Development of Erect Leaves in a Modern Maize Hybrid is Associated with Reduced Responsiveness to Auxin and Light of Young Seedlings In Vitro

Modern corn (Zea mays L.) varieties have been selected for their ability to maintain productivity in dense plantings. We have tested the possibility that the physiological consequence of the selection involves changes in responsiveness to light and auxin.Etiolated seedlings of two older corn hybrids 307 and 3306 elongated significantly more than seedlings of a modern corn hybrid 3394. The level of endogenous auxin and activity of PAT in 307 and 3394 were similar. Hybrid 3394 shows resistance to auxin- and light-induced responses at the seedling, cell and molecular levels. Intact 3394 plants exhibited less responsiveness to the inhibitory effect of R, FR and W, auxin, anti-auxin and inhibitors of PAT. In excised mesocotyl tissue 3394 seedlings also showed essentially low responsiveness to NAA. Cells of 3394 were insensitive to auxin- and light-induced hyperpolarization of the plasma membrane. Expression of ABP4 was much less in 3394 than in 307, and in contrast to 307, it was not upregulated by NAA, R and FR. Preliminary analysis of abp mutants suggests that ABPs may be involved in development of leaf angle in corn.Our results confirm the understanding that auxin interacts with light in the regulation of growth and development of young seedlings and suggest that in corn ABPs may be involved in growth of maize seedlings and development of leaf angle. We hypothesize that ABP4 plays an important role in the auxin- and/or light-induced growth responses. We also hypothesize that in the modern corn hybrid 3394, ABP4 is “mutated,” which may result in the observed 3394 phenotypes, including upright leaves.Key Words: auxin, auxin-binding protein, growth, leaf angle, light, maize     

Mitochondrial heat-shock cognate protein 70 contributes to auxin-mediated embryo development

In Arabidopsis thaliana, mitochondrial-localized heat-shock cognate protein 70-1 (mtHSC70-1) plays an important role in vegetativegrowth. However, whether mtHSC70-1 affects reproductive growth remains unknown. Here, we found that the mtHSC70-1 gene was expressed in the provascular cells of the embryo proper from the early heart stage onward during embryogenesis. Phenotypic analyses of mthsc70-1 mutants revealed that mtHSC70 deficiency leads to defective embryo development and that this effect is mediated by auxin. In addition to a dwarf phenotype, the mthsc70-1 mutant displayed defects in flower morphology, anther development, and embryogenesis. At early developmental stages, the mthsc70-1 embryos exhibited abnormal cell divisions in both embryo proper and suspensor cells. From heart stage onward, they displayed an abnormal shape such as with no or very small cotyledon protrusions, had aberrant number of cotyledons, or were twisted. These embryo defects were associated with reduced or ectopic expression of auxin responsive reporter DR5_rev:GFP. Consistently, the expression of auxin biosynthesis and polar auxin transport genes were markedly altered in mthsc70-1. On the other hand, mitochondrial retrograde regulation (MRR) was enhanced in mthsc70-1. Treatment of wild-type plants with an inhibitor that activates mitochondrial retrograde signaling reduced the expression level of auxin biosynthesis and polar auxin transport genes and induced phenotypes similar to those of mthsc70-1. Taken together, our data reveal that loss of function of mtHSC70-1 induces MRR, which inhibits auxin biosynthesis and polar auxin transport, leading to abnormal auxin gradients and defective embryo development.

mtHSC70-1 dysfunction induces mitochondrial retrograde regulation, which inhibits auxin biosynthesis and polar auxin transport, leading to abnormal auxin gradients and defective embryo development.     

Genetic identification of a second site modifier of ctr1-1 that controls ethylene-responsive and gravitropic root growth in Arabidopsis thaliana

Ethylene controls myriad aspects of plant growth throughout developmental stages in higher plants. It has been well established that ethylene-responsive growth entails extensive crosstalk with other plant hormones, particularly auxin. Here, we report a genetic mutation, named 1-aminocyclopropane carboxylic acid (ACC) resistant root1-1 (are1-1) in Arabidopsis thaliana (L.) Heynh. The CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) encodes a Raf-related protein, functioning as an upstream negative regulator of ethylene signaling in Arabidopsis thaliana. We found that the ctr1-1, a kinase-inactive allele exhibited slightly, but significantly, longer root length, compared to ACC-treated wild-type or ctr1-3, a null allele. Our genetic studies unveiled the existence of are1-1 mutation in the ctr1-1 mutant, as a second-site modifier which confers root-specific ethylene-resistance. Based on well-characterized crosstalk between ethylene and auxin during ethylene-responsive root growth, we performed various physiological analyses. Whereas are1-1 displayed normal sensitivity to synthetic auxins, it showed modest resistance to an auxin transport inhibitor, 1-Nnaphthylphthalamic acid. In addition, are1-1 mutant exhibited ectopically altered DR5:GUS activity upon ethylenetreatment. The results implicated the involvement of are1-1 in auxin-distribution, but not in auxin-biosynthesis, -uptake, or -sensitivity. In agreement, are1-1 mutant exhibited reduced gravitropic root growth and defective redistribution of DR5:GUS activity upon gravi-stimulation. Taken together with genetic and molecular analysis, our results suggest that ARE1 defines a novel locus to control ethylene-responsive root growth as well as gravitropic root growth presumably through auxin distribution in Arabidopsis thaliana.     

Two homologous INDOLE-3-ACETAMIDE (IAM) HYDROLASE genes are required for the auxin effects of IAM in Arabidopsis

Indole-3-acetamide (IAM) is the first confirmed auxin biosynthetic intermediate in some plant pathogenic bacteria. Exogenously applied IAM or production of IAM by overexpressing the bacterial iaaM gene in Arabidopsis causes auxin overproduction phenotypes. However, it is still inconclusive whether plants use IAM as a key precursor for auxin biosynthesis. Herein, we reported the isolation IAM HYDROLASE 1 (IAMH1) gene in Arabidopsis from a forward genetic screen for IAM-insensitive mutants that display normal auxin sensitivities. IAMH1 has a close homolog named IAMH2 that is located right next to IAMH1 on chromosome IV in Arabidopsis. We generated iamh1 iamh2 double mutants using our CRISPR/Cas9 gene editing technology. We showed that disruption of the IAMH genes rendered Arabidopsis plants resistant to IAM treatments and also suppressed the iaaM overexpression phenotypes, suggesting that IAMH1 and IAMH2 are the main enzymes responsible for converting IAM into indole-3-acetic acid (IAA) in Arabidopsis. The iamh double mutants did not display obvious developmental defects, indicating that IAM does not play a major role in auxin biosynthesis under normal growth conditions. Our findings provide a solid foundation for clarifying the roles of IAM in auxin biosynthesis and plant development.     

The AUXIN BINDING PROTEIN 1 Is Required for Differential Auxin Responses Mediating Root Growth

Background

In plants, the phytohormone auxin is a crucial regulator sustaining growth and development. At the cellular level, auxin is interpreted differentially in a tissue- and dose-dependent manner. Mechanisms of auxin signalling are partially unknown and the contribution of the AUXIN BINDING PROTEIN 1 (ABP1) as an auxin receptor is still a matter of debate.

Methodology/Principal Findings

Here we took advantage of the present knowledge of the root biological system to demonstrate that ABP1 is required for auxin response. The use of conditional ABP1 defective plants reveals that the protein is essential for maintenance of the root meristem and acts at least on the D-type CYCLIN/RETINOBLASTOMA pathway to control entry into the cell cycle. ABP1 affects PLETHORA gradients and confers auxin sensitivity to root cells thus defining the competence of the cells to be maintained within the meristem or to elongate. ABP1 is also implicated in the regulation of gene expression in response to auxin.

Conclusions/Significance

Our data support that ABP1 is a key regulator for root growth and is required for auxin-mediated responses. Differential effects of ABP1 on various auxin responses support a model in which ABP1 is the major regulator for auxin action on the cell cycle and regulates auxin-mediated gene expression and cell elongation in addition to the already well known TIR1-mediated ubiquitination pathway.     

The Arabidopsis PLEIOTROPIC DRUG RESISTANCE8/ABCG36 ATP Binding Cassette Transporter Modulates Sensitivity to the Auxin Precursor Indole-3-Butyric Acid

Plants have developed numerous mechanisms to store hormones in inactive but readily available states, enabling rapid responses to environmental changes. The phytohormone auxin has a number of storage precursors, including indole-3-butyric acid (IBA), which is apparently shortened to active indole-3-acetic acid (IAA) in peroxisomes by a process similar to fatty acid β-oxidation. Whereas metabolism of auxin precursors is beginning to be understood, the biological significance of the various precursors is virtually unknown. We identified an Arabidopsis thaliana mutant that specifically restores IBA, but not IAA, responsiveness to auxin signaling mutants. This mutant is defective in PLEIOTROPIC DRUG RESISTANCE8 (PDR8)/PENETRATION3/ABCG36, a plasma membrane–localized ATP binding cassette transporter that has established roles in pathogen responses and cadmium transport. We found that pdr8 mutants display defects in efflux of the auxin precursor IBA and developmental defects in root hair and cotyledon expansion that reveal previously unknown roles for IBA-derived IAA in plant growth and development. Our results are consistent with the possibility that limiting accumulation of the IAA precursor IBA via PDR8-promoted efflux contributes to auxin homeostasis.     

The eta7/csn3-3 Auxin Response Mutant of Arabidopsis Defines a Novel Function for the CSN3 Subunit of the COP9 Signalosome

The COP9 signalosome (CSN) is an eight subunit protein complex conserved in all higher eukaryotes. In Arabidopsis thaliana, the CSN regulates auxin response by removing the ubiquitin-like protein NEDD8/RUB1 from the CUL1 subunit of the SCF^TIR1/AFB ubiquitin-ligase (deneddylation). Previously described null mutations in any CSN subunit result in the pleiotropic cop/det/fus phenotype and cause seedling lethality, hampering the study of CSN functions in plant development. In a genetic screen to identify enhancers of the auxin response defects conferred by the tir1-1 mutation, we identified a viable csn mutant of subunit 3 (CSN3), designated eta7/csn3-3. In addition to enhancing tir1-1 mutant phenotypes, the csn3-3 mutation alone confers several phenotypes indicative of impaired auxin signaling including auxin resistant root growth and diminished auxin responsive gene expression. Unexpectedly however, csn3-3 plants are not defective in either the CSN-mediated deneddylation of CUL1 or in SCF^TIR1-mediated degradation of Aux/IAA proteins. These findings suggest that csn3-3 is an atypical csn mutant that defines a novel CSN or CSN3-specific function. Consistent with this possibility, we observe dramatic differences in double mutant interactions between csn3-3 and other auxin signaling mutants compared to another weak csn mutant, csn1-10. Lastly, unlike other csn mutants, assembly of the CSN holocomplex is unaffected in csn3-3 plants. However, we detected a small CSN3-containing protein complex that is altered in csn3-3 plants. We hypothesize that in addition to its role in the CSN as a cullin deneddylase, CSN3 functions in a distinct protein complex that is required for proper auxin signaling.     

Overexpression of the Auxin Binding PROTEIN1 Modulates PIN-Dependent Auxin Transport in Tobacco Cells

Background

Auxin binding protein 1 (ABP1) is a putative auxin receptor and its function is indispensable for plant growth and development. ABP1 has been shown to be involved in auxin-dependent regulation of cell division and expansion, in plasma-membrane-related processes such as changes in transmembrane potential, and in the regulation of clathrin-dependent endocytosis. However, the ABP1-regulated downstream pathway remains elusive.

Methodology/Principal Findings

Using auxin transport assays and quantitative analysis of cellular morphology we show that ABP1 regulates auxin efflux from tobacco BY-2 cells. The overexpression of ABP1can counterbalance increased auxin efflux and auxin starvation phenotypes caused by the overexpression of PIN auxin efflux carrier. Relevant mechanism involves the ABP1-controlled vesicle trafficking processes, including positive regulation of endocytosis of PIN auxin efflux carriers, as indicated by fluorescence recovery after photobleaching (FRAP) and pharmacological manipulations.

Conclusions/Significance

The findings indicate the involvement of ABP1 in control of rate of auxin transport across plasma membrane emphasizing the role of ABP1 in regulation of PIN activity at the plasma membrane, and highlighting the relevance of ABP1 for the formation of developmentally important, PIN-dependent auxin gradients.     

Complementation of the tomato HWS gene with its Arabidopsis counterpart demonstrates conservation of the gene function between both species

The HAWAIIAN SKIRT (HWS) gene was originally described in Arabidopsis for the characteristic fusion of sepals in the mutant. A tomato line mutated in the putative ortholog gene was isolated in a previous study. The tomato hws-1 mutant showed facultative parthenocarpy and produced fruits with elevated Brix, revealing the gene as a hopeful resource for crop improvement. To confirm the orthology relationship between the Arabidopsis and tomato HWS genes, the hws-1 mutant was complemented with either the tomato wild-type genomic fragment or the Arabidopsis sequence of the gene. In both complementation experiments, defective phenotypes of hws-1 are rescued, albeit to different extents. Recovery of these phenotypes, which include parthenocarpic fruit production, increased Brix, loss of leaflet serration, alteration of bud and petal shape, firmly establishes SlHWS as an ortholog of the originally described HWS in Arabidopsis. This work indicates that the function of HWS is likely to be conserved in a wide range of plant species.     

Role of the Arabidopsis PIN6 Auxin Transporter in Auxin Homeostasis and Auxin-Mediated Development

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.     

Arabidopsis ASA1 Is Important for Jasmonate-Mediated Regulation of Auxin Biosynthesis and Transport during Lateral Root Formation

Plant roots show an impressive degree of plasticity in adapting their branching patterns to ever-changing growth conditions. An important mechanism underlying this adaptation ability is the interaction between hormonal and developmental signals. Here, we analyze the interaction of jasmonate with auxin to regulate lateral root (LR) formation through characterization of an Arabidopsis thaliana mutant, jasmonate-induced defective lateral root1 (jdl1/asa1-1). We demonstrate that, whereas exogenous jasmonate promotes LR formation in wild-type plants, it represses LR formation in jdl1/asa1-1. JDL1 encodes the auxin biosynthetic gene ANTHRANILATE SYNTHASE α1 (ASA1), which is required for jasmonate-induced auxin biosynthesis. Jasmonate elevates local auxin accumulation in the basal meristem of wild-type roots but reduces local auxin accumulation in the basal meristem of mutant roots, suggesting that, in addition to activating ASA1-dependent auxin biosynthesis, jasmonate also affects auxin transport. Indeed, jasmonate modifies the expression of auxin transport genes in an ASA1-dependent manner. We further provide evidence showing that the action mechanism of jasmonate to regulate LR formation through ASA1 differs from that of ethylene. Our results highlight the importance of ASA1 in jasmonate-induced auxin biosynthesis and reveal a role for jasmonate in the attenuation of auxin transport in the root and the fine-tuning of local auxin distribution in the root basal meristem.     

hydra Mutants of Arabidopsis are defective in sterol profiles and auxin and ethylene signaling

The hydra mutants of Arabidopsis are characterized by a pleiotropic phenotype that shows defective embryonic and seedling cell patterning, morphogenesis, and root growth. We demonstrate that the HYDRA1 gene encodes a Delta8-Delta7 sterol isomerase, whereas HYDRA2 encodes a sterol C14 reductase, previously identified as the FACKEL gene product. Seedlings mutant for each gene are similarly defective in the concentrations of the three major Arabidopsis sterols. Promoter::reporter gene analysis showed misexpression of the auxin-regulated DR5 and ACS1 promoters and of the epidermal cell file-specific GL2 promoter in the mutants. The mutants exhibit enhanced responses to auxin. The phenotypes can be rescued partially by inhibition of auxin and ethylene signaling but not by exogenous sterols or brassinosteroids. We propose a model in which correct sterol profiles are required for regulated auxin and ethylene signaling through effects on membrane function.     

NCP1/AtMOB1A Plays Key Roles in Auxin-Mediated Arabidopsis Development

MOB1 protein is a core component of the Hippo signaling pathway in animals where it is involved in controlling tissue growth and tumor suppression. Plant MOB1 proteins display high sequence homology to animal MOB1 proteins, but little is known regarding their role in plant growth and development. Herein we report the critical roles of Arabidopsis MOB1 (AtMOB1A) in auxin-mediated development in Arabidopsis. We found that loss-of-function mutations in AtMOB1A completely eliminated the formation of cotyledons when combined with mutations in PINOID (PID), which encodes a Ser/Thr protein kinase that participates in auxin signaling and transport. We showed that atmob1a was fully rescued by its Drosophila counterpart, suggesting functional conservation. The atmob1a pid double mutants phenocopied several well-characterized mutant combinations that are defective in auxin biosynthesis or transport. Moreover, we demonstrated that atmob1a greatly enhanced several other known auxin mutants, suggesting that AtMOB1A plays a key role in auxin-mediated plant development. The atmob1a single mutant displayed defects in early embryogenesis and had shorter root and smaller flowers than wild type plants. AtMOB1A is uniformly expressed in embryos and suspensor cells during embryogenesis, consistent with its role in embryo development. AtMOB1A protein is localized to nucleus, cytoplasm, and associated to plasma membrane, suggesting that it plays roles in these subcellular localizations. Furthermore, we showed that disruption of AtMOB1A led to a reduced sensitivity to exogenous auxin. Our results demonstrated that AtMOB1A plays an important role in Arabidopsis development by promoting auxin signaling.     

Plasmalemma hyperpolarity and culture of sense and antisense abp transgenic tobacco protoplasts

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Purification and immunohistochemical localization of the auxin binding site i protein from maize coleoptiles

An auxin binding protein fraction prepared by means of affinity chromatography on 2-OH-3,5-diiodobenzoic acid-Sepharose and gel filtration was used as antigen. The obtained rabbit antisera contained antibodies against the auxin, binding protein (ABP) and several contaminating proteins (nonABP). The nonABP could be separated on an appropriate affinity matrix omitting the TIBA analogue. After their immobilization on Sepharose antibodies directed towards contaminating, the proteins were isolated and immobilized, too. This IgGanti nonABP-Sepharose retains almost all contaminating proteins present in the specific eluates of the auxin affinity matrix. In a final affinity chromatography step on IgG-Sepharose a highly purified ABP could be eluted. This ABP was immobilized on Sepharose for the separation of monospecific antibodies against ABP (IgGanti abp). Using these antibodies the ABP could be localized within the outer epidermal cells of the coleoptile by immunofluorescence microscopy. From the inhibition of auxin induced elongation of coleoptile tissue by IgGanti abp it is concluded that the ABP is localized at the plasmalemma of the epidermal cells and that the ABP is involved in auxin action as a true hormone receptor. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.     

Dysfunctional mitochondria regulate the size of root apical meristem and leaf development in Arabidopsis

Mitochondria play an important role in maintaining metabolic and energy homeostasis in the plant cell. Thus, perturbation of mitochondrial structure and function will affect plant growth and development. Arabidopsis slow growth3 (slo3) is defective in At3g61360 that encodes a pentatricopeptide repeat (PPR) protein. Analysis of slo3 mitochondrial RNA metabolism revealed that the splicing of nad7 intron 2 is impaired, which leads to a dramatic reduction in complex I activity. So the SLO3 PPR protein is a splicing factor that is required for the removal of nad7 intron 2 in Arabidopsis. The slo3 mutant plants have obvious phenotypes with severe growth retardation and delayed development. The size of root apical meristem (RAM) is reduced and the production of meristem cells is decreased in slo3. Furthermore, the rosette leaves of slo3 are curled or crinkled, which may be derived from uneven growth of the leaf surface. The underlying mechanisms by which dysfunctional mitochondria affect these growth and developmental phenotypes have yet to be established. Nonetheless, plant hormone auxin is known to play an important role in orchestrating the development of RAM and leaf shape. It is possible that dysfunctional mitochondria may interact with auxin signaling pathways to regulate the boundary of RAM and the cell division arrest front during leaf growth in Arabidopsis.