The transcription factor AtMYB12 is part of a feedback loop regulating cell division orientation in the root meristem vasculature.

Transcriptional networks are crucial to integrate various internal and external signals into optimal responses during plant growth and development. In Arabidopsis thaliana, primary root vasculature patterning and proliferation are controlled by a network centred around the basic Helix-Loop-Helix transcription factor complex, formed by TARGET OF MONOPTEROS 5 (TMO5) and LONESOME HIGHWAY (LHW), which control cell proliferation and division orientation by modulating the cytokinin response and other downstream factors. Despite recent progress, many aspects of the TMO5/LHW pathway are not fully understood. In particular, the upstream regulators of TMO5/LHW activity remain unknown. Here, using a forward genetics approach to identify new factors of the TMO5/LHW pathway, we discovered a novel function of the MYB-type transcription factor, MYB12. MYB12 physically interacts with TMO5 and dampens the TMO5/LHW-mediated induction of direct target gene expression, as well as the periclinal/radial cell divisions. The expression of MYB12 is activated by the cytokinin response, downstream of TMO5/LHW, resulting in a novel MYB12-mediated negative feedback loop that restricts TMO5/LHW activity, to ensure optimal cell proliferation rates during root vascular development.

Means to Quantify Vascular Cell File Numbers in Different Tissues.

Oriented cell divisions are crucial throughout plant development to define the final size and shape of organs and tissues. As most of the tissues in mature roots and stems are derived from vascular tissues, studying cell proliferation in the vascular cell lineage is of great importance. Although perturbations of vascular development are often visible already at the whole plant macroscopic phenotype level, a more detailed characterization of the vascular anatomy, cellular organization, and differentiation status of specific vascular cell types can provide insights into which pathway or developmental program is affected. In particular, defects in the frequency or orientation of cell divisions can be reliably identified from the number of vascular cell files. Here, we provide a detailed description of the different clearing, staining, and imaging techniques that allow precise phenotypic analysis of vascular tissues in different organs of the model plant Arabidopsis thaliana throughout development, including the quantification of cell file numbers, differentiation status of vascular cell types, and expression of reporter genes.

Seedling developmental defects upon blocking CINNAMATE-4-HYDROXYLASE are caused by perturbations in auxin transport.

The phenylpropanoid pathway serves a central role in plant metabolism, providing numerous compounds involved in diverse physiological processes. Most carbon entering the pathway is incorporated into lignin. Although several phenylpropanoid pathway mutants show seedling growth arrest, the role for lignin in seedling growth and development is unexplored. We use complementary pharmacological and genetic approaches to block CINNAMATE-4-HYDROXYLASE (C4H) functionality in Arabidopsis seedlings and a set of molecular and biochemical techniques to investigate the underlying phenotypes. Blocking C4H resulted in reduced lateral rooting and increased adventitious rooting apically in the hypocotyl. These phenotypes coincided with an inhibition in AUX transport. The upstream accumulation in cis-cinnamic acid was found to be likely to cause polar AUX transport inhibition. Conversely, a downstream depletion in lignin perturbed phloem-mediated AUX transport. Restoring lignin deposition effectively reestablished phloem transport and, accordingly, AUX homeostasis. Our results show that the accumulation of bioactive intermediates and depletion in lignin jointly cause the aberrant phenotypes upon blocking C4H, and demonstrate that proper deposition of lignin is essential for the establishment of AUX distribution in seedlings. Our data position the phenylpropanoid pathway and lignin in a new physiological framework, consolidating their importance in plant growth and development.

Vascular transcription factors guide plant epidermal responses to limiting phosphate conditions.

Optimal plant growth is hampered by deficiency of the essential macronutrient phosphate in most soils. Plant roots can, however, increase their root hair density to efficiently forage the soil for this immobile nutrient. By generating and exploiting a high-resolution single-cell gene expression atlas of Arabidopsis roots, we show an enrichment of TARGET OF MONOPTEROS 5/LONESOME HIGHWAY (TMO5/LHW) target gene responses in root hair cells. The TMO5/LHW heterodimer triggers biosynthesis of mobile cytokinin in vascular cells and increases root hair density during low-phosphate conditions by modifying both the length and cell fate of epidermal cells. Moreover, root hair responses in phosphate-deprived conditions are TMO5- and cytokinin-dependent. Cytokinin signaling links root hair responses in the epidermis to perception of phosphate depletion in vascular cells.