GH3-mediated auxin inactivation attenuates multiple stages of lateral root development.

Lateral root (LR) positioning and development rely on the dynamic interplay between auxin production, transport but also inactivation. Nonetheless, how the latter affects LR organogenesis remains largely uninvestigated. Here, we systematically analyze the impact of the major auxin inactivation pathway defined by GRETCHEN HAGEN3-type (GH3) auxin conjugating enzymes and DIOXYGENASE FOR AUXIN OXIDATION1 (DAO1) in all stages of LR development using reporters, genetics and inhibitors in Arabidopsis thaliana. Our data demonstrate that the gh3.1/2/3/4/5/6 hextuple (gh3hex) mutants display a higher LR density due to increased LR initiation and faster LR developmental progression, acting epistatically over dao1-1. Grafting and local inhibitor applications reveal that root and shoot GH3 activities control LR formation. The faster LR development in gh3hex is associated with GH3 expression domains in and around developing LRs. The increase in LR initiation is associated with accelerated auxin response oscillations coinciding with increases in apical meristem size and LR cap cell death rates. Our research reveals how GH3-mediated auxin inactivation attenuates LR development. Local GH3 expression in LR primordia attenuates development and emergence, whereas GH3 effects on pre-initiation stages are indirect, by modulating meristem activities that in turn coordinate root growth with LR spacing.

An LRR receptor kinase controls ABC transporter substrate preferences during plant growth-defense decisions.

The exporter of the auxin precursor indole-3-butyric acid (IBA), ABCG36/PDR8/PEN3, from the model plant Arabidopsis has recently been proposed to also function in the transport of the phytoalexin camalexin. Based on these bonafide substrates, it has been suggested that ABCG36 functions at the interface between growth and defense. Here, we provide evidence that ABCG36 catalyzes the direct, ATP-dependent export of camalexin across the plasma membrane. We identify the leucine-rich repeat receptor kinase, QIAN SHOU KINASE1 (QSK1), as a functional kinase that physically interacts with and phosphorylates ABCG36. Phosphorylation of ABCG36 by QSK1 unilaterally represses IBA export, allowing camalexin export by ABCG36 conferring pathogen resistance. As a consequence, phospho-dead mutants of ABCG36, as well as qsk1 and abcg36 alleles, are hypersensitive to infection with the root pathogen Fusarium oxysporum, caused by elevated fungal progression. Our findings indicate a direct regulatory circuit between a receptor kinase and an ABC transporter that functions to control transporter substrate preference during plant growth and defense balance decisions.

Multi-Knock-a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants.

Plant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programmes. Here we describe the development and validation of Multi-Knock, a genome-scale clustered regularly interspaced short palindromic repeat toolbox that overcomes functional redundancy in Arabidopsis by simultaneously targeting multiple gene-family members, thus identifying genetically hidden components. We computationally designed 59,129 optimal single-guide RNAs that each target two to ten genes within a family at once. Furthermore, partitioning the library into ten sublibraries directed towards a different functional group allows flexible and targeted genetic screens. From the 5,635 single-guide RNAs targeting the plant transportome, we generated over 3,500 independent Arabidopsis lines that allowed us to identify and characterize the first known cytokinin tonoplast-localized transporters in plants. With the ability to overcome functional redundancy in plants at the genome-scale level, the developed strategy can be readily deployed by scientists and breeders for basic research and to expedite breeding efforts.

Restriction of access to the central cavity is a major contributor to substrate selectivity in plant ABCG transporters.

ABCG46 of the legume Medicago truncatula is an ABC-type transporter responsible for highly selective translocation of the phenylpropanoids, 4-coumarate, and liquiritigenin, over the plasma membrane. To investigate molecular determinants of the observed substrate selectivity, we applied a combination of phylogenetic and biochemical analyses, AlphaFold2 structure prediction, molecular dynamics simulations, and mutagenesis. We discovered an unusually narrow transient access path to the central cavity of MtABCG46 that constitutes an initial filter responsible for the selective translocation of phenylpropanoids through a lipid bilayer. Furthermore, we identified remote residue F562 as pivotal for maintaining the stability of this filter. The determination of individual amino acids that impact the selective transport of specialized metabolites may provide new opportunities associated with ABCGs being of interest, in many biological scenarios.

ABCB-mediated shootward auxin transport feeds into the root clock.

Although strongly influenced by environmental conditions, lateral root (LR) positioning along the primary root appears to follow obediently an internal spacing mechanism dictated by auxin oscillations that prepattern the primary root, referred to as the root clock. Surprisingly, none of the hitherto characterized PIN- and ABCB-type auxin transporters seem to be involved in this LR prepatterning mechanism. Here, we characterize ABCB15, 16, 17, 18, and 22 (ABCB15-22) as novel auxin-transporting ABCBs. Knock-down and genome editing of this genetically linked group of ABCBs caused strongly reduced LR densities. These phenotypes were correlated with reduced amplitude, but not reduced frequency of the root clock oscillation. High-resolution auxin transport assays and tissue-specific silencing revealed contributions of ABCB15-22 to shootward auxin transport in the lateral root cap (LRC) and epidermis, thereby explaining the reduced auxin oscillation. Jointly, these data support a model in which LRC-derived auxin contributes to the root clock amplitude.

Chloroplast Auxin Efflux Mediated by ABCB28 and ABCB29 Fine-Tunes Salt and Drought Stress Responses in Arabidopsis.

Photosynthesis is among the first processes negatively affected by environmental cues and its performance directly determines plant cell fitness and ultimately crop yield. Primarily sites of photosynthesis, chloroplasts are unique sites also for the biosynthesis of precursors of the growth regulator auxin and for sensing environmental stress, but their role in intracellular auxin homeostasis, vital for plant growth and survival in changing environments, remains poorly understood. Here, we identified two ATP-binding cassette (ABC) subfamily B transporters, ABCB28 and ABCB29, which export auxin across the chloroplast envelope to the cytosol in a concerted action in vivo. Moreover, we provide evidence for an auxin biosynthesis pathway in Arabidopsis thaliana chloroplasts. The overexpression of ABCB28 and ABCB29 influenced stomatal regulation and resulted in significantly improved water use efficiency and survival rates during salt and drought stresses. Our results suggest that chloroplast auxin production and transport contribute to stomata regulation for conserving water upon salt stress. ABCB28 and ABCB29 integrate photosynthesis and auxin signals and as such hold great potential to improve the adaptation potential of crops to environmental cues.

WAVY GROWTH Arabidopsis E3 ubiquitin ligases affect apical PIN sorting decisions.

Directionality in the intercellular transport of the plant hormone auxin is determined by polar plasma membrane localization of PIN-FORMED (PIN) auxin transport proteins. However, apart from PIN phosphorylation at conserved motifs, no further determinants explicitly controlling polar PIN sorting decisions have been identified. Here we present Arabidopsis WAVY GROWTH 3 (WAV3) and closely related RING-finger E3 ubiquitin ligases, whose loss-of-function mutants show a striking apical-to-basal polarity switch in PIN2 localization in root meristem cells. WAV3 E3 ligases function as essential determinants for PIN polarity, acting independently from PINOID/WAG-dependent PIN phosphorylation. They antagonize ectopic deposition of de novo synthesized PIN proteins already immediately following completion of cell division, presumably via preventing PIN sorting into basal, ARF GEF-mediated trafficking. Our findings reveal an involvement of E3 ligases in the selective targeting of apically localized PINs in higher plants.

Arabidopsis TWISTED DWARF1 regulates stamen elongation by differential activation of ABCB1,19-mediated auxin transport.

Despite clear evidence that a local accumulation of auxin is likewise critical for male fertility, much less is known about the components that regulate auxin-controlled stamen development. In this study, we analyzed physiological and morphological parameters in mutants of key players of ABCB-mediated auxin transport, and spatially and temporally dissected their expression on the protein level as well as auxin fluxes in the Arabidopsis stamens. Our analyses revealed that the FKBP42, TWISTED DWARF1 (TWD1), promotes stamen elongation and, to a lesser extent, anther dehiscence, as well as pollen maturation, and thus is required for seed development. Most of the described developmental defects in twd1 are shared with the abcb1 abcb19 mutant, which can be attributed to the fact that TWD1-as a described ABCB chaperone-is a positive regulator of ABCB1- and ABCB19-mediated auxin transport. However, reduced stamen number was dependent on TWD1 but not on investigated ABCBs, suggesting additional players downstream of TWD1. We predict an overall housekeeping function for ABCB1 during earlier stages, while ABCB19 seems to be responsible for the key event of rapid elongation at later stages of stamen development. Our data indicate that TWD1 controls stamen development by differential activation of ABCB1,19-mediated auxin transport in the stamen.

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.

Ins and outs of AlphaFold2 transmembrane protein structure predictions.

Transmembrane (TM) proteins are major drug targets, but their structure determination, a prerequisite for rational drug design, remains challenging. Recently, the DeepMind's AlphaFold2 machine learning method greatly expanded the structural coverage of sequences with high accuracy. Since the employed algorithm did not take specific properties of TM proteins into account, the reliability of the generated TM structures should be assessed. Therefore, we quantitatively investigated the quality of structures at genome scales, at the level of ABC protein superfamily folds and for specific membrane proteins (e.g. dimer modeling and stability in molecular dynamics simulations). We tested template-free structure prediction with a challenging TM CASP14 target and several TM protein structures published after AlphaFold2 training. Our results suggest that AlphaFold2 performs well in the case of TM proteins and its neural network is not overfitted. We conclude that cautious applications of AlphaFold2 structural models will advance TM protein-associated studies at an unexpected level.

OsRLR4 binds to the OsAUX1 promoter to negatively regulate primary root development in rice.

Root architecture is one of the most important agronomic traits that determines rice crop yield. The primary root (PR) absorbs mineral nutrients and provides mechanical support; however, the molecular mechanisms of PR elongation remain unclear in rice. Here, the two loss-of-function T-DNA insertion mutants of root length regulator 4 (OsRLR4), osrlr4-1 and osrlr4-2 with longer PR, and three OsRLR4 overexpression lines, OE-OsRLR4-1/-2/-3 with shorter PR compared to the wild type/Hwayoung (WT/HY), were identified. OsRLR4 is one of five members of the PRAF subfamily of the regulator chromosome condensation 1 (RCC1) family. Phylogenetic analysis of OsRLR4 from wild and cultivated rice indicated that it is under selective sweeps, suggesting its potential role in domestication. OsRLR4 controls PR development by regulating auxin accumulation in the PR tip and thus the root apical meristem activity. A series of biochemical and genetic analyses demonstrated that OsRLR4 functions directly upstream of the auxin transporter OsAUX1. Moreover, OsRLR4 interacts with the TRITHORAX-like protein OsTrx1 to promote H3K4me3 deposition at the OsAUX1 promoter, thus altering its transcription level. This work provides insight into the cooperation of auxin and epigenetic modifications in regulating root architecture and provides a genetic resource for plant architecture breeding.

ABA homeostasis and long-distance translocation are redundantly regulated by ABCG ABA importers.

The effects of abscisic acid (ABA) on plant growth, development, and response to the environment depend on local ABA concentrations. Here, we show that in Arabidopsis, ABA homeostasis is regulated by two previously unknown ABA transporters. Adenosine triphosphate­binding cassette subfamily G member 17 (ABCG17) and ABCG18 are localized to the plasma membranes of leaf mesophyll and cortex cells to redundantly promote ABA import, leading to conjugated inactive ABA sinks, thus restricting stomatal closure. ABCG17 and ABCG18 double knockdown revealed that the transporters encoded by these genes not only limit stomatal aperture size, conductance, and transpiration while increasing water use efficiency but also control ABA translocation from the shoot to the root to regulate lateral root emergence. Under abiotic stress conditions, ABCG17 and ABCG18 are transcriptionally repressed, promoting active ABA movement and response. The transport mechanism mediated by ABCG17 and ABCG18 allows plants to maintain ABA homeostasis under normal growth conditions.

A novel miR167a-OsARF6-OsAUX3 module regulates grain length and weight in rice.

Grain size is one of the most important factors that control rice yield, as it is associated with grain weight (GW). To date, dozens of rice genes that regulate grain size have been isolated; however, the regulatory mechanism underlying GW control is not fully understood. Here, the quantitative trait locus qGL5 for grain length (GL) and GW was identified in recombinant inbred lines of 9311 and Nipponbare (NPB) and fine mapped to a candidate gene, OsAUX3. Sequence variations between 9311 and NPB in the OsAUX3 promoter and loss of function of OsAUX3 led to higher GL and GW. RNA sequencing, gene expression quantification, dual-luciferase reporter assays, chromatin immunoprecipitation-quantitative PCR, and yeast one-hybrid assays demonstrated that OsARF6 is an upstream transcription factor regulating the expression of OsAUX3. OsARF6 binds directly to the auxin response elements of the OsAUX3 promoter, covering a single-nucleotide polymorphism site between 9311 and NPB/Dongjin/Hwayoung, and thereby controls GL by altering longitudinal expansion and auxin distribution/content in glume cells. Furthermore, we showed that miR167a positively regulate GL and GW by directing OsARF6 mRNA silencing. Taken together, our study reveals that a novel miR167a-OsARF6-OsAUX3 module regulates GL and GW in rice, providing a potential target for the improvement of rice yield.

ABCG transporters export cutin precursors for the formation of the plant cuticle.

The plant cuticle is deposited on the surface of primary plant organs, such as leaves, fruits, and floral organs, forming a diffusion barrier and protecting the plant against various abiotic and biotic stresses. Cutin, the structural polyester of the plant cuticle, is synthesized in the apoplast. Plasma-membrane-localized ATP-binding cassette (ABC) transporters of the G family have been hypothesized to export cutin precursors. Here, we characterize SlABCG42 of tomato representing an ortholog of AtABCG32 in Arabidopsis. SlABCG42 expression in Arabidopsis complements the cuticular deficiencies of the Arabidopsis pec1/abcg32 mutant. RNAi-dependent downregulation of both tomato genes encoding proteins highly homologous to AtABCG32 (SlABCG36 and SlABCG42) leads to reduced cutin deposition and formation of a thinner cuticle in tomato fruits. By using a tobacco (Nicotiana benthamiana) protoplast system, we show that AtABCG32 and SlABCG42 have an export activity for 10,16-dihydroxy hexadecanoyl-2-glycerol, a cutin precursor in vivo. Interestingly, also free ω-hydroxy hexadecanoic acid as well as hexadecanedioic acid were exported, furthering the research on the identification of cutin precursors in vivo and the respective mechanisms of their integration into the cutin polymer.

Early stages of legume-rhizobia symbiosis are controlled by ABCG-mediated transport of active cytokinins.

Growing evidence has highlighted the essential role of plant hormones, notably, cytokinins (CKs), in nitrogen-fixing symbiosis, both at early and late nodulation stages1,2. Despite numerous studies showing the central role of CK in nodulation, the importance of CK transport in the symbiosis is unknown. Here, we show the role of ABCG56, a full-size ATP-binding cassette (ABC) transporter in the early stages of the nodulation. MtABCG56 is expressed in roots and nodules and its messenger RNA levels increase upon treatment with symbiotic bacteria, isolated Nod factor and CKs, accumulating within the epidermis and root cortex. MtABCG56 exports bioactive CKs in an ATP-dependent manner over the plasma membrane and its disruption results in an impairment of nodulation. Our data indicate that ABCG-mediated cytokinin transport is important for proper establishment of N-fixing nodules.