An environmentally responsive transcriptional state modulates cell identities during root development

Abstract

Roots are fundamental organs for plant development and response to their environment: they anchor the plant to its growth substrate, uptake nutrients and water vital to plant growth, and can sense and respond to a variety of biotic and abiotic stresses. The architecture of root systems and their growth are known to be strongly affected by the environmental conditions found in the soil. However, the acquisition of cell identities at the root meristem is still mainly viewed as ontogenetically driven, where a small number of stem cells generate all the cell types through stereotyped divisions followed by differentiation, along a simple developmental trajectory. The extent to which environmental cues precisely shape and affect these developmental trajectories remains an open question. We used single-cell RNA-seq, combined with spatial mapping, to deeply explore the trajectories of cell states at the tip of Arabidopsis roots, known to contain multiple developing lineages. Surprisingly, we found that most lineage trajectories exhibit a stereotyped bifid topology with two developmental trajectories rather than one. The formation of one of the trajectories is driven by a strong and specific activation of genes involved in the responses to various environmental stimuli, that affects only of a subset of the cells in multiple cell types simultaneously, revealing another layer of patterning of cell identities in the root that is independent of cell ontogeny. We demonstrate the robustness of this environmentally-responsive transcriptional state by showing that it is present in a mutant where cell type identities are greatly perturbed, as well as in different Arabidopsis ecotypes. We also show that the root can adapt the proportion of cells that acquire this particular state in response to environmental signals such as nutrient availability. The discovery of this transcriptional signature further highlights the adaptive potential of plant development.

MYB68 regulates suberin patterning and radially distinct endodermal differentiation

“…Within this, we identified two potassium (K) transporters POTASSIUM UPTAKE 8 ( KUP8) and HIGH AFFINITY K+ TRANSPORTER 5 (HAK5) (Osakabe, et al, 2013; Gierth, et al, 2005) as well as the Arabidopsis sodium (Na) transporter HIGH-AFFINITY K+ TRANSPORTER 1 ( HKT1)(Mäser, et al, 2002) ( Figure 5C, Table S6 ) . To probe if these genes are associated with PCs, we created promoter-based transcriptional marker lines or performed whole-mount hybridization chain reaction fluorescent in situ hybridization (HCR-FISH) (Oliva, et al, 2022). Interestingly, in the suberizing part of 6-day-old roots, both KUP8 and HAK5 showed expression in the endodermis, which was biased toward XPAE or NPAE cells and increased in the myb68-2-4 line ( Figure 5D ).…”

“…To probe if these genes are associated with PCs, we created promoter-based transcriptional marker lines or performed whole-mount hybridization chain reaction fluorescent in situ hybridization (HCR-FISH) (Oliva, et al, 2022). Interestingly, in the suberizing part of 6-day-old roots, both KUP8 and HAK5 showed expression in the endodermis, which was biased toward XPAE or NPAE cells and increased in the myb68-2-4 line (Figure 5D).…”

PHYTOMap: Multiplexed single-cell 3D spatial gene expression analysis in plant tissue

“…Five-day-old root tips were mounted on a Poly-D-Lysine coated dish, and all the following steps were conducted on the dish. Samples were fixed, dehydrated, and rehydrated in a similar way as described in previous studies 19,20 with modifications. Arabidopsis root tips were immersed in FAA (16% (v/v) formaldehyde, 5% (v/v) acetic acid, 50% ethanol) for 1 hour at RT.…”

“…A pool of SNAIL probes (500 nM each) was heated at 90ºC for 5 min and cooled down at RT. Samples were incubated in hybridization buffer (2X SSC, 30% formamide, 1% Triton-X,20 mM Ribonucleoside vanadyl complex, and pooled SNAIL probes at 10 nM per oligo) in a 40ºC humidified oven overnight. After hybridization, samples were washed twice in DPBS-TR and once in 4X SSC in DPBS-TR for 30 min at 37ºC and rinsed with DPBS-TR at RT.…”

Fluorescence hybridization chain reaction enables localization of multiple molecular classes combined with plant cell ultrastructure

“…The reagents can be prepared in house or are commercially available through Molecular Instruments (Los Angeles, CA). Recently, HCR FISH has been applied in wholemount tissues of Arabidopsis inflorescences and Arabidopsis, maize and sorghum roots (13,25,26).…”