Mutation of AtPME2, a pH-Dependent Pectin Methylesterase, Affects Cell Wall Structure and Hypocotyl Elongation.

Pectin methylesterases (PMEs) modify homogalacturonan's chemistry and play a key role in regulating primary cell wall mechanical properties. Here, we report on Arabidopsis AtPME2, which we found to be highly expressed during lateral root emergence and dark-grown hypocotyl elongation. We showed that dark-grown hypocotyl elongation was reduced in knock-out mutant lines as compared to the control. The latter was related to the decreased total PME activity as well as increased stiffness of the cell wall in the apical part of the hypocotyl. To relate phenotypic analyses to the biochemical specificity of the enzyme, we produced the mature active enzyme using heterologous expression in Pichia pastoris and characterized it through the use of a generic plant PME antiserum. AtPME2 is more active at neutral compared to acidic pH, on pectins with a degree of 55-70% methylesterification. We further showed that the mode of action of AtPME2 can vary according to pH, from high processivity (at pH8) to low processivity (at pH5), and relate these observations to the differences in electrostatic potential of the protein. Our study brings insights into how the pH-dependent regulation by PME activity could affect the pectin structure and associated cell wall mechanical properties.

Spatial transcriptomics of a lycophyte root sheds light on root evolution.

Plant roots originated independently in lycophytes and euphyllophytes, whereas early vascular plants were rootless. The organization of the root apical meristem in euphyllophytes is well documented, especially in the model plant Arabidopsis. However, little is known about lycophyte roots and their molecular innovations during evolution. In this study, spatial transcriptomics was used to detect 97 root-related genes in the roots of the lycophyte Selaginella moellendorffii. A high number of genes showed expression patterns similar to what has been reported for seed plants, supporting the idea of a highly convergent evolution of mechanisms to control root development. Interaction and complementation data of SHORTROOT (SHR) and SCARECROW (SCR) homologs, furthermore, support a comparable regulation of the ground tissue (GT) between euphyllophytes and lycophytes. Root cap formation, in contrast, appears to be differently regulated. Several experiments indicated an important role of the WUSCHEL-RELATED HOMEOBOX13 gene SmWOX13a in Selaginella root cap formation. In contrast to multiple Arabidopsis WOX paralogs, SmWOX13a is able to induce root cap cells in Arabidopsis and has functionally conserved homologs in the fern Ceratopteris richardii. Lycophytes and a part of the euphyllophytes, therefore, may share a common mechanism regulating root cap formation, which was diversified or lost during seed plant evolution. In summary, we here provide a new spatial data resource for the Selaginella root, which in general advocates for conserved mechanisms to regulate root development but shows a clear divergence in the control of root cap formation, with a novel putative role of WOX genes in root cap formation in non-seed plants.

Trehalose-6-phosphate signaling regulates lateral root formation in Arabidopsis thaliana.

Plant roots explore the soil for water and nutrients, thereby determining plant fitness and agricultural yield, as well as determining ground substructure, water levels, and global carbon sequestration. The colonization of the soil requires investment of carbon and energy, but how sugar and energy signaling are integrated with root branching is unknown. Here, we show through combined genetic and chemical modulation of signaling pathways that the sugar small-molecule signal, trehalose-6-phosphate (T6P) regulates root branching through master kinases SNF1-related kinase-1 (SnRK1) and Target of Rapamycin (TOR) and with the involvement of the plant hormone auxin. Increase of T6P levels both via genetic targeting in lateral root (LR) founder cells and through light-activated release of the presignaling T6P-precursor reveals that T6P increases root branching through coordinated inhibition of SnRK1 and activation of TOR. Auxin, the master regulator of LR formation, impacts this T6P function by transcriptionally down-regulating the T6P-degrader trehalose phosphate phosphatase B in LR cells. Our results reveal a regulatory energy-balance network for LR formation that links the 'sugar signal' T6P to both SnRK1 and TOR downstream of auxin.

Structure-function study of a Ca2+-independent metacaspase involved in lateral root emergence.

Metacaspases are part of an evolutionarily broad family of multifunctional cysteine proteases, involved in disease and normal development. As the structure-function relationship of metacaspases remains poorly understood, we solved the X-ray crystal structure of an Arabidopsis thaliana type II metacaspase (AtMCA-IIf) belonging to a particular subgroup not requiring calcium ions for activation. To study metacaspase activity in plants, we developed an in vitro chemical screen to identify small molecule metacaspase inhibitors and found several hits with a minimal thioxodihydropyrimidine-dione structure, of which some are specific AtMCA-IIf inhibitors. We provide mechanistic insight into the basis of inhibition by the TDP-containing compounds through molecular docking onto the AtMCA-IIf crystal structure. Finally, a TDP-containing compound (TDP6) effectively hampered lateral root emergence in vivo, probably through inhibition of metacaspases specifically expressed in the endodermal cells overlying developing lateral root primordia. In the future, the small compound inhibitors and crystal structure of AtMCA-IIf can be used to study metacaspases in other species, such as important human pathogens, including those causing neglected diseases.

ABCB-mediated shootward auxin transport feeds into the root clock.

Although strongly influenced by environmental conditions, lateral root (LR) positioning along the primary root appears to follow obediently an internal spacing mechanism dictated by auxin oscillations that prepattern the primary root, referred to as the root clock. Surprisingly, none of the hitherto characterized PIN- and ABCB-type auxin transporters seem to be involved in this LR prepatterning mechanism. Here, we characterize ABCB15, 16, 17, 18, and 22 (ABCB15-22) as novel auxin-transporting ABCBs. Knock-down and genome editing of this genetically linked group of ABCBs caused strongly reduced LR densities. These phenotypes were correlated with reduced amplitude, but not reduced frequency of the root clock oscillation. High-resolution auxin transport assays and tissue-specific silencing revealed contributions of ABCB15-22 to shootward auxin transport in the lateral root cap (LRC) and epidermis, thereby explaining the reduced auxin oscillation. Jointly, these data support a model in which LRC-derived auxin contributes to the root clock amplitude.

Cellular and gene expression patterns associated with root bifurcation in Selaginella.

The roots of lycophytes branch through dichotomy or bifurcation, during which the root apex splits into two daughter roots. This is morphologically distinct from lateral root (LR) branching in the extant euphyllophytes, with LRs developing along the root axis at different distances from the apex. Although the process of root bifurcation is poorly understood, such knowledge can be important, because it may represent an evolutionarily ancient strategy that roots recruited to form new stem cells or meristems. In this study, we examined root bifurcation in the lycophyte Selaginella moellendorffii. We characterized an in vitro developmental time frame based on repetitive apex bifurcations, allowing us to sample different stages of dichotomous root branching and analyze the root meristem and root branching in S. moellendorffii at the microscopic and transcriptomic level. Our results showed that, in contrast to previous assumptions, initial cells (ICs) in the root meristem are mostly not tetrahedral but rather show an irregular shape. Tracking down the early stages of root branching argues for the occurrence of a symmetric division of the single IC, resulting in two apical stem cells that initiate root meristem bifurcation. Moreover, we generated a S. moellendorffii root branching transcriptome that resulted in the delineation of a subset of core meristem genes. The occurrence of multiple putative orthologs of meristem genes in this dataset suggests the presence of conserved pathways in the control of meristem and root stem cell establishment or maintenance.

Modulation of Arabidopsis root growth by specialized triterpenes.

Plant roots are specialized belowground organs that spatiotemporally shape their development in function of varying soil conditions. This root plasticity relies on intricate molecular networks driven by phytohormones, such as auxin and jasmonate (JA). Loss-of-function of the NOVEL INTERACTOR OF JAZ (NINJA), a core component of the JA signaling pathway, leads to enhanced triterpene biosynthesis, in particular of the thalianol gene cluster, in Arabidopsis thaliana roots. We have investigated the biological role of thalianol and its derivatives by focusing on Thalianol Synthase (THAS) and Thalianol Acyltransferase 2 (THAA2), two thalianol cluster genes that are upregulated in the roots of ninja mutant plants. THAS and THAA2 activity was investigated in yeast, and metabolite and phenotype profiling of thas and thaa2 loss-of-function plants was carried out. THAA2 was shown to be responsible for the acetylation of thalianol and its derivatives, both in yeast and in planta. In addition, THAS and THAA2 activity was shown to modulate root development. Our results indicate that the thalianol pathway is not only controlled by phytohormonal cues, but also may modulate phytohormonal action itself, thereby affecting root development and interaction with the environment.

Genetic Variability of Arabidopsis thaliana Mature Root System Architecture and Genome-Wide Association Study.

Root system architecture (RSA) has a direct influence on the efficiency of nutrient uptake and plant growth, but the genetics of RSA are often studied only at the seedling stage. To get an insight into the genetic blueprint of a more mature RSA, we exploited natural variation and performed a detailed in vitro study of 241 Arabidopsis thaliana accessions using large petri dishes. A comprehensive analysis of 17 RSA traits showed high variability among the different accessions, unveiling correlations between traits and conditions of the natural habitat of the plants. A sub-selection of these accessions was grown in water-limiting conditions in a rhizotron set-up, which revealed that especially the spatial distribution showed a high consistency between in vitro and ex vitro conditions, while in particular, a large root area in the lower zone favored drought tolerance. The collected RSA phenotype data were used to perform genome-wide association studies (GWAS), which stands out from the previous studies by its exhaustive measurements of RSA traits on more mature Arabidopsis accessions used for GWAS. As a result, we found not only several genes involved in the lateral root (LR) development or auxin signaling pathways to be associated with RSA traits but also new candidate genes that are potentially involved in the adaptation to the natural habitats.

The Phloem Intercalated With Xylem-Correlated 3 Receptor-Like Kinase Constitutively Interacts With Brassinosteroid Insensitive 1-Associated Receptor Kinase 1 and Is Involved in Vascular Development in Arabidopsis.

Leucine-rich repeat receptor-like kinases (LRR-RLKs) play fundamental roles in cell-to-cell and plant-environment communication. LRR-RLKs can function as receptors perceiving endogenous or external ligands, or as coreceptors, which stabilize the complex, and enhance transduction of the intracellular signal. The LRR-RLK BAK1 is a coreceptor for different developmental and immunity pathways. In this article, we identified PXY-CORRELATED 3 (PXC3) as a BAK1-interacting LRR-RLK, which was previously reported to be transcribed in vascular tissues co-expressed with PHLOEM INTERCALATED WITH XYLEM (PXY), the receptor of the TDIF/CLE41 peptide. Characterization of pxc3 loss-of-function mutants revealed reduced hypocotyl stele width and vascular cells compared to wild type, indicating that PXC3 plays a role in the vascular development in Arabidopsis. Furthermore, our data suggest that PXC3 might function as a positive regulator of the CLE41/TDIF-TDR/PXY signaling pathway.

Phosphorylation of MAP65-1 by Arabidopsis Aurora Kinases Is Required for Efficient Cell Cycle Progression.

Aurora kinases are key effectors of mitosis. Plant Auroras are functionally divided into two clades. The alpha Auroras (Aurora1 and Aurora2) associate with the spindle and the cell plate and are implicated in controlling formative divisions throughout plant development. The beta Aurora (Aurora3) localizes to centromeres and likely functions in chromosome separation. In contrast to the wealth of data available on the role of Aurora in other kingdoms, knowledge on their function in plants is merely emerging. This is exemplified by the fact that only histone H3 and the plant homolog of TPX2 have been identified as Aurora substrates in plants. Here we provide biochemical, genetic, and cell biological evidence that the microtubule-bundling protein MAP65-1-a member of the MAP65/Ase1/PRC1 protein family, implicated in central spindle formation and cytokinesis in animals, yeasts, and plants-is a genuine substrate of alpha Aurora kinases. MAP65-1 interacts with Aurora1 in vivo and is phosphorylated on two residues at its unfolded tail domain. Its overexpression and down-regulation antagonistically affect the alpha Aurora double mutant phenotypes. Phospho-mutant analysis shows that Aurora contributes to the microtubule bundling capacity of MAP65-1 in concert with other mitotic kinases.

CEP5 and XIP1/CEPR1 regulate lateral root initiation in Arabidopsis.

Roots explore the soil for water and nutrients through the continuous production of lateral roots. Lateral roots are formed at regular distances in a steadily elongating organ, but how future sites for lateral root formation become established is not yet understood. Here, we identified C-TERMINALLY ENCODED PEPTIDE 5 (CEP5) as a novel, auxin-repressed and phloem pole-expressed signal assisting in the formation of lateral roots. In addition, based on genetic and expression data, we found evidence for the involvement of its proposed receptor, XYLEM INTERMIXED WITH PHLOEM 1 (XIP1)/CEP RECEPTOR 1 (CEPR1), during the process of lateral root initiation. In conclusion, we report here on the existence of a peptide ligand-receptor kinase interaction that impacts lateral root initiation. Our results represent an important step towards the understanding of the cellular communication implicated in the early phases of lateral root formation.

Cyclic programmed cell death stimulates hormone signaling and root development in Arabidopsis.

The plant root cap, surrounding the very tip of the growing root, perceives and transmits environmental signals to the inner root tissues. In Arabidopsis thaliana, auxin released by the root cap contributes to the regular spacing of lateral organs along the primary root axis. Here, we show that the periodicity of lateral organ induction is driven by recurrent programmed cell death at the most distal edge of the root cap. We suggest that synchronous bursts of cell death in lateral root cap cells release pulses of auxin to surrounding root tissues, establishing the pattern for lateral root formation. The dynamics of root cap turnover may therefore coordinate primary root growth with root branching in order to optimize the uptake of water and nutrients from the soil.

Expanding the repertoire of secretory peptides controlling root development with comparative genome analysis and functional assays.

Plant genomes encode numerous small secretory peptides (SSPs) whose functions have yet to be explored. Based on structural features that characterize SSP families known to take part in postembryonic development, this comparative genome analysis resulted in the identification of genes coding for oligopeptides potentially involved in cell-to-cell communication. Because genome annotation based on short sequence homology is difficult, the criteria for the de novo identification and aggregation of conserved SSP sequences were first benchmarked across five reference plant species. The resulting gene families were then extended to 32 genome sequences, including major crops. The global phylogenetic pattern common to the functionally characterized SSP families suggests that their apparition and expansion coincide with that of the land plants. The SSP families can be searched online for members, sequences and consensus (http://bioinformatics.psb.ugent.be/webtools/PlantSSP/). Looking for putative regulators of root development, Arabidopsis thaliana SSP genes were further selected through transcriptome meta-analysis based on their expression at specific stages and in specific cell types in the course of the lateral root formation. As an additional indication that formerly uncharacterized SSPs may control development, this study showed that root growth and branching were altered by the application of synthetic peptides matching conserved SSP motifs, sometimes in very specific ways. The strategy used in the study, combining comparative genomics, transcriptome meta-analysis and peptide functional assays in planta, pinpoints factors potentially involved in non-cell-autonomous regulatory mechanisms. A similar approach can be implemented in different species for the study of a wide range of developmental programmes.

Root Cap-Derived Auxin Pre-patterns the Longitudinal Axis of the Arabidopsis Root.

During the exploration of the soil by plant roots, uptake of water and nutrients can be greatly fostered by a regular spacing of lateral roots (LRs). In the Arabidopsis root, a regular branching pattern depends on oscillatory gene activity to create prebranch sites, patches of cells competent to form LRs. Thus far, the molecular components regulating the oscillations still remain unclear. Here, we show that a local auxin source in the root cap, derived from the auxin precursor indole-3-butyric acid (IBA), modulates the oscillation amplitude, which in turn determines whether a prebranch site is created or not. Moreover, transcriptome profiling identified novel and IBA-regulated components of root patterning, such as the MEMBRANE-ASSOCIATED KINASE REGULATOR4 (MAKR4) that converts the prebranch sites into a regular spacing of lateral organs. Thus, the spatiotemporal patterning of roots is fine-tuned by the root cap-specific conversion pathway of IBA to auxin and the subsequent induction of MAKR4.

A miR169 isoform regulates specific NF-YA targets and root architecture in Arabidopsis.

In plants, roots are essential for water and nutrient acquisition. MicroRNAs (miRNAs) regulate their target mRNAs by transcript cleavage and/or inhibition of protein translation and are known as major post-transcriptional regulators of various developmental pathways and stress responses. In Arabidopsis thaliana, four isoforms of miR169 are encoded by 14 different genes and target diverse mRNAs, encoding subunits A of the NF-Y transcription factor complex. These miRNA isoforms and their targets have previously been linked to nutrient signalling in plants. By using mimicry constructs against different isoforms of miR169 and miR-resistant versions of NF-YA genes we analysed the role of specific miR169 isoforms in root growth and branching. We identified a regulatory node involving the particular miR169defg isoform and NF-YA2 and NF-YA10 genes that acts in the control of primary root growth. The specific expression of MIM169defg constructs altered specific cell type numbers and dimensions in the root meristem. Preventing miR169defg-regulation of NF-YA2 indirectly affected laterial root initiation. We also showed that the miR169defg isoform affects NF-YA2 transcripts both at mRNA stability and translation levels. We propose that a specific miR169 isoform and the NF-YA2 target control root architecture in Arabidopsis.

A new role for glutathione in the regulation of root architecture linked to strigolactones.

Reduced glutathione (GSH) is required for root development, but its functions are not characterized. The effects of GSH depletion on root development were therefore studied in relation to auxin and strigolactone (SL) signalling using a combination of molecular genetic approaches and pharmacological techniques. Lateral root (LR) density was significantly decreased in GSH synthesis mutants (cad2-1, pad2-, rax1-), but not by the GSH synthesis inhibitor, buthionine sulfoximine (BSO). BSO-induced GSH depletion therefore did not influence root architecture in the same way as genetic impairment. Root glutathione contents were similar in the wild-type seedlings and max3-9 and max4-1 mutants that are deficient in SL synthesis and in the SL-signalling mutant, max2-1. BSO-dependent inhibition of GSH synthesis depleted the tissue GSH pool to a similar extent in the wild-type and SL synthesis mutants, with no effect on LR density. The application of the SL analogue GR24 increased root glutathione in the wild-type, max3-9 and max4-1 seedlings, but this increase was absent from max2-1. Taken together, these data establish a link between SLs and the GSH pool that occurs in a MAX2-dependent manner.

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.

A role for the root cap in root branching revealed by the non-auxin probe naxillin.

The acquisition of water and nutrients by plant roots is a fundamental aspect of agriculture and strongly depends on root architecture. Root branching and expansion of the root system is achieved through the development of lateral roots and is to a large extent controlled by the plant hormone auxin. However, the pleiotropic effects of auxin or auxin-like molecules on root systems complicate the study of lateral root development. Here we describe a small-molecule screen in Arabidopsis thaliana that identified naxillin as what is to our knowledge the first non-auxin-like molecule that promotes root branching. By using naxillin as a chemical tool, we identified a new function for root cap-specific conversion of the auxin precursor indole-3-butyric acid into the active auxin indole-3-acetic acid and uncovered the involvement of the root cap in root branching. Delivery of an auxin precursor in peripheral tissues such as the root cap might represent an important mechanism shaping root architecture.

Plasma membrane calcium ATPases are important components of receptor-mediated signaling in plant immune responses and development.

Plasma membrane-resident receptor kinases (RKs) initiate signaling pathways important for plant immunity and development. In Arabidopsis (Arabidopsis thaliana), the receptor for the elicitor-active peptide epitope of bacterial flagellin, flg22, is encoded by FLAGELLIN SENSING2 (FLS2), which promotes plant immunity. Despite its relevance, the molecular components regulating FLS2-mediated signaling remain largely unknown. We show that plasma membrane ARABIDOPSIS-AUTOINHIBITED Ca(2+)-ATPase (ACA8) forms a complex with FLS2 in planta. ACA8 and its closest homolog ACA10 are required for limiting the growth of virulent bacteria. One of the earliest flg22 responses is the transient increase of cytosolic Ca(2+) ions, which is crucial for many of the well-described downstream responses (e.g. generation of reactive oxygen species and the transcriptional activation of defense-associated genes). Mutant aca8 aca10 plants show decreased flg22-induced Ca(2+) and reactive oxygen species bursts and exhibit altered transcriptional reprogramming. In particular, mitogen-activated protein kinase-dependent flg22-induced gene expression is elevated, whereas calcium-dependent protein kinase-dependent flg22-induced gene expression is reduced. These results demonstrate that the fine regulation of Ca(2+) fluxes across the plasma membrane is critical for the coordination of the downstream microbe-associated molecular pattern responses and suggest a mechanistic link between the FLS2 receptor complex and signaling kinases via the secondary messenger Ca(2+). ACA8 also interacts with other RKs such as BRI1 and CLV1 known to regulate plant development, and both aca8 and aca10 mutants show morphological phenotypes, suggesting additional roles for ACA8 and ACA10 in developmental processes. Thus, Ca(2+) ATPases appear to represent general regulatory components of RK-mediated signaling pathways.