CYCLIC NUCLEOTIDE-GATED ION CHANNEL 2 modulates auxin homeostasis and signaling

Cyclic nucleotide-gated ion channels (CNGCs) have been firmly established as Ca²⁺-conducting ion channels that regulate a wide variety of physiological responses in plants. CNGC2 has been implicated in plant immunity and Ca²⁺ signaling due to the autoimmune phenotypes exhibited by null mutants of CNGC2 in Arabidopsis thaliana. However, cngc2 mutants display additional phenotypes that are unique among autoimmune mutants, suggesting that CNGC2 has functions beyond defense and generates distinct Ca²⁺ signals in response to different triggers. In this study, we found that cngc2 mutants showed reduced gravitropism, consistent with a defect in auxin signaling. This was mirrored in the diminished auxin response detected by the auxin reporters DR5::GUS and DII-VENUS and in a strongly impaired auxin-induced Ca²⁺ response. Moreover, the cngc2 mutant exhibits higher levels of the endogenous auxin indole-3-acetic acid, indicating that excess auxin in the cngc2 mutant causes its pleiotropic phenotypes. These auxin signaling defects and the autoimmunity syndrome of the cngc2 mutant could be suppressed by loss-of-function mutations in the auxin biosynthesis gene YUCCA6 (YUC6), as determined by identification of the cngc2 suppressor mutant repressor of cngc2 (rdd1) as an allele of YUC6. A loss-of-function mutation in the upstream auxin biosynthesis gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA1, WEAK ETHYLENE INSENSITIVE8) also suppressed the cngc2 phenotypes, further supporting the tight relationship between CNGC2 and the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS–YUCCA -dependent auxin biosynthesis pathway. Taking these results together, we propose that the Ca²⁺ signal generated by CNGC2 is a part of the negative feedback regulation of auxin homeostasis in which CNGC2 balances cellular auxin perception by influencing auxin biosynthesis.

One-sentence summary: The immunity-related Ca²⁺ channel CYCLIC NUCLEOTIDE-GATED CHANNEL 2 modulates auxin homeostasis and balances cellular auxin perception by influencing auxin biosynthesis.     

Indole-3-Acetic Acid Production by Endophytic Streptomyces sp. En-1 Isolated from Medicinal Plants

Plant-associated actinobacteria are rich sources of bioactive compounds including indole-derived molecules such as phytohormone indole-3-acetic acid (IAA). In view of few investigations concerning the biosynthesis of IAA by endophytic actinobacteria, this study evaluated the potential of IAA production in endophytic streptomycete isolates sourced from medicinal plant species Taxus chinensis and Artemisia annua. By HPLC analysis of IAA combined with molecular screening approach of iaaM, a genetic determinant of streptomycete IAA synthesis via indole-3-acetamide (IAM), our data showed the putative operation of IAM-mediated IAA biosynthesis in Streptomyces sp. En-1 endophytic to Taxus chinensis. Furthermore, using the co-cultivation system of model plant Arabidopsis thaliana and streptomycete, En-1 was found to be colonized intercellularly in the tissues of Arabidopsis, an alternative host, and the effects of endophytic En-1 inoculation on the model plant were also assayed. The phytostimulatory effects of En-1 inoculation suggest that IAA-producing Streptomyces sp. En-1 of endophytic origin could be a promising candidate for utilization in growth improvement of plants of economic and agricultural value.     

Crown-gall-resistant transgenic apple trees that Silence Agrobacterium tumefaciens oncogenes

Crown gall disease is an economically significant problem in fruit and nut orchards, vineyards, and nurseries worldwide. Tumors on stems and leaves result from excessive production of the phytohormones auxin and cytokinin in plant cells genetically transformed by Agrobacterium tumefaciens. High phytohormone levels result from expression of three oncogenes transferred stably into the plant genome from A. tumefaciens: iaaM, iaaH, and ipt. The iaaM and iaaH oncogenes direct auxin biosynthesis, and the ipt oncogene causes cytokinin production. In contrast to other tissues, roots do not respond to high cytokinin levels, and auxin overproduction is sufficient to cause tumor growth on roots. Inactivation of iaaM abolished gall formation on apple tree roots. Transgenes designed to express double-stranded RNA from iaaM and ipt sequences prevented crown gall disease on roots of transgenic apple trees.these authors contributed equally to this workthese authors contributed equally to this work     

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.     

Alterations in Auxin Homeostasis Suppress Defects in Cell Wall Function

The plant cell wall is a highly dynamic structure that changes in response to both environmental and developmental cues. It plays important roles throughout plant growth and development in determining the orientation and extent of cell expansion, providing structural support and acting as a barrier to pathogens. Despite the importance of the cell wall, the signaling pathways regulating its function are not well understood. Two partially redundant leucine-rich-repeat receptor-like kinases (LRR-RLKs), FEI1 and FEI2, regulate cell wall function in Arabidopsis thaliana roots; disruption of the FEIs results in short, swollen roots as a result of decreased cellulose synthesis. We screened for suppressors of this swollen root phenotype and identified two mutations in the putative mitochondrial pyruvate dehydrogenase E1α homolog, IAA-Alanine Resistant 4 (IAR4). Mutations in IAR4 were shown previously to disrupt auxin homeostasis and lead to reduced auxin function. We show that mutations in IAR4 suppress a subset of the fei1 fei2 phenotypes. Consistent with the hypothesis that the suppression of fei1 fei2 by iar4 is the result of reduced auxin function, disruption of the WEI8 and TAR2 genes, which decreases auxin biosynthesis, also suppresses fei1 fei2. In addition, iar4 suppresses the root swelling and accumulation of ectopic lignin phenotypes of other cell wall mutants, including procuste and cobra. Further, iar4 mutants display decreased sensitivity to the cellulose biosynthesis inhibitor isoxaben. These results establish a role for IAR4 in the regulation of cell wall function and provide evidence of crosstalk between the cell wall and auxin during cell expansion in the root.     

The NtAMI1 gene functions in cell division of tobacco BY-2 cells in the presence of indole-3-acetamide

Tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells can be grown in medium containing indole-3-acetamide (IAM). Based on this finding, the NtAMI1 gene, whose product is functionally equivalent to the AtAMI1 gene of Arabidopsis thaliana and the aux2 gene of Agrobacterium rhizogenes, was isolated from BY-2 cells. Overexpression of the NtAMI1 gene allowed BY-2 cells to proliferate at lower concentrations of IAM, whereas suppression of the NtAMI1 gene by RNA interference (RNAi) caused severe growth inhibition in the medium containing IAM. These results suggest that IAM is incorporated into plant cells and converted to the auxin, indole-3-acetic acid, by NtAMI1.     

Evidence that Indole-3-Acetic Acid is Not Synthesized Via the Indole-3-Acetamide Pathway in Pea Roots

The biosynthetic route of the key plant hormone, indole-3-acetic acid (IAA) has confounded generations of biologists. Evidence in higher plants has implicated two auxin intermediates with roles established in bacteria: indole-3-acetamide (IAM) and indole-3-pyruvic acid. Herein, the IAM pathway is investigated in pea (Pisum sativum), a model legume. The compound was not detected in pea tissue, although evidence was obtained for its presence in Arabidopsis, tobacco, and maize. Deuterium-labeled tryptophan was not converted to IAM in pea roots, despite being converted to IAA. After feeds of deuterium-labeled IAM, label was recovered in the IAA conjugate IAA-aspartate (IAAsp), although there was little or no labeling of IAA itself. Plants treated with IAM did not exhibit high-IAA phenotypes, and did not accumulate IAA. This evidence, taken together, indicates that although exogenous IAM may be converted to IAA (and further to IAAsp), the IAM pathway does not operate naturally in pea roots.     

Roles of YUCCAs in auxin biosynthesis and drought stress responses in plants

Auxin, a plant hormone, plays crucial roles in diverse aspects of plant growth and development reacting to and integrating environmental stimuli. Indole-3-acetic acid (IAA) is the major plant auxin that is synthesized by members of the YUCCA (YUC) family of flavin monooxygenases that catalyse a rate-limiting step. Although the paths to IAA biosynthesis are characterized in Arabidopsis, little is known about the corresponding components in potato. Recently, we isolated eight putative StYUC (Solanum tuberosum YUCCA) genes and five putative tryptophan aminotransferase genes in comparison to those found in Arabidopsis.¹ The specific domains of YUC proteins were well conserved in all StYUC amino acid sequences. Transgenic potato (Solanum tuberosum cv. Jowon) overexpressing AtYUC6 showed high-auxin and enhanced drought tolerance phenotypes. The transgenic potatoes also exhibited reduced levels of ROS (reactive oxygen species) compared to control plants. We therefore propose that YUCCA and TAA families in potato would function in the auxin biosynthesis. The overexpression of AtYUC6 in potato establishes enhanced drought tolerance through regulated ROS homeostasis.     

Endophytic fungus Falciphora oryzae promotes lateral root growth by producing indole derivatives after sensing plant signals

The endophytic fungus Falciphora oryzae was initially isolated from wild rice (Oryza granulata) and colonizes many crop species and promotes plant growth. However, the molecular mechanisms underlying F. oryzae-mediated growth promotion are still unknown. We found that F. oryzae was able to colonize Arabidopsis thaliana. The most dramatic change after F. oryzae inoculation was observed in the root architecture, as evidenced by increased lateral root growth but reduced primary root length, similar to the effect of auxin, a significant plant growth hormone. The expression of genes responsible for auxin biosynthesis, transport, and signalling was regulated in Arabidopsis roots after F. oryzae cocultivation. Indole derivatives were detected at significantly higher levels in liquid media after cocultivation compared with separate cultivation of Arabidopsis and F. oryzae. Consistently, the expression of indole biosynthetic genes was highly upregulated in F. oryzae upon treatment with Arabidopsis exudates. Global analysis of Arabidopsis gene expression at the early stage after F. oryzae cocultivation suggested that signals were exchanged to initiate Arabidopsis–F. oryzae interactions. All these results suggest that signalling molecules from Arabidopsis roots are perceived by F. oryzae and induce the biosynthesis of indole derivatives in F. oryzae, consequently stimulating Arabidopsis lateral root growth.     

Over-expression of the <Emphasis Type="Italic">IGI1</Emphasis> leading to altered shoot-branching development related to MAX pathway in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Shoot branching and growth are controlled by phytohormones such as auxin and other components in Arabidopsis. We identified a mutant (igi1) showing decreased height and bunchy branching patterns. The phenotypes reverted to the wild type in response to RNA interference with the IGI1 gene. Histochemical analysis by GUS assay revealed tissue-specific gene expression in the anther and showed that the expression levels of the IGI1 gene in apical parts, including flowers, were higher than in other parts of the plants. The auxin biosynthesis component gene, CYP79B2, was up-regulated in igi1 mutants and the IGI1 gene was down-regulated by IAA treatment. These results indicated that there is an interplay regulation between IGI1 and phytohormone auxin. Moreover, the expression of the auxin-related shoot branching regulation genes, MAX3 and MAX4, was down-regulated in igi1 mutants. Taken together, these results indicate that the overexpression of the IGI1 influenced MAX pathway in the shoot branching regulation.     

Potential but limited redundant roles of MtPIN4, MtPIN5 and MtPIN10/SLM1 in the development of Medicago truncatula

Auxin polar transport is crucial in regulating plant growth and patterning. As auxin efflux carriers, the PIN FORMED (PIN) proteins are responsible for transportation of auxin out of the cell. There are eight and ten PIN members in Arabidopsis (AtPIN) and Medicago truncatula (MtPIN), respectively. Compared with MtPIN10/SMOOTH LEAF MARGIN1 (SLM1), MtPIN4 exhibits a closer relationship with AtPIN1 based phylogenetic analysis. In addition, the gene structure and distribution of transmembrane segments of MtPIN4, MtPIN5 and MtPIN10/SLM1 are similar, implying possible redundant roles among them. However, analysis using Gene Expression Atlas revealed different expression patterns among MtPIN4, MtPIN5 and MtPIN10/SLM1. Loss of function of MtPIN10/SLM1 in M. truncatula resulted in pleiotropic phenotypes in different organs, which are similar with the defects in the pin1 mutant of Arabidopsis, suggesting that the MtPIN10/SLM1 is a putative ortholog of AtPIN1. MtPIN4, MtPIN5 and MtPIN10/SLM1 may have limited redundant functions in the development of M. truncatula. The creation of double and triple mutants will help to elucidate their potential roles in auxin transport and plant development.     

The Pseudomonas savastanoi tryptophan-2-mono-oxygenase is biologically active in Nicotiana tabacum

It has been proposed that the eukaryotic T-DNA-encoded indole-3-acetic acid (IAA) biosynthesis genes of Agrobacterium tumefaciens and their prokaryotic counterpart in Pseudomonas savastanoi originated from common ancestor genes. This paper provides additional evidence for the functional similarity between the gene products. We have demonstrated that a chimeric gene consisting of the coding sequence of the P. savastanoi tryptophan-2-mono-oxygenase (iaaM gene) and a plant promoter encodes an active enzyme in Nicotiana tabacum. Transformants obtained with this chimeric gene grew as a callus on hormone-free media. No stably transformed plantlets could be isolated. The callus tissues contained extremely high levels of indole-3-acetamide and slightly elevated levels of IAA. Either indole-3-acetamide by itself has a low auxin activity or, alternatively, it is converted aspecifically and at low rates into IAA. The P. savastanoi tryptophan-2-mono-oxygenase activity in plants is also able to detoxify the amino-acid analogue 5-methyltryptophan. This property can be used for positive selection of transformed calli.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IAM indole-3-acetamide - NAA naphthalene-1-acetic acid - NPT-II neomycin phosphotransferase II - T-DNA transferred DNA     

Role of AUX1 in the control of organ identity during in vitro organogenesis and in mediating tissue specific auxin and cytokinin interaction in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Classic plant tissue culture experiments have shown that exposure of cell culture to a high auxin to cytokinin ratio promotes root formation and a low auxin to cytokinin ratio leads to shoot regeneration. It has been widely accepted that auxin and cytokinin play an antagonistic role in the control of organ identities during organogenesis in vitro. Since the auxin level is highly elevated in the shoot meristem tissues, it is unclear how a low auxin to cytokinin ratio promotes the regeneration of shoots. To identify genes mediating the cytokinin and auxin interaction during organogenesis in vitro, three allelic mutants that display root instead of shoot regeneration in response to a low auxin to cytokinin ratio are identified using a forward genetic approach in Arabidopsis. Molecular characterization shows that the mutations disrupt the AUX1 gene, which has been reported to regulate auxin influx in plants. Meanwhile, we find that cytokinin substantially stimulates auxin accumulation and redistribution in calli and some specific tissues of Arabidopsis seedlings. In the aux1 mutants, the cytokinin regulated auxin accumulation and redistribution is substantially reduced in both calli and specific tissues of young seedlings. Our results suggest that auxin elevation and other changes stimulated by cytokinin, instead of low auxin or exogenous auxin directly applied, is essential for shoot regeneration. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

ADP1 Affects Plant Architecture by Regulating Local Auxin Biosynthesis

Plant architecture is one of the key factors that affect plant survival and productivity. Plant body structure is established through the iterative initiation and outgrowth of lateral organs, which are derived from the shoot apical meristem and root apical meristem, after embryogenesis. Here we report that ADP1, a putative MATE (multidrug and toxic compound extrusion) transporter, plays an essential role in regulating lateral organ outgrowth, and thus in maintaining normal architecture of Arabidopsis. Elevated expression levels of ADP1 resulted in accelerated plant growth rate, and increased the numbers of axillary branches and flowers. Our molecular and genetic evidence demonstrated that the phenotypes of plants over-expressing ADP1 were caused by reduction of local auxin levels in the meristematic regions. We further discovered that this reduction was probably due to decreased levels of auxin biosynthesis in the local meristematic regions based on the measured reduction in IAA levels and the gene expression data. Simultaneous inactivation of ADP1 and its three closest homologs led to growth retardation, relative reduction of lateral organ number and slightly elevated auxin level. Our results indicated that ADP1-mediated regulation of the local auxin level in meristematic regions is an essential determinant for plant architecture maintenance by restraining the outgrowth of lateral organs.     

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.     

IAA synthesis and root induction with iaa genes under heat shock promoter control

We have devised a heat shock-inducible indole-3-acetic acid (IAA) synthesis system for plant cells, which is based on the iaa genes of the Agrobacterium tumefaciens T-DNA and the heat shock promoter hsp70 of Drosophila melanogaster.Two DNA constructs were tested: one contains the iaaM gene linked to the hsp70 promoter (hsp 70-iaaM) and encodes the production of indoleacetamide (IAM), the other contains hsp 70-iaaM and the wild-type iaaH gene which codes for the conversion of IAM into IAA (hsp 70-iaaM/iaaH). Heat shock-controlled IAM and IAA synthesis was tested on two levels: biochemically by measuring IAM and IAA levels in Kalanchoe stem segments infected with the two constructs, and morphologically by IAA-dependent root formation on Kalanchoe plants, on carrot discs and on tobacco leaf fragments. At both levels the responses were found to be controlled by the heat shock promoter. IAM levels of segments infected with hsp 70-iaaM increased 6-fold upon heat shock induction to 240 pmol IAM per stem segment. The accumulation of IAA in segments infected with hsp 70-iaaM/iaaH and heat-shocked was found to be more variable, possibly due to IAA transport and metabolism. Heat shock treatment of Kalanchoe plants and tobacco leaf fragments infected with hsp 70-iaaM/iaaH led to a strong increase in root formation. On carrot discs, heat shock-specific root induction was also demonstrated, but the responses differed between individual carrots.