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Summary: The phytohormone abscisic acid (ABA) functions in the control of plant stress responses, particularly in drought stress. A significant mechanism in attenuating and terminating ABA signals involves regulated protein turnover, with certain ABA receptors, despite their main presence in the cytosol and nucleus, subjected to vacuolar degradation via the Endosomal Sorting Complex Required for Transport (ESCRT) machinery.Collectively our findings show that discrete TOM1‐LIKE (TOL) proteins, which are functional ESCRT‐0 complex substitutes in plants, affect the trafficking for degradation of core components of the ABA signaling and transport machinery.TOL2,3,5 and 6 modulate ABA signaling where they function additively in degradation of ubiquitinated ABA receptors and transporters.TOLs colocalize with their cargo in different endocytic compartments in the root epidermis and in guard cells of stomata, where they potentially function in ABA‐controlled stomatal aperture.Although the tol2/3/5/6 quadruple mutant plant line is significantly more drought‐tolerant and has a higher ABA sensitivity than control plant lines, it has no obvious growth or development phenotype under standard conditions, making the TOL genes ideal candidates for engineering to improved plant performance. [ABSTRACT FROM AUTHOR]

The environment is continually challenging plants, and in response they 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 heat. Although well understood for shoot organs, the thermomorphogenesis response in roots has only recently received increased research attention. Accordingly, in the past few years, the hormonal responses and underlying molecular players important for root thermomorphogenesis have been revealed. Other responses triggered by high temperature in the root encompass modifications of overall root architecture and interactions with the soil environment, with consequences for the whole plant. Here, we review scientific knowledge and highlight current understanding of root responses to moderately high and extreme temperature. [ABSTRACT FROM AUTHOR]

The phytohormone auxin contributes to virtually every aspect of the plant development. The spatiotemporal distribution of auxin depends on a complex interplay between auxin metabolism and intercellular auxin transport. Intracellular auxin compartmentalization provides another link between auxin transport processes and auxin metabolism. The PIN-LIKES (PILS) putative auxin carriers localize to the endoplasmic reticulum (ER) and contribute to cellular auxin homeostasis. PILS proteins regulate intracellular auxin accumulation, the rate of auxin conjugation and, subsequently, affect nuclear auxin signaling. Here, we investigate sequence diversification of the PILS family in Arabidopsis thaliana and provide insights into the evolution of these novel putative auxin carriers in plants. Our data suggest that PILS proteins are conserved throughout the plant lineage and expanded during higher plant evolution. PILS proteins diversified early during plant evolution into three clades. Besides the ancient Clade I encompassing non-land plant species, PILS proteins evolved into two clades. The diversification of Clade II and Clade III occurred already at the level of non-vascular plant evolution and, hence, both clades contain vascular and non-vascular plant species. Nevertheless, Clade III contains fewer non- and increased numbers of vascular plants, indicating higher importance of Clade III for vascular plant evolution. Notably, PILS proteins are distinct and appear evolutionarily older than the prominent PIN-FORMED auxin carriers. Moreover, we revealed particular PILS sequence divergence in Arabidopsis and assume that these alterations could contribute to distinct gene regulations and protein functions. [ABSTRACT FROM AUTHOR]

Plant vacuoles are essential multifunctional organelles largely distinct from similar organelles in other eukaryotes. Embryo protein storage vacuoles and the lytic vacuoles that perform a general degradation function are the best characterized, but little is known about the biogenesis and transition between these vacuolar types. Here, we designed a fluorescent marker–based forward genetic screen in Arabidopsis thaliana and identified a protein affected trafficking2 (pat2) mutant, whose lytic vacuoles display altered morphology and accumulation of proteins. Unlike other mutants affecting the vacuole, pat2 is specifically defective in the biogenesis, identity, and function of lytic vacuoles but shows normal sorting of proteins to storage vacuoles. PAT2 encodes a putative β-subunit of adaptor protein complex 3 (AP-3) that can partially complement the corresponding yeast mutant. Manipulations of the putative AP-3 β adaptin functions suggest a plant-specific role for the evolutionarily conserved AP-3 β in mediating lytic vacuole performance and transition of storage into the lytic vacuoles independently of the main prevacuolar compartment-based trafficking route. [ABSTRACT FROM AUTHOR]

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The typical parameter used to evaluate the root growth response to gravity is the degree of root bending in time. This employs the quantification of the root tip angle toward gravity and, hence, does not directly assess the actual differential growth process. Here, we describe the cortical cell length as a parameter to quantify cell elongation during the gravitropic response, using median longitudinal confocal sections. This analysis depicts that root organ bending is a consequence of differential cellular elongation on the upper versus lower side of the gravistimulated root. Moreover, we introduce here a simple mounting setup that is suitable to gravistimulate and subsequently image seedlings on upright microscopes.

Time-lapse imaging of roots is highly suitable for depicting gravitropic growth behaviors. However, roots may show faster or slower bending kinetics when compared to control as a result of differences in overall root growth. Accordingly, conditions that cause differential organ growth require growth rate normalization to compare gravitropic curvature. Here, we describe a simple normalization method for gravitropic root growth evaluation. We exemplify this method by exposing seedlings to distinct environmental conditions or disturbing the cellular auxin contents. This data shows that the method is suitable to discriminate between gravitropic and overall organ growth defects.

Microscope images of plant specimens showing expression of GUS markers, besides being very beautiful, provide useful information regarding various biological processes. However, the information extracted from these images is often purely qualitative, and in many publications is not subjected to quantification. Here, we describe a very simple quantification method for GUS histochemical staining that enables detection of subtle differences in gene expression at cellular, tissue, or organ level. The quantification method described is based on the freely available image analysis software ImageJ that is widely used by the scientific community. We exemplify the method by quantifying small and precise changes (at the cellular level) as well as broad changes (at the organ level) in the expression of two previously published reporter lines, such as the pPILS2::GUS and pPILS5::GUS. The method presented here represents an easy tool for converting visual information from GUS histochemical staining images into quantifiable data and is of general importance for plant biologists performing GUS activity-based evaluation of reporter genes.

Plant growth relates to gravity, ensuring that roots grow downwards into the soil and shoots expand aerially. The phytohormone auxin mediates tropistic growth responses, such as root gravitropism. Gravity perception in the very tip of the roots triggers carrier-dependent, asymmetric redistribution of auxin, leading to differential auxin responses and growth regulation at the upper and lower root flanks. This cellular, asymmetry-breaking event will eventually lead to root bending towards the gravity vector. Here, we show how to investigate auxin signaling and auxin carrier dynamics during root gravitropic response, using a chambered cover glass in combination with a confocal live cell imaging approach. To exemplify this method, we used established lines expressing transcriptional and translational green fluorescent protein (GFP) fusions to the auxin responsive promoter element DR5rev and the prominent auxin carrier PIN-FORMED2 (PIN2), respectively. Transgenic seedlings were placed and grown in the chambered cover glasses, enabling defined gravitropic stimulations prior to imaging. This method is optimal for inverted microscopes and significantly reduces stressful manipulations during specimen preparation.