Identification and functional characterization of KNOTTED-like homeobox genes in potato

Abstract

KNOX genes are ubiquitous to green plants and they are known to play vital role in plant development and patterning. KNOX genes belong to a Three Amino acid Loop Extension (TALE) superclass of transcription factors (Burglin et al., 1997). KNOX TFs form a dimer with another family of TFs from TALE superclass, viz. The BELL-TFs and a complex network of KNOX-BELL heterodimers regulate the target genes in a tissue specific manner (Bellaoui et al., 2001; Smith et al., 2002; Hay and Tsiantis, 2010). In lower plants, KNOX genes regulate gamete identity and zygotic development (Lee et al., 2008; Sakakibara et al., 2013). In angiosperms, KNOX-I genes are shown to regulate diverse developmental programs such as meristem maintenance, leaf development, floral development, tuberization and so on (Hay and Tsiantis, 2010; Ragni et al, 2007; Uchida et al, 2010). In spite of an important role KNOX genes play in plant development, only a handful of KNOX target genes are identified so far (Giacomo et al., 2013). To have a deeper understanding about how the KNOX genes function, it is crucial to identify their targets from various plant species. Another important research question is how KNOX gene expression is regulated? Our knowledge about regulation of KNOX gene expression is limited to a few studies, which have shown that KNOX gene expression is repressed outside its expression domain by different epigenetic regulators (Hay and Tsiantis, 2010). It would be interesting to understand how KNOX gene expression is regulated across different developmental pathways to generate diverse plant forms. To investigate the KNOX gene function, the model system chosen in the current study is potato (Solanum tuberosum). The focus of this study is to understand the role of KNOX genes in potato development and tuberization. Previous to the present study, only one potato KNOX gene (Potato Homeobox1, POTH1) was studied and it was demonstrated to regulate vegetative development in potato by modulating GA and cytokinin levels (Rosin et al., 2003; Chen et al., 2004). Notably, the POTH1 transcript was found to be present in phloem sap and phloem cells of potato (Yu et al., 2007; Campbell et al., 2008). Other than the mentioned studies, there were no reports of KNOX gene functions from potato. Hence, the objectives of the current study were: 1. To carry out a thorough literature survey on KNOX genes in plants 2. To identify, validate and obtain full-length sequence for potato KNOX genes 120 Summary 3. To study the regulation of POTH1 expression and to investigate the mobility of POTH1 mRNA 4. To understand the regulation, over-expression and to identify the target genes of POTATO HOMEOBOX15 (POTH15). Chapter 1: Introduction A through literature survey was carried out on KNOX genes discovery, regulation and their functions across plant species. Although, there are numerous studies carried out of KNOX genes in plants, for the purpose of the present study, a short list of 70 reports has been provided as a table (Table 1.1). This literature survey suggested that role of KNOX genes in regulation of tuberization as well as other developmental pathways in potato is not clearly understood. Chapter 2: KNOX genes in potato (Solanum tuberosum ssp Andigena) Previous to current study only one potato KNOX gene, POTH1, was identified from potato (Rosin et al., 2012). To explore the existence and functions of additional KNOX genes in potato, the Potato Genome Sequence Database was mined. In this part of the work, experiments were carried out to identify, validate and clone potato KNOXgenes. The results of these experiments are summarized as follows: (i) Six novel KNOX genes from potato genome were identified, validated and cloned. Thus including POTH1, the number of KNOX genes identified from potato is now seven. (ii) The sequences for all these genes have been deposited to NCBI and their accession numbers are POTH20 (KP335125), POTH15 (KJ477687), StPetroselinum (KJ477688), StKn1 (KJ477691), StHOX1 (KJ477689), and StHox2 (KJ477690). (iii) Further, a phylogenetic analysis of potato KNOX proteins along with tomato and Arabidopsis KNOX proteins revealed that four of them belong to Class-I (POTH1, POTH15, POTH20, and StKn1), two belongs to Class-II (StHox1 and StHox2) and one (StPTS) is a mini-KNOX. 121 Summary Considering the previous reports and the importance of KNOX genes in potato, we choose to continue our work with POTH1 and POTH15, which are summarized in Chapter 3 and 4. Chapter 3: POTH1: Studying the regulation and transcript mobility Before the present study,POTH1 was shown to regulate vegetative development in potato and its transcript was found to be present in phloem. In this part of the work, experiments were conducted to understand the regulation of POTH1 expression and to investigate the mobility of POTH1 transcript across graft-unions. The findings obtained based on these experiments are summarized as follows: (i) The promoter of POTH1 is rich in light regulatory motifs and the POTH1 expression is affected by light. (ii) Micrografts and heterografts with soil-grown plants confirmed that the mRNA of POTH1 is graft transmissible and moves in a rootward direction. (iii) POTH1-UTRs have repressing effect on the translation of marker gene GUS in tobacco protoplasts. (iv) Polypyrimidine tracts as well as putative binding sites for an alba-domain protein were identified in both the 5´ and 3´ UTRs of the POTH1 transcript. (v) Protein/RNA binding assays confirmed interaction of both UTRs with the alba-domain protein and of the 3´ UTR with a polypyrimidine tract-binding protein. Collectively, these results suggest that the full-length mRNA of POTH1 is functional in long-distance trafficking in potato. These findings are published as a paper (Mahajan et al. 2012) Chapter 4: POTH15: Regulation, over-expression and target gene identification. POTATO HOMEOBOX 15 (POTH15) is one of the KNOX-I gene that has been identified and cloned from potato genome. To comprehend the role of POTH15 in potato 122 Summary development, experiments pertaining to regulation, over-expression and target gene identification of POTH15 were conducted. The findings obtained as a part of the POTH15 studies are summarized as follows: (i) Tissue-specific qRT-PCR analysis exhibited a higher abundance of POTH15 mRNA in shoot tips and stolons under tuber inducing short day conditions. (ii) Upstream sequence of POTH15 drives -glucuronidase expression in apical and axillary meristems, petiole, tuber eyes and tuber pith indicating its role in meristem maintenance, leaf development and tuberization. (iii) In accordance with this, over-expression of POTH15 in potato altered multiple morphological traits including leaf development and exhibited early tuberization under in vitro conditions. (iv) Comparative transcriptomic analysis of wild-type and POTH15 over-expression lines identified 435 differentially expressed genes including 79 novel target genes with unknown functions. (v) Functional analysis of these genes revealed their involvement in key biological processes such as metabolism, biotic and abiotic stress responses, regulation of transcription, and signal transduction. (vi) qRT-PCR of selected candidate genes validated their differential expression in wild-type and POTH15 over-expression lines. In conclusion, this is the first report of a tissue-specific photoperiodic regulation of POTH15 and identification of its potential target genes in potato. This study provides new insights into the role of POTH15 and its target genes in regulating plant development and tuberization in potato. The above findings are submitted for a publication (Mahajan et al. 2015).