Auxin triggers pectin modification during rootlet emergence in white lupin.

Emergence of secondary roots through parental tissue is a highly controlled developmental process. Although the model plant Arabidopsis has been useful to uncover the predominant role of auxin in this process, its simple root structure is not representative of how emergence takes place in most plants, which display more complex root anatomy. White lupin is a legume crop producing structures called cluster roots, where closely spaced rootlets emerge synchronously. Rootlet primordia push their way through several cortical cell layers while maintaining the parent root integrity, reflecting more generally the lateral root emergence process in most multilayered species. In this study, we showed that lupin rootlet emergence is associated with an upregulation of cell wall pectin modifying and degrading genes under the active control of auxin. Among them, we identified LaPG3, a polygalacturonase gene typically expressed in cells surrounding the rootlet primordium and we showed that its downregulation delays emergence. Immunolabeling of pectin epitopes and their quantification uncovered a gradual pectin demethylesterification in the emergence zone, which was further enhanced by auxin treatment, revealing a direct hormonal control of cell wall properties. We also report rhamnogalacturonan-I modifications affecting cortical cells that undergo separation as a consequence of primordium outgrowth. In conclusion, we describe a model of how external tissues in front of rootlet primordia display cell wall modifications to allow for the passage of newly formed rootlets.

Tissue-specific inactivation by cytosine deaminase/uracil phosphoribosyl transferase as a tool to study plant biology.

Recent advances in the study of plant developmental and physiological responses have benefited from tissue-specific approaches, revealing the role of some cell types in these processes. Such approaches have relied on the inactivation of target cells using either toxic compounds or deleterious genes; however, both tissue-specific and truly inducible tools are lacking in order to precisely target a developmental window or specific growth response. We engineered the yeast fluorocytosine deaminase (FCY1) gene by creating a fusion with the bacterial uracil phosphoribosyl transferase (UPP) gene. The recombinant protein converts the precursor 5-fluorocytosine (5-FC) into 5-fluorouracyl, a drug used in the treatment of a range of cancers, which triggers DNA and RNA damage. We expressed the FCY-UPP gene construct in specific cell types using enhancer trap lines and promoters, demonstrating that this marker acts in a cell-autonomous manner. We also showed that it can inactivate slow developmental processes like lateral root formation by targeting pericycle cells. It also revealed a role for the lateral root cap and the epidermis in controlling root growth, a faster response. The 5-FC precursor acts systemically, as demonstrated by its ability to inhibit stomatal movements when supplied to the roots in combination with a guard cell-specific promoter. Finally, we demonstrate that the tissular inactivation is reversible, and can therefore be used to synchronize plant responses or to determine cell type-specific functions during different developmental stages. This tool will greatly enhance our capacity to understand the respective role of each cell type in plant physiology and development.

Pangenome of white lupin provides insights into the diversity of the species.

White lupin is an old crop with renewed interest due to its seed high protein content and high nutritional value. Despite a long domestication history in the Mediterranean basin, modern breeding efforts have been fairly scarce. Recent sequencing of its genome has provided tools for further description of genetic resources but detailed characterization of genomic diversity is still missing. Here, we report the genome sequencing of 39 accessions that were used to establish a white lupin pangenome. We defined 32 068 core genes that are present in all individuals and 14 822 that are absent in some and may represent a gene pool for breeding for improved productivity, grain quality, and stress adaptation. We used this new pangenome resource to identify candidate genes for alkaloid synthesis, a key grain quality trait. The white lupin pangenome provides a novel genetic resource to better understand how domestication has shaped the genomic variability within this crop. Thus, this pangenome resource is an important step towards the effective and efficient genetic improvement of white lupin to help meet the rapidly growing demand for plant protein sources for human and animal consumption.

Lateral root emergence in Arabidopsis is dependent on transcription factor LBD29 regulation of auxin influx carrier LAX3.

Lateral root primordia (LRP) originate from pericycle stem cells located deep within parental root tissues. LRP emerge through overlying root tissues by inducing auxin-dependent cell separation and hydraulic changes in adjacent cells. The auxin-inducible auxin influx carrier LAX3 plays a key role concentrating this signal in cells overlying LRP. Delimiting LAX3 expression to two adjacent cell files overlying new LRP is crucial to ensure that auxin-regulated cell separation occurs solely along their shared walls. Multiscale modeling has predicted that this highly focused pattern of expression requires auxin to sequentially induce auxin efflux and influx carriers PIN3 and LAX3, respectively. Consistent with model predictions, we report that auxin-inducible LAX3 expression is regulated indirectly by AUXIN RESPONSE FACTOR 7 (ARF7). Yeast one-hybrid screens revealed that the LAX3 promoter is bound by the transcription factor LBD29, which is a direct target for regulation by ARF7. Disrupting auxin-inducible LBD29 expression or expressing an LBD29-SRDX transcriptional repressor phenocopied the lax3 mutant, resulting in delayed lateral root emergence. We conclude that sequential LBD29 and LAX3 induction by auxin is required to coordinate cell separation and organ emergence.

Anatomical and hormonal description of rootlet primordium development along white lupin cluster root.

Cluster root (CR) is one of the most spectacular plant developmental adaptations to hostile environment. It can be found in a few species from a dozen botanical families, including white lupin (Lupinus albus) in the Fabaceae family. These amazing structures are produced in phosphate-deprived conditions and are made of hundreds of short roots also known as rootlets. White lupin is the only crop bearing CRs and is considered as the model species for CR studies. However, little information is available on CRs atypical development, including the molecular events that trigger their formation. To provide insights on CR formation, we performed an anatomical and cellular description of rootlet development in white lupin. Starting with a classic histological approach, we described rootlet primordium development and defined eight developmental stages from rootlet initiation to their emergence. Due to the major role of hormones in the developmental program of root system, we next focussed on auxin-related mechanisms. We observed the establishment of an auxin maximum through rootlet development in transgenic roots expressing the DR5:GUS auxin reporter. Expression analysis of the main auxin-related genes [TIR, Auxin Response Factor (ARF) and AUX/IAA] during a detailed time course revealed specific expression associated with the formation of the rootlet primordium. We showed that L. albus TRANSPORT INHIBITOR RESPONSE 1b is expressed during rootlet primordium formation and that L. albus AUXIN RESPONSE FACTOR 5 is expressed in the vasculature but absent in the primordium itself. Altogether, our results describe the very early cellular events leading to CR formation and reveal some of the auxin-related mechanisms.

Floral organ abscission peptide IDA and its HAE/HSL2 receptors control cell separation during lateral root emergence.

Throughout their life cycle, plants produce new organs, such as leaves, flowers, and lateral roots. Organs that have served their purpose may be shed after breakdown of primary cell walls between adjacent cell files at the site of detachment. In Arabidopsis, floral organs abscise after pollination, and this cell separation event is controlled by the peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), which signals through the leucine-rich repeat receptor-like kinases HAESA (HAE) and HAESA-LIKE2 (HSL2). Emergence of new lateral root primordia, initiated deep inside the root under the influence of auxin, is similarly dependent on cell wall dissolution between cells in the overlaying endodermal, cortical, and epidermal tissues. Here we show that this process requires IDA, HAE, and HSL2. Mutation in these genes constrains the passage of the growing lateral root primordia through the overlaying layers, resulting in altered shapes of the lateral root primordia and of the overlaying cells. The HAE and HSL2 receptors are redundant in function during floral organ abscission, but during lateral root emergence they are differentially involved in regulating cell wall remodeling genes. In the root, IDA is strongly auxin-inducible and dependent on key regulators of lateral root emergence--the auxin influx carrier LIKE AUX1-3 and AUXIN RESPONSE FACTOR7. The expression levels of the receptor genes are only transiently induced by auxin, suggesting they are limiting factors for cell separation. We conclude that elements of the same cell separation signaling module have been adapted to function in different developmental programs.

Lateral root morphogenesis is dependent on the mechanical properties of the overlaying tissues.

In Arabidopsis, lateral root primordia (LRPs) originate from pericycle cells located deep within the parental root and have to emerge through endodermal, cortical, and epidermal tissues. These overlaying tissues place biomechanical constraints on the LRPs that are likely to impact their morphogenesis. This study probes the interplay between the patterns of cell division, organ shape, and overlaying tissues on LRP morphogenesis by exploiting recent advances in live plant cell imaging and image analysis. Our 3D/4D image analysis revealed that early stage LRPs exhibit tangential divisions that create a ring of cells corralling a population of rapidly dividing cells at its center. The patterns of division in the latter population of cells during LRP morphogenesis are not stereotypical. In contrast, statistical analysis demonstrated that the shape of new LRPs is highly conserved. We tested the relative importance of cell division pattern versus overlaying tissues on LRP morphogenesis using mutant and transgenic approaches. The double mutant aurora1 (aur1) aur2 disrupts the pattern of LRP cell divisions and impacts its growth dynamics, yet the new organ's dome shape remains normal. In contrast, manipulating the properties of overlaying tissues disrupted LRP morphogenesis. We conclude that the interaction with overlaying tissues, rather than the precise pattern of divisions, is most important for LRP morphogenesis and optimizes the process of lateral root emergence.

Root architecture responses: in search of phosphate.

Soil phosphate represents the only source of phosphorus for plants and, consequently, is its entry into the trophic chain. This major component of nucleic acids, phospholipids, and energy currency of the cell (ATP) can limit plant growth because of its low mobility in soil. As a result, root responses to low phosphate favor the exploration of the shallower part of the soil, where phosphate tends to be more abundant, a strategy described as topsoil foraging. We will review the diverse developmental strategies that can be observed among plants by detailing the effect of phosphate deficiency on primary and lateral roots. We also discuss the formation of cluster roots: an advanced adaptive strategy to cope with low phosphate availability observed in a limited number of species. Finally, we will put this work into perspective for future research directions.

AUX/LAX genes encode a family of auxin influx transporters that perform distinct functions during Arabidopsis development.

Auxin transport, which is mediated by specialized influx and efflux carriers, plays a major role in many aspects of plant growth and development. AUXIN1 (AUX1) has been demonstrated to encode a high-affinity auxin influx carrier. In Arabidopsis thaliana, AUX1 belongs to a small multigene family comprising four highly conserved genes (i.e., AUX1 and LIKE AUX1 [LAX] genes LAX1, LAX2, and LAX3). We report that all four members of this AUX/LAX family display auxin uptake functions. Despite the conservation of their biochemical function, AUX1, LAX1, and LAX3 have been described to regulate distinct auxin-dependent developmental processes. Here, we report that LAX2 regulates vascular patterning in cotyledons. We also describe how regulatory and coding sequences of AUX/LAX genes have undergone subfunctionalization based on their distinct patterns of spatial expression and the inability of LAX sequences to rescue aux1 mutant phenotypes, respectively. Despite their high sequence similarity at the protein level, transgenic studies reveal that LAX proteins are not correctly targeted in the AUX1 expression domain. Domain swapping studies suggest that the N-terminal half of AUX1 is essential for correct LAX localization. We conclude that Arabidopsis AUX/LAX genes encode a family of auxin influx transporters that perform distinct developmental functions and have evolved distinct regulatory mechanisms.

Analyzing lateral root development: how to move forward.

Roots are important to plants for a wide variety of processes, including nutrient and water uptake, anchoring and mechanical support, storage functions, and as the major interface between the plant and various biotic and abiotic factors in the soil environment. Therefore, understanding the development and architecture of roots holds potential for the manipulation of root traits to improve the productivity and sustainability of agricultural systems and to better understand and manage natural ecosystems. While lateral root development is a traceable process along the primary root and different stages can be found along this longitudinal axis of time and development, root system architecture is complex and difficult to quantify. Here, we comment on assays to describe lateral root phenotypes and propose ways to move forward regarding the description of root system architecture, also considering crops and the environment.

Modelling of Arabidopsis LAX3 expression suggests auxin homeostasis.

Emergence of new lateral roots from within the primary root in Arabidopsis has been shown to be regulated by the phytohormone auxin, via the expression of the auxin influx carrier LAX3, mediated by the ARF7/19 IAA14 signalling module (Swarup et al., 2008). A single cell model of the LAX3 and IAA14 auxin response was formulated and used to demonstrate that hysteresis and bistability may explain the experimentally observed 'all-or-nothing' LAX3 spatial expression pattern in cortical cells containing a gradient of auxin concentrations. The model was tested further by using a parameter fitting algorithm to match model output with qRT-PCR mRNA expression data following exogenous auxin treatment. It was found that the model is able to show good agreement with the data, but only when the exogenous auxin signal is degraded over time, at a rate higher than that measured in the experimental medium, suggesting the triggering of an endogenous auxin homeostasis mechanism. Testing the model over a more physiologically relevant range of extracellular auxin shows bistability and hysteresis still occur when using the optimised parameters, providing the rate of LAX3 active auxin transport is sufficiently high relative to passive diffusion.

Root gravitropism is regulated by a transient lateral auxin gradient controlled by a tipping-point mechanism.

Gravity profoundly influences plant growth and development. Plants respond to changes in orientation by using gravitropic responses to modify their growth. Cholodny and Went hypothesized over 80 years ago that plants bend in response to a gravity stimulus by generating a lateral gradient of a growth regulator at an organ's apex, later found to be auxin. Auxin regulates root growth by targeting Aux/IAA repressor proteins for degradation. We used an Aux/IAA-based reporter, domain II (DII)-VENUS, in conjunction with a mathematical model to quantify auxin redistribution following a gravity stimulus. Our multidisciplinary approach revealed that auxin is rapidly redistributed to the lower side of the root within minutes of a 90° gravity stimulus. Unexpectedly, auxin asymmetry was rapidly lost as bending root tips reached an angle of 40° to the horizontal. We hypothesize roots use a "tipping point" mechanism that operates to reverse the asymmetric auxin flow at the midpoint of root bending. These mechanistic insights illustrate the scientific value of developing quantitative reporters such as DII-VENUS in conjunction with parameterized mathematical models to provide high-resolution kinetics of hormone redistribution.

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.

pin2 mutant agravitropic root phenotype is conditional and nutrient-sensitive.

Plants have the capacity to sense and adapt to environmental factors using the phytohormone auxin as a major regulator of tropism and development. Among these responses, gravitropism is essential for plant roots to grow downward in the search for nutrients and water. We discovered a new mutant allele of the auxin efflux transporter PIN2 that revealed that pin2 agravitropic root mutants are conditional and nutrient-sensitive. We describe that nutrient composition of the medium, rather than osmolarity, can revert the agravitropic root phenotype of pin2. Indeed, on phosphorus- and nitrogen-deprived media, the agravitropic root defect was restored independently of primary root growth levels. Slow and fast auxin responses were evaluated using DR5 and R2D2 probes, respectively, and revealed a strong modulation by nutrient composition of the culture medium. We evaluated the role of PIN and AUX auxin transporters and demonstrated that neither PIN3 nor AUX1 are involved in this process. However, we observed the ectopic expression of PIN1 in the epidermis in the pin2 mutant background associated with permissive, but not restrictive, conditions. This ectopic expression was associated with a restoration of the asymmetric accumulation of auxin necessary for the reorientation of the root according to gravity. These observations suggest a strong regulation of auxin distribution by nutrients availability, directly impacting root's ability to drive their gravitropic response.

The causal mutation leading to sweetness in modern white lupin cultivars.

Lupins are high-protein crops that are rapidly gaining interest as hardy alternatives to soybean; however, they accumulate antinutritional alkaloids of the quinolizidine type (QAs). Lupin domestication was enabled by the discovery of genetic loci conferring low QA levels (sweetness), but the precise identity of the underlying genes remains uncertain. We show that pauper, the most common sweet locus in white lupin, encodes an acetyltransferase (AT) unexpectedly involved in the early QA pathway. In pauper plants, a single-nucleotide polymorphism (SNP) strongly impairs AT activity, causing pathway blockage. We corroborate our hypothesis by replicating the pauper chemotype in narrow-leafed lupin via mutagenesis. Our work adds a new dimension to QA biosynthesis and establishes the identity of a lupin sweet gene for the first time, thus facilitating lupin breeding and enabling domestication of other QA-containing legumes.

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.

Short-Root regulates primary, lateral, and adventitious root development in Arabidopsis.

Short-Root (SHR) is a well-characterized regulator of radial patterning and indeterminacy of the Arabidopsis (Arabidopsis thaliana) primary root. However, its role during the elaboration of root system architecture remains unclear. We report that the indeterminate wild-type Arabidopsis root system was transformed into a determinate root system in the shr mutant when growing in soil or agar. The root growth behavior of the shr mutant results from its primary root apical meristem failing to initiate cell division following germination. The inability of shr to reactivate mitotic activity in the root apical meristem is associated with the progressive reduction in the abundance of auxin efflux carriers, PIN-FORMED1 (PIN1), PIN2, PIN3, PIN4, and PIN7. The loss of primary root growth in shr is compensated by the activation of anchor root primordia, whose tissues are radially patterned like the wild type. However, SHR function is not restricted to the primary root but is also required for the initiation and patterning of lateral root primordia. In addition, SHR is necessary to maintain the indeterminate growth of lateral and anchor roots. We conclude that SHR regulates a wide array of Arabidopsis root-related developmental processes.

The Highly Repeat-Diverse (Peri) Centromeres of White Lupin (Lupinus albus L.).

Plant genomes are known to be mainly composed of repetitive DNA sequences. Regardless of the non-genic function of these sequences, they are important for chromosome structure and stability during cell-cycle. Based on the recent available whole-genome assembly of white lupin (Lupinus albus L.; WL), we have in silico annotated and in situ mapped the main classes of DNA repeats identified with RepeatExplorer. A highly diverse and an abundance of satellite DNAs were found representing more than 10 families, where three of them were highly associated with CENH3-immunoprecipitated chromatin. Applying a strategy of several re-hybridization steps with different combinations of satDNA, rDNA, and LTR-RTs probes, we were able to construct a repeat-based chromosome map for the identification of most chromosome pairs. Two families of LTR retrotransposons, Ty1/copia SIRE and Ty3/gypsy Tekay, were highly abundant at pericentromeric regions, while the centromeric retrotransposon of WL (CRWL) from the CRM clade showed strong centromere-specific localization in most chromosomes and was also highly enriched with CENH3-immunoprecipitated chromatin. FISH mapping of repeat DNA showed some incongruences with the reference genome, which can be further used for improving the current version of the genome. Our results demonstrate that despite the relatively small genome of WL, a high diversity of pericentromeric repeats was found, emphasizing the rapid evolution of repeat sequences in plant genomes.

Auxin carriers localization drives auxin accumulation in plant cells infected by Frankia in Casuarina glauca actinorhizal nodules.

Actinorhizal symbioses are mutualistic interactions between plants and the soil bacteria Frankia that lead to the formation of nitrogen-fixing root nodules. Little is known about the signaling mechanisms controlling the different steps of the establishment of the symbiosis. The plant hormone auxin has been suggested to play a role. Here we report that auxin accumulates within Frankia-infected cells in actinorhizal nodules of Casuarina glauca. Using a combination of computational modeling and experimental approaches, we establish that this localized auxin accumulation is driven by the cell-specific expression of auxin transporters and by Frankia auxin biosynthesis in planta. Our results indicate that the plant actively restricts auxin accumulation to Frankia-infected cells during the symbiotic interaction.