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Science 323 (5911): 262-265

Copyright © 2009 by the American Association for the Advancement of Science

tasselseed1 Is a Lipoxygenase Affecting Jasmonic Acid Signaling in Sex Determination of Maize

Iván F. Acosta1*, Hélène Laparra1, Sandra P. Romero1, Eric Schmelz2, Mats Hamberg3, John P. Mottinger4, Maria A. Moreno1, and Stephen L. Dellaporta1{dagger}

1 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06511, USA.
2 Center for Medical, Agricultural and Veterinary Entomology, U.S. Department of Agriculture–Agricultural Research Service, Gainesville, FL 32608, USA.
3 Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-17177 Stockholm, Sweden.
4 Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, USA.

Figure 1 Fig. 1.. (A) Structure of the ts1 gene and the ts1 mutant alleles. Hollow boxes at left and right are 5' and 3' untranslated regions (UTRs), respectively; black boxes are exons and angled lines are introns. Mutations in eight ts1 mutant alleles are positioned above the corresponding exons. Insertions are represented by inverted triangles and a single deletion by a triangle. (B) TS1 protein features include a predicted chloroplast transit peptide (cTP, green), the PLAT/LH2 beta-barrel (pink), and the lipoxygenase domain (blue) as well as five conserved residues (H, His; I, Ile; N, Asn) necessary for iron binding (red) and the phenylalanine (F) residue predicting 13-LOX regiospecificity (black). (C) Bayesian and maximum parsimony consensus tree of predicted type 2 13-lipoxygenases in angiosperms. The red arrowhead indicates the position of the maize ts1-encoded lipoxygenase. Posterior probabilities from Bayesian inference and bootstrap support from maximum parsimony analysis less than 100% are displayed below internal nodes to the left and right of a slash, respectively. This subclade is part of a more extensive phylogenetic analysis shown in fig. S4. [View Larger Version of this Image (25K GIF file)]

Figure 2 Fig. 2.. (A) Expression profile of ts1, ts1b, and ts2 in different maize tissues by quantitative RT-PCR on three biological replicates for each tissue. Results are plotted as the ratio to the lowest detected level (ts1b in root) ± SE. Note that the y axis is in logarithmic scale. (B to E) RNA in situ hybridization targeting the 3'UTR of ts1 (dark purple) in developing inflorescences. Scale bars, 200 µm. [(B) and (C)] Wild-type heterozygote male inflorescences (tassels) of 1.6 and 1.5 cm, respectively. (D) Wild-type female inflorescence (ear) of 1.5 cm. (E) Homozygous ts1-Mu01 deletion mutant tassel showing no hybridization signal. (F to I) Colocalization of TS1:mCherry and RbcSnt:GFP fusion proteins in plastids of transfected onion epidermal cells. Scale bars, 50 µm. (F) TS1:mCherry red fluorescence. (G) RbcSnt:GFP green fluorescence. (H) Merge of Ts1:mCherry and RbcSnt:GFP plus two additional channels: 4',6'-diamidino-2-phenylindole (blue fluorescence) for distinguishing nuclei, and differential interference contrast (DIC) for displaying cellular morphology. (I) Scatterplot of pixel gray value frequencies for RbcSnt:GFP (x axis) and Ts1: mCherry (y axis) channels. Frequencies are displayed using a rainbow lookup table (bottom, units between 0 and 255). Region 3 (upper right) contains pixels with signal above background in both channels, and a linear correlation in this region is a qualitative indicator of colocalization (12). [View Larger Version of this Image (55K GIF file)]

Figure 3 Fig. 3.. (A) Partial gas chromatography–MS chromatograms displaying linoleic acid oxidation products generated by crude extracts of wild-type W22 tassels (blue line) but not ts1-ref tassels (red line). HPLC analysis of oxidation products (inset) indicated that the lipid hydroperoxide (HOD) peak was a mixture of 9-hydroxy-10,12-octadecadienoic acid (9-HOD) and 13-hydroxy-9,11-octadecadienoic acid (13-HOD). (B) Box plots summarizing the distribution of jasmonic acid in three tassel sets. Gray circles represent individual measurements. Blue diamonds show the 95% confidence interval of the mean (horizontal blue line). +/+ corresponds to inbred line W22. (C) Blank-treated mutant ts1 tassel. (D) JA-treated ts1 tassel. (E) JA-treated ts2 tassel. [View Larger Version of this Image (68K GIF file)]

Figure 4 Fig. 4.. Biosynthesis of jasmonic acid through the octadecanoid pathway [adapted from (21)]. The first dedicated step in jasmonate biosynthesis is the peroxidation of {alpha}-linolenic acid (18:3) by 13-lipoxygenase to form (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT). This is the putative function of TS1. 13-HPOT is transformed into the specific stereoisomer cis-(+)-12-oxophytodienoic acid (OPDA) through the sequential action of allene oxide synthase [yielding (13S)-12,13-epoxy-octadecatrienoic acid (12,13-EOT)] and allene oxide cyclase. These steps in JA biosynthesis occur in plant plastids (green box), where the corresponding enzymes are localized. Subsequent reactions occur in the peroxisomes (red box). First, the cyclopentenone ring of OPDA is reduced to 12-oxophytoenoic acid (OPC-8) by OPDA reductase. Next, three β-oxidation cycles are proposed to shorten the carboxylic side chain of OPC-8 to produce the 12-carbon JA (21, 24). A β-oxidation cycle is a set of four enzymatic reactions: oxidation, hydration, oxidation, and thiolysis. Not all enzymes acting on β-oxidation during JA biosynthesis have been identified. Because the oxidation in the third step is normally performed by a dehydrogenase activity, it is possible that TS2 may participate in this step of JA biosynthesis. [View Larger Version of this Image (65K GIF file)]

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