Translational profile of developing phellem cells in Arabidopsis thaliana roots.

The phellem is a specialized boundary tissue providing the first line of defense against abiotic and biotic stresses in organs undergoing secondary growth. Phellem cells undergo several differentiation steps, which include cell wall suberization, cell expansion, and programmed cell death. Yet, the molecular players acting particularly in phellem cell differentiation remain poorly described, particularly in the widely used model plant Arabidopsis thaliana. Using specific marker lines we followed the onset and progression of phellem differentiation in A. thaliana roots and further targeted the translatome of newly developed phellem cells using translating ribosome affinity purification followed by mRNA sequencing (TRAP-SEQ). We showed that phellem suberization is initiated early after phellogen (cork cambium) division. The specific translational landscape was organized in three main domains related to energy production, synthesis and transport of cell wall components, and response to stimulus. Novel players in phellem differentiation related to suberin monomer transport and assembly as well as novel transcription regulators were identified. This strategy provided an unprecedented resolution of the translatome of developing phellem cells, giving a detailed and specific view on the molecular mechanisms acting on cell differentiation in periderm tissues of the model plant Arabidopsis.

Transcriptional Analysis in the Arabidopsis Roots Reveals New Regulators that Link rac-GR24 Treatment with Changes in Flavonol Accumulation, Root Hair Elongation and Lateral Root Density.

The synthetic strigolactone (SL) analog, rac-GR24, has been instrumental in studying the role of SLs as well as karrikins because it activates the receptors DWARF14 (D14) and KARRIKIN INSENSITIVE 2 (KAI2) of their signaling pathways, respectively. Treatment with rac-GR24 modifies the root architecture at different levels, such as decreasing the lateral root density (LRD), while promoting root hair elongation or flavonol accumulation. Previously, we have shown that the flavonol biosynthesis is transcriptionally activated in the root by rac-GR24 treatment, but, thus far, the molecular players involved in that response have remained unknown. To get an in-depth insight into the changes that occur after the compound is perceived by the roots, we compared the root transcriptomes of the wild type and the more axillary growth2 (max2) mutant, affected in both SL and karrikin signaling pathways, with and without rac-GR24 treatment. Quantitative reverse transcription (qRT)-PCR, reporter line analysis and mutant phenotyping indicated that the flavonol response and the root hair elongation are controlled by the ELONGATED HYPOCOTYL 5 (HY5) and MYB12 transcription factors, but HY5, in contrast to MYB12, affects the LRD as well. Furthermore, we identified the transcription factors TARGET OF MONOPTEROS 5 (TMO5) and TMO5 LIKE1 as negative and the Mediator complex as positive regulators of the rac-GR24 effect on LRD. Altogether, hereby, we get closer toward understanding the molecular mechanisms that underlay the rac-GR24 responses in the root.

Lateral Root Initiation and the Analysis of Gene Function Using Genome Editing with CRISPR in Arabidopsis.

Lateral root initiation is a post-embryonic process that requires the specification of a subset of pericycle cells adjacent to the xylem pole in the primary root into lateral root founder cells. The first visible event of lateral root initiation in Arabidopsis is the simultaneous migration of nuclei in neighbouring founder cells. Coinciding cell cycle activation is essential for founder cells in the pericycle to undergo formative divisions, resulting in the development of a lateral root primordium (LRP). The plant signalling molecule, auxin, is a major regulator of lateral root development; the understanding of the molecular mechanisms controlling lateral root initiation has progressed tremendously by the use of the Arabidopsis model and a continual improvement of molecular methodologies. Here, we provide an overview of the visible events, cell cycle regulators, and auxin signalling cascades related to the initiation of a new LRP. Furthermore, we highlight the potential of genome editing technology to analyse gene function in lateral root initiation, which provides an excellent model to answer fundamental developmental questions such as coordinated cell division, growth axis establishment as well as the specification of cell fate and cell polarity.

Overexpression of the NMig1 Gene Encoding a NudC Domain Protein Enhances Root Growth and Abiotic Stress Tolerance in Arabidopsis thaliana.

The family of NudC proteins has representatives in all eukaryotes and plays essential evolutionarily conserved roles in many aspects of organismal development and stress response, including nuclear migration, cell division, folding and stabilization of other proteins. This study investigates an undescribed Arabidopsis homolog of the Aspergillus nidulans NudC gene, named NMig1 (for Nuclear Migration 1), which shares high sequence similarity to other plant and mammalian NudC-like genes. Expression of NMig1 was highly upregulated in response to several abiotic stress factors, such as heat shock, drought and high salinity. Constitutive overexpression of NMig1 led to enhanced root growth and lateral root development under optimal and stress conditions. Exposure to abiotic stress resulted in relatively weaker inhibition of root length and branching in NMig1-overexpressing plants, compared to the wild-type Col-0. The expression level of antioxidant enzyme-encoding genes and other stress-associated genes was considerably induced in the transgenic plants. The increased expression of the major antioxidant enzymes and greater antioxidant potential correlated well with the lower levels of reactive oxygen species (ROS) and lower lipid peroxidation. In addition, the overexpression of NMig1 was associated with strong upregulation of genes encoding heat shock proteins and abiotic stress-associated genes. Therefore, our data demonstrate that the NudC homolog NMig1 could be considered as a potentially important target gene for further use, including breeding more resilient crops with improved root architecture under abiotic stress.

GOLVEN peptide signalling through RGI receptors and MPK6 restricts asymmetric cell division during lateral root initiation.

During lateral root initiation, lateral root founder cells undergo asymmetric cell divisions that generate daughter cells with different sizes and fates, a prerequisite for correct primordium organogenesis. An excess of the GLV6/RGF8 peptide disrupts these initial asymmetric cell divisions, resulting in more symmetric divisions and the failure to achieve lateral root organogenesis. Here, we show that loss-of-function GLV6 and its homologue GLV10 increase asymmetric cell divisions during lateral root initiation, and we identified three members of the RGF1 INSENSITIVE/RGF1 receptor subfamily as likely GLV receptors in this process. Through a suppressor screen, we found that MITOGEN-ACTIVATED PROTEIN KINASE6 is a downstream regulator of the GLV pathway. Our data indicate that GLV6 and GLV10 act as inhibitors of asymmetric cell divisions and signal through RGF1 INSENSITIVE receptors and MITOGEN-ACTIVATED PROTEIN KINASE6 to restrict the number of initial asymmetric cell divisions that take place during lateral root initiation.

Give CRISPR a Chance: the GeneSprout Initiative.

Did you know that a group of early-career researchers launched an initiative enabling open dialog on new plant breeding techniques, such as genome editing? We developed a wide-ranging initiative that aims to facilitate public engagement and provide a platform for young plant scientists to encourage participation in science communication.

CRISPR-TSKO: A Technique for Efficient Mutagenesis in Specific Cell Types, Tissues, or Organs in Arabidopsis.

Detailed functional analyses of many fundamentally important plant genes via conventional loss-of-function approaches are impeded by the severe pleiotropic phenotypes resulting from these losses. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a clustered regularly interspaced short palindromic repeats (CRISPR)-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis (Arabidopsis thaliana), CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The modular cloning system developed in this study allows for the efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens avenues for discovering and analyzing gene functions in the spatial and temporal contexts of plant life while avoiding the pleiotropic effects of system-wide losses of gene function.

Lateral Root Inducible System in Arabidopsis and Maize.

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.