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The nematode species Meloidogyne incognita infects a large variety of cultivated crops and is one of nature’s most notorious pests. One cultivated plant which is prone to M. incognita infestation is the tomato. Knowing that in A. thaliana the PIN auxin efflux transporters distribution is being altered upon early invasion by M. incognita, the PIN1 allocation patterns in the giant cells of tomato plants after 15 and 25 days of infection were investigated. PIN1 was absent from the giant cells’ membrane in both assessment timings examined, indicating the maintenance of a local auxin maxima, which was also supported by IAA immunodetection. PIN1 distribution pattern could be attributed on either the nematodes nutritional needs throughout its life cycle or due to the tomato plants differential responses upon M. incognita infection.

Numerous Bacillus spp. in soils show strong plant growth-promotion effects, but the molecular mechanism of most of these bacteria interacting with plants remains unknown. Uncovering the interaction pattern is of considerable importance for potential applications in agriculture. In the present study, we isolated the bacterium Bacillus megaterium strain WW1211 from the rhizosphere of Arabidopsis and investigated the mechanism of WW1211 on plant growth promotion at the cellular and molecular levels. Our results showed that inoculation of WW1211 substantially promoted shoot biomass and lateral root initiation but inhibited primary root growth. The promotion effect of WW1211 was counteracted by the addition of auxin biosynthesis or transport inhibitors, indicating an auxin-dependent pattern. The expression of auxin response and biosynthesis genes in Arabidopsis seedlings and the concentration of indole-3-acetic acid in Arabidopsis roots were enhanced by WW1211 inoculation. Furthermore, the meristematic zone length and the cell division in primary roots were inhibited by WW1211 inoculation. Auxin was enriched in the quiescent center, and the expression of auxin efflux and influx genes in the root tips was suppressed, indicating that WW1211 inhibited the growth of primary roots by modification of the auxin cycle and redistribution in the root meristem. Our results suggest that B. megaterium strain WW1211 induces auxin biosynthesis and simultaneously regulates auxin redistribution in plant shoots and roots, thus stimulating plant growth and lateral root initiation.

Polarity is a feature of life. In higher plants, non-autonomous polarity is largely directed by auxin, the morphogen that drives its own polarized flow, Polar Auxin Transport (PAT), to guide patterning events such as phyllotaxis and tropism. The plasma membrane-localized PIN-FORMED (PIN) auxin efflux carriers are rate-limiting factors in PAT. In yeasts and metazoans, the STE20 kinases are key players in cell polarity. We had previously characterized SIK1 as a STE20/Hippo orthologue in Arabidopsis and confirmed its function in mitotic exit and organ growth. Here we explore the possible link between SIK1, auxin, PIN, and polarity. Abnormal phyllotaxis and gravitropism were observed in sik1. sik1 was more sensitive to exogenous auxin in primary root elongation and lateral root emergence. RNA-Seq revealed reduced expression in auxin biosynthesis genes and induced expression of auxin flux carriers in sik1. However, normal tissue- and sub-cellular localization patterns of PIN1 and PIN2 were observed in sik1. The dark-induced vacuolar degradation of PIN2 also appeared normal in sik1. An additive phenotype was observed in the sik1 pin1 double mutant, indicating that SIK1 does not directly regulate PIN1. The polarity defects of sik1 are hence unlikely mediated by PINs and await future exploration.

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., N‐ethylmaleimide‐sensitive factor (NSF) promotes the fusion of secretory vesicles in eukaryotes. In Arabidopsis, AtNSF negatively regulates leaf serration by affecting trafficking of the PIN‐FORMED1 auxin transporter and interfering with the PIN‐FORMED1 and CUP‐SHAPED COTYLEDON2‐mediated feedback loop for leaf serration.

The unique apical hook in dicotyledonous plants protects the shoot apical meristem and cotyledons when seedlings emerge through the soil. Its formation involves differential cell growth under the coordinated control of plant hormones, especially ethylene and auxin. Microtubules are essential players in plant cell growth that are regulated by multiple microtubule-associated proteins (MAPs). However, the role and underlying mechanisms of MAP-microtubule modules in differential cell growth are poorly understood. In this study, we found that the previously uncharacterized Arabidopsis MAP WAVE-DAMPENED2-LIKE4 (WDL4) protein plays a positive role in apical hook opening. WDL4 exhibits a temporal expression pattern during hook development in dark-grown seedlings that is directly regulated by ethylene signaling. WDL4 mutants showed a delayed hook opening phenotype while overexpression of WDL4 resulted in enhanced hook opening. In particular, wdl4-1 mutants exhibited stronger auxin accumulation in the concave side of the apical hook. Furthermore, the regulation of the auxin maxima and trafficking of the auxin efflux carriers PIN-FORMED1 (PIN1) and PIN7 in the hook region is critical for WDL4-mediated hook opening. Together, our study demonstrates that WDL4 positively regulates apical hook opening by modulating auxin distribution, thus unraveling a mechanism for MAP-mediated differential plant cell growth.

The degree of rice tillering is an important agronomic trait that can be markedly affected by nitrogen supply. However, less is known about how nitrogen-regulated rice tillering is related to polar auxin transport. Compared with nitrate, ammonium induced tiller development and was paralleled with increased 3 H-indole-acetic acid (IAA) transport and greater auxin into the junctions. OsPIN9, an auxin efflux carrier, was selected as the candidate gene involved in ammonium-regulated tillering based on GeneChip data. Compared with wild-type plants, ospin9 mutants had fewer tillers, and OsPIN9 overexpression increased the tiller number. Additionally, OsPIN9 was mainly expressed in vascular tissue of the junction and tiller buds, and encoded a membrane-localised protein. Heterologous expression in Xenopus oocytes and yeast demonstrated that OsPIN9 is a functional auxin efflux transporter. More importantly, its RNA and protein levels were induced by ammonium but not by nitrate, and tiller numbers in mutants did not respond to nitrogen forms. Further advantages, including increased tiller number and grain yield, were observed in overexpression lines grown in the paddy field at a low-nitrogen rate compared with at a high-nitrogen rate. Our data revealed that ammonium supply and an auxin efflux transporter co-ordinately control tiller bud elongation in rice.

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.

Auxin (indole-3-acetic acid, IAA) has been implicated as a susceptibility factor in both beneficial and pathogenic molecular plant−microbe interactions. Previous studies have identified a large number of auxin-related genes underlying quantitative disease resistance loci (QDRLs) for Phytophthora sojae. Thus, we hypothesized that auxin may be involved the P. sojae−soybean interaction. The levels of IAA and related metabolites were measured in mycelia and media supernatant as well as in mock and inoculated soybean roots in a time course assay. The expression of 11 soybean Pin-formed (GmPIN) auxin efflux transporter genes was also examined. Tryptophan, an auxin precursor, was detected in the P. sojae mycelia and media supernatant. During colonization of roots, levels of IAA and related metabolites were significantly higher in both moderately resistant Conrad and moderately susceptible Sloan inoculated roots compared with mock controls at 48 h postinoculation (hpi) in one experiment and at 72 hpi in a second, with Sloan accumulating higher levels of the auxin catabolite IAA-Ala than Conrad. Additionally, one GmPIN at 24 hpi, one at 48 hpi, and three at 72 hpi had higher expression in inoculated compared with the mock control roots in Conrad. The ability of resistant cultivars to cope with auxin accumulation may play an important role in quantitative disease resistance. Levels of jasmonic acid (JA), another plant hormone associated with defense responses, were also higher in inoculated roots at these same time points, suggesting that JA also plays a role during the later stages of infection.

Defects in Rab prenylation in the rgtb1 mutant influence the transport of auxin from ovules to the maternal plant and lead to developmental arrest of the female gametophyte in Arabidopsis., Eukaryotic cells rely on the accuracy and efficiency of vesicular traffic. In plants, disturbances in vesicular trafficking are well studied in quickly dividing root meristem cells or polar growing root hairs and pollen tubes. The development of the female gametophyte, a unique haploid reproductive structure located in the ovule, has received far less attention in studies of vesicular transport. Key molecules providing the specificity of vesicle formation and its subsequent recognition and fusion with the acceptor membrane are Rab proteins. Rabs are anchored to membranes by covalently linked geranylgeranyl group(s) that are added by the Rab geranylgeranyl transferase (RGT) enzyme. Here we show that Arabidopsis plants carrying mutations in the gene encoding the β-subunit of RGT (rgtb1) exhibit severely disrupted female gametogenesis and this effect is of sporophytic origin. Mutations in rgtb1 lead to internalization of the PIN1 and PIN3 proteins from the basal membranes to vesicles in provascular cells of the funiculus. Decreased transport of auxin out of the ovule is accompanied by auxin accumulation in tissue surrounding the growing gametophyte. In addition, female gametophyte development arrests at the uni- or binuclear stage in a significant portion of the rgtb1 ovules. These observations suggest that communication between the sporophyte and the developing female gametophyte relies on Rab-dependent vesicular traffic of the PIN1 and PIN3 transporters and auxin efflux out of the ovule.

Summary To adapt to the diverse array of biotic and abiotic cues, plants have evolved sophisticated mechanisms to sense changes in environmental conditions and modulate their growth. Growth‐promoting hormones and defence signalling fine tune plant development antagonistically. During host–pathogen interactions, this defence–growth trade‐off is mediated by the counteractive effects of the defence hormone salicylic acid (SA) and the growth hormone auxin.Here we revealed an underlying mechanism of SA regulating auxin signalling by constraining the plasma membrane dynamics of PIN2 auxin efflux transporter in Arabidopsis thaliana roots.The lateral diffusion of PIN2 proteins is constrained by SA signalling, during which PIN2 proteins are condensed into hyperclusters depending on REM1.2‐mediated nanodomain compartmentalisation. Furthermore, membrane nanodomain compartmentalisation by SA or Remorin (REM) assembly significantly suppressed clathrin‐mediated endocytosis. Consequently, SA‐induced heterogeneous surface condensation disrupted asymmetric auxin distribution and the resultant gravitropic response.Our results demonstrated a defence–growth trade‐off mechanism by which SA signalling crosstalked with auxin transport by concentrating membrane‐resident PIN2 into heterogeneous compartments.

The basic region/leucine zipper (bZIP) transcription factors play key roles in regulating diverse biological processes in plants. However, their participation in shoot branching has been rarely reported. Here, we isolated a CmbZIP1 transcription factor gene, a member of the bZIP family, from chrysanthemum. Subcellular localization analysis indicated that CmbZIP1 is a nuclear protein. Tissue-specific expression analysis indicated that CmbZIP1 was principally expressed in apical bud and axillary bud. Expression patterns analysis results showed that CmbZIP1 expression was suppressed in axillary buds in response to decapitation but increased in response to shade. Overexpression of CmbZIP1 in Arabidopsis inhibits its shoot branching. In addition, expression of auxin efflux protein PIN-FORMED 1 (PIN1) and auxin signaling components AUXIN RESISTANT 1/3 (AXR1, AXR3) were significantly up-regulated in overexpressing plants in comparison with wild type plants. Moreover, the transcript expression of BRANCHED 2 (AtBRC2) was also significantly up-regulated in overexpressing plants compared with the wild type. Altogether, these results suggest important and negative roles of CmbZIP1 in shoot branching. Our study extends the understanding of the function of bZIP transcription factors in plants and provides valuable gene resources for improving the architectural traits of ornamental plants.

Plants depend on gravity to provide the constant landmark for downward root growth and upward shoot growth. The phytohormone auxin and its cell‐to‐cell transport machinery are central determinants ensuring gravitropic growth. Statolith sedimentation toward gravity is sensed in specialized cells. This positional cue is translated into the polar distribution of PIN auxin efflux carriers at the plasma membrane, leading to asymmetric auxin distribution and consequently, differential growth and organ bending. While we have started to understand the general principles of how primary organs execute gravitropism, we currently lack basic understanding of how lateral plant organs can defy gravitropic responses. Here we briefly review the establishment of the oblique gravitropic set point angle in lateral roots and particularly discuss the emerging role of asymmetric cytokinin signaling as a central anti‐gravitropic signal. Differential cytokinin signaling is co‐opted in gravitropic lateral and hydrotropic primary roots to counterbalance gravitropic root growth.

Glucosinolates (GSLs) are sulfur-containing defense metabolites produced in the Brassicales, including the model plant Arabidopsis (Arabidopsis thaliana). Previous work suggests that specific GSLs may function as signals to provide direct feedback regulation within the plant to calibrate defense and growth. These GSLs include allyl-GSL, a defense metabolite that is one of the most widespread GSLs in Brassicaceae and has also been associated with growth inhibition. Here we show that at least three separate potential catabolic products of allyl-GSL or closely related compounds affect growth and development by altering different mechanisms influencing plant development. Two of the catabolites, raphanusamic acid and 3-butenoic acid, differentially affect processes downstream of the auxin signaling cascade. Another catabolite, acrylic acid, affects meristem development by influencing the progression of the cell cycle. These independent signaling events propagated by the different catabolites enable the plant to execute a specific response that is optimal to any given environment.Allyl-glucosinolate and its catabolites use multiple mechanisms to affect plant growth and development through specific responses that are optimal to any given environment.

Polar auxin transport through plant tissue strictly requires polarly localized PIN proteins and uniformly distributed ABCB proteins. A functional synergy between the two types of membrane protein where their localizations overlap may create the degree of asymmetric auxin efflux required to produce polar auxin transport. We investigated this possibility by expressing ABCB4 and PIN2 in human embryonic kidney cells and measuring whole‐cell ionic currents with the patch‐clamp technique and CsCl‐based electrolytes. ABCB4 activity was 1.81‐fold more selective for Cl− over Cs+ and for PIN2 the value was 2.95. We imposed auxin gradients and determined that ABCB4 and PIN2 were 12‐fold more permeable to the auxin anion (IAA−) than Cl−. This measure of the intrinsic selectivity of the transport pathway was 21‐fold when ABCB4 and PIN2 were co‐expressed. If this increase occurs in plants, it could explain why asymmetric PIN localization is not sufficient to create polar auxin flow. Some form of co‐action or synergy between ABCB4 and PIN2 that increases IAA− selectivity at the cell face where both occur may be important. We also found that auxin stimulated ABCB4 activity, which may contribute to a self‐reinforcement of auxin transport known as canalization.

Functionally divergent paralogs of homomeric proteins do not form potentially deleterious heteromers, which requires distinction between self and non-self (Hochberg et al., 2018; Marchant et al, 2019; Marsh and Teichmann, 2015). In Arabidopsis, two ARF guanine-nucleotide exchange factors (ARF-GEFs) related to mammalian GBF1, named GNOM and GNL1, can mediate coatomer complex (COPI)-coated vesicle formation in retrograde Golgi-endoplasmic reticulum (ER) traffic (Geldner et al., 2003; Richter et al., 2007; Teh and Moore, 2007). Unlike GNL1, however, GNOM is also required for polar recycling of endocytosed auxin efflux regulator PIN1 from endosomes to the plasma membrane. Here we show that these paralogues form homodimers constitutively but no heterodimers. We also address why and how GNOM and GNL1 might be kept separate. These paralogues share a common domain organisation and each N-terminal dimerisation (DCB) domain can interact with the complementary fragment (ΔDCB) of its own and the other protein. However, unlike self-interacting DCBGNOM (Grebe et al., 2000; Anders et al., 2008), DCBGNL1 did not interact with itself nor DCBGNOM. DCBGNOM removal or replacement with DCBGNL1, but not disruption of cysteine bridges that stabilise DCB-DCB interaction, resulted in GNOM-GNL1 heterodimers which impaired developmental processes such as lateral root formation. We propose precocious self-interaction of the DCBGNOM domain as a mechanism to preclude formation of fitness-reducing GNOM-GNL1 heterodimers.

Auxin plays a central role in control of organ abscission, and it is thought that changes in the auxin gradient across the abscission zone are the primary determinant of the onset of abscission. The nature of this gradient, whether in concentration, flow, or perhaps in the response system has not conclusively been determined. We employed a DR5::GUS auxin response reporter system to examine the temporal and spatial distribution of the auxin response activity in response to developmental and environmental cues during pedicel abscission in tomato. In pedicels of young and fully open flowers, auxin response, as indicated by GUS activity, was predominantly detected in the vascular tissues and was almost entirely confined to the abscission zone (AZ) and to the distal portion of the pedicel, with a striking reduction in the proximal tissues below the AZ—a ‘step’, rather than a gradient. Following pollination and during early fruit development, auxin response increased substantially throughout the pedicel. Changes in GUS activity following treatments that caused pedicel abscission (flower removal, high temperature, darkness, ethylene, or N-1-naphthylphthalamic acid (NPA) treatment) were relatively minor, with reduced auxin response in the AZ and some reduction above and below it. Expression of genes encoding some auxin efflux carriers (PIN) and influx carriers (AUX⁄LAX) was substantially reduced in the abscission zone of NPA-treated pedicels, and in pedicels stimulated to abscise by flower removal. Our results suggest that changes in auxin flow distribution through the abscission zone are likely more important than the auxin response system in the regulation of abscission.

Somatic embryogenesis (SE) is the process by which embryos develop from in vitro cultured vegetative tissue explants. The synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) is widely used for SE induction, but SE can also be induced by overexpression of specific transcription factors, such as AT-HOOK MOTIF NUCLEAR LOCALIZED 15 (AHL15). 2,4-D and AHL15 both trigger the biosynthesis of the natural auxin indole-3-acetic acid (IAA). However, the role of this endogenously produced auxin in SE is yet not well understood. In this study we show that the induction of embryonic stem cells from explants does not require IAA biosynthesis, whereas an increase in IAA levels is essential to maintain embryo identity and for embryo formation from these stem cells. Further analysis showed that YUCCA (YUC) genes involved in the IPyA auxin biosynthesis pathway are up-regulated in embryo-forming tissues. Chemical inhibition of the IPyA pathway significantly reduced or completely inhibited the formation of somatic embryos in both 2,4-D-and AHL15-dependent systems. In the latter system, SE could be restored by exogenous IAA application, confirming that the biosynthesis-mediated increase in IAA levels is important. Our analyses also showed that PIN1 and AUX1 are the major auxin carriers that determine respectively auxin efflux and influx during SE. This auxin transport machinery is required for the proper transition of embryonic cells to proembryos and, later, for correct cell fate specification and differentiation. Taken together, our results indicate that auxin biosynthesis in conjunction with its polar transport are required during SE for multicellular somatic proembryo development and differentiation.One sentence summarySomatic embryogenesis in Arabidopsis requires auxin biosynthesis and polar auxin transport only after the acquisition of embryonic competence for somatic proembryo development and differentiation.

To date, synthetic herbicides are the main tools used for weed control, with consequent damage to both the environment and human health. In this respect, searching for new natural molecules and understanding their mode of action could represent an alternative strategy or support to traditional management methods for sustainable agriculture. Protodioscin is a natural molecule belonging to the class of steroid saponins, mainly produced by monocotyledons. In the present paper, protodioscin’s phytotoxic potential was assessed to identify its target and the potential mode of action in the model plant Arabidopsis thaliana. The results highlighted that the root system was the main target of protodioscin, which caused a high inhibitory effect on the primary root length (ED50 50 μM) with morphological alteration, accompanied by a significant increase in the lateral root number and root hair density. Through a pharmacological and microscopic approach, it was underlined that this saponin modified both auxin distribution and transport, causing an auxin accumulation in the region of root maturation and an alteration of proteins responsible for the auxin efflux (PIN2). In conclusion, the saponin protodioscin can modulate the root system of A. thaliana by interfering with the auxin transport (PAT).

With the introduction of the new auxinic herbicide halauxifen-methyl into the oilseed rape (Brassica napus) market, there is a need to understand how this new molecule interacts with indigenous plant hormones (e.g., IAA) in terms of crop response. The aim of this study was to investigate the molecular background by using different growth conditions under which three different auxinic herbicides were administered. These were halauxifen-methyl (Hal), alone and together with aminopyralid (AP) as well as picloram (Pic). Three different hormone classes were determined, free and conjugated indole-3-acetic acid (IAA), aminocyclopropane carboxylic acid (ACC) as a precursor for ethylene, and abscisic acid (ABA) at two different temperatures and growth stages as well as over time (2–168 h after treatment). At 15 °C growth temperature, the effect was more pronounced than at 9 °C, and generally, the younger leaves independent of the developmental stage showed a larger effect on the alterations of hormones. IAA and ACC showed reproducible alterations after auxinic herbicide treatments over time, while ABA did not. Finally, a transcriptome analysis after treatment with two auxinic herbicides, Hal and Pic, showed different expression patterns. Hal treatment leads to the upregulation of auxin and hormone responses at 48 h and 96 h. Pic treatment induced the hormone/auxin response already after 2 h, and this continued for the other time points. The more detailed analysis of the auxin response in the datasets indicate a role for GH3 genes and genes encoding auxin efflux proteins. The upregulation of the GH3 genes correlates with the increase in conjugated IAA at the same time points and treatments. Also, genes for were found that confirm the upregulation of the ethylene pathway.

Novel molecular pinball machines of the plasma membrane control cytosolic Ca2+ levels that regulate plant metabolism. [https://youtu.be/zABg7LiBk88] Essential components involve: 1. an auxin-activated proton pump; 2. arabinogalactan glycoproteins (AGPs); 3. Ca2+ channels; 4. auxin-efflux “PIN” proteins. Typical pinball machines release pinballs that trigger various sound and visual effects. However, in plants “proton pinballs” eject Ca2+ bound by paired glucuronic acid residues of numerous glycomodules in periplasmic AGP-Ca2+. Freed Ca2+ ions flow down the electrostatic gradient through open Ca2+ channels into the cytosol thus activating numerous Ca2+-dependent activities.Clearly cytosolic Ca2+ levels depend on activity of the proton pump, the state of Ca2+ channels and size of the periplasmic AGP-Ca2+ capacitor: Proton pump activation is a major regulatory focal point tightly controlled by the supply of auxin: auxin efflux carriers conveniently known as “PIN” proteins [null mutants are pin-shaped!] pump auxin from cell to cell. Mechanosensitive Ca2+ channels and their activation by reactive oxygen species (ROS) are yet another factor regulating cytosolic Ca2+.Cell expansion also triggers proton pump/pinball activity by mechanotransduction of wall stress via Hechtian adhesion thus forming a Hechtian oscillator that underlies cycles of wall plasticity and oscillatory growth.Finally, Ca2+ homeostasis of plants depends on cell surface external storage as source of dynamic Ca2+, unlike the internal ER storage source of animals where the added regulatory complexities ranging from vitamin D to parathormone contrast with the elegant simplicity of plant life. This paper summarises a sixty year Odyssey.

Novel molecular pinball machines of the plasma membrane control cytosolic Ca2+ levels that regulate plant metabolism. The essential components involve: 1. an auxin-activated proton pump; 2. arabinogalactan glycoproteins (AGPs); 3. Ca2+ channels; 4. auxin-efflux “PIN” proteins. Typical pinball machines release pinballs that trigger various sound and visual effects. However, in plants, “proton pinballs” eject Ca2+ bound by paired glucuronic acid residues of numerous glycomodules in periplasmic AGP-Ca2+. Freed Ca2+ ions flow down the electrostatic gradient through open Ca2+ channels into the cytosol, thus activating numerous Ca2+-dependent activities. Clearly, cytosolic Ca2+ levels depend on the activity of the proton pump, the state of Ca2+ channels and the size of the periplasmic AGP-Ca2+ capacitor; proton pump activation is a major regulatory focal point tightly controlled by the supply of auxin. Auxin efflux carriers conveniently known as “PIN” proteins (null mutants are pin-shaped) pump auxin from cell to cell. Mechanosensitive Ca2+ channels and their activation by reactive oxygen species (ROS) are yet another factor regulating cytosolic Ca2+. Cell expansion also triggers proton pump/pinball activity by the mechanotransduction of wall stress via Hechtian adhesion, thus forming a Hechtian oscillator that underlies cycles of wall plasticity and oscillatory growth. Finally, the Ca2+ homeostasis of plants depends on cell surface external storage as a source of dynamic Ca2+, unlike the internal ER storage source of animals, where the added regulatory complexities ranging from vitamin D to parathormone contrast with the elegant simplicity of plant life. This paper summarizes a sixty-year Odyssey.

Comprehensively controlling phytoplasma-associated jujube witches' broom (JWB) disease is extremely challenging for the jujube industry. Although the pathogenesis of phytoplasma disease has been highlighted in many plant species, the release of lateral buds from dormancy under JWB phytoplasma infection has not been characterized in woody perennial jujube. Here, two 16SrV-B group phytoplasma effectors, SJP1 and SJP2, were experimentally determined to induce witches' broom with increased lateral branches. In vivo interaction and subcellular localization analyses showed that both SJP1 and SJP2 were translocated from the cytoplasm to the nucleus to target the CYC/TB1-TCP transcription factor ZjBRC1. The N- and C-terminal coiled-coil domains of SJP1 and SJP2 were required for the TCP-binding ability. ZjBRC1 bound directly to the auxin efflux carrier ZjPIN1c/3 promoters and down-regulated their expression to promote the accumulation of endogenous auxin indole-3-acetic acid in jujube calli. Furthermore, JWB phytoplasma infection suppressed ZjBRC1 accumulation and induced ZjPIN1c/3 expression to stimulate lateral bud outgrowth. Therefore, SJP1 and SJP2 stimulate lateral bud outgrowth, at least partly, by repressing the ZjBRC1-controlled auxin efflux channel in jujube, representing a potential strategy for comprehensive phytoplasma-associated disease control and a resource for gene editing breeding to create new cultivars with varying degrees of shoot branching.

SUMMARYAuxin and auxin-mediated signaling pathways involved in the regulation of lateral root development are well documented. Although exocytic vesicle trafficking plays an important role in auxin efflux carriers PIN recycling, and polar auxin transport during lateral root formation, however, the mechanistic details of these processes are not well understood. Here, we demonstrate that BYPASS1-LIKE (B1L) regulate lateral root development via exocytic vesicular trafficking-mediated polar auxin transport in Arabidopsis. In b1l mutants, the number of lateral roots increased significantly, and the phenotypes were mainly attributed to lateral root primordium initiation but not to the defects in lateral root primordium development. Furthermore, the auxin signal was stronger in the lateral root primordium of the b1l mutant at stage I than those observed in the wild-type (WT). Moreover, exogenous auxin and auxin transport inhibitory treatments indicated that the phenotype of lateral roots in b1l mutants can be attributed to higher auxin levels and that B1L regulates auxin efflux. Consistently, auxin efflux carriers PIN1-GFP and PIN3-GFP were expressed at higher levels in the lateral root primordium of the b1l mutants. Interestingly, we found that B1L interacted with the exocyst and b1l mutant showed a defect in PIN2 exocytosis. Finally, we found that B1L cooperated with EXO70B1 to regulate lateral root formation. Our findings reveal an essential regulatory mechanism of B1L that interacts with the exocyst to regulate PIN-mediated polar auxin transport and lateral root initiation.

Background and AimsResistance to the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) in wild radish (Raphanus raphanistrum) appears to be due to a complex, multifaceted mechanism possibly involving enhanced constitutive plant defence and alterations in auxin signalling. Based on a previous gene expression analysis highlighting the plasma membrane as being important for 2,4-D resistance, this study aimed to identify the components of the leaf plasma membrane proteome that contribute to resistance.MethodsIsobaric tagging of peptides was used to compare the plasma membrane proteomes of a 2,4-D-susceptible and a 2,4-D-resistant wild radish population under control and 2,4-D-treated conditions. Eight differentially abundant proteins were then targeted for quantification in the plasma membranes of 13 wild radish populations (two susceptible, 11 resistant) using multiple reaction monitoring.Key ResultsTwo receptor-like kinases of unknown function (L-type lectin domain-containing receptor kinase IV.1-like and At1g51820-like) and the ATP-binding cassette transporter ABCB19, an auxin efflux transporter, were identified as being associated with auxinic herbicide resistance. The variability between wild radish populations suggests that the relative contributions of these candidates are different in the different populations.ConclusionsTo date, no receptor-like kinases have been reported to play a role in 2,4-D resistance. The lectin-domain-containing kinase may be involved in perception of 2,4-D at the plasma membrane, but its ability to bind 2,4-D and the identity of its signalling partner(s) need to be confirmed experimentally. ABCB19 is known to export auxinic compounds, but its role in 2,4-D resistance in wild radish appears to be relatively minor.

To clarify the mechanism of gravity-controlled polar auxin transport, we conducted the International Space Station (ISS) experiment “Auxin Transport” (identified by NASA's operation nomenclature) in 2016 and 2017, focusing on the expression of genes related to auxin efflux carrier protein PsPIN1 and its localization in the hook and epicotyl cells of etiolated Alaska pea seedlings grown for three days in the dark under microgravity (μg) and artificial 1 g conditions on a centrifuge in the Cell Biology Experiment Facility (CBEF) in the ISS, and under 1 g conditions on Earth. Regardless of gravity conditions, the accumulation of PsPIN1 mRNA in the proximal side of epicotyls of the seedlings was not different, but tended to be slightly higher as compared with that in the distal side. 2,3,5-Triiodobenzoic acid (TIBA) also did not affect the accumulation of PsPIN1 mRNA in the proximal and distal sides of epicotyls. However, in the apical hook region, TIBA increased the accumulation of PsPIN1 mRNA under μg conditions as compared with that under artificial 1 g conditions in the ISS. The accumulation of PsPIN1 proteins in epicotyls determined by western blotting was almost parallel to that of mRNA of PsPIN1. Immunohistochemical analysis with a specific polyclonal antibody of PsPIN1 revealed that a majority of PsPIN1 in the apical hook and subapical regions of the seedlings grown under artificial 1 g conditions in the ISS localized in the basal side (rootward) of the plasma membrane of the endodermal tissues. Conversely, in the seedlings grown under μg conditions, localization of PsPIN1 was greatly disarrayed. TIBA substantially altered the cellular localization pattern of PsPIN1, especially under μg conditions. These results strongly suggest that the mechanisms by which gravity controls polar auxin transport are more likely to be due to the membrane localization of PsPIN1. This physiologically valuable report describes a close relationship between gravity-controlled polar auxin transport and the localization of auxin efflux carrier PsPIN1 in etiolated pea seedlings based on the μg experiment conducted in space.

Each year, in Europe several hundreds of million cuttings are rooted for production of ornamental young plants. This involves a complex chain including cutting production at low latitude sites and storage and transport of cuttings to rooting stations. Using Petunia hybrida as model, we analysed indole-3-acetic acid (IAA) levels, different metabolites, enzyme activities and gene expression to investigate the contribution of auxin-related pathways, of carbohydrate and nitrogen metabolism and the interaction of both to adventitious root (AR) formation. Blocking of polar auxin transport (PAT) eliminated the 24 h-peak of IAA, reduced the activities of cell wall and vacuolar invertases in the rooting zone and severely inhibited AR formation. Under standard rooting conditions genes controlling PAT and signalling via the Aux/IAA-ARF interaction showed phase-specific regulation indicating particular functions during the induction and differentiation of ARs. Enhanced AR formation after cold dark storage of cuttings was associated with rooting zone-dominated activation of cell wall invertase and induction of one particular member of the cell wall invertase-encoding gene family. Further, amino acids accumulated in cutting tissues under darkness. Moreover, higher levels of IAA were found in the stem base under dark compared to cultivation under light, which was further associated with upregulation of genes controlling one auxin efflux transporter and components of the auxin signalling cascade. These results strongly suggest that a coordinated remobilization of nitrogen within the cutting, establishment of the new sink in the rooting zone and activation of auxin-related pathways contribute to the induction and formation of root meristemoids under the dark phase and to accelerated AR differentiation and growth when the cuttings are subsequently planted.

Plants exhibit diverse polar behaviors in response to directional and non-directional environmental signals, termed tropic and nastic movements, respectively. The ways in which plants incorporate directional information into tropic behaviors is well understood, but it is less well understood how non-directional stimuli, such as ambient temperatures, specify the polarity of nastic behaviors. Here, we demonstrate that a developmentally programmed polarity of auxin flow underlies thermo-induced leaf hyponasty in Arabidopsis (Arabidopsis thaliana). In warm environments, PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) stimulates auxin production in the leaf. This results in the accumulation of auxin in leaf petioles, where PIF4 directly activates a gene encoding the PINOID (PID) protein kinase. PID is involved in polarization of the auxin transporter PIN-FORMED 3 to the outer membranes of petiole cells. Notably, the leaf polarity-determining ASYMMETRIC LEAVES 1 (AS1) directs the induction of PID to occur predominantly in the abaxial petiole region. These observations indicate that the integration of PIF4-mediated auxin biosynthesis and polar transport, and the AS1-mediated developmental shaping of polar auxin flow, coordinate leaf thermonasty, which facilitates leaf cooling in warm environments. We believe that leaf thermonasty is a suitable model system for studying the developmental programming of environmental adaptation in plants.

Plant growth and development are tightly regulated by compounds produced in trace amounts in the plant. Besides the classical phytohormones, many plant metabolites have been described to affect plant development. Among these are several phenylpropanoids, although conclusive evidence for their bioactivity at physiologically relevant concentrations is only available for cinnamic acid. By inhibition of auxin efflux transport, the cis-isoform of cinnamic acid alters auxin homeostasis, resulting in auxin-related growth effects. Despite insight into its mode of action, the molecular target of cis-cinnamic acid is not yet known, and it remains to be determined whether this or other phenylpropanoids have a role to play in regulating plant growth and development under normal or stress conditions.

Pyruvate dehydrogenase is the first enzyme (E1) of the PDH complex (PDC). This multienzyme complex contains E1, E2, and E3 components and controls the entry of carbon into the mitochondrial tricarboxylic acid cycle to enable cellular energy production. The E1 component of the PDC is composed of an E1α catalytic subunit and an E1β regulatory subunit. In Arabidopsis (Arabidopsis thaliana), there are two mitochondrial E1α homologs encoded by IAA-CONJUGATE-RESISTANT 4 (IAR4) and IAR4-LIKE (IAR4L), and one mitochondrial E1β homolog. Although IAR4 was reported to be involved in auxin conjugate sensitivity and auxin homeostasis in root development, its precise role remains unknown. Here, we provide experimental evidence that mitochondrial PDC E1 contributes to polar auxin transport during organ development. We performed genetic screens for factors involved in cotyledon development and identified an uncharacterized mutant, macchi-bou 1 (mab1). MAB1 encodes a mitochondrial PDC E1β subunit that can form both a homodimer and a heterodimer with IAR4. The mab1 mutation impaired MAB1 homodimerization, reduced the abundance of IAR4 and IAR4L, weakened PDC enzymatic activity, and diminished mitochondrial respiration. A metabolomics analysis showed significant changes in metabolites including amino acids in mab1 and, in particular, identified an accumulation of Ala. These results suggest that MAB1 is a component of the Arabidopsis mitochondrial PDC E1. Furthermore, in mab1 mutants and seedlings where the TCA cycle was pharmacologically blocked, we found reduced abundance of the PIN-FORMED (PIN) auxin efflux carriers, possibly due to impaired PIN recycling and enhanced PIN degradation in vacuoles. Therefore, we suggest that mab1 induces defective polar auxin transport via metabolic abnormalities.

A pot experiment was used to evaluate the effects of an arbuscular mycorrhizal fungus (AMF) Funneliformis mosseae on plant growth performance, root-hair growth, and root hormone levels in trifoliat...

Endosomal trafficking plays an important role in regulating plant growth and development both at optimal and stressed conditions. Cold stress response in Arabidopsis root is directly linked to inhibition of the endosomal trafficking of auxin efflux carriers. However, the cellular components that link cold stress and the endosomal trafficking remain elusive. By screening available endosomal trafficking mutants against root growth recovery response under cold stress, we identified GNOM, a SEC7 containing ARF-GEF, as a major modulator of cold response. Contrasting response of partial loss of function mutant gnomB4049/emb30-1 and the engineered Brefeldin A (BFA)-resistant GNOM line, both of which contain mutations within SEC7 domain, to cold stress at the whole-plant level highlights the importance of this domain in modulating the cold response pathway of plants. Cold stress selectively and transiently inhibits GNOM expression. The engineered point mutation at 696 amino acid position (Methionine to Leucine) that makes GNOM resistant to BFA in fact results in overexpression of GNOM both at transcriptional and translational levels, and also alters its subcellular localization. Overexpression and altered cellular localization of GNOM were found to be directly linked to conferring striking cold-resistant phenotype in Arabidopsis. Collectively, these results provide a mechanistic link between GNOM, BFA-sensitive GNOM-regulated trafficking and cold stress.

Auxin is involved in hypocotyl elongation in response to different environmental factors. BIIDXI is a cell wall DUF642 protein that participates in the regulation of the degree of pectin-methylesterification of the cell wall in different tissues, including hypocotyls. Under continuous light, bdx-1 seedlings presented longer hypocotyls than those of WT, while BIIDXI-overexpressed hypocotyls were auxin resistant. Auxin accumulation was observed in epidermal cells from bdx-1 hypocotyls, and the distribution pattern of PIN1 proteins differed. Moreover, the gravitropic response of bdx-1, a process that is highly dependent on auxin flux, was increased. In this study, we determined that BIIDXI is involved in hypocotyl elongation through the regulation of auxin flux.

Meristem is the most functionally dynamic part in a plant body. The shaping of the meristem requires constant cell division and cell elongation, which are influenced by hormones and cell cytoskeletal component, actin. Although the roles of hormones in modulating meristem development have been extensively studied, the role of actin in this process is still elusive. Using the single and double mutants of the vegetative class actin, we demonstrate that actin isovariant ACT7 plays an important role in root meristem development. In absence of ACT7, but not ACT8 and ACT2, depolymerization of actin was observed. Consistently, act7 mutant showed reduced cell division, cell elongation, and meristem length. Intracellular distribution and trafficking of auxin transport proteins in the actin mutants revealed that ACT7 specifically functions in root meristem to facilitate the trafficking of auxin efflux carriers PIN1 and PIN2, and consequently the transport of auxin. Compared with act7, act7act8 double mutant shows slightly enhanced phenotypic response and altered intracellular trafficking. The altered distribution of auxin in act7 and act7act8 affects the response of the roots to ethylene but not to cytokinin. Collectively, our results suggest that ACT7 dependent auxin-ethylene response plays a key role in controlling Arabidopsis root meristem development.