Article Figures & Data
Figures
- Figure 1.
Scheme of Met-derived glucosinolate biosynthesis in Arabidopsis. This process can be divided into the Met chain elongation cycle (I) and the biosynthesis of the core glucosinolate structure (II). The parent methylthioalkyl glucosinolates can undergo further side chain modifications.
- Figure 2.
Immunocytochemical localization of MAM3 protein in Arabidopsis leaves. Cross section of leaves were probed with anti-MAM3 antibody (A and C) or with preimmune serum (E) followed by a fluorescence-labeled secondary antibody. A strong green fluorescence label within chloroplasts in A and C is indicative of the MAM3 protein. Chloroplasts are defined by positive 4,6-diamidino-2-phenylindole staining (B) of the same section as shown in A and by starch granules visualized in D by the differential interference contrast image of C. Negative control performed by treatment with preimmune serum did not exhibit any label (E). Bars = 20 μm in A, B, E, and 5 μm in C and D.
- Figure 3.
Glucosinolate profile of leaves (A) and seeds (B) of MAM3 mutants compared to Col-0 wild type. Glucosinolates were purified as desulfoglucosinolates, fractionated by reverse-phase HPLC, and individually identified and quantified. Individual Met-derived glucosinolates are grouped according to their chain length (no. of methylene carbons in the R group, C2–C8), while indole glucosinolates are depicted separately. Data show the means and ses of three replicate samples.
- Figure 4.
Glucosinolate profile of leaves (A) and seeds (B) of the MAM1 insertion mutant gsm1-3 compared to Col-0 wild type. Aliphatic glucosinolates sum up to 22.4/87.2 μmol g−1 dry weight (leaf/seed) in wild type and 27.3/96.9 μmol g−1 dry weight in the mutant. Glucosinolates were isolated, identified, and quantified as explained in the Figure 3 legend.
- Figure 5.
Steady-state mRNA transcript levels of MAM1 and MAM3 in tissues of Col-0 wild-type and MAM3 mutant lines determined by RT-PCR. DNA fragments for individual transcripts of ACT8 (actin), MAM1, and MAM3 were generated by PCR amplification of products from RT activity primed by oligo(dT) hybridization to the RNA. The PCR products are shown after separation on 1% agarose gels stained with ethidium bromide. Tissue source: 1, roots; 2, mature leaves; 3, expanding leaves; 4, flowers; and 5, siliques.
Tables
- Table I.
Nomenclature for Arabidopsis (Col-0) genes of the IPMS/MAM synthase gene family used in recent publications
Arabidopsis Genome Initiative Locus Code N-Terminal Sequence Bacterial Artificial Chromosomes Kroymann et al. (2001) Junk and Mourad (2002) Field et al. (2004) This Article At1g18500 MASSLLR F15H18 – EST116C2T7 MAML-4 IPMS1 At1g74040 MESSILK F2P9.9 – IMS1 MAML-3 IPMS2 At5g23010 MASSLLT CT20O7 MAM1 IMS3 MAM1/L MAM1 –a MASSLLTb – – – – MAM2 At5g23020 MASLLLT MYJ24 MAM-L IMS2 MAM1/L MAM3 - Table II.
Suitability of 2-oxo acids as substrates for MAM3-mediated condensation with acetyl-CoA
2-Oxo Acid Substrate Structure Resulting Glucosinolate Suitabilitya 4-Methylthio-2-oxobutanoate 
C3 + 2-Oxo-hexanoate C3 + 5-Methylthio-2-oxopentanoate C4 + 2-Oxo-heptanoate C4 + 6-Methylthio-2-oxohexanoate C5 + 2-Oxo-octanoate C5 + 2-Oxo-nonanoate C6 + 8-Methylthio-2-oxooctanoate C7 + 2-Oxo-decanoate C7 + 9-Methylthio-2-oxononanoate C8 + 2-Oxo-undecanoate C8 + 2-Oxo-dodecanoate C9 − ↵a +, Condensation product formed; −, condensation product not formed.
Substrate Km Vmax kcat kcat/Km μm nmol min−1mg−1 s−1 m−1s−1 4-Methylthio-2-oxobutanoic acid (→C3 glucosinolates) 932 ± 56 1,448 ± 299 1.3 ± 0.3 1,380 5-Methylthio-2-oxopentanoic acid (→C4 glucosinolates) 476 ± 199 1,495 ± 679 1.3 ± 0.6 2,730 6-Methylthio-2-oxohexanoic acid (→C5 glucosinolates) 463 ± 210 2,869 ± 768 2.5 ± 0.7 5,400 8-Methylthio-2-oxooctanoic acid (→C7 glucosinolates) 253 ± 123 364 ± 143 0.3 ± 0.1 1,280 9-Methylthio-2-oxononanoic acid (→C8 glucosinolates) 81 ± 21 31 ± 8 0.03 ± 0.01 370 2-Oxoisovalerate 1,000 ± 200 199 ± 38 0.18 ± 0.03 200 Pyruvate 8,600 ± 4,300 191 ± 48 0.17 ± 0.04 23 Acetyl-CoA 2,300 ± 1,200 3,344 ± 1428 3.0 ± 1.3 1,300 ↵a Data presented are means ± sds of at least five replicates per substrate.
- Table IV.
Glucosinolate content (μmol g−1 dry weight) in leaves of gsm2-1 and lines transformed with 35S∷MAM3a
Line C3 C4 C5 C6 C7 C8 ΣCi Col-0 wild type 2.7 16.3 0.5 0.3 0.4 2.2 22.4 gsm2-1 2.5 18.2 0.6 0.2 n.d. n.d. 21.5 T2 Line 1 2.3 6.5 n.d. 1.0 3.2 8.6 21.6 T2 Line 2 3.9 8.0 n.d. 1.6 6.2 15.9 35.5 T2 Line 3 4.1 6.4 n.d. 1.1 3.9 10.1 25.7 ↵a Presented are analyses of Col-0 wild type, the gsm2-1 parent line, and three lines randomly selected from a T2 segregating population that had detectable levels of the introduced MAM3 transcript. n.d., Not detected.
Supplemental Data
Supplemental Figures and Table
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