The first intron of ARF7 is required for expression in root tips.

Auxin regulates plant growth and development through the transcription factors of the AUXIN RESPONSE FACTOR (ARF) gene family. ARF7 is one of five activators that bind DNA and elicit downstream transcriptional responses. In roots, ARF7 regulates growth, gravitropism and redundantly with ARF19, lateral root organogenesis. In this study we analyzed ARF7 cis-regulation, using different non-coding sequences of the ARF7 locus to drive GFP. We show that constructs containing the first intron led to increased signal in the root tip. Although bioinformatics analyses predicted several transcription factor binding sites in the first intron, we were unable to significantly alter expression of GFP in the root by mutating these. We instead observed the intronic sequences needed to be present within the transcribed sequences to drive expression in the root meristem. These data support a mechanism by which intron-mediated enhancement regulates the tissue specific expression of ARF7 in the root meristem.

Systems approaches reveal that ABCB and PIN proteins mediate co-dependent auxin efflux.

Members of the B family of membrane-bound ATP-binding cassette (ABC) transporters represent key components of the auxin efflux machinery in plants. Over the last two decades, experimental studies have shown that modifying ATP-binding cassette sub-family B (ABCB) expression affects auxin distribution and plant phenotypes. However, precisely how ABCB proteins transport auxin in conjunction with the more widely studied family of PIN-formed (PIN) auxin efflux transporters is unclear, and studies using heterologous systems have produced conflicting results. Here, we integrate ABCB localization data into a multicellular model of auxin transport in the Arabidopsis thaliana root tip to predict how ABCB-mediated auxin transport impacts organ-scale auxin distribution. We use our model to test five potential ABCB-PIN regulatory interactions, simulating the auxin dynamics for each interaction and quantitatively comparing the predictions with experimental images of the DII-VENUS auxin reporter in wild-type and abcb single and double loss-of-function mutants. Only specific ABCB-PIN regulatory interactions result in predictions that recreate the experimentally observed DII-VENUS distributions and long-distance auxin transport. Our results suggest that ABCBs enable auxin efflux independently of PINs; however, PIN-mediated auxin efflux is predominantly through a co-dependent efflux where co-localized with ABCBs.

Dual expression and anatomy lines allow simultaneous visualization of gene expression and anatomy.

Studying the developmental genetics of plant organs requires following gene expression in specific tissues. To facilitate this, we have developed dual expression anatomy lines, which incorporate a red plasma membrane marker alongside a fluorescent reporter for a gene of interest in the same vector. Here, we adapted the GreenGate cloning vectors to create two destination vectors showing strong marking of cell membranes in either the whole root or specifically in the lateral roots. This system can also be used in both embryos and whole seedlings. As proof of concept, we follow both gene expression and anatomy in Arabidopsis (Arabidopsis thaliana) during lateral root organogenesis for a period of over 24 h. Coupled with the development of a flow cell and perfusion system, we follow changes in activity of the DII auxin sensor following application of auxin.

Uncovering How Auxin Optimizes Root Systems Architecture in Response to Environmental Stresses.

Since colonizing land, plants have developed mechanisms to tolerate a broad range of abiotic stresses that include flooding, drought, high salinity, and nutrient limitation. Roots play a key role acclimating plants to these as their developmental plasticity enables them to grow toward more favorable conditions and away from limiting or harmful stresses. The phytohormone auxin plays a key role translating these environmental signals into developmental outputs. This is achieved by modulating auxin levels and/or signaling, often through cross talk with other hormone signals like abscisic acid (ABA) or ethylene. In our review, we discuss how auxin controls root responses to water, osmotic and nutrient-related stresses, and describe how the synthesis, degradation, transport, and response of this key signaling hormone helps optimize root architecture to maximize resource acquisition while limiting the impact of abiotic stresses.

Volumetric Segmentation of Cell Cycle Markers in Confocal Images Using Machine Learning and Deep Learning.

Understanding plant growth processes is important for many aspects of biology and food security. Automating the observations of plant development-a process referred to as plant phenotyping-is increasingly important in the plant sciences, and is often a bottleneck. Automated tools are required to analyze the data in microscopy images depicting plant growth, either locating or counting regions of cellular features in images. In this paper, we present to the plant community an introduction to and exploration of two machine learning approaches to address the problem of marker localization in confocal microscopy. First, a comparative study is conducted on the classification accuracy of common conventional machine learning algorithms, as a means to highlight challenges with these methods. Second, a 3D (volumetric) deep learning approach is developed and presented, including consideration of appropriate loss functions and training data. A qualitative and quantitative analysis of all the results produced is performed. Evaluation of all approaches is performed on an unseen time-series sequence comprising several individual 3D volumes, capturing plant growth. The comparative analysis shows that the deep learning approach produces more accurate and robust results than traditional machine learning. To accompany the paper, we are releasing the 4D point annotation tool used to generate the annotations, in the form of a plugin for the popular ImageJ (FIJI) software. Network models and example datasets will also be available online.

Auxin fluxes through plasmodesmata modify root-tip auxin distribution.

Auxin is a key signal regulating plant growth and development. It is well established that auxin dynamics depend on the spatial distribution of efflux and influx carriers on the cell membranes. In this study, we employ a systems approach to characterise an alternative symplastic pathway for auxin mobilisation via plasmodesmata, which function as intercellular pores linking the cytoplasm of adjacent cells. To investigate the role of plasmodesmata in auxin patterning, we developed a multicellular model of the Arabidopsis root tip. We tested the model predictions using the DII-VENUS auxin response reporter, comparing the predicted and observed DII-VENUS distributions using genetic and chemical perturbations designed to affect both carrier-mediated and plasmodesmatal auxin fluxes. The model revealed that carrier-mediated transport alone cannot explain the experimentally determined auxin distribution in the root tip. In contrast, a composite model that incorporates both carrier-mediated and plasmodesmatal auxin fluxes re-capitulates the root-tip auxin distribution. We found that auxin fluxes through plasmodesmata enable auxin reflux and increase total root-tip auxin. We conclude that auxin fluxes through plasmodesmata modify the auxin distribution created by efflux and influx carriers.

Cell Death in Cells Overlying Lateral Root Primordia Facilitates Organ Growth in Arabidopsis.

Plant organ growth is widely accepted to be determined by cell division and cell expansion, but, unlike that in animals, the contribution of cell elimination has rarely been recognized. We investigated this paradigm during Arabidopsis lateral root formation, when the lateral root primordia (LRP) must traverse three overlying cell layers within the parent root. A subset of LRP-overlying cells displayed the induction of marker genes for cell types undergoing developmental cell death, and their cell death was detected by electron, confocal, and light sheet microscopy techniques. LRP growth was delayed in cell-death-deficient mutants lacking the positive cell death regulator ORESARA1/ANAC092 (ORE1). LRP growth was restored in ore1-2 knockout plants by genetically inducing cell elimination in cells overlying the LRP or by physically killing LRP-overlying cells by ablation with optical tweezers. Our results support that, in addition to previously discovered mechanisms, cell elimination contributes to regulating lateral root emergence.

Auxin Metabolism Controls Developmental Decisions in Land Plants.

Unlike animals, whose body plans are set during embryo development, plants maintain the ability to initiate new organs throughout their life cycle. Auxin is a key regulator of almost all aspects of plant development, including morphogenesis and adaptive responses. Cellular auxin concentrations influence whether a cell will divide, grow, or differentiate, thereby contributing to organ formation, growth, and ultimately plant shape. Auxin gradients are established and maintained by a tightly regulated interplay between metabolism, signalling, and transport. Auxin is synthesised, stored, and inactivated by a multitude of parallel pathways that are all tightly regulated. Here we summarise the remarkable progress that has been achieved in identifying some key components of these pathways and the genetic complexity underlying their precise regulation.

PUCHI regulates very long chain fatty acid biosynthesis during lateral root and callus formation.

Lateral root organogenesis plays an essential role in elaborating plant root system architecture. In Arabidopsis, the AP2 family transcription factor PUCHI controls cell proliferation in lateral root primordia. To identify potential targets of PUCHI, we analyzed a time course transcriptomic dataset of lateral root formation. We report that multiple genes coding for very long chain fatty acid (VLCFA) biosynthesis enzymes are induced during lateral root development in a PUCHI-dependent manner. Significantly, several mutants perturbed in VLCFA biosynthesis show similar lateral root developmental defects as puchi-1 Moreover, puchi-1 roots display the same disorganized callus formation phenotype as VLCFA biosynthesis-deficient mutants when grown on auxin-rich callus-inducing medium. Lipidomic profiling of puchi-1 roots revealed reduced VLCFA content compared with WT. We conclude that PUCHI-regulated VLCFA biosynthesis is part of a pathway controlling cell proliferation during lateral root and callus formation.

EXPANSIN A1-mediated radial swelling of pericycle cells positions anticlinal cell divisions during lateral root initiation.

In plants, postembryonic formation of new organs helps shape the adult organism. This requires the tight regulation of when and where a new organ is formed and a coordination of the underlying cell divisions. To build a root system, new lateral roots are continuously developing, and this process requires the tight coordination of asymmetric cell division in adjacent pericycle cells. We identified EXPANSIN A1 (EXPA1) as a cell wall modifying enzyme controlling the divisions marking lateral root initiation. Loss of EXPA1 leads to defects in the first asymmetric pericycle cell divisions and the radial swelling of the pericycle during auxin-driven lateral root formation. We conclude that a localized radial expansion of adjacent pericycle cells is required to position the asymmetric cell divisions and generate a core of small daughter cells, which is a prerequisite for lateral root organogenesis.

Author Correction: A mechanistic framework for auxin dependent Arabidopsis root hair elongation to low external phosphate.

The original version of this Article omitted the following from the Acknowledgements: 'We also thank DBT-CREST BT/HRD/03/01/2002.' This has been corrected in both the PDF and HTML versions of the Article.

A mechanistic framework for auxin dependent Arabidopsis root hair elongation to low external phosphate.

Phosphate (P) is an essential macronutrient for plant growth. Roots employ adaptive mechanisms to forage for P in soil. Root hair elongation is particularly important since P is immobile. Here we report that auxin plays a critical role promoting root hair growth in Arabidopsis in response to low external P. Mutants disrupting auxin synthesis (taa1) and transport (aux1) attenuate the low P root hair response. Conversely, targeting AUX1 expression in lateral root cap and epidermal cells rescues this low P response in aux1. Hence auxin transport from the root apex to differentiation zone promotes auxin-dependent hair response to low P. Low external P results in induction of root hair expressed auxin-inducible transcription factors ARF19, RSL2, and RSL4. Mutants lacking these genes disrupt the low P root hair response. We conclude auxin synthesis, transport and response pathway components play critical roles regulating this low P root adaptive response.

Dioxygenase-encoding AtDAO1 gene controls IAA oxidation and homeostasis in Arabidopsis.

Auxin represents a key signal in plants, regulating almost every aspect of their growth and development. Major breakthroughs have been made dissecting the molecular basis of auxin transport, perception, and response. In contrast, how plants control the metabolism and homeostasis of the major form of auxin in plants, indole-3-acetic acid (IAA), remains unclear. In this paper, we initially describe the function of the Arabidopsis thaliana gene DIOXYGENASE FOR AUXIN OXIDATION 1 (AtDAO1). Transcriptional and translational reporter lines revealed that AtDAO1 encodes a highly root-expressed, cytoplasmically localized IAA oxidase. Stable isotope-labeled IAA feeding studies of loss and gain of function AtDAO1 lines showed that this oxidase represents the major regulator of auxin degradation to 2-oxoindole-3-acetic acid (oxIAA) in Arabidopsis Surprisingly, AtDAO1 loss and gain of function lines exhibited relatively subtle auxin-related phenotypes, such as altered root hair length. Metabolite profiling of mutant lines revealed that disrupting AtDAO1 regulation resulted in major changes in steady-state levels of oxIAA and IAA conjugates but not IAA. Hence, IAA conjugation and catabolism seem to regulate auxin levels in Arabidopsis in a highly redundant manner. We observed that transcripts of AtDOA1 IAA oxidase and GH3 IAA-conjugating enzymes are auxin-inducible, providing a molecular basis for their observed functional redundancy. We conclude that the AtDAO1 gene plays a key role regulating auxin homeostasis in Arabidopsis, acting in concert with GH3 genes, to maintain auxin concentration at optimal levels for plant growth and development.

Dynamic regulation of auxin oxidase and conjugating enzymes AtDAO1 and GH3 modulates auxin homeostasis.

The hormone auxin is a key regulator of plant growth and development, and great progress has been made understanding auxin transport and signaling. Here, we show that auxin metabolism and homeostasis are also regulated in a complex manner. The principal auxin degradation pathways in Arabidopsis include oxidation by Arabidopsis thaliana gene DIOXYGENASE FOR AUXIN OXIDATION 1/2 (AtDAO1/2) and conjugation by Gretchen Hagen3s (GH3s). Metabolic profiling of dao1-1 root tissues revealed a 50% decrease in the oxidation product 2-oxoindole-3-acetic acid (oxIAA) and increases in the conjugated forms indole-3-acetic acid aspartic acid (IAA-Asp) and indole-3-acetic acid glutamic acid (IAA-Glu) of 438- and 240-fold, respectively, whereas auxin remains close to the WT. By fitting parameter values to a mathematical model of these metabolic pathways, we show that, in addition to reduced oxidation, both auxin biosynthesis and conjugation are increased in dao1-1 Transcripts of AtDAO1 and GH3 genes increase in response to auxin over different timescales and concentration ranges. Including this regulation of AtDAO1 and GH3 in an extended model reveals that auxin oxidation is more important for auxin homoeostasis at lower hormone concentrations, whereas auxin conjugation is most significant at high auxin levels. Finally, embedding our homeostasis model in a multicellular simulation to assess the spatial effect of the dao1-1 mutant shows that auxin increases in outer root tissues in agreement with the dao1-1 mutant root hair phenotype. We conclude that auxin homeostasis is dependent on AtDAO1, acting in concert with GH3, to maintain auxin at optimal levels for plant growth and development.

Tonoplast Aquaporins Facilitate Lateral Root Emergence.

Aquaporins (AQPs) are water channels allowing fast and passive diffusion of water across cell membranes. It was hypothesized that AQPs contribute to cell elongation processes by allowing water influx across the plasma membrane and the tonoplast to maintain adequate turgor pressure. Here, we report that, in Arabidopsis (Arabidopsis thaliana), the highly abundant tonoplast AQP isoforms AtTIP1;1, AtTIP1;2, and AtTIP2;1 facilitate the emergence of new lateral root primordia (LRPs). The number of lateral roots was strongly reduced in the triple tip mutant, whereas the single, double, and triple tip mutants showed no or minor reduction in growth of the main root. This phenotype was due to the retardation of LRP emergence. Live cell imaging revealed that tight spatiotemporal control of TIP abundance in the tonoplast of the different LRP cells is pivotal to mediating this developmental process. While lateral root emergence is correlated to a reduction of AtTIP1;1 and AtTIP1;2 protein levels in LRPs, expression of AtTIP2;1 is specifically needed in a restricted cell population at the base, then later at the flanks, of developing LRPs. Interestingly, the LRP emergence phenotype of the triple tip mutants could be fully rescued by expressing AtTIP2;1 under its native promoter. We conclude that TIP isoforms allow the spatial and temporal fine-tuning of cellular water transport, which is critically required during the highly regulated process of LRP morphogenesis and emergence.

The circadian clock rephases during lateral root organ initiation in Arabidopsis thaliana.

The endogenous circadian clock enables organisms to adapt their growth and development to environmental changes. Here we describe how the circadian clock is employed to coordinate responses to the key signal auxin during lateral root (LR) emergence. In the model plant, Arabidopsis thaliana, LRs originate from a group of stem cells deep within the root, necessitating that new organs emerge through overlying root tissues. We report that the circadian clock is rephased during LR development. Metabolite and transcript profiling revealed that the circadian clock controls the levels of auxin and auxin-related genes including the auxin response repressor IAA14 and auxin oxidase AtDAO2. Plants lacking or overexpressing core clock components exhibit LR emergence defects. We conclude that the circadian clock acts to gate auxin signalling during LR development to facilitate organ emergence.

Inference of the Arabidopsis lateral root gene regulatory network suggests a bifurcation mechanism that defines primordia flanking and central zones.

A large number of genes involved in lateral root (LR) organogenesis have been identified over the last decade using forward and reverse genetic approaches in Arabidopsis thaliana. Nevertheless, how these genes interact to form a LR regulatory network largely remains to be elucidated. In this study, we developed a time-delay correlation algorithm (TDCor) to infer the gene regulatory network (GRN) controlling LR primordium initiation and patterning in Arabidopsis from a time-series transcriptomic data set. The predicted network topology links the very early-activated genes involved in LR initiation to later expressed cell identity markers through a multistep genetic cascade exhibiting both positive and negative feedback loops. The predictions were tested for the key transcriptional regulator AUXIN RESPONSE FACTOR7 node, and over 70% of its targets were validated experimentally. Intriguingly, the predicted GRN revealed a mutual inhibition between the ARF7 and ARF5 modules that would control an early bifurcation between two cell fates. Analyses of the expression pattern of ARF7 and ARF5 targets suggest that this patterning mechanism controls flanking and central zone specification in Arabidopsis LR primordia.

Branching out in roots: uncovering form, function, and regulation.

Root branching is critical for plants to secure anchorage and ensure the supply of water, minerals, and nutrients. To date, research on root branching has focused on lateral root development in young seedlings. However, many other programs of postembryonic root organogenesis exist in angiosperms. In cereal crops, the majority of the mature root system is composed of several classes of adventitious roots that include crown roots and brace roots. In this Update, we initially describe the diversity of postembryonic root forms. Next, we review recent advances in our understanding of the genes, signals, and mechanisms regulating lateral root and adventitious root branching in the plant models Arabidopsis (Arabidopsis thaliana), maize (Zea mays), and rice (Oryza sativa). While many common signals, regulatory components, and mechanisms have been identified that control the initiation, morphogenesis, and emergence of new lateral and adventitious root organs, much more remains to be done. We conclude by discussing the challenges and opportunities facing root branching research.

AtMYB93 is a novel negative regulator of lateral root development in Arabidopsis.

Plant root system plasticity is critical for survival in changing environmental conditions. One important aspect of root architecture is lateral root development, a complex process regulated by hormone, environmental and protein signalling pathways. Here we show, using molecular genetic approaches, that the MYB transcription factor AtMYB93 is a novel negative regulator of lateral root development in Arabidopsis. We identify AtMYB93 as an interaction partner of the lateral-root-promoting ARABIDILLO proteins. Atmyb93 mutants have faster lateral root developmental progression and enhanced lateral root densities, while AtMYB93-overexpressing lines display the opposite phenotype. AtMYB93 is expressed strongly, specifically and transiently in the endodermal cells overlying early lateral root primordia and is additionally induced by auxin in the basal meristem of the primary root. Furthermore, Atmyb93 mutant lateral root development is insensitive to auxin, indicating that AtMYB93 is required for normal auxin responses during lateral root development. We propose that AtMYB93 is part of a novel auxin-induced negative feedback loop stimulated in a select few endodermal cells early during lateral root development, ensuring that lateral roots only develop when absolutely required. Putative AtMYB93 homologues are detected throughout flowering plants and represent promising targets for manipulating root systems in diverse crop species.

Delivery of endocytosed proteins to the cell-division plane requires change of pathway from recycling to secretion.

Membrane trafficking is essential to fundamental processes in eukaryotic life, including cell growth and division. In plant cytokinesis, post-Golgi trafficking mediates a massive flow of vesicles that form the partitioning membrane but its regulation remains poorly understood. Here, we identify functionally redundant Arabidopsis ARF guanine-nucleotide exchange factors (ARF-GEFs) BIG1-BIG4 as regulators of post-Golgi trafficking, mediating late secretion from the trans-Golgi network but not recycling of endocytosed proteins to the plasma membrane, although the TGN also functions as an early endosome in plants. In contrast, BIG1-4 are absolutely required for trafficking of both endocytosed and newly synthesized proteins to the cell-division plane during cytokinesis, counteracting recycling to the plasma membrane. This change from recycling to secretory trafficking pathway mediated by ARF-GEFs confers specificity of cargo delivery to the division plane and might thus ensure that the partitioning membrane is completed on time in the absence of a cytokinesis-interphase checkpoint. DOI: http://dx.doi.org/10.7554/eLife.02131.001.