Organ-Specific Microsomes from Dark-Grown Hypocotyls of Arabidopsis thaliana.

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.

Spatially and temporally distinct Ca2+ changes in Lotus japonicus roots orient fungal-triggered signalling pathways towards symbiosis or immunity.

Plants activate an immune or symbiotic response depending on the detection of distinct signals from root-interacting microbes. Both signalling cascades involve Ca2+ as a central mediator of early signal transduction. In this study, we combined aequorin- and cameleon-based methods to dissect the changes in cytosolic and nuclear Ca2+ concentration caused by different chitin-derived fungal elicitors in Lotus japonicus roots. Our quantitative analyses highlighted the dual character of the evoked Ca2+ responses taking advantage of the comparison between different genetic backgrounds: an initial Ca2+ influx, dependent on the LysM receptor CERK6 and independent of the common symbiotic signalling pathway (CSSP), is followed by a second CSSP-dependent and CERK6-independent phase, that corresponds to the well-known perinuclear/nuclear Ca2+ spiking. We show that the expression of immunity marker genes correlates with the amplitude of the first Ca2+ change, depends on elicitor concentration, and is controlled by Ca2+ storage in the vacuole. Our findings provide an insight into the Ca2+-mediated signalling mechanisms discriminating plant immunity- and symbiosis-related pathways in the context of their simultaneous activation by single fungal elicitors.

Gravitropism: The LAZY way of intracellular hitchhiking.

Plant gravitropism has fascinated scientists for centuries. A new study provides a major mechanistic update of the so-called starch/statolith hypothesis, revealing how gravity perception is converted into a physiological response.

Endoplasmic reticulum stress controls PIN-LIKES abundance and thereby growth adaptation.

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.

Auxin transport at the endoplasmic reticulum: roles and structural similarity of PIN-FORMED and PIN-LIKES.

Auxin is a crucial plant hormone that controls a multitude of developmental processes. The directional movement of auxin between cells is largely facilitated by canonical PIN-FORMED proteins in the plasma membrane. In contrast, non-canonical PIN-FORMED proteins and PIN-LIKES proteins appear to reside mainly in the endoplasmic reticulum. Despite recent progress in identifying the roles of the endoplasmic reticulum in cellular auxin responses, the transport dynamics of auxin at the endoplasmic reticulum are not well understood. PIN-LIKES are structurally related to PIN-FORMED proteins, and recently published structures of these transporters have provided new insights into PIN-FORMED proteins and PIN-LIKES function. In this review, we summarize current knowledge on PIN-FORMED proteins and PIN-LIKES in intracellular auxin transport. We discuss the physiological properties of the endoplasmic reticulum and the consequences for transport processes across the ER membrane. Finally, we highlight the emerging role of the endoplasmic reticulum in the dynamics of cellular auxin signalling and its impact on plant development.

K63-linked ubiquitin chains are a global signal for endocytosis and contribute to selective autophagy in plants.

In contrast to other eukaryotic model organisms, the closely related ubiquitin (Ub)-conjugating enzymes UBC35 and UBC36 are the main sources of K63-linked Ub chains in Arabidopsis.1 Although K63-linked chains have been associated with the regulation of vesicle trafficking, definitive proof for their role in endocytosis was missing. We show that the ubc35 ubc36 mutant has pleiotropic phenotypes related to hormone and immune signaling. Specifically, we reveal that ubc35-1 ubc36-1 plants have altered turnover of integral membrane proteins including FLS2, BRI1, and PIN1 at the plasma membrane. Our data indicates that K63-Ub chains are generally required for endocytic trafficking in plants. In addition, we show that in plants K63-Ub chains are involved in selective autophagy through NBR1, the second major pathway delivering cargoes to the vacuole for degradation. Similar to autophagy-defective mutants, ubc35-1 ubc36-1 plants display an accumulation of autophagy markers. Moreover, autophagy receptor NBR1 interacts with K63-Ub chains, which are required for its delivery to the lytic vacuole.2 Together, we show that K63-Ub chains act as a general signal required for the two main pathways delivering cargo to the vacuole and thus, to maintain proteostasis.

Signaling events for photomorphogenic root development.

A germinating seedling incorporates environmental signals such as light into developmental outputs. Light is not only a source of energy, but also a central coordinative signal in plants. Traditionally, most research focuses on aboveground organs' response to light; therefore, our understanding of photomorphogenesis in roots is relatively scarce. However, root development underground is highly responsive to light signals from the shoot and understanding these signaling mechanisms will give a better insight into early seedling development. Here, we review the central light signaling hubs and their role in root growth promotion of Arabidopsis thaliana seedlings.

PILS proteins provide a homeostatic feedback on auxin signaling output.

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.

Endocytic trafficking promotes vacuolar enlargements for fast cell expansion rates in plants.

The vacuole has a space-filling function, allowing a particularly rapid plant cell expansion with very little increase in cytosolic content (Löfke et al., 2015; Scheuring et al., 2016; Dünser et al., 2019). Despite its importance for cell size determination in plants, very little is known about the mechanisms that define vacuolar size. Here, we show that the cellular and vacuolar size expansions are coordinated. By developing a pharmacological tool, we enabled the investigation of membrane delivery to the vacuole during cellular expansion. Our data reveal that endocytic membrane sorting from the plasma membrane to the vacuole is enhanced in the course of rapid root cell expansion. While this 'compromise' mechanism may theoretically at first decelerate cell surface enlargements, it fuels vacuolar expansion and, thereby, ensures the coordinated augmentation of vacuolar occupancy in dynamically expanding plant cells.

Regulation of immune receptor kinase plasma membrane nanoscale organization by a plant peptide hormone and its receptors.

Spatial partitioning is a propensity of biological systems orchestrating cell activities in space and time. The dynamic regulation of plasma membrane nano-environments has recently emerged as a key fundamental aspect of plant signaling, but the molecular components governing it are still mostly unclear. The receptor kinase FERONIA (FER) controls ligand-induced complex formation of the immune receptor kinase FLAGELLIN SENSING 2 (FLS2) with its co-receptor BRASSINOSTEROID-INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1), and perception of the endogenous peptide hormone RAPID ALKALANIZATION FACTOR 23 (RALF23) by FER inhibits immunity. Here, we show that FER regulates the plasma membrane nanoscale organization of FLS2 and BAK1. Our study demonstrates that akin to FER, leucine-rich repeat (LRR) extensin proteins (LRXs) contribute to RALF23 responsiveness and regulate BAK1 nanoscale organization and immune signaling. Furthermore, RALF23 perception leads to rapid modification of FLS2 and BAK1 nanoscale organization, and its inhibitory activity on immune signaling relies on FER kinase activity. Our results suggest that perception of RALF peptides by FER and LRXs actively modulates plasma membrane nanoscale organization to regulate cell surface signaling by other ligand-binding receptor kinases.

A glossary of plant cell structures: Current insights and future questions.

In this glossary of plant cell structures, we asked experts to summarize a present-day view of plant organelles and structures, including a discussion of outstanding questions. In the following short reviews, the authors discuss the complexities of the plant cell endomembrane system, exciting connections between organelles, novel insights into peroxisome structure and function, dynamics of mitochondria, and the mysteries that need to be unlocked from the plant cell wall. These discussions are focused through a lens of new microscopy techniques. Advanced imaging has uncovered unexpected shapes, dynamics, and intricate membrane formations. With a continued focus in the next decade, these imaging modalities coupled with functional studies are sure to begin to unravel mysteries of the plant cell.

Staging of Emerged Lateral Roots in Arabidopsis thaliana.

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.

Xyloglucan Remodeling Defines Auxin-Dependent Differential Tissue Expansion in Plants.

Size control is a fundamental question in biology, showing incremental complexity in plants, whose cells possess a rigid cell wall. The phytohormone auxin is a vital growth regulator with central importance for differential growth control. Our results indicate that auxin-reliant growth programs affect the molecular complexity of xyloglucans, the major type of cell wall hemicellulose in eudicots. Auxin-dependent induction and repression of growth coincide with reduced and enhanced molecular complexity of xyloglucans, respectively. In agreement with a proposed function in growth control, genetic interference with xyloglucan side decorations distinctly modulates auxin-dependent differential growth rates. Our work proposes that auxin-dependent growth programs have a spatially defined effect on xyloglucan's molecular structure, which in turn affects cell wall mechanics and specifies differential, gravitropic hypocotyl growth.

Author Correction: Light triggers PILS-dependent reduction in nuclear auxin signalling for growth transition.

A Correction to this paper has been published: https://doi.org/10.1038/s41477-021-00924-y.

Getting to the Root of Belowground High Temperature Responses in Plants.

The environment is continuously challenging plants. As a response, plants use various coping strategies, such as adaptation of their growth. Thermomorphogenesis is a specific growth adaptation that promotes organ growth in response to moderately high temperature. This would eventually enable plants to cool down by dissipating the heat. Although well understood for shoot organs, the thermomorphogenesis response in roots only recently obtained increasing research attention. Accordingly, in the last few years, the hormonal responses and underlying molecular players important for root thermomorphogenesis were revealed. Other responses triggered by high temperature in the root encompass modifications of overall root architecture and interactions with the soil environment, with consequences on the whole plant. Here, we review the scientific knowledge and highlight the current understanding on roots responding to moderately high and extreme temperature.

Effects of Light Intensity on Root Development in a D-Root Growth System.

Plant development is highly affected by light quality, direction, and intensity. Under natural growth conditions, shoots are directly exposed to light whereas roots develop underground shielded from direct illumination. The photomorphogenic development strongly represses shoot elongation whereas promotes root growth. Over the years, several studies helped the elucidation of signaling elements that coordinate light perception and underlying developmental outputs. Light exposure of the shoots has diverse effects on main root growth and lateral root (LR) formation. In this study, we evaluated the phenotypic root responses of wild-type Arabidopsis plants, as well as several mutants, grown in a D-Root system. We observed that sucrose and light act synergistically to promote root growth and that sucrose alone cannot overcome the light requirement for root growth. We also have shown that roots respond to the light intensity applied to the shoot by changes in primary and LR development. Loss-of-function mutants for several root light-response genes display varying phenotypes according to the light intensity to which shoots are exposed. Low light intensity strongly impaired LR development for most genotypes. Only vid-27 and pils4 mutants showed higher LR density at 40 µmol m-2 s-1 than at 80 µmol m-2 s-1 whereas yuc3 and shy2-2 presented no LR development in any light condition, reinforcing the importance of auxin signaling in light-dependent root development. Our results support the use of D-Root systems to avoid the effects of direct root illumination that might lead to artifacts and unnatural phenotypic outputs.

FRUITFULL Is a Repressor of Apical Hook Opening in Arabidopsis thaliana.

Plants adjust their architecture to a constantly changing environment, requiring adaptation of differential growth. Despite their importance, molecular switches, which define growth transitions, are largely unknown. Apical hook development in dark grown Arabidopsis thaliana (A. thaliana) seedlings serves as a suitable model for differential growth transition in plants. Here, we show that the phytohormone auxin counteracts the light-induced growth transition during apical hook opening. We, subsequently, identified genes which are inversely regulated by light and auxin. We used in silico analysis of the regulatory elements in this set of genes and subsequently used natural variation in gene expression to uncover correlations between underlying transcription factors and the in silico predicted target genes. This approach uncovered that MADS box transcription factor AGAMOUS-LIKE 8 (AGL8)/FRUITFULL (FUL) modulates apical hook opening. Our data shows that transient FUL expression represses the expression of growth stimulating genes during early phases of apical hook development and therewith guards the transition to growth promotion for apical hook opening. Here, we propose a role for FUL in setting tissue identity, thereby regulating differential growth during apical hook development.

Asymmetric cytokinin signaling opposes gravitropism in roots.

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.