Adenosine kinase contributes to cytokinin interconversion in Arabidopsis

Purine salvage enzymes have been implicated, but not proven, to be involved in the interconversion of cytokinin (CK) bases, ribosides, and nucleotides. Here, we use Arabidopsis (Arabidopsis thaliana) lines silenced in adenosine kinase (ADK) expression to understand the contributions of this enzyme activity to in vivo CK metabolism. Both small interfering RNA- and artificial microRNA-mediated silencing of ADK led to impaired root growth, small, crinkled rosette leaves, and reduced apical dominance. Further examination of ADK-deficient roots and leaves revealed their irregular cell division. Root tips had uneven arrangements of root cap cells, reduced meristem sizes, and enlarged cells in the elongation zone; rosette leaves exhibited decreased cell size but increased cell abundance. Expression patterns of the cyclinB1;1::β-glucuronidase and Arabidopsis Response Regulator5::β-glucuronidase reporters in the ADK-deficient background were consistent with altered cell division and an increase in CK activity, respectively. In vivo feeding of ADK-deficient leaves with radiolabeled CK ribosides of isopentenyladenosine and zeatin showed a decreased flux into the corresponding CK nucleotides. Comprehensive high-performance liquid chromatography-tandem mass spectrometry analysis detected significantly higher levels of active CK ribosides in both sense ADK and artificial microADK. Taken together, these metabolic and phenotypic analyses of ADK-deficient lines indicate that ADK contributes to CK homeostasis in vivo.     

Role of adenosine kinase in the control of Streptomyces differentiations: Loss of adenosine kinase suppresses sporulation and actinorhodin biosynthesis while inducing hyperproduction of undecylprodigiosin in Streptomyces lividans

Adenosine kinase (ADK) catalyses phosphorylation of adenosine (Ado) and generates adenosine monophosphate (AMP). ADK gene (adk(Sli), an ortholog of SCO2158) was disrupted in Streptomyces lividans by single crossover-mediated vector integration. The adk(Sli) disruption mutant (Deltaadk(Sli)) was devoid of sporulation and a plasmid copy of adk(Sli) restored sporulation ability in Deltaadk(Sli), thus indicating that loss of adk(Sli) abolishes sporulation in S. lividans. Ado supplementation strongly suppressed sporulation ability in S. lividans wild-type (wt), supporting that disruption of adk(Sli) resulted in Ado accumulation, which in turn suppressed sporulation. Cell-free experiments demonstrated that Deltaadk(Sli) lacked ADK activity and in vitro characterization confirms that adk(Sli) encodes ADK. The intracellular level of Ado was highly elevated while the AMP level was significantly reduced after loss of adk(Sli) while Deltaadk(Sli) displayed no significant derivation from wt in the levels of S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM). Notably, Ado supplementation to wt lowered AMP content, albeit not to the level of Deltaadk(Sli), implying that the reduction of AMP level is partially forced by Ado accumulation in Deltaadk(Sli). In Deltaadk(Sli), actinorhodin (ACT) production was suppressed and undecylprodigiosin (RED) production was dramatically enhanced; however, Ado supplementation failed to exert this differential control. A promoter-probe assay verified repression of actII-orf4 and induction of redD in Deltaadk(Sli), substantiating that unknown metabolic shift(s) of ADK-deficiency evokes differential genetic control on secondary metabolism in S. lividans. The present study is the first report revealing the suppressive role of Ado in Streptomyces development and the differential regulatory function of ADK activity in Streptomyces secondary metabolism, although the underlying mechanism has yet to be elucidated.     

Sustaining S-adenosyl-l-methionine-dependent methyltransferase activity in plant cells

Many biochemical reactions in plants involve the transfer of a methyl group from S -adenosyl- l -methionine (SAM). The transfer of the methyl group from SAM generates S -adenosyl- l -homocysteine (SAH), a potent inhibitor of SAM-dependent methyltransferases (MTs). To mitigate the toxic effects of SAH on MT activity, SAH is removed by SAH hydrolase (SAHH, EC 3.3.1.1) in a reaction generating homocysteine and adenosine (Ado). However, SAHH catalyzes a reversible reaction that is favored to move in the direction of SAH hydrolysis only by removal of these products. Removal of Ado is reported to exert a greater influence on promoting SAH hydrolysis. Whereas animals appear to rely upon Ado deaminase (EC 3.5.4.4) to catabolize Ado, plants appear to use adenosine kinase (EC 2.7.1.20) for this important role. Compounds undergoing methylation represent a broad spectrum of chemically diverse substrates ranging from nucleic acids, lipids and cell wall components to comparatively simpler amines, alcohols and metal halides. Given the diverse nature of methyl acceptor compounds, it is very likely that the demand for SAM synthesis and SAH removal changes both temporally and spatially during the course of plant growth and development. Plants also use SAM as a precursor for the synthesis of ethylene, polyamines, biotin and nicotianamine. These uses are also expected to undergo changes reflective of the metabolic activities of different plants, plant organs, or cells. This review examines the various uses of SAM in plants and addresses how they allocate this resource to satisfy potentially competing needs.     

Inhibition of methylation of DNA and tRNA by adenosine in an adenosine-sensitive mutant of the baby hamster kidney cell line

An adenosine-sensitive (Ados) mutant of baby hamster kidney (BHK) cells, ara-S10d, when treated with a toxic concentration of adenosine (Ado), displayed a substantial elevation of S-adenosylhomocysteine (SAH), S-adenosylmethionine (SAM), and methylthioadenosine (MTA). Wild-type BHK cells treated with the same concentration of Ado (not toxic to these parental cells) produced an elevation of SAH 1.5 times higher than that of ara-S10d cells without a concurrent elevation of SAM or MTA. Inhibition of methylation of DNA and tRNA is greater in ara-S10d cells treated with Ado than that of similarly treated wild-type cells. This inhibition was correlated with the enhanced Ado toxicity, suggesting inhibition of methylation as a possible causal factor for the great increase in Ado sensitivity. Inhibition of methylation may be due to the elevated level of MTA and not solely to the elevation of SAH, a well-known potent inhibitor of numerous methyltransferases.     

Deficiency of adenosine kinase activity affects the degree of pectin methyl-esterification in cell walls of Arabidopsis thaliana

Pectin methyl-esterification is catalysed by S-adenosyl-l-methionine (SAM)-dependent methyltransferases. As deficiency in adenosine kinase (ADK; EC 2.7.1.20) activity impairs SAM recycling and utilization, we investigated the relationship between ADK-deficiency and the degree of pectin methyl-esterification in cell walls of Arabidopsis thaliana. The distribution patterns of epitopes associated with methyl-esterified homogalacturonan in leaves and hypocotyls of wild-type (WT) and ADK-deficient plants were examined using immunolocalization and biochemical techniques. JIM5 and LM7 epitopes, characteristic of low esterified pectins, were more irregularly distributed along the cell wall in ADK-deficient plants than in WT cell walls. In addition, epitopes recognized by JIM7, characteristic of pectins with a higher degree of methyl-esterification, were less abundant in ADK-deficient leaves and hypocotyls. Since de-esterified pectins have enhanced adhesion properties, we propose that the higher abundance and the altered distribution of low methyl-esterified pectin in ADK-deficient cell walls lead to the leaf shape abnormalities observed in these plants.     

High resolution crystal structures of Mycobacterium tuberculosis adenosine kinase: insights into the mechanism and specificity of this novel prokaryotic enzyme

Adenosine kinase (ADK) catalyzes the phosphorylation of adenosine (Ado) to adenosine monophosphate (AMP). It is part of the purine salvage pathway that has been identified only in eukaryotes, with the single exception of Mycobacterium spp. Whereas it is not clear if Mycobacterium tuberculosis (Mtb) ADK is essential, it has been shown that the enzyme can selectively phosphorylate nucleoside analogs to produce products toxic to the cell. We have determined the crystal structure of Mtb ADK unliganded as well as ligand (Ado) bound at 1.5- and 1.9-A resolution, respectively. The structure of the binary complexes with the inhibitor 2-fluoroadenosine (F-Ado) bound and with the adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP) (non-hydrolyzable ATP analog) bound were also solved at 1.9-A resolution. These four structures indicate that Mtb ADK is a dimer formed by an extended beta sheet. The active site of the unliganded ADK is in an open conformation, and upon Ado binding a lid domain of the protein undergoes a large conformation change to close the active site. In the closed conformation, the lid forms direct interactions with the substrate and residues of the active site. Interestingly, AMP-PCP binding alone was not sufficient to produce the closed state of the enzyme. The binding mode of F-Ado was characterized to illustrate the role of additional non-bonding interactions in Mtb ADK compared with human ADK.     

The Regional Concentrations of S-Adenosyl-l-Methionine, S-Adenosyl-l-Homocysteine, and Adenosine in Rat Brain

Abstract: The concentrations of S -adenosyl- l -methionine (SAM), S -adenosyl- l -homocysteine (SAH), and adenosine (Ado) were determined in whole brain and rat brain regions by HPLC. The whole brain contains, respectively, 22 nmol, 1 nmol, and 64 nmol of SAM, SAH, and Ado per g of wet tissue. Their distribution indicated that SAM and SAH levels are highest in brainstem, whereas the Ado level is highest in cortex. With aging the SAM concentrations decrease in whole brain, brainstem, and hypothalamus (–25%) and SAH levels increase by 90% in striatum and by 160% in cerebellum, while Ado levels are increased in all regions by 100–180%.     

Variations in S-adenosylmethionine, S-adenosylhomocysteine and adenosine concentrations in rat liver.

The hepatic concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and adenosine (Ado) in the rat were examined diurnally and as a function of fasting. Ado concentrations increased continuously throughout the fasting period; concentrations after 2 days of fasting were 7.5-fold higher than control values. Diurnally, the concentration of Ado was highest during the light hours. SAM and the ratio of SAM/SAH were reduced greater than 50% due to fasting and exhibited a significant daily rhythm which appeared to be related to dietary methionine availability. Hepatic SAM concentrations decreased continuously during the light hours and increased during the dark period to levels 7.3-fold greater than the lowest light values. The concentration of SAH was altered in a similar fashion yet to a much lesser degree such that the ratio of SAM/SAH paralleled the changes in the concentration of SAM. The SAM/SAH ratio exhibited a 4.5-fold difference between the peak and nadir values.     

The role of two LEAFY paralogs from Idahoa scapigera (Brassicaceae) in the evolution of a derived plant architecture

Idahoa scapigera produces solitary flowers in the axils of rosette leaves without elongation of the shoot axis, a rosette-flowering architecture. Previous work with one of the two I. scapigera LFY paralogs, IscLFY1, showed that this gene caused aerial flowering rosettes in Arabidopsis thaliana. In this paper, we report that after three generations IscLFY1 transgenic lines are phenotypically indistinguishable from wild-type Arabidopsis, indicating that IscLFY1 protein is able to replace normal LFY function. Additionally, we found that ectopic LFY expression late in development can phenocopy aspects of the aerial rosette phenotype, suggesting that shoot compression caused by IscLFY1 could be caused by localized overexpression of a functional IscLFY protein. We also characterized the expression and function of the second I. scapigera LFY paralog, IscLFY2, in A. thaliana. In contrast to IscLFY1, this paralog was expressed in floral meristems and the shoot apical meristem (SAM). In I. scapigera, LFY-specific antibodies detected high protein levels in developing flowers but not in the apex, suggesting trans-regulatory differences between I. scapigera and A. thaliana. Most IscLFY2 transgenic A. thaliana plants were indistinguishable from wild type, but in a minority of lines the SAM was converted to a terminal flower as would be expected from the reporter-expression pattern. Taken together these results show that both I. scapigera paralogs have conserved LFY function, both proteins can rescue lfy and both can modify inflorescence architecture in an A. thaliana background: either by affecting internode elongation (IscLFY1) or by causing homeotic conversion of shoots into flowers (IscLFY2).     

Cell division and morphological changes in the shoot apex of Arabidopsis thaliana during floral transition

Eight-week-old vegetative plants of Arabidopsis thaliana, ecotype Columbia, were induced to flower by a single long day (LD). In this experimental system, it is known that the last component of the floral stimulus moves from the leaves to the apex 24-36 h after the start of the LD, and the first floral meristem is initiated by the shoot apical meristem (SAM) at 44-56 h (Corbesier et al., 1996, The Plant Journal 9: 947-952). Here we show that the rate of cell division is increased at floral transition in all SAM parts but not in the sub-apical pith cells. Mitotic activity starts to increase 24 h after the start of the LD and is two- to three-fold higher at peak times than that in non-induced plants. This activation is followed by the start of SAM enlargement at 44 h, SAM doming at 48 h, and the elongation of apical internodes (bolting) at 52 h.     

The major volatile organic compound emitted from Arabidopsis thaliana flowers, the sesquiterpene (E)-β-caryophyllene, is a defense against a bacterial pathogen

Flowers have a high risk of pathogen attack because of their rich nutrient and moisture content, and high frequency of insect visitors. We investigated the role of (E)-β-caryophyllene in floral defense against a microbial pathogen. This sesquiterpene is a common volatile compound emitted from flowers, and is a major volatile released from the stigma of Arabidopsis thaliana flowers. Arabidopsis thaliana lines lacking a functional (E)-β-caryophyllene synthase or constitutively overexpressing this gene were challenged with Pseudomonas syringae pv. tomato DC3000, which is a bacterial pathogen of brassicaceous plants. Flowers of plant lines lacking (E)-β-caryophyllene emission showed greater bacterial growth on their stigmas than did wild-type flowers, and their seeds were lighter and misshapen. By contrast, plant lines with ectopic (E)-β-caryophyllene emission from vegetative parts were more resistant than wild-type plants to pathogen infection of leaves, and showed reduced cell damage and higher seed production. Based on in vitro experiments, (E)-β-caryophyllene seems to act by direct inhibition of bacterial growth, rather than by triggering defense signaling pathways. (E)-β-Caryophyllene thus appears to serve as a defense against pathogens that invade floral tissues and, like other floral volatiles, may play multiple roles in defense and pollinator attraction.     

超表达AVP1基因提高转基因百脉根的耐盐性和抗旱性

本研究以超表达拟南芥液泡膜H＋-焦磷酸酶编码基因AVPI的转基因百脉根为材料,对其耐盐性和抗旱性进行了检测。结果显示：在200mmol·L^-1 NaCl下处理或自然干旱7d后,转基因植株的生长虽然受到抑制,但受抑程度明显低于野生型植株,前者叶片相对含水量比后者分别高18％和14％,净光合速率分别高20％和21％,而MDA含量则分别低35％和27％,相对质膜透性分别低28％和27％。此外,随着盐和干旱胁迫的加剧,与野生型植株相比,转基因植株体内积累了更多Na＋、K＋和Ca2＋。以上结果表明,AVPI基因的超表达可能提高了百脉根细胞Na＋区域化能力,既减轻了过量Na＋对细胞质的毒害作用,也提高了植株的渗透调节能力,从而增强了百脉根的耐盐性和抗旱性。     

The Arabidopsis homeobox gene, ATHB16, regulates leaf development and the sensitivity to photoperiod in Arabidopsis

This report describes the characterisation of ATHB16, a novel Arabidopsis thaliana homeobox gene, which encodes a homeodomain-leucine zipper class I (HDZip I) protein. We demonstrate that ATHB16 functions as a growth regulator, potentially as a component in the light-sensing mechanism of the plant. Endogenous ATHB16 mRNA was detected in all organs of Arabidopsis, at highest abundance in rosette leaves. Reduced levels of ATHB16 expression in transgenic Arabidopsis plants caused an increase in leaf cell expansion and consequently an increased size of the leaves, whereas leaf shape was unaffected. Transgenic plants with increased ATHB16 mRNA levels developed leaves that were smaller than wild-type leaves. Therefore, we suggest ATHB16 to act as a negative regulator of leaf cell expansion. Furthermore, the flowering time response to photoperiod was increased in plants with reduced ATHB16 levels but reduced in plants with elevated ATHB16 levels, indicating that ATHB16 has an additional role as a suppressor of the flowering time sensitivity to photoperiod in wild-type Arabidopsis. As deduced from the response of transgenic plants with altered levels of ATHB16 expression in hypocotyl elongation assays, the gene may act to regulate plant development as a mediator of a blue light response.     

Identification of a plastid acyl-acyl carrier protein synthetase in Arabidopsis and its role in the activation and elongation of exogenous fatty acids

Plant cells are known to elongate exogenously provided fatty acid (FA), but the subcellular sites and mechanisms for this process are not currently understood. When Arabidopsis leaves were incubated with 14C-FAs with or=20 carbons) but not synthesis of 14C-unsaturated 18-carbon or 16-carbon FAs. Isolated pea chloroplasts were also able to elongate 14C-FAs (相似文献