Constitutive Triple Response Research Articles

Expression of Ethylene Biosynthesis and Signaling Genes during Differential Abscission Responses of Sweet Orange Leaves and Mature Fruit

When applying abscission agents to tree fruit to facilitate harvest, it is desirable to loosen fruit and not leaves or other organs, but mechanisms controlling leaf and fruit drop are not fully understood. The effect of 450 μL·L−1 ethephon (ethylene-releasing agent) alone or in combination with 1-methylcyclopropene [1-MCP (ethylene perception inhibitor)] on leaf and mature fruit abscission of ‘Valencia’ sweet orange (Citrus sinensis) was studied. Leaf abscission increased and fruit detachment force (FDF) decreased significantly especially 4 days after ethephon treatment. Leaf drop rose to over 80% 7 days after application, whereas FDF was only 30% less than untreated control fruit. When 1-MCP was combined with ethephon and applied to ‘Valencia’ sweet orange canopies, leaf abscission was greatly reduced, but reduction in FDF proceeded unabated. We hypothesized that differential response of ‘Valencia’ sweet orange fruit and leaves to 1-MCP was correlated with expression of ethylene biosynthetic and signaling genes and their downstream action. Partial or full-length nucleotide sequences were obtained for ‘Valencia’ sweet orange homologs of 1-amino-cyclopropane-1-carboxylate synthase-1 (CsACS1), 2 (CsACS2), 1-amino-cyclopropane-1-carboxylate oxidase (CsACO), ethylene response sensor-1 (CsERS1), ethylene response-1 (CsETR1), 2 (CsETR2), 3 (CsETR3), constitutive triple response-1 (CsCTR1), ethylene insensitive-2 (CsEIN2), and ethylene insensitive 3-like-1 (CsEIL1) and 2 (CsEIL2). Ethephon application increased expression of biosynthesis genes CsACS1 and CsACO and receptors CsERS1 and CsETR2 in the abscission zones of leaves and mature fruit. Ethephon-induced increase in gene expression was completely suppressed by 1-MCP application in all but CsACS1 and CsACO in fruit abscission zones. Although gene expression was suppressed initially, CsACS1 and CsACO expression in fruit abscission zones treated with 1-MCP in the presence or absence of ethephon increased over the 7-day measurement period, suggesting that CsACS1 and CsACO expression were negatively regulated by basal ethylene production in this tissue. However, 1-MCP treatment alone did not loosen fruit, indicating that CsACS1 and CsACO played minor roles in fruit abscission. To determine if the difference in ethylene sensitivity was the basis of differential response to ethylene within the same organ, potted ‘Valencia’ sweet orange plants were treated with ethylene, and rates of blade and petiole drop and detachment forces at the laminar and petiolar abscission zones were studied. Although leaf blades abscised earlier than petioles, the force of detachment was similar, indicating no differences in ethylene sensitivity. Overall, the most significant difference between fruit and leaf abscission zones was seen in the expression of CsACS1 and CsACO genes, but the expression pattern was poorly correlated with abscission.

Ethylene improves Arabidopsis salt tolerance mainly via retaining K+ in shoots and roots rather than decreasing tissue Na+ content

Ethylene has been reported to play an essential role in the response of Arabidopsis to salinity and K+ deficiency. It was proposed that plant's ability to maintain potassium (K+) and minimize sodium (Na+) in tissues of salinity plants is critical for salt tolerance (ST). It is still unclear how ethylene modulates plant ion homeostasis under saline occasions. We employed Arabidopsis wild type (Col-0), ethylene insensitive mutants (ein2-5 and ein3-1) and constitutive triple response mutant (ctr1-1) plants to compare their phenotypic and physiological responses to salinity. Ethephon applied to plants could convert quickly to ethylene and here was applied exogenously to Col-0 seedlings to validate ethylene role in salt response. We showed that ethylene insensitivity in ein2-5 or ein3-1 plants increased Arabidopsis salt sensitivity than in Col-0. However, the salinity-induced adverse effects on Chlorophyll a/b, photosystem II function (Fv/Fm) and redox state were largely amended in the ctr1-1 than in Col-0 plants with the severe salinity. The compatible solute sucrose and antioxidant system were also up-regulated to improve ST in ctr1-1 plants. The ethephon obviously alleviated the salinity-induced restriction in root length. The subsequent analysis on the Na+ and K+ homeostasis found that ethylene could help plant retain higher shoot or root K+ nutrition in the short- or long-term salt-stressed plants. However, the ethylene did not significantly alter sodium buildup and water relation in the salt-stressed plants. Our observations confirmed the key role of ethylene in improved plant ST and highlighted the ethylene ability to retain K+, rather than decreasing Na+, in shoots and roots to improve Arabidopsis ST.

Exogenously induced expression of ethylene biosynthesis, ethylene perception, phospholipase D, and Rboh-oxidase genes in broccoli seedlings

In higher plants, copper ions, hydrogen peroxide, and cycloheximide have been recognized as very effective inducers of the transcriptional activity of genes encoding the enzymes of the ethylene biosynthesis pathway. In this report, the transcriptional patterns of genes encoding the 1-aminocyclopropane-1-carboxylate synthases (ACSs), 1-aminocyclopropane-1-carboxylate oxidases (ACOs), ETR1, ETR2, and ERS1 ethylene receptors, phospholipase D (PLD)-α1, -α2, -γ1, and -δ, and respiratory burst oxidase homologue (Rboh)-NADPH oxidase-D and -F in response to these inducers in Brassica oleracea etiolated seedlings are shown. ACS1, ACO1, ETR2, PLD-γ1, and RbohD represent genes whose expression was considerably affected by all of the inducers used. The investigations were performed on the seedlings with (i) ethylene insensitivity and (ii) a reduced level of the PLD-derived phosphatidic acid (PA). The general conclusion is that the expression of ACS1, -3, -4, -5, -7, and -11, ACO1, ETR1, ERS1, and ETR2, PLD-γ 1, and RbohD and F genes is undoubtedly under the reciprocal cross-talk of the ethylene and PAPLD signalling routes; both signals affect it in concerted or opposite ways depending on the gene or the type of stimuli. The results of these studies on broccoli seedlings are in agreement with the hypothesis that PA may directly affect the ethylene signal transduction pathway via an inhibitory effect on CTR1 (constitutive triple response 1) activity.

Ethylene regulates ascorbic acid content during dark-induced leaf senescence

The aim of this work was to determine if changes in antioxidant contents are directly controlled by ethylene or are indirect effects associated with modulation of leaf senescence. Detached spinach ( Spinacia oleracea L. cv Bison) leaves were treated with ethephon to induce ethylene production, placed in polyethylene bags and stored under darkness at 23 °C for 3 days. The ethephon treatment produced a rapid decrease on ascorbic acid (AA) content and an increase in its oxidised state before any leaf senescence parameters changed. Both AA synthesis and recovery from oxidised forms were affected by ethylene. In addition, leaves from Arabidopsis ( Arabidopsis thaliana (L.) Heynh) ethylene insensitive mutants ( ein2-1, ein3-1 and ein4) presented higher AA content than leaves from wt plants before dark storage. Furthermore, ein3-1 and ein4 but not ein2-1, showed slower leaf AA content declining than wt plants under dark-induced leaf senescence. The Arabidopsis mutant with constitutive triple response ( ctr1-1) showed lower leaf AA content than wt plants but similar AA degradation rate with a higher oxidised state after dark storage for 3 days. These results demonstrate that ethylene is an important factor controlling AA content in mature leaves.

Ethylene Interacts with Abscisic Acid to Regulate Endosperm Rupture during Germination: A Comparative Approach UsingLepidium sativumandArabidopsis thaliana

The micropylar endosperm cap covering the radicle in the mature seeds of most angiosperms acts as a constraint that regulates seed germination. Here, we report on a comparative seed biology study with the close Brassicaceae relatives Lepidium sativum and Arabidopsis thaliana showing that ethylene biosynthesis and signaling regulate seed germination by a mechanism that requires the coordinated action of the radicle and the endosperm cap. The larger seed size of Lepidium allows direct tissue-specific biomechanical, biochemical, and transcriptome analyses. We show that ethylene promotes endosperm cap weakening of Lepidium and endosperm rupture of both species and that it counteracts the inhibitory action of abscisic acid (ABA) on these two processes. Cross-species microarrays of the Lepidium micropylar endosperm cap and the radicle show that the ethylene-ABA antagonism involves both tissues and has the micropylar endosperm cap as a major target. Ethylene counteracts the ABA-induced inhibition without affecting seed ABA levels. The Arabidopsis loss-of-function mutants ACC oxidase2 (aco2; ethylene biosynthesis) and constitutive triple response1 (ethylene signaling) are impaired in the 1-aminocyclopropane-1-carboxylic acid (ACC)-mediated reversion of the ABA-induced inhibition of seed germination. Ethylene production by the ACC oxidase orthologs Lepidium ACO2 and Arabidopsis ACO2 appears to be a key regulatory step. Endosperm cap weakening and rupture are promoted by ethylene and inhibited by ABA to regulate germination in a process conserved across the Brassicaceae.

Local auxin biosynthesis modulates gradient-directed planar polarity in Arabidopsis

The coordination of cell polarity within the plane of a single tissue layer (planar polarity) is a crucial task during development of multicellular organisms. Mechanisms underlying establishment of planar polarity, however, differ substantially between plants and animals. In Arabidopsis thaliana, planar polarity of root-hair positioning along epidermal cells is coordinated towards maximum concentration of an auxin gradient in the root tip. This gradient has been hypothesized to be sink-driven and computational modelling suggests that auxin efflux carrier activity may be sufficient to generate the gradient in the absence of auxin biosynthesis in the root. Here, we demonstrate that the Raf-like kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1; Refs 8, 9) acts as a concentration-dependent repressor of a biosynthesis-dependent auxin gradient that modulates planar polarity in the root tip. We analysed auxin biosynthesis and concentration gradients in a variety of root-hair-position mutants affected in CTR1 activity, auxin biosynthesis and transport. Our results reveal that planar polarity relies on influx- and efflux-carrier-mediated auxin redistribution from a local biosynthesis maximum. Thus, a local source of auxin biosynthesis contributes to gradient homeostasis during long-range coordination of cellular morphogenesis.

Mitogen-Activated Protein Kinase Cascades and Ethylene: Signaling, Biosynthesis, or Both?: Figure 1.

Mitogen-activated protein kinase (MAPK) cascades serve as important signaling pathways in all eukaryotic cells, as they link the perception of external stimuli to cellular responses. Since the discovery of CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), a Raf-like MAPK kinase kinase (MAPKKK), there has been

PA, a stress-induced short cut to switch-on ethylene signalling by switching-off CTR1?

Constitutive triple response 1 (CTR1) is a protein kinase that represses plant responses to ethylene. Recently, we have shown that CTR1 function is negatively regulated by the lipid second messenger phosphatidic acid (PA) in vitro. PA was shown to inhibit (1) CTR1’s protein kinase activity, (2) the intramolecular interaction between N-terminus and kinase domain, and (3) the interaction of CTR1 with the ethylene receptor ETR1. PA typically accumulates within minutes in response to biotic or abiotic stresses, which are known to induce ethylene formation. Although long-term treatment with ethephon does stimulate PA accumulation, our results show no fast increase in PA in response to ethylene. A speculative model is presented which explains how stress-induced PA formation could switch on downstream ethylene responses via interaction of the lipid with CTR1.

Dual control of nuclear EIN3 by bifurcate MAPK cascades in C2H4 signalling

A principal question in MAP kinase (MAPK/MPK) cascade signalling is how similar components dictate different specificity in the information-processing machineries from yeast to humans and plants. In Arabidopsis, how MPK3/6 modulates distinct outputs in diverse signal transduction pathways remains elusive. By combining systematic cellular and genetic screens, here we uncover a previously unexpected MKK9-MPK3/MPK6 cascade promoting ethylene-insensitive 3 (EIN3)-mediated transcription in ethylene signalling. The mkk9 mutant exhibits a broad spectrum of moderate ethylene-insensitive phenotypes, and translocated MKK9 governs nuclear signalling downstream of receptors. Breaking a linear model and conventional MAPK signalling, ethylene inactivates the negative regulator constitutive triple response 1 (CTR1, a Raf-like MAPK kinase kinase (MAPKKK)) to activate the positive MKK9-MPK3/6 cascade. The bifurcate and antagonistic CTR1 and MKK9 pathways are both critical in determining ethylene-signalling specificity through two MAPK phosphorylation sites with opposite effects on EIN3 stability. The results suggest a new paradigm for linking intertwined MAPK cascades to control quantitative responses and specificity in signalling networks.

Phosphatidic acid binds to and inhibits the activity of Arabidopsis CTR1

Phosphatidic acid (PA) has only recently been identified as an important eukaryotic lipid-signalling molecule. In plants, PA formation is triggered by various biotic and abiotic stresses, including wounding, pathogen attack, drought, salinity, cold, and freezing. However, few molecular targets of PA have been identified so far. One of the best characterized is Raf-1, a mammalian MAPKKK. Arabidopsis thaliana CTR1 (constitutive triple response 1) is one of the plant homologues of Raf-1 and functions as a negative regulator of the ethylene signalling pathway. Here, it is shown that PA binds CTR1 and inhibits its kinase activity. Using different PA-binding assays, the kinase domain of CTR1 (CTR1-K) was found to bind PA directly. Addition of PA resulted in almost complete inhibition of CTR1 kinase activity and disrupted the intramolecular interaction between CTR1-K and the CTR1 N-terminal regulatory domain. Additionally, PA blocked the interaction of CTR1 with ETR1, one of the ethylene receptors. The basic amino acid motif shown to be required for PA binding in Raf-1 is conserved in CTR1-K. However, mutations in this motif did not affect either PA-binding or PA-dependent inhibition of CTR1 activity. Subsequent deletion analysis of CTR1's kinase domain revealed a novel PA-binding region at the C-terminus of the kinase.

A gaseous plant hormone ethylene: the signaling pathway

Phytohormone ethylene has profound effects on growth and development in plants. Molecular genetic studies usingArabidopsis have defined a linear pathway for ethylene signal transduction leading from initial hormone perception to changes in gene expression. Ethylene is perceived by a family of ethylene receptor complex at endoplasmic reticulum (ER), which negatively regulates the ethylene response. Ethylene binding inactivates the receptors and represses the Raf-like kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) that actively represses ethylene response pathway in the absence of ethylene. Consequently, the ETHYLENE INSENSITIVE2 (EIN2), a membrane protein with similarities to Nramp metal ion transporter becomes activated and positively regulates the ethylene signaling pathway by transmitting the signal into the nucleus. Finally, the nuclear signal initiates the transcriptional cascade via the transcription factors ETHYLENE INSENSITIV3/ETHYLENE INSENSITIVE3-LIKE proteins (EIN3/EILs). This review will summarize the up-to-date understanding of ethylene signal transduction, in aiming to illustrate how challenges in hormone biology have been resolved through the power of molecular genetics and to provide references for interested readers searching for further information.Keywords: Arabidopsis, ethylene, hormone, signaling

Dissecting the ethylene pathway of Arabidopsis

The plant hormone ethylene regulates growth, development and stress responses. In recent years, various genomic and proteomic approaches have been initiated to understand both the range of ethylene responses in the plant and the mechanism of signal transduction. Transcriptional profiling experiments reveal broad-ranging effects of ethylene upon gene regulation, with up to 7 per cent of the genes examined demonstrating a significant level of response in one study. Both transcriptional and post-transcriptional mechanisms regulate the expression of components within the ethylene signal transduction pathway. The importance of post-transcriptional regulation via the ubiquitin/proteasome-mediated degradation pathway is apparent in studies on the accumulation of ethylene insensitive 3 (EIN3), a key transcription factor in the pathway. Protein complexes also play a role in modulating ethylene signal transduction, with interactions between the ethylene receptors and the Raf-like kinase constitutive triple response-1 (CTR1) being required for ethylene perception at the endoplasmic reticulum. In this paper, recent developments in unravelling the transcriptional and post-transcriptional regulation of the ethylene signalling and response pathways are considered, along with the latest developments in unravelling the biochemical mechanism behind ethylene perception.

Co-expression of an ethylene receptor gene, ERS1, and ethylene signaling regulator gene, CTR1, in Delphinium during abscission of florets

We are trying to determine the mechanisms responsible for ethylene-induced floret abscission in cut flowers of Delphinium and recently identified an ethylene receptor gene, ERS1, and studied its response to ethylene treatment. In order to identify additional components of the ethylene response network in Delphinium, we performed 3′ and 5′ rapid amplification of cDNA ends (RACE) using the consensus sequence of the serine/threonine kinase domain of the ethylene signaling regulator gene (CTR1) involved in the constitutive triple response (CTR) to ethylene. The full-length cDNA (2754 nt) encoded a protein of 800 amino acids, which contained the expected serine/threonine kinase domain, the consensus ATP-binding site, and the serine/threonine kinase catalytic site. The protein had quite high (>50%) overall identity to CTR1 from Arabidopsis and tomato, and 70––75% identity in the catalytic site. The amount of mRNA encoding both CTR1 and ERS1 more than doubled within 6 h in cut florets incubated in the presence of exogenous ethylene. Similarly, the amount of ERS1 transcript doubled in florets within 6 d of harvesting, presumably in response to endogenous ethylene, while CTR1 mRNA increased to about 40% over the same period. However, in the presence of silver thiosulfate (STS), an ethylene inhibitor, the level of both transcripts remained essentially unchanged for the first 8 d before declining to very low levels. Florets on the control plants had almost completely abscised by 6 d, but the florets on STS-treated plants had not abscised by 20 d, by which time the flowers were almost dead. The data are consistent with the hypothesis that endogenous ethylene evokes the accumulation of both these transcripts (and their encoded proteins), thereby speeding up abscission and reducing the useful shelf life of the cut flowers.

Mutual antagonism of ethylene and jasmonic acid regulates ozone-induced spreading cell death in Arabidopsis.

Ethylene (ET) and jasmonic acid (JA) have opposite effects on ozone (O(3))-induced spreading cell death; ET stimulates, and is required for the spreading cell death, whereas JA protects tissues. We studied the underlying molecular mechanisms with the O(3)-sensitive, JA-insensitive jasmonate resistant 1 (jar1), and the O(3)-tolerant, ET-insensitive ethylene insensitive 2 (ein2) mutants. Blocking ET perception pharmacologically with norbornadiene (NBD) in jar1, or ET signaling genetically in the jar1 ein2 double mutant prevented the spread of cell death. This suggests that EIN2 function is epistatic to JAR1, and that the JAR1-dependent JA pathway halts oxidative cell death by directly inhibiting ET signaling. JAR1-dependent suppression of the ET pathway was apparent also as increased EIN2-dependent gene expression and ET hypersensitivity of jar1. Physiological experiments suggested that the target of JA is upstream of Constitutive Triple Response 1 (CTR1), but downstream of ET biosynthesis. Gene expression analysis of 1-aminocyclopropane-1-carboxylic acid (ACC)-treated and O(3)-exposed ein2 and jar1 revealed reciprocal antagonism: the EIN2-mediated suppression of the JA pathway. The results imply that the O(3)-induced spreading cell death is stimulated by early, rapid accumulation of ET, which can suppress the protecting function of JA thereby allowing cell death to proceed. Extended spreading cell death induces late accumulation of JA, which inhibits the propagation of cell death through inhibition of the ET pathway.

Growth regulators and the control of nucleotide sugar flux.

A small number of plant growth regulators are involved in the control of cell expansion. Despite knowledge of some of their signal transduction cascades, surprisingly little is known of how basic cell expansion-related processes, such as cell wall biosynthesis, are affected during growth. The Arabidopsis (Arabidopsis thaliana) mutant root hair defective1 (rhd1) lacks a functional UDP-glucose 4-epimerase gene, UGE4, which is involved in channeling UDP-D-galactose (UDP-D-Gal) into cell wall polymers. Here, we use rhd1 as a genetic model to analyze the physiological and genetic controls of nucleotide sugar flux. We find that ethylene specifically suppresses all visible aspects of the rhd1 phenotype. The ethylene-triggered suppression of rhd1 is negatively regulated by CONSTITUTIVE TRIPLE RESPONSE1 and requires the function of the wild-type genes ETHYLENE INSENSITIVE2 (EIN2), EIN4, AUXIN-RESISTENT1, and ETHYLENE-INSENSITIVE ROOT1 but does not depend on the activity of wild-type ETHYLENE RECEPTOR1 or EIN3 genes, highlighting the nonlinearity of ethylene signal transduction. Ethylene does not induce the expression of alternative UGE genes but, instead, suppresses the expression of two isoforms, UGE1 and UGE3, in a tissue-specific manner. Ethylene restores the biosynthesis of galactose-containing xyloglucan and arabinosylated galactan cell wall polymers in rhd1 back to wild-type levels. However, the dependence on UGE4 of pectic (1-->4)-beta-D-galactan and glucuronosyl-modified AGP biosynthesis is exacerbated. Our data suggest that ethylene and auxin together participate in the flux control of UDP-D-Gal into cell wall polymers and that the genetic control of this process is qualitatively distinct from previously described responses to ethylene.

Three genes that affect sugar sensing (abscisic acid insensitive 4, abscisic acid insensitive 5, and constitutive triple response 1) are differentially regulated by glucose in Arabidopsis.

Mutant characterization has demonstrated that ABI4 (Abscisic Acid [ABA] Insensitive 4), ABI5 (ABA Insensitive 5), and CTR1 (Constitutive Triple Response 1) genes play an important role in the sugar signaling response in plants. The present study shows that the transcripts of these three genes are modulated by glucose (Glc) independently of the developmental arrest caused by high Glc concentrations. ABI4 and ABI5 transcripts accumulate in response to sugars, whereas the CTR1 transcript is transiently reduced followed by a rapid recovery. The results of our kinetic studies on gene expression indicate that ABI4, ABI5, and CTR1 are regulated by multiple signals including Glc, osmotic stress, and ABA. However, the differential expression profiles caused by these treatments suggest that distinct signaling pathways are used for each signal. ABI4 and ABI5 response to the Glc analog 2-deoxy-Glc supports this conclusion. Glc regulation of ABI4 and CTR1 transcripts is dependent on the developmental stage. Finally, the Glc-mediated regulation of ABI4 and ABI5 is affected in mutants displaying Glc-insensitive phenotypes such as gins, abas, abi4, abi5, and ctr1 but not in abi1-1, abi2-1, and abi3-1, which do not show a Glc-insensitive phenotype. The capacity of transcription factors, like the ones analyzed in this work, to be regulated by a variety of signals might contribute to the ability of plants to respond in a flexible and integral way to continuous changes in the internal and external environment.

Molecular and biochemical characterization of VR-EILs encoding mung bean ETHYLENE INSENSITIVE3-LIKE proteins.

ETHYLENE INSENSITIVE3 (EIN3) is a transcription factor involved in the ethylene signal transduction pathway in Arabidopsis. Two full-length cDNA clones, pVR-EIL1 and pVR-EIL2, encoding EIN3-LIKE proteins were isolated by reverse transcriptase-polymerase chain reaction and by screening the cDNA library of mung bean (Vigna radiata) hypocotyls. VR-EIL1 and VR-EIL2 share 70% identity and display varying degrees of sequence conservation (39%-65%) with previously isolated EIN3 homologs from Arabidopsis, tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum) plants. Gel retardation assay revealed that both VR-EILs were able to interact specifically with optimal binding sequence-1, the recently identified optimal binding sequence for tobacco TEIL, with the binding of VR-EIL2 being more efficient than that of VR-EIL1. Transient expression analysis using a VR-EIL::smGFP fusion gene in onion (Allium cepa) epidermal cells indicated that the VR-EIL proteins were effectively targeted to the nucleus. The fusion protein of VR-EIL2 with GAL4 DNA-binding domain strongly activated transcription of a reporter gene in yeast cells, and an essential domain for transcription-stimulating activity was localized to the amino-terminal acidic region that consists of 50 amino acid residues. In contrast with what has been previously found in EIN3- and TEIL-overexpressing Arabidopsis plants, transgenic tobacco seedlings expressing the VR-EIL genes under the control of cauliflower mosaic virus 35S promoter did not exhibit a constitutive triple response. Instead, they displayed a markedly enhanced proliferation of root hairs, one of the typical ethylene response phenotypes, and increased sensitivity to exogenous ethylene. In addition, the pathogenesis-related (PR) genes encoding beta-1,3-glucanase, osmotin, and PR1 were constitutively expressed in 35S::VR-EIL lines without added ethylene, and were hyperinduced in response to ethylene treatment. These results indicate that VR-EILs are functional in tobacco cells, thereby effectively transactivating the GCC-box-containing PR genes and enhancing sensitivity to ethylene. The possible physiological role of VR-EILs is discussed in the light of the suggestion that they are active components of the ethylene-signaling pathway and their heterologous expressions constitutively turn on a subset of ethylene responses in tobacco plants.

Modulation of CYP79 genes and glucosinolate profiles in Arabidopsis by defense signaling pathways.

Glucosinolates are natural plant products that function in the defense toward herbivores and pathogens. Plant defense is regulated by multiple signal transduction pathways in which salicylic acid (SA), jasmonic acid, and ethylene function as signaling molecules. Glucosinolate content was analyzed in Arabidopsis wild-type plants in response to single or combinatorial treatments with methyljasmonate (MeJA), 2,6-dichloro-isonicotinic acid, ethylene, and 2,4-dichloro-phenoxyacetic acid, or by wounding. In addition, several signal transduction mutants and the SA-depleted transgenic NahG line were analyzed. In parallel, expression of glucosinolate biosynthetic genes of the CYP79 gene family and the UDPG:thiohydroximate glucosyltransferase was monitored. After MeJA treatment, the amount of indole glucosinolates increased 3- to 4-fold, and the corresponding Trp-metabolizing genes CYP79B2 and CYP79B3 were both highly induced. Specifically, the indole glucosinolate N-methoxy-indol-3-ylmethylglucosinolate accumulated 10-fold in response to MeJA treatment, whereas 4-methoxy-indol-3-ylmethylglucosinolate accumulated 1.5-fold in response to 2,6-dichloro-isonicotinic acid. In general, few changes were seen for the levels of aliphatic glucosinolates, although increases in the levels of 8-methylthiooctyl glucosinolate and 8-methylsulfinyloctyl glucosinolate were observed, particularly after MeJA treatments. The findings were supported by the composition of glucosinolates in the coronatine-insensitive mutant coi1, the ctr1 mutant displaying constitutive triple response, and the SA-overproducing mpk4 and cpr1 mutants. The present data indicate that different indole glucosinolate methoxylating enzymes are induced by the jasmonate and the SA signal transduction pathways, whereas the aliphatic glucosinolates appear to be primarily genetically and not environmentally controlled. Thus, different defense pathways activate subsets of biosynthetic enzymes, leading to the accumulation of specific glucosinolates.

Cloning and DNA-binding properties of a tobacco Ethylene-Insensitive3 (EIN3) homolog.

Ethylene-Insensitive3 (EIN3) is a transcription factor that works in the ethylene signaling pathway in Arabidopsis. We isolated a tobacco cDNA encoding an EIN3 homolog as a sequence-specific DNA-binding protein. The encoded protein TEIL (tobaccoEIN3-like) shares 60% identity in amino acid sequence with EIN3. The DNA-binding domain was localized in the N-terminal half, which shows 92% identity in amino acid sequence with the corresponding region of EIN3, suggesting a conserved function in DNA-binding specificity. TEIL was indeed functionally similar to EIN3 because, like EIN3-overexpressing plants, transgenic Arabidopsis seedlings overexpressing TEIL cDNA exhibited constitutive triple response phenotypes. Random binding site selection analysis revealed that the consensus binding sequence for TEIL is AYGWAYCT, where Y and W represent A or C and A or T, respectively. A reporter plasmid containing the TEIL binding sites showed a 7- to 10-fold higher activation relative to that containing a mutated TEIL-binding sequence in tobacco protoplasts. A further 2- to 3-fold increase in activation was observed when a plasmid for TEIL overproduction was co-transfected, indicating that TEIL is a transcriptional activator. Moreover, nuclear extracts from ethylene-treated leaves showed an increase in DNA-binding activity specific to the TEIL-binding sequence, despite the level of the transcripts being unchanged. These observations suggest that TEIL functions as a transcription activator with a relatively redundant DNA-binding specificity, and its function may be regulated at least in part by modulation of the DNA-binding activity through ethylene signaling.

Involvement of ethylene and gibberellin signalings in chromosaponin I-induced cell division and cell elongation in the roots of Arabidopsis seedlings.

Chromosaponin I (CSI), a triterpenoid saponin isolated from pea, stimulates the growth of roots in Arabidopsis thaliana seedlings on wetted filter paper in the light for 14 d. The growth rates of roots in Columbia (Col) and Landsberg erecta (Ler) wild-types were 0.92 and 0.26 mm d(-1), respectively, and they were accelerated to 3.46 (Col) and 2.20 (Ler) mm d(-1) by treating with 300 microM CSI. The length of mature epidermal cells was increased by 1.8-fold (Col) and 2.81-fold (Ler) compared with control and the number of epidermal cells was increased by a factor of 1.65 (Col) and 2.12 (Ler). Treatment with 2-aminoethoxyvinylglycine (AVG), an inhibitor of ethylene biosynthesis, also increased cell length but not cell number. The effects of CSI on root growth were not detected in the ethylene-insensitive mutant ein2-1. CSI did not inhibit ethylene production but stimulated the growth of roots in ctr1-1, the constitutive triple response mutant for ethylene, indicating that CSI inhibits ethylene signaling, especially downstream of CTR1. In the GA-insensitive mutant gai and the mutant spy-3, in which the basal level of GA signaling is activated, CSI did not increase cell number, although both CSI and AVG stimulated cell elongation in these mutants. These results suggest that the inhibition of ethylene signaling is the cause of CSI-induced cell elongation. A possible involvement of both GA and ethylene signalings is discussed for the CSI-induced cell division.