Characterization of the Cytokinin Sensor TCSv2 in Arabidopsis and Tomato

Abstract

Background: Hormones are crucial to plant life and development. Being able to follow the plants hormonal response to various stimuli and throughout developmental processes is an important and increasingly widespread tool. The phytohormone cytokinin (CK) has crucial roles in the regulation of plant growth and development.Results: Here we describe a version of the CK sensor Two Component signaling Sensor (TCS), referred to as TCSv2. TCSv2 has a different arrangement of binding motifs when compared to previous TCS versions, resulting in increased sensitivity in some examined tissues. Here, we examine the CK responsiveness and distribution pattern of TCSv2 in arabidopsis and tomato. Conclusions: the increased sensitivity and reported expression pattern of TCSv2 make it an ideal TCS version to study CK response in particular hosts, such as tomato, and particular tissues, such as leaves and flowers.

Cytokinin-microbiome interactions regulate developmental functions

“…During leaf development, the young leaf undergoes morphogenesis and reaches the mature, differentiated stage of its development simultaneously with the decline in its morphogenetic potential. Compound leaves of tomato are composed of multiple leaflets, which initiate basipetally from a meristematic region at the leaf margin known as the marginal blastozone [ 29 , 51 , 52 ]. The leaf morphogenetic potential is harbored by meristematic cells, which respond to CK and therefore exhibit TCS activation.…”

“…Primer sequences used for the qRT-PCR analyses are detailed in Additional file 1 : Table S1 [ 22 , 30 ]. For developmental gene analysis, we chose genes related to boundary definition, which is important for organ initiation [ 30 , 52 ], meristem maintenance, which is important for increased morphogenesis [ 26 ], and, given the TCSv2 activation results, genes of the CK pathway. Expression of all assayed genes was normalized relative to a geometric mean of the copy number of the three tomato housekeeping genes, ribosomal protein SlRPL8 (Solyc10g006580), Slcyclophilin (Solyc01g111170) and SlEXP (Solyc07g025390).…”

“…The number of leaves was counted by dissecting the shoot under a stereomicroscope and counting all the initiated leaves, starting from P1. Differentiation of the meristem to floral and sympodial follows a predictable pattern in S. lycopersicum M82 [ 52 , 65 ], and was analyzed microscopically in dissected shoots. Leaves are produced successively on the plant, and at a given time point each leaf is at a different developmental stage.…”

“…The TCS sensor was designed using the conserved DNA binding domain in the promoter of type A response regulators (RRs) that is recognized by type-B response regulators. The CK sensor TCSv2 was created based on the CK phosphorelay network, using elements derived from the conserved DNA binding domain in type A RRs [ 52 ]. For TCSv2:3XVENUS, the DNA sequence of TCSv2 was synthesized with flanking NsiI and BamHI restriction site [ 30 ] and the synthetic promoter was then cloned adjacent to 3xVENUS-N7 in the pBJ36 vector [ 52 ].…”

Imaging the living plant cell: From probes to quantification

“…Indeed, plant hormones regulate gene expression via transcription factors specific to each pathway that recognize specific binding sites . Strategies similar to the one used for DR5 were then leveraged to design transcriptional biosensors for cytokinins (Muller and Sheen, 2008;Zurcher et al, 2013;Steiner et al, 2020), ethylene (Stepanova et al, 2007, and abscisic acid (ABA) (Table 5) (Wu et al, 2018). While endogenous promoters of hormone-responsive genes have also been used to analyze the transcriptional responses to hormones, the use of synthetic promoters increases the specificity of the response of the biosensor to a given hormone.…”