Characterization of rpl23aa and rpl23ab mutants
The Arabidopsis genome contains two RPL23a paralogous genes RPL23aA (At2g39460) and RPL23aB (At3g55280), which encode proteins with 95% amino acids identity (see Additional file 1). We acquired T-DNA insertion lines of RPL23aA and RPL23aB, namely SALK_005448 and SAIL-597-B08, respectively (hereafter referred to as rpl23aa and rpl23ab). PCR-genotyping confirmed that both rpl23aa and rpl23ab are homozygous T-DNA insertion alleles (see Additional file 2). Sequencing results revealed that rpl23aa contains a T-DNA insertion in the 3’ UTR region, 10 bp downstream of the stop codon of the RPL23aA gene (Figure 1A), while rpl23ab contains a T-DNA insertion in the second exon of RPL23aB (Figure 1B). A semi-quantitative RT-PCR assay was used to detect transcripts from RPL23aA and RPL23aB in these T-DNA lines. As shown in Figure 1C, the 3’ region around the stop codon of the RPL23aA mRNA was disrupted in the mutant. Because the majority of the RPL23aA mRNA from the T-DNA line was intact, we suspect that SALK_005448 is a hypomorphic allele. rpl23ab is likely a null mutant, because no RPL23aB mRNA was detected (Figure 1D). Absence of dosage compensation by RPL23aA in Arabidopsis was reported following loss of RPL23aB . As shown in Figure 1D, there is also no dosage compensation by RPL23aB in the rpl23aa mutant.
The rpl23aa mutant exhibits pleiotropic defects, including pointed leaves, retarded root growth, and reduced plant size (Figure 2B). These phenotypes are similar to those of a previously reported RNAi line . An incompletely penetrant tricotyledon phenotype (less than 5% of the total population) was observed in rpl23aa mutant plants (see Additional file 3). However, we didn’t observe appreciable defects in terms of growth rate, morphology, flowering or fertility in the rpl23ab mutant (Figure 2D), which is consistent with published work . We amplified genomic DNA encompassing the promoter plus the coding region of RPL23aA from wild-type plants and fused it to the sequence encoding the HA epitope tag. When this transgene was introduced into rpl23aa, the developmental defects were fully rescued (Figure 2C), suggesting that dysfunction of RPL23aA was responsible for the developmental defects in rpl23aa.
RPL23aA and RPL23aB are dosage-dependent genes
In order to study the genetic interaction between RPL23aA and RPL23aB, we crossed rpl23aa with rpl23ab. To our surprise, the doubly heterozygous plants (RPL23aA/rpl23aa; RPL23aB/rpl23ab) in the F1 progeny all have pointed first true leaves (Figure 3B). Siliques of the doubly heterozygous plants are much shorter than siliques of rpl23aa or rpl23ab (Figure 3I). We dissected siliques from RPL23aA/rpl23aa; RPL23aB/rpl23ab plants and found many aborted ovules (Figure 3G and 3H). An F2 population was generated by selfing the above F1 plants. We genotyped 144 F2 plants but did not find double homozygous (rpl23aa /rpl23aa; rpl23ab /rpl23ab) plants. In fact, we did not even detect any genotypes with a single functional allele from either gene (RPL23aA/rpl23aa; rpl23ab /rpl23ab or rpl23aa /rpl23aa; RPL23aB/rpl23ab) (Table 1), although these genotypes are collectively expected to appear in 31.25% (5 out of 16) of the F2 plants. We suspected that this non-allelic non-complementation phenomenon between rpl23aa and rpl23ab is probably due to gene dosage effects.
RPL23aA and RPL23aB genes have similar expression patterns
In order to investigate the expression patterns of RPL23aA and RPL23aB genes, we fused the promoter regions of RPL23aA and RPL23aB genes to the GUS reporter and generated transgenic plants in the Col-0 background. GUS staining of 14 pRPL23aA:GUS and 5 pRPL23aB:GUS independent transgenic lines uncovered a ubiquitous expression pattern for both genes with particularly intense GUS staining in young and actively proliferating tissues, such as developing leaves, floral buds and root apices (Figure 4). Similar expression patterns of RPL23aA and RPL23aB support our hypothesis that the non-allelic non-complementation phenomenon between these two genes is the consequence of overlap in expression (and function) of RPL23aA and RPL23aB in the same cells.
RPL23aA and RPL23aB proteins are functionally equivalent
It has been reported that some paralogous ribosomal proteins have evolved specialized functions in yeast . As mentioned above, dysfunction of RPL23aA results in severe developmental defects, whereas knock-out of RPL23aB has no phenotypic consequences in Arabidopsis ( and this study). It’s natural to assume that these two paralogous ribosomal proteins have undergone functional specialization.
We designed gene complementation experiments to explore whether RPL23aA and RPL23aB have distinct functions. If RPL23aA and RPL23aB have specialized functions, RPL23aB is not expected to complement the rpl23aa mutation. We fused the promoter regions of RPL23aA to the coding region of RPL23aB. The pRPL23aA:RPL23aB transgene was introduced into rpl23aa plants, and 21 independent pRPL23aA:RPL23aB transgene lines were obtained, among which 15 lines rescued the phenotype of rpl23aa (Figure 5 and Additional file 4), indicating that RPL23aA and RPL23aB have equivalent function. The pRPL23aB:RPL23aB transgene was also introduced into rpl23aa plants, and 8 out of 15 independent, homozygous transgenic lines exhibited near wild type morphology (Figure 5 and Additional file 4). However, a portion (about 2%) of the transgenic plants of each line exhibited the tricotyledon phenotype (see Additional file 5). Thus, the pRPL23aB:RPL23aB transgene can largely but not fully rescue the phenotype of rpl23aa.
RPL23aA and RPL23aB genes are transcribed in a concerted manner with higher expression levels of RPL23aA than RPL23aB
Since the above results indicated that RPL23aA and RPL23aB proteins have equivalent function, we suspected that the difference in phenotype between rpl23aa and rpl23ab is due to the difference in the expression levels of these two genes. The expression of RPL23aA may be much higher than RPL23aB, so the impacts on ribosomes by the rpl23aa mutation are higher than the rpl23ab mutation thus leading to much severe morphological defects. We compared the transcript levels of RPL23aA and RPL23aB at different developmental stages and in different organs by analyzing published RNA-seq data . As shown in Figure 6 and Figure S6（Additional file 6）, transcript levels of RPL23aA are much higher than those of RPL23aB at all developmental stages and in all the examined tissues. Strikingly, the spatial and temporal patterns of expression of these two paralogous genes are well matched, suggesting that they are similarly regulated at differently developmental stages in all examined tissues. ACT2, which is a house keeping gene, was included as a control. Transcript levels of RPL23aB are higher than ACT2 in some organs, and total amount of PRL23a transcripts is much higher than ACT2 in most examined organs (Figure 6C, 6E), indicating that RPs are in great demand for plant development.
The polysome/monosome ratio is elevated in the rpl23a mutants
In order to evaluate the effects of the rpl23aa and rpl23ab mutations on polysomes, we fractionated total ribosomes by ultracentrifugation through sucrose density gradients. The polysome profiles from plants of various genotypes are shown in Figure S7 (Additional file7). To our surprise, the ratio between polysomes and the monosome is obviously increased in rpl23aa and slightly increased in rpl23ab compared to wildtype. The increase in the polysome/monosome ratio in rpl23aa was largely rescued by both pRPL23aA:RPL23aA and pRPL23aA:RPL23aB transgenes, whereas the polysome/monosome ratio in pRPL23aB:RPL23aB/rpl23aa transgenic plants is higher than wildtpye and lower than rpl23aa. The changes in ribosomal profile of rpl23aa and rpl23ab indicate that the overall translation state is altered. The higher polysome levels could reflect higher rates of translation or defects in translation, such as slower elongation. While the molecular basis of the higher polysome levels is unknown, the stronger effect of the rpl23aa mutation is consistent with the dominant role of RPL23aA over RPL23aB as suggested by expression levels and mutant phenotypes.