Conservation of polypyrimidine tract binding proteins and their putative target RNAs in several storage root crops

Abstract

Background: Polypyrimidine-tract binding proteins (PTBs) are ubiquitous RNA-binding proteins in plants and animals that play diverse role in RNA metabolic processes. PTB proteins bind to target RNAs through motifs rich in cytosine/uracil residues to fine-tune transcript metabolism. Among tuber and root crops, potato has been widely studied to understand the mobile signals that activate tuber development. Potato PTBs, designated as StPTB1 and StPTB6, function in a long-distance transport system by binding to specific mRNAs (StBEL5 and POTH1) to stabilize them and facilitate their movement from leaf to stolon, the site of tuber induction, where they activate tuber and root growth. Storage tubers and root crops are important sustenance food crops grown throughout the world. Despite the availability of genome sequence for sweet potato, cassava, carrot and sugar beet, the molecular mechanism of root-derived storage organ development remains completely unexplored. Considering the pivotal role of PTBs and their target RNAs in potato storage organ development, we propose that a similar mechanism may be prevalent in storage root crops as well.

Results: Through a bioinformatics survey utilizing available genome databases, we identify the orthologues of potato PTB proteins and two phloem-mobile RNAs, StBEL5 and POTH1, in five storage root crops - sweet potato, cassava, carrot, radish and sugar beet. Like potato, PTB1/6 type proteins from these storage root crops contain four conserved RNA Recognition Motifs (characteristic of RNA-binding PTBs) in their protein sequences. Further, 3´ UTR (untranslated region) analysis of BEL5 and POTH1 orthologues revealed the presence of several cytosine/uracil motifs, similar to those present in potato StBEL5 and POTH1 RNAs. Using RT-qPCR assays, we verified the presence of these related transcripts in leaf and root tissues of these five storage root crops. Similar to potato, BEL5-, PTB1/6- and POTH1-like orthologue RNAs from the aforementioned storage root crops exhibited differential accumulation patterns in leaf and storage root tissues.

Conclusions: Our results suggest that the PTB1/6-like orthologues and their putative targets, BEL5- and POTH1-like mRNAs, from storage root crops could interact physically, similar to that in potato, and potentially, could function as key molecular signals controlling storage organ development in root crops.

Keywords: BEL1-like; KNOX; Phloem mobile; Potato; Signaling; Storage root crops.

Fig. 1

Amino-acid sequence alignment of StPTB1/6-like orthologues in select storage root crops. Gray boxed letters represent the residues in PTB1/6-like orthologues of storage root crops identical to StPTB1 and StPTB6, letters highlighted in red represent the residues identical in at least two PTB1/6-like orthologues, whereas residues not highlighted represent non-conserved residues among these PTB1/6-like orthologues. Four RNA recognition motifs (RRMs) are underlined in red. Potential canonical RNPs in each RRM are highlighted in yellow. Clustal consensus sequences are represented by asterisks below the alignment. The amino-acid sequences of PTB1/6-like orthologues in storage root crops are aligned to StPTB1 and StPTB6 amino-acid sequences in potato as a reference. CmRBP50 RRM and RNP sequences were used for identifying potential RRM and RNPs in these StPTB1/6-like orthologues [5]. Among the different PTB1/6-like variants identified in each storage root crop (Table 1), one protein per crop with the best coverage and identity were considered for the sequence alignment shown here. These accessions for protein sequences were: ItPTB1/6-like (itf09g10450.t1), MePTB1/6-like (Manes.18G093400.1), DcPTB1/6-like (XP_017247842.1), RsPTB1/6-like (XP_018451916.1) and BvPTB1/6-like (XP_010681101.1). PTB, polypyrimidine tract-binding; St, Solanum tuberosum; It, Ipomoea trifida; Me, Manihot esculenta; Bv, Beta vulgaris; Dc, Daucus carota; Rs, Raphanus sativus

Fig. 2

Phylogenetic relationship of RBP50-like PTBs from the Solanaceae family and PTB1/6-like proteins from five storage root crops (sweet potato, cassava, carrot, radish and sugar beet) selected from Table 1. For comparison, the deduced amino-acid sequences for thirteen PTB1/6-like proteins from nine species were analyzed. AtPTB2 (a distant homolog of Arabidopsis AtPTB3), HnRNPI (human PTB) and StBMI1 (a potato non-PTB related protein) amino-acid sequences are included as controls. Conserved RRM (RNA recognition motif) domains characteristic of PTB proteins were also identified using BLAST for all PTB1/6-like proteins from these storage root crops. Amino-acid sequence alignments and phylogenetic analysis were performed using T-COFFEE (hRp://www.ch.embnet.org/soaware/TCoffee.html) and graphical representation of the phylogenetic tree was performed with TreeDyn (v198.3) [42]. Accessions for protein sequences used are written after protein names in the phylogenetic tree. In the phylogenetic tree, the branch length is proportional to the number of substitutions per site and the tree is rerooted using midpoint rooting in TreeDyn. Bv, Beta vulgaris; Cm, Cucurbita maxima; Dc, Daucus carota; It, Ipomoea trifida; Me, Manihot esculenta; Rs, Raphanus sativus; St, Solanum tuberosum; PTB, polypyrimidine tract-binding

Fig. 3

Phylogenetic relationship of POTH1-like proteins from five storage root crops (sweet potato, cassava, carrot, radish and sugar beet). For comparison, the deduced amino-acid sequences for POTH1-like proteins from five storage root crops plus POTH1 of potato were analyzed. StBMI1 (a potato non-POTH1 related protein) amino-acid sequences are included as controls. Amino-acid sequence alignments and phylogenetic analysis were performed using T-COFFEE (hRp://www.ch.embnet.org/ soaware/TCoffee.html) and graphical representation of the phylogenetic tree was performed with TreeDyn (v198.3) [42]. Accessions for protein sequences used are written after protein names in the phylogenetic tree. In the phylogenetic tree, the branch length is proportional to the number of substitutions per site and the tree is rerooted using midpoint rooting in TreeDyn. Bv, Beta vulgaris; Dc, Daucus carota; It, Ipomoea trifida; Me, Manihot esculenta; Rs, Raphanus sativus; St, Solanum tuberosum

Fig. 4

Phylogenetic relationship of BEL5-like proteins from several storage root crops (sweet potato, cassava, carrot, radish and sugar beet). For comparison, the deduced amino-acid sequences for BEL5-like proteins from the five storage root crops plus three from potato (BEL5, − 11, and − 29) were analyzed. StBMI1 (a potato non-BEL5 related protein) amino-acid sequences are included as controls. Amino-acid sequence alignments and phylogenetic analysis were performed using T-COFFEE (hRp://www.ch.embnet.org/soaware/TCoffee.html) and graphical representation of the phylogenetic tree was performed with TreeDyn (v198.3) [42]. Accessions for protein sequences used are written after protein names in the phylogenetic tree. In the phylogenetic tree, the branch length is proportional to the number of substitutions per site and the tree is rerooted using midpoint rooting in TreeDyn. Bv, Beta vulgaris; Dc, Daucus carota; It, Ipomoea trifida; Me, Manihot esculenta; Rs, Raphanus sativus; St, Solanum tuberosum

Fig. 5

Expression analysis of StBEL5, StPTB1/6 and POTH1 orthologues in leaf and storage root samples of the root crops: sweet potato (a), cassava (b), carrot (c), radish (d) and sugar beet (e). Transcript levels of StBEL5, StPTB1, StPTB6 and POTH1 in potato leaf and root tissues are also shown from 3-month old plants (S. tuberosum ssp. andigena) grown under long-day conditions (f). RNA was extracted from leaves and roots and RT-qPCR with gene-specific primers was used to calculate the relative amounts of RNA for each target gene. Three biological samples were measured with three technical replicates and normalized against GAPDH mRNA. The fold change in RNA levels was calculated as the 2^−ΔΔCt value [41] relative to the mean values obtained in the leaf samples (set at a value of 1.0). Standard errors of the means are shown with one, two and three asterisks indicating significant differences (p < 0.05, p < 0.01, p < 0.001, respectively) using a Student’s t-test. Because of their close sequence match (Table 1), quantification of transcripts for PTB1 and PTB6 types in the five storage roots crops was combined as PTB1/6