TCSv2 design and increased sensitivity
TCSv2 is a variant of TCSn, with alternating head-to-head and tail-to-tail orientations of type B ARR-binding sites compared with the tandem tail-to-tail and head-to-head orientation of sites in TCSn (Fig. 1A). TCSv2 demonstrated increased sensitivity in mesophyll protoplast transient assays (Fig. 1A), as well as in arabidopsis floral meristems (Fig. 1B).
CK responsiveness of TCSv2 in various tissues in both arabidopsis and tomato
We cloned TCSv2 according to the description in the methods section, and introduced constructs in which TCSv2 drives VENUS or GUS (β-glucuronidase) expression into tomato and arabidopsis. To examine CK responsiveness of TCSv2 in vivo, we conducted a series of experiments examining TCSv2 driven expression with and without CK treatment in a variety of plant tissues in both arabidopsis and tomato (Fig. 2). For VENUS analyses conducted in arabidopsis, two representative transgenic lines exhibiting moderate (TCSv2:3XVENUS#2) and strong (TCSv2:3XVENUS#7) VENUS expression were selected for the analysis. For VENUS analyses conducted in tomato, several lines were screened, which demonstrated similar VENUS expression levels. One of these was selected for further analyses.
TCS driven VENUS expression is observed primarily in meristematic tissues in the arabidopsis shoot and root, as was reported for previous TCS versions (Fig. 2A,E). Similarly, strong expression was observed in tomato SAM and RAM (Fig. 2G,K). Interestingly, in tomato, strong expression was also observed in the leaf marginal blastozone (Fig. 2G), a meristematic region present in leaf margins and expanded in margin of compound leaves . Upon CK treatment, the VENUS pattern of expression expands to the cotyledons and hypocotyl in arabidopsis (Fig. 2B), and becomes stronger in tomato shoot apexes (Fig. 2H). In roots of both arabidopsis and tomato, TCS driven VENUS expression is observed in the root apex, presumably localized to the root apical meristem, as well as in the columella and internal stele (Fig. 2E,K). This pattern is strengthened and expanded following CK treatment (Fig. 2F,L). TCSv2:NLS-3XVENUS responds to CK treatment in a dose dependent manner in both arabidopsis and tomato (Supplemental Fig. 1), and results obtained in a Ler arabidopsis background were similar (Supplemental Fig. 2). For some tissues, such as shoot apices, tomato may require larger concentration doses of CK to achieve a similar strength response in the same time frame as arabidopsis.
In order to examine the time course of the response of TCSv2 to CK treatment, we utilized a TCSv2:GUS construct (the VENUS construct contains 3 repeats of the VENUS protein and is therefore unsuitable for qPCR analysis). We first characterized GUS expression in arabidopsis and tomato (Fig. 3). Time course experiments show that TCS-driven GUS mRNA peaks 2 hours after CK treatment in arabidopsis (Fig. 3C), and declines soon thereafter. Examination of TypeA ARR genes in the same samples, shows that, as previously reported [28, 29], ARR5 and ARR7 respond earliest, within half an hour of CK treatment (Fig. 3C). In tomato, TCS-driven GUS mRNA peaks 3 hours after CK treatment (Fig. 3D), rising and falling slightly slower than in arabidopsis, perhaps reflecting the higher amount of CK needed to elicit a similar response. When comparing TRR expression in tomato to that of the TCS-GUS mRNA, GUS mRNA expression rises in a manner similar to that of TRR16B and TRR3/4, but to a greater degree, rising slowly and peaking 2–3 hours from CK treatment, while TRR5/6/7 rises more quickly, showing significant increase in expression 30 minutes after CK treatment, similar to arabidopsis ARR7 (Fig. 3C,D). TCSv2 can thus be viewed as an “averaging” output of Type A ARR response in terms of time course, representing a response to CK that is later than the earliest responding ARRs but earlier than the later responding ones. Also of note is that in both arabidopsis and tomato, reflecting perhaps a combined response output that is normally “divided” between several RR genes, TCSv2 responds more strongly to CK treatment than any one individual RR. This should be taken into account when conducting analyses using TCSv2.
TCSv2 driven expression is affected by alterations of endogenous CK level in tomato
To examine whether TCSv2-driven expression responds to alterations in endogenous CK level alterations in tomato, we backcrossed the tomato VENUS line into transgenic plants overexpressing the arabidopsis CK biosynthsis enzyme isopentenyltransferase7 (IPT7) or CK catabolic enzyme CYTOKININ OXIDASE/DEHYDROGENASE-3 (CKX3) , driven by the FIL promoter. As can be seen in Fig. 4, the TCSv2 sensor responds to an increase in endogenous CK with elevation of VENUS expression (Fig. 4D-F) and to a decrease in endogenous CK with a decrease in VENUS expression (Fig. 4G-I), indicating that the sensor is useful for examining both exogenous and endogenous changes in CK levels and CK pathway enzymes. Indeed, we recently successfully utilized the TCSv2 sensor to analyse endogenous effects of different genetic background manipulations on the CK pathway [31–33].
TCSv2 responds specifically to CK treatment
The balance between different hormones is one of the underlying mechanisms serving plant development, growth, and response to various cues. Cytokinin and gibberellin, as well as cytokinin and auxin, can antagonize each other or act in concert in a variety of processes throughout plant development [30, 34–43]. We therefore tested whether the TCS sensor could possibly respond to additional cues other than CK treatment. Figure 5 demonstrates that TCSv2 responds specifically to CK and does not respond to additional tested hormones other than CK, in both tomato (VENUS protein expression 12 hours after CK treatment, Fig. 5A and Supplemental Fig. 3A-E) and arabidopsis (GUS mRNA expression 2 hours after CK treatment, Fig. 5B). This indicates that the TCSv2 sensor is specific and accurate, in addition to being robust, in the detection of CK response in plants.
Characterization of TCSv2 driven expression throughout development
The observation that TCSv2 is primarily visible in meristematic tissues, along with published analyses of previous TCS versions in arabidopsis development, prompted a more in depth examination of TCSv2 in various developmental contexts. Figure 6 presents an analysis of TCSv2 driven expression throughout shoot and leaf development in tomato (Fig. 6A-E) and arabidopsis (Fig. 6F-J). In tomato, TCSv2 is expressed in the SAM and at the margin of young leaf primordia. The TCS expression domain, which likely correlates with the marginal blastozone , appears to be wider in younger primordia (Fig. 6A-C), and becomes localized and quite thin in older primordia (Fig. 6D-E), consistent with the notion that the young leaf undergoes morphogenesis and reaches maturation concurrently with the loss of its morphogenetic potential and meristematic tissues. This is evident in Supplemental Fig. 4, which shows older tomato leaves in which the TCS driven signal is localized to the margins of the developing leaflets only (Supplemental Fig. 4E, the VENUS signal was color coded in dark blue to make it more visible). In contrast, in arabidopsis, which has a simple leaf, the limited morphogenetic potential retained by young leaf primordia serves in the execution of leaf marginal patterning. As such, TCSv2 driven expression can be observed in the SAM (Fig. 6F), at the adaxial side of the leaf base in young leaf primordia (P1-P3, Fig. 6G), becoming restricted as the leaf matures, in-line with the basipetal differentiation of the arabidopsis leaf. TCSv2 is also present throughout the young leaf venation (Fig. 6H-J), and localized to a small number of cells which mark the leaf tip and the peak/tip of a nascent marginal serration (Fig. 6H-J, marked with asterisks), which presumably maintain some form of meristematic qualities to allow for subsequent leaf marginal patterning, which is dependent on CK response. Consistent with the short marginal blastozone activity, TCSv2 is not observed throughout the leaf margin in arabidopsis.
TCSv2 marks the zone of the incipient axillary bud
CK response has been reported to be crucial in the establishment of the axillary bud [44, 45]. Utilizing TCSv2, we followed the generation of the axillary shoot in tomato. We were able to observe CK response signal in the axils of leaf primordia from the P7 stage onward (Fig. 7). At P7, the TCSv2 signal is present in the leaf axil, though no axillary bud or axillary meristem dome has yet formed (Fig. 7B). At later stages, in the P8-P10 axil, an activated bud with a characteristic TCSv2 signal in the meristem and the margins of the developing leaf primordia can be observed (Fig. 7C,D). Interestingly, after induction of flowering, we see TCSv2-driven expression in the axils of younger, P6 primordia (Fig. 7E). When the reproductive transition state is coupled with elevation of endogenous Cytokinin present in pFIL > > IPT7 overexpressing plants, the TCSv2 signal is observed in the axils of even younger primordia – P4 and P5 (Fig. 7F). Notably, TCS driven GFP expression using the first TCS version  was observed in the leaf axils of P6 and older leaf primordia in arabidopsis .
TCSv2 driven expression is observed in all stages of reproductive organ development
As reported previously and here above, CK response and TCSv2 driven expression are primarily observed in meristematic tissues. This is the case also in reproductive organ development (Fig. 8). TCSv2 is observed in the transition to flowering in the domed meristem (Fig. 8A), the transitional meristem (Fig. 8B), and the inflorescence and floral meristems (Fig. 8C). Once reproductive organs have formed, we can observe TCSv2 in the anthers and filaments (Fig. 8D,E) and the ovules (Fig. 8E), indicating that CK response is required for proper reproductive organ development.
In mature embryos, TCSv2 driven expression is observed very strongly in the region of the RAM (Supplemental Fig. 5A-C), but, interestingly, can barely be seen in the progenitor cells of the SAM, both in desiccated and imbibed seeds, even after germination (Supplemental Fig. 5A-C). Following cotyledon expansion and generation of the nascent SAM structure, TCSv2 driven expression can be observed in the meristem and young P1(L2) and P2 (L1) leaves (Supplemental Fig. 5D,E). Different versions of TCS can therefore be used in order to obtain a full picture of CK response in plant tissues. A combination of different TCS promoters could be useful, depending on the exact nature of the processes being observed.