Regulation of the expression of ferredoxin-glutamate synthase in barley

We have investigated the regulation of ferredoxin–glutamate synthase (Fd-GOGAT) in leaves of barley (Hordeum vulgare L. cv. Maris Mink) at the mRNA, protein and enzyme activity levels. Studies of the changes in Fd-GOGAT during plant development showed that the activity in shoots increases rapidly after germination to reach a maximum (on a fresh-weight basis) at day 10 and then declines markedly to less than 50% of the maximal activity by day 30, this decline being correlated with an equivalent loss of Fd-GOGAT protein. Growing the plants in darkness reduced the maximum activity attained in the shoots, but did not affect the overall pattern of the changes or their timing. The activity of Fd-GOGAT increased two- to three-fold within 48 h when etiolated leaves were exposed to light, and Northern blots indicated that the induction occurred at the mRNA level. However, whilst a carbon source could at least partially substitute for light in the induction of nitrate reductase activity, no induction of Fd-GOGAT activity was seen when etiolated leaves were treated with either sucrose or glucose. Interestingly, the levels of Fd-GOGAT mRNA and activity remained high up to a period of 16 h or 72 h darkness, respectively. Compared with plants grown in N-free medium, light-grown plants supplied with nitrate had almost two-fold higher Fd-GOGAT activities and increased Fd-GOGAT mRNA levels, but nitrate had no effect on the abundance of the enzyme or its mRNA in etiolated plants, indicating that light is required for nitrate induction of barley Fd-GOGAT. Received: 23 April 1997 / Accepted: 28 May 1997     

Programme of senescence in petals and carpels of Pisum sativum L. flowers and its control by ethylene

The role of ethylene in the control of senescence of both petals and unpollinated carpels of pea was investigated. An increase in ethylene production accompanied senescence, and the inhibitors of ethylene action were effective in delaying senescence symptoms in different flower verticils. Pollination did not seem to trigger the senescence syndrome in the corolla as deduced from the observation that petals from pollinated and unpollinated flowers and from flowers whose carpels had been removed senesced at the same time. A cDNA clone encoding a putative ethylene-response sensor (psERS) was isolated from pea flowers, and the pattern of expression of its mRNA was studied during development and senescence of different flower tissues. The levels of psERS mRNA paralleled ethylene production (and also levels of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) mRNA) in both petals and styles. Silver thiosulfate treatments were efficient at preventing ACO and psERS mRNA induction in petals. However, the same inhibitor showed no ability to modify expression patterns in pea carpels around the anthesis stage, suggesting different controls for ethylene synthesis and sensitivity in different flower organs. Received: 18 June 1998 / Accepted: 22 December 1998     

Arabidopsis SKP1, a homologue of a cell cycle regulator gene, is predominantly expressed in meristematic cells

The yeast SKP1 gene and its human homolog p19 ^skp1 encode a kinetochore protein required for cell cycle progression at both the DNA synthesis and mitosis phases of the cell cycle. In orchids we identified a cDNA (O108) that is expressed in early stages of ovule development and is homologous to the yeast SKP1. Based on the orchid O108 cDNA clone, we identified and characterized an Arabidopsis thaliana (L.) Heynh. cDNA designated ATskp1 that also has high sequence similarity to yeast SKP1. The Arabidopsis ATskp1 is a single-copy gene that mapped to chromosome 1. The expression of the ATskp1 gene was highly correlated with meristem activity in that its mRNA accumulated in all of the plant meristems including the vegetative shoot meristem, inflorescence and floral meristems, root meristem, and in the leaf and floral organ primordia. In addition, ATskp1 was also highly expressed in the dividing cells of the developing embryo, and in other cells that become multinucleate or undergo endoreplication events such as the endosperm free nuclei, the tapetum and the endothelium. Based on its spatial pattern of expression, ATskp1 is a marker for cells undergoing division and may be required for meristem activity. Received: 6 June 1997 / Accepted: 2 July 1997     

Expression of the phloem lectin is developmentally linked to vascular differentiation in cucurbits

The conducting elements of phloem in angiosperms are a complex of two cell types, sieve elements and companion cells, that form a single developmental and functional unit. During ontogeny of the sieve element/companion cell complex, specific proteins accumulate forming unique structures within sieve elements. Synthesis of these proteins coincides with vascular development and was studied in Cucurbita seedlings by following accumulation of the phloem lectin (PP2) and its mRNA by RNA blot analysis, enzyme-linked immunosorbent assay, immunocytochemistry and in␣situ hybridization. Genes encoding PP2 were developmentally regulated during vascular differentiation in hypocotyls of Cucurbita maxima Duch. Accumulation of PP2 mRNA and protein paralleled one another during hypocotyl elongation, after which mRNA levels decreased, while the protein appeared to be stable. Both PP2 and its mRNA were initially detected during metaphloem differentiation. However, PP2 mRNA was detected in companion cells of both bundle and extrafascicular phloem, but never in differentiating sieve elements. At later stages of development, PP2 mRNA was most often observed in extrafascicular phloem. In developing stems of Cucurbita moschata L., PP2 was immunolocalized in companion cells but not to filamentous phloem protein (P-protein) bodies that characterize immature sieve elements of bundle phloem. In contrast, PP2 was immunolocalized to persistent ␣ P-protein bodies in sieve elements of the extrafascicular phloem. Immunolocalization of PP2 in mature wound sieve elements was similar to that in bundle phloem. It appears that PP2 is synthesized in companion cells, then transported into differentiated sieve elements where it is a component of P-protein filaments in bundle phloem and persistent P-protein bodies in extrafascicular phloem. This differential accumulation in bundle and extrafascicular elements may result from different functional roles of the two types of phloem. Received: 31 July 1996 / Accepted: 27 August 1996     

Carbohydrates modulate the expression of the sunflower cytochrome c gene at the mRNA level

Changes in the steady-state mRNA levels of the gene encoding cytochrome c were analyzed after feeding carbohydrates to detached leaves of sunflower (Helianthus annuus L.). Glucose, fructose and sucrose promoted an increase in mRNA levels, which was not observed with mannitol and other metabolites such as glycerol or acetate. The increase in mRNA levels was proportionally higher in dark-treated leaves. The effect of sugars could be mimicked by compounds that are phosphorylated by hexokinase but not further metabolized, such as mannose or 2-deoxyglucose. This may indicate that hexokinase is involved in the induction of the cytochrome c gene by carbohydrates. The presence of potassium phosphate had no significant effect on the induction by sugars. Our results indicate that the modulation of the expression of nuclear genes encoding mitochondrial components should be added to the list of known effects of carbohydrates on respiration. Received: 5 February 1998 / Accepted: 22 April 1998     

Role of O-acetyl-l-serine in the coordinated regulation of the expression of a soybean seed storage-protein gene by sulfur and nitrogen nutrition

The composition of seed storage proteins is regulated by sulfur and nitrogen supplies. Under conditions of a low sulfur-to-nitrogen ratio, accumulation of the β-subunit of β-conglycinin, a sulfur-poor seed storage protein of soybean (Glycine max [L.] Merr.), is elevated, whereas that of glycinin, a sulfur-rich storage protein, is reduced. Using transgenic Arabidopsis thaliana [L.] Heynh., it was found that the promoter from the gene encoding the β-subunit of β-conglycinin up-regulates gene expression under sulfur deficiency and down-regulates gene expression under nitrogen deficiency. To obtain an insight into the metabolic control of this regulation, the concentrations of metabolites related to the sulfur assimilation pathway were determined. Among the metabolites, O-acetyl-l-serine (OAS), one of the precursors of cysteine biosynthesis, accumulated to higher levels under low-sulfur and high-nitrogen conditions in siliques of transgenic A. thaliana. The pattern of OAS accumulation in response to various levels of sulfur and nitrogen was similar to that of gene expression driven by the β-subunit promoter. Elevated levels of OAS accumulation were also observed in soybean cotyledons cultured under sulfur deficiency. Moreover, OAS applied to in-vitro cultures of immature soybean cotyledons under normal sulfate conditions resulted in a high accumulation of the β-subunit mRNA and protein, whereas the accumulation of glycinin was reduced. These changes were very similar to the responses observed under conditions of sulfur deficiency. Our results suggest that the level of free OAS mediates sulfur- and nitrogen-regulation of soybean seed storage-protein composition. Received: 6 February 1999 / Accepted: 16 March 1999     

Lectin and alliinase are the predominant proteins in nectar from leek (Allium porrum L.) flowers

Analysis of nectar from leek (Allium porrum) flowers by SDS-PAGE revealed the presence of two major polypeptide bands of 50 kDa and 13 kDa, respectively. Using a combination of agglutination tests, enzyme assays and N-terminal sequencing, the polypeptides have been identified as subunits of alliin lyase (alliinase, EC 4.4.1.4) and mannose-binding lectin, respectively. The latter protein is particularly abundant since it represents about 75% of the total nectar protein. Honey produced by bees foraging on flowering leek plants still contains biologically active lectin and alliinase. However, the levels of both proteins are strongly reduced as compared to those in the original nectar. It is evident, therefore, that the lectin as well as the alliinase are inactivated/degraded during the conversion of nectar into honey. Received: 24 May 1996 / Accepted: 19 August 1996     

Salt stress induction of some key antioxidant enzymes and metabolites in eight Iranian wild almond species

The present work describes the changes in the activities of key antioxidant enzymes and the levels of some metabolites in relation to salt tolerance in eight wild almond species. All the species were exposed to four levels of NaCl (control, 40, 80 and 120 mM). Plant fresh biomass, α-, γ- and δ-tocopherol, total soluble proteins, malondialdehyde (MDA_eq), H₂O₂, total phenolics, and the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were analyzed in leaves of salt-stressed and non-stressed plants of the eight almond species. In all the species, salt stress significantly enhanced the activities of SOD and POD, levels of total phenolics and γ- and δ-tocopherols. High levels of salt stress significantly depressed the levels of total soluble proteins, MDA and CAT activity, while salt stress did not significantly affect leaf H₂O₂ contents. Regression analysis showed that the relationship between salt levels and total soluble proteins, CAT, γ-tocopherol, MDA_eq, SOD and POD were statistically significant. Principal component analysis discriminated the almond species based on their degree of tolerance/sensitivity to saline conditions: Prunus reuteri and P. glauca were ranked as salt tolerant, P. lycioides and P. scoparia as moderately tolerant, and P. communis, P. eleagnifolia, P. arabica and P. orientalis as salt sensitive. The results could be used for selecting salt tolerant genotypes to be used as rootstocks for almond cultivation.     

Actin expression in germinating seeds of Phaseolus vulgaris L.

Actin was present at very low levels in the seeds of common bean (Phaseolus vulgaris L.) compared with those from other species, and was observed mostly in the embryo. A time-course of actin expression in germinating bean seeds revealed an induced expression of both the mRNA and protein. Initially, the actin mRNA in seeds was barely detectable by northern blot analysis. However, there was a substantial increase in the expression of the actin mRNA at 24, 48 and 72 h after imbibition, compared with an internal control consisting of a late-embryogenesis-abundant (LEA) type IV gene from P. vulgaris. An increase in the amount of actin in total seed extracts that parallelled that of the mRNA was detected by western blotting starting at 24 h after imbibition. This increase was more apparent when the embryo alone was analyzed. Two-dimensional western blots initially revealed three actin isoforms with isoelectric points (pIs) of approximately 5.6, 5.7 and 5.8, the amounts of which increased within a 48-h period, when a new minor isoform of pI approximately 5.5 appeared; however, after 72 h, the pI-5.8 isoform had almost disappeared and the pI-5.5 isoform had disappeared completely, indicating that these two minor isoforms are expressed transiently. These results indicate that actin is at very low levels in the dry seed but undergoes an increased and differential expression during imbibition, an event probably required to carry out all the necessary functions for germination. Received: 21 July 1998 / Accepted: 1 September 1998     

Transgenic expression of a Δ12-epoxygenase gene in Arabidopsis seeds inhibits accumulation of linoleic acid

The Crepis palaestina cDNA Cpal2 encodes a Δ12-epoxygenase that can catalyse the synthesis of 12,13-epoxy-cis-9-octadecenoic acid (18:1E) from linoleic acid (18:2). When the Cpal2 gene was expressed under the control of the napin seed-specific promoter in Arabidopsis thaliana (L.) Heynh., the seed lipids accumulated only low levels of 18:1E and also 12,13-epoxy-cis-9,15-octadec-2-enoic acid (18:2E). Despite the fact that the levels of these epoxy fatty acids comprised only up to 6.2% of the total fatty acids, there was a very marked increase in oleic acid (18:1) and decrease in linoleic (18:2) and α-linolenic (18:3) acids in these plants, indicating that endogenous Δ12-desaturation was greatly reduced in these plants. Significant between-line differences in the levels of Cpal2 mRNA were observed during seed development, but were not associated with any major variation in mRNA levels for the endogenous ArabidopsisΔ12-desaturase (Fad2). This suggests that if an unfavourable interaction occurs between the transgenic Δ12-epoxygenase and the endogenous Δ12-desaturase, which decreases the level of desaturation, it occurs at either the translational or post-translational level. We further show that the co-expression of a Δ12-desaturase gene from C. palaestina in Cpal2 transgenic Arabidopsis returns the relative proportions of the C18 seed fatty acids to normal levels and results in an almost twofold increase in total epoxy fatty acids. Received: 11 August 2000 / Accepted: 7 September 2000     

Structure and regulation of the Arabidopsis thaliana allene oxide synthase gene

Allene oxide synthase (AOS) is encoded by a single intronless gene in Arabidopsis thaliana (L.) Heynh. The promoter region of the AOS gene exhibits, in addition to the elements of a minimal promoter and the presence of general enhancers, cis-elements that, in other promoters, are responsible for stress- and ethylene-responsiveness. Arabidopsis thaliana and Nicotiana tabacum L. were transformed with a chimaeric gene consisting of a 1.9-kb 5′-upstream sequence and the first 95 nucleotides of the AOS coding sequence translationally fused to uid A encoding β-glucuronidase (GUS). Using histochemistry, GUS activity was seen in older leaves, in the bases of petioles and in stipules, during the early stages of carpel development, in maturing pollen grains and at the base of elongated filaments, as well as in abscission-zone scars. A role for jasmonates in floral organ abscission is suggested by these findings. Furthermore, the AOS promoter was activated both locally as well as systemically upon wounding. Jasmonic acid, 12-oxophytodienoic acid and coronatine strongly induced GUS activity. This induction remained confined to the treated leaf when agonists were applied locally to a leaf, suggesting that neither jasmonic acid nor 12-oxophytodienoic acid are physiologically relevant components of the systemic wound signal complex. Rather, the data show that jasmonates behave as local response regulators produced at or around the sites of action in response to appropriate triggers of their synthesis. Received: 21 September 1998 / Accepted: 30 December 1998     

Cytokines mRNA in bone marrow-derived dendritic cells in asthmatic mouse

By inducing and amplifying dendritic cells (DCs) derived from the bone marrow of asthma murine in vitro, cytokines mRNA were expressed, and the functions of DCs were investigated. Cells isolated from murine bone marrow have been cultured with rmGM-CSF and rmIL-4, and the expression of cytokines mRNA was determined by ribonuclease protection assay combined with multi-probe templates. Large numbers of DCs have been obtained from bone marrow, and they expressed interleukin-13 (IL-13), interleukin-9 (IL-9), and interleukin-3 (IL-3) mRNA. Moreover, the level of IL-13 mRNA and IL-9 mRNA expressed by DCs in asthmatic mice was significantly higher than those in the control groups (P<0.05). But, the level of IL-3 mRNA showed no discrepancy between the two groups (P>0.05). DCs are very important in the forming and developing of asthma, which implies that the therapy targeted at DCs will possibly become a new goal. __________ Translated from Journal of Nanjing Normal University (Natural Science), 2005, 28(2): 96–100 [译自: 南京师范大学学报 (自然科学版), 2005, 28(2): 96–100]     

Phosphoenolpyruvate carboxylase in developing seeds of Vicia faba L.: gene expression and metabolic regulation

To analyze the role of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) during seed development, two cDNA clones encoding two isoforms of PEPCase were isolated from a seed-specific library of Vicia faba. The two sequences (VfPEPCase1 and VfPEPCase2) have a sequence identity of 82 and 89% on the nucleotide and amino acid levels. The VfPEPCase1 mRNA was found to be predominantly expressed in roots and developing cotyledons whereas the VfPEPCase2 mRNA was more abundant in green and maternal tissues. In the cotyledons, PEPCase mRNAs accumulated from early to mid cotyledon stage and decreased thereafter. The PEPCase activity increased continuously during cotyledon development. The enzyme was strongly activated by glucose-6-phosphate, but not by glucose, fructose or sucrose. Asparagine was weakly activating whereas malate, aspartate and glutamate were inhibitory. The inhibitors became less effective with increasing pH. Aspartate was a much stronger inhibitor of cotyledonary PEPCase than glutamate at both pH 7.0 and 7.5. The sensitivity of PEPCase to malate inhibition decreased from early to mid cotyledon stage at a time when storage proteins are synthesized. This indicates activation on the protein level, possibly by protein phosphorylation. Nitrogen starvation in the presence of hexoses but not sucrose decreased mRNA levels of VfPEPCase1 and enzyme activity, indicating control on the mRNA level by both carbon and nitrogen. It is concluded that in developing cotyledons PEPCase is probably important for the synthesis of organic acids to provide carbon skeletons for amino acid synthesis. Received: 15 July 1998 / Accepted: 10 October 1998     

The early light-induced protein is also produced during leaf senescence of Nicotiana tabacum

To better understand the genetic controls of leaf senescence, a tobacco (Nicotiana tabacum L. cv. SR1) mRNA that is up-regulated during senescence was isolated by the cDNA-amplified restriction fragment polymorphism method and the cDNA was cloned. The mRNA coded for the early light-induced protein (ELIP), a member of the chlorophyll a/b-binding protein family that has been implicated in assembly or repair of the photosynthetic machinery during early chloroplast development and abiotic stress. A protein antigenically recognized by antibodies to ELIP appeared during senescence with kinetics similar to those of its mRNA. The mRNA, designated ELIP-TOB, was detected earlier when senescence was enhanced by leaf detachment and treatment with 1-amino-cyclopropane-1-carboxylic acid, and was detected later when senescence was retarded by benzyladenine. However, no ELIP-TOB mRNA was seen in the dark even though senescence was accelerated under these conditions. Furthermore, water stress and anaerobiosis stimulated the appearance of ELIP-TOB mRNA before losses of chlorophyll could be detected. We discuss the conditions that may lead to the up-regulation of ELIP-TOB during senescence and speculate as to the role of the gene product in this terminal phase of leaf development. Received: 18 May 2000 / Accepted: 24 June 2000     

Activation of latent origins of DNA replication in florally determined shoot meristems of long-day and short-day plants: Silene coeli-rosa and Pharbitis nil

Replicon spacing was measured during the S-phase of the cell cycle in shoot meristems of Silene coeli-rosa L., a long-day (LD) plant, and Pharbitis nil Chois, a short-day (SD) plant to examine the hypothesis that activation of latent origins of DNA replication is a feature of floral determination. Silene coeli-rosa was germinated and grown in SD for 28 d and then exposed to either a florally inductive combination of 7 LD + 2 SD, the last day of which coincides with determination of the sepal and stamen whorls, or was germinated and grown in 37 non-inductive SD. Pharbitis nil was germinated and grown in continuous light (CL) for 5 d and then given either 48 h of inductive darkness followed by 1 d of CL, the last day of which coincides with determination of the sepal, petal and stamen whorls, or given one of two independent non-inductive treatments: 48 h dark interrupted by red light (R) + 1 d of CL, or 8 d of CL. Following these treatments, each batch of plants was exposed to tritiated [methyl-³H]thymidine for 30, 60, 90 or 120 min. Apical domes were dissected, nuclei lysed and prepared as fibre autoradiographs from which replicon size was recorded. In S. coeli-rosa, replicon size was in the range 10–15 μm in SD (non-inductive) and 0–5 μm in LD (inductive) while in P. nil it was 10–15 μm in the 48 h dark interrupted by R, 5–10 μm in CL (both non-inductive) but was reduced to 0–5 μm in the 48 h dark treatment (inductive). Therefore, the recruitment of additional initiation points for DNA replication occurred in both a LD and a SD plant immediately before the appearance of floral organs. The data are consistent in showing that a shortening of S-phase, which is a characteristic feature of florally determined shoot meristems for both species, is brought about by the activation of latent origins of DNA replication. Received: 14 May 1998 / Accepted: 20 August 1998     

Identification of quantitative trait loci associated with self-compatibility in a Prunus species

Self-compatibility in Rosaceous fruit species is based on a single-locus qualitative trait. However, the evidence observed in different species has indicated the presence of modifier genes outside the S locus affecting the expression of self-compatibility/self-incompatibility. The study of a progeny obtained from the cross of the almond genotypes ‘Vivot’× ‘Blanquerna’ has allowed the construction of a genetic map based on microsatellite markers and the identification for the first time in the Rosaceae family of two additional loci located outside the S locus and affecting the expression of self-compatibility/self-incompatibility. A quantitative trait locus (QTL) was located relatively close to the S locus, on linkage group 6 (G6), whereas the second one was located on G8. These QTLs appear to be involved in conferring self-compatibility to genotypes not possessing the S _f allele. These results are consistent with almond being a self-incompatible species with a genetic background of pseudo-self-compatibility controlled by modifier genes. The effect of the S _f allele and the two QTLs may contribute to explain the wide range of fruit sets observed when self-pollinating different almond genotypes.