Substrate recognition and transport mechanism of the PIN-FORMED auxin exporters.

Auxins are pivotal plant hormones that regulate plant growth and transmembrane polar auxin transport (PAT) direct patterns of development. The PIN-FORMED (PIN) family of membrane transporters mediate auxin export from the plant cell and play crucial roles in PAT. Here we describe the recently solved structures of PIN transporters, PIN1, PIN3, and PIN8, and also their mechanisms of substrate recognition and transport of auxin. We compare structures of PINs in both inward- and outward-facing conformations, as well as PINs with different binding configurations for auxin. By this comparative analysis, a model emerges for an elevator transport mechanism. Central structural elements necessary for function are identified, and we show that these are shared with other distantly related protein families.

Diverse branching forms regulated by a core auxin transport mechanism in plants.

Diverse branching forms have evolved multiple times across the tree of life to facilitate resource acquisition and exchange with the environment. In the vascular plant group, the ancestral pattern of branching involves dichotomy of a parent shoot apex to form two new daughter apices. The molecular basis of axillary branching in Arabidopsis is well understood, but few regulators of dichotomous branching are known. Through analyses of dichotomous branching in the lycophyte, Selaginella kraussiana, we identify PIN-mediated auxin transport as an ancestral branch regulator of vascular plants. We show that short-range auxin transport out of the apices promotes dichotomy and that branch dominance is globally coordinated by long-range auxin transport. Uniquely in Selaginella, angle meristems initiate at each dichotomy, and these can develop into rhizophores or branching angle shoots. We show that long-range auxin transport and a transitory drop in PIN expression are involved in angle shoot development. We conclude that PIN-mediated auxin transport is an ancestral mechanism for vascular plant branching that was independently recruited into Selaginella angle shoot development and seed plant axillary branching during evolution.

The nitrate transporter NRT2.1 directly antagonizes PIN7-mediated auxin transport for root growth adaptation.

As a crucial nitrogen source, nitrate (NO3-) is a key nutrient for plants. Accordingly, root systems adapt to maximize NO3- availability, a developmental regulation also involving the phytohormone auxin. Nonetheless, the molecular mechanisms underlying this regulation remain poorly understood. Here, we identify low-nitrate-resistant mutant (lonr) in Arabidopsis (Arabidopsis thaliana), whose root growth fails to adapt to low-NO3- conditions. lonr2 is defective in the high-affinity NO3- transporter NRT2.1. lonr2 (nrt2.1) mutants exhibit defects in polar auxin transport, and their low-NO3--induced root phenotype depends on the PIN7 auxin exporter activity. NRT2.1 directly associates with PIN7 and antagonizes PIN7-mediated auxin efflux depending on NO3- levels. These results reveal a mechanism by which NRT2.1 in response to NO3- limitation directly regulates auxin transport activity and, thus, root growth. This adaptive mechanism contributes to the root developmental plasticity to help plants cope with changes in NO3- availability.

Suppression of pinoid mutant phenotypes by mutations in PIN-FORMED 1 and PIN1-GFP fusion.

Disruption of either the auxin transporter PIN-FORMED 1 (PIN1) or the protein kinase PINOID (PID) leads to the development of pin-like inflorescences. Previous studies have shown that phosphoregulation of PIN1 by AGC kinases including PID directs auxin flux to drive organ initiation. Here, we report unexpected findings on the genetic interactions between these two genes. We deleted the first 2/3 of the PIN1 coding sequence using CRISPR/Cas9, and the resulting pin1 mutant (pin1-27) was a strong allele. Surprisingly, heterozygous pin1-27 suppressed two independent pid null mutants, whereas homozygous pin1-27 enhanced the phenotypes of the pid mutants during embryogenesis. Furthermore, we show that deletion of either the hydrophilic loop or the second half of PIN1 also abolished PIN1 function, yet those heterozygous pin1 mutants were also capable of rescuing pid nulls. Moreover, we inserted green fluorescent protein (GFP) into the hydrophilic loop of PIN1 through CRISPR-mediated homology-directed repair (HDR). The GFP signal and pattern in the PIN1-GFPHDR line are similar to those in the previously reported PIN1-GFP transgenic lines. Interestingly, the PIN1-GFPHDR line also rescued various pid null mutant alleles in a semidominant fashion. We conclude that decreasing the number of functional PIN1 copies is sufficient to suppress the pid mutant phenotype, suggesting that PIN1 is likely part of a larger protein complex required for organogenesis.

FAB1C, a phosphatidylinositol 3-phosphate 5-kinase, interacts with PIN-FORMEDs and modulates their lytic trafficking in Arabidopsis.

PIN-FORMEDs (PINs) are auxin efflux carriers that asymmetrically target the plasma membrane (PM) and are critical for forming local auxin gradients and auxin responses. While the cytoplasmic hydrophilic loop domain of PIN (PIN-HL) is known to include some molecular cues (e.g., phosphorylation) for the modulation of PIN's intracellular trafficking and activity, the complexity of auxin responses suggests that additional regulatory modules may operate in the PIN-HL domain. Here, we have identified and characterized a PIN-HL-interacting protein (PIP) called FORMATION OF APLOID AND BINUCLEATE CELL 1C (FAB1C), a phosphatidylinositol-3-phosphate 5-kinase, which modulates PIN's lytic trafficking. FAB1C directly interacts with PIN-HL and is required for the polarity establishment and vacuolar trafficking of PINs. Unphosphorylated forms of PIN2 interact more readily with FAB1C and are more susceptible to vacuolar lytic trafficking compared to phosphorylated forms. FAB1C also affected lateral root formation by modulating the abundance of periclinally localized PIN1 and auxin maximum in the growing lateral root primordium. These findings suggest that a membrane-lipid modifier can target the cargo-including vesicle by directly interacting with the cargo and modulate its trafficking depending on the cargo's phosphorylation status.

Transient efflux inhibition improves plant regeneration by natural auxins.

Plant genome editing and propagation are important tools in crop breeding and production. Both rely heavily on the development of efficient in vitro plant regeneration systems. Two prominent regeneration systems that are widely employed in crop production are somatic embryogenesis (SE) and de novo shoot regeneration. In many of the protocols for SE or shoot regeneration, explants are treated with the synthetic auxin analog 2,4-dichlorophenoxyacetic acid (2,4-D), since natural auxins, such as indole-3-acetic acid (IAA) or 4-chloroindole-3-acetic acid (4-Cl-IAA), are less effective or even fail to induce regeneration. Based on previous reports that 2,4-D, compared to endogenous auxins, is not effectively exported from plant cells, we investigated whether efflux inhibition of endogenous auxins could convert these auxins into efficient inducers of SE in Arabidopsis immature zygotic embryos (IZEs). We show that natural auxins and synthetic analogs thereof become efficient inducers of SE when their efflux is transiently inhibited by co-application of the auxin transport inhibitor naphthylphthalamic acid (NPA). Moreover, IZEs of auxin efflux mutants pin2 or abcb1 abcb19 show enhanced SE efficiency when treated with IAA or efflux-inhibited IAA, confirming that auxin efflux reduces the efficiency of Arabidopsis SE. Importantly, in contrast to the 2,4-D system, where only 50-60% of the embryos converted to seedlings, all SEs induced by transport-inhibited natural auxins converted to seedlings. Efflux-inhibited IAA, like 2,4-D, also efficiently induced SE from carrot suspension cells, whereas IAA alone could not, and efflux-inhibited 4-Cl-IAA significantly improved de novo shoot regeneration in Brassica napus. Our data provides new insights into the action of 2,4-D as an efficient inducer of plant regeneration but also shows that replacing this synthetic auxin for efflux-inhibited natural auxin significantly improves different types of plant regeneration, leading to a more synchronized and homogenous development of the regenerated plants.

Ocimum kilimandscharicum 4CL11 negatively regulates adventitious root development via accumulation of flavonoid glycosides.

4-Coumarate-CoA Ligase (4CL) is an important enzyme in the phenylpropanoid biosynthesis pathway. Multiple 4CLs are identified in Ocimum species; however, their in planta functions remain enigmatic. In this study, we independently overexpressed three Ok4CL isoforms from Ocimum kilimandscharicum (Ok4CL7, -11, and -15) in Nicotiana benthamiana. Interestingly, Ok4CL11 overexpression (OE) caused a rootless or reduced root growth phenotype, whereas overexpression of Ok4CL15 produced normal adventitious root (AR) growth. Ok4CL11 overexpression in N. benthamiana resulted in upregulation of genes involved in flavonoid biosynthesis and associated glycosyltransferases accompanied by accumulation of specific flavonoid-glycosides (kaempferol-3-rhamnoside, kaempferol-3,7-O-bis-alpha-l-rhamnoside [K3,7R], and quercetin-3-O-rutinoside) that possibly reduced auxin levels in plants, and such effects were not seen for Ok4CL7 and -15. Docking analysis suggested that auxin transporters (PINs/LAXs) have higher binding affinity to these specific flavonoid-glycosides, and thus could disrupt auxin transport/signaling, which cumulatively resulted in a rootless phenotype. Reduced auxin levels, increased K3,7R in the middle and basal stem sections, and grafting experiments (intra and inter-species) indicated a disruption of auxin transport by K3,7R and its negative effect on AR development. Supplementation of flavonoids and the specific glycosides accumulated by Ok4CL11-OE to the wild-type N. benthamiana explants delayed the AR emergence and also inhibited AR growth. While overexpression of all three Ok4CLs increased lignin accumulation, flavonoids, and their specific glycosides were accumulated only in Ok4CL11-OE lines. In summary, our study reveals unique indirect function of Ok4CL11 to increase specific flavonoids and their glycosides, which are negative regulators of root growth, likely involved in inhibition of auxin transport and signaling.

Modulation of plant root growth by nitrogen source-defined regulation of polar auxin transport.

Availability of the essential macronutrient nitrogen in soil plays a critical role in plant growth, development, and impacts agricultural productivity. Plants have evolved different strategies for sensing and responding to heterogeneous nitrogen distribution. Modulation of root system architecture, including primary root growth and branching, is among the most essential plant adaptions to ensure adequate nitrogen acquisition. However, the immediate molecular pathways coordinating the adjustment of root growth in response to distinct nitrogen sources, such as nitrate or ammonium, are poorly understood. Here, we show that growth as manifested by cell division and elongation is synchronized by coordinated auxin flux between two adjacent outer tissue layers of the root. This coordination is achieved by nitrate-dependent dephosphorylation of the PIN2 auxin efflux carrier at a previously uncharacterized phosphorylation site, leading to subsequent PIN2 lateralization and thereby regulating auxin flow between adjacent tissues. A dynamic computer model based on our experimental data successfully recapitulates experimental observations. Our study provides mechanistic insights broadening our understanding of root growth mechanisms in dynamic environments.

High temperature perception in leaves promotes vascular regeneration and graft formation in distant tissues.

Cellular regeneration in response to wounding is fundamental to maintain tissue integrity. Various internal factors including hormones and transcription factors mediate healing, but little is known about the role of external factors. To understand how the environment affects regeneration, we investigated the effects of temperature upon the horticulturally relevant process of plant grafting. We found that elevated temperatures accelerated vascular regeneration in Arabidopsis thaliana and tomato grafts. Leaves were crucial for this effect, as blocking auxin transport or mutating PHYTOCHROME INTERACTING FACTOR 4 (PIF4) or YUCCA2/5/8/9 in the cotyledons abolished the temperature enhancement. However, these perturbations did not affect grafting at ambient temperatures, and temperature enhancement of callus formation and tissue adhesion did not require PIF4, suggesting leaf-derived auxin specifically enhanced vascular regeneration in response to elevated temperatures. We also found that elevated temperatures accelerated the formation of inter-plant vascular connections between the parasitic plant Phtheirospermum japonicum and host Arabidopsis, and this effect required shoot-derived auxin from the parasite. Taken together, our results identify a pathway whereby local temperature perception mediates long distance auxin signaling to modify regeneration, grafting and parasitism. This article has an associated 'The people behind the papers' interview.

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.

Polar auxin transport modulates early leaf flattening.

The flattened leaf form is an important adaptation for efficient photosynthesis, and the developmental process of flattened leaves has been intensively studied. Classic microsurgery studies in potato and tomato suggest that the shoot apical meristem (SAM) communicates with the leaf primordia to promote leaf blade formation. More recently, it was found that polar auxin transport (PAT) could mediate this communication. However, it is unclear how the expression of leaf patterning genes is tailored by PAT routes originating from SAM. By combining experimental observations and computer model simulations, we show that microsurgical incisions and local inhibition of PAT in tomato interfere with auxin transport toward the leaf margins, reducing auxin response levels and altering the leaf blade shape. Importantly, oval auxin responses result in the bipolar expression of SlLAM1 that determines leaf blade formation. Furthermore, wounding caused by incisions promotes degradation of SlREV, a known regulator of leaf polarity. Additionally, computer simulations suggest that local auxin biosynthesis in early leaf primordia could remove necessity for external auxin supply originating from SAM, potentially explaining differences between species. Together, our findings establish how PAT near emerging leaf primordia determines spatial auxin patterning and refines SlLAM1 expression in the leaf margins to guide leaf flattening.

Biosynthesis- and transport-mediated dynamic auxin distribution during seed development controls seed size in Arabidopsis.

Auxin is indispensable to the fertilization-induced coordinated development of the embryo, endosperm, and seed coat. However, little attention has been given to the distribution pattern, maintenance mechanism, and function of auxin throughout the process of seed development. In the present study, we found that auxin response signals display a dynamic distribution pattern during Arabidopsis seed development. Shortly after fertilization, strong auxin response signals were observed at the funiculus, chalaza, and micropylar integument where the embryo attaches. Later, additional signals appeared at the middle layer of the inner integument (ii1') above the chalaza and the whole inner layer of the outer integument (oi1). These signals peaked when the seed was mature, then declined upon desiccation and disappeared in the dried seed. Auxin biosynthesis genes, including ASB1, TAA1, YUC1, YUC4, YUC8, and YUC9, contributed to the accumulation of auxin in the funiculus and seed coat. Auxin efflux carrier PIN3 and influx carrier AUX1 also contributed to the polar auxin distribution in the seed coat. PIN3 was expressed in the ii1 (innermost layer of the inner integument) and oi1 layers of the integument and showed polar localization. AUX1 was expressed in both layers of the outer integument and the endosperm and displayed a uniform localization. Further research demonstrated that the accumulation of auxin in the seed coat regulates seed size. Transgenic plants that specifically express the YUC8 gene in the oi2 or ii1 seed coat produced larger seeds. These results provide useful tools for cultivating high-yielding crops.

Boron supply restores aluminum-blocked auxin transport by the modulation of PIN2 trafficking in the root apical transition zone.

The supply of boron (B) alleviates the toxic effects of aluminum (Al) on root growth; however, the mechanistic basis of this process remains elusive. This study filled this knowledge gap, demonstrating that boron modifies auxin distribution and transport in Al-exposed Arabidopsis roots. In B-deprived roots, treatment with Al induced an increase in auxin content in the root apical meristem zone (MZ) and transition zone (TZ), whereas in the elongation zone (EZ) the auxin content was decreased beyond the level required for adequate growth. These distribution patterns are explained by the fact that basipetal auxin transport from the TZ to the EZ was disrupted by Al-inhibited PIN-FORMED 2 (PIN2) endocytosis. Experiments involving the modulation of protein biosynthesis by cycloheximide (CHX) and transcriptional regulation by cordycepin (COR) demonstrated that the Al-induced increase of PIN2 membrane proteins was dependent upon the inhibition of PIN2 endocytosis, rather than on the transcriptional regulation of the PIN2 gene. Experiments reporting on the profiling of Al3+ and PIN2 proteins revealed that the inhibition of endocytosis of PIN2 proteins was the result of Al-induced limitation of the fluidity of the plasma membrane. The supply of B mediated the turnover of PIN2 endosomes conjugated with indole-3-acetic acid (IAA), and thus restored the Al-induced inhibition of IAA transport through the TZ to the EZ. Overall, the reported results demonstrate that boron supply mediates PIN2 endosome-based auxin transport to alleviate Al toxicity in plant roots.

Endogenous auxin maintains embryonic cell identity and promotes somatic embryo development in Arabidopsis.

Somatic embryogenesis (SE), or embryo development from in vitro cultured vegetative explants, can be induced in Arabidopsis by the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) or by overexpression of specific transcription factors, such as AT-HOOK MOTIF NUCLEAR LOCALIZED 15 (AHL15). Here, we explored the role of endogenous auxin [indole-3-acetic acid (IAA)] during 2,4-D and AHL15-induced SE. Using the pWOX2:NLS-YFP reporter, we identified three distinct developmental stages for 2,4-D and AHL15-induced SE in Arabidopsis, with these being (i) acquisition of embryo identity; (ii) formation of pro-embryos; and (iii) somatic embryo patterning and development. The acquisition of embryo identity coincided with enhanced expression of the indole-3-pyruvic acid auxin biosynthesis YUCCA genes, resulting in an enhanced pDR5:GFP-reported auxin response in the embryo-forming tissues. Chemical inhibition of the indole-3-pyruvic acid pathway did not affect the acquisition of embryo identity, but significantly reduced or completely inhibited the formation of pro-embryos. Co-application of IAA with auxin biosynthesis inhibitors in the AHL15-induced SE system rescued differentiated somatic embryo formation, confirming that increased IAA levels are important during the last two stages of SE. Our analyses also showed that polar auxin transport, with AUXIN/LIKE-AUX influx and PIN-FORMED1 efflux carriers as important drivers, is required for the transition of embryonic cells to proembryos and, later, for correct cell fate specification and differentiation. Taken together, our results indicate that endogenous IAA biosynthesis and its polar transport are not required for the acquisition of embryo identity, but rather to maintain embryonic cell identity and for the formation of multicellular proembryos and their development into histodifferentiated embryos.

Alternate bearing in 'Hass' avocado - Fruit load-induced changes in bud auxin homeostasis are associated with flowering repression.

In 'Hass' avocado (Persea americana), fruit presence reduces next season flowering. Recent fruit tree studies proposed that heavy fruit load (HFL) generates an auxin (IAA) signal in the buds, which represses flowering. However, the nature of this signal remains unknown. Here, we investigated the effect of avocado HFL on bud IAA accumulation and flowering transition. We found that IAA-aspartate and IAA-glutamate conjugate levels were significantly higher in buds from 'on' (fully loaded) than 'off' (low-loaded) trees, hinting that free IAA levels were higher in the former. Expression analysis showed that coinciding with flowering reduction, HFL induced the floral repressor PaTFL1, and suggested that accumulation of IAA in buds as imposed by HFL was associated with its conjugation to aspartate and glutamate and resulted both from de novo IAA synthesis, as well as from reduced IAA export. Accordingly, experiments involving radiolabelled 14C-IAA demonstrated that HFL reduced shoot basipetal IAA transport. Lastly, we confirmed the negative effects of IAA on flowering, showing that IAA and PAT blocker (TIBA) treatments delayed 'off' trees inflorescence development, reducing their inflorescence axis and inducing PaTFL1 transcript. Together, our data suggest that avocado HFL generates IAA signalling in buds that induces PaTFL1, which represses inflorescence development.

Naphthylphthalamic acid associates with and inhibits PIN auxin transporters.

N-1-naphthylphthalamic acid (NPA) is a key inhibitor of directional (polar) transport of the hormone auxin in plants. For decades, it has been a pivotal tool in elucidating the unique polar auxin transport-based processes underlying plant growth and development. Its exact mode of action has long been sought after and is still being debated, with prevailing mechanistic schemes describing only indirect connections between NPA and the main transporters responsible for directional transport, namely PIN auxin exporters. Here we present data supporting a model in which NPA associates with PINs in a more direct manner than hitherto postulated. We show that NPA inhibits PIN activity in a heterologous oocyte system and that expression of NPA-sensitive PINs in plant, yeast, and oocyte membranes leads to specific saturable NPA binding. We thus propose that PINs are a bona fide NPA target. This offers a straightforward molecular basis for NPA inhibition of PIN-dependent auxin transport and a logical parsimonious explanation for the known physiological effects of NPA on plant growth, as well as an alternative hypothesis to interpret past and future results. We also introduce PIN dimerization and describe an effect of NPA on this, suggesting that NPA binding could be exploited to gain insights into structural aspects of PINs related to their transport mechanism.

OsPDCD5 negatively regulates plant architecture and grain yield in rice.

Plant architecture is an important agronomic trait that affects crop yield. Here, we report that a gene involved in programmed cell death, OsPDCD5, negatively regulates plant architecture and grain yield in rice. We used the CRISPR/Cas9 system to introduce loss-of-function mutations into OsPDCD5 in 11 rice cultivars. Targeted mutagenesis of OsPDCD5 enhanced grain yield and improved plant architecture by increasing plant height and optimizing panicle type and grain shape. Transcriptome analysis showed that OsPDCD5 knockout affected auxin biosynthesis, as well as the gibberellin and cytokinin biosynthesis and signaling pathways. OsPDCD5 interacted directly with OsAGAP, and OsAGAP positively regulated plant architecture and grain yield in rice. Collectively, these findings demonstrate that OsPDCD5 is a promising candidate gene for breeding super rice cultivars with increased yield potential and superior quality.

OsSPL14 acts upstream of OsPIN1b and PILS6b to modulate axillary bud outgrowth by fine-tuning auxin transport in rice.

As a multigenic trait, rice tillering can optimize plant architecture for the maximum agronomic yield. SQUAMOSA PROMOTER BINDING PROTEIN-LIKE14 (OsSPL14) has been demonstrated to be necessary and sufficient to inhibit rice branching, but the underlying mechanism remains largely unclear. Here, we demonstrated that OsSPL14, which is cleaved by miR529 and miR156, inhibits tillering by fine-tuning auxin transport in rice. RNA interference of OsSPL14 or miR529 and miR156 overexpression significantly increased the tiller number, whereas OsSPL14 overexpression decreased the tiller number. Histological analysis revealed that the OsSPL14-overexpressing line had normal initiation of axillary buds but inhibited outgrowth of tillers. Moreover, OsSPL14 was found to be responsive to indole-acetic acid and 1-naphthylphthalamic acid, and RNA interference of OsSPL14 reduced polar auxin transport and increased 1-naphthylphthalamic acid sensitivity of rice plants. Further analysis revealed that OsSPL14 directly binds to the promoter of PIN-FORMED 1b (OsPIN1b) and PIN-LIKE6b (PILS6b) to regulate their expression positively. OsPIN1b and PILS6b were highly expressed in axillary buds and proved involved in bud outgrowth. Loss of function of OsPIN1b or PILS6b increased the tiller number of rice. Taken together, our findings suggested that OsSPL14 could control axillary bud outgrowth and tiller number by activating the expression of OsPIN1b and PILS6b to fine-tune auxin transport in rice.

Mapping of the Classical Mutation rosette Highlights a Role for Calcium in Wound-Induced Rooting.

Removal of the root system induces the formation of new roots from the remaining shoot. This process is primarily controlled by the phytohormone auxin, which interacts with other signals in a yet unresolved manner. Here, we study the classical tomato mutation rosette (ro), which lacks shoot-borne roots. ro mutants were severely inhibited in formation of wound-induced roots (WiRs) and had reduced auxin transport rates. We mapped ro to the tomato ortholog of the Arabidopsis thaliana BIG and the mammalians UBR4/p600. RO/BIG is a large protein of unknown biochemical function. In A. thaliana, BIG was implicated in regulating auxin transport and calcium homeostasis. We show that exogenous calcium inhibits WiR formation in tomato and A. thaliana ro/big mutants. Exogenous calcium antagonized the root-promoting effects of the auxin indole-3-acetic-acid but not of 2,4-dichlorophenoxyacetic acid, an auxin analog that is not recognized by the polar transport machinery, and accumulation of the auxin transporter PIN-FORMED1 (PIN1) was sensitive to calcium levels in the ro/big mutants. Consistent with a role for calcium in mediating auxin transport, both ro/big mutants and calcium-treated wild-type plants were hypersensitive to treatment with polar auxin transport inhibitors. Subcellular localization of BIG suggests that, like its mammalian ortholog, it is associated with the endoplasmic reticulum. Analysis of subcellular morphology revealed that ro/big mutants exhibited disruption in cytoplasmic streaming. We suggest that RO/BIG maintains auxin flow by stabilizing PIN membrane localization, possibly by attenuating the inhibitory effect of Ca2+ on cytoplasmic streaming.

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.