Rather than increasing the fluorescent signal in the deep cell layers, the entire triangular pattern of pWUS:eGFP-WUS shifted down by one cell layer. This occurred with little or no loss in fluorescent amplitude, though the slope of the upper gradient became slightly shallower in proportion to greater distance separating L1 cells from the new concentration peak.WUS proteins are known to occur in a nuclear localized pattern, which suggests involvement with nuclear pore trafficking mechanisms. To determine if cytokinin regulate WUS movements through the nuclear pore, two modified version of the pWUS:eGFP-WUS reporter were obtained, which contained nuclear import and nuclear export sequences tags: pWUS:NLS-eGFP-WUS, which was previously described by [20], and pWUS:eGFP-NES-WUS. If cytokinin favors nuclear import over export, or vice versa, one of these constructs should amplify the nuclear/cytoplasmic distribution of the fluorescent signal, producing a significantly different fluorescent pattern. 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 modifiedreporters 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, black plastic planting pots 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 , 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.The absence of cytokinin responses in the CZ is a novel feature of SAM organization, whose existence was clearly revealed by the distinct absence of pTCSn1:mGFP5-ER expression in response to exogenous cytokinin . Although it has been shown that this tissue lacks significant expression of hormone-response genes, the centripetal spread of pTCSn1:mGFP5- ER expression following pCLV3:GR-LhG4 x p6xOP: ARR1ΔDDK-GR induction indicates that the absence of a cytokinin response is also accompanied by a repressive mechanism as well. Presumably this mechanism works on a protein level, as the ARR1ΔDDK-GR protein is unaffected by cytokinin signaling pathways. Given the appearance of cytokinin activity in the peripheral zone of pCLV3:GR-LhG4 x p6xOP: ARR1ΔDDK-GR plants following just 6 hours of dexamethasone treatment, this also argues against a radial degradation gradient, because the ARR1ΔDDK-GR hybrid protein would likely be destroyed before it ever reached the PZ, and degradation alone would likely still allow faint pTCSn1:mGFP5-ER activity in the CZ. Instead, it seems more likely that the ARR1ΔDDK-GR simply can’t bind to its DNA target sites, perhaps due to chromatin silencing, which might also explain the lack of hormone signalling pathway expression in these cells. However, a degradation model is consistent with the linear pWUS:eGFP-WUS concentration gradients observed in the L1-L3 tissues, whose slopes were rigidly maintained despite fluctuations in total protein concentrations . While stem cell identity is known to require the migration of WUS proteins into the overlying CZ cells, the response-free zone continues to be clearly visible even in wus-1 mutants. The zone was also present in clv3-2 mutants, and the lack of response by pCLV3:mGFP5-ER to a variety of different cytokinin treatments strongly suggests that CLV3 expression is not regulated by cytokinin responses. One possible exception is the reduced expression levels of CLV3 in ahk2/3/4 plants, though the near-absence of WUS proteins in this mutant background might suggest that CLV3 is simply not strongly activated. The potential link between cytokinin and WUS transcription is a bit harder to dis-entangle though, as ectopic cytokinin responses produced by the pCLV3:GR-LhG4 x p6xOP: ARR1ΔDDK-GR system did significantly increase the number of WUS-expressing cells, all ofwhich also had strong cytokinin responses by 48 hours. Close examination of RNA in-situ images however, reveals that WUS is expressed even in the complete absence of cytokinin responses, both in subsets of SAM tissue in the two-component system, and in the ahk2/3/4 mutant. However, this pattern may be tissue-specific, as the RNA in-situ images also show that WUS is not strongly expressed in the L1 and L2 of the pCLV3:GR-LhG4 x p6xOP: ARR1ΔDDK-GR system, drainage pot which is true whether or not cytokinin responses occur in those cells. In addition, the pWUS-eGFP-WUS fluorescence level was also largely unchanged when cytokinin levels were reduced with the p35S:GR-LhG4::p6xOP:CKX3 construct. Based on these observations, it seems quite likely that WUS transcription responds directly to cytokinin responses. Although the number of WUS-expressing cells does dramatically increase following prolonged induction of cytokinin responses in the response-free zone, this appears to be a secondary effect that occurs after the cells have acquired stem cell identity.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 non-cell 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 signalactually 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, 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.