CYP720A1 function in roots is required for flowering time and systemic acquired resistance in the foliage of Arabidopsis.

Systemic acquired resistance (SAR) is an inducible defense mechanism that systemically enhances resistance against pathogens in foliar tissues. SAR, which engages salicylic acid (SA) signaling, shares molecular components with the autonomous pathway, which is involved in controlling flowering time in Arabidopsis thaliana. FLOWERING LOCUS D (FLD) is one such autonomous pathway component that is required for flowering time and the systemic accumulation of SA during SAR. Here, we show that CYP720A1, a putative cytochrome P450 monoxygenase, controls FLD expression and is required for the timing of flowering and the manifestation of SAR. The delayed flowering time in the cyp720a1 mutant correlated with the elevated transcript level of the floral repressor FLC, while the SAR deficiency phenotype of the cyp720a1 mutant correlated with the inability to systemically accumulate SA. CYP720A1 transcript abundance in shoots is poor compared with roots. Reciprocal root-shoot grafting confirmed that CYP720A1 function in the roots is critical for flowering time and SAR. We therefore suggest that root to shoot communication involving a CYP720A1-dependent factor contributes to the timing of reproductive development and defense in the foliage.

Dehydroabietinal promotes flowering time and plant defense in Arabidopsis via the autonomous pathway genes FLOWERING LOCUS D, FVE, and RELATIVE OF EARLY FLOWERING 6.

Abietane diterpenoids are tricyclic diterpenes whose biological functions in angiosperms are largely unknown. Here, we show that dehydroabietinal (DA) fosters transition from the vegetative phase to reproductive development in Arabidopsis thaliana by promoting flowering time. DA's promotion of flowering time was mediated through up-regulation of the autonomous pathway genes FLOWERING LOCUS D (FLD), RELATIVE OF EARLY FLOWERING 6 (REF6), and FVE, which repress expression of FLOWERING LOCUS C (FLC), a negative regulator of the key floral integrator FLOWERING LOCUS T (FT). Our results further indicate that FLD, REF6, and FVE are also required for systemic acquired resistance (SAR), an inducible defense mechanism that is also activated by DA. However, unlike flowering time, FT was not required for DA-induced SAR. Conversely, salicylic acid, which is essential for the manifestation of SAR, was not required for the DA-promoted flowering time. Thus, although the autonomous pathway genes FLD, REF6, and FVE are involved in SAR and flowering time, these biological processes are not interdependent. We suggest that SAR and flowering time signaling pathways bifurcate at a step downstream of FLD, REF6, and FVE, with an FLC-dependent arm controlling flowering time, and an FLC-independent pathway controlling SAR.

Photochemical formation of chitosan-stabilized near-infrared-absorbing silver Nanoworms: A "Green" synthetic strategy and activity on Gram-negative pathogenic bacteria.

A facile, single-step, non-seeded photochemical protocol for producing a new type of anisotropic silver nanostructure, "nanoworms", with curved longer dimensions and smooth, rounded edges. The nanoworms exhibit surface plasmon resonance (SPR) absorption in the near-infrared window (NIRW) region and are stabilized using biocompatible polymer chitosan, rendering biocompatibility and amplified safety for biological utility of the composition. Both NIRW-absorbing nanoworms and visible-absorbing nanospheres herein are attained exclusively by employing green chemistry principles. Contrary to seed-mediated or polyol techniques, the protocol demonstrates the feasibility to selectively synthesize NIRW-absorbing silver nanostructures in a single step and in complete absence of any known reducing agent. The effect of irradiation, pH, and concentration of starting materials on the formation of nanoworms vs nanospheres is investigated in detail and analyzed by optical spectroscopy and electron microscopy. The dominant SPR obtained in the NIRW region of the nanoworms results from anisotropic AgNPs, as opposed to agglomeration. From TEM images, it is also very clear that a strong correlation exists between the SPR peak maximum and the size distribution of the anisotropic nanoworm structures, with SPR peak maximum exhibiting red shift with the increase in the size of the nanoworm population. Although there is significant size variation of different nanoworms of a given population, all samples exhibit remarkable stability. The nanoworms retained their NIRW-absorbing features even at physiological pH and at a constant ionic strength. The nanodispersions also retained their SPR features in King's B medium. Antipathogenic assays reveal that the anisotropic NIRW-absorbing nanoworms exhibit the highest growth inhibition compared to other spherical nanosilver and molecular silver forms on Gram-negative pathogenic bacteria, Pseudomonas syringae pv. maculicola ES4326 and P. syringae pv. tomato DC3000. These results underscore shape effects of AgNPs and suggest that nanoworms favor the adhesion to (curved) rod-shaped Gram-negative bacteria, resulting in the highest inhibition compared to isotropic AgNPs (smaller spheres), sulfa antibiotics (silver sulfadiazine), and silver ions (AgNO3).

Signaling by small metabolites in systemic acquired resistance.

Plants can retain the memory of a prior encounter with a pest. This memory confers upon a plant the ability to subsequently activate defenses more robustly when challenged by a pest. In plants that have retained the memory of a prior, localized, foliar infection by a pathogen, the pathogen-free distal organs develop immunity against subsequent infections by a broad-spectrum of pathogens. The long-term immunity conferred by this mechanism, which is termed systemic acquired resistance (SAR), is inheritable over a few generations. Signaling mediated by the phenolic metabolite salicylic acid (SA) is critical for the manifestation of SAR. Recent studies have described the involvement of additional small metabolites in SAR signaling, including methyl salicylate, the abietane diterpenoid dehydroabietinal, the lysine catabolite pipecolic acid, a glycerol-3-phosphate-dependent factor and the dicarboxylic acid azelaic acid. Many of these metabolites can be systemically transported through the plant and probably facilitate communication by the primary infected tissue with the distal tissues, which is essential for the activation of SAR. Some of these metabolites have been implicated in the SAR-associated rapid activation of defenses in response to subsequent exposure to the pathogen, a mechanism termed priming. Here, we summarize the role of these signaling metabolites in SAR, and the relationship between them and SA signaling in SAR.

Arabidopsis thaliana FLOWERING LOCUS D is required for systemic acquired resistance.

Localized infection in plants often induces systemic acquired resistance (SAR), which provides long-term protection against subsequent infections. A signal originating in the SAR-inducing organ is transported to the distal organs, where it stimulates salicylic acid (SA) accumulation and priming, a mechanism that results in more robust activation of defenses in response to subsequent pathogen infection. In recent years, several metabolites that promote long-distance SAR signaling have been identified. However, the mechanism or mechanisms by which plants perceive and respond to the SAR signals are largely obscure. Here, we show that, in Arabidopsis thaliana, the FLOWERING LOCUS D (FLD) is required for responding to the SAR signals leading to the systemic accumulation of SA and enhancement of disease resistance. Although the fld mutant was competent in accumulating the SAR-inducing signal, it was unable to respond to the SAR signal that accumulates in petiole exudates of wild-type leaves inoculated with a SAR-inducing pathogen. Supporting FLD's role in systemic SAR signaling, we observed that dehydroabietinal and azelaic acid, two metabolites that, in wild-type plants, promote SAR-associated systemic accumulation of SA and priming, respectively, were unable to promote SAR in the fld mutant. FLD also participates in flowering, where it functions to repress expression of the flowering repressor FLOWERING LOCUS C (FLC). However, epistasis analysis indicates that FLD's function in SAR is independent of FLC.

Biochemical and Molecular-Genetic Characterization of SFD1's Involvement in Lipid Metabolism and Defense Signaling.

The Arabidopsis thaliana SFD1 (suppressor of fatty acid desaturase deficiency1) gene (also known as GLY1) is required for accumulation of 34:6 (i.e., 18:3-16:3) monogalactosyldiacylglycerol (MGDG) and for the activation of systemic acquired resistance (SAR), an inducible defense mechanism that confers resistance against a broad spectrum of pathogens. SFD1, which has been suggested to be involved in lipid-based signaling in SAR, contains a putative chloroplast transit peptide and has glycerol-3-phosphate synthesizing dihydroxyacetone phosphate (DHAP) reductase (also referred as glycerol-3-phosphate dehydrogenase) activity. The goals of this study were to determine if the DHAP reductase activity and chloroplast localization are required for SFD1's involvement in galactolipid metabolism and SAR signaling. The crystal structure of a Leishmania mexicana glycerol-3-phosphate dehydrogenase was used to model SFD1 structure and identify Lys194, Lys279, and Asp332 as potential catalytic site residues in SFD1. Mutational analysis of SFD1 confirmed that Lys194, Lys279, and Asp332 are critical for SFD1's DHAP reductase activity, and its involvement in SAR. SFD1 proteins with these residues individually substituted by Ala lacked DHAP reductase activity and were unable to complement the SAR defect of the sfd1 mutant. The SFD1-Ala279 protein was also unable to restore 34:6-MGDG content when expressed in the sfd1 mutant. In vivo imaging of a green fluorescent protein-tagged SFD1 protein demonstrated that SFD1 is targeted to the chloroplast. The N-terminal 43 amino acids, which are required for proper targeting of SFD1 to the chloroplast, are also required for SFD1's function in lipid metabolism and SAR. Taken together, these results demonstrate that SFD1's DHAP reductase activity is required in the chloroplast for lipid metabolism and defense signaling.

An abietane diterpenoid is a potent activator of systemic acquired resistance.

Abietane diterpenoids are major constituents of conifer resins that have important industrial and medicinal applications. However, their function in plants is poorly understood. Here we show that dehydroabietinal (DA), an abietane diterpenoid, is an activator of systemic acquired resistance (SAR), which is an inducible defense mechanism that is activated in the distal, non-colonized, organs of a plant that has experienced a local foliar infection. DA was purified as a SAR-activating factor from vascular sap of Arabidopsis thaliana leaves treated with a SAR-inducing microbe. Locally applied DA is translocated through the plant and systemically induces the accumulation of salicylic acid (SA), an important activator of defense, thus leading to enhanced resistance against subsequent infections. The NPR1 (NON-EXPRESSOR OF PR GENES1), FMO1 (FLAVIN-DEPENDENT MONOOXYGENASE1) and DIR1 (DEFECTIVE IN INDUCED RESISTANCE1) genes, which are critical for biologically induced SAR, are also required for the DA-induced SAR, which is further enhanced by azelaic acid, a defense priming molecule. In response to the biological induction of SAR, DA in vascular sap is redistributed into a SAR-inducing 'signaling DA' pool that is associated with a trypsin-sensitive high molecular weight fraction, a finding that suggests that DA-orchestrated SAR involves a vascular sap protein(s).

Direct infusion mass spectrometry of oxylipin-containing Arabidopsis membrane lipids reveals varied patterns in different stress responses.

Direct infusion electrospray ionization triple quadrupole precursor scanning for three oxidized fatty acyl anions revealed 86 mass spectral peaks representing polar membrane lipids in extracts from Arabidopsis (Arabidopsis thaliana) infected with Pseudomonas syringae pv tomato DC3000 expressing AvrRpt2 (PstAvr). Quadrupole time-of-flight and Fourier transform ion cyclotron resonance mass spectrometry provided evidence for the presence of membrane lipids containing one or more oxidized acyl chains. The membrane lipids included molecular species of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, digalactosyldiacylglycerol, monogalactosyldiacylglycerol, and acylated monogalactosyldiacylglycerol. The oxidized chains were identified at the level of chemical formula and included C(18)H(27)O(3) (abbreviated 18:4-O, to indicate four double bond equivalents and one oxygen beyond the carbonyl group), C(18)H(29)O(3) (18:3-O), C(18)H(31)O(3) (18:2-O), C(18)H(29)O(4) (18:3-2O), C(18)H(31)O(4) (18:2-2O), and C(16)H(23)O(3) (16:4-O). Mass spectral signals from the polar oxidized lipid (ox-lipid) species were quantified in extracts of Arabidopsis leaves subjected to wounding, infection by PstAvr, infection by a virulent strain of P. syringae, and low temperature. Ox-lipids produced low amounts of mass spectral signal, 0.1% to 3.2% as much as obtained in typical direct infusion profiling of normal-chain membrane lipids of the same classes. Analysis of the oxidized membrane lipid species and normal-chain phosphatidic acids indicated that stress-induced ox-lipid composition differs from the basal ox-lipid composition. Additionally, different stresses result in the production of varied amounts, different timing, and different compositional patterns of stress-induced membrane lipids. These data form the basis for a working hypothesis that the stress-specific signatures of ox-lipids, like those of oxylipins, are indicative of their functions.

Involvement of salicylate and jasmonate signaling pathways in Arabidopsis interaction with Fusarium graminearum.

Fusarium graminearum is the principal causative agent of Fusarium head blight (FHB), a devastating disease of wheat and barley. This fungus can also colonize Arabidopsis thaliana. Disease resistance was enhanced in transgenic wheat and Arabidopsis plants that constitutively overexpress the NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) gene, which regulates salicylic acid (SA) signaling and modulates the activation of jasmonic acid (JA)-dependent defenses. Here, we provide several lines of evidence that reveal an important role for SA and JA signaling in Arabidopsis defense against F. graminearum. SA level was elevated in fungus-inoculated leaves, and SA application and biologically activated systemic acquired resistance enhanced resistance. Furthermore, the disruption of SA accumulation and signaling in the sid2 mutant and NahG transgenic plant, and the npr1 and wrky18 mutants, respectively, resulted in heightened susceptibility to this fungus in leaves and inflorescence. JA signaling was activated in parallel with SA signaling in the fungus-challenged plants. However, the hyperresistance of the JA pathway mutants opr3, coi1, and jar1 indicates that this pathway contributes to susceptibility. Genetic and biochemical experiments indicate that the JA pathway promotes disease by attenuating the activation of SA signaling in fungus-inoculated plants. However, the hypersusceptibility of the jar1 npr1 double mutant compared with the npr1 mutant suggests that JAR1 also contributes to defense, signifying a dichotomous role of JA and a JAR1-dependent mechanism in this interaction.

Plastid omega3-fatty acid desaturase-dependent accumulation of a systemic acquired resistance inducing activity in petiole exudates of Arabidopsis thaliana is independent of jasmonic acid.

Systemic acquired resistance (SAR) is an inducible defense mechanism that is activated throughout the plant, subsequent to localized inoculation with a pathogen. The establishment of SAR requires translocation of an unknown signal from the pathogen-inoculated leaf to the distal organs, where salicylic acid-dependent defenses are activated. We demonstrate here that petiole exudates (PeXs) collected from Arabidopsis leaves inoculated with an avirulent (Avr) Pseudomonas syringae strain promote resistance when applied to Arabidopsis, tomato (Lycopersicum esculentum) and wheat (Triticum aestivum). Arabidopsis FATTY ACID DESATURASE7 (FAD7), SUPPRESSOR OF FATTY ACID DESATURASE DEFICIENCY1 (SFD1) and SFD2 genes are required for accumulation of the SAR-inducing activity. In contrast to Avr PeX from wild-type plants, Avr PeXs from fad7, sfd1 and sfd2 mutants were unable to activate SAR when applied to wild-type plants. However, the SAR-inducing activity was reconstituted by mixing Avr PeXs collected from fad7 and sfd1 with Avr PeX from the SAR-deficient dir1 mutant. Since FAD7, SFD1 and SFD2 are involved in plastid glycerolipid biosynthesis and SAR is also compromised in the Arabidopsis monogalactosyldiacylglycerol synthase1 mutant we suggest that a plastid glycerolipid-dependent factor is required in Avr PeX along with the DIR1-encoded lipid transfer protein for long-distance signaling in SAR. FAD7-synthesized lipids provide fatty acids for synthesis of jasmonic acid (JA). However, co-infiltration of JA and methylJA with Avr PeX from fad7 and sfd1 did not reconstitute the SAR-inducing activity. In addition, JA did not co-purify with the SAR-inducing activity confirming that JA is not the mobile signal in SAR.