Local anesthetic-induced microscopic and mesoscopic effects in micelles. A fluorescence,spin label and SAXS study

The interaction of the local anesthetic tetracaine (TTC) with anionic sodium lauryl sulfate (SLS) and zwitterionic 3-(N-hexadecyl-N,N-dimethylammonio)propanesulfonate (HPS) micelles was investigated by fluorescence, spin labeling EPR and small angle X-ray scattering (SAXS). Fluorescence pH titrations allowed the choice of adequate pHs for the EPR and SAXS experiments, where either charged or uncharged TTC would be present. The data also indicated that the anesthetic is located in a less polar environment than its charged counterpart in both micellar systems. EPR spectra evidenced that both anesthetic forms increased molecular organization within the SLS micelle, the cationic form exerting a more pronounced effect. The SAXS data showed that protonated TTC causes an increase in the SLS polar shell thickness, hydration number, and aggregation number, whereas the micellar features are not altered upon incorporation of the uncharged drug. The combined results suggest that the electrostatic interaction between charged TTC and SLS, and the intercalation of the drug in the micellar polar region induce a change in molecular packing with a decrease in the mean cross-sectional area, not observed when the neutral drug sinks more deeply into the micellar hydrophobic domain. In the case of HPS micelles, the EPR spectral changes were small for the charged anesthetic and the SAXS data did not evidence any change in micellar structure, suggesting that this species protrudes more into the aqueous phase due to the lack of electrostatic attractive forces in this system.     

Organization and dynamics in micellar structural transition monitored by pyrene fluorescence

Structural transitions involving shape changes play an important role in cellular physiology. Such transition can be induced in charged micelles at a given temperature by increasing ionic strength of the medium. We have monitored the change in organization and dynamics associated with sphere-to-rod transition of SDS micelles utilizing pyrene fluorescence. We report here, utilizing changes in the ratio of pyrene vibronic peak intensities (I₁/I₃), the apparent dielectric constant experienced by pyrene in spherical SDS micelles (in absence of salt) to be ∼32. Interestingly, the apparent micellar dielectric constant exhibits a reduction with increasing NaCl concentration. The dielectric constant in rod-shaped micelles of SDS (in presence of 0.5 M NaCl) turns out to be ∼22. To the best of our knowledge, these results constitute one of the early reports on polarity estimates in rod-shaped micelles. In addition, pyrene excimer/monomer ratio shows increase in SDS micelles with increasing NaCl concentration. We interpret this increase due to an increase in average number of pyrene molecules per micelle associated with the sphere-to-rod structural transition. These results could be significant in micellar drug solubilization and delivery, and in membrane morphology changes.     

Phase equilibria of mixtures of plant galactolipids. The formation of a bicontinuous cubic phase

The chloroplast galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were isolated from wheat leaves. The phase equilibria of galactolipid-water systems with MGDG / DGDG molar ratios equal to 0:1, 1:2, 1.2:1, 2:1 and 1:0 were investigated, using nuclear magnetic resonance (NMR) methods. MGDG and DGDG form reversed hexagonal and lamellar phases, respectively, at temperatures between 10 and 40°C at all water contents studied (up to about 14 mol ²H₂O per mol lipid). The galactolipid mixtures show a complex phase forming reversed hexagonal, lamellar and reversed cubic phases, depending on water content and temperature. It was found that the water hydration is similar for the lamellar and hexagonal phases formed by DGDG and MGDG, respectively. The non-lamellar phase areas increase with increasing content of MGDG. Small-angle X-ray measurements show that the cubic phase belongs to the Ia3d space group. From translational diffusion studies by NMR it is concluded that the structure of this cubic phase is bicontinuous.     

Synthesis of novel pH-sensitive chitosan graft copolymers and micellar solubilization of paclitaxel

Herein, highly pH-sensitive graft copolymers, N-octyl-N-(2-carboxylbenzoyl) chitosan derivatives, were synthesized and characterized by FTIR, ¹H NMR, ¹³C NMR, differential scanning calorimetry and X-ray diffraction spectrometry. The polymers can form micelles solublizing paclitaxel, with critical micellar concentrations ranged from 0.07 to 0.32 mg/ml, drug-loading rate ranged from 30.7% to 65.3% and entrapment efficiency ranged from 44.2% to 61.4%. Additionally, the result shows that the micelles display highly sensitivity to mildly acidic pH while reasonably stable at physiologic pH, which might pave the way for building pharmaceutical nanocarriers specifically releasing cargo at certain pathological sites of body.     

State Transitions in the Green Alga Scenedesmus Obliquus Probed by Time-Resolved Chlorophyll Fluorescence Spectroscopy and Global Data Analysis

Decay-associated fluorescence spectra of the green alga Scenedesmus obliquus have been measured by single-photon timing with picosecond resolution in various states of light adaptation. The data have been analyzed by applying a global data analysis procedure. The amplitudes of the decay-associated spectra allow a determination of the relative antenna sizes of the photosystems. We arrive at the following conclusions: (a) The fluorescence kinetics of algal cells with open PS II centers (F₀ level) have to be described by a sum of three exponential components. These decay components are attributed to photosystem (PS) I (τ ≈ 85 ps, λ_max^em ≈ 695-700 nm), open PS II α-centers (τ ≈ 300 ps, λ_max^em = 685 nm), and open PS II β-centers (τ ≈ 600 ps, λ_max^em = 685 nm). A fourth component of very low amplitude (τ ≈ 2.2-2.3 ns, λ_max^em = 685 nm) derives from dead chlorophyll. (b) At the F_max level of fluorescence there are also three decay components. They originate from PS I with properties identical to those at the F₀ level, from closed PS II α-centers (τ ≈ 2.2 ns, λ_max^em = 685 nm) and from closed PS β-centers (τ ≈ 1.2 ns, λ_max^em = 685 nm). (c) The major effect of light-induced state transitions on the fluorescence kinetics involves a change in the relative antenna size of α- and β-units brought about by the reversible migration of light-harvesting complexes between α-centers and β-centers. (d) A transition to state II does not measurably increase the direct absorption cross-section (antenna size) of PS I. Our data can be rationalized in terms of a model of the antenna organization that relates the effects of state transitions and light-harvesting complex phosphorylation with the concepts of PS II α,β-heterogeneity. We discuss why our results are in disagreement with those of a recent lifetime study of Chlorella by M. Hodges and I. Moya (1986, Biochim. Biophys. Acta., 849:193-202).     

Fluctuations, exchange processes, and water diffusion in aqueous protein systems: A study of bovine serum albumin by diverse NMR techniques

Experimental frequency, concentration, and temperature dependences of the deuteron relaxation times T₁ and T₂ of D₂O solutions of bovine serum albumin are reported and theoretically described in a closed form without formal parameters. Crucial processes of the theoretical concept are material exchange, translational diffusion of water molecules on the rugged surfaces of proteins, and tumbling of the macromolecules. It is also concluded that, apart from averaging of the relaxation rates in the diverse deuteron phases, material exchange contributes to transverse relaxation by exchange modulation of the Larmor frequency. The rate limiting factor of macromolecular tumbling is determined by the free water content. In a certain analogy to the classical free-volume theory, a “free-water-volume theory” is presented. There are two characteristic water mass fractions indicating the saturation of the hydration shells (C_s ≈ 0.3) and the onset of protein tumbling (C₀ ≈ 0.6). The existence of the translational degrees of freedom of water molecules in the hydration shells has been verified by direct measurement of the diffusion coefficient using an NMR field-gradient technique. The concentration and temperature dependences show phenomena indicating a percolation transition of clusters of free water. The threshold water content was found to be C_c^w ≈ 0.43.     

Osmosensitivity of Sucrose Uptake by Immature Pea Cotyledons Disappears during Development

Sucrose uptake was studied in isolated, immature pea cotyledons (Pisum sativum L. cv Marzia) in relation to their developmental stage. During the developmental period examined the water content of the cotyledons decreased from ≈80% “stage 1” to ≈55% “stage 2”. When assayed in an isotonic medium (400 osmoles per cubic meter) the influx capacity per gram fresh weight for sucrose was almost constant during this developmental period. The influx could be analyzed into a saturable component (K_m ≈ 9 moles per cubic meter; V_max ≈ 150 nanomoles per minute per gram fresh weight) and an unsaturable component (k_i ≈ 0.5 nanomoles per minute per gram fresh weight [per mole per cubic meter]). Incubation in a hypotonic medium reduced the sucrose influx in stage 1 cotyledons, up to 80% reduction at 0 milliosmole (medium without mannitol), but had no effect on sucrose uptake by stage 2 cotyledons. Reduced uptake in a hypotonic medium (100 osmoles per cubic meter) could be attributed to a lowering of the V_max from 150 to 36 nanomoles per minute per gram fresh weight. During incubation of stage 1 cotyledons and stage 2-cotyledons in a hypotonic medium (200 osmoles per cubic meter) their volume increased by 16% and 5.6%, respectively, while the calculated turgor pressure increased from 0.2 to 0.6 megapascal for cotyledons of both developmental stages. Reduced sucrose influx in hypotonic medium, therefore, seems to be related to cell swelling (membrane stretching) rather than to increased turgor pressure.     

Relationship between three-dimensional arrays of "lipidic particles" and bicontinuous cubic lipid phases

Several lipid-water mixtures form phases that give rise to freeze-fracture replicas exhibiting three-dimensional regular arrays of closely packed globular elements, often called "lipidic particles". These phases have often been poorly classified with respect to long-range organization and symmetry and have in most cases been asserted to be built up by closed lipid aggregates, such as reversed micelles. However, studies of phases giving rise to the above-mentioned freeze-fracture replicas, with X-ray diffraction and the nuclear magnetic resonance pulsed field gradient diffusion technique, have revealed that they are cubic liquid-crystalline phases and with one exception bicontinuous phases, i.e., cubic phases in which both the hydrocarbon and the water regions are continuous. Up to now the only known exception is a cubic phase composed of closed rod-shaped micelles of the normal type. Thus it is not possible to decide from a freeze-fracture image of a cubic phase, showing three-dimensional arrays of "lipidic particles", if the phase is bicontinuous or composed of closed lipid aggregates. Hitherto, it has not been shown that a biological membrane lipid-water system is able to form a cubic liquid-crystalline phase consisting of reversed micelles. The existence of such a phase is also improbable considering the location in the phase diagrams of cubic phases formed by biological membrane lipid-water systems.     

A comparative study of lysozyme conformation in various reverse micellar systems

The activity and conformation of lysozyme solubilized in apolar solvents via reverse micelles was investigated. The systems used were sodium di-2-ethylhexylsulfosuccinate (AOT)/isooctane/H₂O, cetyltrioctylammoniumbromide (CTAB)/CHCl₃, isooctane/H₂O; tetraethyleneglycoldodecylether (EO₄C₁₂)/isooctane/H₂O, and bulk water. CD spectra of lysozyme in reverse micellar solutions were investigated as a function of w₀ (= [H₂O]/[AOT]) and were compared to the spectra in aqueous solutions. No marked changes were found in the EO₄C₁₂ or in the CTAB systems with respect to water, which indicates that no sizeable conformational changes of the enzyme occurred upon solubilization in the reverse micellar systems. In agreement with previous studies [C. Grandi, R. E. Smith, and P. L. Luisi (1981) J. Biol. Chem. 256 , 837–843] dramatic conformational changes can be inferred in the AOT system on the basis of CD studies. This is taken as an indication that the enzyme denatures in this micellar system. This is particularly striking because the enzyme is fully active in AOT reverse micelles. The apparent paradox is solved by the observation that the native CD spectrum (and by inference, the native conformation) is maintained when lysozyme is bound to NAG or NAG₃, and by inference, when the substrate is bound, e.g., during enzyme turnover. However, in the absence of added NAG, NAG₃, or substrate, the enzyme in the AOT reverse micellar system rapidly denatures. Together with CD studies, fluorescence and nmr data confirm the hypothesis of an irreversible denaturation of lysozyme in the AOT system, the denaturation being slowed down when the substrate is present. The activity of the enzyme has been studied as a function of pH and w₀ using the chromophoric substrate 3,4-dinitrophenyl-tetra-N-acetyl-β-D -chitotetraoside (3,4-DNP-NAG₄). Generally speaking, the kinetic parameters are comparable to those found in bulk water solution. More detailed, in the CTAB system, k_cat tends to be smaller than in aqueous solution (with quite similar K_M), whereas in the EO₄C₁₂ system (at pH 7.0) the turnover number is larger and K_M is smaller than in water. In the AOT system, the kinetic parameters at pH 7.0 are also quite comparable to those found in water.     

Kinetic models for the dynamical behavior of polyacrylamide (PAAm)–κ-carrageenan (κC) composite gels

A fluorescence method was employed for studying the drying and swelling of PAAm–κC composite gels, which were formed from acrylamide (AAm) and N, N’- methylenebisacrylamide (BIS) with various κ–carrageenan (κC) contents by free radical crosslinking copolymerization in water. Composite gels were prepared at 80 °C with pyranine (Py) as a fluorescence probe. Scattered light, I_sc, and fluorescence emission intensities, I_em, were monitored during drying and swelling of these gels. The fluorescence intensity of pyranine increased and decreased as drying and swelling time are increased, respectively, for all gel samples. The Stern–Volmer equation combined with moving boundary and Li-Tanaka models were used to explain the behavior of I_em during drying and swelling processes respectively. It is found that the desorption coefficient D_d decreased as κC contents were increased for a given temperature during drying. However, the cooperative diffusion coefficient, D_s presented exactly the opposite case. Conventional gravimetrical and volumetric experiments were also carried out during drying and swelling of PAAm–κC composite gels. It was observed that D_d and D_s values measured with the fluorescence method were found to be much larger than they were measured with the conventional methods.     

Growth, gas exchange, and water-use efficiency response of two young apple cultivars to drought stress in two scion-one rootstock grafting system

The combination of two scion-one rootstock was used for two apple cultivars, ‘Pink Lady’ and ‘Qinguan’, budded on the same, one-year-old Malus hupehensis (Pamp.) Rehd. to reduce the impact of root and pot size and in order to understand the growth, water-use efficiency (WUE), and chlorophyll fluorescence characteristics. The two-scion grafted trees were planted in plastic pots under two water regimes, i.e. 70% field capacity (FC) and 55% FC. Results indicated that different scions were affected differently by drought stress. ‘Pink Lady’ had higher net photosynthetic rate (P _N), stomatal conductance (g _s), and transpiration rate (E) compared with ‘Qinguan’ under both water treatments. However, ‘Qinguan’ had lower minimal fluorescence (F₀), higher maximum fluorescence (F_m), and higher maximum photochemical efficiency of photosystem II (F_v/F_m) than ‘Pink Lady’ at 55% FC. Moreover, ‘Qinguan’ had larger shoot dry mass (ShDM) and higher intrinsic WUE_I than ‘Pink Lady’ under both water status. Gas-exchange and growth parameters, except for P _N and scion diameter, were significantly affected by the cultivar and water treatment. At 70% FC, ShDM was significantly correlated with WUE_I. Moreover, WUE_I was negatively linearly correlated with g _s at either 70 or 55% FC. These results might indicate that ‘Pink Lady’ was more sensitive to drought than ‘Qinguan’. ‘Qinguan’ apple was able to improve WUE more than ‘Pink Lady’ under both well-watered and drought conditions. The growth parameters and photosynthetic capacity of two different scions showed that the combination of double scion-one rootstock might eliminate the influences of the rootstock and pot size.     

Tryptophan fluorescence reveals the presence of long-range interactions in the denatured state of ribonuclease Sa

Characterizing the denatured state ensemble is crucial to understanding protein stability and the mechanism of protein folding. The aim of this research was to see if fluorescence could be used to gain new information on the denatured state ensemble. Ribonuclease Sa (RNase Sa) contains no Trp residues. We made five variants of RNase Sa by adding Trp residues at locations where they are found in other members of the microbial ribonuclease family. To better understand the protein denatured state, we also studied the fluorescence properties of the following peptides: N-acetyl-Trp-amide (NATA), N-acetyl-Ala-Trp-Ala-amide (AWA), N-acetyl-Ala-Ala-Trp-Ala-Ala-amide (AAWAA), and the five pentapeptides with the same sequence as the Trp substitution sites in RNase Sa. The major conclusions are: 1), the wavelength of maximum fluorescence intensity, λ_max, does not differ significantly for the peptides and the denatured proteins; 2), the fluorescence intensity at λ_max, I_F, differs significantly for the five Trp containing variants of RNase Sa; 3), the I_F differences for the denatured proteins are mirrored in the peptides, showing that the short-range effects giving rise to the I_F differences in the peptides are also present in the proteins; 4) the I_F values for the denatured proteins are more than 30% greater than for the peptides, showing the presence of long-range effects in the proteins; 5), fluorescence quenching of Trp by acrylamide and iodide is more than 50% greater in the peptides than in the denatured proteins, showing that long-range effects limit the accessibility of the quenchers to the Trp side chains in the proteins; and 6), these results show that nonlocal effects in the denatured states of proteins influence Trp fluorescence and accessibility significantly.     

Hyperfine shift NMR studies of hydrated phospholipid inverted micelles

The ³¹P nuclear magnetic resonance (NMR) spectra of benzene solutions of hydrated dipalmitoyl lecithin (DPL) inverted micelles, with and without incorporated paramagnetic lanthanide ions, have been recorded. Individual resonances for micelles containing none, one, and two ions can be resolved and observed in the presence of one another. The relative intensities of these peaks yield some information on the state of aggregation of lipid inverted micelles prepared by ultrasonic irradiation. The relative intensities and chemical shifts of resonances of unsonicated mixtures of preformed micelles containing different numbers of ions per micelle indicate that some kind of equilibration occurs. The data are consistent with a selective fusion of multi-ion micelles with ion-free micelles. The NMR spectra place constraints on the lifetimes of metal ions and lipid and water molecules within a micelle before transfer to another.     

Lutein-chlorophyll-a energy transfer in detergent micelles

Absorption, fluorescence and fluorescence excitation spectra were determined for equimolar mixed micellar detergent solutions of lutein and chlorophyll-a in the concentration range from 9·10^?6 to 1.8·10^?4 M, with detergent (triton-X100) concentrations from 3·-10^?4 to 7·10^?3 M. In the range of detergent concentrations studied the pigments incorporated into the detergent micelles attained a high local concentration (0.1 to 0.01 M), reminiscent of pigment concentration within the chloroplast. A lutein → chlorophyll-a energy transfer with an efficiency of about 15% was found in these systems. In dilute (9·10^?6 M) pigment solution with concentrated (7·10^?3 M) detergent practically no transfer is observed. The extent of aggregation and the efficiency of transfer depend on the composition of the system. The aggregation of chlorophyll-a is partly inhibited by lutein molecules. It is shown that the energy transfer efficiency as function of distance follows anr ^?3 relationship,R ₀ being 22 å.     

Interactions between biliary lipid micelles and intestinal brush border membranes investigated by 1H and 31P nuclear magnetic resonance

The effect of taurocholate and lecithincholesterol-taurocholate mixed micelles on the structure of isolated intestinal brush border membranes was investigated by nuclear magnetic resonance (NMR). Rabbit brush border membranes isolated by a Mg²⁺ precipitation step were chosen for this study because of their stability and integrity as revealed by ³¹P NMR. Incubation of taurocholate with the brush border membranes does not induce significant solubilization of these membranes even when the taurocholate/phospholipid ratio reaches 3.0 ¹H NMR studies indicate that taurocholate is included in the membrane bilayer at low concentration (3 mM). However this biliary salt produces a size diminution of the vesicles when its concentration increases. Incorporation of lecithin or lecithin-cholesterol in micelles of taurocholate and subsequent incubation with brush border membranes lead simultaneously to a decrease in the ³¹P NMR isotropic/bilayer line ratio, and to an increase in . These results indicate a protective effect of these compounds against lytic damage of taurocholate. Futhermore the equilibrium distribution of lecithin between mixed micelles and the membrane bilayer is strongly in favour of complete integration of micellar components in the bilayer. These data suggest that uptake of lipids from the micellar phase by isolated brush border membranes involves an interaction of the micelles with membranes followed by a fusion process.     

Thermal Characterization,Crystal Field Analysis and In-Band Pumped Laser Performance of Er Doped NaY(WO4)2 Disordered Laser Crystals

Undoped and Er-doped NaY(WO₄)₂ disordered single crystals have been grown by the Czochralski technique. The specific heat and thermal conductivity (κ) of these crystals have been characterized from T = 4 K to 700 K and 360 K, respectively. It is shown that κ exhibits anisotropy characteristic of single crystals as well as a κ(T) behavior observed in glasses, with a saturation mean free phonon path of 3.6 Å and 4.5 Å for propagation along a and c crystal axes, respectively. The relative energy positions and irreducible representations of Stark Er³⁺ levels up to ⁴G_7/2 multiplet have been determined by the combination of experimental low (<10 K) temperature optical absorption and photoluminescence measurements and simulations with a single-electron Hamiltonian including both free-ion and crystal field interactions. Absorption, emission and gain cross sections of the ⁴I_13/2↔⁴I_15/2 laser related transition have been determined at 77 K. The ⁴I_13/2 Er³⁺ lifetime (τ) was measured in the temperature range of 77–300 K, and was found to change from τ (77K) ≈ 4.5 ms to τ (300K) ≈ 3.5 ms. Laser operation is demonstrated at 77 K and 300 K by resonantly pumping the ⁴I_13/2 multiplet at λ≈1500 nm with a broadband (FWHM≈20 nm) diode laser source perfectly matching the 77 K crystal ⁴I_15/2 → ⁴I_13/2 absorption profile. At 77 K as much as 5.5 W of output power were obtained in π-polarized configuration with a slope efficiency versus absorbed pump power of 57%, the free running laser wavelength in air was λ≈1611 nm with the laser output bandwidth of 3.5 nm. The laser emission was tunable over 30.7 nm, from 1590.7 nm to 1621.4 nm, for the same π-polarized configuration.     

On the conformations of poly-N-methyl-L-alanine (PNMA) in solution

High-resolution nuclear magnetic resonance spectra at 100 MHz have been obtained on poly-N-methyl-L -alanine in the chloroform–trifluoroacetic acid system under various conditions of solvent composition. Different spectra are observed for the CH₃–C_α, C_α–H, and N–CH₃ protons. On the basis of conformational analysis an interpretation of the NMR results was attempted.     

Structural and dielectric properties of synthetic glycolipids in mixtures with water

Three synthetically produced glycolipids, N-(β-D-glucopyranosyl)-N-octadecyl-stearoylamide (OSGA), N-(β-D-glucopyranosyl-N-octadecyl-oleoylamide (OOGA), N-(β-D-galactopyranosyl)-N-octadecyl-lauroylamide (OLGA) have been studied in different mixtures with water by x-ray diffraction and dielectric measurements with microwaves at 9.4 GHz. The measurements were performed in the temperature range -50-70°C. X-Ray diffraction revealed a direct L_β' → H transition at 20°C, 60°C, and 45°C depending on the glycolipid species but nearly not on the water content. The hexagonal phases are saturated at a water content of ≈20 wt%. The lamellar phase absorbs even less water (< 10 wt%). The dielectric data show that in the H phase the binding of water is stronger than in the L_β' phase. In the temperature range below 0°C, OSGA and OOGA show a “subzero transition” due to the freeze-out of water in a separate ice phase. This transition can be seen in an abrupt decrease of the dielectric function because the dielectric response of ice is much smaller at microwave frequencies. OLGA does not show the subzero transition but an additional transition, hexagonal → distorted hexagonal at 60°C.     

Photosynthesis in Drought-Adapted Cassava

After 45 d of limited water supply, cassava (Manihot esculenta Crantz) exhibited pronounced reduction in shoot growth, high leaf fall, and decreased stomatal conductance. However, the water status of the remaining leaves was unaffected. This was combined with an amplified heliotropic response and drooping which minimises radiant energy interception at mid-day, suggesting that leaves are sensitive to high irradiance (I). In well-irrigated plants, CO₂-saturated oxygen evolution and net photosynthetic rate (P _N) in air were markedly higher (5-fold) in young (expanding) leaves than in mature leaves. Water limitation did not strongly modify CO₂-saturated oxygen evolution but it altered P _N in air for both types of leaves, although differently. The mature leaves of drought-adapted plants displayed residual rate of P _N and deteriorated photosystem 2 (PS2) photochemistry estimated from chlorophyll (Chl) a fluorescence measurements. In young leaves at moderate I, P _N was depressed by only 66 % in stressed plants. Moreover, the photochemical quenching of Chl a fluorescence and the quantum efficiency of PS2 photochemistry in young leaves were comparable in both control and stressed plants. In contrast at high I, P _N was almost null and marked decreases in the two fluorescence parameters were apparent. Hence the strong heliotropic response and drooping displayed by young leaves under water limitation is an important strategy for avoiding inactivation of P _N by high I and therefore for cassava tolerance to drought.