Flavonol-mediated stabilization of PIN efflux complexes regulates polar auxin transport.

The transport of auxin controls the rate, direction and localization of plant growth and development. The course of auxin transport is defined by the polar subcellular localization of the PIN proteins, a family of auxin efflux transporters. However, little is known about the composition and regulation of the PIN protein complex. Here, using blue-native PAGE and quantitative mass spectrometry, we identify native PIN core transport units as homo- and heteromers assembled from PIN1, PIN2, PIN3, PIN4 and PIN7 subunits only. Furthermore, we show that endogenous flavonols stabilize PIN dimers to regulate auxin efflux in the same way as does the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). This inhibitory mechanism is counteracted both by the natural auxin indole-3-acetic acid and by phosphomimetic amino acids introduced into the PIN1 cytoplasmic domain. Our results lend mechanistic insights into an endogenous control mechanism which regulates PIN function and opens the way for a deeper understanding of the protein environment and regulation of the polar auxin transport complex.

Role of reactive oxygen species in the modulation of auxin flux and root development in Arabidopsis thaliana.

Reactive oxygen species (ROS) play a dual role in plant biology, acting as important signal transduction molecules and as toxic byproducts of aerobic metabolism that accumulate in cells upon exposure to different stressors and lead to cell death. In plants, root architecture is regulated by the distribution and intercellular flow of the phytohormone auxin. In this study, we identified ROS as an important modulator of auxin distribution and response in the root. ROS production is necessary for root growth, proper tissue patterning, cell growth, and lateral root (LR) induction. Alterations in ROS balance led to altered auxin distribution and response in SOD and RHD2 loss-of-function mutants. Treatment of Arabidopsis seedlings with additional sources of ROS (hydrogen peroxide) or an ROS production inhibitor (diphenylene iodonium) induced phenocopies of the mutants studied. Simultaneous application of auxin and ROS increased LR primordia induction, and PIN-FORMED protein immunolocalization further demonstrated the existing link between auxin and ROS in orchestrating cell division and auxin flux during root development. In Arabidopsis roots, genetic alterations in ROS balance led to defective auxin distribution and growth-related responses in roots. Exogenous hydrogen peroxide alters the establishment of the endogenous auxin gradient in the root meristem through regulation of PIN-FORMED polarity, while the simultaneous application of hydrogen peroxide and auxin enhanced LR induction in a dose- and position-dependent manner through activation of cell division.

Regulation of early seedling establishment and root development in Arabidopsis thaliana by light and carbohydrates.

MAIN CONCLUSION: Root development is regulated by sucrose and light during early seedling establishment through changes in the auxin response and chromatin topology. Light is a key environmental signal that regulates plant growth and development. The impact of light on development is primarily analyzed in the above-ground tissues, but little is known about the mechanisms by which light shapes the architecture of underground roots. Our study shows that carbohydrate starvation during skotomorphogenesis is accompanied by compaction of nuclei in the root apical meristem, which prevents cell cycle progression and leads to irreversible root differentiation in the absence of external carbohydrates, as evidenced by the lack of DNA replication and increased numbers of nuclei with specific chromatin characteristics. In these conditions, induction of photomorphogenesis was unable to restore seedling growth, as overall root growth was compromised. The addition of carbohydrates, either locally or systemically by transferring seedlings to sugar-containing medium, led to the induction of adventitious root formation with rapid recovery of seedling growth. Conversely, transferring in vitro carbohydrate-grown seedlings from light to dark transiently promoted cell elongation and significantly reduced root meristem size, but did not primarily affect cell cycle kinetics. We show that, in the presence of sucrose, dark incubation does not affect zonation in the root apical meristem but leads to shortening of the proliferative and transition zones. Sugar starvation led to a rapid increase in lysine demethylation of histone H3 at position K9, which preceded a rapid decline in cell cycle activity and activation of cell differentiation. In conclusion, carbohydrates are required for cell cycle activity, epigenetics reprogramming and for postmitotic cell elongation and auxin-regulated response in the root apical meristem.

AZG1 is a cytokinin transporter that interacts with auxin transporter PIN1 and regulates the root stress response.

An environmentally responsive root system is crucial for plant growth and crop yield, especially in suboptimal soil conditions. This responsiveness enables the plant to exploit regions of high nutrient density while simultaneously minimizing abiotic stress. Despite the vital importance of root systems in regulating plant growth, significant gaps of knowledge exist in the mechanisms that regulate their architecture. Auxin defines both the frequency of lateral root (LR) initiation and the rate of LR outgrowth. Here, we describe a search for proteins that regulate root system architecture (RSA) by interacting directly with a key auxin transporter, PIN1. The native separation of Arabidopsis plasma membrane protein complexes identified several PIN1 co-purifying proteins. Among them, AZG1 was subsequently confirmed as a PIN1 interactor. Here, we show that, in Arabidopsis, AZG1 is a cytokinin (CK) import protein that co-localizes with and stabilizes PIN1, linking auxin and CK transport streams. AZG1 expression in LR primordia is sensitive to NaCl, and the frequency of LRs is AZG1-dependent under salt stress. This report therefore identifies a potential point for auxin:cytokinin crosstalk, which shapes RSA in response to NaCl.

A new gene for auxin synthesis.

There is much interest in understanding the pathways that trigger biosynthesis of the plant hormone auxin. In this issue, Stepanova et al. (2008) and Tao et al. (2008) reveal that a small family of tryptophan aminotransferases catalyze formation of indole-3-pyruvic acid (IPA) from L-tryptophan (L-Trp), the first step in a pathway for auxin biosynthesis.

Author Correction: U-Net: deep learning for cell counting, detection, and morphometry.

In the version of this paper originally published, one of the affiliations for Dominic Mai was incorrect: "Center for Biological Systems Analysis (ZBSA), Albert-Ludwigs-University, Freiburg, Germany" should have been "Life Imaging Center, Center for Biological Systems Analysis, Albert-Ludwigs-University, Freiburg, Germany." This change required some renumbering of subsequent author affiliations. These corrections have been made in the PDF and HTML versions of the article, as well as in any cover sheets for associated Supplementary Information.

U-Net: deep learning for cell counting, detection, and morphometry.

U-Net is a generic deep-learning solution for frequently occurring quantification tasks such as cell detection and shape measurements in biomedical image data. We present an ImageJ plugin that enables non-machine-learning experts to analyze their data with U-Net on either a local computer or a remote server/cloud service. The plugin comes with pretrained models for single-cell segmentation and allows for U-Net to be adapted to new tasks on the basis of a few annotated samples.

A simple pipeline for cell cycle kinetic studies in the root apical meristem.

Root system architecture ultimately depends on precise signaling between different cells and tissues in the root apical meristem (RAM) and integration with environmental cues. This study describes a simple pipeline to simultaneously determine cellular parameters, nucleus geometry, and cell cycle kinetics in the RAM. The method uses marker-free techniques for nucleus and cell boundary detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining for DNA replication quantification. Based on this approach, we characterized differences in cell volume, nucleus volume, and nucleus shape across different domains of the Arabidopsis RAM. We found that DNA replication patterns were cell layer and region dependent. G2 phase duration, which varied from 3.5 h in the pericycle to more than 4.5 h in the epidermis, was found to be associated with some features of nucleus geometry. Endocycle duration was determined as the time required to achieve 100% EdU-positive cells in the elongation zone and, as such, it was estimated to be in the region of 5 h for the epidermis and cortex. This experimental pipeline could be used to precisely map cell cycle duration in the RAM of mutants and in response to environmental stress in several plant species without the need for introgressing molecular cell cycle markers.

The role of AUX1 during lateral root development in the domestication of the model C4 grass Setaria italica.

C4 photosynthesis increases the efficiency of carbon fixation by spatially separating high concentrations of molecular oxygen from Rubisco. The specialized leaf anatomy required for this separation evolved independently many times. The morphology of C4 root systems is also distinctive and adapted to support high rates of photosynthesis; however, little is known about the molecular mechanisms that have driven the evolution of C4 root system architecture. Using a mutant screen in the C4 model plant Setaria italica, we identify Siaux1-1 and Siaux1-2 as root system architecture mutants. Unlike in S. viridis, AUX1 promotes lateral root development in S. italica. A cell by cell analysis of the Siaux1-1 root apical meristem revealed changes in the distribution of cell volumes in all cell layers and a dependence of the frequency of protophloem and protoxylem strands on SiAUX1. We explore the molecular basis of the role of SiAUX1 in seedling development using an RNAseq analysis of wild-type and Siaux1-1 plants and present novel targets for SiAUX1-dependent gene regulation. Using a selection sweep and haplotype analysis of SiAUX1, we show that Hap-2412TT in the promoter region of SiAUX1 is an allele which is associated with lateral root number and has been strongly selected for during Setaria domestication.

The Systems Architecture of Molecular Memory in Poplar after Abiotic Stress.

Throughout the temperate zones, plants face combined drought and heat spells in increasing frequency and intensity. Here, we compared periodic (intermittent, i.e., high-frequency) versus chronic (continuous, i.e., high-intensity) drought-heat stress scenarios in gray poplar (Populus× canescens) plants for phenotypic and transcriptomic effects during stress and after recovery. Photosynthetic productivity after stress recovery exceeded the performance of poplar trees without stress experience. We analyzed the molecular basis of this stress-related memory phenotype and investigated gene expression responses across five major tree compartments including organs and wood tissues. For each of these tissue samples, transcriptomic changes induced by the two stress scenarios were highly similar during the stress phase but strikingly divergent after recovery. Characteristic molecular response patterns were found across tissues but involved different genes in each tissue. Only a small fraction of genes showed similar stress and recovery expression profiles across all tissues, including type 2C protein phosphatases, the LATE EMBRYOGENESIS ABUNDANT PROTEIN4-5 genes, and homologs of the Arabidopsis (Arabidopsis thaliana) transcription factor HOMEOBOX7. Analysis of the predicted transcription factor regulatory networks for these genes suggested that a complex interplay of common and tissue-specific components contributes to the coordination of post-recovery responses to stress in woody plants.

Color recycling: metabolization of apocarotenoid degradation products suggests carbon regeneration via primary metabolic pathways.

KEY MESSAGE: Analysis of carotenoid-accumulating roots revealed that oxidative carotenoid degradation yields glyoxal and methylglyoxal. Our data suggest that these compounds are detoxified via the glyoxalase system and re-enter primary metabolic pathways. Carotenoid levels in plant tissues depend on the relative rates of synthesis and degradation. We recently identified redox enzymes previously known to be involved in the detoxification of fatty acid-derived reactive carbonyl species which were able to convert apocarotenoids into corresponding alcohols and carboxylic acids. However, their subsequent metabolization pathways remain unresolved. Interestingly, we found that carotenoid-accumulating roots have increased levels of glutathione, suggesting apocarotenoid glutathionylation to occur. In vitro and in planta investigations did not, however, support the occurrence of non-enzymatic or enzymatic glutathionylation of ß-apocarotenoids. An alternative breakdown pathway is the continued oxidative degradation of primary apocarotenoids or their derivatives into the shortest possible oxidation products, namely glyoxal and methylglyoxal, which also accumulated in carotenoid-accumulating roots. In fact, combined transcriptome and metabolome analysis suggest that the high levels of glutathione are most probably required for detoxifying apocarotenoid-derived glyoxal and methylglyoxal via the glyoxalase pathway, yielding glycolate and D-lactate, respectively. Further transcriptome analysis suggested subsequent reactions involving activities associated with photorespiration and the peroxisome-specific glycolate/glyoxylate transporter. Finally, detoxified primary apocarotenoid degradation products might be converted into pyruvate which is possibly re-used for the synthesis of carotenoid biosynthesis precursors. Our findings allow to envision carbon recycling during carotenoid biosynthesis, degradation and re-synthesis which consumes energy, but partially maintains initially fixed carbon via re-introducing reactive carotenoid degradation products into primary metabolic pathways.

The unconventional prefoldin RPB5 interactor mediates the gravitropic response by modulating cytoskeleton organization and auxin transport in Arabidopsis.

Gravity-induced root curvature involves the asymmetric distribution of the phytohormone auxin. This response depends on the concerted activities of the auxin transporters such as PIN-FORMED (PIN) proteins for auxin efflux and AUXIN RESISTANT 1 (AUX1) for auxin influx. However, how the auxin gradient is established remains elusive. Here we identified a new mutant with a short root, strong auxin distribution in the lateral root cap and an impaired gravitropic response. The causal gene encoded an Arabidopsis homolog of the human unconventional prefoldin RPB5 interactor (URI). AtURI interacted with prefoldin 2 (PFD2) and PFD6, two ß-type PFD members that modulate actin and tubulin patterning in roots. The auxin reporter DR5rev :GFP showed that asymmetric auxin redistribution after gravistimulation is disordered in aturi-1 root tips. Treatment with the endomembrane protein trafficking inhibitor brefeldin A indicated that recycling of the auxin transporter PIN2 is disrupted in aturi-1 roots as well as in pfd mutants. We propose that AtURI cooperates with PFDs to recycle PIN2 and modulate auxin distribution.

CDC48B facilitates the intercellular trafficking of SHORT-ROOT during radial patterning in roots.

CELL DIVISION CONTROL PROTEIN48 (CDC48) is essential for membrane fusion, protein degradation, and other cellular processes. Here, we revealed the crucial role of CDC48B in regulating periclinal cell division in roots by analyzing the recessive gen1 mutant. We identified the GEN1 gene through map-based cloning and verified that GEN1 encodes CDC48B. gen1 showed severely inhibited root growth, increased periclinal cell division in the endodermis, defective middle cortex (MC) formation, and altered ground tissue patterning in roots. Consistent with these phenotypes, CYCLIND 6;1(CYCD6;1), a periclinal cell division marker, was upregulated in gen1 compared to Col-0. The ratio of SHRpro :SHR-GFP fluorescence in pre-dividing nuclei versus the adjacent stele decreased by 33% in gen1, indicating that the trafficking of SHORT-ROOT (SHR) decreased in gen1 when endodermal cells started to divide. These findings suggest that the loss of function of CDC48B inhibits the intercellular trafficking of SHR from the stele to the endodermis, thereby decreasing SHR accumulation in the endodermis. These findings shed light on the crucial role of CDC48B in regulating periclinal cell division in roots.

Retrograde Induction of phyB Orchestrates Ethylene-Auxin Hierarchy to Regulate Growth.

Exquisitely regulated plastid-to-nucleus communication by retrograde signaling pathways is essential for fine-tuning of responses to the prevailing environmental conditions. The plastidial retrograde signaling metabolite methylerythritol cyclodiphosphate (MEcPP) has emerged as a stress signal transduced into a diverse ensemble of response outputs. Here, we demonstrate enhanced phytochrome B protein abundance in red light-grown MEcPP-accumulating ceh1 mutant Arabidopsis (Arabidopsis thaliana) plants relative to wild-type seedlings. We further establish MEcPP-mediated coordination of phytochrome B with auxin and ethylene signaling pathways and uncover differential hypocotyl growth of red light-grown seedlings in response to these phytohormones. Genetic and pharmacological interference with ethylene and auxin pathways outlines the hierarchy of responses, placing ethylene epistatic to the auxin signaling pathway. Collectively, our findings establish a key role of a plastidial retrograde metabolite in orchestrating the transduction of a repertoire of signaling cascades. This work positions plastids at the zenith of relaying information coordinating external signals and internal regulatory circuitry to secure organismal integrity.

Small molecules mediate cellular reprogramming across two kingdoms.

The fertilized egg is the single totipotent cell from which multicellular organisms arise through the processes of cell division and differentiation. While animals typically lose their capacity to redifferentiate cells that are already fully differentiated, plant cells are thought to remain totipotent (Su et al., 2020). Every gardener knows well that plants can regenerate a full array of plant tissues from already differentiated organs. This also seems to be true for single plant cells such as protoplasts, which, under proper in vitro culture conditions, served as the initial source for generation of transgenic plants (Skoog and Miller, 1957; Birnbaum and Sánchez Alvarado, 2008). However, the mechanisms behind the totipotency of plant cells remain elusive, with the exception of the knowledge that the developmental fate of regenerating tissues can be directed by the ratio of two plant hormones, auxin and cytokinin (Skoog and Miller, 1957).

Auxin biosynthesis and cellular efflux act together to regulate leaf vein patterning.

Our current understanding of vein development in leaves is based on canalization of the plant hormone auxin into self-reinforcing streams which determine the sites of vascular cell differentiation. By comparison, how auxin biosynthesis affects leaf vein patterning is less well understood. Here, after observing that inhibiting polar auxin transport rescues the sparse leaf vein phenotype in auxin biosynthesis mutants, we propose that the processes of auxin biosynthesis and cellular auxin efflux work in concert during vein development. By using computational modeling, we show that localized auxin maxima are able to interact with mechanical forces generated by the morphological constraints which are imposed during early primordium development. This interaction is able to explain four fundamental characteristics of midvein morphology in a growing leaf: (i) distal cell division; (ii) coordinated cell elongation; (iii) a midvein positioned in the center of the primordium; and (iv) a midvein which is distally branched. Domains of auxin biosynthetic enzyme expression are not positioned by auxin canalization, as they are observed before auxin efflux proteins polarize. This suggests that the site-specific accumulation of auxin, as regulated by the balanced action of cellular auxin efflux and local auxin biosynthesis, is crucial for leaf vein formation.

Salicylic Acid Affects Root Meristem Patterning via Auxin Distribution in a Concentration-Dependent Manner.

The phytohormone salicylic acid (SA) is well known for its induction of pathogenesis-related proteins and systemic acquired resistance; SA also has specific effects on plant growth and development. Here we analyzed the effect of SA on Arabidopsis (Arabidopsis thaliana) root development. We show that exogenous SA treatment at low (below 50 µM) and high (greater than 50 µM) concentrations affect root meristem development in two different PR1-independent ways. Low-concentration SA promoted adventitious roots and altered architecture of the root apical meristem, whereas high-concentration SA inhibited all growth processes in the root. All exposures to exogenous SA led to changes in auxin synthesis and transport. A wide range of SA treatment concentrations activated auxin synthesis, but the effect of SA on auxin transport was dose dependent. Mathematical modeling of auxin synthesis and transport predicted auxin accumulation or depletion in the root tip following low- or high-concentration SA treatments, respectively. SA-induced auxin accumulation led to the formation of more layers of columella initials, an additional cortical cell layer (middle cortex), and extra files of epidermis, cortex, and endodermis cells. Suppression of SHORT ROOT and activation of CYCLIN D6;1 mediated the changes in radial architecture of the root. We propose that low-concentration SA plays an important role in shaping root meristem structure and root system architecture.

AtNSF regulates leaf serration by modulating intracellular trafficking of PIN1 in Arabidopsis thaliana.

In eukaryotes, N-ethylmaleimide-sensitive factor (NSF) is a conserved AAA+ ATPase and a key component of the membrane trafficking machinery that promotes the fusion of secretory vesicles with target membranes. Here, we demonstrate that the Arabidopsis thaliana genome contains a single copy of NSF, AtNSF, which plays an essential role in the regulation of leaf serration. The AtNSF knock-down mutant, atnsf-1, exhibited more serrations in the leaf margin. Moreover, polar localization of the PIN-FORMED1 (PIN1) auxin efflux transporter was diffuse around the margins of atnsf-1 leaves and root growth was inhibited in the atnsf-1 mutant. More PIN1-GFP accumulated in the intracellular compartments of atnsf-1 plants, suggesting that AtNSF is required for intracellular trafficking of PIN between the endosome and plasma membrane. Furthermore, the serration phenotype was suppressed in the atnsf-1 pin1-8 double mutant, suggesting that AtNSF is required for PIN1-mediated polar auxin transport to regulate leaf serration. The CUP-SHAPED COTYLEDON2 (CUC2) transcription factor gene is up-regulated in atnsf-1 plants and the cuc2-3 single mutant exhibits smooth leaf margins, demonstrating that AtNSF also functions in the CUC2 pathway. Our results reveal that AtNSF regulates the PIN1-generated auxin maxima with a CUC2-mediated feedback loop to control leaf serration. This article is protected by copyright. All rights reserved.

Root Gravitropism Is Regulated by a Crosstalk between para-Aminobenzoic Acid, Ethylene, and Auxin.

Plants respond to gravitational force through directional growth along the gravity vector. Although auxin is the central component of the root graviresponse, it works in concert with other plant hormones. Here, we show that the folate precursor para-aminobenzoic acid (PABA) is a key modulator of the auxin-ethylene interplay during root gravitropism in Arabidopsis (Arabidopsis thaliana). In gravistimulated roots, PABA promotes an asymmetric auxin response, which causes the asymmetric growth responsible for root curvature. This activity requires the auxin response transcription factors AUXIN RESPONSE FACTOR7 (ARF7) and ARF19 as well as ethylene biosynthesis and signaling, indicating that PABA activity requires both auxin and ethylene pathways. Similar to ethylene, exogenous PABA reverses the agravitropic root growth of the auxin transport mutant pin-formed2 (pin2) and the auxin biosynthetic double mutant with loss of function of weak ethylene insensitive (wei) genes, wei8wei2, but not the pin2wei8wei2 triple mutant. This finding suggests that PABA regulates the ethylene-dependent reciprocal compensation between auxin transport and biosynthesis. Furthermore, manipulation of endogenous free PABA levels by modulating the expression of the gene encoding its glucosylation enzyme, UDP-GLYCOSYL TRANSFERASE75B1, impacts the root graviresponse, suggesting that endogenous free PABA levels may play a crucial role in modulating the auxin-ethylene cross talk necessary for root gravitropism.

Converging Light, Energy and Hormonal Signaling Control Meristem Activity, Leaf Initiation, and Growth.

The development of leaf primordia is subject to light control of meristematic activity. Light regulates the expression of thousands of genes with roles in cell proliferation, organ development, and differentiation of photosynthetic cells. Previous work has highlighted roles for hormone homeostasis and the energy-dependent Target of Rapamycin (TOR) kinase in meristematic activity, yet a picture of how these two regulatory mechanisms depend on light perception and interact with each other has yet to emerge. Their relevance beyond leaf initiation also is unclear. Here, we report the discovery that the dark-arrested meristematic region of Arabidopsis (Arabidopsis thaliana) experiences a local energy deprivation state and confirm previous findings that the PIN1 auxin transporter is diffusely localized in the dark. Light triggers a rapid removal of the starvation state and the establishment of PIN1 polar membrane localization consistent with auxin export, both preceding the induction of cell cycle- and cytoplasmic growth-associated genes. We demonstrate that shoot meristematic activity can occur in the dark through the manipulation of auxin and cytokinin activity as well as through the activation of energy signaling, both targets of photomorphogenesis action, but the organ developmental outcomes differ: while TOR-dependent energy signals alone stimulate cell proliferation, the development of a normal leaf lamina requires photomorphogenesis-like hormonal responses. We further show that energy signaling adjusts the extent of cell cycle activity and growth of young leaves non-cellautonomously to available photosynthates and leads to organs constituted of a greater number of cells developing under higher irradiance. This makes energy signaling perhaps the most important biomass growth determinant under natural, unstressed conditions.