Phylogeny of nucleolar Pumilio proteins
Pumilio proteins are ubiquitous in eukaryotic organisms, albeit in different numbers [1, 5]. Among the organisms whose whole genome sequences are available, higher plants have a higher number of Pumilio proteins than photosynthetic single-cell organisms and nonplant organisms; for example, 25 Pumilio proteins are found in Arabidopsis thaliana, 20 in Oryza sativa, 14 in Physcomitrella patens, 5 in Chlamydomonas reinhardtii, 11 in Caenorhabditis elegans, 7 in Saccharomyces cerevisiae, and 2 in humans . Based on a similarity search using Arabidopsis nucleolar Pumilio proteins (APUM23 and APUM24) as queries and the existence of a nucleolar localization signal(s) (NoLS) as a requirement , the green plants whose genomes have been sequenced (Phytozome v12.1; https://phytozome.jgi.doe.gov) were shown to have two putative nucleolar Pumilio proteins. Using PacBio Iso-Seq analysis, we also identified two putative nucleolar Pumilio proteins out of four Pumilio proteins in C. corallina. Consistent with our transcriptome analysis, four Pumilio proteins were predicted in the Chara genome data, including 2 nucleolar forms . When compared with Arabidopsis APUMs, comprising 25 Pumilio proteins, ChPUM2 and ChPUM3 displayed high homology with APUM23 and APUM24, respectively, while ChPUM1 and ChPUM4 belonged to other distinct clades (Additional file 1: Figure S1).
To gain insight into the evolutionary relationship of putative nucleolar Pumilio proteins, we constructed a phylogenetic tree with the homologous proteins of 14 species of green plants together with 5 outgroup species that contain a NoLS(s)  (Fig. 1a and b). All green plants analyzed in this study had two proteins belonging to the APUM23 and APUM24 clades. The ChPUM2 of C. corallina was closer to APUM23 than APUM24, whereas ChPUM3 was categorized in the APUM24 clade. Phylogenetic analyses using ChPUM2 and ChPUM3 indicated that C. corallina is closer to land plants than other green algae examined in this study, suggesting that the evolution of nucleolar Pumilio proteins is consistent with previously determined phylogenetic positioning [21-25].
We then compared the number and position of Puf domains in ChPUM2, ChPUM3, APUM23, and APUM24 using the SMART web program (http://smart.embl-heidelberg.de) . APUM23 and APUM24 have six and five Puf domains, respectively, and ChPUM2 and ChPUM3 have one and five Puf domains, respectively. As each Puf domain has been known to recognize a single RNA base , this observation raised the possibility that ChPUM2 may have a distinct RNA binding property from APUM23 and that ChPUM3 may bind similar, if not identical, RNA motifs as APUM24 (Fig. 1c).
Structure of ChPUM2 and ChPUM3
Classic structural analysis of Pumilio proteins shows a tandem repeat of 8 Puf domains in the C-terminal region [30, 31]. However, a recent analysis of human Puf-A and yeast Puf6 identified 11 Puf domains, including 3 additional domains, in these Pumilio proteins involved in pre-rRNA processing . A similar analysis previously performed for APUM23 showed 10 Puf domains instead of the six previously known domains [10, 12]. Consistent with a close phylogenetic relationship between APUM23 and ChPUM2 (Fig. 1a and Additional file 1: Figure S1), ChPUM2 also contained 10 Puf domains that showed an identical distribution as in APUM23 (Fig. 2a and Additional file 2: Figure S2a). Each domain of ChPUM2 showed an average 26% identity and 41% homology with the corresponding domain of APUM23. Notably, a high degree of homology was found in the 1st, 2nd, and 5th residues of the 2nd a-helix of each Puf domain (red boxes in Fig. 2a and Additional file 2: Figure S2a). These three residues have been known to play a pivotal role in the recognition of RNA bases .
Using the same approach, ChPUM3 was shown to possess 11 Puf domains (N-R1 to N-R3 at the N-terminus and C-R1 to C-R8 at the C-terminus) (Fig. 2b and Additional file 2: Figure S2b), as reported in human Puf-A and APUM24 . Comparison of amino acids in 3 N-terminal Puf domains displayed an average identity of 47% and homology of 67% between APUM24 and ChPUM3, and that of 8 C-terminal domains showed 39% identity and 55% homology. Out of the 11 Puf domains, two domains (C-R5 and C-R7) had lower identities than the other domains (Fig. 2b).
Comparison of amino acid sequences among Puf domains suggested that ChPUM2 and ChPUM3 may be functional homologs of Arabidopsis APUM23 and APUM24, respectively (Fig. 2a and b), which is consistent with the observation obtained from phylogenetic analysis. However, since ChPUM2 and APUM23 contain different numbers of Puf domains, unlike ChPUM3 and APUM24, in the classic domain analysis (Fig. 1c), they may bind distinct RNA substrates. Therefore, to determine the structural relationship between nucleolar ChPUM and APUM proteins, we predicted the tertiary structure of these proteins using the SWISS-MODEL web server (https://swissmodel.expasy.org/) . A previous high-resolution structural study demonstrated that the C-shaped structure of APUM23 has a long chain between the 2nd and 3rd a-helix of the R3 domain that participates in the recognition of RNA bases . Homology modeling revealed a high similarity between ChPUM2 and the APUM23 reference protein, as well as the C-shaped structure similar to APUM23, in the 3-dimensional structure (Fig. 3a). Compared with APUM23, ChPUM2 has a long random coil in the R3 domain (red colored lines in the bottom panels of Fig. 3a), but it maintains uninterrupted 2nd and 3rd a-helical structures in this domain, similar to APUM23. Therefore, analyses of consensus amino acid sequences and homology modeling suggest that ChPUM2 may recognize similar, if not identical, RNA bases.
In contrast to the C-shaped configuration of ChPUM2 and APUM23, an L-shaped structure was predicted for ChPUM3 and APUM24, similar to the human Puf-A reference protein  (Fig. 3b). The most marked structural differences between ChPUM3 and APUM24 were found in the C-R5, and N-R2 and N-R3 domains. ChPUM3 had a longer random coil in the C-R5 domain than APUM24 (Fig. 2b and the dotted circles in Fig. 3b). Additionally, ChPUM3 contained negatively charged (E210) and uncharged (Q249) amino acids in the N-R2 and N-R3 domains (Fig. 2b), instead of the basic amino acids that are known to be involved in RNA binding and found at both sites of APUM24 and the human Puf-A reference . Thus, it appeared that ChPUM3 has different RNA binding specificity from APUM24, considering the chain length of C-R5 and the lack of basic amino acids in 2 N-terminal Puf domains.
Subcellular localization of ChPUM2 and ChPUM3
Next, we examined the subcellular localization of ChPUM2-RFP and ChPUM3-RFP. Previously, the GFP fusions of APUM23 and APUM24 were known to preferentially localize in the nucleoli of Arabidopsis root and tobacco leaf cells [10, 15]. We performed Agrobacterium-mediated coinfiltration into N. benthamiana leaf cells using 35S:APUM23-GFP and 35S:ChPUM2-RFP and 35S:APUM24-GFP and 35S:ChPUM3-RFP. All GFP- and RFP-tagged Pumilio proteins were found in the nucleoli and were weakly detected in the nucleoplasm (Fig. 4). The colocalization results suggest that ChPUM2 and ChPUM3 may play similar roles in pre-rRNA recognition and processing to APUM23 and APUM24.
ChPUM2 rescued the apum23 mutant phenotype, but ChPUM3 did not rescue apum24
To assess whether ChPUM2 and ChPUM3 are involved in nucleolar functions similar to APUM23 and APUM24, complementation assays were performed on homozygous apum23 (Fig. 5) and heterozygous apum24 (Fig. 6) mutants by transforming the 35S:ChPUM2 and 35S:ChPUM3 constructs. While the apum23-2 mutant showed the phenotypes of delayed germination (Fig. 5a and b), short roots (bottom panel in Fig. 5b), short inflorescence stems (upper panel in Fig. 5c), light green leaves (bottom panel in Fig. 5c), and streptomycin resistance (Fig. 5d), the 35S:ChPUM2/apum23-2-/- plants exhibited normal phenotypes (Fig. 5a-d). Notably, the recovered streptomycin susceptibility of 35S:ChPUM2/apum23-2-/- plants indicates the normal ribosomal functions of complemented plants (Fig. 5d). In contrast to 35S:ChPUM2/apum23-2-/- plants, 35S:ChPUM3/apum23-2-/- plants maintained an apum23-2 mutant phenotype (Fig. 5a-d). The failure to restore the apum23-2 phenotype with 35S:ChPUM3 excluded the possibility that ChPUM3 and APUM23 are orthologous proteins.
In addition to the restoration of morphological phenotypes, 35S:ChPUM2/apum23-2-/- rescued the defects observed in the apum23-2-/- mutant that accumulates poly(A)-tailed 5’ETS-18S-ITS1 and 5.8S-ITS2 pre-rRNAs (Fig. 5e). The poly(A) pre-rRNAs were detected using quantitative reverse transcriptase-PCR (qRT-PCR) and three combinations of primers. In the apum23-2-/- mutant, all three qRT-PCR products (5’ETS-18S, 18S-ITS1, and 5.8S-ITS2) were accumulated, but in 35S:ChPUM2/apum23-2-/- plants, the amounts of poly(A)-tailed 18S-ITS1 and 5.8S-ITS2 pre-rRNAs were greatly reduced compared with those in apum23-2-/-, although the amount of poly(A) 5.8S-ITS2 was slightly decreased. In contrast to 35S:ChPUM2/apum23-2-/-, 35S:ChPUM3/apum23-2-/- showed a nearly identical amount of poly(A) pre-rRNAs to the apum23-2-/- mutant (Fig. 5e, right panel). The qRT-PCR results indicate that ChPUM2 is involved in a similar pre-rRNA processing pathway as APUM23, although it was not fully functional in the removal of the 5.8S pre-rRNA byproducts.
In contrast to the restoration of apum23 by the ChPUM2 transgene, the apum24 phenotype was not restored by the ChPUM2 or ChPUM3 transgenes. As the homozygous apum24-/- mutant is lethal [13, 15, 33], the heterozygous apum24-1+/- mutant was used for complementation analysis. The 35S:ChPUM3/apum24-1+/- plants set normal and abnormal seeds at similar rates as the apum24+/- mutant (Fig. 6a-c and Table 1), showing 30.7% and 33.2% abnormal seeds for the 35S:ChPUM3/apum24-1+/- and apum24-1+/- plants, respectively. As expected, the 35S:ChPUM2/apum24-1+/- plants produced abnormal seeds (32.5%) at a similar ratio to the 35S:ChPUM3/apum24-1+/- plants. Consistent with this result for the morphological phenotype, 35S:ChPUM2/apum24-1+/- and 35S:ChPUM3/apum24-1+/- plants accumulated poly(A)-tailed 18S-ITS1 and 5.8S-ITS2 pre-rRNA similar to the apum24-1+/- plants (Fig. 6d), indicating that ChPUM3 is not a functional ortholog of Arabidopsis APUM24.
ChPUM2 restored the salt- and glucose-hypersensitive phenotypes of apum23, but ChPUM3 did not restore the apum23 and apum24 phenotypes
apum23 and apum24 showed changes in the expression levels of ribosomal biogenesis-related genes, which in turn resulted in hypersensitivity to high concentrations of salt in apum23  and glucose in a weak apum24 mutant . We therefore examined whether ChPUM2 and ChPUM3 could restore the altered physiological phenotypes of apum23-/- and apum24+/-. The 35S:ChPUM2/apum23-2-/- seedlings exhibited a similar degree of resistance to 150 mM NaCl and 200 mM glucose as wild-type Col-0 seedlings, while the 35S:ChPUM3/apum23-2-/- seedlings showed NaCl and glucose susceptibility similar to that of apum23-2-/- seedlings (left panel in Fig. 7a). However, 35S:ChPUM2/apum24-1+/- and 35S:ChPUM3/apum24-1+/- failed to recover the salt sensitivity of apum24-1+/- (right panel in Fig. 7a). Unexpectedly, when 35S:ChPUM2 or 35S:ChPUM3 was overexpressed in apum24-1+/-, their transgenic seedlings showed a similar glucose resistance as that found in wild-type and apum24-1+/- seedlings (right panel in Fig. 7a). It was previously reported that APUM24 gene expression was greatly increased in wild-type plants by exogenously supplied glucose [13, 15]. Our data showed similar expression levels of the APUM24 transcript in the Col-0 (pB2GW7) control, apum24-1+/-, 35S:ChPUM2/apum24-1+/-, and 35S:ChPUM3/apum24-1+/- in the presence of 200 mM glucose (Fig. 7b). The normal growth of apum24-1+/- suggests that heterozygotic expression of APUM24 might be sufficient for the glucose-induced phenotype. Similar to apum24-1+/-, the apum24-1+/- plants transformed with 35S:ChPUM2 or 35S:ChPUM3 grew normally under glucose treatment, probably owing to the expression of heterozygotic APUM24. Moreover, all the plants showed similar amounts of unprocessed 5.8S rRNAs as Col-0 (pB2GW7) control under 200 mM glucose treatment (Fig. 7c), while they displayed 5.08- to 6.26-fold higher amounts of unprocessed 5.8S rRNAs than the Col-0 (pB2GW7) control (Fig. 6d). This result suggests that glucose supplementation of heterozygous apum24+/- increased the level of heterozygous APUM24 up to the homozygous APUM24 level. Therefore, the normal phenotype and 5.8S pre-rRNA processing that were observed in 35S:ChPUM2/apum24-1+/- and 35S:ChPUM3/apum24-1+/- plants resulted from increased levels of Arabidopsis APUM24 caused by exogenously supplied glucose.