Endogenous ABA-levels correlated with susceptibility of cultivars but were not affected by infection
Our experiments revealed a higher susceptibility of bar- ley cv. Steptoe to infection with M. oryzae as compared to cv. Ingrid (Figure 4) and an increase in susceptibility to infection in both cultivars after ABA treatment. En- dogenous ABA level in Steptoe was twice as high as in Ingrid (Figure 7A), corroborating the role of ABA in sus- ceptibility. ABA analysis in further cultivars and in the mutant Az34 showed that the ABA level in Steptoe was not unique and that the mutant Az34 contained a com- parable level of ABA to cv. Ingrid (Figure 7A). Because ABA is known to suppress the SA-dependent defence pathway [2,9,23,24] as well as SA-mediated induction of systemic acquired resistance , we determined the content of free SA in leaf extracts of the same cultivars as used for ABA analysis. Interestingly, SA and ABA concentrations were correlated in the different barley ge- notypes (Figure 7B) as indicated by a correlation coeffi- cient of 0.8967 (p-value 0.039, both calculated by Pearson Product Moment Correlation using SigmaStat). To eluci- date changes in endogenous levels of ABA after inocu- lation with M. oryzae, leaf samples of inoculated and Figure 4 Cultivar-specific differences in disease severity after ABA-treatment. Abscisicacid (20 μM) or mock-solution were sprayed onto seven-days- old primary leaves of barley cultivars Ingrid, Steptoe, Morex, Pallas, Golden Promise, Hannah and Sultan5. Inoculation with M. oryzae isolate TH6772 (200,000 conidia mL −1 ) took place one hour after treatment. Disease severity was evaluated six days after inoculation by counting blast lesions. Means
Z. Naturforsch. 47c, 7 0 1 -7 0 4 (1 9 9 2 ); received June 10/July 21, 1992 A lgae, AbscisicAcid, Extracellular Production, Stress Factors
The com m on soil green algae Chlorella vulgaris and Stichococcus hacillaris increase extra cellular abscisicacid (A BA ) production under salt, acid or drought stress 5 - 1 0 times. Produc tion o f A BA also increases from young to senescent cultures 2 - 3 times.
Abscisic A cid, Betacyanin. Phytolacca am ericana. Suspension Culture, Tyrosine
Growth o f cells and the accumulation of betacyanin were suppressed by the addition of abscisicacid (A B A ) to suspension cultures of Phytolacca americana. The decrease in the accumulation o f betacyanin was overcom e by exogenously supplied tyrosine which is a pre cursor of betacyanin. A B A decreased the level o f free tyrosine in the cells. Feeding experi ments using labeled tyrosine revealed that A B A reduced the incorporation of labeled tyro sine into betacyanins (to about 50% of the control rate). These results suggest that both the availability of tyrosine and the biosynthetic activity of the pathway from tyrosine to the betacyanins are involved in the inhibition o f the accumulation of betacyanins by A B A in Phytolacca americana cells.
the plant. An alternative interpretation could be that the ABA-treatment directly influenced biosynthesis or accumulation of autofluorescent material. Strikingly, the frequency of interaction sites assigned to category “infection hyphae in epidermal cell without fluores- cence” declined dramatically from 48 to 72 h p.i. for ABA-treated plants whereas the frequency of inter- action sites found for the category “autofluorescence, collapsed mesophyll cells” increased at the same magni- tude (Figure 3). This indicates a correlation of dimin- ished autofluorescent response in attacked epidermal cells with accelerated pathogen spreading into the mesophyll and is in accordance with observations pre- viously reported by Zellerhoff et al. [13,21]. Concomi- tantly, the frequency of interaction sites assigned to the Figure 2 Effect of phytohormone application on the infection of barley with M. oryzae. Primary leaves of barley cultivar Ingrid were sprayed with the following solutions seven days after sawing: sodium salicylate (SA, 0.1 mM), 1-aminocyclopropane-1-carboxylic acid (ACC, 20 μM), indole-3-acetic acid (IAA, 20 μM), gibberellic acid (GA 3 , 20 μM), abscisicacid (ABA, 20 μM) or mock solution, respectively. One hour after
acidification of the cell wall, which according to the acid growth theory decreases the cell- wall extensibility and cell elongation. However cell-wall acidification of a newly developed maize hybrid SR 03 does not change and therefore accounts for its resistance during the first phase of salt stress. It has been proposed in some studies that the events that limit leaf growth following osmotic stress are controlled by root-sourced signals. Inhibition of leaf growth by salt stress is a complex mechanism and may involve multiple signals. So it was assumed that the salt resistance of SR 03 may involve a signal distinct from that of salt-sensitive ones or differs in pathway of a common signal. The hormonal signal abscisicacid (ABA) accumulates in plant tissues under salt stress and the maize genotypes differing in salt resistance have shown variations in ABA synthesis. In addition there are also some indications that the stress hormone jasmonic acid (JA) also takes part in osmotic stress signaling.
For many years abscisicacid has been postulated to have a role in the development of lateral branches or tillers. Previous studies indicated that the hormone abscisicacid (ABA) inhibits bud outgrowth as shown after exogenous ABA treatment in pea, Arabidopsis, Ipomoea spp., tomato, or sunflower (Chatfield et al. 2000, Cline and Oh 2006), or by the application of the ABA biosynthesis inhibitor fluridone in rose (Rosa spp.) (Le Bris et al. 1999), or in excised nodes of transgenic ABA-insensitive poplars (Populus spp.) (Arend et al. 2009). A negative correlation between ABA levels and the developmental status of buds was found in a wide range of plant species (Gocal et al. 1991, Knox and Wareing 1984, Mader et al. 2003, Tamas et al. 1979, Tucker 1977, Tucker and Mansfield 1971). The possibility that ABA may control branch development has been more widely explored. After decapitation of plants in a variety of species, ABA abundance in dormant ABs has been found to decrease (Gocal et al. 1991, Knox and Wareing 1984, Mader et al. 2003). When plants were exposed to light with high
Correspondence to: Y. K oshita , NARO Institute of Fruit Tree Science, 2-1, Fujimoto, Tsukuba, Ibaraki, 305-8605, Japan. Fax: +81-298-
838-6437. E-mail: email@example.com
The effect of short-term low and high temperature treatment to clusters before and after the onset of color development on anthocyanin and abscisicacid (ABA) content in the peel of 'Aki Queen' (Vitis labruscana L.), a tetraploid grape cultivar with red peel was investi- gated. Grape clusters were exposed to different tem- perature conditions, either from the beginning of the berry softening to the onset of color development, 47 to 56 days after full bloom (DAFB), or from the onset of color development onwards for 10 days, 56 to 66 DAFB. Low-temperature (2-5 °C lower than control) treat- ments in both, 47 to 56 DAFB and 56 to 66 DAFB had the tendency to increase anthocyanin concentration and the concentration in the peel was higher than that in the control. The difference in the ABA content after low and high temperature treatment in the 47 to 56 DAFB suggests that temperature from the beginning of berry softening to the onset of color development might af- fect the ABA content of the peel. On the other hand, treatment from the onset of color development onwards for 10 days might affect the anthocyanin concentration, but the effect on ABA content is relatively low. These results suggest that temperature-dependent accumula- tion of anthocyanin during maturation correlate with ABA content at the onset of color development in 'Aki Queen' grape.
AbscisicAcid, Diffusion, Indole Acetic Acid, Phaseolus coccineus, Plasm alem m a, Transport Uptake o f abscisicacid by 5 mm long decapped root tips is a linear function o f the external ABA concentration in the range o f 2.9 x 1 0 - 8 m to 1 0 ~ 4 m and decreases dram atically with in creasing pH. At pH 8 . 0 uptake rate is extremely low, even at high ABA concentrations. This indicated that nearly all o f the ABA is taken up as the undissociated m olecule ABAH. U ptake o f ABA is influenced by agents m odifying the pH gradients betw een the m edium and the tissue such as salts o f weak acids incubated at low external pH (inhibition o f uptake and stim ulation o f ABA release by abolishing the pH gradients), protonophores such as CC CP (in h ib ition o f uptake) and fusicoccin (stim ulation o f uptake by increasing the pH betw een m edium and cytoplasm). It is concluded that ABA distributes betw een the com partm ents o f the root cells according to the pH gradients with the undissociated m olecule as the only penetrating species. Uptake and release occur w ithout participation o f a saturable com ponent by diffusion. In contrast IAA permeates the plasm alem m a as both IAAH and IAA- .
M. Baier an d W. H a rtu n g • ABA T ra n sp o rt an d C ytoplasm ic pH 107
täte) is th at the viability of the guard cells is not af fected by these acids. Therefore we incubated epi dermal disks over a period up to 24 h in a buffer ing medium (10 m o lm “3 KCl, 20 m olm “3 MES, 1 m o lm “3 C aC l2, pH 5.0, adjusted with N aO H ) in the presence of different concentrations of sodium nitrite (0 .5 -2 m o lm “3) and sodium acetate ( 2 - 6 m o lm -3). At regular time intervals two disks were taken out and rinsed for 20 s with an identical buffering medium, but without the weak acid. The viability o f the guard cell complexes was examined by their ability to show stomatal movement (open ing with fusicoccin) and to accumulate a vital stain in their vacuoles. To test whether the stom ata are still functioning, the epidermal disk was placed on the surface o f 2 ml buffering medium (10 m o lm “3 KC1, 20 m o ltr r 3 MES, 1 m olm “3 CaCl2 and 15 m m o lm “3 fusicoccin, pH 5.8/NaOH), with addi tional halogen light (500 (imol photons x m “2s“ '). These are conditions which prom ote the stom atal opening. After 20 min incubation time the aper ture o f 50 stom atal complexes was measured microscopically.
Plant material and analysis o f secondary metabolites
Barley (Hordeum vulgare L. cv. Wasedori, Yu- kijirusi Shubyo, Sapporo, Japan) seeds were soaked in distilled water for 24 h to facilitate ger mination. The soaked seeds were then sown in wet vermiculite and m aintained at 20 °C for 6 d under continuous artificial light (15 W m -2) in growth chambers. The lower epidermis of 7-d-old primary leaves was peeled off, and 5 cm segments were taken 1 - 6 cm from the leaf tip. The segments were floated on 10 ml of solution of JA or ABA in Petri dishes with the peeled surface in contact with the solution. A fter incubation for an appropriate period, the leaf segments were extracted with 10 volumes of m ethanol for 24 h. The extracts were subjected to reversed-phase H PLC analyses [col umn: Wakosil II 5C18 HG, 150 mm x 4.6 mm i.d., Wako Pure Chemical, Osaka, Japan; linear gradi ent: 5 % -1 0 % B/A for 20 min (solvent A: H zO containing 0.1% trifluoroacetic acid, solvent B: acetonitrile); flow rate: 0 .8 ml/min; detection 280 nm].
The sulfonation process of camphor and its hal- ogenated derivatives is well understood, and dif ferent products are obtained depending on the re action conditions [22-24], In contrast, even under very different conditions (gaseous S 0 3  or sulfuric acid/acetanhydride ), fenchone always yields the same sulfonic acid, without ac companying products of rearrangem ent, provided that no heating of the reaction mixture occurs. We first confirmed the structure of this sulfonic acid by two-dimensional C -C -correlation (IN A D E Q U A T E ) [25, 26] of the cyclic imine (5), obtained by the reaction of the sulfonyl chloride with ammonia, followed by acid-induced cycli- zation . The spectrum shows crosspeaks be tween all connected carbons, and in particular, the subunit C H 3- C q- C H 3 can be identified, which means that the original assumption of Treibs and Lorenz about the structure of 2 is defi nitely confirmed. Obviously, no rearrangem ent occurs during sulfonation. Oxidation of the imine (5) with 3-chloroperbenzoic acid cleanly yields the oxaziridine ( 6 ). Im proved procedures for the prep aration of 6 and its precursors are given in the experim ental part.
Fatty acid biotransformation activity of E. coli BL21(DE3) pCOLA-RcsB-DsrA
The effect of the GDAR system engineering on the whole-cell fatty acid biotransformation activity was investigated by conducting the bioconversion of ricinoleic acid (i.e., 12-hydroxyoc- tadec-9-enoic acid (1)) into n-heptanoic acid (5) and 11-hydroxyundec-9-enoic acid (4) ( S1 Scheme ), which was described in our previous study [ 15 ]. When ricinoleic acid was added into the culture broth of the recombinant E. coli BL21(DE3) pACYC-ADH, pET-BVMO, pCOLA or E. coli BL21(DE3) pACYC-ADH, pET-BVMO, pCOLA-RcsB-DsrA expressing the alcohol dehydrogenase (ADH) of Micrococcus luteus and the Baeyer-Villiger monooxygenase (BVMO) of Pseudomonas putida, the final product formation rates of the both cells were similar to at t < 4 h ( Fig 6 ). However, the final product formation rate of E. coli BL21(DE3) pACYC-ADH, pET-BVMO, pCOLA ceased resulting in an accumulation of the reaction intermediate (2) in the culture medium at t > 4 h, when the product concentration and bioconversion yield reached over 6 mM and 45%, respectively ( Fig 6A ). This might be ascribed to the toxicity of n- heptanoic acid ( Fig 5B ). In contrast, the final product formation rate of E. coli BL21(DE3) pACYC-ADH, pET-BVMO, pCOLA-RcsB-DsrA was further maintained resulting in a final product concentration of over 10 mM (conversion yield, 68%), which was 1.6-fold higher as compared to the E. coli BL21(DE3) pACYC-ADH, pET-BVMO, pCOLA. This value was also Fig 4. The relationship between GadA/B activity and cultivation pH (A) or n-heptanoic acid concentration (B). The GadA/B
First, selective reduction of the carbonic acid to a prim ary alcohol with diborane or borane-dim ethyl- sulfide complex , and second, oxidation of the alcohol to an aldehyde function with pyridinium dichrom ate  or other approved reagents (Fig. 4). The reduction of 1 to the alcohol 6 was accomplished easily in 65% yield but the oxidation of 6 failed. T reatm ent of 6 with pyridinium dichrom ate yielded only overoxidized product 1 whereas 6 rem ained un changed by treatm ent with pyridiniumchlorochro- m ate . O th er reagents were not tried with regard to the acid lability of the protective groups.
20 g of 4 were refluxed with 200 ml o f 6 M HC1 for 8 - 1 0 h. A fter cooling to —20 °C, the precipitated phthalic acid was rem oved by suction filtration and the clear filtrate brought to dryness in a vacuum d e siccator over KOH. The dry product was recrystal lized from m ethanol/isopropanol. Yield: 11.2 g (92%) o f white crystals, m .p. 147 °C.
discussed . According to the “preferential exclusion hypothesis”, established for protein stabilization, solutes are preferentially excluded from the surface resulting in the formation of a stabilizing solvent layer [17, 178-179]. However, it is questionable if this hypothesis can be adapted to nucleic acid nanoparticles, as the relatively high amounts of cryoprotectants required point to a nonspecific bulk stabilization [17, 178-179]. Based on the “glass formation or vitrification hypothesis”, nucleic acid nanoparticles are entrapped in the amorphous gassy matrix when the sample is cooled below the glass transition temperature (Tg`) limiting particle mobility and thus, preventing particle aggregation [17, 178]. As some sugars were able to preserve particle size at temperatures well above Tg` vitrification cannot be the only stabilization mechanism . The “particle isolation hypothesis” states that particles have to be sufficiently separated in the freeze-concentrate in order to inhibit particle aggregation, which is observed above a critical excipient to complex ratio . However, these three mechanisms are not suitable to solely explain the stabilization of nucleic acid nanoparticles during freezing. Thus, the influence of freezing on nucleic acid nanoparticles and underlying stabilization mechanisms during freezing will be addressed in detail in Chapter 6.
In this context it has been proposed that the human genetic profile was originally established on a n-6 to n-3 PUFA ratio of approximately 1:1 as found in “ancient” diets, whereas today’s Western diet has been estimated to provide n-6 to n-3 PUFAs in a ratio of 15:1–20:1. It has been hypothesized that this may contribute to many serious health issues typically found in Western societies, including CRC. However, previous in vitro observations have led to some uncertainty regarding differential roles of n-3 and n-6 PUFAs in CRC cells. While the majority of investigations conducted in this field addressed neither the effects of n-6 PUFAs nor the impact of a balanced n-6 to n-3 PUFA ratio, several other studies reported n-3 and n-6 PUFAs to exert anti-cancerous effects in vitro. Hence, it was the aim of the present study to investigate the impact of n-3 PUFA docosahexaenoic acid (DHA) and n-6 PUFA AA and their combination on CRC cell line LS 174T in vitro.
Electron spin resonance (E P R ) sp ectra d e m o n stra te the form ation o f a com plex betw een the a sco rb ate ion and disulfides such as o x id ized g lu ta th io n e , cystine, g am m a globulin, bovine serum alb u m in , 4 -4 ' d ith io p y rid in e , and d ith io b u ty ric acid. H ow ever, except in th e case o f 4 -4 ' dith io p y rid in e, asco rb ic acid (A H 2) is not c ap a b le o f reducing the disu lfid e b o n d in these co m pounds. T hese results can be exp lain ed in term s o f th e redox po ten tial o f ascorbic acid and its rad ical se m id eh y d ro asco rb ic acid (SDA). These co m p o u n d s can red u ce the S - S b ond o f 4 -4 ' d ith io p y rid in e, th e redox p o ten tial o f w hich is £o = — 0. IV and, thus, m o re p ositive th a n the ones d e te r m ined for the ascorbic acid system .