The chromosomal locations and linkage relationships of the structural genes for the prolamin storage proteins (secalins) of rye

Summary Rye secalins are a polymorphic mixture of polypeptides which are classified into four major groups. Previous studies have shown that the structural genes for two of the groups (the -secalins and 40K -secalins) are located on the short arm of chromosome 1R and those for a third group (the high molecular weight secalins) on the long arm of the same chromosome. Analysis of F₂ grain from crosses between inbred lines of S. cereale shows that the structural genes for the -secalins (designated Sec 1) and the high molecular weight secalins (designated Sec 3) are loosely linked (40.8 ±3.76% recombination, 57.4 ± 11.30 cM). Analysis of wheat rye addition lines shows that the structural genes for the 75K -secalins are present on chromosome 2R. This locus is provisionally designated Sec 2. These genes are probably derived from those for the 40K -secalins by duplication, divergence and translocation. Analysis of secalin fractions from wild species of rye shows that all contain 75K -secalins, indicating that the duplication and divergence, if not the translocation, occurred before speciation of the genus.     

The regulation of NADPH-protochlorophyllide oxidoreductases A and B in green barley plants kept under a diurnal light/dark cycle

In etiolated barley (Hordeum vulgare L.) seedlings the light-induced accumulation of chlorophyll is controlled by two light-dependent NADPH-proto-chlorophyllide oxidoreductase (POR; EC 1.6.99.1) enzymes. While the concentration of one of these enzymes (POR A) and its mRNA rapidly decline during illumination, the second POR protein (POR B) and its mRNA remain at an approximately constant level during the transition from dark growth to the light. These results may suggest that only one of the enzymes, POR B, operates throughout the greening process and in light-adapted mature plants while the second enzyme, POR A, is active only in etiolated seedlings at the beginning of illumination. The fate of the two POR proteins and their mRNAs in fully green plants, however, has not been studied yet. In the present work we determined changes in the level of POR A and POR B proteins and mRNAs in green barley plants kept under a diurnal 12 h light/12 h dark cycle. In green barley plants, not only POR B is present but also trace amounts of POR A continue to reappear transiently at the end of a night period and seem to be involved in the synthesis and accumulation of chlorophyll at the beginning of each day.Abbreviations Chl chlorophyll - Chlide chlorophyllide - Lhcb light-harvesting chlorophyll a/b protein - Pchlide protochlorophyllide - POR NADPH-protochlorophyllide oxidoreductase Dedicated to Horst Senger on the occasion of his 65th birthday.We thank Dr. Dieter Rubli for photography and Renate Langjahr for typing. This work was supported by the Swiss National Science Foundation and the ETH-Zürich.     

A role for glutamate decarboxylase during tomato ripening: the characterisation of a cDNA encoding a putative glutamate decarboxylase with a calmodulin-binding site

A tomato fruit cDNA library was differentially screened to identify mRNAs present at higher levels in fruit of the tomato ripening mutant rin (ripening inhibitor). Complete sequencing of a unique clone ERT D1 revealed an open reading frame with homology to several glutamate decarboxylases. The deduced polypeptide sequence has 80% overall amino acid sequence similarity to a Petunia hybrida glutamate decarboxylase (petGAD) which carries a calmodulin-binding site at its carboxyl terminus and ERT D1 appears to have a similar domain. ERT D1 mRNA levels peaked at the first visible sign of fruit colour change during normal tomato ripening and then declined, whereas in fruit of the ripening impaired mutant, rin, accumulation of this mRNA continued until at least 14 days after the onset of ripening. This mRNA was present at much lower levels in other tissues, such as leaves, roots and stem, and was not increased by wounding. Possible roles for GAD, and its product -aminobutyric acid (GABA) in fruit, are discussed.     

ADP/ATP and protein phosphorylation dependence of phototransformable protochlorophyllide in isolated etioplast membranes

The effects of modulated ADP/ATP and NADPH/NADP⁺ ratios, and of protein kinase inhibitors, on the in vitro reformation of phototransformable protochlorophyllide, i.e. the aggregated ternary complexes between NADPH, protochlorophyllide, and NADPH-protochlorophyllide oxidoreductase (POR, EC 1.3.1.33), in etioplast membranes isolated from dark-grown wheat (Triticum aestivum) were investigated. Low temperature fluorescence emission spectra (–196 °C) were used to determine the state of the pigments. The presence of spectral intermediates of protochlorophyllide and the reformation of phototransformable protochlorophyllide were reduced at high ATP, but favoured by high ADP. Increased ADP level partly prevented the chlorophyllide blue-shift. The protein kinase inhibitor K252a prevented reformation of phototransformable protochlorophyllide without showing any effect on the chlorophyllide blue-shift. Addition of NADPH did not overcome the inhibition. The results indicate that protein phosphorylation plays a role in the conversion of the non-phototransformable protochlorophyllide to POR-associated phototransformable protochlorophyllide. The possible presence of a plastid ADP-dependent kinase, the activity of which favours the formation of PLBs, is discussed. Reversible protein phosphorylation is suggested as a regulatory mechanism in the prolamellar body formation and its light-dependent dispersal by affecting the membrane association of POR. By the presence of a high concentration of phototransformable protochlorophyllide, prolamellar bodies can act as light sensors for plastid development. The modulation of plastid protein kinase and protein phosphatase activities by the NADPH/NADP⁺ ratio is suggested. This revised version was published online in June 2006 with corrections to the Cover Date.     

Detection of a 65 kDaras binding protein in rat and sheep brain cytosol using a chemical cross linking agent

The ability of aras protein to associate with proteins present in rat brain cytosolin vitro was investigated using chemical cross-linking agents and the¹²⁵I-labelled v-H-ras protein. Two iodinated protein complexes with apparent molecular weights of 40 and 85 kDa were observed when a mixture of rat brain cytosol and [¹²⁵I]ras was treated with the cross-linking agent disuccinimidyl suberate and subjected to SDS-PAGE. Formation of the [¹²⁵I] 85 kDa complex was enhanced by a high concentration of EDTA while generation of the 40 kDa species was abolished by this treatment. Formation of the [¹²⁵I] 85 kDa complex was inhibited by unlabelledras protein, GTP, GTPS, and GDP but not by ATPS and GMP.Chromatography of the cross-linked brain cytosol-[¹²⁵I]ras mixture on DEAE cellulose partially resolved the [¹²⁵I] 85 kDa complex from the [¹²⁵I]ras protein. The [¹²⁵I] 85 kDa complex (formed using ethyleneglycolbis (succinimidylsuccinate) as the cross-linking agent) could be immunoprecipitated using a rabbit anti-ras polyclonal antibody. Treatment of the immunoprecipitate with hydroxylamine to cleave the cross-link yielded [¹²⁵I]-labelledras. A substantial enrichment of the proportion of the [¹²⁵I] 85 kDa complex in the cross-linked extract was achieved by preparative SDS-PAGE. It is concluded that thein vitro chemical cross-linking approach employed here has detected tworas binding proteins in rat brain cytosol: a 65 kDa heat-sensitive and a 20 kDa heat-stable protein. The possibility that the 65 kDaras binding protein is aras regulatory orras effector protein which has not so far been characterised is briefly discussed.Abbreviations DSS disuccinimidyl suberate - EGS ethyleneglycolbis (succinimidylsuccinate) - GTPS guanosine 5-[-thio] triphosphate - ATPS adenosine 5-[-thio] triphosphate     

Isolation of a cDNA encoding a 70 kDa heat-shock cognate protein expressed in vegetative tissues ofArabidopsis thaliana

A cDNA encoding a 70 kDa heat shock cognate protein (hsc70) was isolated fromArabidopsis thaliana by using a rat hsc70 cDNA as probe. Sequence analysis demonstrated the conservation of functional domains and important amino acid residues among hsc70s in plants and animals. The expression of this gene was stress-inducible, and was found at a substantial level during normal growth in root, stem, leaf and flower tissues, but not in siliques. Multiple copies of this gene exist in theArabidopsis genome.     

Isolation of an Arabidopsis cDNA sequence encoding a 22 kDa calcium-binding protein (CaBP-22) related to calmodulin

Complementary DNA sequences were isolated from a library of cloned Arabidopsis leaf mRNA sequences in gt10 that encoded a 21.7 kDa polypeptide (CaBP-22), which shared 66% amino acid sequence identity with Arabidopsis calmodulin. The putative Ca²⁺-binding domains of CaBP-22 and calmodulin, however, were more conserved and shared 79% sequence identity. Ca²⁺ binding by CaBP-22, which was inferred from its amino acid sequence similarity with calmodulin, was demonstrated indirectly by Ca²⁺-induced mobility shifting of in vitro translated CaBP-22 during SDS-polyacrylamide gel electrophoresis. CaBP-22 is encoded by a ca. 0.9 kb mRNA that was detected by northern blotting of leaf poly(A)⁺ RNA; this mRNA was slightly larger than the 809 bp CaBP-22 cDNA insert, indicating that the deduced amino acid sequence of CaBP-22 is near full-length. CaBP-22 mRNA was detected in RNA fractions isolated from leaves of both soil-grown and hydroponically grown Arabidopsis, but below the limits of detection in RNA isolated from roots, and developing siliques. Thus, CaBP-22 represents a new member of the EF-hand family of Ca²⁺-binding proteins with no known animal homologue and may participate in transducing Ca²⁺ signals to a specific subset of response elements.     

EDTA-assisted heavy-metal uptake by poplar and sunflower grown at a long-term sewage-sludge farm

Little information is available concerning the efficacy of chelates applied to biosolids (sewage-sludge)-treated soil for heavy-metal removal. The purpose of the experiment was to determine the availability to sunflower (Helianthus annuus L.) and hybrid poplar (Populus deltoides Marsh. × P. nigra L.) seedlings, of non-essential (Cd, Ni, Pb) and essential heavy metals (Cu, Fe, Mn, Zn) in field soil injected with biosolids since 1976 and treated with ethylenediamine-tetraacetic acid (EDTA) in 2001. Sunflower was grown at two densities, 20000 and 60000 plants/ha, and poplar at 10000 plants/ha. The tetrasodium salt of EDTA was applied at rates of 0, 0.5, 1, and 2 g EDTA salt per kg surface (25-cm depth) soil. The EDTA did not affect uptake by poplar of the three non-essential (Cd, Ni, Pb) and four essential (Cu, Fe, Mn, Zn) heavy metals. For sunflower, the 1.0 g/kg rate of chelate addition resulted in maximal removal of the three non-essential heavy metals (Cd, Ni, Pb). Uptake of the essential heavy metals by sunflower was little affected by the EDTA. At the 20000 plants/ha density, leaves of sunflower grown with 1.0 g EDTA Na₄2H₂O per kg soil accumulated more Cd, Ni, and Pb than leaves of sunflower grown without the EDTA salt. At this density, concentrations of Cd in leaves of sunflower without EDTA and with 1.0 g/kg EDTA salt were 2.2 and 6.5 g/g, respectively; for Ni, they were 6.7 and 19.2 g/g, respectively; and for Pb, they were 15.6 and 46.9 g/g, respectively. At the 60000 plants/ha density, stems of sunflower grown with 1.0 g EDTA Na₄2H₂O per kg soil accumulated more Cd, Ni, and Pb than stems of sunflower grown without the EDTA salt. At this density, concentrations of Cd in stems of sunflower without EDTA and with 1.0 g/kg EDTA salt were 0.6 and 4.6 g/g, respectively; for Ni, they were 1.7 and 17.6 g/g, respectively; and for Pb, they were 5.2 and 42.8 g/g, respectively. Removal of the non-essential heavy metals by sunflower was greater at the higher plant density (60000 plants/ha) compared to the lower one (20000 plants/ha).     

Identification of two differentially regulated isoforms of protochlorophyllide oxidoreductase (POR) from tobacco revealed a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms

NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide a in the chlorophyll biosynthetic pathway. Here, we identified two distinct POR cDNAs from tobacco. Both POR isoforms are encoded by a respective single copy gene in tobacco genome. The overall deduced amino acid sequences of two tobacco cDNAs, designated here POR1 and POR2, displayed significant identities (∼75%), but showed different patterns of light and developmental regulation. In contrast to the previously isolated POR isoforms of Arabidopsis thaliana and barley, the expression of both tobacco POR isoforms were not negatively regulated by light and persisted in matured green tissues. Furthermore, the expression of both genes appeared to be regulated by a diurnal regulation. These results show a wide variety of light- and development-dependent regulations of POR gene expression among angiosperms. Furthermore, phylogenetic analysis including tobacco revealed that POR gene family is differentially represented by angiosperms, most of which is probably caused by independent gene duplication in individual plant. Present results imply a modification of the previous concept that chlorophyll biosynthesis and chloroplast differentiation in angiosperms are ubiquitously controlled by unique functions of two POR isoforms. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of cell turgor on hydraulic conductivity and elastic modulus of Elodea leaf cells

Water relation parameters of leaf cells of the aquatic plant Elodea densa have been measured using the pressure probe. For cells in both the upper and lower epidermis it was found that the elastic modulus () and the hydraulic conductivity (Lp) were dependent on cell turgor (P). Lp was (7.8±5.5)·10^-7 cm s^-1 bar^-1 (mean±SD; n=22 cells) for P>4 bar in cells of the upper epidermis and was increasing by a factor of up to three for P0 bar. No polarity of water movement or concentration dependence of Lp was observed. For cells of the lower epidermis the Lp-values were similar and the hydraulic conductivity also showed a similar dependence on turgor. No wall ingrowth or wall labyrinths (as in transfer cells) could be found in the cells of the lower epidermis. The elastic modulus () of cells of the upper epidermis could be measured over the whole pressure range (P=0–7 bar) by changing the osmotic pressure of the medium. increased linearly with increasing turgor and ranged between 10 and 150 bar. For cells of the lower epidermis the dependence of on P was similar, although the pressure dependence could not be measured on single cells. The Lp-values are compared with literature data obtained for Elodea by a nuclear magnetic resonance (NMR)-technique. The dependence of Lp on P is discussed in terms of pressure dependent structural changes of the cell membranes and interactions between solute and water transport.Abbreviations P cell turgor pressure - Lp hydraulic conductivity - volumetric elastic modulus - T _1/2 half-time of water exchange of individual cell     

Anthraquinones production,hydrogen peroxide level and antioxidant vitamins in Morinda elliptica cell suspension cultures from intermediary and production medium strategies

The effects of medium strategies [maintenance (M), intermediary (G), and production (P) medium] on cell growth, anthraquinone (AQ) production, hydrogen peroxide (H₂O₂) level, lipid peroxidation, and antioxidant vitamins in Morinda elliptica cell suspension cultures were investigated. These were compared with third-stage leaf and 1-month-old callus culture. With P medium strategy, cell growth at 49 g l^–1, intracellular AQ content at 42 mg g^–1 DW, and H₂O₂ level at 9 mol g^–1 FW medium were the highest as compared to the others. However, the extent of lipid peroxidation at 40.4 nmol g^–1 FW and total carotenoids at 13.3 mg g^–1 FW for cultures in P medium were comparable to that in the leaf, which had registered sevenfold lower AQ and 2.2-fold lower H₂O₂ levels. Vitamin C content at 30–120 g g^–1 FW in all culture systems was almost half the leaf content. On the other hand, vitamin E content was around 400–500 g g^–1 FW in 7-day-old cultures from all medium strategies and reduced to 50–150 g g^–1 FW on day 14 and 21; as compared to 60 g g^–1 FW in callus and 200 g g^–1 FW in the leaf. This study suggests that medium strategies and cell growth phase in cell culture could influence the competition between primary and secondary metabolism, oxidative stresses and antioxidative measures. When compared with the leaf metabolism, these activities are dynamic depending on the types and availability of antioxidants.Abbreviations AQ Anthraquinone - DW Dry cell weight - FW Fresh cell weight - G Intermediary medium - M Maintenance medium - MDA Malondialdehyde - P Production medium - ROS Reactive oxygen species - TBA Thiobarbituric acid - t_d Doubling time     

Haploid production in durum wheat by the interaction of Aegilops kotschyi cytoplasm and 1BL/1RS chromosomal interchange

The present study describes the development of an alloplasmic haploid-inducer in durum wheat cv Cando. This cultivar possesses the homozygous wheat-rye translocation 1BL/1RS from the 6x-wheat cv Veery. The nucleus of 4x-Cando-Veery 1BL/1RS was introduced into Aegilops kotschyi cytoplasm by initially using (kotschyi)-Salmon as the maternal parent. In the cross of this alloplasmic durum line with Cando-Veery 1BL/1RS, which was used as the recurrent pollen parent, haploids (n=14) were produced. The frequency of haploids increased from 5.7% in the F₁ generation to 14% in the BC₁ generation. The presence of rye chromosome arm 1RS and the concomitant loss of 1BS in (kotschyi)-Cando-Veery 1BL/1RS are necessary for haploid induction. Proposals are made which may enable the use of haploids produced by nucleo-cytoplasmic interactions in future wheat breeding programs.     

Phosphorylation of a putative myosin light chain inChara by calcium-dependent protein kinase

Summary Ca²⁺-dependent protein kinase (CDPK) has been proposed to mediate inhibition by Ca²⁺ of cytoplasmic streaming in the green algaChara. We have identified the in vivo substrate(s) of CDPK inChara by using vacuolar perfusion of individual internodal cells with [-³²P]ATP. Phosphorylation of several polypeptides is enhanced when perfusions are performed at 10^–4M free Ca²⁺ compared to <10^–9M free Ca²⁺. The Ca²⁺-stimulated phosphorylation of these proteins is inhibited by the presence of a monoclonal antibody to soybean CDPK. One of these proteins is 16 to 18kDa and is recognized by an antibody against gizzard myosin light chains. These results demonstrate that inChara, several polypeptides are phophorylated by CDPK and one of these proteins has been tentatively identified as a myosin light chain. These observations support the hypothesis that Ca²⁺-regulated phosphorylation of myosin is involved in the regulation of cytoplasmic streaming.Abbreviations CDPK calcium-dependent protein kinase - mAb monoclonal antibody     

Lime-induced chlorosis inPinus radiata

Summary Addition of marl (CaCO₃) and/or manure to an acid soil in pots caused lime-induced chlorosis inPinus radiata seedlings, especially after excessive irrigation for 15 days. Chlorotic symptoms and their intensity were found to be related more to soil moisture and to the HCO₃ ^– concentrations, than to percentage of free CaCO₃, in the soil mixtures.Comparative chemical analysis showed lower total Fe and Mn concentrations and higher concentrations of cations and organic anions in the needles of seedlings with chlorotic symptoms than in the needles of healthy ones.     

Structure and expression of a nuclear gene for the PSI-D subunit of photosystem I inNicotiana sylvestris

The PSI-D subunit is the ferredoxin-binding site of photosystem I, and is encoded by the nuclear genepsaD. We isolated apsaD genomic clone fromNicotiana sylvestris, by screening a genomic library with apsaD cDNA which we previously cloned fromN. sylvestris (Yamamotoet al., Plant Mol Biol 17: 1251, 1991). Nucleotide sequence analysis revealed that this genomic clone contains apsaD gene, which does not correspond to thepsaD cDNA, so we designated these genespsaDb andpsaDa, respectively. ThepsaDb clone encodes a protein of 214 amino acids uninterrupted by introns. The N-terminal sequence determined for theN. sylvestris PSI-D protein encoded bypsaDb begins at the 49th residue. The products ofpsaDa andpsaDb share 82.7% and 79.5% identity at the amino acid and nucleotide levels, respectively. Genomic Southern analysis showed that two copies ofpsaD are present in theN. sylvestris genome. Ribonuclease protection assays and immunoblot analysis inN. sylvestris indicate that both genes are expressed in leaves, stems and flower buds, but neither is expressed in roots. During leaf development, the ratio ofpsaDb topsaDa mRNA increases from 0.12 in leaf buds to 0.36 in mature leaves. The relative abundance of the corresponding proteins decreased over the same developmental period. These results indicate that differential regulation mechanisms controlpsaDa andpsaDb expression at both the mRNA and protein levels during leaf development.     

Isolation and classification of chlorophyll-deficient xantha mutants of Arabidopsis thaliana

Mutant lines of Arabidopsis thaliana that are either blocked at various steps of the biosynthetic pathway of chlorophyll (Chl) or that are disturbed in one of the subsequent steps leading to the assembly of an active photosynthetic membrane were isolated by screening for Chl-deficient xantha (xan) mutants. Only mutants that segregated in a 31 ratio, that contained the same carotenoid spectrum as etiolated wild-type seedlings and less than 2% of the Chl of wild-type control seedlings, and whose Chl content was not affected by the addition of sucrose to the growth medium were selected for a more detailed analysis. As a final test for the classification of the selected mutants, light-grown xan mutants were vacuum-infiltrated and incubated with the common precursor of tetrapyrroles, -aminolevulinic acid (ALA), in the dark. Two major groups of mutants could be distinguished. Some of the mutants were blocked at various steps of the Chl pathway between ALA and protochlorophyllide (Pchlide) and did not accumulate the latter in the dark. The other mutants accumulated Pchlide in the dark regardless of whether exogenous ALA was added. This latter group could be subdivided into mutants with a biochemical lesion in a recently discovered second light-dependent Pchlide reduction step that occurs in green plants and mutants that have blocks in the assembly of Chl protein complexes. In the present work a total of seven different loci could be defined genetically in Arabidopsis that affect the synthesis of Chl and its integration into the growing photosynthetic membrane.Abbreviations ALA -aminolevulinic acid - Chl chlorophyll - Chlide chlorophyllide - Pchlide protochlorophyllide - POR NADPH-Protochlorophyllide oxidoreductase - xan xantha This study was initiated while one of the authors (K.A.) was on sabbatical leave in the laboratory of Dr. C. Somerville (MSU, East Lansing, Mich., USA). We are extremely grateful to Dr. Somerville and his coworkers for advice and support during this time. This research was supported by the Deutsche Forschungsgemeinschaft and the Schweizerischer Nationalfonds.