- binding proteins and their putative target RNAs in several storage root crops. - Storage tubers and root crops are important sustenance food crops grown throughout the world.. - 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. - 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: Phloem mobile, Signaling, Potato, BEL1-like, KNOX, Storage root crops. - Most PTB proteins are present in the nucleus, but in some systems, PTBs shuttle rapidly between the nucleus and cytoplasm [8]. - The interaction between RRMs and target RNAs brings separated pyrimidine tracts into close proxim- ity making loops in the structure of target RNAs [1].. - Of the three PTBs in Arabidopsis (AtPTB1. - Binding and movement assays have shown that cytosine/uracil motifs predominately present in the 3´ UTRs of StBEL5 appeared to be most critical in transporting its RNA from leaves into the stolon tips in potato . - We will now discuss in more detail the role of specific PTB proteins of potato that function as chaperones in the delivery of a key mobile signal that activates tuber formation.. - This movement is enhanced under short-day conditions and is mediated by motifs present in the UTRs of StBEL . - Potato PTBs in the same class as CmRBP50, designated StPTB1 and StPTB6, were confirmed to bind to StBEL5 mRNA through CU motifs present in its 3´ UTR. - In addition, many tuber and root crops ex- hibit antioxidative, hypoglycemic, hypocholesterolemic, antimicrobial, and immunomodulatory properties [35].. - They also have diverse numerous applications in the paper, fabric and starch adhesives industries [36]. - Several root crops exhibit an immense potential as functional foods and nutraceutical ingredients to be explored in disease risk re- duction and wellness [35]. - There has been no attempt, however, to address the question of whether or not the StBEL and PTB compo- nents are conserved in the genome of any other storage organ food crops. - In this study, our goal was to establish the conservation of BEL5, POTH1, and PTB orthologues in the genomes of five storage root crops.. - If they do exist, it would be compelling to explore their po- tential role (similar to potato) as signals in the regulation of storage organ development.. - Identifying orthologues of StBEL5, POTH1, StPTB1 and − 6 in storage root crops. - RNA and protein sequences of POTH1, StBEL5, StPTB1 and − 6 orthologue genes in storage root crops, carrot (Daucus carota), radish (Raphanus sativus) and sugar beet (Beta vulgaris), were obtained from NCBI by pro- tein BLAST suite using respective potato protein se- quences as queries (https://blast.ncbi.nlm.nih.gov/. - Sequence alignment analysis for StPTB1 and − 6 orthologues in storage root crops. - Protein sequences of PTB1/6-like orthologues from the storage root crops, sweet potato, cassava, carrot, radish and sugar beet were aligned to potato StPTB1 and StPTB6 amino-acid sequences as reference. - 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. - 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. - 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. - as references in identifying putative RRM and RNPs in the PTB1/6-like orthologues from the five storage root crops.. - Leaf and storage root samples of sweet potato, cas- sava, carrot, radish and sugar beet were harvested from the agricultural farm near the Pune institute.. - Total RNA from leaf and root tissues was iso- lated using RNAiso Plus (Takara-Clontech) with three biological replicates from the aforementioned storage root crops, except cassava. - For StBEL5-, StPTB1/6- and POTH1-like orthologues in the storage root crops, qPCR reactions were carried out with three biological replicates and three technical replicates. - In the phylogenetic tree, the branch length is proportional to the number of substitutions per site and the tree was rerooted using midpoint rooting in TreeDyn. - Conserved RRM (RNA recognition motif ) do- mains characteristic of PTB proteins were also identified using BLAST for all PTB1/6-like proteins from the five storage root crops. - Similarly, phylogenetic trees were built for POTH1- and BEL5-like orthologues in these five storage root crops.. - Table 1 Potato PTB orthologues in five storage root crops. - Sweet potato (Ipomoea trifida). - Orthologues of the potato PTB1/6 proteins in storage root crops. - Among the PTB orthologues in each storage root crop, proteins with the best coverage and identity (highlighted in bold) were used in the multiple sequence alignment (Fig. - Identification of BEL5-, POTH1- and PTB1/6-like genes in storage root crops. - In order to characterize PTBs and two target RNAs in storage root crops, we sought putative orthologues through a BLAST search. - Our data mining revealed that orthologues of both target RNAs and StPTB1/6-like pro- teins are present in the genomes of the root crops exam- ined here. - Multiple sequence alignment of StPTB1 and StPTB6 with select orthologues from these storage root crops revealed a high overall sequence match and a high level of concordance in the conserved sequence of the RRMs and RNPs (Fig. - Like CmRBP50, StPTB1 and StPTB6, the orthologues in these storage root crops contain two putative RNPs in each RRM (Fig. - Because of StBEL5 ’ s role as a mobile signal for tuber de- velopment [19] and the involvement of KNOTTED1-like homeobox genes in root development using a BLAST search with StBEL5 and POTH1 as queries, we identified several orthologues for both types in the ge- nomes of the five root crops. - 3 and 4) and for scoring CU motifs in the 3´ UTR of their transcript se- quences (Table 3). - In mammals, such CU motifs in the RNA sequence function to interact with PTB-like proteins to regulate RNA metabolism [1, 2, 9]. - In potato and pump- kin, PTB proteins bind specifically to the conserved CU motifs in the 3´ UTRs of select mobile RNAs to mediate their transport from source tissues to target sites . - 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. - 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. - 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. - Similarly, a CU motif search in the orthologues of StBEL5 and POTH1 for the five storage root crops revealed the presence of several CU motifs in the 3´ UTRs of their RNA se- quences (Table 3. - There are 20 such motifs in the 3´ UTR of the POTH1-like tran- script of cassava and 15 motifs in the 3´ UTR of the BEL5-like transcript of sweet potato. - Transcript detection of BEL5-, POTH1- and PTB1/6-like genes in storage root crop organs. - To validate the activity of the conserved genes in this study, RT-qPCR with gene-specific primers was utilized to meas- ure levels of transcripts for POTH1, StBEL5, and StPTB1/6 orthologues in leaf and storage root samples of sweet po- tato, cassava, carrot, radish and sugar beet (Fig. - Be- cause of their close sequence match, quantification of transcripts for PTB1 and PTB6 types in the five roots crops was combined. - Using RNA levels in leaves as a stand- ard, more abundant accumulation of all three types oc- curred in the storage roots of sweet potato, carrot, and. - 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. - 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. - PTB1/6-like gene activity in storage root tissues was higher in sweet potato, carrot and radish, whereas it was lower in cassava and sugar beet, compared to leaf tissues (Fig. - Except for the low level of StBEL5 RNA in roots, all other target genes measured here were relatively abundant in the potato organs (Fig. - Overall, transcripts of the BEL5-, PTB1/6- and POTH1-like orthologues in the five storage root crops exhibited a significant difference in accumulation patterns in leaves compared to storage root tissues (Fig. - Conserved elements of the StBEL/StPTB complex in a sample of storage root crops. - Comprehensive searches in the genomes of several storage root crops revealed conservation of key components in RNA/protein complexes that function in potato as regula- tors of both root and tuber development. - Se- quence identity matches among the proteins in the PTB1/. - 6 family (with four RRMs) present in the several genomes searched here ranged from 72% for sugar beet to 88% for. - sweet potato. - The ex- istence of PTB1/6 proteins and the conserved target RNAs with their abundant CU motifs in 3´ UTRs suggest the possibility that a PTB-chaperone/BEL-RNA complex may be functional in the development of storage roots in a range of important crop species. - It is even conceivable that PTB/RNA signal complexes are phloem mobile in these root crops.. - StBEL5 mRNA originates in the leaf, and its move- ment to both stolons and roots is induced by a short-day photoperiod and mediated by two RNA-binding proteins, StPTB1 and − 6 [16]. - The presence of cytosine/uracil (CU) motifs in the 3´ UTR of BEL5-like and POTH1-like mRNAs from a range of storage root crops. - Among POTH1- and BEL5-like orthologues in each storage root crop, protein with the best coverage and identity (highlighted in bold) were considered for identification of CU motifs in the 3´ UTRs of respective transcript sequences (Additional file 5: Table S3). - Three root stage-specific BEL1-like proteins were also identified in the storage roots of sweet potato [49, 50]. - Four class-I KNOX genes (designated Ibkn1–4) were identified that were active in the storage roots of sweet potato [44, 45]. - Ibkn2 and Ibkn4 were highly expressed in the developing storage roots of cultivars with a higher capacity for storage root formation [45]. - Seven class-I KNOX genes have been identified in the genome of cassava [46]. - Several of these KNOX genes were differ- entially expressed in storage root tissues suggesting they play an important role in their development. - 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). - 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). - 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. - During sweet potato storage root development, KNOXI gene expression and cytokinin levels were positively correlated [44]. - Consistent with this, BEL5-, PTB1/6- and POTH1-like genes from five storage root crops - cassava, sweet potato, carrot, radish and sugar beet also exhibited differential patterns of ex- pression in leaves and storage root tissues (Fig. - Conservation of the PTB1/6 proteins and members of the StBEL family was observed in genomic searches for several storage root crops. - Using the RNA/PTB protein complexes of potato that function in long-distance signaling as a model, it is conceivable that similar complexes may function during storage root development. - Are their RNAs phloem mobile? Can these PTB types mediate transcript stability and transport? Comparable to potato, can transgenic ex- pression of the genes encoding the PTB1/6 proteins and the BEL5-like mRNAs affect storage root yields? Future experi- mental analyses will be critical to confirm the role of these components in storage root development and to assess their potential for enhancing root crop production.. - Catalog and alignments of PTB1/6 types in five root crops species. - Cytosine/uracil motifs in the 3´ UTRs of BEL5- and POTH1-like mRNAs. - AK helped in sample harvest as well as performed trnS analysis for storage root crop species authentification.. - Structure-function relationships of the polypyrimidine tract binding protein. - Characterization of the nuclear export signal of polypyrimidine tract-binding protein. - Expression of class I knotted1-like homeobox genes in the storage roots of sweet potato (Ipomoea batatas). - Isolation and functional characterization of the genes expressed during storage root formation of sweet potato: Okayama University. - Cassava (Manihot esculenta Krantz) genome harbors KNOX genes differentially expressed during storage root development. - Transcriptional profiling of sweet potato (Ipomoea batatas) roots indicates down-regulation of lignin biosynthesis and up-regulation of starch biosynthesis at an early stage of storage root formation. - Changes in the endogenous level of zeatin riboside, abscisic acid and indole acetic acid during formation and thickening of tuberous roots in sweet potato
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