Final Report Summary - CASDEV (The role of Casparian strip formation and degradation in primary and lateral root development) In all vascular plants the endodermis functions as a crucial barrier, as it separates the outer apoplastic space of the cortical cell layers, often in direct contact with the soil, from the inner apoplastic space of the vascular bundle. To create this barrier, endodermal cells secrete hydrophobic material in a highly localised and coordinated manner at all four lateral sides of the cell, resulting in the formation of the 'Casparian strip' (CS). The CS was believed to be composed of cork-like suberin, cross-linked into an extensive supra-molecular network. However, recent data from the host lab now indeed confirms that in Arabidopsis thaliana the (early) CS is made out of lignin and not suberin (Naseer et al., 2012). Moreover, the plasma membrane (PM) underlying the CS is more electron dense and ordered, suggesting the presence of protein scaffolds that are tightly attached to the extracellular matrix. We have shown CS domain protein 1-5 (CASP1-5) localise and probably organise the formation of this diffusion barrier in the PM of endodermal cells. Moreover, we hypothesised that the CASP proteins form the direct the deposition of lignin-like material in the cell wall of endodermal cells. (Roppolo et al., 2011). The presence of this ring of lignin-like material in the longitudinal plane of endodermal cells should have strong impact on different aspects of root growth and branching. The objectives of this proposal were set out to address these questions. The goal of the first objective was to determine whether the CS affects the overall root growth, as its presence would make it impossible for those cells to elongate any further. It was reported that interfering with gibberellic acid (GA) signalling specifically in the endodermis caused severe retardation of root growth (Ubeda-Tomas et al., 2009). To determine if this effect was caused by the presence of the CS, these mutant lines where GA-signalling was only affected in the endodermis, were crossed with novel CS-mutants isolated in the host lab. Soon we will know if these double mutants still display the extreme growth retardation. As the CASP proteins are the first proteins described to localise to the CS domain (CSD) they provided us for the first time with tools to study the organisation of the CSD in living cells using green fluorescent protein (GFP) fusions and live cell imaging. Therefore, the second objective was aimed to describe the organisation of the CSD and the CS itself. We could show that the CASP1 proteins first localise homogenously at the PM around the cell and then gradually start to localise to form the ring-like structure. This is a gradual process as the CASP proteins first form a dotted ring-like structure in the middle of the cell, which in 2 hours becomes a completely closed ring. In addition, through the use of photo-activatable fluorescent fusions with CASP1, we could show that as soon as the CASP proteins start to localise to the CSD, they are immobile. This supports the hypothesis that they form large protein scaffolds. We are now pursuing super-resolution imaging to get more insight into this process. Root branching through the formation of lateral roots (LRs) is important for proper development of plants, as this facilitates enhanced water and nutrient uptake. Interestingly, LRs originate in the pericycle, a single cell layer below the endodermis, and consecutively need to grow through all above-lying cell layers in order to emerge on the surface of the root. However, at the time of LR initiation, the fully elongated endodermal cells already contain a CS. This would suggest that in order to emerge, plants have devised a mechanism to locally and temporally undo the CS, so that the LR can emerge. This process would need to be tightly regulated, since breaking of the CS will result in loss of nutrients from the vascular tissue into the apoplast, but also would make the plant more vulnerable to pathogens that normally would be blocked by the CS. The last 2 objectives of this proposal deal with this process. Using live-cell imaging using CASP1-GFP together with different organelle markers (PM, tonoplast nucleus), we investigated the fate of the endodermis during LR formation. Our results strongly support our initial hypothesis that LR emergence is a very regulated process. Moreover, endodermal cells overlying a LR primordium do not seem to die in the process of emergence. Instead, we observed very regulated changes in cellular architecture of endodermal cells allowing passage of the newly formed LR. In addition, we also could show that when the newly-formed LR will pass through the endodermis, small holes seem to appear in the CS network overlying the LRP. The next step was to identify plant factors that regulate CS modification allowing emergence. To do this we undertook a transcriptomics approach. It was previously suggested that endodermal auxin signalling was involved LR emergence. To test this hypothesis, we specifically blocked auxin signalling in the endodermis through expression of shy2-2, a dominant repressor of auxin signalling, in elongating endodermal cells using the CASP1 promoter. This resulted in plants with no emerged LRs at all, confirming our hypothesis that SHY2 regulates CS-modifying enzymes. However, upon closer inspection we observed that the proCASP1::shy2-2 plants did not even initiate LRs at all. Exogenous addition of auxin resulted in the formation of new LRs that displayed a strong defect in emergence compared to wild-type plants. Through these experiments we have discovered a completely new module in LR formation. This reveals that early auxin signalling between the endodermis and the pericycle is required for LR formation. We hypothesise that impaired mechanical sensing between the pericycle and endodermis is causing this phenotype. In parallel, we are using this mutant for microarrays studies to identify new factors regulating the early communication between the pericycle and the endodermis, as well as factors responsible for the modification of the CS. Overall, we have created a lot of new insight in how the CS gets organised and soon will know the answer whether the CS limits primary root growth. In addition, we have created a lot of new insight regarding the role of the endodermis during LR formation, something that had been largely ignored till now. Through this proposal we have revealed a whole signalling pathway that is crucial for LR formation and now provides an excellent working model to study the role of mechanical sensing during organ formation. We expect that the results of the microarray experiments will lead to the discovery of novel proteins involved in lignin modification and/or degradation. These finding will further strengthen Europe's leading role in research on root development. The newly discovered mechanical sensing module will fuel this emerging field in plant biology in Europe. The potential discovery of lignin-modifying enzymes could lead to novel patent applications and strengthen the research on second-generation biofuels in Europe.
