ARP2/3 complex associates with peroxisomes to participate in pexophagy in plants.

Actin-related protein (ARP2/3) complex is a heteroheptameric protein complex, evolutionary conserved in all eukaryotic organisms. Its conserved role is based on the induction of actin polymerization at the interface between membranes and the cytoplasm. Plant ARP2/3 has been reported to participate in actin reorganization at the plasma membrane during polarized growth of trichomes and at the plasma membrane-endoplasmic reticulum contact sites. Here we demonstrate that individual plant subunits of ARP2/3 fused to fluorescent proteins form motile spot-like structures in the cytoplasm that are associated with peroxisomes in Arabidopsis and tobacco. ARP2/3 is found at the peroxisome periphery and contains the assembled ARP2/3 complex and the WAVE/SCAR complex subunit NAP1. This ARP2/3-positive peroxisomal domain colocalizes with the autophagosome and, under conditions that affect the autophagy, colocalization between ARP2/3 and the autophagosome increases. ARP2/3 subunits co-immunoprecipitate with ATG8f and peroxisome-associated ARP2/3 interact in vivo with the ATG8f marker. Since mutants lacking functional ARP2/3 complex have more peroxisomes than wild type, we suggest that ARP2/3 has a novel role in the process of peroxisome degradation by autophagy, called pexophagy.

CRISPR-Cas9 Arabidopsis mutants of genes for ARPC1 and ARPC3 subunits of ARP2/3 complex reveal differential roles of complex subunits.

Protein complex Arp2/3 has a conserved role in the nucleation of branched actin filaments. It is constituted of seven subunits, including actin-like subunits ARP2 and ARP3 plus five other subunits called Arp2/3 Complex Component 1 to 5, which are not related to actin. Knock-out plant mutants lacking individual plant ARP2/3 subunits have a typical phenotype of distorted trichomes, altered pavement cells shape and defects in cell adhesion. While knock-out mutant Arabidopsis plants for most ARP2/3 subunits have been characterized before, Arabidopsis plant mutants missing ARPC1 and ARPC3 subunits have not yet been described. Using CRISPR/Cas9, we generated knock-out mutants lacking ARPC1 and ARPC3 subunits. We confirmed that the loss of ARPC1 subunits results in the typical ARP2/3 mutant phenotype. However, the mutants lacking ARPC3 subunits resulted in plants with surprisingly different phenotypes. Our results suggest that plant ARP2/3 complex function in trichome shaping does not require ARPC3 subunit, while the fully assembled complex is necessary for the establishment of correct cell adhesion in the epidermis.

Throttling Growth Speed: Evaluation of aux1-7 Root Growth Profile by Combining D-Root system and Root Penetration Assay.

Directional root growth control is crucial for plant fitness. The degree of root growth deviation depends on several factors, whereby exogenous growth conditions have a profound impact. The perception of mechanical impedance by wild-type roots results in the modulation of root growth traits, and it is known that gravitropic stimulus influences distinct root movement patterns in concert with mechanoadaptation. Mutants with reduced shootward auxin transport are described as being numb towards mechanostimulus and gravistimulus, whereby different growth conditions on agar-supplemented medium have a profound effect on how much directional root growth and root movement patterns differ between wild types and mutants. To reduce the impact of unilateral mechanostimulus on roots grown along agar-supplemented medium, we compared the root movement of Col-0 and auxin resistant 1-7 in a root penetration assay to test how both lines adjust the growth patterns of evenly mechanostimulated roots. We combined the assay with the D-root system to reduce light-induced growth deviation. Moreover, the impact of sucrose supplementation in the growth medium was investigated because exogenous sugar enhances root growth deviation in the vertical direction. Overall, we observed a more regular growth pattern for Col-0 but evaluated a higher level of skewing of aux1-7 compared to the wild type than known from published data. Finally, the tracking of the growth rate of the gravistimulated roots revealed that Col-0 has a throttling elongation rate during the bending process, but aux1-7 does not.

Lessons Learned from the Studies of Roots Shaded from Direct Root Illumination.

The root is the below-ground organ of a plant, and it has evolved multiple signaling pathways that allow adaptation of architecture, growth rate, and direction to an ever-changing environment. Roots grow along the gravitropic vector towards beneficial areas in the soil to provide the plant with proper nutrients to ensure its survival and productivity. In addition, roots have developed escape mechanisms to avoid adverse environments, which include direct illumination. Standard laboratory growth conditions for basic research of plant development and stress adaptation include growing seedlings in Petri dishes on medium with roots exposed to light. Several studies have shown that direct illumination of roots alters their morphology, cellular and biochemical responses, which results in reduced nutrient uptake and adaptability upon additive stress stimuli. In this review, we summarize recent methods that allow the study of shaded roots under controlled laboratory conditions and discuss the observed changes in the results depending on the root illumination status.

Exogenous carbon source supplementation counteracts root and hypocotyl growth limitations under increased cotyledon shading, with glucose and sucrose differentially modulating growth curves.

Plant growth is continuously modulated by endogenous and exogenous stimuli. By no means the only, but well described, signaling molecules produced in plants and distributed through the plant body to orchestrate efficient growth are photosynthates. Light is a potent exogenous stimulus that determines, first, the rate of photosynthesis, but also the rate of plant growth. Root meristem activity is reduced with direct illumination but enhanced with increased sugar levels. With reduced cotyledon illumination, the seedling increases hypocotyl elongation until adequate light exposure is again provided. If endogenous carbon sources are limited, this leads to a temporary inhibition of root growth. Experimental growth conditions include exogenous supplementation of sucrose or glucose in addition to culturing seedlings under light exposure in Petri dishes. We compared total root length and hypocotyl elongation of Arabidopsis thaliana wild type Col-0 in response to illumination status and carbon source in the growth medium. Overall, sucrose supplementation promoted hypocotyl and root length to a greater extent than glucose supplementation. Glucose promoted root length compared to non-supplemented seedlings especially when cotyledon illumination was greatly reduced.

Dissecting Hierarchies between Light, Sugar and Auxin Action Underpinning Root and Root Hair Growth.

Plant roots are very plastic and can adjust their tissue organization and cell appearance during abiotic stress responses. Previous studies showed that direct root illumination and sugar supplementation mask root growth phenotypes and traits. Sugar and light signaling where further connected to changes in auxin biosynthesis and distribution along the root. Auxin signaling underpins almost all processes involved in the establishment of root traits, including total root length, gravitropic growth, root hair initiation and elongation. Root hair plasticity allows maximized nutrient uptake and therefore plant productivity, and root hair priming and elongation require proper auxin availability. In the presence of sucrose in the growth medium, root hair emergence is partially rescued, but the full potential of root hair elongation is lost. With our work we describe a combinatory study showing to which extent light and sucrose are antagonistically influencing root length, but additively affecting root hair emergence and elongation. Furthermore, we investigated the impact of the loss of PIN-FORMED2, an auxin efflux carrier mediating shootward auxin transporter, on the establishment of root traits in combination with all growth conditions.

Bundling up the Role of the Actin Cytoskeleton in Primary Root Growth.

Primary root growth is required by the plant to anchor in the soil and reach out for nutrients and water, while dealing with obstacles. Efficient root elongation and bending depends upon the coordinated action of environmental sensing, signal transduction, and growth responses. The actin cytoskeleton is a highly plastic network that constitutes a point of integration for environmental stimuli and hormonal pathways. In this review, we present a detailed compilation highlighting the importance of the actin cytoskeleton during primary root growth and we describe how actin-binding proteins, plant hormones, and actin-disrupting drugs affect root growth and root actin. We also discuss the feedback loop between actin and root responses to light and gravity. Actin affects cell division and elongation through the control of its own organization. We remark upon the importance of longitudinally oriented actin bundles as a hallmark of cell elongation as well as the role of the actin cytoskeleton in protein trafficking and vacuolar reshaping during this process. The actin network is shaped by a plethora of actin-binding proteins; however, there is still a large gap in connecting the molecular function of these proteins with their developmental effects. Here, we summarize their function and known effects on primary root growth with a focus on their high level of specialization. Light and gravity are key factors that help us understand root growth directionality. The response of the root to gravity relies on hormonal, particularly auxin, homeostasis, and the actin cytoskeleton. Actin is necessary for the perception of the gravity stimulus via the repositioning of sedimenting statoliths, but it is also involved in mediating the growth response via the trafficking of auxin transporters and cell elongation. Furthermore, auxin and auxin analogs can affect the composition of the actin network, indicating a potential feedback loop. Light, in its turn, affects actin organization and hence, root growth, although its precise role remains largely unknown. Recently, fundamental studies with the latest techniques have given us more in-depth knowledge of the role and organization of actin in the coordination of root growth; however, there remains a lot to discover, especially in how actin organization helps cell shaping, and therefore root growth.

Arp2/3 Complex Is Required for Auxin-Driven Cell Expansion Through Regulation of Auxin Transporter Homeostasis.

The Arp2/3 complex is an actin nucleator shown to be required throughout plant morphogenesis, contributing to processes such as cell expansion, tissue differentiation or cell wall assembly. A recent publication demonstrated that plants lacking functional Arp2/3 complex also present defects in auxin distribution and transport. This work shows that Arp2/3 complex subunits are predominantly expressed in the provasculature, although other plant tissues also show promoter activity (e.g., cotyledons, apical meristems, or root tip). Moreover, auxin can trigger subunit expression, indicating a role of this phytohormone in mediating the complex activity. Further investigation of the functional interaction between Arp2/3 complex and auxin signaling also reveals their cooperation in determining pavement cell shape, presumably through the role of Arp2/3 complex in the correct auxin carrier trafficking. Young seedlings of arpc5 mutants show increased auxin-triggered proteasomal degradation of DII-VENUS and altered PIN3 distribution, with higher levels of the protein in the vacuole. Closer observation of vacuolar morphology revealed the presence of a more fragmented vacuolar compartment when Arp2/3 function is abolished, hinting a generalized role of Arp2/3 complex in endomembrane function and protein trafficking.

Arabidopsis thaliana plants lacking the ARP2/3 complex show defects in cell wall assembly and auxin distribution.

Background and Aim: The cytoskeleton plays an important role in the synthesis of plant cell walls. Both microtubules and actin cytoskeleton are known to be involved in the morphogenesis of plant cells through their role in cell wall building. The role of ARP2/3-nucleated actin cytoskeleton in the morphogenesis of cotyledon pavement cells has been described before. Seedlings of Arabidopsis mutants lacking a functional ARP2/3 complex display specific cell wall-associated defects. Methods: In three independent Arabidopsis mutant lines lacking subunits of the ARP2/3 complex, phenotypes associated with the loss of the complex were analysed throughout plant development. Organ size and anatomy, cell wall composition, and auxin distribution were investigated. Key Results: ARP2/3-related phenotype is associated with changes in cell wall composition, and the phenotype is manifested especially in mature tissues. Cell walls of mature plants contain less cellulose and a higher amount of homogalacturonan, and display changes in cell wall lignification. Vascular bundles of mutant inflorescence stems show a changed pattern of AUX1-YFP expression. Plants lacking a functional ARP2/3 complex have decreased basipetal auxin transport. Conclusions: The results suggest that the ARP2/3 complex has a morphogenetic function related to cell wall synthesis and auxin transport.