Large-scale reverse genetics in Arabidopsis: case studies from the Chloroplast 2010 Project

Abstract

Traditionally, phenotype-driven forward genetic plant mutant studies have been among the most successful approaches to revealing the roles of genes and their products and elucidating biochemical, developmental, and signaling pathways. A limitation is that it is time consuming, and sometimes technically challenging, to discover the gene responsible for a phenotype by map-based cloning or discovery of the insertion element. Reverse genetics is also an excellent way to associate genes with phenotypes, although an absence of detectable phenotypes often results when screening a small number of mutants with a limited range of phenotypic assays. The Arabidopsis Chloroplast 2010 Project (www.plastid.msu.edu) seeks synergy between forward and reverse genetics by screening thousands of sequence-indexed Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutants for a diverse set of phenotypes. Results from this project are discussed that highlight the strengths and limitations of the approach. We describe the discovery of altered fatty acid desaturation phenotypes associated with mutants of At1g10310, previously described as a pterin aldehyde reductase in folate metabolism. Data are presented to show that growth, fatty acid, and chlorophyll fluorescence defects previously associated with antisense inhibition of synthesis of the family of acyl carrier proteins can be attributed to a single gene insertion in Acyl Carrier Protein4 (At4g25050). A variety of cautionary examples associated with the use of sequence-indexed T-DNA mutants are described, including the need to genotype all lines chosen for analysis (even when they number in the thousands) and the presence of tagged and untagged secondary mutations that can lead to the observed phenotypes.

Figure 1.

Mutation of gene At1g10310 causes decreased seed 18:1Δ⁹ fatty acids. A, Schematic representation of the At1g10310 gene model and two insertion alleles. White rectangles represent exons, black rectangles represent 5′ and 3′ untranslated regions, solid lines represent introns and intergenic regions, and gray triangles represent a T-DNA insertion for SALK_125505C and a Ds element for RIKEN line 15-1699-1. B, At1g10310 steady-state mRNA levels in wild-type Columbia (WT Col), a line derived from a wild-type segregant plant (WT Segregant), SALK_125505C (T-DNA), and RIKEN mutant line 15-1699-1 (Ds). C, Seed fatty acid composition of a line derived from a wild-type segregant plant (WT Segregant), the Columbia ecotype (WT Col), and the two mutant lines, T-DNA and Ds, as determined by GC-FID of FAMEs. Error bars represent sd values for five biological replicates (each replicate consisted of 30 seeds). Differences between the mutant and the wild type of greater than 15% that are statistically significant (Student's t test, P < 0.01) compared with the wild type are marked by asterisks.

Figure 2.

35S:At1g10310 mRNA overexpresser lines have more 18:1Δ⁹ and less 18:3 seed fatty acids than the wild type. A, Eighteen-carbon seed fatty acid composition for the wild type (WT Col) and four transgenic lines (35S:line-1 to -4) transformed with a 35S:At1g10310 construct. Each replicate consisted of 30 seeds, and the error bars represent sd values for three biological replicates. Considerable changes (>15%) relative to the wild type that are statistically significant (Student's t test, P < 0.01) are marked by asterisks. B, Semiquantitative PCR shows that the four lines are high expressers of At1g10310 mRNA. Plants were grown under 16-h-light/8-h-dark conditions. [See online article for color version of this figure.]

Figure 3.

Molecular basis and associated phenotypes for two ACP4 (At4g25050) T-DNA alleles. A, SALK_099519 contains an insertion in intron 1 of At4g25050, and SAIL_104_H07 contains an insertion in exon 2 of At4g25050. Schematic representation of the gene model and alleles is as described in the Figure 1 legend. B, Small size and pale green appearance of SALK_099519 and SAIL_104_H07 compared with a Columbia wild-type plant (WT Col). C, F_v/F_m values were calculated before high light (BHL), after plants were exposed to 3 h of high light (1,500−1,700 μE m⁻² s⁻¹; AHL), and after a 2-d recovery period (Recovery) using the MAXI version of the IMAGING-PAM M-series chlorophyll fluorescence system (Heinz-Walz Instruments). Wild-type control plants were used to assign cutoff values for F_v/F_m under each condition. Red coloring in the false-color images indicates tissues that were below the wild-type cutoff value for F_v/F_m. D, Altered leaf fatty acid composition of SALK_099519 and SAIL_104_H07 compared with wild-type plants. Error bars represent sd values for two biological replicates.

Figure 4.

Inconsistent phenotypes of three At1g64400 T-DNA insertion lines. A, Insertion sites and photographs of representative plants for each mutant line. Schematic representation of the gene model and alleles is as described in the Figure 1 legend. B, 16:1Δ⁷ and 16:2 FAME levels of wild-type (WT Col), SALK_027707C, SALK_084299, and SAIL_868_G02 lines as measured by GC-FID. Statistically significant differences between each mutant and the wild type (Student's t test, P < 0.01) are depicted by asterisks. Error bars represent sd values of four biological replicates for SALK_027707C and SAIL_868_G02 and three biological replicates for the wild type and SALK_084299.

Figure 5.

Summary genotyping information for T-DNA lines genotyped by the Chloroplast 2010 pipeline. A, A total of 3,673 independent T-DNA lines annotated as homozygous for specific alleles were genotyped; 74% were homozygous (Homo), 12% were wild type (WT), and 14% had individuals with a combination of any of the three genotypes but were not all homozygous (Seg) for the proposed insertion. These could include seed stocks contaminated with wild-type seed or with seeds of other mutants. B, Out of the 2,733 homozygous SALK lines, 70% of loci have a single allele (One), 26% of genes have two independent homozygous mutants (Two), and 4% of genes have more than two homozygous mutants (>2).

Figure 6.

Molecular basis of the altered FAME phenotype of mutant SALK_006881C. A, Locations and details of two mutations identified in SALK_006881C on chromosomes (Chr) II and III (centromeres are depicted by black ovals; gene models and mutation sites are schematically represented as described in the Fig. 1 legend). Based on gene model At3g15850.1, the 8-bp deletion (8 bp del; indicated with an arrow) occurs 1,600 bp downstream of the FAD5 translation start site. B, GC-FID chromatogram traces of total leaf FAME extracts of wild-type (WT Col), SALK_006881C, and fad5-1 plants (Heilmann et al., 2004a). Peaks for 16:1Δ⁷, 16:2, and 16:3 are indicated by arrows. Based upon these results, the FAD5 allele found in SALK_006881C is now designated fad5-2. The insertional mutation in At2g37290 has been renamed rgc1-1.

Figure 7.

Molecular basis of the reduced starch phenotype of mutant SALK_133788C. A, Two T-DNA insertions were detected in the starch-deficient mutant SALK_133788C: one insertion lies in the single exon of At5g65300 and the other in the last exon of ADG1. Starch-proficient SALK_069313C carries an insertion in At5g65300, with no T-DNA in ADG1. Schematic representation of chromosomes (Chr), gene models, and mutations is as described in the Figure 1 and 6 legends. B, The top row shows whole plant images for the wild type (WT Col), SALK_133788C, and SALK_069313C. The bottom row shows iodine staining of leaf discs (from the same plants as in the top row) harvested 8 h into the 12-h light period.

Figure 8.

Molecular basis of the high free Thr and Cys in mutant SALK_134724C. A, Mutations identified in SALK_134724C include a T-DNA insertion in the promoter region of IAA33 and deletion (illustrated with a dash) of a T residue 1,209 nucleotides downstream from the translational start site of THA1 (based on gene model At1g08630.1). Exonic nucleotides are in uppercase, while intronic nucleotides are in lowercase. Schematic representation of chromosomes (Chr), gene models, and mutations is as for Figure 6. B, Liquid chromatography-mass spectrometry analysis of Thr and Cys levels (mol %) in wild-type (WT Col), SALK_134724C, and tha1-1 seeds. Error bars represent se values of four replicates. Based upon these results, the At1g08630 allele found in SALK_134724C is now designated tha1-3 and the insertional mutation in IAA33 is referred to as iaa33-2.