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Studying the developmental genetics of plant organs, requires following gene expression in specific tissues. To facilitate this, we have developed the Dual Expression Anatomy Lines (DEAL), 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 24h,. and cCoupled with the development of a flow cell and perfusion system, we follow changes in activity of the DII auxin sensor following application of auxin.

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 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-localised with ABCBs.

© 2021 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd. The phytohormone cytokinin plays a significant role in nearly all aspects of plant growth and development. Cytokinin signaling has primarily been studied in the dicot model Arabidopsis, with relatively little work done in monocots, which include rice (Oryza sativa) and other cereals of agronomic importance. The cytokinin signaling pathway is a phosphorelay comprised of the histidine kinase receptors, the authentic histidine phosphotransfer proteins (AHPs) and type-B response regulators (RRs). Two negative regulators of cytokinin signaling have been identified: the type-A RRs, which are cytokinin primary response genes, and the pseudo histidine phosphotransfer proteins (PHPs), which lack the His residue required for phosphorelay. Here, we describe the role of the rice PHP genes. Phylogenic analysis indicates that the PHPs are generally first found in the genomes of gymnosperms and that they arose independently in monocots and dicots. Consistent with this, the three rice PHPs fail to complement an Arabidopsis php mutant (aphp1/ahp6). Disruption of the three rice PHPs results in a molecular phenotype consistent with these elements acting as negative regulators of cytokinin signaling, including the induction of a number of type-A RR and cytokinin oxidase genes. The triple php mutant affects multiple aspects of rice growth and development, including shoot morphology, panicle architecture, and seed fill. In contrast to Arabidopsis, disruption of the rice PHPs does not affect root vascular patterning, suggesting that while many aspects of key signaling networks are conserved between monocots and dicots, the roles of at least some cytokinin signaling elements are distinct.

The regulation of signalling capacity, combined with the spatiotemporal distribution of developmental signals themselves, is pivotal in setting developmental responses in both plants and animals. The hormone auxin is a key signal for plant growth and development that acts through the AUXIN RESPONSE FACTOR (ARF) transcription factors. A subset of these, the conserved class A ARFs, are transcriptional activators of auxin-responsive target genes that are essential for regulating auxin signalling throughout the plant lifecycle. Although class A ARFs have tissue-specific expression patterns, how their expression is regulated is unknown. Here we show, by investigating chromatin modifications and accessibility, that loci encoding these proteins are constitutively open for transcription. Through yeast one-hybrid screening, we identify the transcriptional regulators of the genes encoding class A ARFs from Arabidopsis thaliana and demonstrate that each gene is controlled by specific sets of transcriptional regulators. Transient transformation assays and expression analyses in mutants reveal that, in planta, the majority of these regulators repress the transcription of genes encoding class A ARFs. These observations support a scenario in which the default configuration of open chromatin enables a network of transcriptional repressors to regulate expression levels of class A ARF proteins and modulate auxin signalling output throughout development.

Pattern formation is typically controlled through the interaction between molecular signals within a given tissue. During early embryonic development, roots of the model plant Arabidopsis thaliana have a radially symmetric pattern, but a heterogeneous input of the hormone auxin from the two cotyledons forces the vascular cylinder to develop a diarch pattern with two xylem poles. Molecular analyses and mathematical approaches have uncovered the regulatory circuit that propagates this initial auxin signal into a stable cellular pattern. The diarch pattern seen in Arabidopsis is relatively uncommon among flowering plants, with most species having between three and eight xylem poles. Here, we have used multiscale mathematical modelling to demonstrate that this regulatory module does not require a heterogeneous auxin input to specify the vascular pattern. Instead, the pattern can emerge dynamically, with its final form dependent upon spatial constraints and growth. The predictions of our simulations compare to experimental observations of xylem pole number across a range of species, as well as in transgenic systems in Arabidopsis in which we manipulate the size of the vascular cylinder. By considering the spatial constraints, our model is able to explain much of the diversity seen in different flowering plant species.

Plants adapt to heterogeneous soil conditions by altering their root architecture. For example, roots branch when in contact with water using the hydropatterning response. We report that hydropatterning is dependent on auxin response factor ARF7. This transcription factor induces asymmetric expression of its target gene LBD16 in lateral root founder cells on the side of the root in contact with water. This differential expression pattern is regulated by post-translational modification of ARF7 with the SUMO protein. SUMOylation negatively regulates ARF7 DNA binding activity. ARF7 SUMOylation is required to recruit the Aux/IAA repressor protein IAA3. Blocking ARF7 SUMOylation disrupts IAA3 recruitment and hydropatterning. We conclude that SUMO-dependent regulation of auxin response controls root branching pattern in response to water availability.

Phosphorus is a crucial macronutrient for plants playing a critical role in many cellular signaling and energy cycling processes. In light of this, phosphorus acquisition efficiency is an important target trait for crop improvement, but it also provides an ecological adaptation for growth of plants in low nutrient environments. Increased root hair density has been shown to improve phosphorus uptake and plant health in a number of species.In several plant families, including Brassicaceae, root hair bearing cells are positioned on the epidermis according to their position in relation to cortex cells, with hair cells positioned in the cleft between two underlying cortex cells. Thus the number of cortex cells determines the number of epidermal cells in the root hair position. Previous research has associated phosphorus-limiting conditions with an increase in the number of cortex cell files in Arabidopsis thaliana roots, but they have not investigated the spatial or temporal domains in which these extra divisions occur or explored the consequences this has had on root hair formation.In this study, we use 3D reconstructions of root meristems to demonstrate that the anticlinal cell divisions seen under low phosphate are exclusive to the cortex. When grown on media containing replete levels of phosphorous, Arabidopsis thaliana plants almost invariably show 8 cortex cells; however when grown in phosphate limited conditions, seedlings typically develop 12-16 cortex cells, resulting in a significant increase in the number of epidermal cells at hair forming positions. These anticlinal divisions occur within the initial cells and can be seen within 24 hours of transfer of plants to low phosphorous conditions. We show that these changes in the underlying cortical cells feed into epidermal patterning by altering the regular spacing of root hairs.