In-silico, evolutionary, and functional analysis of CHUP1 and its related proteins in Bienertia sinuspersici-a comparative study across C3, C4, CAM, and SCC4 model plants

Abstract

Single-cell C₄ (SCC₄) plants with bienertioid anatomy carry out photosynthesis in a single cell. Chloroplast movement is the underlying phenomenon, where chloroplast unusual positioning 1 (CHUP1) plays a key role. This study aimed to characterize CHUP1 and CHUP1-like proteins in an SCC₄ photosynthetic plant, Bienertia sinuspersici. Also, a comparative analysis of SCC4 CHUP1 was made with C₃, C₄, and CAM model plants including an extant basal angiosperm, Amborella. The CHUP1 gene exists as a single copy from the basal angiosperms to SCC₄ plants. Our analysis identified that Chenopodium quinoa, a recently duplicated allotetraploid, has two copies of CHUP1. In addition, the numbers of CHUP1-like and its associated proteins such as CHUP1-like_a, CHUP1-like_b, HPR, TPR, and ABP varied between the species. Hidden Markov Model analysis showed that the gene size of CHUP1-like_a and CHUP1-like_b of SCC₄ species, Bienertia, and Suaeda were enlarged than other plants. Also, we identified that CHUP1-like_a and CHUP1-like_b are absent in Arabidopsis and Amborella, respectively. Motif analysis identified several conserved and variable motifs based on the orders (monocot and dicot) as well as photosynthetic pathways. For instance, CAM plants such as pineapple and cactus shared certain motifs of CHUP1-like_a irrespective of their distant phylogenetic relationship. The free ratio model showed that CHUP1 maintained purifying selection, whereas CHUP1-like_a and CHUP1-like_b have adaptive functions between SCC₄ plants and quinoa. Similarly, rice and maize branches displayed functional diversification on CHUP1-like_b. Relative gene expression data showed that during the subcellular compartmentalization process of Bienertia, CHUP1 and actin-binding proteins (ABP) genes showed a similar pattern of expression. Altogether, the results of this study provide insight into the evolutionary and functional details of CHUP1 and its associated proteins in the development of the SCC₄ system in comparison with other C₃, C₄, and CAM model plants.

Keywords: Bienertia sinuspersici; CHUP1 protein; CHUP1-like proteins; Phylogenetic analysis; Single-Cell C4 plants; Subcellular compartmentalization.

Figure 1. Phylogenetic tree of CHUP1, CHUP1-like, and its associated proteins.

Am.t (Amborella trichopoda, purple up triangle); Zm (Zea mays, dark green square); Sb (Sorghum bicolor, pink diamond); Os (Oryza sativa, light green down triangle); Ac (Ananas cosmosus, orange up triangle); At (Arabidopsis thaliana, brown circle); Cg (Carnegiea gigantea, orange-outlined up triangle); Cq (Chenopodium quinoa, purple-outline diamond); Ah (Amaranthus hypochondriacis, brown-outlined circle); Sa (Suaeda aralocaspica, aqua-outlined diamond); and Bs (Bienertia sinuspersici, aqua diamond). The percentage of 1,000 bootstrap replicates was represented in branches. ABP, actin-binding protein; HPR, hydroxyproline-rich glycoproteins; TPR, tetratricopeptide repeat. Dicots, monocots, and Amborella are highlighted in bold dark orange-colored branches, bold dark blue-colored branches, and thin light blue-colored branches, respectively.

Figure 2. Functional domain prediction of CHUP 1 and CHUP 1_like proteins from the C₃, C₄, CAM, and SCC₄ model plants.

HD, hydrophobic domain; CCD, coiled-coil domain; LZ, leucine zipper region; ABD, actin-binding domain; PRM, proline-rich motif. At, A. thaliana; Zm, Z. mays; Ac, A. cosmosus; and Bs, B. sinuspersici. The blue star represents the position of the LZ region that has been manually curated based on the motif pattern.

Figure 3. Gene structure and motif of CHUP1-like_a from C₃, C₄, CAM, and SCC₄ plants.

(A) The gene structure of CHUP1 was analyzed using GENE STRUCTURE DISPLAY SERVER (GSDS) v. 2.0 (http://gsds.gao-lab.org/index.php). The CDS and gene sequences were used as input. (B) The motif of the CHUP1 was identified using the MULTIPLE EM for MOTIF ELICITATION (MEME) server v. 4.9.1 (https://meme-suite.org/meme/tools/meme). The protein sequence was used as input. The motif numbers are labeled in order. The black stars denote the differential motif either between species, lineages, types, or clades. The square box indicates the shared motifs among lineages. The circle (blue) around the motifs specifies the unique motifs present in monocots. Note: The sequences of each motif are provided at the bottom of the figure in a black square box.

Figure 4. Gene structure and motif of CHUP1-like_a from C₃, C₄, CAM, and SCC₄ plants.

(A) The gene structure of CHUP1-like_a was analyzed using GENE STRUCTURE DISPLAY SERVER (GSDS) v. 2.0 (http://gsds.gao-lab.org/index.php). The CDS and gene sequences were used as input. (B) The motif of the CHUP1 was identified using the MULTIPLE EM for MOTIF ELICITATION (MEME) server v. 4.9.1 (https://meme-suite.org/meme/tools/meme). The protein sequence was used as input. The motif numbers are labeled in order. The black stars denote the differential motif either between species, lineages, types, or clades. The square box indicates the shared motifs among lineages. Note: The sequences of each motif are provided at the bottom of the figure in a black square box.

Figure 5. Gene structure and motif of CHUP1-like_b from C₃, C₄, CAM, and SCC₄ plants.

(A) The gene structure of CHUP1-like_b was analyzed using GENE STRUCTURE DISPLAY SERVER (GSDS) v. 2.0 (http://gsds.gao-lab.org/index.php). The CDS and gene sequences were used as input. (B) The motif of the CHUP1 was identified using the MULTIPLE EM for MOTIF ELICITATION (MEME) server v. 4.9.1 (https://meme-suite.org/meme/tools/meme). The protein sequence was used as input. The motif numbers are labeled in order. The black star denotes the differential motif either between species, lineages, types, or clades. Note: The sequences of each motif are provided at the bottom of the figure in a black square box.

Figure 6. Phylogenetic analysis by maximum likelihood.

The unrooted images show the dN/dS ratio of (A) CHUP1, (B) CHUP1-like_a, and (C) CHUP1-like_b proteins from plant species included in the study. A free-ratio model was used to calculate independent ω for each branch of C3, C4, CAM, and SCC4 plants. The ratio of dN/dS was mentioned on the branches and lineages. Branches with an estimated ω ratio > 1 were emphasized in thick black color lines.

Figure 7. Eexpression studies of CHUP1, CHUP1-like, and its associated genes in B. sinuspersici.

(A) Leaf samples of B. sinuspersici from young (Stage 1), intermediate (Stage 2), and mature (Stage 3) stage plants (details in materials and methods) (B) Development of central chloroplasts and peripheral chloroplasts in the different stages of leaves. Chloroplast autofluorescence (top panels) and bright-field (bottom panels) images, scale bar = 10 µm. Relative expression of (C) CHUP1, (D) CHUP1-like_a, (E) CHUP1-like_b, (F) ABP, and (G) TPR was analyzed using qRT-PCR in three different developmental stages. ABP, actin-binding protein; TPR, tetratricopeptide repeat. Different letters indicate that treatments are significantly different at p ≤ 0.05. The error bar indicates the standard error of the mean.