Developing a platform for production of the oxylipin KODA in plants.

KODA (9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid) is a plant oxylipin involved in recovery from stress. As an agrichemical, KODA helps maintain crop production under various environmental stresses. In plants, KODA is synthesized from α-linolenic acids via 9-lipoxygenase (9-LOX) and allene oxide synthase (AOS), although the amount is usually low, except in the free-floating aquatic plant Lemna paucicostata. To improve KODA biosynthetic yield in other plants such as Nicotiana benthamiana and Arabidopsis thaliana, we developed a system to overproduce KODA in vivo via ectopic expression of L. paucicostata 9-LOX and AOS. The transient expression in N. benthamiana showed that the expression of these two genes is sufficient to produce KODA in leaves. However, stable expression of 9-LOX and AOS (with consequent KODA production) in Arabidopsis plants succeeded only when the two proteins were targeted to plastids or the endoplasmic reticulum/lipid droplets. Although only small amounts of KODA could be detected in crude leaf extracts of transgenic Nicotiana or Arabidopsis plants, subsequent incubation of the extracts increased KODA abundance over time. Therefore, KODA production in transgenic plants stably expressing 9-LOX and AOS requires specific sub-cellular localization of these two enzymes and incubation of crude leaf extracts, which liberates α-linolenic acid via breakdown of endogenous lipids.

Ultrafine bubble water mitigates plant growth in damaged soil.

Water containing ultrafine/nano bubbles (UFBs) promoted the growth of tomato (Solanum lycopersicum) in soil damaged by cultivation of tomato in the previous year or bacterial wilt-like disease and also promoted the growth of lettuce (Lactuca sativa) when lettuce was grown in the soil damaged by repeated cultivation of lettuce. On the other hand, UFB supply did not affect plant growth in rock wool or healthy soil. Furthermore, the growth of lettuce was not affected by UFB water treatment in the soil damaged by the cultivation of tomato. UFB water partly suppressed the growth of the pathogen of bacteria wilt disease, Ralstonia solanacearum in vitro. These data suggest that UFB water is effective to recover the plant growth from soil damage.

α-Ketol linolenic acid (KODA) application affects endogenous abscisic acid, jasmonic acid and aromatic volatiles in grapes infected by a pathogen (Glomerella cingulata).

Effects of α-ketol linolenic acid (KODA) application on endogenous abscisic acid (ABA), jasmonic acid (JA), and aromatic volatiles were investigated in 'Kyoho' grapes (Vitis labrusca×Vitis vinifera) infected by a pathogen (Glomerella cingulata). The expressions of 9-cis-epoxycarotenoid dioxygenase (VvNCED1), ABA 8'-hydroxylase (VvCYP707A1), lipoxygenase (VvLOX), and allene oxide synthase (VvAOS) were also examined. The grape berries were dipped in 0.1mM KODA solution before inoculation with the pathogen and stored at 25°C for 12 days. The development of infection was significantly suppressed upon KODA treatment. Endogenous ABA, JA and phaseic acid (PA) were induced in inoculated berries. KODA application before inoculation increased endogenous ABA, PA and JA through the activation of VvNCED1, VvCYP707A1 and VvAOS genes, respectively. In addition, terpenes, methyl salicylate (Me-SA) and C6-aldehydes such as (E)-2-hexenal and cis-3-hexenal associated with fungal resistance also increased in KODA-treated berries during storage. These results suggest that the synergistic effect of JA, ABA, and some aromatic volatiles induced by KODA application may provide resistance to pathogen infection in grape berries.

Large-scale production of saikosaponins through root culturing of Bupleurum falcatum L. using modified airlift reactors.

Modification of internal configuration of a bubble column, airlift and stirred tank reactor (10-200 L) was made for root cultures of Bupleurum falcatum L. Agitation with an impeller covered with partition mesh was ineffective for a 10-L modified reactor, because it caused intensive foaming and subsequent overflow of the culture medium even at a low rotation speed of 50 rpm and a low aeration rate of 0.1 vvm (volume per volume of medium). In contrast, efficient aeration through a ceramic sparger placed at the bottom of a 20-L bubble column reactor yielded approximately 25 g/L of dry roots and 500 mg/L of saikosaponin-a and saikosaponin-d over 42 days. On a 200-L scale, however, the roots became flocculated under the upper perforated plate initially positioned near the middle of the reactor, forming a firm disk of roots and a large empty space between the disk and the medium. Thus, the roots had poor contact with the medium, which severely suppressed their growth. To avoid this flocculation, a bottom perforated plate and draft tube were installed as a partitioning device separating the culturing area (outside the draft tube) from the aeration area (inside the draft tube). The draft tube was made of a stainless steel mesh rather than a solid material, and the tube greatly increased the root yield in the 20-L reactor. This configuration was successfully applied at the 200-L scale, yielding 500-600 mg/L of saikosaponin-a and saikosaponin-d over 56 days.

C14-oxylipin glucosides isolated from Lemna paucicostata.

Oxylipin glucosides (2-4) were isolated from Lemna paucicostata with their structures and absolute configurations elucidated by spectroscopic and chemical methods. Compounds 2-4 were glucosides of C14 oxylipin which were synthesized from alpha-linolenic acid via the 9-lipoxygenase pathway.

Structure and biological activity of novel FN analogs as flowering inducers.

(12Z,15Z)-9-Hydroxy-10-oxooctadeca-12,15-dienoic acid (1) and norepinephrine (2) undergo cycloaddition to afford FN1 (3) and FN2 (4), both of which induce flowering in Lemna paucicostata. Although the derivatives of 1 were suggested to also yield FN-like compounds after reacting with 2, their structures have not been elucidated. In this report, we investigated the structure and stereochemistry of seven novel FN analogs. These analogs were shown to be formed in the same regio- and stereocontrolled manner as FNs. Moreover, the activity of FN analogs on flowering induction was investigated, and we determined that all analogs, except for compound 8, were effective flowering inducers for L. paucicostata.

A new 9-lipoxygenase cDNA from developing rice seeds.

We isolated a novel C9 position specific lipoxygenase (r9-LOX1) cDNA from developing rice seeds. The enzymatic features of r9-LOX1 resembled those of rice LOX-L3 known to be contained in rice germ and to have C9-specific LOX activity. However, the expression level of the r9-LOX1 gene was higher in imbibed seeds rather than developing seeds. A homology search against the rice nucleotide database revealed the r9-LOX1 gene to be on rice chromosome 3 (accession number AC093017). The restriction enzyme map of the reported genomic sequence agreed with the result of the Southern blot analysis for the r9-LOX1. The enzyme could be useful for in vitro synthesis of 9,10-ketol-octadecadienoic acid.

Changes in catecholamine levels in short day-induced cotyledons of Pharbitis nil.

We investigated the effects of catecholamine on flower-induction in P. nil (cv. Violet). GC-SIM analysis identified dopamine for the first time in P. nil seedlings. Dopamine levels in the cotyledons did not show a significant change during the inducing dark treatment. The dopamine content of cotyledons exposed to various durations of darkness were 0.1-0.2 nmol/g fresh weight. The same content was found when cotyledons were exposed to continuous light.

Endogenous alpha-ketol linolenic acid levels in short day-induced cotyledons are closely related to flower induction in Pharbitis nil.

Alpha-ketol linolenic acid [KODA, 9,10-ketol-octadecadienoic acid, that is 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid] is a signal compound found in Lemna paucicostata after exposure to stress, such as drought, heat or osmotic stress. KODA reacts with catecholamines to generate products that strongly induce flowering, although KODA itself is inactive [Yokoyama et al. (2000) Plant Cell Physiol. 41: 110; Yamaguchi et al. (2001) Plant Cell Physiol. 42: 1201]. We examined the role of KODA in the flower-induction process of Pharbitis nil (violet). KODA was identified for the first time in seedlings of P. nil grown under a flower-inductive condition (16-h dark exposure), by means of LC-SIM and LC-MS/MS. In addition, the changes in endogenous KODA levels (evaluated after esterification of KODA with 9-anthryldiazomethane) during the flower-inductive phase in short day-induced cotyledons were closely related to flower induction. The KODA concentration sharply increased in seedlings during the last 2 h of a 16-h dark period, while the KODA level showed no significant elevation under continuous light. The increase of KODA level occurred in cotyledonal blades, but not in other parts (petiole, hypocotyls and shoot tip). When the 16-h dark period was interrupted with a 10-min light exposure at the 8th h, flower induction was blocked and KODA level also failed to increase. The degree of elevation of KODA concentration in response to 16-h dark exposure was the highest when the cotyledons had just unfolded, and gradually decreased in seedlings grown under continuous light for longer periods, reaching the basal level at the 3rd day after unfolding. Flower-inducing ability also decreased in a similar manner. These results suggest that KODA may be involved in flower induction in P. nil.