Sequential induction of auxin efflux and influx carriers regulates lateral root emergence

In Arabidopsis, lateral roots originate from pericycle cells deep within the primary root. New lateral root primordia (LRP) have to emerge through several overlaying tissues. Here, we report that auxin produced in new LRP is transported towards the outer tissues where it triggers cell separation by inducing both the auxin influx carrier LAX3 and cell‐wall enzymes. LAX3 is expressed in just two cell files overlaying new LRP. To understand how this striking pattern of LAX3 expression is regulated, we developed a mathematical model that captures the network regulating its expression and auxin transport within realistic three‐dimensional cell and tissue geometries. Our model revealed that, for the LAX3 spatial expression to be robust to natural variations in root tissue geometry, an efflux carrier is required—later identified to be PIN3. To prevent LAX3 from being transiently expressed in multiple cell files, PIN3 and LAX3 must be induced consecutively, which we later demonstrated to be the case. Our study exemplifies how mathematical models can be used to direct experiments to elucidate complex developmental processes.     

The novel cyst nematode effector protein 19C07 interacts with the Arabidopsis auxin influx transporter LAX3 to control feeding site development

Plant-parasitic cyst nematodes penetrate plant roots and transform cells near the vasculature into specialized feeding sites called syncytia. Syncytia form by incorporating neighboring cells into a single fused cell by cell wall dissolution. This process is initiated via injection of esophageal gland cell effector proteins from the nematode stylet into the host cell. Once inside the cell, these proteins may interact with host proteins that regulate the phytohormone auxin, as cellular concentrations of auxin increase in developing syncytia. Soybean cyst nematode (Heterodera glycines) Hg19C07 is a novel effector protein expressed specifically in the dorsal gland cell during nematode parasitism. Here, we describe its ortholog in the beet cyst nematode (Heterodera schachtii), Hs19C07. We demonstrate that Hs19C07 interacts with the Arabidopsis (Arabidopsis thaliana) auxin influx transporter LAX3. LAX3 is expressed in cells overlying lateral root primordia, providing auxin signaling that triggers the expression of cell wall-modifying enzymes, allowing lateral roots to emerge. We found that LAX3 and polygalacturonase, a LAX3-induced cell wall-modifying enzyme, are expressed in the developing syncytium and in cells to be incorporated into the syncytium. We observed no decrease in H. schachtii infectivity in aux1 and lax3 single mutants. However, a decrease was observed in both the aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant. In addition, ectopic expression of 19C07 was found to speed up lateral root emergence. We propose that Hs19C07 most likely increases LAX3-mediated auxin influx and may provide a mechanism for cyst nematodes to modulate auxin flow into root cells, stimulating cell wall hydrolysis for syncytium development.     

Ethylene–auxin interactions regulate lateral root initiation and emergence in Arabidopsis thaliana

Plant root systems display considerable plasticity in response to endogenous and environmental signals. Auxin stimulates pericycle cells within elongating primary roots to enter de novo organogenesis, leading to the establishment of new lateral root meristems. Crosstalk between auxin and ethylene in root elongation has been demonstrated, but interactions between these hormones in root branching are not well characterized. We find that enhanced ethylene synthesis, resulting from the application of low concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), promotes the initiation of lateral root primordia. Treatment with higher doses of ACC strongly inhibits the ability of pericycle cells to initiate new lateral root primordia, but promotes the emergence of existing lateral root primordia: behaviour that is also seen in the eto1 mutation. These effects are correlated with decreased pericycle cell length and increased lateral root primordia cell width. When auxin is applied simultaneously with ACC, ACC is unable to prevent the auxin stimulation of lateral root formation in the root tissues formed prior to ACC exposure. However, in root tissues formed after transfer to ACC, in which elongation is reduced, auxin does not rescue the ethylene inhibition of primordia initiation, but instead increases it by several fold. Mutations that block auxin responses, slr1 and arf7 arf19, render initiation of lateral root primordia insensitive to the promoting effect of low ethylene levels, and mutations that inhibit ethylene-stimulated auxin biosynthesis, wei2 and wei7 , reduce the inhibitory effect of higher ethylene levels, consistent with ethylene regulating root branching through interactions with auxin.     

Systems Analysis of Auxin Transport in the Arabidopsis Root Apex

Auxin is a key regulator of plant growth and development. Within the root tip, auxin distribution plays a crucial role specifying developmental zones and coordinating tropic responses. Determining how the organ-scale auxin pattern is regulated at the cellular scale is essential to understanding how these processes are controlled. In this study, we developed an auxin transport model based on actual root cell geometries and carrier subcellular localizations. We tested model predictions using the DII-VENUS auxin sensor in conjunction with state-of-the-art segmentation tools. Our study revealed that auxin efflux carriers alone cannot create the pattern of auxin distribution at the root tip and that AUX1/LAX influx carriers are also required. We observed that AUX1 in lateral root cap (LRC) and elongating epidermal cells greatly enhance auxin’s shootward flux, with this flux being predominantly through the LRC, entering the epidermal cells only as they enter the elongation zone. We conclude that the nonpolar AUX1/LAX influx carriers control which tissues have high auxin levels, whereas the polar PIN carriers control the direction of auxin transport within these tissues.     

Cytokinins act directly on lateral root founder cells to inhibit root initiation

In Arabidopsis thaliana, lateral roots are formed from root pericycle cells adjacent to the xylem poles. Lateral root development is regulated antagonistically by the plant hormones auxin and cytokinin. While a great deal is known about how auxin promotes lateral root development, the mechanism of cytokinin repression is still unclear. Elevating cytokinin levels was observed to disrupt lateral root initiation and the regular pattern of divisions that characterizes lateral root development in Arabidopsis. To identify the stage of lateral root development that is sensitive to cytokinins, we targeted the expression of the Agrobacterium tumefaciens cytokinin biosynthesis enzyme isopentenyltransferase to either xylem-pole pericycle cells or young lateral root primordia using GAL4-GFP enhancer trap lines. Transactivation experiments revealed that xylem-pole pericycle cells are sensitive to cytokinins, whereas young lateral root primordia are not. This effect is physiologically significant because transactivation of the Arabidopsis cytokinin degrading enzyme cytokinin oxidase 1 in lateral root founder cells results in increased lateral root formation. We observed that cytokinins perturb the expression of PIN genes in lateral root founder cells and prevent the formation of an auxin gradient that is required to pattern lateral root primordia.     

Expression studies on AUX1-like genes in Medicago truncatula suggest that auxin is required at two steps in early nodule development

Medicago truncatula contains a family of at least five genes related to AUX1 of Arabidopsis thaliana (termed MtLAX genes for Medicago truncatula-like AUX1 genes). The high sequence similarity between the encoded proteins and AUX1 implies that the MtLAX genes encode auxin import carriers. The MtLAX genes are expressed in roots and other organs, suggesting that they play pleiotropic roles related to auxin uptake. In primary roots, the MtLAX genes are expressed preferentially in the root tips, particularly in the provascular bundles and root caps. During lateral root and nodule development, the genes are expressed in the primordia, particularly in cells that were probably derived from the pericycle. At slightly later stages, the genes are expressed in the regions of the developing organs where the vasculature arises (central position for lateral roots and peripheral region for nodules). These results are consistent with MtLAX being involved in local auxin transport and suggest that auxin is required at two common stages of lateral root and nodule development: development of the primordia and differentiation of the vasculature.     

Mutations in the Diageotropica (Dgt) gene uncouple patterned cell division during lateral root initiation from proliferative cell division in the pericycle

In angiosperms, root branching requires a continuous re-initiation of new root meristems. Through some unknown mechanism, in most eudicots pericycle cells positioned against the protoxylem change identity and initiate patterned division, leading to formation of lateral root primordia that further develop into lateral roots. This process is auxin-regulated. We have observed that three mutations in the Diageotropica (Dgt) gene in tomato prevent primordium formation. Detailed analysis of one of these mutants, dgt1-1, demonstrated that the mutation does not abolish the proliferative capacity of the xylem-adjacent pericycle in the differentiated root portion. Files of shortened pericycle cells found in dgt1-1 roots were unrelated to primordium formation. Auxin application stimulated this unusual proliferation, leading to formation of a multi-layered xylem-adjacent pericycle, but did not rescue the primordium formation. In contrast to wild type, auxin could not induce any cell divisions in the pericycle of the most distal dgt1-1 root-tip portion. In wild-type roots, the Dgt gene promoter was expressed strongly in lateral root primordia starting from their initiation, and on auxin treatment was induced in the primary root meristem. Auxin level and distribution were altered in dgt1-1 root tissues, as judged by direct auxin measurements, and the tissue-specific expression of an auxin-response reporter was altered in transgenic plants. Together, our data demonstrate that the Dgt gene product, a type-A cyclophilin, is essential for morphogenesis of lateral root primordia, and that the dgt mutations uncouple patterned cell division in lateral root initiation from proliferative cell division in the pericycle.     

Auxin distribution and transport during embryogenesis and seed germi-nation of Arabidopsis

INTRODUCTIONAuxin plays an important role in regu1ating celldivision, e1ongation and differentiatiou, vascular tis-sue fOrmation[1], pollen deve1opment[2] and 1eafyhead fOrmation[3]. Adrin polar transport is be-1ieved to invohe in a variety of important growthand developmenial processes, including the patternfOrmation of eInbryO, leaf morphogenesis and theroot gravity response[4--8]. Auxin po1ar transportinhibitor has been proved essential illterference ofataln transport leading to patte…     

The Control of Lateral Root Development in Cultured Pea Seedlings. II. Root Fasciation Induced by Auxin Inhibitors

Lateral root development in cultured seedlings of Pisum sativum (cv. Alaska) was modified by the application of auxin transport inhibitors or antagonists. When applied either to replace the root tip or beneath the cotyledonary node, two auxin transport inhibitors, 2,3,5-triiodobenzoic acid (TIBA) and 3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo[5,1-α]isoindol-8-one (DPX-1840), increased cell division activity opposite the protoxylem poles. This resulted in the formation of masses of cells, which we are calling root primordial masses (RPMs), 2 to 3 days after treatment. RPMs differed from lateral root primordia in that they lacked apical organization. Some roots however developed both RPMs and lateral roots indicating that both structures were similar in terms of the timing and location of cell division in the pericycle and endodermis leading to their initiation. Removal of the auxin transport inhibitors allowed many of the RPMs to organize later into lateral root primordia and to emerge in clusters. When the auxin, indoleacetic acid (IAA) was added to the growth medium along with DPX-1840, 3 ranks of RPMs now in the form of fasciated lateral roots emerged from the primary root. The auxin antagonist, p-chlorophenoxy-isobutyric acid (PCIB), also induced RPM formation. In contrast to DPX-1840 treatment, the addition of IAA during PCIB treatment caused normal lateral root development.     

Mutations in Arabidopsis multidrug resistance-like ABC transporters separate the roles of acropetal and basipetal auxin transport in lateral root development

Auxin affects the shape of root systems by influencing elongation and branching. Because multidrug resistance (MDR)-like ABC transporters participate in auxin transport, they may be expected to contribute to root system development. This reverse genetic study of Arabidopsis thaliana roots shows that MDR4-mediated basipetal auxin transport did not affect root elongation or branching. However, impaired acropetal auxin transport due to mutation of the MDR1 gene caused 21% of nascent lateral roots to arrest their growth and the remainder to elongate 50% more slowly than the wild type. Reporter gene analyses indicated a severe auxin deficit in the apex of mdr1 but not mdr4 lateral roots. The mdr1 deficit was explained by 40% less acropetal auxin transport within the mdr1 lateral roots. The slow elongation of mdr1 lateral roots was rescued by auxin and phenocopied in the wild type by an inhibitor of polar auxin transport. Confocal microscopy analysis of a functional green fluorescent protein-MDR1 translational fusion showed the protein to be auxin inducible and present in the tissues responsible for acropetal transport in the primary root. The protein also accumulated in lateral root primordia and later in the tissues responsible for acropetal transport within the lateral root, fully supporting the conclusion that auxin levels established by MDR1-dependent acropetal transport control lateral root growth rate to influence root system architecture.     

BYPASS1-LIKE regulates lateral root initiation via exocytic vesicular trafficking-mediated PIN recycling in Arabidopsis

Auxin and auxin-mediated signaling pathways are known to regulate lateral root development. Although exocytic vesicle trafficking plays an important role in recycling the PIN-FORMED (PIN) auxin efflux carriers and in polar auxin transport during lateral root formation, the mechanistic details of these processes are not well understood. Here, we demonstrate that BYPASS1-LIKE (B1L) regulates lateral root initiation via exocytic vesicular trafficking-mediated PIN recycling in Arabidopsis thaliana. b1l mutants contained significantly more lateral roots than the wild type, primarily due to increased lateral root primordium initiation. Furthermore, the auxin signal was stronger in stage I lateral root primordia of b1l than in those of the wild type. Treatment with exogenous auxin and an auxin transport inhibitor indicated that the lateral root phenotype of b1l could be attributed to higher auxin levels and that B1L regulates auxin efflux. Indeed, compared to the wild type, C-terminally green fluorescent protein-tagged PIN1 and PIN3 accumulated at higher levels in b1l lateral root primordia. B1L interacted with the exocyst, and b1l showed defective PIN exocytosis. These observations indicate that B1L interacts with the exocyst to regulate PIN-mediated polar auxin transport and lateral root initiation in Arabidopsis.     

A role for auxin redistribution in the responses of the root system architecture to phosphate starvation in Arabidopsis

The changes in root system architecture (RSA) triggered by phosphate (P) deprivation were studied in Arabidopsis (Arabidopsis thaliana) plants grown for 14 d on 1 mM or 3 microM P. Two different temporal phases were observed in the response of RSA to low P. First, lateral root (LR) development was promoted between days 7 and 11 after germination, but, after day 11, all root growth parameters were negatively affected, leading to a general reduction of primary root (PR) and LR lengths and of LR density. Low P availability had contrasting effects on various stages of LR development, with a marked inhibition of primordia initiation but a strong stimulation of activation of the initiated primordia. The involvement of auxin signaling in these morphological changes was investigated in wild-type plants treated with indole-3-acetic acid or 2,3,5-triiodobenzoic acid and in axr4-1, aux1-7, and eir1-1 mutants. Most effects of low P on RSA were dramatically modified in the mutants or hormone-treated wild-type plants. This shows that auxin plays a major role in the P starvation-induced changes of root development. From these data, we hypothesize that several aspects of the RSA response to low P are triggered by local modifications of auxin concentration. A model is proposed that postulates that P starvation results in (1) an overaccumulation of auxin in the apex of the PR and in young LRs, (2) an overaccumulation of auxin or a change in sensitivity to auxin in the lateral primordia, and (3) a decrease in auxin concentration in the lateral primordia initiation zone of the PR and in old laterals. Measurements of local changes in auxin concentrations induced by low P, either by direct quantification or by biosensor expression pattern (DR5::beta-glucuronidase reporter gene), are in line with these hypotheses. Furthermore, the observation that low P availability mimicked the action of auxin in promoting LR development in the alf3 mutant confirmed that P starvation stimulates primordia emergence through increased accumulation of auxin or change in sensitivity to auxin in the primordia. Both the strong effect of 2,3,5-triiodobenzoic acid and the phenotype of the auxin-transport mutants (aux1, eir1) suggest that low P availability modifies local auxin concentrations within the root system through changes in auxin transport rather than auxin synthesis.     

Auxin Influx Carriers Control Vascular Patterning and Xylem Differentiation in Arabidopsis thaliana

Auxin is an essential hormone for plant growth and development. Auxin influx carriers AUX1/LAX transport auxin into the cell, while auxin efflux carriers PIN pump it out of the cell. It is well established that efflux carriers play an important role in the shoot vascular patterning, yet the contribution of influx carriers to the shoot vasculature remains unknown. Here, we combined theoretical and experimental approaches to decipher the role of auxin influx carriers in the patterning and differentiation of vascular tissues in the Arabidopsis inflorescence stem. Our theoretical analysis predicts that influx carriers facilitate periodic patterning and modulate the periodicity of auxin maxima. In agreement, we observed fewer and more spaced vascular bundles in quadruple mutants plants of the auxin influx carriers aux1lax1lax2lax3. Furthermore, we show AUX1/LAX carriers promote xylem differentiation in both the shoot and the root tissues. Influx carriers increase cytoplasmic auxin signaling, and thereby differentiation. In addition to this cytoplasmic role of auxin, our computational simulations propose a role for extracellular auxin as an inhibitor of xylem differentiation. Altogether, our study shows that auxin influx carriers AUX1/LAX regulate vascular patterning and differentiation in plants.     

AUX1 promotes lateral root formation by facilitating indole-3-acetic acid distribution between sink and source tissues in the Arabidopsis seedling

Arabidopsis root architecture is regulated by shoot-derived signals such as nitrate and auxin. We report that mutations in the putative auxin influx carrier AUX1 modify root architecture as a result of the disruption in hormone transport between indole-3-acetic acid (IAA) source and sink tissues. Gas chromatography-selected reaction monitoring-mass spectrometry measurements revealed that the aux1 mutant exhibited altered IAA distribution in young leaf and root tissues, the major IAA source and sink organs, respectively, in the developing seedling. Expression studies using the auxin-inducible reporter IAA2::uidA revealed that AUX1 facilitates IAA loading into the leaf vascular transport system. AUX1 also facilitates IAA unloading in the primary root apex and developing lateral root primordium. Exogenous application of the synthetic auxin 1-naphthylacetic acid is able to rescue the aux1 lateral root phenotype, implying that root auxin levels are suboptimal for lateral root primordium initiation in the mutant.     

Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal

Re-orientation of Arabidopsis seedlings induces a rapid, asymmetric release of the growth regulator auxin from gravity-sensing columella cells at the root apex. The resulting lateral auxin gradient is hypothesized to drive differential cell expansion in elongation-zone tissues. We mapped those root tissues that function to transport or respond to auxin during a gravitropic response. Targeted expression of the auxin influx facilitator AUX1 demonstrated that root gravitropism requires auxin to be transported via the lateral root cap to all elongating epidermal cells. A three-dimensional model of the root elongation zone predicted that AUX1 causes the majority of auxin to accumulate in the epidermis. Selectively disrupting the auxin responsiveness of expanding epidermal cells by expressing a mutant form of the AUX/IAA17 protein, axr3-1, abolished root gravitropism. We conclude that gravitropic curvature in Arabidopsis roots is primarily driven by the differential expansion of epidermal cells in response to an influx-carrier-dependent auxin gradient.     

Root meristems in Medicago truncatula tissue culture arise from vascular-derived procambial-like cells in a process regulated by ethylene

Leaf explants of Medicago truncatula were used to investigate the origins of auxin-induced root formation. On the application of auxin there is some callus formation (not the massive amount that occurs in response to auxin plus cytokinin) and roots appear shortly after the first visible callus. Histological examination reveals morphologically distinctive sheets of callus cells that emanate from the veins of the leaf explants and, within this cell type, root primordia are produced as well as some vascular tissue cells. What is suggested is that the vein-derived cells (VDCs) are procambial-like and function as pluripotent stem cells with a propensity to form root meristems or vascular tissues in response to added auxin. The development of root primordia from these pluripotent cells was clearly up-regulated by the use of the sickle (skl) mutant, which is a mutant impaired in ethylene signal transduction while the wild type and the sunn mutant, defective in auxin polar transport, produced similar numbers of roots. The skl mutant in generating many more roots concomitantly formed fewer vascular tissues. The root meristems differentiate similarly to normal roots producing a central cylinder of vascular tissue, which connects with the leaf explant veins. The VDCs appear to be derived from the cells of or near the phloem. The leaf observations suggest that a pool of stem cells exist in vascular tissue that, in combination with auxin and perhaps other factors, drive a diversity of plant development outcomes that is species specific. The way auxin interacts with other hormones is a key factor in determining the stem cell fate. The histological data in this study also assist in the interpretation of the molecular analysis of auxin-induced root formation in cultured leaves of M. truncatula.