Ana Campilho

Cell signalling by microRNA165/6 directs gene dose-dependent root cell fate

A key question in developmental biology is how cells exchange positional information for proper patterning during organ development. In plant roots the radial tissue organization is highly conserved with a central vascular cylinder in which two water conducting cell types, protoxylem and metaxylem, are patterned centripetally. We show that this patterning occurs through crosstalk between the vascular cylinder and the surrounding endodermis mediated by cell-to-cell movement of a transcription factor in one direction and microRNAs in the other. SHORT ROOT, produced in the vascular cylinder, moves into the endodermis to activate SCARECROW. Together these transcription factors activate MIR165a and MIR166b. Endodermally produced microRNA165/6 then acts to degrade its target mRNAs encoding class III homeodomain-leucine zipper transcription factors in the endodermis and stele periphery. The resulting differential distribution of target mRNA in the vascular cylinder determines xylem cell types in a dosage-dependent manner.

The RETINOBLASTOMA-RELATED Gene Regulates Stem Cell Maintenance in Arabidopsis Roots

The maintenance of stem cells in defined locations is crucial for all multicellular organisms. Although intrinsic factors and signals for stem cell fate have been identified in several species, it has remained unclear how these connect to the ability to reenter the cell cycle that is one of the defining properties of stem cells. We show that local reduction of expression of the RETINOBLASTOMA-RELATED (RBR) gene in Arabidopsis roots increases the amount of stem cells without affecting cell cycle duration in mitotically active cells. Conversely, induced RBR overexpression dissipates stem cells prior to arresting other mitotic cells. Overexpression of D cyclins, KIP-related proteins, and E2F factors also affects root stem cell pool size, and genetic interactions suggest that these factors function in a canonical RBR pathway to regulate somatic stem cells. Expression analysis and genetic interactions position RBR-mediated regulation of the stem cell state downstream of the patterning gene SCARECROW.

Callose Biosynthesis Regulates Symplastic Trafficking during Root Development

Plant cells are connected through plasmodesmata (PD), membrane-lined channels that allow symplastic movement of molecules between cells. However, little is known about the role of PD-mediated signaling during plant morphogenesis. Here, we describe an Arabidopsis gene, CALS3/GSL12. Gain-of-function mutations in CALS3 result in increased accumulation of callose (β-1,3-glucan) at the PD, a decrease in PD aperture, defects in root development, and reduced intercellular trafficking. Enhancement of CALS3 expression during phloem development suppressed loss-of-function mutations in the phloem abundant callose synthase, CALS7 indicating that CALS3 is a bona fide callose synthase. CALS3 alleles allowed us to spatially and temporally control the PD aperture between plant tissues. Using this tool, we are able to show that movement of the transcription factor SHORT-ROOT and microRNA165 between the stele and the endodermis is PD dependent. Taken together, we conclude that regulated callose biosynthesis at PD is essential for cell signaling.

The NAC Domain Transcription Factors FEZ and SOMBRERO Control the Orientation of Cell Division Plane in Arabidopsis Root Stem Cells

Because plant cells do not migrate, cell division planes are crucial determinants of plant cellular architecture. In Arabidopsis roots, stringent control of cell divisions leads to a virtually invariant division pattern, including those that create new tissue layers. However, the mechanisms that control oriented cell divisions are hitherto poorly understood. Here, we reveal one such mechanism in which FEZ and SOMBRERO (SMB), two plant-specific NAC-domain transcription factors, control the delicately tuned reorientation and timing of cell division in a subset of stem cells. FEZ is expressed in root cap stem cells, where it promotes periclinal, root cap-forming cell divisions. In contrast, SMB negatively regulates FEZ activity, repressing stem cell-like divisions in the root cap daughter cells. FEZ becomes expressed in predivision stem cells, induces oriented cell division, and activates expression of its negative regulator, SMB, thus generating a feedback loop for controlled switches in cell division plane.

SOMBRERO, BEARSKIN1, and BEARSKIN2 Regulate Root Cap Maturation in Arabidopsis

This work demonstrates that three closely related Arabidopsis transcription factors are involved in activating the specific modifications to cell walls that are required for a fully functional root cap. These transcription factors share a generic transcriptional activity with other closely related proteins, which are involved in different aspects of cell wall modification. The root cap has a central role in root growth, determining the growth trajectory and facilitating penetration into the soil. Root cap cells have specialized functions and morphologies, and border cells are released into the rhizosphere by specific cell wall modifications. Here, we demonstrate that the cellular maturation of root cap is redundantly regulated by three genes, SOMBRERO (SMB), BEARSKIN1 (BRN1), and BRN2, which are members of the Class IIB NAC transcription factor family, together with the VASCULAR NAC DOMAIN (VND) and NAC SECONDARY WALL THICKENING PROMOTING FACTOR (NST) genes that regulate secondary cell wall synthesis in specialized cell types. Lateral cap cells in smb-3 mutants continue to divide and fail to detach from the root, phenotypes that are independent of FEZ upregulation in smb-3. In brn1-1 brn2-1 double mutants, columella cells fail to detach, while in triple mutants, cells fail to mature in all parts of the cap. This complex genetic redundancy involves differences in expression, protein activity, and target specificity. All three genes have very similar overexpression phenotypes to the VND/NST genes, indicating that members of this family are largely functionally equivalent. Our results suggest that Class IIB NAC proteins regulate cell maturation in cells that undergo terminal differentiation with strong cell wall modifications.

CHOLINE TRANSPORTER-LIKE1 is required for sieve plate development to mediate long-distance cell-to-cell communication

Phloem, a plant tissue responsible for long-distance molecular transport, harbours specific junctions, sieve areas, between the conducting cells. To date, little is known about the molecular framework related to the biogenesis of these sieve areas. Here we identify mutations at the CHER1/AtCTL1 locus of Arabidopsis thaliana. The mutations cause several phenotypic abnormalities, including reduced pore density and altered pore structure in the sieve areas associated with impaired phloem function. CHER1 encodes a member of a poorly characterized choline transporter-like protein family in plants and animals. We show that CHER1 facilitates choline transport, localizes to the trans-Golgi network, and during cytokinesis is associated with the phragmoplast. Consistent with its function in the elaboration of the sieve areas, CHER1 has a sustained, polar localization in the forming sieve plates. Our results indicate that the regulation of choline levels is crucial for phloem development and conductivity in plants.

AHP6 Inhibits Cytokinin Signaling to Regulate the Orientation of Pericycle Cell Division during Lateral Root Initiation

In Arabidopsis thaliana, lateral roots (LRs) initiate from anticlinal cell divisions of pericycle founder cells. The formation of LR primordia is regulated antagonistically by the phytohormones cytokinin and auxin. It has previously been shown that cytokinin has an inhibitory effect on the patterning events occurring during LR formation. However, the molecular players involved in cytokinin repression are still unknown. In a similar manner to protoxylem formation in Arabidopsis roots, in which AHP6 (ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6) acts as a cytokinin inhibitor, we reveal that AHP6 also functions as a cytokinin repressor during early stages of LR development. We show that AHP6 is expressed at different developmental stages during LR formation and is required for the correct orientation of cell divisions at the onset of LR development. Moreover, we demonstrate that AHP6 influences the localization of the auxin efflux carrier PIN1, which is necessary for patterning the LR primordia. In summary, we show that the inhibition of cytokinin signaling through AHP6 is required to establish the correct pattern during LR initiation.

Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI

Summary N6‐adenosine methylation (m6A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m6A writer proteins in Arabidopsis thaliana. The components required for m6A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m6A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m6A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m6A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences.

Automatic Tracking of Arabidopsis thaliana Root Meristem in Confocal Microscopy

The Arabidopsis thaliana is a well defined and a suited system to study plant development at the cellular level. To follow in vivo the root meristem activity under a confocal microscope the image acquisition process was automated through a coherent observation of a fixed point of the root tip. This position information allows the microscope stage control to track the root tip. Root tip estimation is performed following two approaches: computing the root central line intersection with the contour or the maximum filtered contour curvature point. The first method fits the root border with lines, using the Radon transform and a classification procedure. The central line is defined as the line that bisects the angle between these lines. The intersection of the central line with the root contour provides an estimate for the root tip position. The second method is based on contour traversing, followed by convolution of the contour coordinates with a Gaussian kernel. Curvature is computed for this filtered contour. The maximum curvature point provides another root tip estimate. A third method, based on a Kalman estimator is used to select between the previous two outputs. The system allowed the tracking of the root meristem for more than 20 hours in several experiments.

The Arabidopsis HP6 gene is expressed in Medicago truncatula lateral roots and root nodule primordia

Expression patterns of orthologous genes can be similar between distantly related species, suggesting that developmental programs can be conserved between organisms. Here, we show that the promoter of AHP6, a gene which is involved in Arabidopsis lateral root development, also drives the expression of the reporter GUS gene in lateral roots of Medicago truncatula suggesting that similar regulatory elements are involved in lateral root organogenesis in these species. Interestingly, the AHP6 promoter was able to drive GUS expression in root nodules and nodule primordia, structures that are absent in Arabidopsis. We found two AHP6 orthologous genes in the M. truncatula genome and we speculate that these putative cytokinin inhibitors may play a role during lateral root and nodule development in this species.