Interestingly, suppression of endodermal ABA signalling seems to contribute to the inactivation of aquaporin-mediated Lpr in a wild-type Scheduling low but frequent NO3 − applications, at-tuned to crop demand, allows the crop to take up most of the NO3 − before it passes through the low-salinity zone into the saline fringes. Figure 7 simulates continuous NO3 − application and a scenario which applies NO3 − only every 10 d, while the total amount of NO3 − applied is the same for both simulations. High-frequency applications of NO3 − using drip irrigation in-creased N uptake efficiency in some cases .Both Casparian strips and suberin lamellae, two extracellular hydrophobic barriers located in the wall of endodermal cells of the root, are thought to play important roles in restricting the free diffusion of solutes and water . Casparian strips act as apoplastic barriers not only to block solutes moving into the xylem through the free space between cells, but also to prevent their backfow from the stele to the apoplast of the cor-tex. Suberin lamellae, due to their deposition between the endodermal plasma membrane and secondary cell wall, do not block aploplastic transport but rather limit transcellular transport of nutrients and possibly water at the endodermis. Cross talk between the Casparian strip and suberin lamellae exists, with suberin being deposited in response to disruption of Casparian strips . Tese extracellular barriers are therefore at a cross-road between control of mineral nutrient and water uptake. However, the mechanisms that allow plants to integrate both these barrier functions to enable the simultaneous uptake of sufcient water and mineral nutrients remain under explored. Te dirigent-like protein Enhanced Suberin1 functions in the correct formation of Casparian strips by allowing the lignin, deposited at the Casparian Strip Domain through the action of Peroxidase64 and the Respiratory Burst Oxidase Homolog F ,blueberry packaging to form into a continuous ring. In the absence of this dirigent-like protein defective Casparian strips are formed along with enhanced and early deposition of suberin in the endodermis.
A similar pattern of Casparian strip disruption and response is also observed when the Casparian Strip Domain is disrupted through the loss of Casparian Strip Domain Proteins. Tese changes lead to systematic alterations in the profile of mineral nutrients and trace elements accumulating in leaves, and this phenotype provided the first tool for identification of genes involved in Casparian strip development. Detection of the diffusible vasculature-derived peptides CASPARIAN STRIP INTEGRITY FACTORS 1 & 2 through interaction with the SCHENGEN3 receptor-like kinase is what drives this endodermal response to loss of Casparian strip integrity. Here, we report that detection of a loss of Casparian strip integrity at the root endodermis by the CIFs/SGN3 pathway leads to an integrated local and long-distance response. This response rebalances water and mineral nutrient uptake, compensating for breakage of the Casparian strip apoplastic seal between the stele and the cor-tex. This rebalancing involves both a reduction in root hydraulic conductivity driven by deactivation of aquapor-ins, and limitation of ion leakage through deposition of suberin in endodermal cell walls. This local root-based response is also coupled to a reduction in water demand in the shoot driven by ABA-mediated stomatal closure.Te dirigent-like protein Enhanced Suberin1 functions in the formation of Casparian strips by allowing the correct deposition of lignin at the Casparian strip domain. Te enhanced deposition of suberin in the esb1-1 mutant with disrupted Casparian strips can clearly be observed using the lipophilic stain Fluorol Yellow 088 close to the root tip , and this can be quantified by counting the number of endodermal cells afer the onset of cell expansion to the first appearance of yellow fuorescence . This early deposition of suberin is also verifed by the clear correspondence of FY 088 staining with enhanced promoter activity of known suberin biosynthetic genes, including GPAT5 monitored through both GUS staining and GFP fuorescence , and also others through GUS staining . This is further reinforced by the enhanced expression of known suberin biosynthetic genes in esb1-1 relative to wild-type . To better understand the causal link between Casparian strip integrity and enhanced deposition of suberin, we performed a reciprocal grafing experiment that revealed that the esb1-1 mutation is only required in the root to drive enhanced deposition of suberin at the endodermis, placing the function of ESB1 and the driver for increased suberin in the same tissue .
To determine the cause and effect rela-tionship between damaged Casparian strips and enhanced suberin we carefully monitored the first appearance of both Casparian strips and enhanced suberin in esb1-1. Using lignin staining in the Casparian strip marker line pCASP1::CASP1::GFP, we are able to observe that damaged Casparian strips are visible 2.5 days afer sowing . This is at least 12hr before the first indication of enhanced suberin biosynthesis, which we monitor using promoter activity of suberin biosynthetic genes GPAT5, FAR4, FAR1 and FAR5 . This was also verified by the direct observation of suberin deposition with FY 088 . Te observation that treatment with the CIF2 peptide, normally leaked from the stele through loss of Casparian strip integrity, can enhance suberin deposition in wild-type plants supports our interpretation that enhanced suberin deposition is a response to loss of integrity of the Casparian strip-based apoplastic diffusion barrier. Furthermore, loss-of-function of the receptor-like kinase SGN3, required for sensing of CIFs, blocks the enhanced deposition of suberin in esb1-1 and casp1-1casp3-1 based on a chemical analysis of suberin in esb1-1 , and also on FY 088 staining. We conclude that Casparian strip defects sensed by the CIFs/SGN3 surveillance system lead to enhanced deposition of suberin in the endodermis.Te observation that enhanced suberin is deposited as a response to loss of integrity of the endodermal-based diffusion barrier between stele and cortex, raises the question, what is the function of this increased suberin deposition? Previously, the extent of endodermal suberin has been shown to be part of the response to nutrient status . We therefore tested the selectivity to solutes σNaCl, in roots varying in the extent of suberin deposition and the functionality of Casparian strips. For this, we measured solute leakage into xylem sap of pressurized roots at increasing sodium chloride concentrations in the solution bathing the roots. Taken individually, σNaCl of roots of esb1-1, sgn3-3 and wild-type were not significantly different from one another , which is surprising given the disruption of the Casparian strip-based apoplastic diffusion barrier in both mutants.
However, removal of suberin in esb1-1, by endodermal-specific ectopic expression of a cutinase ,blueberry packaging box caused a significant decrease in σNaCl compared to wild-type plants , and a similar tendency when compared to esb1-1 . This supports the notion that enhanced suberin deposition at the endodermis helps prevent passive solute leakage caused by defects in the Casparian strips of the esb1-1 mutant. We also observed a significant decrease in σNaCl in the double mutant esb1-1sgn3-3 compared to both wild-type and sgn3-3 . It is known that SGN3 is required for the enhanced deposition of suberin that occurs at the endodermis in esb1-1 . Our observation that removal of this enhanced suberin in esb1-1sgn3-3 decreases σNaCl further supports our conclusion that the role of this increased suberin deposition is to limit solute leakage where Casparian strip barriers are disrupted.It has also been suggested that endodermal suberin may impact water permeability, though how is still unclear. To further address the role of enhanced endodermal suberin, we investigated root hydraulic conductivity of esb1-1 and observed a significant reduction by 62% with respect to wild-type . Importantly, this difference in esb1-1 Lpr originates mainly from a reduction in an aquaporin-mediated water transport pathway . We also observed that the azide-resistant water transport pathway was lower in esb1-1 than in wild-type , yet to a lesser extent than the aquaporin mediated pathway. Te dra-matic reduction in aquaporin-mediated Lpr in esb1-1 we observe is an intriguing fnding, which led us to consider if this lack of aquaporin activity in esb1-1 roots is due to a direct output from the CIFs/SGN3 signalling pathway, or if it represents an efect downstream of enhanced suberin deposition. We found that removal of endodermal suberin in esb1-1 through expression of CDEF1 in the endodermis had no further efect on Lpr . This rules out a role for suberin in the reduced aquaporin-mediated Lpr of esb1-1. However, in the esb1-1sgn3-3 dou-ble mutant, as compared to esb1-1, we observed a full recovery of Lpr back to wild-type levels . Loss of Casparian strip integrity in esb1-1 therefore appears to be sensed by the CIFs/SGN3 signalling pathway, which leads to the inactivation of aquaporins, thereby reducing Lpr . To support this conclusion, we show that exogenous application of CIF2 to wild-type plants for 3h induces a reduction in Lpr, and only in the presence of a functional SGN3 . We have established the existence of two critical outputs of the CIFs/SGN3 diffusion-barrier surveillance system. Tese are enhanced deposition of endodermal suberin limiting solute leakage, and the inactivation of root aquaporin activity reducing Lpr. Do these two independent outputs of the CIFs/SGN3 diffusion barrier surveillance system work in parallel, or in series with one response leading to the other? Te fact that removal of endodermal suberin in esb1-1 does not compensate for its reduced Lpr suggests that enhanced suberin deposition is not the cause of the reduced aquaporin-mediated Lpr. However, reduced activity of aquaporins through loss-of-function of the two major aquaporins PIP2;1 and PIP2;2 in the pip2;1pip2;2 double mutant, does cause significant increases in endodermal suberin deposition . A similar increase in suberin is also observed afer treatment with the aquaporin inhibitor sodium azide through observation of the activity of the transcriptional reporter pGPAT5::mCITRINE-SYP122 for suberin biosynthesis. GPAT5 expression is observed to expand toward the root tip after 6 hours only of sodium azide treatment .
Based on this evidence, we propose the following sequence of events. Casparian strip defects are detected by the apoplastic leakage of CIFs from the stele, being sensed by SGN3. Once activated, SGN3 signals the inactivation of aqua-porins thereby reducing Lpr which in turn leads to the early and enhanced deposition of endodermal suberin. Insuch a model, SGN3 would inhibit aquaporin function, which may appear at variance with the usual activation of aquaporins through phosphorylation. Yet, such an inhibition was recently described in the case of FERONIA, a protein kinase inactivating PIP2; 1 function through an as yet unknown mechanism.Abscisic acid has been shown to be involved in regulating both aquaporin activity reviewed in and suberin deposition, making ABA an interesting can-didate worth exploring for a role in downstream CIFs/SGN3 signalling. To probe this possibility we expressed the dominant negative allele of the regulator of ABA signalling ABA-INSENSITIVE 1 in the endoder-mis of esb1-1 using pELTP::abi1. This abi1 construct specifically blocks ABA signalling at the endodermis and delays suberisation in a wild-type background as previously shown in . In esb1-1, we observed abi1 to have no effect on either the inactivation of aquaporins or the enhanced deposition suberin . We also observe that aquaporin inhibition with sodium azide in the pELTP::abi1-1 line still induces expression of the suberin biosynthesis gene GPAT5 toward the root tip in the pGPAT::mCITRINE-SYP122 line, as observed in wild-type . Based on this, activation of ABA signalling in the endodermis does not link perception of Casparian strip defects with the downstream responses of reduced aquaporin-mediated Lpr or suberin deposition. Suppbackground .The esb1-1 mutant is known to have reduced stomatal apertures and enhanced wilting resistance. This suggests that the CIFs/SGN3 sensing system not only initiates a local root response to Casparian strip integrity but is also involved in initiating long-distance responses in the shoot. We observe reduced stomatal apertures in esb1-1 , and an analysis of the expression of a set of known ABA signalling and response genes in leaves suggest that this stomatal closure is part of an ABA driven response. The aba1 mutation confers a strong ABA deficiency.By generating an esb1-1aba1 double mutant, we investigated the ABA-dependent component in the leaf response we observe in esb1-1. ABA-defciency in esb1-1aba1 suppressed both the reduced stomatal aperture and the activation of expression of ABA signalling and response genes that we observe in esb1-1 .