Article Figures & Data
Figures
- Figure 1.
Characterization of a T-DNA insertion allele of Arabidopsis HCS1 (SAIL_1277_E03; line hcs1-1). A, Structure of the HCS1 gene carrying the inverted tandem insert of T-DNA. Black boxes and lines indicate exons and introns, respectively. The size of T-DNA is not drawn to scale. The locations of primer sequences used for PCR genotyping are marked with arrows. B, Characterization of the T-DNA insertion locus by PCR. Genomic DNA from a wild-type (Wt) and a heterozygous HCS1/hcs1-1 mutant (Het) plant was amplified using the primer combinations indicated. Three PCR reactions per plant were performed. HCS1.for and Hcs1.rev amplify a 0.46-kb product from the wild-type allele, LB3 and Hcs1.rev amplify a 0.29-kb product from the disrupted allele, and Hcs1.for and LB3 amplify a 0.41-kb product from the disrupted allele. Left and right flanking lanes (M) show DNA size markers. C, Light microscopy analysis of a wild-type immature silique showing uniform seed development and heterozygous HCS1/hcs1-1 immature siliques containing aborted, white, and shrunken ovules (asterisks) and aborted, white or brown, and shriveled seeds (arrowheads). Scale bars = 500 μm. D, Seed production from wild-type (HCS1/HCS1) and heterozygous (HCS1/hcs1-1) Arabidopsis plants. Three types of seed/ovules were identified in the siliques and counted: normal seeds containing full-size embryos, abnormal seeds containing aborted embryos, and aborted white ovules.
- Figure 2.
Identification and characterization of a T-DNA insertion allele of Arabidopsis HCS2. A, Structure of the HCS2 gene carrying the T-DNA insertion. Black boxes and lines indicate exons and introns, respectively. The size of T-DNA is not drawn to scale. The locations of primer sequences used for molecular analyses (screening of the T-DNA insertion lines collection, PCR genotyping, and mRNA quantification) are marked with arrows. B, Characterization of the T-DNA insertion locus by PCR. Genomic DNA from a wild-type (Wt), a heterozygous HCS2/hcs2 mutant (Het) plant, and a homozygous hcs2/hcs2 mutant (Hom) plant was amplified using primers Exon 7, Hcs2.stop, and Tag5. Exon 7 and Hcs2.stop amplify a 0.84-kb product from the wild-type allele, and Tag5 and Hcs2.stop amplify a 0.32-kb product from the disrupted allele. C, Real-time RT-PCR quantification of HCS1 and HCS2 gene expression in the wild-type (Wt), heterozygous (Het), and homozygous (Hom) mutant plants. The relative amount of HCS1 and HCS2 mRNAs in aerial parts of Arabidopsis plants was determined using specific primers (Table I) as described in “Materials and Methods.” Data (relative transcripts abundance normalized to the expression levels in wild-type plants) are means of three independent experiments performed with three cDNA dilutions ± sd. ACTIN1 was used as an internal control to normalize for variation in the amount of cDNA template. D, HCS activity measurements in protein extracts from wild-type (Wt) and homozygous (Hom) mutant plants using recombinant ACC138 (ACC), MCC220 (MCC), BCCP1, and BCCP2 as the apocarboxylase substrates. Data are means ± sd of four independent determinations. E, ACCase and MCCase activity measurements in protein extracts from wild-type (Wt) and homozygous (Hom) mutant plants. Data are means ± sd of three independent determinations. F, Western-blot analysis of biotinylated proteins in protein extracts from wild-type (Wt) and homozygous (Hom) mutant plant leaves using streptavidin coupled to peroxidase for detection. Each fraction contained 50 μg proteins. ACC1, MCCα, BCCP1, and BCCP2 proteins are indicated to the left. Position of molecular mass markers is given on the right.
- Figure 3.
The 5′ upstream region of Arabidopsis HCS1.un and HCS1.s cDNA species. The 5′ sequence of the HCS1 gene (At2g25710) is reported. Sequences of cDNAs obtained by RACE or RT-PCR are shaded. Initiation codons are in uppercase letters and in bold. Arrows indicate the experimentally obtained transcription start site (Denis et al., 2002). A, Nucleotide sequence of the 5′-UTR and of the first four exons of the HCS1 cDNA spliced isoform. B, Nucleotide sequence of the 5′-UTR and of the first four exons of the HCS1 cDNA unspliced isoform. The uORF is boxed.
- Figure 4.
The nature of the 5′-UTR of HCS1 mRNA influences AUG choice in vitro and controls the targeting of HCS1 protein in vivo. A, In vitro transcription-translation experiments. Polypeptides radioactively labeled with [35S]Met were subjected to SDS-PAGE and analyzed by phosphorimaging as described in “Materials and Methods.” Fluorography of translation products obtained with 1 μg of pPCRscript-HCS1.s (lane 1), 1 μg of pPCRscript-HCS1.un (lane 2), and 1 μg of pPCRscript-HCS1.ATG0m (lane 3). The apparent molecular masses of the polypeptides produced are indicated on the right. B and C, Transient expression of GFP-fusion proteins in Arabidopsis protoplasts. B, GFP alone (35S-GFP) and chimera between GFP and the transit peptides of the small subunit of Rubisco (ATS1A-GFP) or dihydropterin pyrophosphokinase/dihydropteroate synthase (HPPK DHPS-GFP) were used as controls for the targeting of the reporter protein to the cytosol, chloroplasts, and mitochondria, respectively. C, Various plasmid constructs engineered with HCS1 cDNA sequences fused upstream and in frame with GFP sequence were introduced into Arabidopsis protoplasts as described in “Materials and Methods.” Schematic representations of cDNA constructs used are shown on the right of corresponding images. TP, Transit peptide. Images are optical photomicrographs (bright field), GFP fluorescence (GFP; green pseudocolor), and chlorophyll fluorescence (chlorophyll; red pseudocolor). Scale bars = 10 μm.
- Figure 5.
Relative abundance of HCS1.s and HCS1.un mRNA species in Arabidopsis organs. A, Schematic representation of left plus, left minus, and right oligonucleotide positions on HCS1 cDNA variants. Left plus oligonucleotide enabled the quantification of HCS1.un mRNA. Left minus oligonucleotide enabled the quantification of HCS1.s mRNA. B, Real-time RT-PCR experiment on poly(A+) RNA from various Arabidopsis organs (roots [R], stems [St], leaves [L], flowers [F], siliques [Si], and seeds [S]) using HCS1.s- and HCS1.un-specific primers. Data (relative transcripts abundance normalized to the expression level of HCS1.un in seeds) are means of three independent experiments performed with three cDNA dilutions ± sd. ACTIN1 was used as an internal control to normalize for variation in the amount of cDNA template. Note the log scale in y axis.
- Figure 6.
Evidence for multiple subcellular localizations of HCS1 in Arabidopsis. A, Soluble proteins (100 μg per lane) from a total leaf extract (T), purified mitochondria (Mi), purified chloroplasts (Cp), and a cytosolic-enriched fraction (Cy) were analyzed by western-blot analysis with affinity-purified polyclonal antibodies raised against recombinant HCS1 (Tissot et al., 1998). The same fractions were assayed for HCS specific activity. Pure recombinant HCS1-ATG1 (HCS1 41-kD precursor form, 10 ng), HCS1-ATG2 (HCS1 37-kD mature form, 10 ng), and HCS2 (37-kD gene product, 25 ng) proteins were run on the same gel as controls. B, Specific activities of cytosolic (pyrophosphate:Fru-6-P 1-phosphotransferase; PFP), chloroplast stroma (NADP-dependent glyceraldehyde-3-P dehydrogenase; GraPDH), and mitochondrial matrix (fumarase) markers were measured (Tissot et al., 1997) to analyze cross-contaminations between subfractions. Data are means ± sd of three replicates.
Tables
Primer Name Primer Sequence Annealing Temperaturea Genotyping of hcs1 mutant Hcs1.for 5′-CTGCCTCAGGTTCGAAACTATTAGGAC-3′ Hcs1.rev 5′-TAACACACAAAGGTAATCAACCAACC-3′ LB3 5′-TAGCATCTGAATTTCATAACCAATCTCG-3′ Isolation and genotyping of hcs2 mutant Hcs2.Met 5′-CGTTATTGAACAGTTTCGTGGTCATGC-3′ Hcs2.Stop 5′-CATCTGCGTTCACCAAAAGATGCTTC-3′ Tag3 5′-CTGATACCAGACGTTGCCCGCATAA-3′ Tag5 5′-CTACAAATTGCCTTTTCTTATCGACCATG-3′ Exon7 5′-GGAGCTTTACTATAGGACATGGC-3′ Real-time PCR Hcs2.Q5 5′-CCAGTTGGTTCAGTTTGTGTCTCTGATATACAAC-3′ 60°C Hcs2.Q3 5′-GGCACTACTCGACCATCTTCCATTTCTAAT-3′ 60°C Hcs1.Q5 5′-CCAGTTGGTTCAGTTTGTGTCACTGATATCCAGT-3′ 64°C Hcs1.Q3 5′-GGCACGACTCGACCATCTTCCATTTCTAGA-3′ 64°C Left minus 5′-GAACGAACTGCTTCCATTAAACTGTTGT-3′ 60°C Left plus 5′-GAACGAACTGCTTCCATTAAACTATAA-3′ 60°C Right 5′-AGCTCTCCTCTGCATCTGAGCATTGC-3′ 60°C Actin.5 5′-GTTTTGCTGGGGATGATGC-3′ 55°C Actin.3 5′-GGATTGAGCTTCATCGCC-3′ 55°C 5′-RACE experiment Hcs1.E4 5′-CCATACATTCTTTGTTCTGC-3′ BCCP2, ACC138, cloning and expression 5′-bccp2 5′-AAATCTGAACATATGGCTAAAGTCTCTGG-3′ 3′-bccp2 5′-GCAGCTAAAGAGCTCCTTCTT-3′ 5′-acc 5′-AGTAAATATCATATGGATGTAGTCC-3′ 3′-acc 5′-TTATTTTGAGCTCAATCAAGATCAAGATTGGC-3′ Mutagenesis ATG0.for 5′-CGAATAGCAGCAAGATCTTTGATTTGGCTAC-3′ ATG0.rev 5′-GTAGCCAAATCAAAGATCTTGCTGCTATTCG-3′ Cloning of HCS1 cDNA variants RACE.KPN 5′-TTAAATTAAAGGTACCGCTCTCCTCTGCATC-3′ STOP1.SAC 5′-AAAATCTTGAGCTCATATTTTTCTTCGTCGAAC-3′ HCS1-GFP fusion cloning ATG1.XBA 5′-GATCTAGATGGAAGCAGTTCGTTCAAC-3′ Hcs1s.XBA 5′-ACAGCTCTAGACTGCATCTGAGCATTG C-3′ ATG2.XBA 5′-CAACCTTATCTAGATTTCATCTACTG-3′ Hcs1un.XBA 5′-GTCTTCTAGATTAAAAATTGCAACTTTAAC-3′ Hcs1.BAM 5′-CCCTTAAACTGGATCCCAGTGACAC-3′ ↵a The annealing temperature used for the real-time PCR is indicated.
Supplemental Data
Supplemental Figure
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