Article Figures & Data
Figures
- Figure 1.
jai1-1 Plants Are Insensitive to MeJA.
(A) Morphology of 4-week-old Micro-Tom (wild type [WT]; left) and jai1-1 (right) plants.
(B) Rapid assay for MeJA-induced PPO activity in tomato leaves. Leaf juice from individual leaflets of 15-day-old plants was expressed on a nitrocellulose membrane and assayed for PPO activity as described by Howe and Ryan (1999). Wild-type plants were treated with MeJA (+) or ethanol (−) as a control. F2 plants derived from a cross between jai1-1 and the wild type were treated with MeJA before testing for PPO activity. Dark brown staining indicates the presence of PPO activity.
(C) Response of germinating seedlings to MeJA. Wild-type, heterozygous (J/j), and homozygous (j/j) seeds were germinated on moist filter paper and exposed to MeJA (+) for 2 days. A wild-type seedling grown in the absence of MeJA (−) is shown as a control.
- Figure 2.
Reproductive Phenotypes of jai1-1 Plants.
(A) and (B) Developmental progression of wild-type (A) and jai1-1 (B) flowers. Arrows in (B) show the stigma protruding from the anther cone.
(C) and (D) Developing fruit on wild-type (C) and jai1-1 (D) plants.
(E) Mature wild-type (top) and jai1-1 (bottom) fruit.
(F) Enlargement of jai1-1 fruit in (E), showing small undeveloped seeds.
(G) and (H) Fluorescein diacetate/propidium iodide costaining showing viable (green) and nonviable (red) pollen from wild-type (G) and jai1-1 (H) anthers.
(I) and (J) Light microscopic images (×100 magnification) of germinated wild-type (I) and jai1-1 (J) pollen.
- Figure 3.
jai1-1 Plants Exhibit Defects in Trichome Development.
(A) to (C) Photographs of developing green fruit from wild-type (A), jai1-1 (B), and 35S-LeCoi1–complemented jai1-1 (C) plants.
(D) and (E) Scanning electron micrographs of the surface of developing green fruit from wild-type (D) and jai1-1 (E) plants. Arrows in (D) denote type-VI (t-VI) and type-I (t-I) trichomes. The tissue fixation procedure used for scanning electron microscopy affected the structure of type-I trichomes, which were 1 to 2.5 mm long on intact tissue (see [A] and [D]).
(F) and (G) Scanning electron micrographs of wild-type (F) and jai1-1 (G) sepals.
(H) and (I) Scanning electron micrographs of the adaxial (upper) side of young leaves from wild-type (H) and jai1-1 (I) plants.
Bars in (D) to (I) = 0.2 mm.
- Figure 4.
Monoterpene Content in Trichome Exudates.
Trichome exudates obtained by brief extraction of tissues with MTBE were analyzed for monoterpene content by gas chromatography–mass spectrometry.
(A) Wild-type (WT) green fruit.
(B) jai1-1 green fruit.
(C) Wild-type sepal.
(D) jai1-1 sepal.
(E) Before extraction with MTBE, wild-type fruit was wiped with a cotton swab to remove trichome contents.
(F) Exudate collected directly from type-VI trichome glands of wild-type green fruit. The y axis indicates arbitrary absorbance units (AU).
- Figure 5.
MeJA-Induced Gene Expression in Wild-Type and jai1-1 Plants.
Three-week-old Micro-Tom and jai1-1 plants were exposed to MeJA vapor for various lengths of time (hours) in an enclosed box. Leaves from similarly treated plants of the same genotype were pooled for RNA extraction. RNA isolated from untreated plants (0-h time point) also was analyzed as a control. RNA gel blots were hybridized to cDNA probes representing four late genes (PI-I, PI-II, CATHEPSIN D INHIBITOR [CDI], and THREONINE DEAMINASE [TD]) and four early genes (LIPOXYGENASE D [LoxD], ALLENE OXIDE SYNTHASE2 [AOS2], OPDA REDUCTASE3 [OPR3], and PROSYSTEMIN [PSYS]). Blots also were hybridized to the LeCoi1 cDNA and, as a loading control, to an eIF4A probe.
- Figure 6.
Resistance of jai1-1 Plants to the Two-Spotted Spider Mite Is Severely Compromised.
(A) to (D) Fifteen adult female mites were transferred to a single leaf on 3-week-old wild-type ([A] and [C]) and jai1-1 ([B] and [D]) plants. Photographs of the infested leaves were taken 6 days ([A] and [B]) and 11 days ([C] and [D]) after challenge. The boxed area in (B) shows the initial effects of feeding damage on jai1-1.
(E) and (F) Photographs of representative 7-week-old wild-type (E) and jai1-1 (F) plants at 30 days after infestation.
(G) Two-choice assay to measure the preference of spider mites for wild-type (WT) or jai1-1 plants. Ten adult female spider mites were placed in an arena equidistant from wild-type and jai1-1 leaflets. The number of mites that moved to one or the other of the leaflets was determined 1 h after initiating the assay, as was the number of mites that failed to make a choice (nc). Data represent means and standard errors from 16 repetitions (total of 160 mites).
(H) Fecundity of two-spotted spider mites on wild-type (black bar), jai1-1 (red bar), and 35S-LeCoi1–complemented jai1-1 (green bar) plants. Data represent means and standard deviations from 12 experimental repetitions in which five teneral female mites were reared for 4 days on leaf discs of the indicated host genotype.
- Figure 7.
Deduced Amino Acid Sequence of LeCOI1 Compared with Homologs in Arabidopsis and Rice.
Sequence alignments were performed using the GCG sequence analysis package (Genetics Computer Group, Madison, WI). Amino acids that are either identical or similar between the three sequences are indicated in black and gray, respectively. Arrows denote the approximate positions of the 16 imperfect LRR domains. The arrow and LRR domain number are indicated beneath the corresponding LRR. The asterisk denotes the Gly residue that is changed to a Cys in jai1-2 plants. GenBank accession numbers are given at the end of Methods. The N-terminal end of the rice sequence contains 35 additional amino acids that are not included in the alignment.
- Figure 8.
The jai1-1 Mutation Is a 6.2-kb Deletion in LeCoi1.
(A) Scheme of a 9.4-kb region of genomic DNA containing LeCoi1 and a gene encoding a putative MYB transcription factor (pMYB). The three exons (E1 to E3) and intervening sequences that constitute LeCoi1 are drawn to scale. Only the translated portion of the first and last exons are shown, together with the start (ATG) and stop (TAG) codons within the respective exons. The 6.2-kb region of DNA that is deleted in jai1-1 plants is shown by the horizontal hatched line. B indicates BglII restriction sites used for the DNA gel blot analysis shown in (C), and B* indicates the presence of two BglII sites that are separated by 24 bp. cDNA probes used to detect the 5′ and 3′ ends of the gene in (C) are shown as thick horizontal bars.
(B) Structures of the Arabidopsis (AtCOI1) and rice (OsCOI1) homologous genes drawn to the same scale as LeCoi1 in (A).
(C) DNA gel blot analysis of BglII-restricted genomic DNA from the wild type (lanes 1), jai1-1 (lanes 2), and an F1 hybrid produced from a cross between the wild type and jai1-1 (lanes 3). Three identical blots were hybridized to a full-length (FL) LeCoi1 cDNA probe or to probes corresponding to the 5′ and 3′ ends of the cDNA (see [A]). Numbers at left indicate the positions of DNA size standards (in kb).
(D) RNA gel blot analysis of LeCoi1 transcript levels in wild-type (WT) and jai1-1 plants. Five micrograms of total RNA from root (R), petiole (P), leaf (L), unopened flower bud (F), sepal (S), and immature green fruit (G) was immobilized to a membrane and hybridized to a LeCoi1 cDNA probe (top gel). A duplicate blot was hybridized to an eIF4A cDNA probe as a loading control.
- Figure 9.
MeJA-Induced Gene Expression in 35S-Coi1–Complemented Transgenic Lines.
Total RNA was prepared from wild-type (WT), jai1-1 (jai1), and T2 siblings from two 35S-Coi1–complemented lines (T2-08 and T2-13) either before (−) or after (+) exposure to MeJA vapor for 12 h. For each transgenic line tested, a PCR assay was used to identify siblings that either harbor (+) or lack (−) the 35S-LeCoi1 transgene. RNA gel blots were hybridized to cDNA probes for PI-II and LeCoi1 and to eIF4A as a loading control.
Tables
Expression Ratio Accession No. Gene Product (Best BLAST Hit) Wild Type jai1 AI485116 Thr deaminase 41.9 – a Q10712 Leu aminopeptidase A (LAP-A) 33.0 1.3 AI485529 Putative acyltransferase 25.9 0.7 AI486173 Protein translation inhibitor 19.8 – a AI897750 Kunitz-type trypsin inhibitor 14.8 0.8 AW037833 Metallocarboxypeptidase inhibitor 12.7 – a AI487422 Pto-responsive gene 1 protein 12.5 1.0 AI488657 Cathepsin D inhibitor protein (CDI) 12.4 0.7 K03291 Proteinase inhibitor II (PI-II) 11.2 – a AW649914 Leu aminopeptidase N (LAP-N) 11.0 1.3 AW624058 Allene oxide cylase (AOC) 10.8 1.4 U09026 Lipoxygenase A (LOXA) 9.2 0.8 Z12838 Polyphenol oxidase F (PPO-F) 8.4 1.3 K03290 Proteinase inhibitor (PI-I) 8.2 1.0 AW092579 Nucleoside diphosphate kinase 5.7 1.0 AI897184 Glyoxylase family protein 5.5 0.9 AI490318 NAC domain protein 5.5 0.8 AI486025 4-Coumarate:CoA ligase 5.2 1.1 AW040669 Thioredoxin M-type 3 chloroplast precursor 5.1 0.9 AI486916 Kunitz-type enzyme inhibitor 4.5 1.1 AI486546 Wound-inducible carboxypeptidase (WIC) 4.5 1.1 AF198389 Cystatin 4.4 1.1 AI489221 Wound-inducible WRKY transcription factor 4.3 1.0 AI483527 Kunitz-type trypsin inhibitor 4.2 0.7 Z21793 DAHP-synthase 2 4.2 1.2 AW034958 12-Oxo-phytodienoate reductase3 (OPR3) 4.2 1.1 AI771886 RD2 auxin-regulated protein 4.0 1.1 AW032472 Unknown protein, similar to PnFL-2 4.0 0.9 AW220064 Glutathione S-transferase 3.9 1.2 AF230371 Allene oxide synthase2 (AOS2) 3.8 1.0 AW648326 Adenosylmethionine decarboxylase 3.5 1.1 U37840 Lipoxygenase D (LOXD) 3.4 1.0 AI895589 Allene oxide synthase1 (AOS1) 3.0 0.9 M84800 Prosystemin 2.9 1.2 AI897620 Putative chorismate mutase 2.9 1.2 AI483536 TMV response-related gene product 2.7 0.9 BE459901 Putative caffeoyl-CoA O-methyltransferase 2.6 0.7 ↵a Expression was not detectable, indicating that the microarray signal was below background levels.
Three-week-old Castlemart (wild-type) and jai1-1 plants were treated with either MeJA or ethanol (mock control) for 8 h. Leaf tissue was harvested for RNA isolation after the treatment. A custom cDNA microarray slide representing ∼500 tomato genes was hybridized simultaneously to probes derived from RNA isolated from ethanol- and MeJA-treated plants of the same genotype (i.e., wild type or jai1-1). Numbers represent the mean expression ratio (MeJA:ethanol) of two independent biological replicates for each experiment. Genes (GenBank accession number and putative function) that were differentially regulated by >2.5-fold in response to MeJA in wild-type plants are listed.
Supplemental Data
The following material is provided as Supplemental Data, and is also available online at http://www.prl.msu.edu/howe/miame2/index.html.
Files in this Data Supplement:
- Supplemental Table 1 - A complete list of cDNA microarray clones with average expression ratios (+/-SE) for all four hybridization experiments.
- Supplemental Table 2 - List of all genes that were differentially regulated >2.5-fold in wild-type and jai1-1 plants in response to MeJA.
