Xanthophyll cycle and energy-dependent fluorescence quenching in leaves from pea plants grown under intermittent light

The possible role of zeaxanthin formation and antenna proteins in energy-dependent chlorophyll fluorescence quenching (qE) has been investigated. Intermittent-light-grown pea (Pisum sativum L.) plants that lack most of the chlorophyll a/b antenna proteins exhibited a significantly reduced qE upon illumination with respect to control plants. On the other hand, the violaxanthin content related to the number of reaction centers and to xanthophyll cycle activity, i.e. the conversion of violaxanthin into zeaxanthin, was found to be increased in the antenna-protein-depleted plants. Western blot analyses indicated that, with the exception of CP 26, the content of all chlorophyll a/b-binding proteins in these plants is reduced to less than 10% of control values. The results indicate that chlorophyll a/b-binding antenna proteins are involved in the energy-dependent fluorescence quenching but that only a part of qE can be attributed to quenching by chlorophyll a/b-binding proteins. It seems very unlikely that xanthophylls are exclusively responsible for the qE mechanism.Abbreviations CAB chlorophyll a/b-binding - Chl chlorophyll - F_V variable fluorescence - IML intermittent light - LHC light harvesting complex - PFD photon flux density - qP photochemical quenching of chlorophyll fluoresence - qN non-photochemical quenching - qE energy-dependent quenching - qI photoinhibitory quenching - qT quenching by state transition     

Carotenoid synthesis and pleiotropic effects in carotenoid-deficient seedlings of maize

Plastid-envelope membranes from seedlings ofZea mays L. made carotenoid-deficient by either norflurazon treatment or mutation lack an activity permitting conversion of phytoene to -carotene. This activity in membrane fractions was measured by coincubation in vitro with a soluble system from spinach chloroplasts capable of converting [¹⁴C]isopentenyl pyrophosphate into phytoene. When grown in light, the carotenoid-deficient seedlings lack many soluble chloroplast proteins, including NADP-dependent malic enzyme (EC 1.1.1.40), pyruvate phosphate dikinase (EC 2.7.9.1), and ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), but apparently still contain the soluble activities permitting synthesis of phytoene.Abbreviations IPP isopentenyl pyrophosphate - LHCP light-harvesting chlorophylla/b-binding protein - norflurazon 4-chloro-5(methylamine)-2-(,,-trifluoro-m-tolyl)-3-(2H)-pyrazinone - TLC thin-layer chromatography - Tris 3-amino-2-(dihydroxymethyl)-1,3-propanediol     

Phototropism of maize coleoptiles Influences of light gradients

The lateral fluence-rate gradients in unilaterally irradiated maize (Zea mays L.) coleoptiles were calculated on the basis of the proportions of P _fr (far-red-absorbing form of phytochrome) measured spectroscopically in transverse slices of the coleoptiles (top 1 cm). The results showed the occurrence of significant gradients that are wavelength-dependent. The gradient at 449 nm was steeper than those measured at 516, 534 and 551 nm, which were steeper than that measured at 665 nm. The ratios between the sides proximal and distal to the light source were, for example, 1:0.12 (449 nm), 1:0.23 (534 nm), and 1:0.28 (665 nm). Fluence-response curves for coleoptile phototropism (first positive curvature produced by less than 100 s unilateral irradiation) were measured at 449, 516, 534 and 551 nm. Comparison of the threshold fluences indicated that the responsiveness to 551 nm is about 10^4.8 less than that to 449 nm. Increasing wavelengths led to a decrease in maximal curvature, which correlated with the decrease of the fluence-rate ratios between the proximal and distal sides. Phototropic fluence-response curves were also measured using bilateral irradiation (449 nm). In one set of experiments, the fluence ratio was kept constant (either 1:1/2, 1:1/4 or 1:1/16) and the total fluence was varied, and in the other set the fluence applied to one side was kept constant and the fluence ratio was varied. A simple model based on the assumption that only one photoreaction occurs, and that the response is a function of the difference between the proximal and distal sides in the local photoreceptor action was tested. A fluence-response curve for this local photoreceptor action was calculated based on the fluence-rate ratio and the phototropic fluence-response curve measured for 449 nm. This curve was used, in conjunction with the measured fluence-rate ratios, as a basis for calculating phototropic fluence-response curves for other wavelengths and those for 449 nm obtained with bilateral irradiation. The calculated fluence-response curves showed excellent agreement with the experimental data. It is concluded that the threshold for maize coleoptile phototropism reflects the apparent photoconversion cross-section of the blue-light receptor whereas the maximal curvature depends on the steepness of the light gradient across the coleoptile.Abbreviations and symbols I(x) fluence rate at the depth x - P _fr phytochrome (far-red absorbing) - P _r phytochrome (red absorbing) - P _tot total phytochrome (P _r+P _fr) - photoconversion cross-section     

Quantitative correlation of peripheral and intrinsic core polypeptides of photosystem II with photosynthetic electron-transport activity ofAcetabularia mediterranea in red and blue light

The high photosynthetic activity (O₂ production and CO₂ consumption) ofAcetabularia mediterranea Lamour. (=A. acetabulum (L.) Silva) characteristic of cells cultured in white light decreases slowly when cells are kept in continuous red light, and is less than 20% of the original activity after three weeks. Subsequent blue irradiation restores the original activity completely within 3–5 d. The polypeptide composition of the thylakoids from cells grown in either red or blue light and after transfer from red to blue light was analyzed mainly with regards to photosystem II (PSII). The P700-containing reaction-centre complex of photosystem I, CPI, showed only minor quantitative alterations as a consequence of the growth-light quality, which correlated well with the activity of photosystem I under these conditions. In PSII, no drastic changes occurred in the quantity of the reaction-centre components D1 (herbicide-binding polypeptide) and D2, as determined by immunoblots. Likewise, the proteins associated with the water-splitting apparatus did not change detectably in thylakoids from red- or blue-light-treated cells (the 16-kDa component could not be found inAcetabularia thylakoids). The level of the major light-harvesting complex was completely unaffected by the light quality. In contrast, the quantities of the chlorophyll a-protein complexes of the core antenna, CP43 and CP47 (and probably CP29), changed, with kinetics similar to those of total photosynthetic activity. We postulate that the function of the PSII antenna became increasingly impaired in the absence of blue light (i.e. in red light), while blue light had a restoring effect. The peripheral antenna, comprising the light-harvesting complexes, is probably functionally connected with the reaction-centre chlorophylls via the core antenna chlorophyll-protein complexes (CP43, CP47 and probably CP29). A deficiency of these complexes would lead to uncoupling of antenna and reaction centre in the majority of PSII complexes after long periods of red-light treatment.     

Microcallus growth from maize protoplasts

Maize (Zea mays L.) protoplasts obtained from Type I and Type II calli from several genotypes were shown to be capable of synthesizing cell walls and forming small clusters of cells. The medium used also supported cluster formation from protoplasts obtained from root tips. The effects of various additions to the medium (such as casein hydrolysate, coconut water, amino acids, sugars, phytohormones, nitrate, calcium, and dimethylsulfoxide as well as pH variations on cellcluster formation were determined. The method of culture (protoplasts plated in agarose or supported in alginate beads in liquid medium) as well as several components of the medium were found to be critical for microcallus formation. Protoplasts obtained from embryogenic Type I callus and cultured in the medium of C. Nitsch and J.P. Nitsch (1967, Planta 72, 355–370) modified by various additions (NN 67-mod medium) were affected most by various sugars, casein hydrolysate, coconut water, and a combination of the auxins napthalene-1-acetic acid (2 mg/l) and 2,4-dichlorophenoxyacetic acid (0.1 mg/l), and the cytokinin N⁶-benzylaminopurine (0.5 mg/l). Cluster size in the agarose culture system was from 0.1 to 0.5 mm diameter and in the alginate culture system, up to 2.0 mm diameter.     

Regulation of electron transport in maize mesophyll chloroplasts: The relationship between chlorophyll a fluorescence quenching and O2 evolution

The relationship between the redox state of the primary electron acceptor of photosystem II (Q_A) and the rate of O₂ evolution in isolated mesophyll chloroplasts from Zea mays L. is examined using pulse-modulated chlorophyll a fluorescence techniques. A linear relationship between photochemical quenching of chlorophyll fluorescence (q_Q) and the rate of O₂ evolution is evident under most conditions with either glycerate 3-phosphate or oxaloacetate as substrates. There appears to be no effect of the transthylakoid pH gradient on the rate of electron transfer from photosystem II into Q_A in these chloroplasts. However, the proportion of electron transport occurring through cyclic-pseudocyclic pathways relative to the non-cyclic pathway appears to be regulated by metabolic demand for ATP. The majority of non-photochemical quenching in these chloroplasts at moderate irradiances appeared to be energy-dependent quenching.Abbreviations and symbols PSII photosystem II - Fm maximum fluorescence obtained on application of a saturating light pulse - Fo basal fluorescence recorded in the absence of actinic light (i.e. all PSII traps are open) - Fv Fm-Fo - q_Q photochemical quenching - q_NP non-photochemical quenching - q_E energy-dependent quenching of chlorophyll fluorescence     

Desensitization by red and blue light of phototropism in maize coleoptiles

The effects of pretreatments with red and blue light (RL, BL) on the fluence-response curve for the phototropism induced by a BL pulse (first positive curvature) were investigated with darkadapted maize (Zea mays L.) coleoptiles. A pulse of RL, giving a fluence sufficient to saturate phytochrome-mediated responses in this material, shifted the bell-shaped phototropic fluence-response curve to higher fluences and increased its peak height. A pulse of high-fluence BL given immediately prior to this RL treatment temporarily suppressed the phototropic fluence-response curve, and shifted the curve to higher fluences than induced by RL alone. The shift by BL progressed rapidly compared to that by RL. The results indicate (1) that first positive curvature is desensitized by both phytochrome and a BL system, (2) that desensitization by BL occurs with respect to both the maximal response and the quantum efficiency, and (3) that the desensitization responses mediated by phytochrome and the BL system can be induced simultaneously but develop following different kinetics. It is suggested that theses desensitization responses contribute to the induction of second positive curvature, a response induced by prolonged irradiation.Abbreviations BL blue light - RL red light CIW-DPB Publication No. 1001     

Inversion of gravitropism by symmetric blue light on the clinostat

Gravitropic stimulation of maize (Zea mays L.) seedlings resulted in a continuous curvature of the coleoptiles in a direction opposing the vector of gravity when the seedlings were rotated on a horizontal clinostat. The orientation of this response, however, was reversed when the gravitropic stimulation was preceeded by symmetric preirradiation with blue light (12.7 mol photons·m^–2). The fluence-response curve of this blue light exhibited a lower threshold at 0.5 mol·m^–2, and could be separated into two parts: fluences exceeding 5 mol·m^–2 reversed the direction of the gravitropic response, whereas for a range between the threshold and 4 mol·m^–2 a split population was obtained. In all cases a very strong curvature resulted either in the direction of gravity or in the opposite orientation. A minor fraction of seedlings, however, curved towards the caryopsis. Furthermore, the capacity of blue light to reverse the direction of the gravitropic response disappeared with the duration of gravitropic stimulation and it depended on the delay time between both stimulations. Thistonic blue-light influence appears to be transient, which is in contrast to the stability observed fortropistic blue-light effects.This work was supported by the Deutsche Forschungsgemeinschaft.     

Comparison of the kinetic properties,inhibition and labelling of the phosphate translocators from maize and spinach mesophyll chloroplasts

The kinetic properties of the phosphate translocator from maize (Zea mays L.) mesophyll chloroplasts have been determined. We have used a double silicone-oil-layer centrifugation system in order to obtain true initial uptake rates in forward-reaction experiments. In addition, it was possible to perform back-exchange experiments and to study the effects of illumination and of preloading the chloroplasts with different substrates on transport. It is shown that the phosphate translocator from mesophyll chloroplasts of maize, a C₄ plant, transports inorganic phosphate and phosphorylated C₃ compounds in which the phosphate group is linked to the C₃ atom (e.g. 3-phosphoglycerate and triose phosphate). The affinities of the transported metabolites towards the translocator protein are about one order of magnitude higher than in mesophyll chloroplasts from the C₃ plant, spinach. In contrast to the phosphate translocator from C₃-mesophyll chloroplasts, that of C₄-mesophyll chloroplasts catalyzes in addition the transport of C₃ compounds where the phosphate group is attached to the C₂ atom (e.g. 2-phosphoglycerate, phosphoenolpyruvate). The phosphate translocator from both chloroplast types is strongly inhibited by pyridoxal-5-phosphate (PLP), 2,4,6-trinitrobenzenesulfonic acid and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS). In the case of the spinach translocator protein these inhibitors were shown to react with the same amino-acid residue at the substrate binding site, and one molecule of either DIDS or PLP is obviously required per substrate binding site for the inactivation of the translocation process. In the functionally active dimeric translocator protein only one substrate-binding site appears to be accessible at a particular time, indicating that the site might be exposed to each side of the membrane in turn. Using [³H]-H₂DIDS for the labelling of maize mesophyll envelopes the radioactivity was found to be associated with two polypeptides of about 29 and 30 kDa. Since Western-blot analysis showed that only the 30 kDa polypeptide reacted with an antiserum directed against the spinach phosphate translocator protein it is suggested that this polypeptide presumably represents the phosphate translocator from maize mesophyll chloroplasts.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - PEP phosphoenolpyruvate - 2-,3-PGA 2-,3-phosphoglycerate - PLP pyridoxal-5-phosphate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TNBS 2,4,6-trinitrobenzenesulfonic acid - triose P triose phosphate This work was supported by the Deutsche Forschungsgemeinschaft     

Evidence for an essential role of carotenoids in the assembly of an active photosystem II

Dark-grown cells of mutant C-6D of the green alga Scenedesmus obliquus exhibit a high activity of photosystem I (PSI) but lack activity of photosystem II (PSII). These cells contain only the pigment-protein complex CPI, representing the reaction-center of PSI. Only chlorophyll a and precursors of carotenoids (lycopene, neurosporene, -carotene, -zeacarotene) could be detected in dark-grown cells by analysis using high-performance liquid chromatography.Activity of PSII and the corresponding pigment-protein complex, CPa, develop immediately upon transfer to light. Light-harvesting complexes and higher molecular forms of PSI are synthesized only in the later stages of light-induced chloroplast differentiation. During illumination the amounts of carotenoid precursors decrease and carotenes, xanthophylls and chlorophylls a and b are formed. -Carotene and lutein are synthesized without a lag-phase. Their kinetics are similar to those of CPa formation and development of PSII activity. In contrast, all other xanthophylls are synthesized only after a lag-phase of about 30 min.Inhibition of the transformation of precursors into carotenoids by nicotine prevents the light-inducible development of PSII activity and CPa formation. During illumination under anaerobic conditions no xanthophylls are synthesized but high amounts of - and -carotene accumulate. Such cells exhibit no PSII activity and show only traces of CPa. After subsequent transfer to aerobic conditions the xanthophylls are synthesized and simultaneously active PSII units are formed.The results prove that carotenoids are essential components for the assembly of active PSII units. Strong evidence is given that lutein is the absolute necessary prerequisite for this process. Whether -carotene is also an absolute necessary prerequisite for a functioning PSII unit cannot be deduced from our experiments.Abbreviations CP pigment-protein complex - HPLC high-performance liquid chromatography - LHCP light-harvesting chlorophyll-protein complex - PAGE polyacrylamide gel electrophoresis - PCV packed cell volume - PS photosystem     

Cation-activated ATPase activity of plasmalemma-enriched membrane preparations from maize coleoptiles

The ATPase activity present in plasmalemma-enriched preparations from maize coleoptiles shows an optimum at pH 6, a strong dependence on Mg²⁺, and is stimulated by K⁺ and other monovalent cations, both organic and inorganic. The activation of ATPase by K⁺ obeys Michaelis Menten kinetics, saturation being reached at 50 mM K⁺ concentration. K⁺, Mg²⁺-stimulated ATPase activity is strongly inhibited by N,N-dicyclohexylcarbodiimide and by diethylstilbestrol and, to a lesser extent, by octylguanidine.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - DTE dithioerythritol - Ellmans r 5-5 dithiobis (2 nitrobenzoic) acid - FC fusicoccin - NPA naphthylphthalamic acid - OG octylguanidine - PCMBS p-chloromercuribenzensulphonate     

Embryogenic callus formation from maize protoplasts

Maize (Zea mays L.) protoplasts have been obtained which divide rapidly and produce a callus that differentiates to form somatic embryos. The somatic embryos can be induced to form roots and small leaf-like structures. The genotype was the hybrid A188xBlack Mexican Sweet. Protoplasts were prepared from an embryogenic suspension culture derived from a Type II callus which had been selected from Type I callus produced by immature zygotic embryos. The basal medium for the suspension culture was N6 (C.C. Chu et al., 1975, Scientia Sinica 18, 659–668). The 2,4-dichlorophenoxyacetic acid concentration of the suspension culture was critical for subsequent protoplast growth and was optimal at 4.0 mg.l. Protoplasts had to be cultured in a low-osmoticum medium (0.3 M mannitol) for subsequent cell divisions to occur. The protoplasts have been transformed transiently with the gene chloramphenicol acetyltransferase (CAT) containing the 35S promoter obtained from cauliflower mosaic virus (CaMV-35S).Abbreviations FDA fluorescein diacetate - ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Chloroplast proliferation based on their distribution and arrangement inAdiantum protonemata growing under red light

Chloroplast proliferation was investigated inAdiantum protonemata growing under continuous red light. Cell division is absent when cells are grown under red light. The chloroplast number increases as the cell length increases, therefore the chloroplasts divide in the absence of cell division. Chloroplasts in the basal part of the filamentous protonemal cell migrate gradually toward the cell apex, but there is no large net migration from the tip to the base or vice versa, indicating that chloroplast division takes place in the apical part of the protonemata. Chloroplast number in the apical 100 μm was maintained at about 200 during cell growth at least over eight days. The chloroplasts were either dumbbell- or ellipsoid-shaped. Dumbbell-shaped chloroplasts are abundant everywhere in a protonema, ranging from 30 to 50% of the total chloroplasts. The dumbbell-shaped chloroplasts attached to or very close to the plasma membrane seem to be the ones that are dividing but the dumbbell-shaped ones in the other regions do not divide. These data support the hypothesis that a signal from the plasma membrane induces the dumbbell-shaped chloroplasts to divide.     

Plastid development in seedlings of Echinochloa crus-galli var. oryzicola under anoxic germination conditions

Plastid development in the primary leaf of Echinochloa crus-galli (L.) Beauv. var. oryzicola (Vasing.) Ohwi was followed during 5 d of anoxic germination and growth. Plastids develop slowly from simple spheroidal proplastids into larger pleomorphic plastids with several stromal membranes and many peripheral membrane vesicles. A small prolamellar body is present at 96 h with perforated (pro)thylakoids extending into the stroma. Changes in starch grains and plastoglobuli are evidence of carbohydrate and lipid metabolism. Plastid division is indicated by dumbbell plastid profiles after 4 d of anoxia. These results demonstrate that plastids not only maintain their integrity during anaerobic germination but also show developmental changes involving an increase in internal membrane complexity, although to a lesser extent than in etiolated shoots.Abbreviation PLB prolamellar body Scientific paper No. 6167. College of Agriculture, Washington State University, Pullman     

Loss or retention of chloroplast DNA in maize seedlings is affected by both light and genotype

We examined the chloroplast DNA (cpDNA) from plastids obtained from wild type maize (Zea mays L.) seedlings grown under different light conditions and from photosynthetic mutants grown under white light. The cpDNA was evaluated by real-time quantitative PCR, quantitative DNA fluorescence, and blot-hybridization following pulsed-field gel electrophoresis. The amount of DNA per plastid in light-grown seedlings declines greatly from stalk to leaf blade during proplastid-to-chloroplast development, and this decline is due to cpDNA degradation. In contrast, during proplastid-to-etioplast development in the dark, the cpDNA levels increase from the stalk to the blade. Our results suggest that DNA replication continues in the etioplasts of the upper regions of the stalk and in the leaves. The cpDNA level decreases rapidly, however, after dark-grown seedlings are transferred to light and the etioplasts develop into photosynthetically active chloroplasts. Light, therefore, triggers the degradation of DNA in maize chloroplasts. The cpDNA is retained in the leaf blade of seedlings grown under red, but not blue light. We suggest that light signaling pathways are involved in mediating cpDNA levels, and that red light promotes replication and inhibits degradation and blue light promotes degradation. For five of nine photosynthetic mutants, cpDNA levels in expanded leaves are higher than in wild type, indicating that nuclear genotype can affect the loss or retention of cpDNA.     

Factors affecting the binding between fusicoccin and plasma membranes from maize roots

Plasma membranes have been purified from roots of maize (Zea mays L.) using a two-phase aqueous polymer system, dextran-polyethylene glycol. The plant material was homogenized in the presence of a mixture of natural protease inhibitors from potato (Solanum tuberosum L.); these inhibitors have been shown to be more effective than phenylmethylsulfonyl fluoride in suppressing the endogenous proteases in maize roots. Inhibition of proteolysis in the homogenization medium markedly increased (about tenfold) the number of lowaffinity binding sites for fusicoccin (FC). In addition, storage of plasma membranes at −20° C decreased both the number of the low-affinity sites and their dissociation constant (K_D); this effect was in all probability caused by lipid peroxidation. The presence of EDTA throughout isolation and storage of the plasma membranes stabilized the parameters of FC binding to the membranes. The kinetics of binding of [³H]dihydroFC and the competition between [³H]dihydroFC and FCs A, C, J, and H were determined for the low-affinity sites. It was found that (i) the rate constant of association between FC and the low-affinity binding sites is about two orders of magnitude lower than that for the high-affinity sites; (ii) different FCs can be arranged in the order of decreasing avidity for the low-affinity FCbinding site: FC A>FC C>FC J>FC H. The authors are indebted to Dr. L.M. Krasnopolskaya (Institute of Agricultural Biotechnology, Moscow, Russia) for fusicoccins A, C, J, and H, and to Dr. A.V. Galkin (Institute of Agricultural Biotechnology, Moscow, Russia) for valuable comments and ren dering the paper into English.     

Development of the Casparian strip in primary roots of maize under salt stress

The Casparian strip in the endodermis of vascular plant roots appears to play an important role in preventing the influx of salts into the stele through the apoplast under salt stress. The effects of salinity on the development and morphology of the Casparian strip in primary roots of maize (Zea mays L.) were studied. Compared to the controls, the strip matured closer to the root tip with increase in the ambient concentration of NaCl. During growth in 200 mM NaCl, the number and the length of the endodermal cells in the region between the root tip and the lowest position of the endodermal strip decreased, as did the apparent rate of production of cells in single files of endodermal cells (the rate of cell formation being equal to the rate at which cells are lost from the meristem). The estimated time required for an individual cell to complete the formation of the strip after generation of the cell in the presence of 200 mM NaCl was not very different from that required in controls. Thus, salinity did not substantially affect the actual process of formation of the strip in individual cells. The radial width of the Casparian strip, a morphological parameter that should be related to the effectiveness of the strip as a barrier, increased in the presence of 200 mM NaCl. The mean width of the lignified region was 0.92 m in distilled water and 1.33 m in 200 mM NaCl at the lowest position of the strip. The mean width of the strip relative to that of the radial wall at this position was significantly greater after growth in the presence of 200 mM NaCl than in the controls, namely, 20.5% in distilled water and 33.9% in 200 mM NaCl. These observations suggest that the function of the strip is enhanced under salt stress.     

Microcallus formation from maize protoplasts prepared from embryogenic callus

Conditions have been developed that induce maize (Zea mays L.) protoplasts to re-synthesize cell walls and to initiate cell divisions. Two types of embryogenic maize callus were used as a source of protoplasts: a heterogeneous callus (Type I) derived from immature embryos after three weeks in culture, and a friable, rapidly growing callus (Type II) selected from portions of the Type I callus. Many variables in the growth conditions of the donor tissue (type of medium, transfer schedule, age of callus), protoplast isolation solutions (pH, osmolarity, type and concentration of cell wall hydrolyzing enzymes, addition of polyamines) and conditions (amount of time in enzyme, amount of tissue per volume of enzyme incubation medium, agitation, preplasmolysis of source tissue, type of callus), and purification procedures (filtration and-or flotation), were found to affect both yield and viability of protoplasts (based upon fluorescein-diacetate staining). Our isolation procedure yielded high numbers of viable, uninucleated maize callus protoplasts which were densely cytoplasmic and varied in size from 20 to 50 m in diameter. Protoplasts plated in solid medium formed walls and divided several times. Of several gelling agents tested for protoplast propagation, only agarose resulted in protoplasts capable of sustained divisions leading to the formation of microcalli. Plating efficiency was established over a wide range of protoplast densities (10³–10⁷ protoplasts/ml). Highest plating efficiency (25%) was obtained at 1·10⁶ protoplasts/ml). The resulting microcalli grew to be dense clusters of about 0.1–0.5 mm in diameter and then stopped growing. Nurse cultures of maize and carrot (Daucus carota L.), were used to establish that individual protoplasts (not contaminating cells or cell clusters) formed walls and divided. Nurse cultures also increased the efficiency of microcallus formation from protoplasts.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) salts - MS 1D Murashige and Skoog salts with 1 mg/l 2,4-D - MS 2D Murashige and Skoog salts with 2 mg/l 2,4-D - N₆ medium of Chu et al. (1975) - NN67-mod medium of Nitsch and Nitsch (1967) as modified in the present paper - FDA fluorescein diacetate - LMP low melting point     

Rapid,localized light effect on root growth in maize

A method using optical microfibers permitted localized exposure of the cap or the elongating part of growing maize (Zea mays L.) roots to white light. When the cap was illuminated, a strong and very rapid inhibition of the elongation rate of the roots was found. When the light microbeam was directed at the elongating region, the roots continued to grow at the same rate as before the illumination.