were studied by analyzing the measured chlorophyll fluorescence signals. Both the applied stresses influenced photosystem II (PSII) activity. However, the effects depend on the stress type and the duration of its application. Phenomenological parameters were shifted shortly after the application of both stresses, whereas fluorescence ratios and yield values were altered most significantly after 7 days of stress imposition. The earliest changes in PSII activity of both cultivars were observed in the case of high temperature treatment. The maximal quantum efficiency of the photosynthetic apparatus (F(v)/F(M)) did not alter after stress application. Therefore, we could not recommend this parameter for early detection of such stress. In contrast, the results from the present investigation strongly indicate that the most significantly changed chlorophyll a fluorescence parameters could be used as an efficient tool for the early diagnosis of temperature stress in barley.     

Alkylresorcinols in barley (Hordeum vulgare L. distichon) grains

This study was carried out to compare grains of barley (Hordeum vulgare L. distichon) regarding contents and compositions of 5-n-alkylresorcinols. Mixtures of resorcinol homologues were isolated from acetone extracts from five barley cultivars. These polyketide metabolites were identified by chromatographic and spectroscopic means. The content and homologue patterns among different varieties were similar. The predominant compounds were 1,3-dihydroxy-5-n-heneicosylbenzene (C21:0), 1,3-dihydroxy-5-n-nonadecylbenzene (C19:0) and 1,3-dihydroxy-5-n-pentacosylbenzene (C25:0). The alkylresorcinol concentrations, in contrast to their compositions, depended on environmental and agricultural factors.     

Ring chromosomes in barley (Hordeum vulgare L.)

A plant with 2n = 14 + 1 ring chromosomes was obtained in the progeny of a primary trisomie for chromosome 7 of a two-rowed cultivar, Shin Ebisu 16. The morphological characteristics of the trisomic plants with an extra ring chromosome were similar to the primary trisomic for chromosome 7 (Semierect), which suggests that it originated from this chromosome. The ring chromosomes were not completely stable in mitotic cells because of abnormal behavior. Chromosome complements varied in different plants and in different roots within a plant. Root tip cells and spikes with 2n = 14 and 14 + 2 ring chromosomes were observed on plants with 14 + 1 ring chromosomes. Breakage-fusion-bridge cycle was inferred. The ring chromosome was associated with two normal homologues forming a trivalent in 17.6% sporocytes at metaphase I. The transmission of the extra ring chromosome was 23.1% in the progeny of the plant with 14 + 1 ring chromosomes. Trivalent formation may have been much higher at early prophase stages which were difficult to analyze in barley; only 4 of 120 sporocytes analyzed showed an isolated ring at pachytene. The ring chromosome moved to one pole without separation in 24.7% of the sporocytes at AI, and divided in 27.1% sporocytes giving rise to 8-8 separation. Only 10% of the sporocytes showed bridge formation at AI.     

The proteolytic degradation in vitro of the NADPH-protochlorophyllide oxidoreductase of barley (Hordeum vulgare L.)

A cell-free membrane system has been developed from isolated barley etioplasts which displays a highly selective decrease of the NADPH-protochlorophyllide oxidoreductase in vitro which is indistinguishable from that observed previously in the intact plant. The rapid breakdown of the enzyme protein in vitro is caused by a membrane-bound proteolytic activity. The protease is essentially independent of pH in the physiological pH range of 6 to 8.5. The optimum temperature for the reaction is approximately 40 degrees C. In the presence of excessive protochlorophyllide the enzyme is no longer degraded or inactivated during illumination of dark-grown plants. In the isolated membrane fraction protochlorophyllide also enhances the stability of the enzyme, a similar effect is exerted by NADPH but not by NADH. The results suggest that the inactivation of the NADPH-protochlorophyllide oxidoreductase is influenced by the interaction of the enzyme with protochlorophyllide and NADPH. In the absence of these two components the enzyme becomes susceptible to proteolytic degradation.     

Lysine Catabolism in Barley (Hordeum vulgare L.)

Lysine catabolism in seedlings of barley (Hordeum vulgare L. var. Emir) was studied by direct injection of the following tracers into the endosperm of the seedlings: aspartic acid-3-(14)C, 2-aminoadipic acid-1-(14)C, saccharopine-(14)C, 2,6-diaminopimelic acid-1-(7)-(14)C, and lysine-1-(14)C. Labeled saccharopine was formed only after the administration of either labeled 2,6-diaminopimelic acid or labeled lysine to the seedlings. The metabolic fate of the other tracers administered also supported a catabolic lysine pathway via saccharopine, and apparently proceeding by a reversal of some of the biosynthetic steps of the 2-aminoadipic acid pathway known from lysine biosynthesis in most fungi. Pipecolic acid seems not to be on the main pathway of l-lysine catabolism in barley seedlings.     

Cloning and mapping of telomere-associated sequences from Hordeum vulgare L.

Summary We present a novel approach to the efficient cloning of telomere-associated sequences and demonstrate its application to the cloning and mapping of these sequences from barley. The method is a modification of the Vectorette PCR technique and allows specific amplification and cloning of subtelomeric sequences. Telomere-associated sequences isolated from barley include hypervariable, repetitive sequences. Polymorphisms detected by subtelomeric markers behaved as Mendelian factors and were mapped to the most distal positions of barley chromosomes 1, 2, and 4.     

Identification of a phytase gene in barley (Hordeum vulgare L.)

Background

Endogenous phytase plays a crucial role in phytate degradation and is thus closely related to nutrient efficiency in barley products. The understanding of genetic information of phytase in barley can provide a useful tool for breeding new barley varieties with high phytase activity.

Methodology/Principal Findings

Quantitative trait loci (QTL) analysis for phytase activity was conducted using a doubled haploid population. Phytase protein was purified and identified by the LC-ESI MS/MS Shotgun method. Purple acid phosphatase (PAP) gene was sequenced and the position was compared with the QTL controlling phytase activity. A major QTL for phytase activity was mapped to chromosome 5 H in barley. The gene controlling phytase activity in the region was named as mqPhy. The gene HvPAP a was mapped to the same position as mqPhy, supporting the colinearity between HvPAP a and mqPhy.

Conclusions/Significance

It is the first report on QTLs for phytase activity and the results showed that HvPAP a, which shares a same position with the QTL, is a major phytase gene in barley grains.     

Characterization and mapping of a Prbs gene controlling spike development in Hordeum vulgare L.

The barley mutant, poly-row-and-branched spike (prbs) showed altered inflorescence morphology: complete conversion of the rudimentary lateral spikelets in two-rowed barley into fully developed fertile spikelets similar to the six-rowed phenotype, and additional spikelets in the middle of spike. Moreover, branched spikes emerged in progeny from a cross between the mutant and a six-rowed barley cultivar. Morphological observation of the development of immature spikes of the mutant and descendants with branched spikes showed that the Prbs gene is involved in spikelet development in the triple-mound stage. In mutant prbs, new meristems initiated at the flanks of lateral spikelets and middle spikelet meristems were converted to branch meristems, developing branched spikes. These observations suggested that the Prbs gene plays a crucial role in spikelet initiation and identity maintenance. The Prbs gene may be an important modifier in inflorescence differentiation from a panicle into a spike. The branched spikes emerging in hybrids from a cross between the mutant and six-rowed barley cultivar were not conferred by the gene vrs1 or Int-c, which decide spike morphology in six-rowed barley. These results imply that although six-row genes vrs1 and Int-c and prbs have similar effects on lateral spikelet development, they have different functions in branched spikes. The Prbs gene was mapped to chromosome 3H between SSR marker Bmag0023 and marker Cbic60 at a genetic distance of 3.3 and 5.4 centimorgans (cM), respectively.     

Clonal analysis of the development of the barley (Hordeum vulgare L.) leaf using periclinal chlorophyll chimeras

Barley seedlings that show mosaic expression of chlorophyll were selected from the progenies of mutagenized seeds. The sectored plants were grown under conditions that lead to the formation of lateral tillers, and a fraction of these had different kinds of leaf variegation. These sectorially and periclinally chimeric shoots were used to analyze the cellular organization of the barley shoot apex and the clonal development of the leaf. The shoot apex is organized in two cell lineages, L1 and L2. As well as giving rise to the epidermis, the L1 layer contributes to leaf mesophyll, particularly at the margins, but, on the adaxial side of leaf laminae, also in more central positions. The L1 layer alone is responsible for the formation of the hood, a flower homologue structure present in strains homozygoous for the dominant allele at the K (hooded ) locus. The relative contribution of L2 to leaf formation decreases in younger tillers and during tiller development from the basal to the flag leaf. Chimerism of the plants was generated by non-transmissible somatic events or by nuclear mutations. Received: 28 May 1998 / Accepted: 20 July 1998