Overexpression of Arabidopsis MAP kinase kinase 7 leads to activation of plant basal and systemic acquired resistance

There is a growing body of evidence indicating that mitogen-activated protein kinase (MAPK) cascades are involved in plant defense responses. Analysis of the completed Arabidopsis thaliana genome sequence has revealed the existence of 20 MAPKs, 10 MAPKKs and 60 MAPKKKs, implying a high level of complexity in MAPK signaling pathways, and making the assignment of gene functions difficult. The MAP kinase kinase 7 (MKK7) gene of Arabidopsis has previously been shown to negatively regulate polar auxin transport. Here we provide evidence that MKK7 positively regulates plant basal and systemic acquired resistance (SAR). The activation-tagged bud1 mutant, in which the expression of MKK7 is increased, accumulates elevated levels of salicylic acid (SA), exhibits constitutive pathogenesis-related (PR) gene expression, and displays enhanced resistance to both Pseudomonas syringae pv. maculicola (Psm) ES4326 and Hyaloperonospora parasitica Noco2. Both PR gene expression and disease resistance of the bud1 plants depend on SA, and partially depend on NPR1. We demonstrate that the constitutive defense response in bud1 plants is a result of the increased expression of MKK7, and requires the kinase activity of the MKK7 protein. We found that expression of the MKK7 gene in wild-type plants is induced by pathogen infection. Reducing mRNA levels of MKK7 by antisense RNA expression not only compromises basal resistance, but also blocks the induction of SAR. Intriguingly, ectopic expression of MKK7 in local tissues induces PR gene expression and resistance to Psm ES4326 in systemic tissues, indicating that activation of MKK7 is sufficient for generating the mobile signal of SAR.     

The mitogen-activated protein kinase cascade MKK3-MPK6 is an important part of the jasmonate signal transduction pathway in Arabidopsis

The plant hormone jasmonic acid (JA) plays a key role in the environmental stress responses and developmental processes of plants. Although ATMYC2/JASMONATE-INSENSITIVE1 (JIN1) is a major positive regulator of JA-inducible gene expression and essential for JA-dependent developmental processes in Arabidopsis thaliana, molecular mechanisms underlying the control of ATMYC2/JIN1 expression remain largely unknown. Here, we identify a mitogen-activated protein kinase (MAPK) cascade, MAPK KINASE 3 (MKK3)-MAPK 6 (MPK6), which is activated by JA in Arabidopsis. We also show that JA negatively controls ATMYC2/JIN1 expression, based on quantitative RT-PCR and genetic analyses using gain-of-function and loss-of-function mutants of the MKK3-MPK6 cascade. These results indicate that this kinase unit plays a key role in JA-dependent negative regulation of ATMYC2/JIN1 expression. Both positive and negative regulation by JA may be used to fine-tune ATMYC2/JIN1 expression to control JA signaling. Moreover, JA-regulated root growth inhibition is affected by mutations in the MKK3-MPK6 cascade, which indicates important roles in JA signaling. We provide a model explaining how MPK6 can convert three distinct signals - JA, pathogen, and cold/salt stress - into three different sets of responses in Arabidopsis.     

An Arabidopsis kinase cascade influences auxin‐responsive cell expansion

Mitogen‐activated protein kinase (MPK) cascades are conserved mechanisms of signal transduction across eukaryotes. Despite the importance of MPK proteins in signaling events, specific roles for many Arabidopsis MPK proteins remain unknown. Multiple studies have suggested roles for MPK signaling in a variety of auxin‐related processes. To identify MPK proteins with roles in auxin response, we screened mpk insertional alleles and identified mpk1‐1 as a mutant that displays hypersensitivity in auxin‐responsive cell expansion assays. Further, mutants defective in the upstream MAP kinase kinase MKK3 also display hypersensitivity in auxin‐responsive cell expansion assays, suggesting that this MPK cascade affects auxin‐influenced cell expansion. We found that MPK1 interacts with and phosphorylates ROP BINDING PROTEIN KINASE 1 (RBK1), a protein kinase that interacts with members of the Rho‐like GTPases from Plants (ROP) small GTPase family. Similar to mpk1‐1 and mkk3‐1 mutants, rbk1 insertional mutants display auxin hypersensitivity, consistent with a possible role for RBK1 downstream of MPK1 in influencing auxin‐responsive cell expansion. We found that RBK1 directly phosphorylates ROP4 and ROP6, supporting the possibility that RBK1 effects on auxin‐responsive cell expansion are mediated through phosphorylation‐dependent modulation of ROP activity. Our data suggest a MKK3 ? MPK1 ? RBK1 phosphorylation cascade that may provide a dynamic module for altering cell expansion.     

Strigolactone Acts Downstream of Auxin to Regulate Bud Outgrowth in Pea and Arabidopsis

During the last century, two key hypotheses have been proposed to explain apical dominance in plants: auxin promotes the production of a second messenger that moves up into buds to repress their outgrowth, and auxin saturation in the stem inhibits auxin transport from buds, thereby inhibiting bud outgrowth. The recent discovery of strigolactone as the novel shoot-branching inhibitor allowed us to test its mode of action in relation to these hypotheses. We found that exogenously applied strigolactone inhibited bud outgrowth in pea (Pisum sativum) even when auxin was depleted after decapitation. We also found that strigolactone application reduced branching in Arabidopsis (Arabidopsis thaliana) auxin response mutants, suggesting that auxin may act through strigolactones to facilitate apical dominance. Moreover, strigolactone application to tiny buds of mutant or decapitated pea plants rapidly stopped outgrowth, in contrast to applying N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, which significantly slowed growth only after several days. Whereas strigolactone or NPA applied to growing buds reduced bud length, only NPA blocked auxin transport in the bud. Wild-type and strigolactone biosynthesis mutant pea and Arabidopsis shoots were capable of instantly transporting additional amounts of auxin in excess of endogenous levels, contrary to predictions of auxin transport models. These data suggest that strigolactone does not act primarily by affecting auxin transport from buds. Rather, the primary repressor of bud outgrowth appears to be the auxin-dependent production of strigolactones.     

Activities of auxin polar transport in inflorescence axes of flower mutants ofArabidopsis thaliana: Relevance to flower formation and growth

Relationships between the activity of auxin polar transport and flower formation were studied using several flower mutants ofArabidopsis thaliana. The activity of auxin polar transport in the upper portion of inflorescence axis of wildtype plants ofArabidopsis thaliana was significantly lower than that of the basal part. The activities of auxin polar transport in the upper portion of inflorescence axes ofap1 andclv1 mutants were significantly higher than that of wild-type plant. However, those of other flower mutants tested,ap3-1, ag, pi, Fl-40, Fl-54, Fl-89 andpin-formed, were extremely low as compared with that of wild one. We got some evidence that the reduction of the activity of auxin polar transport is concerned with the growth and development of plants. We could mimic it by the removal of all flowers and pods including mature or immature seeds. Moreover, artificial pollination inap3-1 andpi mutants, in which no seeds are found naturally, resulted in the partial recovery of the activity of auxin polar transport in inflorescence axis. Considering these results in this study together with the fact that inhibitors of auxin polar transport generated almost same disruptions ofpin-formed orpinoid mutants which normally had no flowers in inflorescence axis (Okadaet al. 1991, Uedaet al. 1992, Bennettet al. 1995), the systern of auxin polar transport and its activity in inflorescence axis seems to be essential for the development of flower bud in early stage ofArabidopsis thaliana, and the activity of auxin polar transport is also regulated by the formation of flowers and seeds in inflorescence axis.     

拟南芥矮小丛生突变体的分离与分子鉴定

顶端优势是指侧生分生组织的生长被主茎或主花序所抑制.最近的研究通过分离和鉴定顶端优势发生改变的突变体开始揭示顶端优势的分子机制.通过T-DNA标签法分离了拟南芥矮小丛生(bushy and dwarf 1, bud1 )突变体.突变体植株的表型包括顶端优势丧失、株型矮小,表明bud1 突变体存在生长素代谢、运输或信号传导的缺陷.一个对生长素特异反应的启动子驱动的报告基因在bud1 中表达模式改变.生长素敏感性和运输能力的测定表明这两个过程在 bud1中均正常.以上结果显示bud1 表型是生长素代谢缺陷的结果.遗传分析表明BUD1 为半显性突变且与一个T-DNA插入共分离,可通过iPCR方法分离.     

拟南芥矮小丛生突变体的分离与分子鉴定

顶端优势是指侧生分生组织的生长被主茎或主花序所抑制。最近的研究通过分离和鉴定顶端优势发生改变的突变体开始揭示顶端优势的分子机制。通过T-DNA标签法分离了拟南芥矮小丛生(bushy and dwarf l,budl)突变体。突变体植株的表型包括顶端优势丧失、株型矮小,表明budl突变体存在生长素代谢、运输或信号传导的缺陷。一个对生长素特异反应的启动子驱动的报告基因在budl中表达模式改变。生长素敏感性和运输能力的测定表明这两个过程在budl中均正常。以上结果显示budl表型是生长素代谢缺陷的结果。遗传分析表明BUDI为半显性突变且与一个T-DNA插入共分离,可通过iPCR方法分离。     

A Pseudomonas syringae ADP-Ribosyltransferase Inhibits Arabidopsis Mitogen-Activated Protein Kinase Kinases

The successful recognition of pathogen-associated molecular patterns (PAMPs) as a danger signal is crucial for plants to fend off numerous potential pathogenic microbes. The signal is relayed through mitogen-activated protein kinase (MPK) cascades to activate defenses. Here, we show that the Pseudomonas syringae type III effector HopF2 can interact with Arabidopsis thaliana MAP KINASE KINASE5 (MKK5) and likely other MKKs to inhibit MPKs and PAMP-triggered immunity. Inhibition of PAMP-induced MPK phosphorylation was observed when HopF2 was delivered naturally by the bacterial type III secretion system. In addition, HopF2 Arg-71 and Asp-175 residues that are required for the interaction with MKK5 are also necessary for blocking MAP kinase activation, PAMP-triggered defenses, and virulence function in plants. HopF2 can inactivate MKK5 and ADP-ribosylate the C terminus of MKK5 in vitro. Arg-313 of MKK5 is required for ADP-ribosylation by HopF2 and MKK5 function in the plant cell. Together, these results indicate that MKKs are important targets of HopF2.     

D6PK AGCVIII Kinases Are Required for Auxin Transport and Phototropic Hypocotyl Bending in Arabidopsis

Phototropic hypocotyl bending in response to blue light excitation is an important adaptive process that helps plants to optimize their exposure to light. In Arabidopsis thaliana, phototropic hypocotyl bending is initiated by the blue light receptors and protein kinases phototropin1 (phot1) and phot2. Phototropic responses also require auxin transport and were shown to be partially compromised in mutants of the PIN-FORMED (PIN) auxin efflux facilitators. We previously described the D6 PROTEIN KINASE (D6PK) subfamily of AGCVIII kinases, which we proposed to directly regulate PIN-mediated auxin transport. Here, we show that phototropic hypocotyl bending is strongly dependent on the activity of D6PKs and the PIN proteins PIN3, PIN4, and PIN7. While early blue light and phot-dependent signaling events are not affected by the loss of D6PKs, we detect a gradual loss of PIN3 phosphorylation in d6pk mutants of increasing complexity that is most severe in the d6pk d6pkl1 d6pkl2 d6pkl3 quadruple mutant. This is accompanied by a reduction of basipetal auxin transport in the hypocotyls of d6pk as well as in pin mutants. Based on our data, we propose that D6PK-dependent PIN regulation promotes auxin transport and that auxin transport in the hypocotyl is a prerequisite for phot1-dependent hypocotyl bending.     

Flavonols accumulate asymmetrically and affect auxin transport in Arabidopsis

Flavonoids represent a class of secondary metabolites with diverse functions in plants including ultraviolet protection, pathogen defense, and interspecies communication. They are also known as modulators of signaling processes in plant and animal systems and therefore are considered to have beneficial effects as nutraceuticals. The rol1-2 (for repressor of lrx1) mutation of Arabidopsis (Arabidopsis thaliana) induces aberrant accumulation of flavonols and a cell-growth phenotype in the shoot. The hyponastic cotyledons, aberrant shape of pavement cells, and deformed trichomes in rol1-2 mutants are suppressed by blocking flavonoid biosynthesis, suggesting that the altered flavonol accumulation in these plants induces the shoot phenotype. Indeed, the identification of several transparent testa, myb, and fls1 (for flavonol synthase1) alleles in a rol1-2 suppressor screen provides genetic evidence that flavonols interfere with shoot development in rol1-2 seedlings. The increased accumulation of auxin in rol1-2 seedlings appears to be caused by a flavonol-induced modification of auxin transport. Quantification of auxin export from mesophyll protoplasts revealed that naphthalene-1-acetic acid but not indole-3-acetic acid transport is affected by the rol1-2 mutation. Inhibition of flavonol biosynthesis in rol1-2 fls1-3 restores naphthalene-1-acetic acid transport to wild-type levels, indicating a very specific mode of action of flavonols on the auxin transport machinery.     

Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects.

Polar auxin transport plays a key role in the regulation of plant growth and development. To identify genes involved in this process, we have developed a genetic procedure to screen for mutants of Arabidopsis that are altered in their response to auxin transport inhibitors. We recovered a total of 16 independent mutants that defined seven genes, called TRANSPORT INHIBITOR RESPONSE (TIR) genes. Recessive mutations in one of these genes, TIR3, result in altered responses to transport inhibitors, a reduction in polar auxin transport, and a variety of morphological defects that can be ascribed to changes in indole-3-acetic acid distribution. Most dramatically, tir3 seedlings are strongly deficient in lateral root production, a process that is known to depend on polar auxin transport from the shoot into the root. In addition, tir3 plants display a reduction in apical dominance as well as decreased elongation of siliques, pedicels, roots, and the inflorescence. Biochemical studies indicate that tir3 plants have a reduced number of N-1-naphthylphthalamic (NPA) binding sites, suggesting that the TIR3 gene is required for expression, localization, or stabilization of the NPA binding protein (NBP). Alternatively, the TIR3 gene may encode the NBP. Because the tir3 mutants have a substantial defect in NPA binding, their phenotype provides genetic evidence for a role for the NBP in plant growth and development.     

Requirement of the Auxin Polar Transport System in Early Stages of Arabidopsis Floral Bud Formation

The pin-formed mutant pin 1-1, one of the Arabidopsis flower mutants, has several structural abnormalities in inflorescence axes, flowers, and leaves. In some cases, pin1-1 forms a flower with abnormal structure (wide petals, no stamens, pistil-like structure with no ovules in the ovary) at the top of inflorescence axes. In other cases, no floral buds are formed on the axes. An independently isolated allelic mutant (pin1-2) shows similar phenotypes. These mutant phenotypes are exactly the same in wild-type plants cultured in the presence of chemical compounds known as auxin polar transport inhibitors: 9-hydroxyfluorene-9-carboxylic acid or N-(1-naphthyl)phthalamic acid. We tested the polar transport activity of indole-3-acetic acid and the endogenous amount of free indole-3-acetic acid in the tissue of inflorescence axes of the pin1 mutants and wild type. The polar transport activity in the pin 1-1 mutant and in the pin1-2 mutant was decreased to 14% and 7% of wild type, respectively. These observations strongly suggest that the normal level of polar transport activity in the inflorescence axes is required in early developmental stages of floral bud formation in Arabidopsis and that the primary function of the pin1 gene is auxin polar transport in the inflorescence axis.     

Alkoxy-auxins are selective inhibitors of auxin transport mediated by PIN, ABCB, and AUX1 transporters

Polar auxin movement is a primary regulator of programmed and plastic plant development. Auxin transport is highly regulated at the cellular level and is mediated by coordinated transport activity of plasma membrane-localized PIN, ABCB, and AUX1/LAX transporters. The activity of these transporters has been extensively analyzed using a combination of pharmacological inhibitors, synthetic auxins, and knock-out mutants in Arabidopsis. However, efforts to analyze auxin-dependent growth in other species that are less tractable to genetic manipulation require more selective inhibitors than are currently available. In this report, we characterize the inhibitory activity of 5-alkoxy derivatives of indole 3-acetic acid and 7-alkoxy derivatives of naphthalene 1-acetic acid, finding that the hexyloxy and benzyloxy derivatives act as potent inhibitors of auxin action in plants. These alkoxy-auxin analogs inhibit polar auxin transport and tropic responses associated with asymmetric auxin distribution in Arabidopsis and maize. The alkoxy-auxin analogs inhibit auxin transport mediated by AUX1, PIN, and ABCB proteins expressed in yeast. However, these analogs did not inhibit or activate SCF(TIR1) auxin signaling and had no effect on the subcellular trafficking of PIN proteins. Together these results indicate that alkoxy-auxins are inactive auxin analogs for auxin signaling, but are recognized by PIN, ABCB, and AUX1 auxin transport proteins. Alkoxy-auxins are powerful new tools for analyses of auxin-dependent development.     

Enhanced Polar Auxin Transport in Tomato Polycotyledon Mutant Seems to be Related to Glutathione Levels

Glutathione-dependent root growth in Arabidopsis is linked to polar auxin transport (PAT). Arabidopsis mutants with reduced glutathione (GSH) levels also show reduced PAT. To gain an insight into the relationship between PAT and GSH level, we analyzed tomato polycotyledon mutant, pct1-2, which has enhanced PAT. Microarray analysis of gene expression in pct1-2 mutant revealed underexpression of several genes related to glutamate and glutathione metabolism. In consonance with microarray analysis, enzymatic as well as in vivo assay revealed higher glutathione levels in the early phase of pct1-2 seedling growth than WT. The inhibition of auxin transport by 2,3,5-triiodobenzoic acid (TIBA) reduced both GSH level and PIN1 expression in pct1-2 root tips. The reduction of in vivo GSH accumulation in pct1-2 root tips by buthionine sulfoximine (BSO) stimulated elongation of the short root of pct1-2 mutant akin to TIBA. The rescue of the short root phenotype of the pct1-2 mutant was restricted to TIBA and BSO. The other auxin transport inhibitors 1-N-naphthylphthalamic acid (NPA), 2-[4-(diethylamino)-2-hydroxybenzoyl] benzoic acid (BUM), 3-chloro-4-hydroxyphenylacetic acid (CHPAA), brefeldin and gravacin inhibited root elongation in both WT and pct1-2 mutant. Our results indicate a relationship between PAT and GSH levels in tomato akin to Arabidopsis. Our work also highlights that TIBA rescues the short root phenotype of the pct1-2 mutant by acting on a PAT component distinct from the site of action of other PAT inhibitors.

     

An auxin transport independent pathway is involved in phosphate stress-induced root architectural alterations in Arabidopsis. Identification of BIG as a mediator of auxin in pericycle cell activation

Arabidopsis (Arabidopsis thaliana) plants display a number of root developmental responses to low phosphate availability, including primary root growth inhibition, greater formation of lateral roots, and increased root hair elongation. To gain insight into the regulatory mechanisms by which phosphorus (P) availability alters postembryonic root development, we performed a mutant screen to identify genetic determinants involved in the response to P deprivation. Three low phosphate-resistant root lines (lpr1-1 to lpr1-3) were isolated because of their reduced lateral root formation in low P conditions. Genetic and molecular analyses revealed that all lpr1 mutants were allelic to BIG, which is required for normal auxin transport in Arabidopsis. Detailed characterization of lateral root primordia (LRP) development in wild-type and lpr1 mutants revealed that BIG is required for pericycle cell activation to form LRP in both high (1 mm) and low (1 microm) P conditions, but not for the low P-induced alterations in primary root growth, lateral root emergence, and root hair elongation. Exogenously supplied auxin restored normal lateral root formation in lpr1 mutants in the two P treatments. Treatment of wild-type Arabidopsis seedlings with brefeldin A, a fungal metabolite that blocks auxin transport, phenocopies the root developmental alterations observed in lpr1 mutants in both high and low P conditions, suggesting that BIG participates in vesicular targeting of auxin transporters. Taken together, our results show that auxin transport and BIG function have fundamental roles in pericycle cell activation to form LRP and promote root hair elongation. The mechanism that activates root system architectural alterations in response to P deprivation, however, seems to be independent of auxin transport and BIG.     

Over-expression of OsAGAP, an ARF-GAP, interferes with auxin influx, vesicle trafficking and root development

Development and organogenesis in both dicot and monocot plants are highly dependent on polar auxin transport (PAT), which requires the proper asymmetric localization of both auxin influx and efflux carriers. In the model dicot plant Arabidopsis thaliana, the trafficking and localization of auxin efflux facilitators such as PIN-FORMED1 (PIN1) are mediated by GNOM, a guanine-nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, but molecular regulators of the auxin influx facilitators remain unknown. Here, we show that over-expression of OsAGAP, an ARF-GTPase-activating protein (ARF-GAP) in rice, impaired PAT and interfered with both primary and lateral root development. The lateral root phenotype could be rescued by the membrane-permeable auxin 1-naphthyl acetic acid, but not by indole 3-acetic acid (IAA) or by 2,4-dichloro-phenoxyacetic acid, which require influx facilitators to enter the cells. OsAGAP-over-expressing plants had alterations in vesicle trafficking and localization of the presumptive A. thaliana auxin-influx carrier AUX1, but not in the localization of the auxin efflux facilitators. Together, our data suggest that OsAGAP has a specific role in regulating vesicle trafficking pathways such as the auxin influx pathway, which in turn controls auxin-dependent root growth in plants.     

Auxin signaling and transport promote susceptibility to the root-infecting fungal pathogen Fusarium oxysporum in Arabidopsis

Fusarium oxysporum is a root-infecting fungal pathogen that causes wilt disease on a broad range of plant species, including the model plant Arabidopsis thaliana. Currently, very little is known about the molecular or physiological processes that are activated in the host during infection and the roles these processes play in resistance and susceptibility to F. oxysporum. In this study, we analyzed global gene expression profiles of F. oxysporum-infected Arabidopsis plants. Genes involved in jasmonate biosynthesis as well as jasmonate-dependent defense were coordinately induced by F. oxysporum. Similarly, tryptophan pathway genes, including those involved in both indole-glucosinolate and auxin biosynthesis, were upregulated in both the leaves and the roots of inoculated plants. Analysis of plants expressing the DR5:GUS construct suggested that root auxin homeostasis was altered during F. oxysporum infection. However, Arabidopsis mutants with altered auxin and tryptophan-derived metabolites such as indole-glucosinolates and camalexin did not show an altered resistance to this pathogen. In contrast, several auxin-signaling mutants were more resistant to F. oxysporum. Chemical or genetic alteration of polar auxin transport also conferred increased pathogen resistance. Our results suggest that, similarly to many other pathogenic and nonpathogenic or beneficial soil organisms, F. oxysporum requires components of auxin signaling and transport to colonize the plant more effectively. Potential mechanisms of auxin signaling and transport-mediated F. oxysporum susceptibility are discussed.