Characterization of Tomato Cytokinin Response Factor Genes SlCRF1 and SlCRF2 in Tomato Growth and Development

Abstract

Cytokinin is a plant hormone that plays a myriad of roles in plant growth and development. Cytokinin is perceived in plants by a multiple-step phosphorelay that is similar to bacterial two component system (TCS). In addition to this main cytokinin signaling pathway, a branch pathway has been identified of which Cytokinin Response Factors (CRFs) are important components that act as transcription factors. The Arabidopsis CRFs have been shown to play a role in leaf and embryo development, although almost nothing is known about the role of CRFs in other species. This study addresses this in tomato using Micro-Tom, a miniature model variety to investigate the role of CRFs in this system: known as Solanum lycopersium CRFs or SlCRFs. Eleven different SlCRFs were identified and examined with particular attention here directed to understanding SlCRF1 and SlCRF2, in growth and development of tomato plants with a focus on the leaf-related processes. Examination of these SlCRF responses to hormone (especially cytokinin) and abiotic stress treatments indicates that these genes may play a variety of roles in the regulation of these processes in tomato. qRT-PCR analysis revealed that both SlCRF1 and SlCRF2 are inducible by cytokinin in leaves and stems, with SlCRF2 induced to a much greater level in leaves. Examination of the expression of these two genes in leaf, stem, and roots of different ages indicates that SlCRF2 transcript levels are higher in younger than older organs while SlCRF1 shows an opposite expression pattern. Analysis of promoter::GUS reporter lines revealed that both SlCRF1 and SlCRF2 are predominantly expressed in the vasculature of leaf, stem, roots, and fruits. To gain a general understanding of the tomato leaf transcriptome and how it might be regulated by cytokinin, next-generation RNA sequencing has been performed on leaf samples of two ages, 13d and 35d, treated with cytokinin or the solvent vehicle control dimethyl sulfoxide (DMSO). This analysis revealed a large number of novel cytokinin regulated transcripts and provides a solid foundation for the future study of cytokinin and cytokinin regulated genes involved in compound leaf development and other developmental processes in tomato.