In vitro enzymatic chlorophyll catabolism in wheat elicited by cereal aphid feeding

Chlorophyll degradation is a complex phenomenon that often accompanies insect feeding damage to plants. Loss of chlorophyll can be initiated by several reactions, including oxidative bleaching, chlorophyllase activity, and Mg-dechelatase activity. Extracts from the Russian wheat aphid [Diuraphis noxia (Mordvilko)], the bird cherry-oat aphid [Rhopalosiphum padi (L.)], and aphid-infested and uninfested wheat plants were assayed in vitro for activities involved in chlorophyll degradation. Although the initial infestation was the same (10 apterous adults) for both aphid species, D. noxia weight was significantly higher than R. padi after feeding for 12 days. Consequently, D. noxia feeding caused greater fresh leaf weight reduction than R. padi feeding. Chlorophyll degradation assays showed no activity from either D. noxia or R. padi extracts. Plant extract assays showed a significant difference in Mg-dechelatase activity, while no difference was detected in either the chlorophyllase or oxidative bleaching pathways among the aphid-infested or uninfested plant extracts. Diuraphis noxia-infested leaf extracts showed a greater increase of Mg-dechelatase activity than either R. padi-infested or the uninfested plants. The findings suggest that leaf chlorosis elicited by D. noxia feeding is different from the chlorophyll degradation that occurs in natural plant senescence. Aphid-elicited chlorosis might be the result of a Mg-dechelatase-driven catabolism of chlorophyll in challenged wheat seedlings, however, the factor(s) from D. noxia that elicited the increase of Mg-dechelatase activity still remain to be determined.     

Chlorophyllase activities and chlorophyll degradation during leaf senescence in non-yellowing mutant and wild type of Phaseolus vulgaris L

The activities of chlorophyllase, contents of pigments including chlorophyll a and b, chlorophyllide a and b, and phaeophorbide a during leaf senescence under low oxygen (0.5% O2) and control (air) were investigated in a non-yellowing mutant and wild-type leaves of snap beans (Phaseolus vulgaris L.). Chlorophyllase from leaf tissues had maximum activity when incubated at 40C in a mixture containing 50% acetone. In both mutant and wild type, chlorophyllase activity was the highest in freshly harvested non-senescent leaves and decreased sharply in the course of senescence, indicating that the loss of chlorophylls in senescing leaves is not directly related to the activity of chlorophyllase and that chlorophyllase activity is not altered in the mutant. The wild type had higher ratios of chlorophyll a to chlorophyll b than the mutant and chlorophyll a : b ratios increased during senescence in both types. In the senescent mutant leaves, accumulations of chlorophyllide a and chlorophyllide b were detected, but no phaeophorbide a was found. Chlorophyllide b had a greater accumulation than chlorophyllide a in the early stage of senescence. Low oxygen treatment not only delayed chlorophyll degradation but also enhanced the accumulations of chlorophyllide a and b and lowered the ratios of chlorophyll a to chlorophyll b.     

Prevalences of fungal pathogens and othernatural enemies of cereal aphids (Homoptera:Aphididae) in wheat under dryland and irrigatedconditions in South Africa

Surveys were conducted in the summer andwinter rainfall wheat producing regions of SouthAfrica in a first attempt to investigate theidentity and impact of entomopathogenic fungi withinthe cereal aphid complex. Wheat produced underdryland and irrigated conditions was surveyed duringthe 1996 and 1997 seasons. Six cereal aphid specieswere recorded of which the Russian wheat aphid, Diuraphis noxia, was the most abundantunder dryland conditions in the summer rainfallregion as opposed to the oat aphid, Rhopalosiphum padi, in the winter rainfallregion. Rose grain aphid, Metopolophiumdirhodum, was most prevalent underirrigated conditions in the summer rainfall region.Five species of entomopathogenic fungi were recordedincluding four entomophthorales and the hyphomycete,Beauveria bassiana. TheEntomophthorales included Pandora neoaphidis, Conidiobolus obscurus, C.thromboides, and Entomophthoraplanchoniana. Pandora neoaphidis wasthe most important etiological agent recorded fromD. noxia, with up to 50% mycosis recordedunder dryland conditions in the Bethlehem summerrainfall region. Similarly, P. neoaphidis wasthe most prevalent species within populations ofM. dirhodum. under irrigated conditions in theBergville/Winterton summer rainfall region (up to77% mycosis). However, mycoses of R. padi didnot exceed 1.7% in samples from these areas,suggesting that R. padi may be lesssusceptible to P. neoaphidis than M.dirhodum. Epizootics in populations of D.noxia under dryland conditions in both the winterand summer rainfall regions indicated a high levelof susceptibility to P. neoaphidis.Occurrences of hymenopterous parasitoids andpredators in populations of D. noxia were low,although a parasitism level of 25% was recorded inone small sample of R. padi collected from anirrigated field in the summer rainfall region.     

Enzymatic chlorophyll degradation in wheat near-isogenic lines elicited by cereal aphid (Homoptera: Aphididae) feeding

Chlorophyll degradation enzyme (i.e., chlorophyllase, Mg-dechelatase, and chlorophyll oxidase) activities of aphid-infested and uninfested 'Tugela' and Tugela near-isogenic wheat lines (i.e., Tugela-Dn1, Tugela-Dn2, and Tugela-Dn5) were assayed. Chlorophyllase activity was higher in bird cherry-oat aphid, Rhopalosiphum padi (L.) (Homoptera: Aphididae),-infested wheat lines compared with Russian wheat aphid, Diuraphis noxia (Mordvilko) (Homoptera: Aphididae)]-infested and uninfested plants. Mg-dechelatase activity was higher in D. noxia-infested wheat lines than in R. padi-infested and uninfested plants. Also, Mg-dechelatase activity was lower in Tugela wheat infested with D. noxia than in Tugela near-isogenic lines with Dn genes. Based on the in vitro assays of chlorophyll degradation enzyme (i.e., chlorophyllase and Mg-dechelatase) activities, we proposed that the chlorotic symptoms observed on D. noxia-infested Tugela wheat were most likely to be elicited by unbalanced chlorophyll biosynthesis and degradation.     

Chlorophyll fluorescence imaging of photosynthetic activity in sun and shade leaves of trees

The differences in pigment levels, photosynthetic activity and the chlorophyll fluorescence decrease ratio R _Fd (as indicator of photosynthetic rates) of green sun and shade leaves of three broadleaf trees (Platanus acerifolia Willd., Populus alba L., Tilia cordata Mill.) were compared. Sun leaves were characterized by higher levels of total chlorophylls a + b and total carotenoids x + c as well as higher values for the weight ratio chlorophyll (Chl) a/b (sun leaves 3.23–3.45; shade leaves: 2.74–2.81), and lower values for the ratio chlorophylls to carotenoids (a + b)/(x + c) (with 4.44–4.70 in sun leaves and 5.04–5.72 in shade leaves). Sun leaves exhibited higher photosynthetic rates P _N on a leaf area basis (mean of 9.1–10.1 μmol CO₂ m⁻² s⁻¹) and Chl basis, which correlated well with the higher values of stomatal conductance G _s (range 105–180 mmol m⁻² s⁻¹), as compared to shade leaves (G _s range 25–77 mmol m⁻² s⁻¹; P _N: 3.2–3.7 μmol CO₂ m⁻² s⁻¹). The higher photosynthetic rates could also be detected via imaging the Chl fluorescence decrease ratio R _Fd, which possessed higher values in sun leaves (2.8–3.0) as compared to shade leaves (1.4–1.8). In addition, via R _Fd images it was shown that the photosynthetic activity of the leaves of all trees exhibits a large heterogeneity across the leaf area, and in general to a higher extent in sun leaves than in shade leaves.     

Measurement of serum levels of natalizumab, an immunoglobulin G4 therapeutic monoclonal antibody

Chlorophyllide a is a metabolite late in the biosynthesis of chlorophylls and bacteriochlorophylls. Isolation procedures for chlorophyllide a from Rhodobacter capsulatus CB1200 and barley (Hordeum vulgare L.) are described and compared. R. capsulatus CB1200 is a double mutant in the bacteriochlorophyllide a biosynthetic pathway, and chlorophyllide a is excreted by the cells when grown in Tween 80-containing liquid medium. It was purified by liquid or solid phase extraction, yielding 7 mg of chlorophyllide a from 1 L of culture. In a second approach, intrinsic chlorophyllase activity was used to dephytylate chlorophyll in an acetonic preparation of leaves of wild-type or chlorophyll b-deficient barley. Purification was achieved by liquid phase extraction, yielding 14 μg of chlorophyllide a per gram of barley leaves. Chlorophyllide a was identified by thin layer chromatography, absorption spectroscopy, and mass spectrometry.     

The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b

Over the last half century, the most frequently used assay for chlorophylls in higher plants and green algae, the Arnon assay [Arnon DI (1949) Plant Physiol 24: 1–15], employed simultaneous equations for determining the concentrations of chlorophylls a and b in aqueous 80% acetone extracts of chlorophyllous plant and algal materials. These equations, however, were developed using extinction coefficients for chlorophylls a and b derived from early inaccurate spectrophotometric data. Thus, Arnon's equations give inaccurate chlorophyll a and b determinations and, therefore, inaccurate chlorophyll a/b ratios, which are always low. This paper describes how the ratios are increasingly and alarmingly low as the proportion of chlorophyll a increases. Accurate extinction coefficients for chlorophylls a and b, and the more reliable simultaneous equations derived from them, have been published subsequently by many research groups; these new post-Arnon equations, however, have been ignored by many researchers. This Minireview records the history of the development of accurate simultaneous equations and some difficulties and anomalies arising from the retention of Arnon's seriously flawed equations. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of Aluminium on Pigments and Pigment-Protein Complexes of Soybean

The effect of aluminium (0.5 –1.0 mM) on contents of phosphorus, pigments, and pigment-protein complexes was studied in soybean (Glycine max Merril.) grown in different nutrient medium with and without P. Increased Al concentrations led to the decrease in the contents of chlorophylls (Chl) a and b, and carotenoids (Car) in soybean leaves, but Chl a/b ratio did not vary significantly. In long-term experiments, P ameliorates the negative effects of Al.     

Spectral Properties of the Green Alga Trebouxia,a Phycobiont of Cryptoendolithic Lichens in the Antarctic Dry Valley

An algologically pure culture of the green alga Trebouxia, a phycobiont of cryptoendolithic lichens, was isolated from sandstone samples collected in the high-altitude polar regions of Antarctica. The absorption and second-derivative absorption spectra of acetone extract of the Antarctic phycobiont cells were studied in comparison with those of a Trebouxia phycobiont isolated recently from a Parmeliaceae lichen in the Mid-European climatic zone. The cells of the Antarctic phycobiont were characterized by a lower content of chlorophyll a and a higher ratio of chlorophyll b and carotenoids to chlorophyll a as compared to the Mid-European phycobiont. Furthermore, the carotenoids of the Antarctic phycobiont were more diverse. The low-temperature fluorescence spectra of the Antarctic phycobiont were characterized by an increased intensity of the short-wavelength fluorescence peak of chlorophyll aand a diminished intensity of fluorescence in the long-wavelength spectral region.     

Photosynthetic Characteristics of Chloroplasts of Primary Wheat Leaves Grown under Different Irradiance

The rate of accumulation of total chlorophyll (Chl) and carotenoids (Car) of leaves grown under high irradiance, HI (30 and 45 W m^–2) was faster than at moderate irradiance, MI (15 W m^–2). However, the senescence phase started earlier in the samples and proceeded at a faster rate. Chl a/b and Chl (a+b)/Car values showed faster loss of Chl a (compared to Chl b) and Chl (a+b) (compared to Car) in HI leaves. Protein accumulation and loss were also similar to that of Chl (a+b) content. Increase in Chl fluorescence during the development phase may suggest a gradual change in thylakoid organisation, however, the temporal kinetics were different in HI and MI samples. Increase in fluorescence polarisation during senescence of HI leaves compared to the control (MI) suggests conversion of thylakoid membranes to gel phase. Chloroplasts prepared from HI seedlings showed higher rate of photochemical activities, however, the activity declined earlier and at faster rate compared to the control.     

枫香秋季变色期叶色变化及其生理基础

为探究秋季枫叶呈色的关键生理因素,该文以转色期叶色为绿色、黄色和红色的枫香单株为试材,研究了L^*、a^*、b^*值变化与叶片色素、可溶性糖及可溶性蛋白质含量变化的相关性。结果表明:(1)在变色期,3种色彩枫香叶片叶绿素a、叶绿素b、总叶绿素和类胡萝卜素均大量降解,花色素苷不同程度积累。(2)绿色叶单株叶绿素和类胡萝卜素始终保持较高含量,花色素苷含量上升4.2倍,叶片内色素含量比值始终保持稳定; 黄色叶单株叶绿素和类胡萝卜素含量最低,花色素苷含量上升4.4倍,b^*值与叶绿素含量极显著负相关,与类胡萝卜素含量显著负相关,与花色素苷/类胡萝卜素含量比值极显著正相关; 红色叶单株叶绿素和类胡萝卜素含量略高于黄色叶单株,花色素苷含量上升27.2倍,a^*值与叶绿素含量、类胡萝卜素含量极显著负相关,与花色素苷含量显著正相关,与色素含量比值无显著相关性。(3)红色叶单株具有较高的可溶性糖含量和可溶性蛋白质含量。因此,在枫香叶片变色期,保持较高的叶绿素和类胡萝卜素含量,维持色素含量比值稳定使叶片呈现绿色; 叶绿素和类胡萝卜素的大量降解,以及花色素苷/类胡萝卜素含量比值的升高使叶片呈现黄色; 叶绿素的降解和花色素苷的大量合成使叶片呈现红色。     

Physiological responses of wheat and barley to Russian wheat aphid, Diuraphis noxia (Mordvilko) and bird cherry-oat aphid, Rhopalosiphum padi (L.) (Hemiptera: Aphididae)

Although aphids are among the most injurious of all agronomic insect pests, much remains unknown about how their feeding alters plant physiology. Two experiments were conducted to examine the physiological responses of wheat, Triticum aestivum L. and barley, Hordeum vulgare L. to injury by Diuraphis noxia (Mordvilko) and Rhopalosiphum padi (L.) (Hemiptera: Aphididae). Gas-exchange parameters, chlorophyll fluorescence, and chlorophyll content were examined at 3, 6, and 9 days post-infestation on control and aphid (D. noxia and R. padi) infested treatments. In general, chlorophyll content and chlorophyll fluorescence parameters (non-variable minimal fluorescence, maximal fluorescence, and variable fluorescence) were not significantly affected by either aphid species. Photochemical and non-photochemical quenching coefficients were significantly impacted by both aphid species, suggesting that aphid feeding may influence the photoprotective xanthophyll cycle altering the thylakoid membrane pH gradient. Feeding by both aphid species resulted in an increase in electron transport rate, but at different time periods. Wheat plants infested with D. noxia had accelerated declines in photosynthetic capacity when compared to R. padi-infested and control plants. These plants exhibited decreased values for A_max, which was accompanied by decreased values for V_cmax and J_max Neither aphid species negatively affected the photosynthetic capacity of the barley plants until day 9. At this time, aphid-infested plants had decreased values for A_max which was accompanied by decreased values in J_max. Although R. padi feeding does not typically result in visual damage symptoms as previously demonstrated, clearly this aphid does have an impact on the gas-exchange and chlorophyll fluorescence of its host plants. Handling editor: Heikki Hokkanen     

Photosystem 2-activity and thylakoid membrane polypeptides of <Emphasis Type="Italic">in vitro</Emphasis> cultured chrysanthemum as affected by NaCl

Long-term (30 d) effects of 100, 200, 300, and 400 mM NaCl on photosystem 2 (PS 2)-mediated electron transport activity and content of D1 protein in the thylakoid membranes of chrysanthemum (Dendranthema grandiflorum) cultured in vitro at low irradiance 20 μmol(photon) m⁻² s⁻¹ were investigated. 100 mM NaCl increased contents of chlorophylls (Chl) a and b, carotenoids (Car; xanthophylls + carotenes), and the ratio of Chl a/b, and Car/Chl a+b. However, further increase in NaCl concentration led to the significant reduction in the contents of Chl a, and Chl b, and increase in the ratio of Chl a/b and Car/Chl a+b. NaCl treatment decreased the PS 2-mediated electron transport activity and contents of various thylakoid membrane polypeptides including D1 protein.     

The development of antenna complexes of barley (Hordeum vulgare cv. Akcent) under different light conditions as judged from the analysis of 77 K chlorophyll a fluorescence spectra

Using 77 K chlorophyll a (Chl a) fluorescence spectra in vivo, the development was studied of Photosystems II (PS II) and I (PS I) during greening of barley under intermittent light followed by continuous light at low (LI, 50 μmol m⁻² s⁻¹) and high (HI, 1000 μmol m⁻² s⁻¹) irradiances. The greening at HI intermittent light was accompanied with significantly reduced fluorescence intensity from Chl b excitation for both PS II (F685) and PS I (F743), in comparison with LI plants, indicating that assembly of light-harvesting complexes (LHC) of both photosystems was affected to a similar degree. During greening at continuous HI, a slower increase of emission from Chl b excitation in PS II as compared with PS I was observed, indicating a preferred reduction in the accumulation of LHC II. The following characteristics of 77 K Chl a fluorescence spectra documented the photoprotective function of an elevated content of carotenoids in HI leaves: (1) a pronounced suppression of Soret region of excitation spectra (410–450 nm) in comparison with the red region (670–690 nm) during the early stage of greening indicated a strongly reduced excitation energy transfer from carotenoids to the Chl a fluorescing forms within PS I and PS II; (2) changes in the shape of the excitation band of Chl b and carotenoids (460–490 nm) during greening under continuous light confirmed that the energy transfer from carotenoids to Chl a within PS II remained lower as compared with the LI plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Changes in the chlorophylls and carotenoids of leaves of Nicotiana tabacum during senescence

In addition to chlorophylls a and b, β-carotene, lutein, violaxanthin and neoxanthin, leaves of tobacco (Nicotiana tabacum L. cv. Virginia Gold) contain antheraxanthin in some harvests. In lower leaves, chlorophylls decreased more rapidly than carotenoids during senescence, but both types of pigment decreased at equal rates in upper leaves. The chlorophyll a:b ratio decreased only in post-mature leaves. Total carotenoid decreased with age, with the relative proportion of β-carotene increasing in lower leaves. Seasonal influences rather than age of leaf determines whether antheraxanthin is present. No esterified xanthophylls were found in senescent leaves.     

Changes in the chlorophyll and carotenoid contents in the leaves of steppe plants along a latitudinal gradient in South Ural

We studied the content of chlorophylls and carotenoids in the leaves of steppe plants of South Ural growing along a latitudinal gradient from southern steppe to forest-steppe. The content of chlorophylls (a + b) was 5–6 mg per 1 g of the leaf dry weight and did not depend on the latitude, whereas the content of carotenoids in the leaves increased northward from 1.0 to 1.5 mg/g dry wt. At the same time, the greatest changes occurred in the ratios between the forms of pigments: the chlorophyll a/b ratio increased from 1.8 to 2.8, and the chlorophyll/carotenoid ratio decreased from 5.6 to 3.5. The obtained results indicate that adaptation of the pigment apparatus of steppe plants growing along the latitudinal gradient occurs due to the transformation of the light-harvesting complex.     

Optical spectroscopic studies of light-harvesting by pigment-reconstituted peridinin-chlorophyll-proteins at cryogenic temperatures

Low temperature, steady-state, optical spectroscopic methods were used to study the spectral features of peridinin-chlorophyll-protein (PCP) complexes in which recombinant apoprotein has been refolded in the presence of peridinin and either chlorophyll a (Chl a), chlorophyll b (Chl b), chlorophyll d (Chl d), 3-acetyl-chlorophyll a (3-acetyl-Chl a) or bacteriochlorophyll a (BChl a). Absorption spectra taken at 10 K provide better resolution of the spectroscopic bands than seen at room temperature and reveal specific pigment–protein interactions responsible for the positions of the Q_y bands of the chlorophylls. The study reveals that the functional groups attached to Ring I of the two protein-bound chlorophylls modulate the Q_y and Soret transition energies. Fluorescence excitation spectra were used to compute energy transfer efficiencies of the various complexes at room temperature and these were correlated with previously reported ultrafast, time-resolved optical spectroscopic dynamics data. The results illustrate the robust nature and value of the PCP complex, which maintains a high efficiency of antenna function even in the presence of non-native chlorophyll species, as an effective tool for elucidating the molecular details of photosynthetic light-harvesting.     

Spatio-temporal variation of photosynthetic pigments in the CAM tree <Emphasis Type="Italic">Clusia hilariana</Emphasis> Schlechtendal associated with dry spells during the rainy season in southeastern Brazil

In this work we assessed spatial and temporal variation of leaf pigment content of Clusia hilariana, a dominant CAM tree on the sandy coastal plains of SE Brazil. Chlorophyll a + b concentration, chlorophyll a/b ratio, and total carotenoid concentration were measured three times during the wet season. Chlorophyll a + b and total carotenoids decreased while the chlorophyll a/b ratio increased after a series of dry spells. We discuss our results in the context of Clusia literature, and argue that leaf-level pigmentation changes are an important source of phenotypic flexibility during acclimation to fluctuating light levels and groundwater availability.     

Attraction of the predatory mites Typhlodromalus manihoti and Typhlodromalus aripo to cassava plants infested by cassava green mite

The attraction of the predatory mites, Typhlodromalus manihoti and Typhlodromalus aripo, to the host plant-spider mite complex, Manihot esculenta – Mononychellus tanajoa, was investigated with a Y-tube olfactometer. Factors examined included predator starvation period, several combinations of cassava leaf biomass and initial M. tanajoa infestations, M. tanajoa-damaged leaves with mites and/or their residues removed, M. tanajoa alone, and mechanically damaged cassava leaves. We found that females of T. manihoti and T. aripo were significantly attracted to M. tanajoa-infested cassava leaves when the predators were starved for 2, 6, or 10 h. Satiated T. aripo was significantly attracted to infested cassava leaves whereas satiated T. manihoti did not discriminate between infested and non-infested leaves. When a choice was given between either two or four leaves infested with 200 female M. tanajoa and an equivalent number of non-infested leaves, 2 h-starved T. manihoti and T. aripo were significantly attracted to each of the infested groups of cassava leaves. At a density of 12 female M. tanajoa per leaf on four leaves, 2 h-starved T. manihoti was still attracted to M. tanajoa-infested leaves whereas 2 h-starved T. aripo was not attracted. When a choice was given between non-infested cassava leaves and either infested leaves from which only M. tanajoa females had been removed, or infested leaves from which all M. tanajoa and their visible products (web, feces) had been wiped off, T. aripo preferred odors from both types of previously infested leaves. Typhlodromalus manihoti was only attracted to infested leaves from which the M. tanajoa females only had been removed. Finally, the two predators were not attracted to 400 female M. tanajoa on clean cotton wool or to mechanically wounded leaves. This supports the hypothesis that M. tanajoa damage induces volatile cues in cassava leaves that attract T. manihoti and T. aripo to M. tanajoa-infested leaves.     

Purification and Characterization of Chlorophyllase from Alga (Phaeodactylum tricornutum) by Preparative Isoelectric Focusing

Chlorophyllase from a diatom alga (Phaeodactylum tricornutum) was obtained and the partially purified extract has been further purified using preparative isoelectric focusing on a Rotofor cell. Three fractions, FI, FII, and FIII, were separated from the Rotofor cell and salt and ampholytes were removed to give fractions FI′, FII′, and FIII′, respectively. Enzyme fractions FI′, FII′, and FIII′, respectively. Enzyme fractions FI′, FII′, and FIII′ showed specific activities of 15.2 × 10^?4, 226.7 ×10^?4 and 33.8 × 10^?4 µmol/mg protein/min, respectively. Most of the enzyme activity (84%) was in fraction FII′. The optimum pH for chlorophyllase activity was 8.0 for FI′ and 8.5 for both FII′ and FIII′. Apparent K_m values for enzyme fractions FI′, FII′, and FIII′ were 2.1nM, 2.3nM, and 2.0 nM, respectively. Enzyme fractions FII′ and FIII′ showed higher chlorophyllase activity towards the partially purified chlorophyll when it was compared to that with the crude chlorophyll as well as with both chlorophylls a and b. However, the enzyme fraction FI′ had higher activity towards the crude chlorophyll when it was compared to that with both chlorophylls a and b, but with a preference for chlorophyll a over chlorophyll b. The inhibitory effect of diisopropyl flurophosphate (DIFP) on chlorophyllase activity demonstrates a noncompetitive inhibitor kinetics with K_i values of 1.29mM, 2.14mM, and 0.71mM for FI′. FII′, and FIII′, respectively.