As seen in Figure 3.6 however, the only significant change produced by 24 hour cytokinin treatments was a doubling of the fluorescent signal, which affected both modified reporters and the pWUS:eGFP-WUS control alike. Expression of pWUS:NLS-eGFP-WUS was more clearly nuclear localized than pWUS:eGFP-WUS as expected, while pWUS:eGFP-NESWUS produced very little fluorescent signal under either treatment. To more accurately estimate the nuclear/cytoplasmic ratio, these resulting confocal images were further analyzed in order to estimate the relative concentration of fluorescent molecules in each subcellular compartment. First the average fluorescent concentration was measured in a portion of each subcellular compartment, and these figures were then multiplied by the volume of the cytoplasm and nucleus, respectively. Nuclei were assumed to be spherical, and their volume was calculated directly from the largest observable diameter. The volume of the whole cell was more difficult to obtain however, as optical sections often only allowed measurements of their length and width, as 3-D optical reconstructions frequently did not include the entire cell volume. Instead, “depth” was estimated using the average of the length and width measurements, reflecting the approximately cubic-rectangular shape of the cells in L1-L3. However, when compared to presumably more accurate cell volumes measured using a tessellation method, the volumes calculated by the present study were on average 2x larger than expected. The present volume estimates did not change significantly when “depth” values were substituted with measurements from unrelated SAM images ,drainage for plants in pots suggesting that these volume estimates are at least reasonably accurate, even if they lack precision.

Curiously, the smallest volumes produced by the tessellation method closely approach the largest nuclear volumes obtained in the present analysis , raising the possibility that this computer-automated method may have occasionally measured nuclei and/or vacuoles instead of entire cells. Overall, pWUS:NLS-eGFP-WUS plants were found to produce about 15% smaller cells, 15% smaller nuclei, and 15% less total fluorescence when compared to pWUS:eGFP-WUS plants, but otherwise both reporters displayed similar subcellular distribution patterns: The L1 and L2 cells had identical nuclear/cytoplasmic ratios, and these values were independent of the total fluorophore concentration in each cell. In contrast, L3 cells had a distinctly elevated nuclear/cytoplasmic ratio that was on average almost twice as large as the upper two cell layers, and as much as 5x more in a outlier data. All nuclei held 2-4x more fluorescent units than would be expected based on their volume alone, yet counter-intuitively, this was just a small fraction of the total number of fluorescent units within the cell. Instead, the majority of fluorescent units were found in the larger volume of the cytoplasm, though at a lower concentration than what occurs inside the nucleus. Cytokinin treatments did not significant change the nuclear/cytoplasmic ratios for either reporter, nor were any layer-specific patterns induced by this treatment. The only clear response to cytokinin treatment was a change in nuclear volume, which increased by an average of 154% in all three layers in both backgrounds. The change in nuclear volume apparently occurred at the expense of the cytoplasm, as the total cell volume remained constant . Unexpectedly, the pWUS:NLS-eGFP-WUS reporter was found to have nuclear/cytoplasmic ratios that was essentially identical to those produced by pWUS:eGFP-WUS.

This is inconsistent with the idea that the NLS tag drives nuclear import, though analysis of the pWUS:NLS-eGFPWUS longitudinal gradient did find that protein movements into the L1 and L2 was slightly restricted , consistent with NLS trapping it inside the nucleus. However, this data also suggests an interesting paradox, as it implies that nuclear trapping occurs without significant nuclear enrichment. Another possible way in which cytokinin responses might affect the distribution of WUS protein is by regulating WUS translation and degradation rates. To study this possibility, the pWUS:eGFP-WUS reporter was exposed to the chemical inhibitors cyclohexmide and MG132, blocking protein translation and proteosome-mediated decay, respectively. Following 4 hour treatments with cycloheximide, no significant loss of fluorescence was found. Unexpectedly, the comparable treatment with MG132 led to the rapid loss of the fluorescent signal. When these chemical treatments were supplemented with exogenous 6-benzylaminopurine to boost the cytokinin response above a basal level however, these patterns were completely reversed. In the conditions used by the present study, elimination of the cytokinin-response free zone could only be achieved with the pCLV3:GR-LhG4 x p6xOP: ARR1ΔDDK-GR system. This does not rule out a negative regulatory pathway though, as weak pTCSn1:mGFP5-ER expression patterns were found in the L1 and L2 cells of clv3-2 mutants . In addition, the weak expression pattern also produced a gradient from L3 up to L1 cells, which is consistent with noncell autonomous movement of cytokinin response proteins. Although the present data cannot identify which proteins might be involved, the most likely candidates would be members of the cytokinin signal transduction pathway, including Arabidopsis Histidine Phosphatase and ARR family proteins. However, in most cases the movements of these native proteins have not yet been studied. The sole exception is ARR7, which when ectopically expressed in L1 cells, was found to move by at least one cell layer.

Presumably, if exogenous cytokinin were applied at high enough concentrations, such non-cell autonomous movement might be sufficient to repress the response-free zone even in WT plants, eventually inducing cell proliferation and WUS expression. Although this experiment was not attempted by the present study, it is interesting to note when extremely high cytokinin concentrations have been used, the SAM has been shown to have higher WUS transcript levels. Exogenous cytokinin applications have even been found to produce a downward expansion of the WUS-expressing cell volume, similar to the results of the pCLV3:GRLhG4 x p6xOP: ARR1ΔDDK-GR system in the present study. Thus it would thus be of interest to determine if the cytokinin-induced increase in WUS transcript levels is due to an increase in the number of cells expressing WUS, or if this reflects an increase in the amount of WUS transcripts per individual cell.From a developmental standpoint, the cytokinin-response free zone appears to be required in order suppress cell division in the underlying RM. This is supported by the massive cell proliferation observed following the loss of the response-free zone in induced pCLV3:GR-LhG4 x ARR1ΔDDK-GR plants. While it is tempting to speculate that the response-free zone is needed to produce a downwardly mobile morphogen that stimulates such proliferation, the elimination of the source would likely produce shoot-ward patterns as the morphogen concentration gradient decays, rather than the rootward pattern that is actually observed. Instead, the suppression of both WUS and CLV3 expression around lateral anlagen even after prolonged dexamethasone treatment in the pCLV3:GR-LhG4 x ARR1ΔDDK-GR background suggests that the repressive signal actually originates in the PZ. As the CZ is known to produce auxin biosynthesis genes CZ, and that cytokinin has repeatedly been found to reduce auxin transport, a likely model suggests that the ectopic cytokinin response in the CZ blocks auxin transport to the PZ. The subsequent failure to activate repressive auxin response factors in the PZ might then favor proliferation over cell elongation.In a developmental context, nuclear trapping has repeatedly been shown to restrict the movement of transcription factors to a single cell layer. The extended range of WUS protein movement over 3-5 cell layers is somewhat inconsistent with a full nuclear trapping model, though the pWUS:eGFP-WUS reporter does clearly show a moderate nuclear pattern. However, the nuclear localization of WUS was found to be largely independent of cytokinin responses, though two other patterns were found instead. The first of these was the enlarged nuclear volume, which was clearly cytokinin-dependent. Similar enlarged nuclei in other angiosperms have been correlated with endo-reduplication, and this is consistent with the enhanced cell proliferation rates seen under prolonged chemical treatments. The absence of any change in the WUS nuclear/cytoplasmic ratio is most easily explained a passive process,30 litre pot as dilution of WUS in an enlarged nucleus would be precisely balanced by an increase in WUS concentration in the cytoplasm, so long as the total cell volume itself did not change. The failure of protein re-distribution to occur following the nuclear volume is harder to explain, as active transport mechanisms through the nuclear pore should presumably restore the original concentrations within a few minutes. No such equilibrium adjustment was detected in the present study, which counter-intuitively suggests that WUS only has a limited ability to move through the nuclear pore. This may reflect the mass of the eGFP-WUS protein, which at 64kDa, is much larger than the 40kDa passive diffusion limit of the nuclear pore. The presence of nuclear fluorescent patterns however, clearly shows that at least some hybrid fluorescent proteins are actively transported into the nucleus Surprisingly, the addition of an extra NLS tag in the pWUS:NLS-eGFP-WUS reporter did not significantly enrich the nuclear localized pattern compared to pWUS:eGFP-WUS. Such a pattern might be expected to occur if WUS has a native NLS motif, which would make the added NLS tag is functionally redundant. The present data however, cannot rule out the possibility that NLS tag is blocked by some aspect of WUS structure.

The NLS-eGFP-WUS protein is 8% larger than eGFP-WUS, and this figure is reminiscent of the 15% reduction found in pWUS:NLS-eGFP-WUS meristems, and might suggest that the limited mobility of the NLS construct reflects a size-dependent fractionation process, rather than nuclear trapping. The continued presence of WUS in the cytoplasm however, requires the existence of a nuclear export mechanism to balance out the effects of nuclear import. Although such a function has not been attributed to the WUS protein, the EAR-like motif present in its C-terminus closely resembles a lysine rich NES motif. The same motif is also recognized by TPL , though it is not clear which function predominates in WUS. Even if EAR-like motif functions as a nuclear exit sequence however, a system based exclusively on nuclear pore transport would likely shift the equilibrium to one extreme state or another rather than be perfectly balanced at an intermediate state. One clue about how a stable intermediate is achieved comes from the pWUS:eGFP-NES-WUS construct, whose fluorescent pattern is very weak, but is not zero , suggesting that WUS is rapidly degraded in the cytoplasm. This observation also indicates that WUS EAR-like motif has at best a weak NES function, as the added NES tag could only have such a strong impact if the native WUS molecule lacked a strong native NES function.Together these observations indicate that only small fraction of WUS proteins are transported into the nucleus, while those that remain in the cytoplasm are degraded. Rather than being nuclear enriched, this situation is probably more accurately described as cytoplasmic depletion. Interestingly, the WUS subcellular pattern closely parallels a similar situation for Cytidylyltransferase proteins in the Mouse model, where mono-ubiquitination of the NLS motif was demonstrated to prevent nuclear import, and was further associated with higher rates of proteolytic degradation of CCTα in the cytoplasm.On the basis of current evidence, it seems likely that cytokinin responses are necessary for WUS protein stability. This may reflect a common trend for SAM expressed genes, as cytokinin exposure is also known to increase the stability of ACS and ARR1. Evidence of a positive relationship is perhaps best seen in the ahk2/3/4 receptor mutant background, where WUS mRNA is transcribed normally in the complete absence of cytokinin responses , yet the translated GFP reporter is barely detectable . Conversely, when cytokinin responses are induced by pCLV3:GR-LhG4 x ARR1ΔDDK-GR, we see an increase in WUS proteins in the L1 and L2 cells after 48 hours, but little or no WUS transcription in these cells . This suggests that the WUS proteins that move into the L1 and L2 cells are rapidly degraded in the absence of strong cytokinin responses, but become protected following ectopic cytokinin activation. However, the idea of a positive correlation begins to break down when p35S:GRLhG4::p6xOP:CKX3 induction is considered, because this did not obviously reduce pWUS:eGFP-WUS fluorescent levels . The fact that the peak fluorescence shifted down by one layer might indicate that the response-free zone became larger, but the variability of this construct makes it difficult to draw firm conclusions. Another potential problem can be found in pCLV3:GR-LhG4 x ARR1ΔDDK-GR x pWUS:eGFP-WUS plants that have been induced with dexamethasone for 48 hours.