Developmentally regulated HEART STOPPER, a mitochondrially targeted L18 ribosomal protein gene, is required for cell division, differentiation, and seed development in Arabidopsis

Highlight An Arabidopsis L18 mitochondrial ribosomal protein modulates cell division, differentiation, and development, potentially with other diverse L18 family members, by forming heterogeneous ribosomes in mitochondria.


Genetic mapping, mutation detection and complementation
HES/hes was crossed to Columbia. In the F1 population, only the heterozygotes were harvested for further analysis. As homozygous hes/hes plants did not occur, the genotype of each F2 plant was scored with seed phenotypes. Selected SSLP markers (http//:www.arabidopsis.org) from five chromosomes were used to determine the genotypes for each F2 plant and the map location of HES was able to be calculated based on the association between seed phenotypes and markers. The HES gene was located close to a SSLP marker F16J7-TRB (http//:www.arabidopsis.org) on Chromosome 1. For detailed mapping and cloning, a range of indel markers covering the HES region were designed based on the Monsato SNP data base (http//:www.arabidopsis.org) ( Table S11). The genetic distance as shown by the crossover frequency (total crossovers out of total chromosomes) for each marker is indicated in Fig. S2. Primers for mapping are listed in Table S11. The HES is located between 2764k FR and 2867k FR, a 103kb region containing 25 annotated genes (At1g08700 to At1g08960; www.arabidopsis.org). Lines with T-DNA insertion in exons of 16 of the 25 genes were examined but no hes-like seed phenotypes were observed.
We then PCR amplified the other nine candidate genes from heterozygous HES/hes plants into individual overlapping fragments. A heteroduplex DNA PCR fragment between WT and hes DNA should have a mismatch that can be cleaved by the nuclease CEL1 at the mismatch site (Oleykowski et al., 1998). The PCR fragments, after denaturing and annealing, were digested by adding 0.5µl CEL1 enzyme mixture for 30 minute at 40ºC and visualized on a 1.5% Agarose gel. The PCR fragments overlapped each other by at least 200 bp to avoid the mismatch site being too close to the end of a particular fragment. A mismatch was identified from a fragment derived from At1g08845 and confirmed by Sanger sequencing (Fig. S2B). The At1g08845 gene contains two introns and produces a ~700 nucleotide transcript based on ESTs from this region of the genome. The longest transcript contains a 179 nucleotide 5' UTR, a 39 nucleotide 3'UTR with a 567 nucleotide coding region.
Although another At1g08845 transcript was predicted (longer at the 5' end with two additional introns and an extra 40 amino acids), this was not supported by ESTs or by comparison to the deduced sequences of orthologs in other plant species (Fig. S3). The hes mutant contains a single nucleotide substitution of G to A at nucleotide 421 of the At1g08845 coding region, which is predicted to lead to a conserved glycine (G) at position 141 being replaced by an arginine (R) (Fig. S3). The plants derived from HES/hes heterozygous plants were floral dipped with Agrobacterium stain GV3101 containing binary plasmids carrying At1g08845 genomic sequence (Clough and Bent, 1998).
Primary transformants were selected on MS medium supplemented with 50µg/ml Kanamycin. Three types of transgenic kanamycin-resistant T1 plants were obtained (Table S1). Type I plants, like wild type, had no small-sized aborted seeds; type II showed reduced ratios of small seeds compared to HES/hes (Fig. S2C); and type III showed the ratio of small size seeds comparable to HES/hes (Fig.   S2C). Sequencing of type II and III plants showed both had the A to G mutation (Fig. S2C)

Phylogenetic analysis
The amino acid sequences of ribosomal L18/L5 family proteins were obtained by blasting from the NCBI database (http://www.ncbi.nhn.nih.gov) using the HES protein sequence. Mega4.1 was used to make sequence alignments and construct phylogenetic trees using the Neighbour Joining algorithm.
The protein IDs (or accession numbers) and species names can be identified in the phylogenetic tree. with Agrobacterium stain GV3101 containing binary plasmids carrying HES:GFP genomic sequence.
Three types of transgenic kanamycin-resistant T1 plants were also obtained ( The homozygous plants carrying HES:GUS were used for examining HES expression in more details. Twenty randomly-selected genes with decreased expression and eighteen up-regulated genes were used for verification of microarray results. Reference genes ACTIN 2 , APC2 (At2g04660) and HBT (At2g20000) showed stable expression in hes and wt seeds in microarray assay (Table S2). Seven upregulated genes in "mitochondrial dysfunction regulon" were verified with the three reference genes (Table S5). The rest were only tested with ACTIN2.