Carbon allocation in developing spruce needles. Enzymes and intermediates of sucrose metabolism

In lyophilized needles of Norway spruce ( Picea abies [L.] Karsten) and starting from bud break, we determined enzyme activities (sucrose phosphate synthase [SPS; EC 2.4,1.14]. sucrose synthase [SS; EC 2.4,1.13]. acid invertase [AI; EC 3.2,1.26]) and intermediates (starch, sucrose, glucose, fructose; fructose 6-phosphate, fructose 2.6-bisphosphate [F26BP]) of carbohydrate metabolism together with needle weight, shoot length, chlorophyll and protein. For up to 110 days after bud break, samples were taken twice a week from about 25-year-old trees under field conditions. At least three periods can be distinguished during needle maturation. During the first period (up to 45 days after bud break) Al showed the highest extractable activity. This coincided with very high levels of F26BP (up to 11 pmol [mg dry weight]⁻¹) and a transient increase of starch in parallel to a decrease of sucrose. The interval between 45 and 70 days after bud break was characterized by high SS activity (ratio of fructose/glucose >1), much decreased levels of F26BP (down to below 1 pmol [mg dry weight]⁻¹), and a pronounced increase in the dry weight/fresh weight ratio. In parallel, starch declined and soluble carbohydrates increased. Finally, needle maturation was characterized by decreasing SS and continuously increasing SPS activities, so that the ratio of SPS/SS increased more than 6-fold. AI. however, did not decline with maturation. Changes in pool sizes of metabolites and enzyme activities (AI. SPS) are consistent with current concepts on sink/source transition. SS is obviously important with regard to the synthesis of structural polysaccharides.     

Presence of β-cyanoalanine synthase in unimbibed dry seeds and its activation by ethylene during pre-germination

Evolution of HCN from both rice ( Oryza sativa ) and cocklebur ( Xanthium pennsylvanicum ) seeds increased during a pre-germination period and preceded the evolution of (C₂H₄). These two species were adopted as the representatives of starchy and fatty seeds, respectively. Ethylene promotes seed germination of many species. However, HCN evolution declined abruptly when the radicles emerged and before the peak in C₂H₄ evolution. More-over, both rice and soybean ( Glycine max ) seeds showed some activity of β-cyanoalanine synthase (CAS, EC 4.4.1.9) even in the unimbibed dry state. The activities of CAS in the lower seed of cocklebur and in soybean seeds increased rapidly after emergence of the radicle. However, the CAS of rice seeds, with high activity in the dry state, exhibited a bimodal change, gradually decreasing until radicle emergence had occurred, but then increaing. It is thus likly that HCN evolution during initial imbibition may be derived from cyanogenic reserves and controlled by both pre-existing and subsequently-developing CAS. The exogenous application of C₂H₄ stimulated the activities of CAS in both rice and upper cocklebur seeds and reduced their cyanogen contents. Therefore, the decline of HCN evolution after germination seems to be due to the increased activities of CAS by endogenously produced C₂H₄.     

Anthranilate synthase and chorismate mutase activities in transgenic tobacco plants overexpressing tryptophan decarboxylase fromCatharanthus roseus

TransgenicNicotiana tabacum L. Petit Havana SR1 F₁-plants expressing tryptophan decarboxylase cDNA (tdc) fromCatharanthus roseus (L.) G. Don under the control of the CaMV 35S promoter and terminator exhibited tryptophan decarboxylase (TDC) enzyme activity and accumulated tryptamine. The plants with the highest TDC activity contained 19 pkat per mg of protein. The influence of transgenic expression oftdc on the activities of anthranilate synthase (AS) and chorismate mutase (CM) were examined in 10 transgenic tobacco plants. The specific activities of these two chorismate-utilizing enzymes were not significantly affected by expression oftdc, despite their important functions as branch point enzymes in the shikimate pathway. The results indicate that the normal route of tryptophan biosynthesis in plants is sufficient to supply a considerable amount of this essential amino acid for the biosynthesis of secondary metabolites. Despite their increased tryptamine content, the growth and development of the transgenic tobacco plants expressingtdc appeared normal.     

Cloning and analysis of the polyhydroxyalkanoic acid synthase gene from an Acinetobacter sp.: Evidence that the gene is both plasmid and chromosomally located

Abstract The polyhydroxyalkanoic acid (PHA) synthase gene ( phaC_Ac ) of a species of Acinetobacter isolated from an activated sludge treatment plant was cloned by heterologous complementation in a poly-β-hydroxybutyrate (PHB) negative mutant of Alcaligenes eutrophus . Nucleotide sequence analysis of phaC_Ac revelaed an open reading frame of 1770 bp with potential to encode a 67.7 kDa protein. The deduced amino acid sequence displays high similarity to other PHA synthase proteins. Probing with an internal region of phaC_Ac revealed that the PHA sythase gene may be present in more than one copy and may occur at both plasmid and chromosomal locations in Acinetobacter spp. This is the first organisms for which evidence has been presented to suggest that a gene involved in PHA metabolism is plasmid-encoded. Purification of PHB granules from sucrose gradients identified proteins of 38 kDa, 41 kDa and 64 kDa which may have a role in PHB metabolism.     

An autonomously propagating luciferase-encoding vector for Dictyostelium discoideum

We have constructed a luc reporter vector for Dictyostelium discoideum using a 626-bp fragment from the nuclear-associated plasmid Ddp2. The ori from Ddp2 is localized within this fragment and was used to provide an autonomous replication sequence for the reporter vector. This reporter vector was stably retained in D. discoideum AX3K cells without alteration. The vector molecule was also found to exist in relatively low copy number compared to other Dictyostelium vectors in the transformed cells. We demonstrated the utility of this vector as a reporter vector with glycogen synthase promoter/luc fusions of varying sizes.     

鸭肝脂肪酸合成酶的胍变性与失活

报道了鸭肝脂肪酸俣成酶在胍变性过程中构变性过程中构象变化和活性变化的关系,首次验证了邹承鲁提出的酶活性部位构象的理论适用于多功能复合酶,同时该酶变性及复性均可测定出多个阶段,且证明有无活性稳态酶存在,在低浓度盐酸胍溶液中该酶的全反应活性和其中两个还原部位单独活性被同步可逆抑制,随着胍浓度增高,出现不可逆失活且程度和速度均迅速提高,在０．５４ｍｏｌ／Ｌ胍中该酶全反应活性在１．５分种内已有一半不可逆失     

Characterization and expression in Streptomyces lividans of cefD and cefE genes from Nocardia lactamdurans: the organization of the cephamycin gene cluster differs from that in Streptomyces clavuligerus

Summary The cefD and cefE genes of Nocardia lactamdurans, which encode isopenicillin N epimerase and deacetoxycephalosporin C synthase respectively, have been located 0.63 kb upstream from the lysine-6-amino-transferase (lat) gene. cefD contains an open reading frame (ORF) of 1197 nucleotides (nt) encoding a protein of 398 amino acids with a M_r of 43 622. The deduced amino acid sequence exhibits 62.2% identity to the cefD gene product of Streptomyces clavuligerus. The sequence SXHKXL in isopenicillin N epimerase resembles the consensus sequence for pyridoxal phosphate binding found in several amino acid decarboxylases from Enterobacteria. cefE contains an ORF of 945 nt encoding a protein of 314 amino acids with a M_r of 34532, which is similar to the deacetoxycephalosporin C synthase of S. clavuligerus. Expression of both genes, cefD and cefE, in S. lividans transformants, results in deacetoxycephalosporin C synthase and isopenicillin N epimerase activities that are 10–12 times higher than those in N. lactamdurans. The cefD and cefE genes of N. lactamdurans are closely linked but the overall organization of the cephamycin gene cluster differs in N. lactamdurans and S. clavuligerus.     

Sucrose and fructan metabolism in wheat roots at chilling temperatures

Sucrose and fructan metabolism were studied in wheat ( Triticuin aotiirum L. cv. Tribal 800) roots during a period at chilling temperature. Enzyme activities related to fructan and sucrose metabolism were measured. Sucrose-sucrose fructosyl transfer-ase (EC 2.4.1.99) activity increased more than 25-fold when plants were cooled to 4°C. Sucrose synthase (EC 2.4.1.13) and sucrose-phosphate synthase (EC 2.4.1.14) activities also increased, but low temperatures had no significant effect on invertaso (EC 3.2.1.26) or on fructan hydrolase (EC 3.2.1.26) activities. The accumulation pattern of fructan in roots was different to that in leaves. In roots chilling stimulated the synthesis of fructans of high degree of polymerization.     

Chromium-induced inhibition of ethylene evolution in bean (Phaseolus vulgaris) leaves

The influence of chromium concentration on ethylene production in bean plants ( Phaseolus vulgaris L. cv. Contender) was investigated. A Cr ion-induced inhibition of ethylene synthesis from endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) was observed within both leaf discs floated on 2 m M CrO²⁻₄ or Cr³⁺ and leaf discs from plants cultured in nutrient solutions containing 10, 20 or 40 μ M CrO²⁻₄. However, Cr ions supplied either to plants with the nutrient solution or to discs with the incubation medium rather increased the conversion of exogenous ACC to ethylene. Primary leaves of plants exposed to CrO²⁻₄-containing nutrient solutions showed a statistically insignificant decrease of ACC-synthase activity. In the trifoliolate leaves of plants exposed to 10 μ M CrO²⁻₄, in which a significant decrease of ethylene production from endogenous ACC was observed, a substantial increase of ACC synthase was found. These results indicate that Cr ion-induced inhibition of ethylene production is not due to a breakdown of membrane integrity, which is necessary for ethylene forming enzyme activity, but caused by metabolic alterations leading to decreased ACC availability. Chromium ions may act by inhibiting ACC synthase activity or by diverting a metabolic step prior to the ACC synthase catalyzed reaction.