Photoperiodic control of growth, development and phytohormone balance in Solanum tuberosum

Some lines of Solanum tuberosum ssp. andigena are strictly photoperiodic, forming tubers only in short days and flowers only in long days. We used this advantageous phenomenon to study phytohormone involvement in the development of the plants, mainly that of tuber formation. Plants grown for 2 months under short days (SD) of 14 h darkness, night break (1 h white light in the middle of the 14‐h dark period) and continuous light (LD) were compared. Short day‐grown plants formed tubers, while plants in LD flowered. Night break prevented tuber formation, but caused flowering, although it was weaker than in LD. Plants grown under night break displayed many growth characteristics intermediate between SD and LD. Under LD and night break regimes ABA levels in all organs were about one‐fourth of those under SD. An opposite trend was found for gibberellin content: it was very low in SD‐grown plants and 4‐10 times higher under both other conditions with the exception of roots and stolons in night break. Thus, the ratio of ABA/GA, known to be important for tuber formation, was high in SD and low in night break and LD. The level of free indoleacetic acid (IAA) was high in LD‐ and night break‐grown plants and it was much lower in SD‐grown plants, including tubers. Cytokinin (CK) levels were also high in LD‐ and night break‐grown plants. In SD, leaves had slightly decreased and stems and roots + stolons had more pronouncedly decreased cytokinin levels. The CK/IAA ratio was thus higher in SD in stems and roots + stolons; it was also high in tubers and no difference was found in leaves. These results indicate that the effect of photoperiod on tuber formation and development may be mediated by photoperiod‐induced changes in hormone levels.     

Occurrence of jasmonic acid in organs of Solanum tuberosum L. and its effect on tuberization

The aims of this study were to demonstrate the endogenous presence of jasmonic acid (JA) in roots, stolons and periderm of new formed tubers, by means of bioassays, ELISA and GC-MS, and to test a microdrop bioassay using the leaflets of potato cuttings cultured in vitro. Our results confirm the existence of JA by bioassays and GC-MS in foliage, stolons, roots and tuber periderm.Abbreviations DW dry weight - GC-MS gas chromatography-mass spectrometry - JA jasmonic acid - MeOH methanol - SD short day     

Hydroxylated jasmonates are commonly occurring metabolites of jasmonic acid and contribute to a partial switch-off in jasmonate signaling

In potato 12-hydroxyjasmonic acid (12-OH-JA) is a tuber-inducing compound. Here, it is demonstrated that 12-OH-JA, as well as its sulfated and glucosylated derivatives, are constituents of various organs of many plant species. All accumulate differentially and usually to much higher concentrations than jasmonic acid (JA). In wounded tomato leaves, 12-OH-JA and its sulfated, as well as glucosylated, derivative accumulate after JA, and their diminished accumulation in wounded leaves of the JA-deficient mutants spr2 and acx1 and also a JA-deficient 35S::AOCantisense line suggest their JA-dependent formation. To elucidate how signaling properties of JA/JAME (jasmonic acid methyl ester) are affected by hydroxylation and sulfation, germination and root growth were recorded in the presence of the different jasmonates, indicating that 12-OH-JA and 12-hydroxyjasmonic acid sulfate (12-HSO(4)-JA) were not bioactive. Expression analyses for 29 genes showed that expression of wound-inducible genes such as those coding for PROTEINASE INHIBITOR2, POLYPHENOL OXIDASE, THREONINE DEAMINASE or ARGINASE was induced by JAME and less induced or even down-regulated by 12-OH-JA and 12-HSO(4)-JA. Almost all genes coding for enzymes in JA biosynthesis were up-regulated by JAME but down-regulated by 12-OH-JA and 12-HSO(4)-JA. The data suggest that wound-induced metabolic conversion of JA/JAME into 12-OH-JA alters expression pattern of genes including a switch off in JA signaling for a subset of genes.     

Allene oxide cyclase is essential for theobroxide-induced jasmonic acid biosynthesis in Pharbitis nil

Theobroxide, a natural product, strongly stimulates the biosynthesis of jasmonic acid (JA) in Pharbitis nil. In this study, we investigated the accumulation of protein by the immunoblot analysis of lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC), key enzymes in JA biosynthesis, and how the endogenous levels of JA in P. nil are affected by theobroxide. The effect of JA on the accumulations of these proteins was monitored simultaneously. The results show that theobroxide treatment led to a high level accumulation of JA, which is due to high accumulations of LOX, AOS, and AOC proteins induced by theobroxide treatment both under short day (SD) and long day (LD) conditions. However, under SD conditions AOS and AOC proteins are not enhanced by JA treatment. Kinetic analysis of protein levels shows that a biphasic activation of AOC protein by theobroxide is displayed and the first activation of AOC protein together with elevated JA levels is observed within 30min after treatment. Meanwhile, AOS and LOX proteins are activated by theobroxide later than AOC protein, suggesting that AOC plays an essential role in the initial JA formation induced by theobroxide. Since theobroxide-increased JA levels also show a biphasic manner similar to AOC activation and AOS, LOX proteins are activated later than AOC, and thus we propose a positive JA feedback regulation. Interestingly, AOS protein, which is also the enzyme for the biosynthesis of 9,10-ketol-octadecadienoic acid (KODA, a flowering inducing factor), accumulates markedly due to the simultaneous involvement of theobroxide and SD conditions, suggesting that AOS probably plays a role in flower bud formation in P. nil.     

Relation Between Environmental Factors and the LOX Activities Upon Potato Tuber Formation and Flower-bud Formation in Morning Glory

Life cycles of plants including tuberization and flowering are strongly related to environmental factors such as photoperiod and temperature. Theobroxide induces potato tuber formation and flower bud formation of morning glory under non-inductive conditions and stimulates the activity of lipoxygenase (LOX). In this study, to understand the LOX activity more systematically, the relationships between LOX activity and light and temperature, which effects potato tuber and flower-bud formation, have been investigated. The results showed that LOX activity in morning glory was greatly enhanced up to 30 min and then declined after switching from the light to the dark condition, while the activity did not vary when switching from the dark to the light condition. In addition, the temperature profile of measured LOX activity in the potato and morning glory plants was nearly consistent with the time taken to form potato tubers and flower buds in morning glory, respectively, at different growing temperatures. These results strongly suggest that LOX activity is directly connected with light and temperature to regulate the formation of tubers and flower-buds.     

Theobroxide induces tubers in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) and flower buds in morning glory (<Emphasis Type="Italic">Pharbitis nil</Emphasis>) under non-inductive high temperatures

Induction of some plant organs including tubers and flower buds begins with sensing environmental cues, such as photoperiod and temperature in the leaves. Theobroxide has been shown to induce potato tuberization and flower-bud formation in morning glory under non-inductive photoperiodic conditions, stimulating the activity of lipoxygenase (LOX) and the synthesis of jasmonic acid (JA). In the present study, the ability of theobroxide to overcome the inhibitory effect of unfavorable high temperature on the induction of tubers in potato and flower buds in morning glory was examined. Both tuber induction and flower-bud formation under non-inductive high temperatures were promoted by the application of theobroxide at a high concentration. However, although theobroxide treatment resulted in an increase in fresh weight during potato tuber growth at 30°C, morning glory plants treated with theobroxide at 35°C failed to bloom, implying that theobroxide may assist only in flower-bud formation.     

Temperature regulates tuber-inducing lipoxygenase-derived metabolites in potato (Solanum tuberosum)

Temperature is one of the major environmental factors affecting potato tuberization. It has been suggested that lipoxygenase (LOX) mediates between temperature and tuber induction. In this study, the contents of the LOX-derived metabolites hydroperoxylinolenic acid (HPOT), jasmonic acid (JA), tuberonic acid (TA) and tuberonic acid glucoside (TAG) were analyzed in leaves of potatoes growing at different temperatures. At low, tuber-inducing temperature, endogenous levels of JA, TA and TAG rise, indicating their crucial role in tuber induction. The concentration of 13(S)-HPOT seems not to be directly affected by temperature. Instead, the molecule has only a short half-life in leaves and is readily metabolized.     

Jasmonic Acid is Induced in a Biphasic Manner in Response of Pea Seedlings to Wounding

The role of jasmonic acid (JA) in plant wounding response has been demonstrated. However, the source of JA in wound signaling remains unclear. In the present study, pea seedlings were used as material to investigate the systemic induction of JA and the activation of lipoxygenase (LOX)-dependent octadecanoid pathway upon wounding. The results showed that endogenous JA could induce two peaks in the wounded leaves and the stalks, while only one peak in the systemic leaves.LOX activity and its protein amount were also induced and the stimulation mainly occurred in the late phase, while one peak of induction was present after pretreatment with JA. Applied nordihydroguaiaretic acid (NDGA), an inhibitor of LOX activity, only inhibited the induction of JA in the late phase, and the resistance of pea was impaired. Furthermore, 13(S)-hydroperoxy-9(Z), 11 (E)-octadecadienoic acid (13(S)-H(P)ODE) was confirmed to be the main product of LOX throughout the experimental time. In addition, immunocytochemical analysis also revealed the occurrence of JA biosynthesis and transport upon wounding. These results demonstrated that wound-induced JA in wounded leaves resulted from Its biosynthesis and conversion from its conjugates, while in systemic leaves resulted from its transport and biosynthesis; and proved that the LOX pathway was vital to the wound-induced defense response involved in JA biosynthesis.     

Use of the growth retardant tetcyclacis for potato tuber formation in vitro

The effects of the plant growth retardant tetcyclacis on in vitro tuber formation in potatoes was studied, using two different approaches: 1. tuber formation in various lines that did not or hardly form tubers under control conditions, and 2. tuber formation by the variety Bintje, which readily forms tubers. The ABA-deficient (droopy) lines of S. phureja hardly formed tubers without the addition of tetcyclacis. In the presence of this growth retardant tuberization was nearly 100%, within three weeks of in vitro culture, even in the absence of cytokinin. A series of somatic hybrids between S. tuberosum and S. brevidens, that did not form tubers in field and pot experiments, were tested. They all formed tubers in vitro in the presence of tetcyclacis. Stoloniferous shoots formed on single-node cuttings from in vitro grown Solanum tuberosum var Bintje plantlets were transferred to media containing a high level of sucrose. In the presence of tetcyclacis, tuber formation started after 4 days, reaching a maximum level of 80% at day 7. Tubers formed in the presence of tetcyclacis, accumulated starch and expressed several tuber-specific genes. These effects were fully antagonized by gibberellic acid. It is concluded that the growth retardant tetcyclacis is a potent tool in the study of tuber formation in potatoes.Abbreviations ABA abscisic acid - BAP benzylaminopurine - GA₃ gibberellic acid - STS silver thiosulphate - TET tetcyclacis     

Initial Anatomical Changes Associated with Tuber Formation on Single-node Potato (Solanum tuberosum L.) Cuttings: A Re-evaluation

Cell division and cell expansion during early stages of tuberdevelopment were studied using developing axillary buds on single-leafcuttings from potato (Solanum tuberosum L.). Cuttings takenfrom plants induced to form tubers, by short day (SD) treatment,were compared with cuttings from non-induced (long day, LD)plants. In the apical zone of the buds, cell division occurredfrom the first day after cutting, in both LD and SD cuttings.The planes of these divisions were transverse, associated withelongation of the buds. At day 5, a new orientation of celldivision was observed in the subapical zone of SD cuttings only.These divisions were longitudinal, associated with radial growth.Cell expansion occurred in both SD and LD cuttings, and wasnot uniquely related to the onset of tuber formation. Copyright1999 Annals of Botany Company Solanum tuberosum L., potato, tuber formation, cell division, cell expansion.     

Coordinate expression of AOS genes and JA accumulation: JA is not required for initiation of closing layer in wound healing tubers

Wounding induces a series of coordinated physiological responses essential for protection and healing of the damaged tissue. Wound-induced formation of jasmonic acid (JA) is important in defense responses in leaves, but comparatively little is known about the induction of JA biosynthesis and its role(s) in tuber wound-healing. In this study, the effects of wounding on JA content, expression of JA biosynthetic genes, and the involvement of JA in the initiation of closing layer formation in potato tubers were determined. In addition, the role of abscisic acid (ABA) in wound-induced JA accumulation was examined. The basal JA content in non-wounded tuber tissues was low (<3 ng g⁻¹ FW). Two hours after wounding, the JA content increased by >5-fold, reached a maximum between 4 and 6 h after wounding, and declined to near-basal levels thereafter. Tuber age (storage duration) had little effect on the pattern of JA accumulation. The expressions of the JA biosynthetic genes (StAOS2, StAOC, and StOPR3) were greatly increased by wounding reaching a maximum 2-4 h after wounding and declining thereafter. A 1-h aqueous wash of tuber discs immediately after wounding resulted in a 94% inhibition of wound-induced JA accumulation. Neither JA treatment nor inhibition of JA accumulation affected suberin polyphenolic accumulation during closing layer development indicating that JA was not essential for the initiation of primary suberization. ABA treatment did not restore JA accumulation in washed tuber tissues suggesting that leaching of endogenous ABA was either not involved or not solely involved in this loss of JA accumulation by washing. Collectively, these results indicate that JA is not required for the induction of processes essential to the initiation of suberization during closing layer development, but do not exclude the possibility that JA may be involved in other wound related responses.     

Metabolism and Transport of [2-14C]({+/-}) Jasmonic Acid in the Potato Plant

Metabolism and transport of [2-¹⁴C](±) jasmonic acid(JA) were studied using 2-week-old potato plants. Plants grownunder tuber inducing conditions (short days) and non-inducingconditions (long days) were assayed 1, 5, 10, 15, 20, and 25days after substance application. The incorporated [2-¹⁴C](±)JAwas metabolized to tuberonic acid glucoside (TAG) within 2 weeks.The radioactivity of the ethanol extract from each plant partwas measured. More than half of the total radioactivity wasstill associated with the treated leaves up to 25 days afterthe application of [2-¹⁴C](±)JA. In the other parts ofthe plants, the upper leaves contained the most total radioactivity,followed by the stems and lower leaves. Under long days no tuberswere produced, and the radioactivity of the stolons was observed.In contrast, under short days tuber were produced, and the radioactivitieswere present in stolons and tubers. Flower buds and flowerswere produced more under long days and accumulated high radioactivity.It is concluded from the combined data that JA is metabolizedto TAG and transported to all parts of the plant. A high accumulationof TAG in tubers and flower buds may induce the formation oftubers and flower buds. (Received January 22, 1996; Accepted April 15, 1996)     

Possible role of jasmonic acid in the regulation of floral induction, evocation and floral differentiation in Lemna minor L.

Jasmonic acid (JA) is implicated in a wide variety of developmental and physiological processes in plants. Here, we studied the effects of JA and the combination of JA and ethylenediamine-dio-hydroxyphenyl-acetic acid (EDDHA) on flowering in Lemna minor in axenical cultures. JA (0.475-47.5 nmol l(-1)) enhanced floral induction in L. minor under long-day (LD) conditions. Under the same conditions, at a concentration of 237.5 nmol l(-1), JA inhibited floral induction, and at a concentration of 475 nmol l(-1) it prevented floral induction. Under LD conditions with LD preculture, a combination of EDDHA (20,500 nmol l(-1)) and JA (47.5 nmol l(-1)) had a synergistic effect on the promotion of floral induction. Floral induction was enhanced to the greatest extent in experiments with LD precultures. Microscopic examination of microphotographs of histological sections showed that JA and, to an even greater extent, JA+EDDHA at optimal concentrations promote apical floral induction (evocation). Furthermore, JA, and to an even greater extent JA in combination with EDDHA in an optimal concentration, also promote flower differentiation, especially the development of stamens, as is evident from the microphotographs. The experimental results show that JA promotes floral induction in other species of Lemnaceae from various groups according to their photoperiodic response. The results support our hypothesis that, in addition to previously ascribed functions, JA may regulate floral induction, evocation and floral differentiation. Our hypothesis is supported also by the results obtained by quantitative determination of endogenous JA levels in L. minor at three growth stages. The levels of endogenous JA decreased from 389 ng JA g(-1) (fresh weight) of L. minor during the vegetative stage to 217 ng JA g(-1) during the evocation stage, and to 37.5 ng JA g(-1) during the flowering stage, which proves that JA is used for flowering.     

The effect of thaumatin gene overexpression on the properties of H(+)-ATPase from the plasmalemma of potato tuber cells

The introduction of the thaumatin gene into potato plants was accompanied by a decrease in the activity of H(+)-ATPase in the plasmalemma (PL) of tuber cells. When tubers were released from dormancy, the enzyme was activated in the tuber cells of both the original and transgenic plants. Experiments performed in vitro demonstrated that sensitivities to ambiol (AM) and jasmonic acid (JA) of H(+)-ATPase in the PL of tubers from the original plants were lower after the release from a period of deep dormancy. In preparations from the tubers of transgenic plants, the situation was reversed. The differences between the activities of H(+)-ATPase in the PL preparations produced from the original and transgenic tubers that sprouted under the action of AM and JA were detected. Thus, the overexpression of the thaumatin gene in potato plants changed the properties of H(+)-ATPase from PL.     

Dihydroxydocosahexaenoic acids of the neuroprotectin D family: synthesis, structure, and inhibition of human 5-lipoxygenase

During aerobic oxidation of docosahexaenoic acid (DHA), soybean lipoxygenase (sLOX) has been shown to form 7,17(S)-dihydro(pero)xydocosahexaenoic acid [7,17(S)-diH(P)DHA] along with its previously described positional isomer, 10,17(S)-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid. 7,17(S)-diH(P)DHA was also obtained via sLOX-catalyzed oxidation of either 17(S)-hydroperoxydocosahexaenoic acid [17(S)-HPDHA] or 17(S)-hydroxydocosahexaenoic acid [17(S)-HDHA]. The structures of the products were elucidated by normal-phase, reverse-phase, and chiral-phase HPLC analyses and by ultraviolet, NMR, and tandem mass spectroscopy and GC-MS. 7,17(S)-diH(P)DHA was shown to have 4Z,8E,10Z,13Z,15E,19Z geometry of the double bonds. In addition, a compound apparently identical to the sLOX-derived 7,17(S)-diH(P)DHA was produced by another enzyme, potato tuber LOX, in the reactions of oxygenation of either 17(S)-HPDHA or 17(S)-HDHA. All of the dihydroxydocosahexaenoic acids (diHDHAs) formed by either of the enzymes were clearly produced through double lipoxygenation of the corresponding substrate. 7,17(S)-diHDHA inhibited human recombinant 5-lipoxygenase in the reaction of arachidonic acid (AA) oxidation. In standard conditions with 100 microM AA as substrate, the IC(50) value for 7,17(S)-diHDHA was found to be 7 microM, whereas IC(50) for 10,17(S)-DiHDHA was 15 microM. Similar inhibition by the diHDHAs was observed with sLOX, a quintessential 15LOX, although the strongest inhibition was produced by 10,17(S)-diHDHA (IC(50) = 4 microM). Inhibition of sLOX by 7,17(S)-diHDHA was slightly less potent, with an IC(50) value of 9 microM. These findings suggest that 7,17(S)-diHDHA along with its 10,17(S) counterpart might have anti-inflammatory and anticancer activities, which could be exerted, at least in part, through direct inhibition of 5LOX and 15LOX.     

Gibberellin-induced inhibition and promotion of sprouting in aerial tubers of Begonia evansiana Andr. in relation to photoperiodic treatment and tuber stage

Gibberellic-acid (GA₃) treatment, when applied within a period ranging from the start of short-day (SD) treatment until about 10 SD, GA₃ strongly inhibited formation of aerial tubers in response to SD and brought about sprouting of developing aerial tubers. In contrast, when applied after about 10 SD or more, GA₃ hastened the completion of the dormant state in the tubers and prolonged their dormancy. The dormancy-promoting effect of GA₃ on detached tubers increased with their degree of maturation. Application of growth retardants N-dimethylaminosuccinamic acid (B-9), 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine carboxylate methyl chloride (AMO-1618) and 2-chloroethyltrimethylammonium chloride (CCC) to the cuttings delayed the onset of dormancy in the aerial tuber. When the retardants were applied to detached aerial tubers, however, such a delay of dormancy was not observed, and GA₃ application did not inhibit sprouting in aerial tubers detached from CCC-treated cuttings.Abbreviations GA gibberellin - GA₃ gibberellic acid - SD short day(s) - LD long day(s) - SDP short-day plant - LDP long-day plant - CCC 2-chloroethyltrimethylammonium chloride - B-9 N-dimethylaminosuccinamic acid - AMO-1618 2-isopropyl-4-dimethyl-amino-5-methylphenyl-1-piperidine carboxylate methyl chloride     

Analysis of the subcellular localisation of lipoxygenase in legume and actinorhizal nodules

Plant lipoxygenases (LOXs; EC 1.13.11.12) catalyse the oxygenation of polyunsaturated fatty acids, linoleic (18:2) and α-linolenic acid (18:3(n-3)) and are involved in processes such as stress responses and development. Depending on the regio-specificity of a LOX, the incorporation of molecular oxygen leads to formation of 9- or 13-fatty acid hydroperoxides, which are used by LOX itself as well as by members of at least six different enzyme families to form a series of biologically active molecules, collectively called oxylipins. The best characterised oxylipins are the jasmonates: jasmonic acid (JA) and its isoleucine conjugate that are signalling compounds in vegetative and propagative plant development. In several types of nitrogen-fixing root nodules, LOX expression and/or activity is induced during nodule development. Allene oxide cyclase (AOC), a committed enzyme of the JA biosynthetic pathway, has been shown to localise to plastids of nodules of one legume and two actinorhizal plants, Medicago truncatula, Datisca glomerata and Casuarina glauca, respectively. Using an antibody that recognises several types of LOX interspecifically, LOX protein levels were compared in roots and nodules of these plants, showing no significant differences and no obvious nodule-specific isoforms. A comparison of the cell-specific localisation of LOXs and AOC led to the conclusion that (i) only cytosolic LOXs were detected although it is generally assumed that the (13S)-hydroperoxy α-linolenic acid for JA biosynthesis is produced in the plastids, and (ii) in cells of the nodule vascular tissue that contain AOC, no LOX protein could be detected.     

Tuber morphology and starch accumulation are independent phenomena: Evidence from ipt-transgenic potato lines

Tuber formation and carbohydrate metabolism in potatoes were studied using transgenic potato plants carrying the Agrobacterium tumefaciens ipt gene, involved in cytokinin biosynthesis. Three independent transformants, viz. clones 1, 11 and 13, whose cytokinin and auxin content had previously been shown to be different from each other and from the wild-type, were analysed in vitro. Clones 11 and 13 showed a higher ability to form stolons and tubers, as evident from: (1) stolon development in whole plants grown under non-inductive conditions, (2) total number and weight of tubers formed by cuttings of this clone in darkness, (3) tubers appeared earlier than tubers of wild-type plants and at a lower sucrose concentration in the medium. Clone 1 did not form stolons or tubers under any conditions tested, but rather formed short shoots. A series of metabolic changes, known to be characteristic for tubers, were analysed in leaves, stems and developing buds. It was found that the short type of shoots, formed by clone 1, had metabolic characteristics very similar to tubers formed in wild-type or clones 11 and 13, including glucose, fructose, sucrose, and starch levels, and activities of invertase, sucrose synthase and fructokinase. It is concluded that the regulation of the stolon swelling and of carbohydrate metabolism, normally occurring simultaneously, can be uncoupled, and are thus, at least partly independent phenomena. The present data obtained with a high-cytokinin line indicate that cytokinins (probably in concert with auxins) might be mainly involved in the regulation of tuber morphology.     

A one-step method of 10,17-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid synthesis by soybean lipoxygenase

A product of lipoxygenase (LOX) oxidation of docosahexaenoic acid (DHA), 10,17-dihydro(pero)xydocosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid [10,17(S)-diH(P)DHA] was obtained through various reaction pathways that involved DHA, 17(S)-hydro(pero)xydocosahexa-4Z,7Z,11Z,13Z,15E,19Z-enoic acid [17(S)-H(P)DHA], soybean lipoxygenase (sLOX), and potato tuber lipoxygenase (ptLOX) in various combinations. The structure of the product was confirmed by HPLC, ultraviolet (UV) light spectrometry, GC-MS, tandem MS, and NMR spectroscopy. It has been found that 10,17(S)-diH(P)DHA formed by sLOX through direct oxidation of either DHA or 17(S)-H(P)DHA was apparently identical to the product of ptLOX oxidation of the latter. The sLOX- and ptLOX-derived samples of 10,17-diHDHAs coeluted under the conditions of normal, reverse, and chiral phase HPLC analyses, displayed identical UV absorption spectra with maxima at 260, 270, and 280 nm, and had similar one-dimensional and two-dimensional proton NMR spectra. Analysis of their NMR spectra led to the conclusion that 10,17-diHDHA formed by sLOX had solely 11E,13Z,15E configuration of the conjugated triene fragment, which was identical to the previously published structure of its ptLOX-derived counterpart. Based on the cis,trans geometry of the reaction products, the conclusion is made that in the tested conditions sLOX catalyzed direct double dioxygenation of DHA. Compared with the previously described two-enzyme method that involved sLOX and ptLOX, the current simplified one-enzyme procedure uses only sLOX as the catalyst of both dioxygenation steps.