Transverse sections of immature and mature sugarcane internodes were investigated anatomically with white and fluorescence light microscopy. The pattern of lignification and suberization was tested histo‐chemically. Lignification began in the xylem of vascular bundles and progressed through the sclerenchymatic bundle sheath into the storage parenchyma. Suberization began in parenchyma cells adjacent to vascular bundle sheaths and spread to the storage parenchyma and outer sheath cells. In mature internodes most of the storage parenchyma was lignified and suberized to a significant degree, except in portions of walls of isolated cells. The pattern of increasing lignification and suberization in maturing internodes more or less paralleled an increase of sucrose in stem tissue. In mature internodes having a high sucrose concentration, the vascular tissue was surrounded by thick‐walled, lignified and suberized sclerenchyma cells. The apoplastic tracer dyes triso‐dium 3‐hydroxy‐5,8,10‐pyrenetrisulfonate (PTS) and amido black 10 B, fed into cut ends of the stalk, wereconfined to the vascular bundles in all internodes above the one that was cut — with no dye apparently in storage parenchyma tissue. Thus both structural and experimental evidence is consistent with vascular tissue being increasingly isolated from the storage parenchyma as maturation of the tissue proceeds. We conclude that in mature internodes the pathway for sugars from the phloem to the storage parenchyma is symplastic. The data suggest that an increasingly greater role for a symplastic pathway of sugar transfer occurs as the tissue undergoes lignification/suberization.

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Sugarcane cell suspensions took up sugar from the medium at rates comparable to or greater than sugarcane tissue slices or plants in the field. This system offers an opportunity for the study of kinetic and energetic mechanisms of sugar transport in storage parenchyma-like cells in the absence of heterogeneity introduced by tissues. The following results were obtained: (a) The sugar uptake system was specific for hexoses; as previously proposed, sucrose was hydrolyzed by an extracellular invertase before the sugar moieties were taken up; no evidence for multiple sugar uptake systems was obtained. - (b) Uptake of the glucose-analog 3-O-methylglucose (3-OMG) reached a plateau value with an intracellular concentration higher than in the medium (approximately 15-fold). - (c) There was a balance of influx and efflux during steady state; the rate of exchange influx was lower than the rate of net influx; the Km value was higher (70 μM) than for net influx (24 μM); the exchange efflux is proposed to be mediated by the same transport system with a Km value of approximately 2.6 mM for internal 3-OMG; the rate of net efflux of hexoses was less than a third of the rate of exchange efflux. - (d) The uptake of hexoses proceeded as proton-symport with a stoichiometry of 0.87 H(+) per sugar; during the onset of hexose transport there was a K(+) exit of 0.94 K(+) per sugar for charge compensation. (It was assumed that the "real" stoichiometries are 1 H(+) and 1 K(+) per sugar.) The Km values for sugar transport and sugar-induced proton uptake were identical. Sucrose induced proton uptake only in the presence of cell wall invertase. - (e) There was no net proton uptake with 3-OMG by cells which were preloaded with glucose though there was significant sugar uptake. It is assumed, therefore, that the exit of hexose occurs together with protons. - (f) The protonmotive potential of sugarcane cells corresponded to about 120 mV: pH-gradient 1.1 units, membrane potential of-60 mV (these values increased if vacuolar pH and membrane potential were also considered). It was abolished by uncouplers, and the magnitude of the components depended on the external pH value. We present evidence for the operation of a proton-coupled sugar transport system in cell suspensions that were derived from, and have characteristics of, storage parenchyma. The quantitative rates of sugar transport suggest that the role of this transport system is not limiting for sugar storage.

Although D-galactose is normally toxic to sugarcane (Saccharm sp.) cells, a cell line that grows on 100 mM galactose has been propagated. Nonadapted cells in a medium containing galactose instead of sucrose accumulate UDP-galactose; these cells also have much lower UDP-galactose 4epimerase (EC plants (e.g. 4, 8, 26, 30). The exact cause of this toxicity is still in doubt. Although galactose is taken up by wheat roots (4), tomato roots (9), and sugarcane cells (16), and is respired by oat coleoptiles (26) and tomato roots (22), it or its metabolites (I 1, 30) prevent synthesis ofcell walls (26) and prevent cell expansion (14), possibly by feedback regulation (1). Galactose also promotes auxin-dependent ethylene evolution, and it has been suggested that ethylene might be the cause of its toxic symptoms (6).In cell cultures galactose appears to support limited growth in some instances, even when it is the only carbohydrate in the medium (17). However, growth rates have been measured only with Lolium (20) 2 Abbreviations: Gal-l-P: galactose 1-phosphate; UDP-Gal: UDP-galactose; G-6-P: glucose 6-phosphate; G-l-P: glucose 1-phosphate; F-6-P: This cell line has lost the ability to differentiate. The stock culture was maintained by transferring an inoculum at 14-day intervals to fresh M-3 medium (25) containing 50 mm sucrose and modified only in 2,4-dichlorophenoxyacetic acid concentration (9 gm instead of 27 ,M).Galactose adaptation of the cell line described above was accomplished over a period of approximately 5 months without mutagenic treatment. The sucrose cell line was transferred to a medium containing 100 mM galactose instead of sucrose. After 6 days the normally clear culture fluid turned opaque. Cells were kept on this medium for an additional week before they were transferred to a similar medium containing 1% agar. At time of transfer to the agar medium, respiration rates were determined (model 53 oxygen monitor, Yellow Springs Instrument Co., Yellow Springs, Ohio). An undetermined proportion of cells in the culture was viable. However, these cells remained quiescent for several weeks. Upon transfer to a similar agar medium with fresh galactose, a few small colonies gradually formed among the cell clumps. These colonies were removed and repeatedly subcultured on fresh galactose medium containing agar. When sufficient callus mass developed, pieces of calli were transferred back to a galactose liquid medium to establish the galactose-adapted suspension culture.Following several transfers to fresh liquid medium, the galactose-adapted cells subsequently were subcultured at regular 14-day intervals. Although cells continued to grow well on 100 mm galactose, 50 mm galactose was substituted for the higher concentration used during the adaptation period. Except as otherwise noted, experiments reported here were carried out in the presence of either 25 mM sucrose or 50 mm galactose.We will refer to cell types throughout the text as follows: stock sucrose cultures (S); stock galactose-propagated cultures...

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Abstract. Growth characteristics and nutrient changes in medium and cells of batchgrown sugarcane cultures were investigated over a period of 14 days. Amino acids, PO~-and K ÷ were substantially removed from the medium during the first seven days of culture; a strong preference for uptake of organic nitrogen over inorganic nitrogen was observed. Sodium uptake increased during the time when K + was becoming deficient in the medium. The main anions taken up were SOl-and PO]-. Strong acidification and a virtually total extraceilular hydrolysis of sucrose in the medium during the first seven days of culture were also observed.Tapering off of the rapid growth phase was accompanied by an increase of intracellular sucrose and a decrease of intracellular protein. As cells went from rapid growth into stationary phase, cytoplasmic space of the cells decreased slightly in favor of vacuolar space. Overall cell volume stayed constant throughout the growth cycle, except during a short period before onset of rapid growth.Transport of the glucose analog 3-O-methyl glucose remained constant in terms of K m value but the Vma x was slightly higher in rapidly growing ceils.

Two spatialy separated forms of ornithine transcarbamoylase (EC 2.1.3.3) of different molecular weights coexist in sugarcane (Saccharum sp.). The smaller form of the enzyme (mol wt 79,000) appears to be cytoplasmic, while a laer form (mol wt 224,000) sedimented with mitochondria. The Km of the cytoplasmic enzyme for ornithine was 3.11 mM, while the enzyme in the mitochondrial fraction had a Km of 0.50 mm for this substrate; both enzymes had similar affinity for carbamoyl phosphate (0.12 mM). Characteristics of the smailer ornithine transcarbamoylase are in keeping with a predominantly catabolic function, those of the enzyme which sediments with mitochondria, with an anabolic function. Only the mitochondrial enzyme was regulated in vivo by exogenous arginine.Ornithine transcarbamoylase (EC 2.1.3.3) (OTC)2 is of physiological importance for the carbamoylation of ornithine, as well as for the decarbamoylation of citrulline. The first reaction is significant because of its role in the arginine biosynthetic pathway, while for some organisms the reverse reaction has a function in the arginine dihydrolase pathway. In mammals (4) and fungi (24), OTC is located exclusively in the mitochondria, while in plants, OTCs of cytoplasmic (6) Mitochondria in the pellet were washed with extracting buffer and resuspended in 0.01 M tris (pH 8) for 1 hr. During this period the mitochondria were permitted to break, and debris was subsequently removed by centrifugation (15,000g for 5 min). Protein in the supernatant was precipitated, resolubilized, and dialyzed as described for the soluble fraction. This preparation was designated as OTCM.Determination of OTC. In the direction of citrulline synthesis, OTC was assayed essentially by the method of Ong and Jackson (12), using 1 ml of 0.1 M tris (pH 8), 5 mm ornithine, and freshly prepared 5 mm carbamoyl-P. Enzyme preparations were added to produce between 30 and 100 nmol of citrulline during a 10-min incubation period at 37 C. Citrulline was determined according to Prescott and Jones, method II (13).OTC in the direction of ornithine formation was assayed by the radioisotope procedure of Reichard (14) which measures 14CO2 released from 14C-ureido-labeled citrulline (New England Nuclear) in the presence of arsenate and enzyme. Unlabeled citrulline was added as shown under "Results," and 0.5 ml of extract was used with 1 ml of the reaction mixture, containing 0.1 M citrate buffer (pH 7) and 50 mm arsenate. Incubations in side armed flasks were for 1 hr at 30 C, and the reaction was terminated by addition of 0.5 ml of 6 N HCl and 15% trichloroacetic acid from the side arm. Radioactivity was measured in a liquid scintillation counter (model LS-150, Beckman Instruments Inc., Palo Alto, Calif.) in scintillation fluid containing PPO, POPOP, and toluene. TLC of the assay mixture after incubation showed unreacted citrulline as the only radioactive substance.Determination of Succinic Dehydrogenase (SDH). SDH was assayed according to the method described by Veeger et al. (22