GmPIN-dependent polar auxin transport is involved in soybean nodule development

To overcome nitrogen deficiency, legume roots establish symbiotic interactions with nitrogen-fixing rhizobia that are fostered in specialized organs (nodules). Similar to other organs, nodule formation is determined by a local maximum of the phytohormone auxin at the primordium site. However, how auxin regulates nodule development remains poorly understood. Here, we found that in soybean, (Glycine max), dynamic auxin transport driven by PIN-FORMED (PIN) transporter GmPIN1 is involved in nodule primordium formation. GmPIN1 was specifically expressed in nodule primordium cells and GmPIN1 was polarly localized in these cells. Two nodulation regulators, (iso)flavonoids trigger expanded distribution of GmPIN1b to root cortical cells, and cytokinin rearranges GmPIN1b polarity. Gmpin1abc triple mutants generated with CRISPR-Cas9 showed the impaired establishment of auxin maxima in nodule meristems and aberrant divisions in the nodule primordium cells. Moreover, overexpression of GmPIN1 suppressed nodule primordium initiation. GmPIN9d, an ortholog of Arabidopsis thaliana PIN2, acts together with GmPIN1 later in nodule development to acropetally transport auxin in vascular bundles, fine-tuning the auxin supply for nodule enlargement. Our findings reveal how PIN-dependent auxin transport modulates different aspects of soybean nodule development and suggest that the establishment of auxin gradient is a prerequisite for the proper interaction between legumes and rhizobia.

In soybean, nodule primordium formation involves GmPIN1-mediated polar auxin transport within primordium cells, and nodule enlargement involves the collaboration of GmPIN9d and GmPIN1-dependent auxin transport within nodule vasculature.     

Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation.     

Temporal and spatial order of events during the induction of cortical cell divisions in white clover by Rhizobium leguminosarum bv. trifolii inoculation or localized cytokinin addition

We examined the timing and location of several early root responses to Rhizobium leguminosarum bv. trifolii infection, compared with a localized addition of cytokinin in white clover, to study the role of cytokinin in early signaling during nodule initiation. Induction of ENOD40 expression by either rhizobia or cytokinin was similar in timing and location and occurred in nodule progenitor cells in the inner cortex. Inoculation of rhizobia in the mature root failed to induce ENOD40 expression and cortical cell divisions (ccd). Nitrate addition at levels repressing nodule formation inhibited ENOD40 induction by rhizobia but not by cytokinin. ENOD40 expression was not induced by auxin, an auxin transport inhibitor, or an ethylene precursor. In contrast to rhizobia, cytokinin addition was not sufficient to induce a modulation of the auxin flow, the induction of specific chalcone synthase genes, and the accumulation of fluorescent compounds associated with nodule initiation. However, cytokinin addition was sufficient for the localized induction of auxin-induced GH3 gene expression and the initiation of ccd. Our results suggest that rhizobia induce cytokinin-mediated events in parallel to changes in auxin-related responses during nodule initiation and support a role of ENOD40 in regulating ccd. We propose a model for the interactions of cytokinin with auxin, ENOD40, flavonoids, and nitrate during nodulation.     

The Diageotropica Gene Differentially Affects Auxin and Cytokinin Responses throughout Development in Tomato

The interactions between the plant hormones auxin and cytokinin throughout plant development are complex, and genetic investigations of the interdependency of auxin and cytokinin signaling have been limited. We have characterized the cytokinin sensitivity of the auxin-resistant diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) in a range of auxin- and cytokinin-regulated responses. Intact, etiolated dgt seedlings showed cross-resistance to cytokinin with respect to root elongation, but cytokinin effects on hypocotyl growth and ethylene synthesis in these seedlings were not impaired by the dgt mutation. Seven-week-old, green wild-type and dgt plants were also equally sensitive to cytokinin with respect to shoot growth and hypocotyl and internode elongation. The effects of cytokinin and the dgt mutation on these processes appeared additive. In tissue culture organ regeneration from dgt hypocotyl explants showed reduced sensitivity to auxin but normal sensitivity to cytokinin, and the effects of cytokinin and the mutation were again additive. However, although callus induction from dgt hypocotyl explants required auxin and cytokinin, dgt calli did not show the typical concentration-dependent stimulation of growth by either auxin or cytokinin observed in wild-type calli. Cross-resistance of the dgt mutant to cytokinin thus was found to be limited to a small subset of auxin- and cytokinin-regulated growth processes affected by the dgt mutation, indicating that auxin and cytokinin regulate plant growth through both shared and separate signaling pathways.     

Auxin transport in maize roots in response to localized nitrate supply

Background and Aims

Roots typically respond to localized nitrate by enhancing lateral-root growth. Polar auxin transport has important roles in lateral-root formation and growth; however, it is a matter of debate whether or how auxin plays a role in the localized response of lateral roots to nitrate.

Methods

Treating maize (Zea mays) in a split-root system, auxin levels were quantified directly and polar transport was assayed by the movement of [³H]IAA. The effects of exogenous auxin and polar auxin transport inhibitors were also examined.

Key Results

Auxin levels in roots decreased more in the nitrate-fed compartment than in the nitrate-free compartment and nitrate treatment appeared to inhibit shoot-to-root auxin transport. However, exogenous application of IAA only partially reduced the stimulatory effect of localized nitrate, and auxin level in the roots was similarly reduced by local applications of ammonium that did not stimulate lateral-root growth.

Conclusions

It is concluded that local applications of nitrate reduced shoot-to-root auxin transport and decreased auxin concentration in roots to a level more suitable for lateral-root growth. However, alteration of root auxin level alone is not sufficient to stimulate lateral-root growth.     

The influence of cytokinin-auxin cross-regulation on cell-fate determination in Arabidopsis thaliana root development

Root growth and development in Arabidopsis thaliana are sustained by a specialised zone termed the meristem, which contains a population of dividing and differentiating cells that are functionally analogous to a stem cell niche in animals. The hormones auxin and cytokinin control meristem size antagonistically. Local accumulation of auxin promotes cell division and the initiation of a lateral root primordium. By contrast, high cytokinin concentrations disrupt the regular pattern of divisions that characterises lateral root development, and promote differentiation. The way in which the hormones interact is controlled by a genetic regulatory network. In this paper, we propose a deterministic mathematical model to describe this network and present model simulations that reproduce the experimentally observed effects of cytokinin on the expression of auxin regulated genes. We show how auxin response genes and auxin efflux transporters may be affected by the presence of cytokinin. We also analyse and compare the responses of the hormones auxin and cytokinin to changes in their supply with the responses obtained by genetic mutations of SHY2, which encodes a protein that plays a key role in balancing cytokinin and auxin regulation of meristem size. We show that although shy2 mutations can qualitatively reproduce the effect of varying auxin and cytokinin supply on their response genes, some elements of the network respond differently to changes in hormonal supply and to genetic mutations, implying a different, general response of the network. We conclude that an analysis based on the ratio between these two hormones may be misleading and that a mathematical model can serve as a useful tool for stimulate further experimental work by predicting the response of the network to changes in hormone levels and to other genetic mutations.     

RcRR1, a Rosa canina Type-A Response Regulator Gene,Is Involved in Cytokinin-Modulated Rhizoid Organogenesis

In vitro, a new protocol of plant regeneration in rose was achieved via protocorm-like bodies (PLBs) induced from the root-like organs named rhizoids that developed from leaf explants. The development of rhizoids is a critical stage for efficient regeneration, which is triggered by exogenous auxin. However, the role of cytokinin in the control of organogenesis in rose is as yet uncharacterized. The aim of this study was to elucidate the molecular mechanism of cytokinin-modulated rhizoid formation in Rosa canina. Here, we found that cytokinin is a key regulator in the formation of rhizoids. Treatment with cytokinin reduced callus activity and significantly inhibited rhizoid formation in Rosa canina. We further isolated the full-length cDNA of a type-A response regulator gene of cytokinin signaling, RcRR1, from which the deduced amino acid sequence contained the conserved DDK motif. Gene expression analysis revealed that RcRR1 was differentially expressed during rhizoid formation and its expression level was rapidly up-regulated by cytokinin. In addition, the functionality of RcRR1 was tested in Arabidopsis. RcRR1 was found to be localized to the nucleus in GFP-RcRR1 transgenic plants and overexpression of RcRR1 resulted in increased primary root length and lateral root density. More importantly, RcRR1 overexpression transgenic plants also showed reduced sensitivity to cytokinin during root growth; auxin distribution and the expression of auxin efflux carriers PIN genes were altered in RcRR1 overexpression plants. Taken together, these results demonstrate that RcRR1 is a functional type-A response regulator which is involved in cytokinin-regulated rhizoid formation in Rosa canina.     

Studies of aberrant phyllotaxy1 Mutants of Maize Indicate Complex Interactions between Auxin and Cytokinin Signaling in the Shoot Apical Meristem

One of the most fascinating aspects of plant morphology is the regular geometric arrangement of leaves and flowers, called phyllotaxy. The shoot apical meristem (SAM) determines these patterns, which vary depending on species and developmental stage. Auxin acts as an instructive signal in leaf initiation, and its transport has been implicated in phyllotaxy regulation in Arabidopsis (Arabidopsis thaliana). Altered phyllotactic patterns are observed in a maize (Zea mays) mutant, aberrant phyllotaxy1 (abph1, also known as abphyl1), and ABPH1 encodes a cytokinin-inducible type A response regulator, suggesting that cytokinin signals are also involved in the mechanism by which phyllotactic patterns are established. Therefore, we investigated the interaction between auxin and cytokinin signaling in phyllotaxy. Treatment of maize shoots with a polar auxin transport inhibitor, 1-naphthylphthalamic acid, strongly reduced ABPH1 expression, suggesting that auxin or its polar transport is required for ABPH1 expression. Immunolocalization of the PINFORMED1 (PIN1) polar auxin transporter revealed that PIN1 expression marks leaf primordia in maize, similarly to Arabidopsis. Interestingly, maize PIN1 expression at the incipient leaf primordium was greatly reduced in abph1 mutants. Consistently, auxin levels were reduced in abph1, and the maize PIN1 homolog was induced not only by auxin but also by cytokinin treatments. Our results indicate distinct roles for ABPH1 as a negative regulator of SAM size and a positive regulator of PIN1 expression. These studies highlight a complex interaction between auxin and cytokinin signaling in the specification of phyllotactic patterns and suggest an alternative model for the generation of altered phyllotactic patterns in abph1 mutants. We propose that reduced auxin levels and PIN1 expression in abph1 mutant SAMs delay leaf initiation, contributing to the enlarged SAM and altered phyllotaxy of these mutants.     

DAR2 acts as an important node connecting cytokinin,auxin, SHY2 and PLT1/2 in root meristem size control

Cytokinin and auxin antagonistically affect cell proliferation and differentiation and thus regulate root meristem size by influencing the abundance of SHORT HYPOCOTYL2 (SHY2/IAA3). SHY2 affects auxin distribution in the root meristem by repressing the auxin-inducible expression of PIN-FORMED (PIN) auxin transport genes. The PLETHORA (PLT1/2) genes influence root meristem growth by promoting stem cells and transit-amplifying cells. However, the factors connecting cytokinin, auxin, SHY2 and PLT1/2 are largely unknown. In a recent study, we have shown that the DA1-related protein 2 (DAR2) acts downstream of cytokinin and SHY2 but upstream of PLT1/2 to affect root meristem size. Here, we discuss the possible molecular mechanisms by which Arabidopsis DAR2 controls root meristem size.     

Multiple AUX/IAA�CARF modules regulate lateral root formation: the role of Arabidopsis SHY2/IAA3-mediated auxin signalling

In Arabidopsis thaliana, lateral root (LR) formation is regulated by multiple auxin/indole-3-acetic acid (Aux/IAA)–AUXIN RESPONSE FACTOR (ARF) modules: (i) the IAA28–ARFs module regulates LR founder cell specification; (ii) the SOLITARY-ROOT (SLR)/IAA14–ARF7–ARF19 module regulates nuclear migration and asymmetric cell divisions of the LR founder cells for LR initiation; and (iii) the BODENLOS/IAA12–MONOPTEROS/ARF5 module also regulates LR initiation and organogenesis. The number of Aux/IAA–ARF modules involved in LR formation remains unknown. In this study, we isolated the shy2-101 mutant, a gain-of-function allele of short hypocotyl2/suppressor of hy2 (shy2)/iaa3 in the Columbia accession. We demonstrated that the shy2-101 mutation not only strongly inhibits LR primordium development and emergence but also significantly increases the number of LR initiation sites with the activation of LATERAL ORGAN BOUNDARIES-DOMAIN16/ASYMMETRIC LEAVES2-LIKE18, a target gene of the SLR/IAA14–ARF7–ARF19 module. Genetic analysis revealed that enhanced LR initiation in shy2-101 depended on the SLR/IAA14–ARF7–ARF19 module. We also showed that the shy2 roots contain higher levels of endogenous IAA. These observations indicate that the SHY2/IAA3–ARF-signalling module regulates not only LR primordium development and emergence after SLR/IAA14–ARF7–ARF19 module-dependent LR initiation but also inhibits LR initiation by affecting auxin homeostasis, suggesting that multiple Aux/IAA–ARF modules cooperatively regulate the developmental steps during LR formation.     

Microglial calcium signal acts as a rapid sensor of single neuron damage in vivo

In the healthy adult brain microglia, the main immune-competent cells of the CNS, have a distinct (so-called resting or surveying) phenotype. Resting microglia can only be studied in vivo since any isolation of brain tissue inevitably triggers microglial activation. Here we used in vivo two-photon imaging to obtain a first insight into Ca²⁺ signaling in resting cortical microglia. The majority (80%) of microglial cells showed no spontaneous Ca²⁺ transients at rest and in conditions of strong neuronal activity. However, they reliably responded with large, generalized Ca²⁺ transients to damage of an individual neuron. These damage-induced responses had a short latency (0.4-4 s) and were localized to the immediate vicinity of the damaged neuron (< 50 μm cell body-to-cell body distance). They were occluded by the application of ATPγS as well as UDP and 2-MeSADP, the agonists of metabotropic P2Y receptors, and they required Ca²⁺ release from the intracellular Ca²⁺ stores. Thus, our in vivo data suggest that microglial Ca²⁺ signals occur mostly under pathological conditions and identify a Ca²⁺ store-operated signal, which represents a very sensitive, rapid, and highly localized response of microglial cells to brain damage. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Auxin distribution is differentially affected by nitrate in roots of two rice cultivars differing in responsiveness to nitrogen

Background and Aims

Although ammonium (NH₄⁺) is the preferred form of nitrogen over nitrate (NO₃⁻) for rice (Oryza sativa), lateral root (LR) growth in roots is enhanced by partial NO₃⁻ nutrition (PNN). The roles of auxin distribution and polar transport in LR formation in response to localized NO₃⁻ availability are not known.

Methods

Time-course studies in a split-root experimental system were used to investigate LR development patterns, auxin distribution, polar auxin transport and expression of auxin transporter genes in LR zones in response to localized PNN in ‘Nanguang’ and ‘Elio’ rice cultivars, which show high and low responsiveness to NO₃⁻, respectively. Patterns of auxin distribution and the effects of polar auxin transport inhibitors were also examined in DR5::GUS transgenic plants.

Key Results

Initiation of LRs was enhanced by PNN after 7 d cultivation in ‘Nanguang’ but not in ‘Elio’. Auxin concentration in the roots of ‘Nanguang’ increased by approx. 24 % after 5 d cultivation with PNN compared with NH₄⁺ as the sole nitrogen source, but no difference was observed in ‘Elio’. More auxin flux into the LR zone in ‘Nanguang’ roots was observed in response to NO₃⁻ compared with NH₄⁺ treatment. A greater number of auxin influx and efflux transporter genes showed increased expression in the LR zone in response to PNN in ‘Nanguang’ than in ‘Elio’.

Conclusions

The results indicate that higher NO₃⁻ responsiveness is associated with greater auxin accumulation in the LR zone and is strongly related to a higher rate of LR initiation in the cultivar ‘Nanguang’.     

Distribution and change patterns of free IAA, ABP 1 and PM H⁺-ATPase during ovary and ovule development of Nicotiana tabacum L

Auxin plays key roles in flower induction, embryogenesis, seed formation and seedling development, but little is known about whether auxin regulates the development of ovaries and ovules before pollination. In the present report, we measured the content of free indole-3-acetic (IAA) in ovaries of Nicotiana tabacum L., and localized free IAA, auxin binding protein 1 (ABP1) and plasma membrane (PM) H⁺-ATPase in the ovaries and ovules. The level of free IAA in the developmental ovaries increased gradually from the stages of ovular primordium to the functional megaspore, but slightly decreased when the embryo sacs formed. Immunoenzyme labeling clearly showed that both IAA and ABP1 were distributed in the ovules, the edge of the placenta, vascular tissues and the ovary wall, while PM H⁺-ATPase was mainly localized in the ovules. By using immunogold labeling, the subcellular distributions of IAA, ABP1 and PM H⁺-ATPase in the ovules were also shown. The results suggest that IAA, ABP1 and PM H⁺-ATPase may play roles in the ovary and ovule initiation, formation and differentiation.     

Auxin and cytokinin control formation of the quiescent centre in the adventitious root apex of arabidopsis

Background and Aims

Adventitious roots (ARs) are part of the root system in numerous plants, and are required for successful micropropagation. In the Arabidopsis thaliana primary root (PR) and lateral roots (LRs), the quiescent centre (QC) in the stem cell niche of the meristem controls apical growth with the involvement of auxin and cytokinin. In arabidopsis, ARs emerge in planta from the hypocotyl pericycle, and from different tissues in in vitro cultured explants, e.g. from the stem endodermis in thin cell layer (TCL) explants. The aim of this study was to investigate the establishment and maintenance of the QC in arabidopsis ARs, in planta and in TCL explants, because information about this process is still lacking, and it has potential use for biotechnological applications.

Methods

Expression of PR/LR QC markers and auxin influx (LAX3)/efflux (PIN1) genes was investigated in the presence/absence of exogenous auxin and cytokinin. Auxin was monitored by the DR5::GUS system and cytokinin by immunolocalization. The expression of the auxin-biosynthetic YUCCA6 gene was also investigated by in situ hybridization in planta and in AR-forming TCLs from the indole acetic acid (IAA)-overproducing superroot2-1 mutant and its wild type.

Key Results

The accumulation of auxin and the expression of the QC marker WOX5 characterized the early derivatives of the AR founder cells, in planta and in in vitro cultured TCLs. By determination of PIN1 auxin efflux carrier and LAX3 auxin influx carrier activities, an auxin maximum was determined to occur at the AR tip, to which WOX5 expression was restricted, establishing the positioning of the QC. Cytokinin caused a restriction of LAX3 and PIN1 expression domains, and concomitantly the auxin biosynthesis YUCCA6 gene was expressed in the apex.

Conclusions

In ARs formed in planta and TCLs, the QC is established in a similar way, and auxin transport and biosynthesis are involved through cytokinin tuning.