Vascular associated death1, a novel GRAM domain-containing protein, is a regulator of cell death and defense responses in vascular tissues

The hypersensitive response (HR) is a programmed cell death that is commonly associated with plant disease resistance. A novel lesion mimic mutant, vad1 (for vascular associated death1), that exhibits light conditional appearance of propagative HR-like lesions along the vascular system was identified. Lesion formation is associated with expression of defense genes, production of high levels of salicylic acid (SA), and increased resistance to virulent and avirulent strains of Pseudomonas syringae pv tomato. Analyses of the progeny from crosses between vad1 plants and either nahG transgenic plants, sid1, nonexpressor of PR1 (npr1), enhanced disease susceptibility1 (eds1), or non-race specific disease resistance1 (ndr1) mutants, revealed the vad1 cell death phenotype to be dependent on SA biosynthesis but NPR1 independent; in addition, both EDS1 and NDR1 are necessary for the proper timing and amplification of cell death as well as for increased resistance to Pseudomonas strains. VAD1 encodes a novel putative membrane-associated protein containing a GRAM domain, a lipid or protein binding signaling domain, and is expressed in response to pathogen infection at the vicinity of the hypersensitive lesions. VAD1 might thus represent a new potential function in cell death control associated with cells in the vicinity of vascular bundles.     

Ozone-induced ethylene production is dependent on salicylic acid,and both salicylic acid and ethylene act in concert to regulate ozone-induced cell death

Ethylene is known to influence plant defense responses including cell death in response to both biotic and abiotic stress factors. However, whether ethylene acts alone or in conjunction with other signaling pathways is not clearly understood. Ethylene overproducer mutants, eto1 and eto3, produced high levels of ethylene and developed necrotic lesions in response to an acute O3 exposure that does not induce lesions in O3-tolerant wild-type Col-0 plants. Treatment of plants with ethylene inhibitors completely blocked O3-induced ethylene production and partially attenuated O3-induced cell death. Analyses of the responses of molecular markers of specific signaling pathways indicated a relationship between salicylic acid (SA)- and ethylene-signaling pathways and O3 sensitivity. Both eto1 and eto3 plants constitutively accumulated threefold higher levels of total SA and exhibited a rapid increase in free SA and ethylene levels prior to lesion formation in response to O3 exposure. SA pre-treatments increased O3 sensitivity of Col-0, suggesting that constitutive high SA levels prime leaf tissue to exhibit increased magnitude of O3-induced cell death. NahG and npr1 plants compromised in SA signaling failed to produce ethylene in response to O3 and other stress factors suggesting that SA is required for stress-induced ethylene production. Furthermore, NahG expression in the dominant eto3 mutant attenuated ethylene-dependent PR4 expression and rescued the O3-induced HR (hypersensitive response) cell death phenotype exhibited by eto3 plants. Our results suggest that both SA and ethylene act in concert to influence cell death in O3-sensitive genotypes, and that O3-induced ethylene production is dependent on SA.     

Ethylene signaling is required for the acceleration of cell death induced by the activation of AtMEK5 in Arabidopsis

Mitogen-activated protein kinases (MAPKs) are involved in the regulation of plant growth, development and responses to a wide variety of stimuli. In a conditional gain-of-function transgenic system, the activation of AtMEK5, a MAPK kinase, can in turn activate endogenous AtMAPK3 and AtMAPK6, and can lead to a striking increase in ethylene production and induce hypersensitive response (HR)-like cell death in Arabidopsis. However, the role of the increased ethylene production in regulating this HR-like cell death remains unknown. Using Arabidopsis transgenic plants that express AtMEK5(DD), an active mutant of AtMEK5 that is under the control of a steroid-inducible promoter, we tested the contribution of ethylene to cell death. We found that ethylene biosynthesis occurs before cell death. Cell death was delayed by inhibiting AtMEK5-induced ethylene production using inhibitors of ACC-synthases, ACC-oxidases or ethylene receptors. In the mutants AtMEK5(DD)/etr1-1 and AtMEK5(DD)/ein2-1, both of which showed insensitivity to ethylene, the expression of AtMEK5(DD) protein, activity of AtMAPK3 and AtMAPK6, and ethylene production were the same as those seen in AtMEK5(DD) transgenic plants, but cell death was also delayed. These data suggest that ethylene signaling perception is required to accelerate cell death that is induced by AtMEK5 activation.     

Ethylene inhibits abscisic acid-induced stomatal closure in Arabidopsis

To examine the cross talk between the abscisic acid (ABA) and ethylene signal transduction pathways, signaling events during ABA-induced stomatal closure were examined in Arabidopsis (Arabidopsis thaliana) wild-type plants, in an ethylene-overproducing mutant (eto1-1), and in two ethylene-insensitive mutants (etr1-1 and ein3-1). Using isolated epidermal peels, stomata of wild-type plants were found to close within a few minutes in response to ABA, whereas stomata of the eto1-1 mutant showed a similar but less sensitive ABA response. In addition, ABA-induced stomatal closure could be inhibited by application of ethylene or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). In contrast, stomata of the etr1-1 and ein3-1 mutants were able to close in response to concomitant ABA and ACC application, although to a lesser extent than in wild-type plants. Moreover, expression of the ABA-induced gene RAB18 was reduced following ACC application. These results indicate that ethylene delays stomatal closure by inhibiting the ABA signaling pathway. The same inhibitive effects of ethylene on stomatal closure were observed in ABA-irrigated plants and the plants in drought condition. Furthermore, upon drought stress, the rate of transpiration was greater in eto1-1 and wild-type plants exposed to ethylene than in untreated wild-type control plants, indicating that the inhibitive effects of ethylene on ABA-induced stomatal closure were also observed in planta.     

Ethylene signaling may be involved in the regulation of tocopherol biosynthesis in Arabidopsis thaliana

Tocopherol biosynthesis was investigated in ein3-1, etr1-1 and eto1-1 mutants of Arabidopsis thaliana, which show a defect in ethylene signaling, perception and over-produce ethylene, respectively. A mutation in the EIN3 gene delayed the water-stress related increase in α-tocopherol and caused a reduction in the levels of this antioxidant by ca. 30% compared to the wild type. In contrast to the wild type and ein3-1 mutants, both etr1-1 and eto1-1 mutants showed a sharp (up to 5-fold) increase in α-tocopherol levels during leaf aging. It is concluded that ethylene perception and signaling may be involved in the regulation of tocopherol biosynthesis during water stress and leaf aging.     

Exploiting the triple response of Arabidopsis to identify ethylene-related mutants.

Alterations in the response of dark-grown seedlings to ethylene (the "triple response") were used to isolate a collection of ethylene-related mutants in Arabidopsis thaliana. Mutants displaying a constitutive response (eto1) were found to produce at least 40 times more ethylene than the wild type. The morphological defects in etiolated eto1-1 seedlings reverted to wild type under conditions in which ethylene biosynthesis or ethylene action were inhibited. Mutants that failed to display the apical hook in the absence of ethylene (his1) exhibited reduced ethylene production. In the presence of exogenous ethylene, hypocotyl and root of etiolated his1-1 seedlings were inhibited in elongation but no apical hook was observed. Mutants that were insensitive to ethylene (ein1 and ein2) produced increased amounts of ethylene, displayed hormone insensitivity in both hypocotyl and root responses, and showed an apical hook. Each of the "triple response" mutants has an effect on the shape of the seedling and on the production of the hormone. These mutants should prove to be useful tools for dissecting the mode of ethylene action in plants.     

Ethylene signaling pathway and MAPK cascades are required for AAL toxin-induced programmed cell death

Programmed cell death (PCD), known as hypersensitive response cell death, has an important role in plant defense response. The signaling pathway of PCD remains unknown. We employed AAL toxin and Nicotiana umbratica to analysis plant PCD. AAL toxin is a pathogenicity factor of the necrotrophic pathogen Alternaria alternata f. sp. lycopersici. N. umbratica is sensitive to AAL toxin, susceptible to pathogens, and effective in Tobacco rattle virus-based virus-induced gene silencing (VIGS). VIGS analyses indicated that AAL toxin-triggered cell death (ACD) is dependent upon the mitogen-activated protein (MAP) kinase kinase MEK2, which is upstream of both salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK) responsible for ethylene (ET) synthesis. ET treatment of MEK2-silenced N. umbratica re-established ACD. In SIPK- and WIPK-silenced N. umbratica, ACD was compromised and ET accumulation was not observed. However, in contrast to the case of MEK2-silenced plants, ET treatment did not induce cell death in SIPK- and WIPK-silenced plants. This work showed that ET-dependent pathway and MAP kinase cascades are required in ACD. Our results suggested that MEK2-SIPK/WIPK cascades have roles in ET biosynthesis; however, SIPK and WIPK have other roles in ET signaling or another pathway leading to cell death by AAL toxin.     

Signaling pathways that regulate the enhanced disease resistance of Arabidopsis "defense, no death" mutants

Arabidopsis dnd1 and dnd2 mutants lack cyclic nucleotide-gated ion channel proteins and carry out avirulence or resistance gene-mediated defense with a greatly reduced hypersensitive response (HR). They also exhibit elevated broad-spectrum disease resistance and constitutively elevated salicylic acid (SA) levels. We examined the contributions of NPR1, SID2 (EDS16), NDR1, and EIN2 to dnd phenotypes. Mutations that affect SA accumulation or signaling (sid2, npr1, and ndr1) abolished the enhanced resistance of dnd mutants against Pseudomonas syringae pv. tomato and Hyaloperonospora parasitica but not Botrytis cinerea. When SA-associated pathways were disrupted, the constitutive activation of NPR1-dependent and NPR1-independent and SA-dependent pathways was redirected toward PDF1.2-associated pathways. This PDF1.2 overexpression was downregulated after infection by P. syringae. Disruption of ethylene signaling abolished the enhanced resistance to B. cinerea but not P. syringae or H. parasitica. However, loss of NPR1, SID2, NDR1, or EIN2 did not detectably alter the reduced HR in dnd mutants. The susceptibility of dnd ein2 plants to B. cinerea despite their reduced-HR phenotype suggests that cell death repression is not the primary cause of dnd resistance to necrotrophic pathogens. The partial restoration of resistance to B. cinerea in dnd1 npr1 ein2 triple mutants indicated that this resistance is not entirely EIN2 dependent. The above findings indicate that the broad-spectrum resistance of dnd mutants occurs due to activation or sensitization of multiple defense pathways, yet none of the investigated pathways are required for the reduced-HR phenotype.     

Ethylene and shoot regeneration: hookless1 modulates de novo shoot organogenesis in Arabidopsis thaliana

We have investigated the role of ethylene in shoot regeneration from cotyledon explants of Arabidopsis thaliana. We examined the ethylene sensitivity of five ecotypes representing both poor and prolific shoot regenerators and identified Dijon-G, a poor regenerator, as an ecotype with dramatically enhanced ethylene sensitivity. However, inhibiting ethylene action with silver nitrate generally reduced shoot organogenesis in ecotypes capable of regeneration. In ecotype Col-0, we found that ethylene-insensitive mutants (etr1-1, ein2-1, ein4, ein7) exhibited reduced shoot regeneration rates, whereas constitutive ethylene response mutants (ctr1-1, ctr1-12) increased the proportion of explants producing shoots. Our experiments with ethylene over-production mutants (eto1, eto2 and eto3) indicate that the ethylene biosynthesis inhibitor gene, ETO1, can act as an inhibitor of shoot regeneration. Pharmacological elevation of ethylene levels was also found to significantly increase the proportion of explants regenerating shoots. We determined that the hookless1 (hls1-1) mutant, a suppressor of the ethylene response phenotypes of ctr1 and eto1 mutants, is capable of dramatically enhancing shoot organogenesis. The effects of ACC and loss of HLS1 function on shoot organogenesis were found to be largely additive.     

Ethylene advances the transition from vegetative growth to flowering in Arabidopsis thaliana

The transition from vegetative growth to flowering is the most drastic change in plant development. In order to examine the involvement of ethylene in growth transition, we compared the development of ethylene-related mutants, eto1, etr1, ein2-1 and ein3-1, with the wild type (WT) in Arabidopsis thaliana. The ethylene sensitivity of two WT and the mutants is decreased in the following order: eto1 = WT < ein3-1 < ein2-1 = etr1-1. Bolting time was also delayed in nearly the same order: eto1 < WT < ein3-1 < ein2-1 < etr1. Leaf numbers increased according to the delay of bolting time, indicating that the delay of bolting time was caused by the delay of transition from vegetative to reproductive growth. Other growth parameters, including leaf area and number of flowers opening at the same time, increased in the same order, indicating that these changes were caused by a single factor, the amount of ethylene signal which was transferred though an ethylene signal transduction pathway. These results suggest that ethylene is involved in the transition from vegetative to reproductive growth in Arabidopsis thaliana.     

Susceptibility to the sugar beet cyst nematode is modulated by ethylene signal transduction in Arabidopsis thaliana

Previously, we identified Arabidopsis thaliana mutant rhd1-4 as hypersusceptible to the sugar beet cyst nematode Heterodera schachtii. We assessed rhd1-4 as well as two other rhd1 alleles and found that each exhibited, in addition to H. schachtii hypersusceptibility, decreased root length, increased root hair length and density, and deformation of the root epidermal cells compared with wild-type A. thaliana ecotype Columbia (Col-0). Treatment of rhd1-4 and Col-0 with the ethylene inhibitors 2-aminoethoxyvinylglycine and silver nitrate and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid suggests that the rhd1-4 hypersusceptibility and root morphology phenotypes are the result of an increased ethylene response. Assessment of known ethylene mutants further support the finding that ethylene plays a role in mediating A. thaliana susceptibility to H. schachtii because mutants that overproduce ethylene (eto1-1, eto2, and eto3) are hypersusceptible to H. schachtii and mutants that are ethylene-insensitive (etr1-1, ein2-1, ein3-1, eir1-1, and axr2) are less susceptible to H. schachtii. Because the ethylene mutants tested show altered susceptibility and altered root hair density and length, a discrimination between the effects of altered ethylene signal transduction and root hair density on susceptibility was accomplished by analyzing the ttg and gl2 mutants, which produce ectopic root hairs that result in greatly increased root hair densities while maintaining normal ethylene signal transduction. The observed normal susceptibilities to H. schachtii of ttg and g12 indicate that increased root hair density, per se, does not cause hypersusceptibility. Furthermore, the results of nematode attraction assays suggest that the hypersusceptibility of rhd1-4 and the ethylene-overproducing mutant eto3 may be the result of increased attraction of H. schachtii-infective juveniles to root exudates of these plants. Our findings indicate that rhd1 is altered in its ethylene response and that ethylene signal transduction positively influences plant susceptibility to cyst nematodes.     

Identification of an essential component of the elicitation active site of the EIX protein elicitor

Defense mechanisms of plants against pathogens often entail cell wall strengthening, ethylene biosynthesis, expression of pathogen-related proteins and hypersensitive responses (HR). Pathogen-derived elicitors trigger these defense responses. The Elicitor Ethylene-inducing Xylanase (EIX) elicits HR and other plant defense responses in some tobacco and tomato cultivars independently of its xylan degradation activity. The elicitation epitope on the EIX protein responsible for inducing the HR response has been elucidated. Through the generation of EIX-specific polyclonal antibodies and screening of combinatorial phage display peptide libraries an essential sequence of the EIX elicitation activity has been identified. This sequence consists of the pentapeptide TKLGE mapped to an exposed beta-strand of the EIX protein. Substitution of the pentapeptide TKLGE to VKGT inhibited the elicitation activity but not the beta-1-4-endoxylanase activity of the EIX protein further demonstrating that elicitation and enzyme activity are independent properties. Elucidation of a peptide sequence that is essential for elicitation of HR creates the opportunity to understand the control and signaling of plant defense.     

Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis.

Activation of the plant defensin gene PDF1.2 in Arabidopsis by pathogens has been shown previously to be blocked in the ethylene response mutant ein2-1 and the jasmonate response mutant coi1-1. In this work, we have further investigated the interactions between the ethylene and jasmonate signal pathways for the induction of this defense response. Inoculation of wild-type Arabidopsis plants with the fungus Alternaria brassicicola led to a marked increase in production of jasmonic acid, and this response was not blocked in the ein2-1 mutant. Likewise, A. brassicicola infection caused stimulated emission of ethylene both in wild-type plants and in coi1-1 mutants. However, treatment of either ein2-1 or coi1-1 mutants with methyl jasmonate or ethylene did not induce PDF1.2, as it did in wild-type plants. We conclude from these experiments that both the ethylene and jasmonate signaling pathways need to be triggered concomitantly, and not sequentially, to activate PDF1.2 upon pathogen infection. In support of this idea, we observed a marked synergy between ethylene and methyl jasmonate for the induction of PDF1.2 in plants grown under sterile conditions. In contrast to the clear interdependence of the ethylene and jasmonate pathways for pathogen-induced activation of PDF1.2, functional ethylene and jasmonate signaling pathways are not required for growth responses induced by jasmonate and ethylene, respectively.     

Sublethal concentrations of salicylic acid decrease the formation of reactive oxygen species but maintain an increased nitric oxide production in the root apex of the ethylene-insensitive Never ripe tomato mutants

The pattern of salicylic acid (SA)-induced production of reactive oxygen species (ROS) and nitric oxide (NO) were different in the apex of adventitious roots in wild-type and in the ethylene-insensitive Never ripe (Nr) mutants of tomato (Solanum lycopersicum L. cv Ailsa Craig). ROS were upregulated, while NO remained at the control level in apical root tissues of wildtype plants exposed to sublethal concentrations of SA. In contrast, Nr plants expressing a defective ethylene receptor displayed a reduced level of ROS and a higher NO content in the apical root cells. In wild-type plants NO production seems to be ROS(H₂O₂)-dependent at cell death-inducing concentrations of SA, indicating that ROS and NO may interact to trigger oxidative cell death. In the absence of significant ROS accumulation, the increased NO production caused moderate reduction in cell viability in root apex of Nr plants exposed to 10⁻³ M SA. This suggests that a functional ethylene signaling pathway is necessary for the control of ROS and NO production induced by SA.Key words: ethylene receptor mutant, never ripe, nitric oxide, reactive oxygen species, root apex, salicylic acid, tomatoSeveral signal molecules, including salicylic acid (SA) have been implicated in the response of plants to biotic^1–3 and abiotic stressors.^4–6 SA was identified as a central regulator of local defense against (hemi)biotophic pathogens inducing a hypersensitive response (HR), which is characterized by the development of lesions that restrict pathogen spread. It has also emerged as a possible signaling component involved in the activation of certain plant defense responses in non-infected part of the plants establishing the systemic acquired resistance (SAR).⁷The SA-induced biotic and abiotic stress adaptation most likely involves reactive oxygen species (ROS) and nitric oxide (NO) in primary signaling events that activate multiple signal transduction pathways. SA-induced ROS is required for the activation of antioxidant defense mechanisms⁴ and if the generation of ROS exceeds the capacity of antioxidant systems, the cells die.⁸ NO is another important player that is required for the induction of defense mechanisms⁹ or for ROS-induced cell death.¹⁰Accumulation of SA, and two other plant hormones, ethylene (ET) and jasmonic acid (JA) are intimately associated with the initiation or spread of cell death. In HR SA and ROS have been proposed to be on a positive feedback loop that amplifies signals and leads to programmed cell death (PCD). Ethylene caused increased spreading of cell death, while lesion containment can be achieved by JA through decreasing the sensitivity of the cells to ethylene and through the suppression of SA biosynthesis and signaling.⁸Ethylene evolution is associated with diverse physiological processes such as leaf and flower senescence, abscission of organs and fruit ripening.¹¹ The biosynthesis of ethylene is stimulated by a variety of abiotic and biotic stress factors. Ethylene overproducing mutants (eto1 and eto3) of Arabidopsis were found to be more sensitive to O₃, an abiotic stressor which induces ROS-dependent cell death.¹² Cadmium-induced cell death was also accompanied by increased production of ethylene and simultaneously by H₂O₂ accumulation in tomato cell suspension, and based on the effect of specific inhibitors of ethylene biosynthesis and action the authors concluded that the cell death process required H₂O₂ production and a functional ethylene signaling pathway.¹³ Ethylene signaling is also required for the susceptible disease response of tomato plants infected with Xanthomonas campestris pv vesicatoria.¹⁴ It was found that the accumulation of SA and increased production of ethylene were important components of the disease symptoms of this pathogen in wild-type plants, while in Never ripe (Nr) mutants, which have a non-functional ethylene receptor, the infected plants failed to accumulate SA, produced less ethylene, and the leaves exhibited reduced necrotic lesions.It has been also shown that SA enhances NO synthesis in a dose-dependent manner.¹⁵ ROS, such as ^·O₂⁻ and H₂O₂ as well as NO can act together in the cell death regulation and propagation.^8,16 The compartment-specific (down)regulation of ROS can be controlled by NO, accordingly, ROS and NO homeostasis may be essential for the induction or for the avoidance of cell death.     

Reduced expression of the tomato ethylene receptor gene LeETR4 enhances the hypersensitive response to Xanthomonas campestris pv. vesicatoria

The hypersensitive response (HR) involves rapid death of cells at the site of pathogen infection and is thought to limit pathogen growth through the plant. Ethylene regulates senescence and developmental programmed cell death, but its role in hypersensitive cell death is less clear. Expression of two ethylene receptor genes, NR and LeETR4, is induced in tomato (Lycopersicon esculentum cv. Mill) leaves during an HR to Xanthomonas campestris pv. vesicatoria, with the greatest increase observed in LeETR4. LeETR4 antisense plants previously were shown to exhibit increased sensitivity to ethylene. These plants also exhibit greatly reduced induction of LeETR4 expression during infection and an accelerated HR at inoculum concentrations ranging from 10(5) to 10(7) CFU/ml. Increases in ethylene synthesis and pathogenesis-related gene expression are greater and more rapid in infected LeETR4 antisense plants, indicating an enhanced defense response. Populations of avirulent X. campestris pv. vesicatoria decrease more quickly and to a lower level in the transgenic plants, indicating a greater resistance to this pathogen. Because the ethylene action inhibitor 1-methylcyclopropene alleviates the enhanced HR phenotype in LeETR4 antisense plants, these changes in pathogen response are a result of increased ethylene sensitivity.     

Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana

Lateral root branching is a genetically defined and environmentally regulated process. Auxin is required for lateral root formation, and mutants that are altered in auxin synthesis, transport or signaling often have lateral root defects. Crosstalk between auxin and ethylene in root elongation has been demonstrated, but interactions between these hormones in the regulation of Arabidopsis lateral root formation are not well characterized. This study utilized Arabidopsis mutants altered in ethylene signaling and synthesis to explore the role of ethylene in lateral root formation. We find that enhanced ethylene synthesis or signaling, through the eto1-1 and ctr1-1 mutations, or through the application of 1-aminocyclopropane-1-carboxylic acid (ACC), negatively impacts lateral root formation, and is reversible by treatment with the ethylene antagonist, silver nitrate. In contrast, mutations that block ethylene responses, etr1-3 and ein2-5 , enhance root formation and render it insensitive to the effect of ACC, even though these mutants have reduced root elongation at high ACC doses. ACC treatments or the eto1-1 mutation significantly enhance radiolabeled indole-3-acetic acid (IAA) transport in both the acropetal and the basipetal directions. ein2-5 and etr1-3 have less acropetal IAA transport, and transport is no longer regulated by ACC. DR5-GUS reporter expression is also altered by ACC treatment, which is consistent with transport differences. The aux1-7 mutant, which has a defect in an IAA influx protein, is insensitive to the ethylene inhibition of root formation. aux1-7 also has ACC-insensitive acropetal and basipetal IAA transport, as well as altered DR5-GUS expression, which is consistent with ethylene altering AUX1-mediated IAA uptake, and thereby blocking lateral root formation.     

Riboflavin-induced Priming for Pathogen Defense in Arabidopsis thaliana

Riboflavin (vitamin B2) participates in a variety of redox processes that affect plant defense responses. Previously we have shown that riboflavin induces pathogen resistance in the absence of hypersensitive cell death (HCD) in plants. Herein, we report that riboflavin induces priming of defense responses in Arabidopsis thaliana toward infection by virulent Pseudomonas syringae pv. Tomato DC3000 (Pst). Induced resistance was mechanistically connected with the expression of defense response genes and cellular defense events, including H2O2 burst, HCD, and callose deposition in the plant. Riboflavin treatment and inoculation of plants with Pst were neither active but both synergized to induce defense responses. The priming process needed NPR1 (essential regulator of systemic acquired resistance) and maintenance of H2O2 burst but was independent of salicylic acid, jasmonic acid, ethylene, and abscisic acid. Our results suggest that the role of riboflavin in priming defenses is subject to a signaling process distinct from the known pathways of hormone signal transduction.