植物4-香豆酸:辅酶A连接酶

就植物中4-香豆酸：辅酶A连接酶的基因表达调控和底物结合功能域及其与木质素的关系以及其基因家族的进化的研究进展做了介绍。     

Alterations in the Biosynthesis of Lignin in Transgenic Plants with Chimeric Genes for 4-Coumarate: Coenzyme A Ligase

The introduction of chimeric sense and antisense gene constructsfor 4-coumarate:coenzyme A ligase into tobacco plants causedthe reduction of the 4CL activity in the transgenic plants.In the transgenic plants, the cell walls of the xylem tissuein stems were brown and the molecular structure of lignin inthe colored cell walls was dramatically different from thatin the control plants. Analysis with different types of stainrevealed that levels of cinnamyl aldehyde residues and syringylunits in lignin were depressed in the brownish cell walls. Furthermore,the lignin content in colored tissue was lower than that inthe normal tissue. Our results indicate that 4CL has importantroles in the determination of the composition and the amountof lignin in tobacco plants. (Received December 27, 1995; Accepted July 23, 1996)     

Structural Characterization of Modified Lignin in Transgenic Tobacco Plants in Which the Activity of 4-Coumarate:Coenzyme A Ligase Is Depressed

Transgenic tobacco (Nicotiana tabacum L.) plants in which the activity of 4-coumarate:coenzyme A ligase is very low contain a novel lignin in their xylem. Details of changes in hydroxycinnamic acids bound to cell walls and in the structure of the novel lignin were identified by base hydrolysis, alkaline nitrobenzene oxidation, pyrolysis-gas chromatography, and 13C-nuclear magnetic resonance analysis. In the brownish tissue of the transgenic plants, the levels of three hydroxycinnamic acids, p-coumaric, ferulic, and sinapic, which were bound to cell walls, were apparently increased as a result of down-regulation of the expression of the gene for 4-coumarate:coenzyme A ligase. Some of these hydroxycinnamic acids were linked to cell walls via ester and ether linkages. The accumulation of hydroxycinnamic acids also induced an increase in the level of condensed units in the novel lignin of the brownish tissue. Our data indicate that the behavior of some of the incorporated hydroxycinnamic acids resembles lignin monomers in the brownish tissue, and their accumulation results in dramatic changes in the biosynthesis of lignin in transgenic plants.     

4-Coumarate:Coenzyme A Ligase and Isoperoxidase Expression in Zinnia Mesophyll Cells Induced to Differentiate into Tracheary Elements

When cultured in inductive medium containing adequate auxin and cytokinin, isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate into tracheary elements with lignified secondary wall thickenings. Differentiation does not occur when cells are cultured in control medium, which has reduced levels of auxin and/or cytokinin. The activities of two enzymes involved in lignin synthesis, 4-coumarate:coenzyme A ligase and peroxidase, were examined. An induction-specific cationic isoperoxidase, visualized by low pH polyacrylamide gel electrophoresis, is detectable in soluble and wall fractions of cultured Zinnia cells long before tracheary elements visibly differentiate and is thus an early marker of differentiation. Compounds (such as antiauxins, anticytokinins, and tunicamycin) that inhibit or delay differentiation alter the expression of this isoperoxidase. 4-Coumarate:coenzyme A ligase activity increases dramatically only as cells differentiate. Together, these results suggest that the onset of lignification in differentiating Zinnia cells might be controlled by the availability of precursors synthesized by way of 4-coumarate:coenzyme A ligase. These precursors would then be polymerized into lignin in the cell wall by the induction-specific isoperoxidase.     

Two Divergent Members of 4-Coumarate： Coenzyme A Ligase from Salvia miltiorrhiza Bunge： cDNA Cloning and Functional Study

4-Coumarate ： coenzyme A Ilgase （4CL） Is one of the key enzymes In phenylpropanoid metabolism leading to series of phenollcs, Including water-soluble phenolic acids, which are important compounds determining the medicinal quality of Danshen （Salvia miltiorrhiza Bunge）, a traditional Chinese medicinal herb. To Investigate the function of 4CL in the biosynthesis of water-soluble phenolic acid in Danshen, we have cloned two cDNAs （Sm4CL1 and Sm4CL2） encoding divergent 4CL members by applying nested reverse transcrlptlon-polymerase chain reaction （RT-PCR） with degenerate primers followed by 5′/3′rapid amplification of cDNA ends （RACE） （Note, these sequence data have been submitted to the GenBank database under accession numbers AY237163 and AY237164）. Either of the coding regions was inserted into a pRSET vector and a kinetic assay was performed with purified recombinant proteins. The substrate utilization profile of Sm4CL1 was distinct from that of Sm4CL2. The Km values of Sm4CL1 and Sm4CL2 to 4-coumarlc acid were （72.20±4.10） and （6.50±1.45） μmol/L, respectively. These results, In conjunction with Northern blotting and other information, imply that Sm4CL2 may play an Important role in the biosynthesis of watersoluble phenolic compounds, whereas Sm4CL1 may play a minor role in the pathway. Southern blotting analysis suggested that both Sm4CL1 and Sm4CL2 genes are present as a single copy and are located at different sites In the genome.     

枇杷4-香豆酸CoA连接酶的某些特性

以枇杷品种‘解放钟’果实为试材,用硫酸铵分级盐析方法提取4-香豆酸CoA连接酶,其最适温度为10和40℃,40和10℃下的热稳定性较好;最适pH为8．0且较稳定;最适底物为咖啡酸。     

Isolation and Characterization of Two cDNAs Encoding 4-Coumarate:CoA Ligase in Lithospermum Cell Cultures

Two near full-length cDNAs (LE4CL-1, LE4CL-2), which encode4-coumarate:CoA ligase (4CL), were cloned from a library ofLithospermum erythrorhizon cell suspension cultures by the useof heterologous probe of potato 4CL. These cDNAs are 2.1 kband 2.2 kb in length, respectively. LE4CL-1 encodes 636 aminoacids, whose homologies to the 4CL protein sequences known topotato, parsley, pine and rice, were found to be 68%, 66%, 56%and 50% (identities on amino acid level), respectively, whereasthose of the predicted translation product of LE4CL-2 (594 aminoacids) to the above 4CL proteins were 49{small tilde}54%. Thesimilarity of the deduced amino acid sequences between the two4CLs from Lithospermum cell cultures was 49% in identity. Northernanalyses showed that the mRNA levels of both LE4CL-1 and LE4CL-2were much higher under illumination than in the dark, as reportedfor the 4CL genes of such plants as parsley. In comparison ofmRNA levels of LE4CL-1 and LE4CL-2, the former was demonstratedto be generally higher than the latter by means of an applicationof RT-PCR. The genomic southern blot experiments suggested thatthere are probably three copies of LE4CL-1 in the Lithospermumgenome DNA, whereas only one copy was detected for LE4CL-2. (Received May 26, 1995; Accepted August 16, 1995)     

p18~(INK4C)调控小鼠T细胞的发育及功能

p18INK4C属于细胞周期蛋白激酶抑制剂,其突变或缺失与某些肿瘤的发生密切相关,如T细胞白血病,但目前关于p18调控T细胞发育及功能的研究还鲜有报道,其调控机制仍不明确.本研究利用p18基因敲除(p18KO)小鼠,系统地研究了胸腺中T细胞的早期发育及成熟T细胞的增殖和活化功能,并利用逆转录病毒的方法在Lin?造血干祖细胞上过表达p18,移植4个月后检测其对T细胞的影响.结果表明,p18的缺失对胸腺T细胞的早期发育影响不明显,但随着p18KO小鼠周龄的增加会促进CD4+CD8+双阳性T细胞的数量,此外,p18还通过影响CD3+成熟T细胞的细胞周期进程及IFN-?,GATA3,Tbx21和Foxp3等的表达增强脾脏T细胞的增殖和活化;进一步在造血干祖细胞上过表达p18后会影响T细胞的发育和成熟,进而纠正T细胞在数量上的异常.本研究阐释了p18在T细胞早期发育及后期活化中的调控机制,并证实可通过在干祖细胞水平改变p18的表达进而影响T细胞的分化,这对p18调控T细胞功能异常及参与T细胞白血病的发生提供了新的理论依据和重要的研究价值.     

Differential Properties of 4-Coumarate: CoA Ligase Related to Growth Suppression by Chalcone in Maize and Rice

Lignin and related metabolites have diverse and important functions for plant growth and development. 4-Coumarate: CoA ligase (4CL, EC 6.2.1.12) is one of the key enzymes in phenylpropanoid metabolism and lignin biosynthesis. In a previous study, maize (Zea maize L. cv. Yellowcorn) growth was suppressed to a greater extent by root-applied chalcone than rice (Oryza sativa L. cv. Nipponbare). The objective of this study is to clarify the relationship between the growth suppression and 4CL properties. In crude extracts, total 4CL activity and total protein content of rice were higher 1.8- and 2.7-fold than that of maize, respectively. After a gel-filtration chromatography, a single peak of 4CL activity from maize and rice was evident coincidently for both species. After anion-exchange chromatography, a single peak of 4CL activity was also apparent for both species; however, the peak of maize did not coincide with that of rice. The enzyme activity of maize and rice exhibited similar order of substrate specificities when using p-coumaric, cinnamic, caffeic, ferulic and sinapic acids substrates. Chalcone inhibited 4CL activity in maize more strongly than in rice, and 4CL kinetic data in the presence and absence of chalcone exhibited uncompetitive inhibition in both maize and rice. These results suggest that total activity and the inhibitory property of 4CL contributes to differences in growth suppression by chalcone between maize and rice, although further efforts are needed to clarify the potential of 4CL as a novel action site of the growth suppression.     

Acid:Coenzyme A Ligase in Brain: Fatty Acid Specificity in Cellular and Subcellular Fractions

Abstract: We measured long-chain fatty acid:coenzyme A (CoA) ligase (EC 6.2.1.3) activity with four fatty acids in brain homogenates, and cellular and subcellular fractions to determine whether there are differences in activity that could be correlated with differences in fatty acid composition and metabolism. In rat brain homogenates, ligase activity varied appreciably with the four acids, with 18:2 > 18:1 > 16:0 > 22:1 (nmol acyl-CoA formed/min/mg protein; 1.46, 1.20, 0.96, and 0.57, respectively). This order was similar under all incubation conditions tested, including variable pH and fatty acid concentrations. The relative specific activities (RSA, 16:0 = 1.0) with the four substrates were similar in rat brain homogenate, mitochondria, and microsomes, with the highest specific activities in the latter fraction. The RSA were also similar in ox brain homogenates, in rabbit brain microsomes prepared from gray and white matter, in neurons isolated from rat brain, and in cultured neuroblastoma cells. Rat liver homogenates had a significantly different pattern of RSA. These results indicate that the ligase(s) has a preference for certain fatty acids, but suggest that the major control of fatty acid composition and metabolism is a function of subsequent metabolic steps.     

IP3 Responsiveness Is Regulated in a Meiotic Cell Cycle Dependent Manner: Implications for Fertilization Induced Calcium Signalling

Fertilization of mammalian eggs is known to trigger a series of transient rises in cytosolic calcium, known as calcium oscillations, the initiation and duration of which are crucial for meiotic exit and subsequent entry into embryogenesis. It is not known how these calcium oscillations are terminated when the zygote exits meiosis; it is thought that responsiveness to inositol 1,4,5-trisphosphate (IP3) is involved, since the oscillations are known to be mediated by an IP3 dependent mechanism. Here we report that IP3 responsiveness is maintained throughout meiotic maturation and falls very rapidly after meiotic exit. We also show that inhibition of the major cell cycle kinase, CDK1, has no effect on responsiveness, but that prolonging CDK1 activity prevents the decline in responsiveness normally seen at meiotic exit. We conclude that CDK1 plays a role, but is not the only factor involved in controlling IP3 responsiveness during the meiotic cell cycle.     

Phloem-Specific Methionine Recycling Fuels Polyamine Biosynthesis in a Sulfur-Dependent Manner and Promotes Flower and Seed Development

The Yang or Met Cycle is a series of reactions catalyzing the recycling of the sulfur (S) compound 5′-methylthioadenosine (MTA) to Met. MTA is produced as a by-product in ethylene, nicotianamine, and polyamine biosynthesis. Whether the Met Cycle preferentially fuels one of these pathways in a S-dependent manner remained unclear so far. We analyzed Arabidopsis (Arabidopsis thaliana) mutants with defects in the Met Cycle enzymes 5-METHYLTHIORIBOSE-1-PHOSPHATE-ISOMERASE1 (MTI1) and DEHYDRATASE-ENOLASE-PHOSPHATASE-COMPLEX1 (DEP1) under different S conditions and assayed the contribution of the Met Cycle to the regeneration of S for these pathways. Neither mti1 nor dep1 mutants could recycle MTA but showed S-dependent reproductive failure, which was accompanied by reduced levels of the polyamines putrescine, spermidine, and spermine in mutant inflorescences. Complementation experiments with external application of these three polyamines showed that only the triamine spermine could specifically rescue the S-dependent reproductive defects of the mutant plants. Furthermore, expressing gene-reporter fusions in Arabidopsis showed that MTI1 and DEP1 were mainly expressed in the vasculature of all plant parts. Phloem-specific reconstitution of Met Cycle activity in mti1 and dep1 mutant plants was sufficient to rescue their S-dependent mutant phenotypes. We conclude from these analyses that phloem-specific S recycling during periods of S starvation is essential for the biosynthesis of polyamines required for flowering and seed development.Sulfur (S) deficiency greatly impacts flower development and seed yield of different plant species (Hell, 2008; Marschner and Marschner, 2012; D’Hooghe et al., 2013). Shoots and flowers of S-deprived plants appear pale yellow and seeds show reduced germination efficiency (Higgins et al., 1986; Nikiforova et al., 2003). In particular, Brassica species have high S demands, presumably because of the large amounts of Cys-rich storage proteins in their cotyledons (Shewry and Casey, 1999) and the production of glucosinolates, which mostly derive from Met (Windsor et al., 2005). Both sulfate transport and assimilation pathways are highly regulated by S availability, and the expression and activity levels of the corresponding proteins are efficiently adjusted under low S availability (Saito, 2004; Koprivova and Kopriva, 2014). S deficiency promotes the synthesis of transport proteins of the SULFATE TRANSPORTER (SULTR) family to increase root sulfate uptake (Shinmachi et al., 2010; Maruyama-Nakashita et al., 2015) or sulfate efflux from storage vacuoles (Kataoka et al., 2004), which supports the remobilization of sulfate from source to sink tissues. Moreover, plants increase the efficiency of S utilization by inducing S recycling pathways. The Met Cycle, also known as Yang cycle or 5′-methylthioadenosine (MTA) cycle, is the major S recycling pathway in plants and consists of a series of reactions that convert MTA back to Met (Sauter et al., 2013). MTA is generated as a by-product during ethylene, polyamine, and nicotianamine synthesis. However, the quantitative contribution of these three pathways to MTA formation and their relative importance for Met regeneration via the Met Cycle are still unclear.The existence of a recycling pathway for Met was first postulated by Baur and Yang (1972), and the first enzymatic activities of plant Met Cycle enzymes, 5′-methylthioribose kinase (MTK) and 5′-methylthioadenosine nucleosidase (MTN), were found 5 years later in extracts from lupin seeds (Guranowski, 1983). The first genes encoding plant Met Cycle enzymes (MTK from Arabidopsis [Arabidopsis thaliana] and MTK1 and MTK2 from rice [Oryza sativa]) were cloned 20 years later (Sauter et al., 2004). In the following years, genes for acidoreductone dioxygenases (ARD1-4) as well as for MTA nucleosidases (MTN1 and MTN2) were identified (Sauter et al., 2005; Rzewuski et al., 2007). The number of the remaining enzymatic steps and genes of the Met Cycle in Arabidopsis remained unclear, as the conversion of 5-methyl-thioribulose-1-P (MTRu-1-P) to 1,2-dihydroxy-3-keto-5-methylthiopentene (DHKMP) is catalyzed by two enzymes in certain organisms and by three enzymes in others (Sekowska et al., 2004). Recently, two novel higher plant Met Cycle genes, 5-METHYLTHIORIBOSE-1-PHOSPHATE ISOMERASE1 (MTI1) and DEHYDRATASE-ENOLASE-PHOSPHATASE-COMPLEX1 (DEP1), have been identified (Pommerrenig et al., 2011). DEP1 is a trifunctional enzyme that catalyzes the conversion of MTRu-1-P to DHKMP. MTI1 has a surprising and high similarity to eukaryotic translation initiation factor eIF-2B family proteins. Phylogenetic analyses and complementation of a yeast strain with a defect in the corresponding MRI1 gene confirmed the enzymatic conversion of MTR-1P to MTRu-1-P by the MTI1 protein (Pommerrenig et al., 2011).An essential role of the Met Cycle for plant S nutrition has been proposed for plants and plant organs exhibiting high ethylene synthesis, for example, during germination and seedling growth, but also during periods of hypoxia or fruit ripening. During these phases, elevated Met Cycle activity helps to replenish the Met pool (Baur and Yang, 1972; Bürstenbinder et al., 2007; Rzewuski et al., 2007). Utilizing a mtk mutant, the ethylene overproducing mutant eto3, and the mtk/eto3 double mutant, Bürstenbinder et al. (2007) could show that the Met Cycle is important during periods of high ethylene synthesis in seedlings. In contrast, in adult plants, the overall ethylene synthesis is low; thus, an elevated S requirement for ethylene may be restricted to plants that naturally produce or need to produce large quantities of the hormone for a prolonged period of time (Rzewuski et al., 2007; Sauter et al., 2013).However, Met Cycle activities are not restricted to seedlings and fruits, since the levels of both mRNA of Met Cycle genes and Met Cycle-related metabolites were found to accumulate in the vasculature of adult rosette leaves of Arabidopsis and Plantago major (Pommerrenig et al., 2011). The specific expression of Met Cycle genes in the vasculature is in line with the second essential function of the Met Cycle, which is the degradation of MTA, the by-product of ethylene, nicotianamine, and polyamine biosynthesis. Mutants lacking MTA nucleosidase activity (Bürstenbinder et al., 2010; Waduwara-Jayabahu et al., 2012) also showed hyperproliferation of xylem elements in their vasculature and impaired flower development. These effects have been attributed to elevated MTA levels and inhibited polyamine and nicotianamine (NA) biosynthesis.Polyamines are positively charged polycations, which occur in all living organisms and fulfill important functions in cellular metabolism. In Arabidopsis, the main polyamines are putrescine, spermidine, spermine, and thermospermine. All polyamines have the ability to bind DNA but also contribute to plant tolerance to biotic and abiotic stresses (Jimènez-Bremont et al., 2014; Minocha et al., 2014). Spermine synthase (SPMS) has been shown to protect plants during salt stress. Additionally, thermospermine, which is synthesized by thermospermine synthase (ACL5), functions in vascular development by repressing xylem differentiation (Vera-Sirera et al., 2010; Takano et al., 2012), and spermidine has been proven important for plant reproduction (Imai et al., 2004; Deeb et al., 2010). The acl5/spms double mutant was shown to be hypersensitive to salt stress but could be rescued by the exogenous application of spermine (Yamaguchi et al., 2006). Overexpression of spermidine and spermine biosynthesis or exogenous supply of spermine have been reported to increase the tolerance to drought (Capell et al., 2004) or heat stress (Sagor et al., 2013).Whether the Met Cycle has a role apart from the degradation of MTA, most notably in sustaining S supply for efficient polyamine biosynthesis during reproductive growth, and to what extent the Met Cycle contributes to S nutrition remained unclear so far. For clarification of this issue, we isolated Arabidopsis mutants defective in the expression of MTI1 and DEP1 and studied their growth and metabolism under S-limiting conditions. To this end, we were able to characterize MTA-independent effects of altered Met Cycle activities. During our analyses we discovered strong S-dependent reproductive defects in mti1 and dep1 mutants that had not been reported for other genes of the Met Cycle. Our physiological analyses of mti1 and dep1 mutants further illustrate the importance of sustained polyamine synthesis for plant reproduction during S deficiency.     

Dynamic Transcription of Long Non-Coding RNA Genes during CD4+ T Cell Development and Activation

Background

Recent evidence shows that long non-coding RNA (LncRNA) play important regulatory roles in many biology process, including cell development, activation and oncogenesis. However, the roles of these LncRNAs in the development and activation of CD4⁺ T cells, which is an important component of immune response, remain unknown.

Results

To predict the function of LncRNA in the development and activation of CD4⁺ T cells, first, we examined the expression profiles of LncRNAs and mRNAs in CD4⁻CD8⁻ (DN), CD4⁺CD8⁺ (DP), CD4⁺CD8⁻, and activated CD4⁺CD8⁻ T cells in a microarray analysis and verified these results by real time PCRs (qPCR). We found that the expression of hundreds of LncRNAs significantly changed in each process of developmental transition, including DN into DP, DP into CD4⁺CD8⁻, and CD4⁺CD8⁻ into activated CD4⁺ T cells. A Kendall distance analysis suggested that the expression of LncRNAs in DN, DP, CD4⁺CD8⁻ T cells and activated CD4⁺ T cells were correlated with the expression of mRNAs in these T cells. The Blat algorithm and GO analysis suggested that LncRNAs may exert important roles in the development and activation of CD4⁺ T cells. These roles included proliferation, homeostasis, maturation, activation, migration, apoptosis and calcium ion transportation.

Conclusion

The present study found that the expression profiles of LncRNAs in different stages of CD4⁺ T cells are distinguishable. LncRNAs are involved in the key biological process in CD4⁺ T cell development and activation.