The LysM receptor-like kinase SlLYK10 controls lipochitooligosaccharide signaling in inner cell layers of tomato roots.

Establishment of arbuscular mycorrhiza (AM) relies on a plant signaling pathway that can be activated by fungal chitinic signals such as short chain chitooligosaccharides (CO) and lipo-chitooligosaccharides (LCOs). The tomato LysM receptor-like kinase (LysM RLK) SlLYK10 has high affinity for LCOs and is involved in root colonization by arbuscular mycorrhizal fungi (AMF), however its role in LCO responses has not yet been studied. Here, we show that SlLYK10 proteins produced by the Sllyk10-1 and Sllyk10-2 mutant alleles, which both cause decreases in AMF colonization, and carry mutations in LysM1 and 2 respectively, have similar LCO binding affinities compared to the WT SlLYK10. However, the mutant forms were no longer able to induce cell death in Nicotiana benthamiana when co-expressed with MtLYK3, a Medicago truncatula LCO co-receptor, while they physically interacted with MtLYK3 in co-purification experiments. This suggests that the LysM mutations affect the ability of SlLYK10 to trigger signaling through a potential co-receptor rather than its ability to bind LCOs. Interestingly, tomato lines that contain a calcium (Ca2+) concentration reporter (Genetically Encoded Ca2+ indicators, GECO), showed Ca2+ spiking in response to LCO applications, but this occurred only in inner cell layers of the roots, while short chain COs also induced Ca2+ spiking in the epidermis. Moreover, LCO-induced Ca2+spiking was decreased in Sllyk10-1*GECO plants, suggesting that the decrease in AMF colonization in Sllyk10-1 is due to abnormal LCO signaling.

A dirigent protein complex directs lignin polymerization and assembly of the root diffusion barrier.

Functionally similar to the tight junctions present in animal guts, plant roots have evolved a lignified Casparian strip as an extracellular diffusion barrier in the endodermis to seal the root apoplast and maintain nutrient homeostasis. How this diffusion barrier is structured has been partially defined, but its lignin polymerization and assembly steps remain elusive. Here, we characterize a family of dirigent proteins (DPs) essential for both the localized polymerization of lignin required for Casparian strip biogenesis in the cell wall and for attachment of the strip to the plasma membrane to seal the apoplast. We reveal a Casparian strip lignification mechanism that requires cooperation between DPs and the Schengen pathway. Furthermore, we demonstrate that DPs directly mediate lignin polymerization as part of this mechanism.

Physiological roles of Casparian strips and suberin in the transport of water and solutes.

The formation of Casparian strips (CS) and the deposition of suberin at the endodermis of plant roots are thought to limit the apoplastic transport of water and ions. We investigated the specific role of each of these apoplastic barriers in the control of hydro-mineral transport by roots and the consequences on shoot growth. A collection of Arabidopsis thaliana mutants defective in suberin deposition and/or CS development was characterized under standard conditions using a hydroponic system and the Phenopsis platform. Mutants altered in suberin deposition had enhanced root hydraulic conductivity, indicating a restrictive role for this compound in water transport. In contrast, defective CS directly increased solute leakage and indirectly reduced root hydraulic conductivity. Defective CS also led to a reduction in rosette growth, which was partly dependent on the hydro-mineral status of the plant. Ectopic suberin was shown to partially compensate for defective CS phenotypes. Altogether, our work shows that the functionality of the root apoplastic diffusion barriers greatly influences the plant physiology, and that their integrity is tightly surveyed.

Non-invasive hydrodynamic imaging in plant roots at cellular resolution.

A key impediment to studying water-related mechanisms in plants is the inability to non-invasively image water fluxes in cells at high temporal and spatial resolution. Here, we report that Raman microspectroscopy, complemented by hydrodynamic modelling, can achieve this goal - monitoring hydrodynamics within living root tissues at cell- and sub-second-scale resolutions. Raman imaging of water-transporting xylem vessels in Arabidopsis thaliana mutant roots reveals faster xylem water transport in endodermal diffusion barrier mutants. Furthermore, transverse line scans across the root suggest water transported via the root xylem does not re-enter outer root tissues nor the surrounding soil when en-route to shoot tissues if endodermal diffusion barriers are intact, thereby separating 'two water worlds'.

Two chemically distinct root lignin barriers control solute and water balance.

Lignin is a complex polymer deposited in the cell wall of specialised plant cells, where it provides essential cellular functions. Plants coordinate timing, location, abundance and composition of lignin deposition in response to endogenous and exogenous cues. In roots, a fine band of lignin, the Casparian strip encircles endodermal cells. This forms an extracellular barrier to solutes and water and plays a critical role in maintaining nutrient homeostasis. A signalling pathway senses the integrity of this diffusion barrier and can induce over-lignification to compensate for barrier defects. Here, we report that activation of this endodermal sensing mechanism triggers a transcriptional reprogramming strongly inducing the phenylpropanoid pathway and immune signaling. This leads to deposition of compensatory lignin that is chemically distinct from Casparian strip lignin. We also report that a complete loss of endodermal lignification drastically impacts mineral nutrients homeostasis and plant growth.

Magnesium and calcium overaccumulate in the leaves of a schengen3 mutant of Brassica rapa.

Magnesium (Mg) and calcium (Ca) are essential mineral nutrients poorly supplied in many human food systems. In grazing livestock, Mg and Ca deficiencies are costly welfare issues. Here, we report a Brassica rapa loss-of-function schengen3 (sgn3) mutant, braA.sgn3.a-1, which accumulates twice as much Mg and a third more Ca in its leaves. We mapped braA.sgn3.a to a single recessive locus using a forward ionomic screen of chemically mutagenized lines with subsequent backcrossing and linked-read sequencing of second back-crossed, second filial generation (BC2F2) segregants. Confocal imaging revealed a disrupted root endodermal diffusion barrier, consistent with SGN3 encoding a receptor-like kinase required for normal formation of Casparian strips, as reported in thale cress (Arabidopsis thaliana). Analysis of the spatial distribution of elements showed elevated extracellular Mg concentrations in leaves of braA.sgn3.a-1, hypothesized to result from preferential export of excessive Mg from cells to ensure suitable cellular concentrations. This work confirms a conserved role of SGN3 in controlling nutrient homeostasis in B. rapa, and reveals mechanisms by which plants are able to deal with perturbed shoot element concentrations resulting from a "leaky" root endodermal barrier. Characterization of variation in leaf Mg and Ca accumulation across a mutagenized population of B. rapa shows promise for using such populations in breeding programs to increase edible concentrations of essential human and animal nutrients.

A Novel Signaling Pathway Required for Arabidopsis Endodermal Root Organization Shapes the Rhizosphere Microbiome.

The Casparian strip (CS) constitutes a physical diffusion barrier to water and nutrients in plant roots, which is formed by the polar deposition of lignin polymer in the endodermis tissue. The precise pattern of lignin deposition is determined by the scaffolding activity of membrane-bound Casparian Strip domain proteins (CASPs), but little is known of the mechanism(s) directing this process. Here, we demonstrate that Endodermis-specific Receptor-like Kinase 1 (ERK1) and, to a lesser extent, ROP Binding Kinase1 (RBK1) are also involved in regulating CS formation, with the former playing an essential role in lignin deposition as well as in the localization of CASP1. We show that ERK1 is localized to the cytoplasm and nucleus of the endodermis and that together with the circadian clock regulator, Time for Coffee (TIC), forms part of a novel signaling pathway necessary for correct CS organization and suberization of the endodermis, with their single or combined loss of function resulting in altered root microbiome composition. In addition, we found that other mutants displaying defects in suberin deposition at the CS also display altered root exudates and microbiome composition. Thus, our work reveals a complex network of signaling factors operating within the root endodermis that establish both the CS diffusion barrier and influence the microbial composition of the rhizosphere.

Coordination between microbiota and root endodermis supports plant mineral nutrient homeostasis.

Plant roots and animal guts have evolved specialized cell layers to control mineral nutrient homeostasis. These layers must tolerate the resident microbiota while keeping homeostatic integrity. Whether and how the root diffusion barriers in the endodermis, which are critical for the mineral nutrient balance of plants, coordinate with the microbiota is unknown. We demonstrate that genes controlling endodermal function in the model plant Arabidopsis thaliana contribute to the plant microbiome assembly. We characterized a regulatory mechanism of endodermal differentiation driven by the microbiota with profound effects on nutrient homeostasis. Furthermore, we demonstrate that this mechanism is linked to the microbiota's capacity to repress responses to the phytohormone abscisic acid in the root. Our findings establish the endodermis as a regulatory hub coordinating microbiota assembly and homeostatic mechanisms.

Uclacyanin Proteins Are Required for Lignified Nanodomain Formation within Casparian Strips.

Casparian strips (CSs) are cell wall modifications of vascular plants restricting extracellular free diffusion into and out of the vascular system [1]. This barrier plays a critical role in controlling the acquisition of nutrients and water necessary for normal plant development [2-5]. CSs are formed by the precise deposition of a band of lignin approximately 2 µm wide and 150 nm thick spanning the apoplastic space between adjacent endodermal cells [6, 7]. Here, we identified a copper-containing protein, Uclacyanin1 (UCC1), that is sub-compartmentalized within the CS. UCC1 forms a central CS nanodomain in comparison with other CS-located proteins that are found to be mainly accumulated at the periphery of the CS. We found that loss-of-function of two uclacyanins (UCC1 and UCC2) reduces lignification specifically in this central CS nanodomain, revealing a nano-compartmentalized machinery for lignin polymerization. This loss of lignification leads to increased endodermal permeability and, consequently, to a loss of mineral nutrient homeostasis.

Surveillance of cell wall diffusion barrier integrity modulates water and solute transport in plants.

The endodermis is a key cell layer in plant roots that contributes to the controlled uptake of water and mineral nutrients into plants. In order to provide such functionality the endodermal cell wall has specific chemical modifications consisting of lignin bands (Casparian strips) that encircle each cell, and deposition of a waxy-like substance (suberin) between the wall and the plasma membrane. These two extracellular deposits provide control of diffusion enabling the endodermis to direct the movement of water and solutes into and out of the vascular system in roots. Loss of integrity of the Casparian strip-based apoplastic barrier is sensed by the leakage of a small peptide from the stele into the cortex. Here, we report that such sensing of barrier integrity leads to the rebalancing of water and mineral nutrient uptake, compensating for breakage of Casparian strips. This rebalancing involves both a reduction in root hydraulic conductivity driven by deactivation of aquaporins, and downstream limitation of ion leakage through deposition of suberin. These responses in the root are also coupled to a reduction in water demand in the shoot mediated by ABA-dependent stomatal closure.

Iron- and ferritin-dependent reactive oxygen species distribution: impact on Arabidopsis root system architecture.

Iron (Fe) homeostasis is integrated with the production of reactive oxygen species (ROS), and distribution at the root tip participates in the control of root growth. Excess Fe increases ferritin abundance, enabling the storage of Fe, which contributes to protection of plants against Fe-induced oxidative stress. AtFer1 and AtFer3 are the two ferritin genes expressed in the meristematic zone, pericycle and endodermis of the Arabidopsis thaliana root, and it is in these regions that we observe Fe stained dots. This staining disappears in the triple fer1-3-4 ferritin mutant. Fe excess decreases primary root length in the same way in wild-type and in fer1-3-4 mutant. In contrast, the Fe-mediated decrease of lateral root (LR) length and density is enhanced in fer1-3-4 plants due to a defect in LR emergence. We observe that this interaction between excess Fe, ferritin, and root system architecture (RSA) is in part mediated by the H2O2/O2·- balance between the root cell proliferation and differentiation zones regulated by the UPB1 transcription factor. Meristem size is also decreased in response to Fe excess in ferritin mutant plants, implicating cell cycle arrest mediated by the ROS-activated SMR5/SMR7 cyclin-dependent kinase inhibitors pathway in the interaction between Fe and RSA.

Iron and ferritin dependent ROS distribution impact Arabidopsis root system architecture.

Iron (Fe) homeostasis is integrated with the production of Reactive Oxygen Species (ROS) whose distribution at the root tip participates in the control of root growth. Excess Fe increases ferritin abundance, enabling the storage of Fe which contributes to protection of plants against Fe-induced oxidative stress. AtFer1 and AtFer3 are the two ferritin genes expressed in the meristematic zone, pericycle and endodermis of the Arabidopsis thaliana (Arabidopsis) root, and it is in these regions that we observe Fe stained dots. This staining disappears in the triple fer1-3-4 ferritin mutant. Fe excess decreases primary root length in the same way in wild-type and in fer1-3-4 mutant. In contrast, the Fe mediated decrease of lateral root (LR) length and density is enhanced in fer1-3-4 plants due to a defect in LR emergence. We observe that this interaction between excess Fe, ferritin and RSA is in part mediated by the H2O2/O2.- balance between the root cell proliferation and differentiation zones regulated by the UPB1 transcription factor. Further, meristem size is also decreased in response to Fe excess in ferritin mutant plants, implicating cell cycle arrest mediated by the ROS-activated SMR5/SMR7 cyclin-dependent kinase inhibitors pathway in the interaction between Fe and RSA.

Iron around the clock.

Carbon assimilation, a key determinant of plant biomass production, is under circadian regulation. Light and temperature are major inputs of the plant clock that control various daily rhythms. Such rhythms confer adaptive advantages to the organisms by adjusting their metabolism in anticipation of environmental fluctuations. The relationship between the circadian clock and nutrition extends far beyond the regulation of carbon assimilation as mineral nutrition, and specially iron homeostasis, is regulated through this mechanism. Conversely, iron status was identified as a new and important input regulating the central oscillator, raising the question of the nature of the Fe-dependent signal that modulates the period of the circadian clock. Several lines of evidence strongly suggest that fully developed and functional chloroplasts as well as early light signalling events, involving phytochromes, are essential to couple the clock to Fe responses. Nevertheless, the exact nature of the signal, which most probably involves unknown or not yet fully characterized elements of the chloroplast-to-nucleus retrograde signalling pathway, remains to be identified. Finally, this regulation may also involves epigenetic components.

Involvement of the ABCG37 transporter in secretion of scopoletin and derivatives by Arabidopsis roots in response to iron deficiency.

Studies of Iron (Fe) uptake mechanisms by plant roots have focussed on Fe(III)-siderophores or Fe(II) transport systems. Iron deficency also enhances root secretion of flavins and phenolics. However, the nature of these compounds, their transport outside the roots and their role in Fe nutrition are largely unknown. We used HPLC/ESI-MS (TOF) and HPLC/ESI-MS/MS (ion trap) to characterize fluorescent phenolic-type compounds accumulated in roots or exported to the culture medium of Arabidopsis plants in response to Fe deficiency. Wild-type and mutant plants altered either in phenylpropanoid biosynthesis or in the ABCG37 (PDR9) ABC transporter were grown under standard or Fe-deficient nutrition conditions and compared. Fe deficiency upregulates the expression of genes encoding enzymes of the phenylpropanoid pathway and leads to the synthesis and secretion of phenolic compounds belonging to the coumarin family. The ABCG37 gene is also upregulated in response to Fe deficiency and coumarin export is impaired in pdr9 mutant plants. Therefore it can be concluded that: Fe deficiency induces the secretion of coumarin compounds by Arabidopsis roots; the ABCG37 ABC transporter is required for this secretion to take place; and these compounds improved plant Fe nutrition.

Iron and ROS control of the DownSTream mRNA decay pathway is essential for plant fitness.

A new regulatory pathway involved in plant response to oxidative stress was revealed using the iron-induced Arabidopsis ferritin AtFER1 as a model. Using pharmacological and genetic approaches, the DownSTream (DST) cis-acting element in the 3'-untranslated region of the AtFER1 mRNA was shown to be involved in the degradation of this transcript, and oxidative stress triggers this destabilization. In the two previously identified trans-acting mutants (dst1 and dst2), AtFER1 mRNA stability is indeed impaired. Other iron-regulated genes containing putative DST sequences also displayed altered expression. Further physiological characterization identified this oxidative stress-induced DST-dependent degradation pathway as an essential regulatory mechanism to modulate mRNA accumulation patterns. Alteration of this control dramatically impacts plant oxidative physiology and growth. In conclusion, the DST-dependent mRNA stability control appears to be an essential mechanism that allows plants to cope with adverse environmental conditions.