Plants strategically respond to gravity and actively set defined growth angles. These gravitropic set-point angles (GSAs) shape the width and depth of the root system, determining soil exploration for water and nutrient uptake. Despite its physiological importance, the concept by which lateral roots establish their growth angle has remained largely unclear. Here, we show that temporal regulation of gravity responsiveness within a defined developmental phase sets the initial GSA in lateral roots. Stage-specific transcriptome profiling revealed that SHOOT GRAVITROPISM 9 (SGR9) is upregulated in emerged lateral roots as they first respond to gravity. Loss of SGR9 slows amyloplast sedimentation and delays the PIN3-dependent establishment of asymmetric auxin distribution. Notably, the delay in the gravity response alters, rather than merely postpones, the initial stable growth angle. Our findings reveal that a developmental time window enables transient sensory dynamics to be translated into stable initial root growth angles, illustrating how developmental timing can convert short-lived signals into lasting form.

Auxin is a crucial phytohormone that regulates plant development and facilitates dynamic responses to environmental changes through subcellular control mechanisms. PIN-LIKES (PILS) are auxin transport facilitators at the endoplasmic reticulum (ER) that mediate nuclear auxin abundance and signaling. Although the posttranslational regulation of PILS is important for acclimating growth responses, the molecular mechanisms involved remain largely unknown. This study demonstrates that components of the ER-associated degradation (ERAD) machinery regulate the proteasome-dependent degradation of functional PILS proteins under nonstressed conditions. We further reveal that both internal and external signals use the ERAD complex to differentially modulate the turnover rates of PILS proteins. Our findings uncover an additional physiological role of the ERAD complex in regulating PILS protein turnover. This finding uncovers the interplay between protein homeostasis at the ER and growth regulation, opening unexplored molecular avenues into how plants acclimate to internal and external cues.

In this book chapter, we present a method for microsome isolation from the hypocotyl tissue of dark-grown Arabidopsis thaliana. Microsomes are heterogeneous, vesicle-like membranes, which are, not exclusively, derived but enriched with membranes of the endoplasmic reticulum (ER). Here, we describe the experimental setup, including sample preparation, homogenization, differential centrifugation steps, and quality control measures after microsome isolation.

Extreme environmental conditions eventually limit plant growth (1, 2). Here we reveal an unprecedented mechanism that enables multiple external cues to get integrated into auxin-dependent growth programs in Arabidopsis thaliana . Our forward genetics approach on dark grown hypocotyls uncovered that an imbalance in membrane lipids enhances the protein abundance of PIN-LIKES (PILS) (3–5) auxin transport facilitators at the endoplasmic reticulum (ER), which thereby limits nuclear auxin signaling and growth rates. We show that this subcellular response relates to ER stress signaling, which directly impacts on PILS protein turnover in a tissue-dependent manner. This mechanism allows PILS proteins to integrate environmental input with phytohormone auxin signaling, contributing to stress-induced growth adaptation in plants.

Extreme environmental conditions eventually limit plant growth [J. R. Dinneny, Annu. Rev. Cell Dev. Biol. 35, 1-19 (2019), N. Gigli-Bisceglia, C. Testerink, Curr. Opin. Plant Biol. 64, 102120 (2021)]. Here, we reveal a mechanism that enables multiple external cues to get integrated into auxin-dependent growth programs in Arabidopsis thaliana. Our forward genetics approach on dark -grown hypocotyls uncovered that an imbalance in membrane lipids enhances the protein abundance of PIN -LIKES (PILS) [E. Barbez et al., Nature 485, 119 (2012)] auxin transport facilitators at the endoplasmic reticulum (ER), which thereby limits nuclear auxin signaling and growth rates. We show that this subcellular response relates to ER stress signaling, which directly impacts PILS protein turnover in a tissue-dependent manner. This mechanism allows PILS proteins to integrate environmental input with phytohormone auxin signaling, contributing to stress-induced growth adaptation in plants.

Chromatin dynamics enable the precise control of transcriptional programmes. The balance between restricting and opening of regulatory sequences on the DNA needs to be adjusted to prevailing conditions and is fine-tuned by chromatin remodelling proteins. DEK is an evolutionarily conserved chromatin architectural protein regulating important chromatin-related processes. However, the molecular link between DEK-induced chromatin reconfigurations and upstream signalling events remains unknown. Here, we show that ASKβ/AtSK31 is a salt stress-activated glycogen synthase kinase 3 (GSK3) from Arabidopsis thaliana that phosphorylates DEK3. This specific phosphorylation alters nuclear DEK3 protein complex composition and affects nucleosome occupancy and chromatin accessibility that is translated into changes in gene expression, contributing to salt stress tolerance. These findings reveal that DEK3 phosphorylation is critical for chromatin function and cellular stress response and provide a mechanistic example of how GSK3-based signalling is directly linked to chromatin, facilitating a transcriptional response.

Multiple internal and external signals modulate the metabolism, intercellular transport and signaling of the phytohormone auxin. Considering this complexity, it remains largely unknown how plant cells monitor and ensure the homeostasis of auxin responses. PIN-LIKES (PILS) intracellular auxin transport facilitators at the endoplasmic reticulum are suitable candidates to buffer cellular auxin responses because they limit nuclear abundance and signaling of auxin. We used forward genetics to identify gloomy and shiny pils (gasp) mutants that define the PILS6 protein abundance in a post-translational manner. Here, we show that GASP1 encodes an uncharacterized RING/U-box superfamily protein that impacts on auxin signaling output. The low auxin signaling in gasp1 mutants correlates with reduced abundance of PILS5 and PILS6 proteins. Mechanistically, we show that high and low auxin conditions increase and reduce PILS6 protein levels, respectively. Accordingly, non-optimum auxin concentrations are buffered by alterations in PILS6 abundance, consequently leading to homeostatic auxin output regulation. We envision that this feedback mechanism provides robustness to auxin-dependent plant development.

The root system in plants plays a fundamental role in water and nutrient uptake. Lateral roots emerge from the primary root (PR) and its directional organ growth allows the plant to strategically explore the surrounding area. Compared to the main root, lateral roots initially display a distinct gravitropic set point angle, which is established shortly after emergence. Here, we describe a unifying protocol for the morphological description and classification of emerged, young lateral roots.

Directional intercellular transport of the phytohormone auxin mediated by PIN-FORMED (PIN) efflux carriers has essential roles in both coordinating patterning processes and integrating multiple external cues by rapidly redirecting auxin fluxes. PIN activity is therefore regulated by multiple internal and external cues, for which the underlying molecular mechanisms are not fully elucidated. Here, we demonstrate that 3'-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1 (PDK1), which is conserved in plants and mammals, functions as a molecular hub that perceives upstream lipid signalling and modulates downstream substrate activity through phosphorylation. Using genetic analysis, we show that the loss-of-function Arabidopsis pdk1.1 pdk1.2 mutant exhibits a plethora of abnormalities in organogenesis and growth due to defective polar auxin transport. Further cellular and biochemical analyses reveal that PDK1 phosphorylates D6 protein kinase, a well-known upstream activator of PIN proteins. We uncover a lipid-dependent phosphorylation cascade that connects membrane-composition-based cellular signalling with plant growth and patterning by regulating morphogenetic auxin fluxes. PMID: 32393881

Plants depend on gravity to provide the constant landmark for downward root growth and upward shoot growth. The phytohormone auxin and its cell-to-cell transport machinery are central determinants ensuring gravitropic growth. Statolith sedimentation toward gravity is sensed in specialized cells. This positional cue is translated into the polar distribution of PIN auxin efflux carriers at the plasma membrane, leading to asymmetric auxin distribution and consequently, differential growth and organ bending. While we have started to understand the general principles of how primary organs execute gravitropism, we currently lack basic understanding of how lateral plant organs can defy gravitropic responses. Here we briefly review the establishment of the oblique gravitropic set point angle in lateral roots and particularly discuss the emerging role of asymmetric cytokinin signaling as a central anti-gravitropic signal. Differential cytokinin signaling is co-opted in gravitropic lateral and hydrotropic primary roots to counterbalance gravitropic root growth.

The root system architecture describes the shape and spatial arrangement of roots within the soil. Its spatial distribution depends on growth and branching rates as well as directional organ growth. The embryonic primary root gives rise to lateral (secondary) roots, and the ratio of both root types changes over the life span of a plant. Most studies have focused on the growth of primary roots and the development of lateral root primordia. Comparably less is known about the growth regulation of secondary root organs. Here, we review similarities and differences between primary and lateral root organ growth, and emphasize particularly how external stimuli and internal signals differentially integrate root system growth.

The 3-phosphoinositide-dependent protein kinase 1 (PDK1) is a conserved master regulator of AGC kinases in eukaryotic organisms. pdk1 loss of function causes a lethal phenotype in animals and yeasts, but only mild phenotypic defects in Arabidopsis thaliana (Arabidopsis). The Arabidopsis genome contains two PDK1-encoding genes, PDK1 and PDK2. Here, we used clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to generate true loss-of-function pdk1 alleles, which, when combined with pdk2 alleles, showed severe developmental defects including fused cotyledons, a short primary root, dwarf stature and defects in male fertility. We obtained evidence that PDK1 is responsible for AGC1 kinase PROTEIN KINASE ASSOCIATED WITH BRX (PAX) activation by phosphorylation during vascular development, and that the PDK1 phospholipid-binding Pleckstrin Homology domain is not required for this process. Our data indicate that PDK1 regulates polar auxin transport by activating AGC1 clade kinases, resulting in PIN phosphorylation. PMID: 32393878

Auxin and brassinosteroids (BR) are crucial growth regulators and display overlapping functions during plant development. Here, we reveal an alternative phytohormone crosstalk mechanism, revealing that brassinosteroid signaling controls nuclear abundance of auxin. We performed a forward genetic screen for imperial pils ( imp ) mutants that enhance the overexpression phenotypes of PIN-LIKES (PILS) putative intracellular auxin transport facilitator. Here we report that the imp1 mutant is defective in the brassinosteroid-receptor BRI1. Our data reveals that BR signaling transcriptionally and posttranslationally represses accumulation of PILS proteins at the endoplasmic reticulum, thereby increasing nuclear abundance and signaling of auxin. We demonstrate that this alternative phytohormonal crosstalk mechanism integrates BR signaling into auxin-dependent organ growth rates and likely has widespread importance for plant development.

Directional organ growth allows the plant root system to strategically cover its surroundings. Intercellular auxin transport is aligned with the gravity vector in the primary root tips, facilitating downward organ bending at the lower root flank. Here we show that cytokinin signaling functions as a lateral root specific anti-gravitropic component, promoting the radial distribution of the root system. We performed a genome-wide association study and reveal that signal peptide processing of Cytokinin Oxidase 2 (CKX2) affects its enzymatic activity and, thereby, determines the degradation of cytokinins in natural Arabidopsis thaliana accessions. Cytokinin signaling interferes with growth at the upper lateral root flank and thereby prevents downward bending. Our interdisciplinary approach proposes that two phytohormonal cues at opposite organ flanks counterbalance each other’s negative impact on growth, suppressing organ growth towards gravity and allow for radial expansion of the root system.

The decision of whether to grow and proliferate or to restrict growth and develop resilience to stress is a key biological trade-off. In plants, constitutive growth results in increased sensitivity to environmental stress. The underlying mechanisms controlling this decision are however not well understood. We used temperature as a cue to discover regulators of this process in plants, as it both enhances growth and development rates within a specific range and is also a stress at extremes. We found that the conserved chromatin-associated protein DEK plays a central role in balancing the response between growth and arrest in Arabidopsis, and it does this via H2A.Z-nucleosomes. DEK target genes show two distinct categories of chromatin architecture based on the distribution of H2A.Z in +1 nucleosome and gene body, and these predict induction or repression by DEK. We show that these chromatin signatures of DEK target genes are conserved in human cells, suggesting that DEK may act through an evolutionarily conserved mechanism to control the balance between growth and arrest in plants and animals.

Global warming is threatening plant productivity, because plant growth is highly sensitive to elevated temperatures. High temperature (HT) triggers the auxin biosynthesis-dependent growth in aerial tissues. On the other hand, the contribution of auxin to HT-induced root growth is currently under debate. Here we show that the putative intracellular auxin carrier PIN-LIKES 6 (PILS6) is a negative regulator of organ growth and that its abundance is highly sensitive to HT. PILS6 localises to the endoplasmic reticulum (ER) and limits the nuclear availability of auxin, consequently reducing the auxin signalling output. HT represses the transcription and protein abundance of PILS6 specifically in the root tip, which impacts on PILS6-dependent root organ growth rates. Accordingly, we hypothesize that PILS6 is part of a novel mechanism, linking HT to auxin responses in roots.

Single-cell RNA sequencing (scRNA-seq) has been used extensively to study cell-specific gene expression in animals, but it has not been widely applied to plants.Here, we describe the use of a commercially available droplet-based microfluidics platform for high-throughput scRNA-seq to obtain single-cell transcriptomes from protoplasts of more than 10,000 Arabidopsis (Arabidopsis thaliana) root cells.We find that all major tissues and developmental stages are represented in this single-cell transcriptome population.Further, distinct subpopulations and rare cell types, including putative quiescent center cells, were identified.A focused analysis of root epidermal cell transcriptomes defined developmental trajectories for individual cells progressing from meristematic through mature stages of root-hair and nonhair cell differentiation.In addition, single-cell transcriptomes were obtained from root epidermis mutants, enabling a comparative analysis of gene expression at single-cell resolution and providing an unprecedented view of the impact of the mutated genes.Overall, this study demonstrates the feasibility and utility of scRNA-seq in plants and provides a first-generation gene expression map of the Arabidopsis root at single-cell resolution.