Allene oxide synthases of barley (Hordeum vulgare cv. Salome): tissue specific regulation in seedling development

Abstract

Summary Allene oxide synthase (AOS) is the first enzyme in the lipoxygenase (LOX) pathway which leads to formation of jasmonic acid (JA). Two full‐length cDNAs of AOS designated as AOS1 and AOS2, respectively, were isolated from barley (H. vulgare cv. Salome) leaves, which represent the first AOS clones from a monocotyledonous species. For AOS1, the open reading frame encompasses 1461 bp encoding a polypeptide of 487 amino acids with calculated molecular mass of 53.4 kDa and an isoelectric point of 9.3, whereas the corresponding data of AOS2 are 1443 bp, 480 amino acids, 52.7 kDa and 7.9. Southern blot analysis revealed at least two genes. Despite the lack of a putative chloroplast signal peptide in both sequences, the protein co‐purified with chloroplasts and was localized within chloroplasts by immunocytochemical analysis. The barley AOSs, expressed in bacteria as active enzymes, catalyze the dehydration of LOX‐derived 9‐ as well as 13‐hydroperoxides of polyenoic fatty acids to the unstable allene oxides. In leaves, AOS mRNA accumulated upon treatment with jasmonates, octadecanoids and metabolizable carbohydrates, but not upon floating on abscisic acid, NaCl, Na‐salicylate or infection with powdery mildew. In developing seedlings, AOS mRNA strongly accumulated in the scutellar nodule, but less in the leaf base. Both tissues exhibited elevated JA levels. In situ hybridizations revealed the preferential occurrence of AOS mRNA in parenchymatic cells surrounding the vascular bundles of the scutellar nodule and in the young convoluted leaves as well as within the first internode. The properties of both barley AOSs, their up‐regulation of their mRNAs and their tissue specific expression suggest a role during seedling development and jasmonate biosynthesis.

Lipid metabolism in arbuscular mycorrhizal roots of Medicago truncatula

“…So far, all published data about the subcellular localization of AOS forms and AOS activity showed an association with chloroplast envelopes Maucher et al, 2000;Froehlich et al, 2001). This is in agreement with amino acid sequence data indicating that nearly all AOS proteins contain a putative chloroplast transit peptide.…”

“…Most of them prefer 13-hydroperoxides as substrates and are involved in JA production. Immunocytochemical analysis in barley revealed the association of AOS with chloroplasts (Maucher et al, 2000) and confirmed the localization of AOS activity within chloroplass in spinach and tomato leaves Froehlich et al, 2001).…”

“…To characterize the nature of the products of the enzyme reaction of StAOS3, we used [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]-9-HPOTE as substrate. The reaction product, eluting at about 7 min, showed the same retention time in the HPLC-chromatograms as the α-ketol synthesized as control by unspecific 9/13-AOS from barley (Maucher et al, 2000). Formation of 9-oxo-nonanoic acid, eluting at about 4 min, the product of a HPL, and colneleic acid (data not shown), the product of a DES, was not observed (Figure 2a).…”

“…This is in agreement with amino acid sequence data indicating that nearly all AOS proteins contain a putative chloroplast transit peptide. Also the two AOS enzymes from barley that do not contain a transit peptide could be shown to occur preferentially, though not exclusively, in chloroplasts (Maucher et al, 2000). Using the StAOS3 antibody for immunolocalization of StAOS3 in sections from roots, stolons, tubers and leaves from potato, a specific signal was detected in amyloplasts and proplastids from the subterranean organs, but not in mesophyll chloroplasts ( Figure 7).…”

“…In order to see whether LOXs are present in the plastids labelled by the StAOS3 antibody, LOX immunolocalization was performed using an antibody raised against lipid body LOX isolated from cucumber seedlings which had been shown to recognize interspecifically cytosolic and plastidic LOX and not to distinguish between 9-LOXs and 13-LOXs or membrane-bound and soluble isoforms (Feussner et al, 1995;Hause et al, 2000). In roots, stolons, tubers and leaves, LOX-specific labelling was restricted to the cytoplasm; no signal could be detected in plastids ( Figure 8).…”