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Agrobacterium tumefaciens increases cytokinin production in plastids by modifying the biosynthetic pathway in the host plant Hitoshi Sakakibara*, Hiroyuki Kasaharas, Nanae Ueda*, Mikiko Kojima*, Kentaro Takei, Shojiro Hishiyama Tadao Asami, Kazunori Okada*, Yuji Kamiya, Tomoyuki Yamaya, and Shinjiro Yamaguchi Laboratories for-communication Mechanisms and Cellular Growth and Developmert, p ant Sc ence Center, RKEN, Sale。1-7ง2, Turnu Yokohama 2300045, Japan Forestry and Forest Products Research Institute, baraki 305-8687, Japan Pant Functions Laboratony RİKEN, Birosawa 2. 1, wako 351-0190, Japan; and ..Botechnology Research Center, University of Tokyo, 1-5-1 Yayo, ayoku, Tokyo 113-4657, pan Edited by ake MadMilan, University of Bristol, Bristol, United Kingdom, and approved May 24, 2005recelved for eviw January 31, 200 Agrobacterium tumefaciens infects plants and induces the forma2-methyl-2-(E)-butemyl 4-diphosphate (HMBDP) (10), which tion of tumors called crown galls by integrating the transferned has recently been shown to occur as an intermediate in the MEP DNA (T-DNA) region of the Ti-plasmid into the plant nucear pathway (11)(Fig. 1). However, the participation of HMBDP in genome. Tumors are formed because the T-DNA encodes enzymes CK biosynthesis has never been demonstrated in planta. It should that modify the synthesis of two plant growth hormones, auxin also be noted that a fraction of tZtype CKs would be produced and cytokinin (CK). Here, we show that a CK biosynthesis enzyme in an iPRMP-independent manner through the isomerization of Tmr, which is encoded by the Agrobacterium T-DNA region, is cis-zeatin (cZ)-type CKs, which are thought to be formed by the targeted to and functions in plastids of infected plant cells, despite degradation of tRNA containing cZ-aype premylation(1), but having no typical plastid-targeting sequence. Evidence is provided the extcnt to which this occurs in Anabilopis is unknown that Tme is an adenosine that creates a new CK biosynthesis bypass by using 1-hydroxy-2- methyl 2-E)-butenyl 4-diphosphate 0HMBDP) as a substrate. Un like in the conventional CK biosynthesis pathway in plants, trans zeatin-type CKs are produced directly without the requirement for P450 the plastid localization of Tmr, HMBDP is an intermediate in the ОРТ) Tmr is an IPT that is encoded on the transfersed-DNA (T-DNA) segion of the Ti plasmid ol Agrobucteri amefuciens Therefore, this bacterial enzyme functions in plant cels upon indicated that engineered expression of Tmr in plants elevated the synthesis of Z-type CKs without a substantial increase in the production of iP derivatives (9, 13, 14). These observations imply that Tmr may be involved in the PRMP-independent synthesis of tz by using HMBDP in in- infection. Previous reports hydroxylation. Consistent with These results thesis route for universal isoprenoid precursors. demonstrate that A tumefaciens modifies CK biosynthesis by sending a key enryme into plastids of the host plant to promote fected plant cells In this model however, it is unclear how Tm recruits HMBDP as a substrate from the plastid-specific MEP pathway, because this bacterial protcin does not have an appar- supporting informatioe on the PNAS web site) To address how Tmr modaics CK osymthesis in the host, we investigated the substrate preference and subcellular localization cellsEvidence is provided that Tmr is targeted to the plastid of infected plant cells and uses HMBDP in the MEP pathway as a major substrate. The plastid localization of Tmr explains how this bacterial enryme utilizes the substrate in the plastid-localized isoprenoid pathway, but is surprising be- cause it is a bacterial protein and does not have amy apparent inins (CKs) are a group of plant hormones essential torof Tmr in plant cells. Evidence is Il division and differentiation in plants (1). Most natural CKs, including isopentenyladenine (iP) and trans-zeatin (tZ) (Fig. I), are derivatives of N-prenylated adenine (1). The premyl side chain of CKs can be derived from the methylerythritol phosphate (MEP) pathway or the mevalonate (MVA) pathway, both of which supply common Cs units for isoprenoid biosyn- thesis (2, 3). The MEP pathway widely occurs in the hacterial kingdom and the plastids of plants, whereas the MVA pathway Materials and Methods is commonly found in the cytosol of eukaryotes (2. 3). Thus plants have the two different isoprenoid pathways in separate subcellular compartments. Recent work has shown that the majority of the prenyl side chain of iP and tZ is derived from the MEP pathway in Arabidopsis scedlings (4). Plant MaterialsAaidopsis shaliane ecotype Columbia was used în this study. A ส0wn gall cell line of periwinkle V208 (Catha- nanthus rs G. Doa) (15) was cultured in hormone-free MS) iquid medium (16) supplemented with 3% sucrose. A wid-type cell lite of periwinkle CRA was cultured in MS liquid medium supplemented with 3% sucrose (IPT, EC25.1.27) (Fig. 1S), At least and 03 pg/ml 24-dichlorophenosyacetic acid. Both cell lines To initiate CK biosynthesis, an isoprenoid precursor is trans- ferred to AMP. ADPor ATP by adenosine phosphate- Itransferase two routes have been proposed for the formation of 1Z, an active CK. in plants. In the conventional iP riboside 5-monophosphate iPRMP)-dependent pathway, dimethylallyl DMAPP) is used as the side chain precursor for iPRMP,which is then converted to 12 riboside 5 (8) (CYP73SAI idopsis; Fig 1). The other proposed pathway is an iPRMP ndepondent bypass, in which an unidentified hydroxylatednk derivative of DMAPP is directly transferred to the adenine To…emnmdeN_brawe ed.6.akuia enteramosp moiety (9). A candidate for this putative substrate is 1-hydroxy by The National Acadeny ofScoff tU 棘72-9977 I PNAS I July 12, S vol. 102 l n028
GAP DXS Fig. 1. 鈊hematK representation of isotope labeling of СК biosynthess through the Mtp pathway Red arrows ndate th pos ble metabolism of IpCpx klassical pathway) Blue arrows ป ow possible conveniom e#13,ANOJDX through HMBOP multiple metabolic steps. S-Ketoclomazone 0C) was included in all incubations to inhibit the endogemous ILP pathway PyR pyruvate GAP,glyceraldehyde 3. phosphate: P, menephospholic PP, dighespho4c acid CYP735A1 and CYP735A2 are cytochrome P450 monooygerwes in Arabo psis pathway) into Cks Dashed arrows denote were provided by the Experimental Plant Division, RIKEN were introduced into Arabidopsis by the floral dip method (25) Bioresource Center (Tsukuba, Japan). progenies were used for all Chemicals. HMBDP was synthesized by Wako Pure Chemical in Vive Labeling Experiments. Seedlings of transgenic Anaidopsis for 3 days on Murashipe-Skoog (MS)-agar media and then transferred to MS liquid media (15 ml) con- taining KC (I M; added as a 15-l methanol solution) and Synthesis of 13.A-w0JDX. [ 3,4-101DX (95% atom-labeled) was isotope-labeled DX (08 mM). Ten days later, soedings were synthesized as reported ( 17) by using H-О (95% atom-labeled) treated with 30 μΜ dexamethasone for 24 h, then harvested Sor for Sharpless asymmetric dehydroxylation of 1 benzyloxy-2따 CK aaalysis. For i-vivo labeling, V208 and CRA cells were DX (08 mM) in its IH and C NMR spectral data (18). To determine the a 100-ml flask for 1 week. Wild-type strains of A. fmfaciens O-incorporation into 13,4-O IDX, authentic DX and 34 MAFF302307 and MAFF302376 (native to Japan) were ob OİDX were analyzed by gas chromatography-mass spectrum. tained from Ministry of Agriculture, Forestry and Faberes ctry after comversion to trimethysilyl (TMS) derivatives as GenBank at the National Institute of Agrobiological Scicnoes reported (19) 3,4.1OİDX-TMS derivative was as follows mass (Tsukula. Japan). Colonics of wild-type Agndadenum were spectrum (EI, 70 cV) m/z: 31 1 <M-43., 20%), 220 (100), 207 inoculated onto wounded stem tissues of tomato (Lycuperaron (50). I 47( 70). 1 19 (40). DX-TMS derivative was as follows: mass es alena鼎Ailsa Craig) plants. When crown galls formed (4-6 spectrum (EI. 70 cv) m/z: 307 (M-43-20%). 218 (100). 205 weeks after incubation), they were injected with a mature of 3 aal of KC ( 1 μΜ) and 30 μ1 of Botope-labeled DX (0.8 mM ) by using a syringe. Galls were harvested 3 days after the injection synthesis ofS-Ketocamazone (KC), КС (20) was synthesized from for CK analysis. Extraction, purification, and analysis of CKs 2-chlorophenylmethylhydroxylamine (21). To a solution of were performed as described (4). The andO incorporation (788 mg, 5 mmol) and tri levels were calculated by using the molecular ion clasier ethylamine (1.01 g 10 tamol)an CH2C12 (10 ml) was added qM-N and +1 to +4 is top mers) after subtraction of (Osaka). 11-ieji-deoxy-D.aylulose (DX) (99% prepared as reported (4) labeled) was plants were penten-4-one. The structure of [34 DX was confirmed by cultured with KC (1 M) and dimethylmaronyl dichloride (845 mg, 5mmol) in CH Cl: (2.5ml) natural C abundance (26). at GC. After the solution was stirred for 30 min, IN-HCl was added to the reaction mixture, and then repeatedly extracted C Analysis Extraction and determination of CKs from trans- with ethyl acetate and then with CH Cl. The combined organic genic Anabidepsis lines overexpressing IPTs were performed as layers were washed with NaHCO, solution, dried with NaSO· and concentrated. The product was purified by silica gel column described (27). chromatography to give KC (924 mg yield 85%, colorless oil). Particle Bombard ent. Full-length or partial coding tep as of The H NMR data of KC was identical with those of authenticTmr were fused to the amino terminus of the GFP gene, which was controlled by the caulif lower mosaic virus 35S promoter (35SsGFPIS65TD (28). As the control markers for plastids and Recombinant Enryme. The coding region of Tmr from pTi- mitochondria, signal peptides of Arahidopis geray lpcranyl SAKURA (23) was ligated inte pOE60 (Oiagen, Valencia, CA) diphosphate synthase 1 and 6 (GGPSI and GGPSh, respectivcly to express the His-tagged recombinant proteins. Detailed pro (29) were fused to the amino terminus of GFP gene driven by the cedures for preparation of the recombinant proteins have been 35S promoter. The DNA constructs were introduced into noots of 2-week-old Arabidopsis seedlings by particle bombardment PDU-1000/He, Bio-Rad). Transient expression was observed Transgenic Arabidepsis-Expressing IPT. The coding region of Ter by laser confocal-scanning fluorescence microscopy after over- and AtiPTs was ligated into the pTA700I vector (24) to yield construct containing the dexamethasome-inducible IPT gene a night incubation (Fluoview IXS, Olympus, Melville, NY pTA-Tms, pTA-AIPTI, pTA-AtIPT2, pTA-AIPTS pTA- Western Analysis. Ahout 400 mg of scedlings were homogenined AtIPT4, PTA-AtIPTS, or PTA-APT7) The chimeric genes in two volumes of ice-cold extraction buffer (s0 mM TrisHCL PNAS uly 12, 2005 ol 902I no 289973
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