Growth Changes in Disks Cut from Young Leaves of Phaseolus

Disks cut from leaves of different ages were cultured on mediawith and without added carbohydrate. Relative growth-rates forchanges in area of disks were found to be similar to those forintact leaves, and to decline as the age of the parent materialincreased. Cellular changes occurring concurrently with thisdecline were followed. Protein per cell was found to increasein disks cultured in the light on sucrose but not in the absenceof supplied carbohydrate. This requirement for carbohydratecould not be satisfied by materials produced endogenously inthe light, even though under these conditons starch was formedand there were large increases in cell-wall material. Changesin disks and in intact leaves of the same age were broadly similar.The large effects of light and carbohydrate supply are discussedin relation to disk expansion and leaf growth in general.     

Some Effects of Temperature and Irradiance on Growth of the First Four Leaves of Wheat, Triticum aestivum

Plants of Heron wheat were grown at 20 and 15 °C and inquantum flux densities of 400 and 200 µmol m^–2 s^–1.At completion of expansion of the first or second leaf, plantswere transferred between temperatures and quantum flux densities.Final size and cell number were measured for each of the firstfour main-stem leaves. Leaf area was affected only slightlyby treatment and effects on leaf length and width were alsosmall. It was concluded that leaf extension rate, which waslower at the lower temperature and in the lower light regime,is inversely related to the duration of leaf expansion. Leafdry wt was higher for plants grown in high light and for plantsgrown at 15 °C; transfer treatments led to readjustmentswhereby dry wts of leaves expanded after transfer resembledthose of leaves on plants kept throughout in the post-transferconditions. Leaf cell number was not affected by treatment but mean drywt per cell was significantly greater in high light, and forthe first two leaves, at 15 °C. There was a major and highlysignificant effect of treatment on the ratio of dry: fresh wtper cell, this being larger for leaves in high light. Transfertreatments between light regimes led to rapid changes in expandingleaves as was found for leaf dry wt. It was concluded that theexpanding grass leaf is much less dependent on older leavesto provide the necessary materials for cell division and expansionthan is the dicotyledon leaf. It is suggested that the increasein cell dry wt in high light is associated with an increasein cell wall material which is under photomorphogenic control. Triticum aestivum, wheat, leaf growth, cell division, cell expansion, cell size     

Photosynthetic Determinants of Growth in Maize Plants: Effects of Nitrogen Nutrition on Growth, Carbon Fixation and Photochemical Features

Maize(Zea mays L.) plants were grown in a greenhouse with differentlevels of nitrate-N (2 to 20 millimolar). Nitrogen nutritionhad dramatic effects on plant growth and photosynthetic characteristicsof mature leaves. Increasing nitrogen resulted in greater biomassproduction, shoot/root ratios, and rates of leaf expansion duringthe day. The elongating zone of high-N plants had higher activities(per gram fresh weight) of sucrose synthase and neutral invertasethan low-N plants, suggesting that increased leaf growth wasrelated to a greater biochemical capacity for sucrose metabolism. Mature leaves of high-N plants had higher rates of photosynthesisand assimilate export (sucrose formation), and partitioned morecarbon into sucrose relative to starch. Increased photosyntheticrates (leaf area basis) were associated with higher levels ofribulose-l,5-bisphosphate carboxylase, phosphoenolpyruvate carboxylaseand pyruvate, phosphate dikinase (determined immunochemically).In addition, N-nutrition affected the functional organizationof chlorophyll in the leaves. Large increases in the numberof PS I reaction centers were observed which fully accountedfor increases in leaf chlorophyll content with increasing nitratesupply. Collectively, the results suggest that increased growth of maizeplants at high light and optimal nitrogen nutrition is relatedto greater capacity for photosynthesis and translocation inmature leaves, and possibly increased capacity for sucrose metabolismin expanding leaves. (Received May 22, 1989; Accepted August 28, 1989)     

Nucleic acid and protein synthesis in discs cut from mature leaves of Nicotiana tabacum L. and cultured on nutrient agar with and without kinetin

The effect of kinetin on aspects of the metabolism of discs cut from mature leaves of Nicotiana tabacum and cultured in the light on agar containing mineral salts and sucrose was studied. In the first few days of culture there was a rapid decline in chlorophyll content. Discs treated with kinetin in the light began to resynthesise chlorophyll after 3–4 days and this was correlated with chloroplast replication. Kinetin promoted chloroplast replication but was not always essential. An increase in fresh weight also occurred, due mainly to cell expansion. Nitrate reductase activity increased rapidly during the first few hours after placing discs on the culture medium but kinetin had no effect on this reponse. Subsequently there were dramatic increases in RNA and protein content which were largely independent of kinetin. Gel electrophoresis showed that cytoplasmic and chloroplast ribosomal RNA and a large amount of soluble RNA were synthesised during culture of the discs. These results are discussed in relation to the role of kinetin in delaying leaf sensescence.     

Leaf Growth in Phaseolus vulgaris: I. Growth of the first pair of leaves under constant conditions

The growth of the first pair of leaves of Phaseolus vulgaris(French bean) has been studied during germination and followingemergence of the seedling. The leaves are well developed inthe embryo and, at 22.5° C, show an exponential increasein fresh weight, dry weight, and leaf area up until about eightdays from planting. Cell division commences about two days afterplanting and is exponential for a short period. Considerablechanges in cell volume occur during the period over which celldivision occurs. Cell division ceases soon after emergence andunfolding, when the leaf has reached only 17 per cent of itsfinal area. Cessation of cell division is followed by a phaseof growth which is due entirely to cell expansion. The significanceof these findings is discussed in relation to recent work onother genera.     

Separation of cell enlargement and division in bean leaves

A method is presented for inducing cell enlargement in intact leaves and leaf strips of Phaseolus vulgaris L. without the complication of cell division. Primary bean leaves complete cell division and stop growing after 10 d in dim red light. Transfer to white light induces expansion (50% in 24 h) which is entirely the consequence of cell enlargement. Leaf strips from red-light-grown seedlings placed in white light and provided external solutes (10 mM KCl+10 mM sucrose) expand at the same rate as intact leaves in the light. This system makes possible future investigation of the mechanism of leaf cell enlargement.     

Multiple steps of leaf thickening during sun‐leaf formation in Arabidopsis

Plant morphological and physiological traits exhibit plasticity in response to light intensity. Leaf thickness is enhanced under high light (HL) conditions compared with low light (LL) conditions through increases in both cell number and size in the dorsoventral direction; however, the regulation of such phenotypic plasticity in leaf thickness (namely, sun‐ or shade‐leaf formation) during the developmental process remains largely unclear. By modifying observation techniques for tiny leaf primordia in Arabidopsis thaliana, we analysed sun‐ and shade‐leaf development in a time‐course manner and found that the process of leaf thickening can be divided into early and late phases. In the early phase, anisotropic cell elongation and periclinal cell division on the adaxial side of mesophyll tissue occurred under the HL conditions used, which resulted in the dorsoventral growth of sun leaves. Anisotropic cell elongation in the palisade tissue is triggered by blue‐light irradiation. We discovered that anisotropic cell elongation processes before or after periclinal cell division were differentially regulated independent of or dependent upon signalling through blue‐light receptors. In contrast, during the late phase, isotropic cell expansion associated with the endocycle, which determined the final leaf thickness, occurred irrespective of the light conditions. Sucrose production was high under HL conditions, and we found that sucrose promoted isotropic cell expansion and the endocycle even under LL conditions. Our analyses based on this method of time‐course observation addressed the developmental framework of sun‐ and shade‐leaf formation.     

The Mechanism of Abscisic Acid-induced Proline Accumulation in Barley Leaves

When leaf blades of fully expanded second leaves of barley (cv. Prior) were excised and incubated with the cut end in a 20 milligram per liter solution of abscisic acid, they accumulated proline at the rate of about 1 micromole per hour per gram fresh weight after a 3- to 4-hour lag. This accumulation occurred reproducibly only if leaves were pretreated by placing the cut end in a solution consisting of 50 millimolar sucrose and 1 millimolar glutamate. Treated leaves were taken from plants which had been in the light for 24 hours.     

Opportunities for regulation of sugar beet storage root growth

The percentage of sucrose in sugar beet storage root fresh and dry matter is closely related to root structure. It has been suggested that the sucrose content might be increased by using plant growth regulators to modify storage root structure through control of cambial development, cell division and cell expansion. During storage root development correlations were found between the changing phytohormone profiles and the formation of secondary cambia and their subsequent cell division and expansion. Sugar beet root derived cell suspension cultures were used for detailed studies of the roles of endogenous phytohormones. The gibberellin synthesis inhibitor paclobutrazol was tested in cell cultures and whole plants. The observations provide a basis for development of plant growth regulator regimes to optimise sucrose yield from sugar beet.     

Studies on the Growth in Culture of Plant Cells: I. GROWTH PATTERNS IN BATCH PROPAGATED SUSPENSION CULTURES

Techniques are described for following increases in total cellnumber, fresh weight and dry weight, and changes in mean cellsize, and in the relative number of free cells to cell aggregatesduring the growth of batch-propagated suspension cultures oftissues derived from several species of angiosperms. When totalcell number is plotted against time it is seen that there canbe distinguished in sequence a lag phase, phases of acceleration,maximum rate, and negative acceleration of cell division and,finaly, a stationary phase. Studies with Parthenocissus tricuspidatacrown-gall tissue, growing in a synthetic liquid medium, haveshown that the total cell production per culture in the firstinstance is limited by nitrate supply rather than by the supplyof other inorganic ions, sucrose supply aeration, or the releaseof endogenous inhibibors. Studies, particularly with Acer pseudoplatanustissue, have shown that during the period of high cell-divisionrate, mean cell size reached its minimum value and average numberof cells per cell aggregate its maximum value. Cell separationdoes not occur to a significant extent until cell-division activityhas almost ceased and it is dependent upon cell expansion. Thebalance between cell division and cell expansion determinesthe ‘cellular unit’ composition of the cultures.Refinement of the control of growth patterns in plant suspensioncultures calls for further study of the ‘conditioning’of media, of factors which limit the duration of the periodof high mitotic activity, and of the conditions necessary forfull and rapid cell expansion.     

The Dynamics of Leaf Growth and Photosynthetic Capacity in Capsicum frutescens L.

In Capsicum frutescens L. cv. California Wonder the specificleaf weight (dry weight per unit laminar area) at leaf unfoldingis three times higher in the eighth leaf than in the first leafproduced. Intermediate leaves exhibit a trend between the twoThe change in specific leaf weight during laminar expansionis greatest in leaf 1 and least (sometimes zero) in leaf 8.Large changes in specific leaf weight during laminar expansionare associated with a large degree of palisade cell expansion,while leaves showing smaller rates of change have less palisadecell expansion but cell division is more evident. At leaf unfoldingthe fraction I protein content per unit laminar area is higherin upper than in lower leaves. Ribulose diphosphate carboxylaseactivity per unit laminar area and ¹⁴CO₂ fixation per unit laminararea have a similar pattern of development in all leaves andshow no correlation with the changes in specific leaf weight.The peak of activity in all leaves occurs when the laminar areais 10 cm². These results are compared with previous data onlaminar expansion and are seen as in accord with current ideason leaf growth.     

The different growth responses of the Arabidopsis thaliana leaf blade and the petiole during shade avoidance are regulated by photoreceptors and sugar

During the shade-avoidance response, leaf blade expansion is inhibited and petiole elongation is enhanced. In this study, we examined the roles of photoreceptors and sugar on the differential growth of the leaf blade and petiole in shade conditions. Under the conditions examined, cell expansion, not cell division, played a major role in the differential leaf growth. The enhanced cell expansion in the leaf blade is associated with an increase in the ploidy level, whereas cell elongation was stimulated in the petiole in dark conditions without an increase in the ploidy level. Analysis of phytochrome, cryptochrome and phototropin mutants revealed that phytochromes and cryptochromes specifically regulate the contrasting growth patterns of the leaf blade and petiole in shade. Examination of the effects of photo-assimilated sucrose on the growth of the leaf blade and petiole revealed growth-promotional effects of sucrose that are highly dependent on the light conditions. The leaf blades of abscisic acid-deficient and sugar-insensitive mutants did not expand in blue light, but expanded normally in red light. These results suggest that both the regulation of light signals and the modulation of responses to sugar are important in the control of the differential photomorphogenesis of the leaf blade and petiole.     

The Effects of Kinetin, Gibberellic Acid, and Light on Expansion and Cell Division in Leaf Disks of Dwarf Bean (Phaseolus vulgaris)

The effects of kinetin (Kn), gibberellic acid (G), and light(L) on cell expansion and division in disks from 6-day-old etiolatedprimary leaves of dwarf bean are described. Cell number wasdetermined by direct counting after disks were digested in apectinase/EDTA mixture to separate the cells. Kn increased leafexpansion wholly by increasing cell size. G also increased cellsize; it increased cell division in the dark but not in thelight. Light also increased cell size and cell division; iteliminated the effect of G on cell division but enhanced theeffect of G on cell expansion.     

Co-Ordination of Cell Division and Tissue Expansion in Sunflower, Tobacco, and Pea Leaves: Dependence or Independence of Both Processes?

Abstract Temporal analyses of cell division and tissue expansion in pea, tobacco, and sunflower leaves reveal that both processes follow similar patterns during leaf development. Relative cell division and relative tissue expansion rates are maximal and constant during early leaf development, but they decline later. In contrast, relative cell expansion rate follows a bell-shaped curve during leaf growth. Cell division and tissue expansion have common responses to temperature, intercepted radiation, and water deficit. As a consequence, final leaf area and cell number remain highly correlated throughout a large range of environmental conditions for these different plant species, indicating that cell division and tissue expansion are co-ordinated during leaf development. This co-ordination between processes has long been explained by dependence between both processes. Most studies on dicotyledonous leaf development indicate that leaf expansion rate depends on the number of cells in the leaf. We tested this hypothesis with a large range of environmental conditions and different plant species. Accordingly, we found a strong correlation between both absolute leaf expansion rate and leaf cell number. However, we showed that this relationship is not necessarily causal because it can be simulated by the hypothesis of independence between cell division and tissue expansion according to Green's theory of growth (1976). Received 23 February 2000; accepted 3 March 2000     

Effects of azadirachtin after systemic uptake intoBrassica oleracea on larvae ofPieris brassicae

Petioles of intact cabbage leaves were placed in azadirachtin solutions for 24 h. Azadirachtin or active metabolites of this compound appeared to be translocated systemically as performance of first instar larvae ofPieris brassicae L. placed on disks from those leaves was affected. The position within a leaf from which disks were excised did not affect the extent of these effects, indicating a homogeneous distribution of active compounds in the laminar tissue. At doses ranging from 1–10 ppm of azadirachtin in the solution taken up by the leaves, gain of larval fresh weight and leaf area consumed were reduced. These effects were dose dependent up to 60–90 ppm. At 10 ppm and higher doses no larva reached the second instar within 72 h. Mortality during the 72 h period was highest at 10 ppm, due to the coincidence of lethal action with ecdysis in combination with the effect on developmental rate. Azadirachtin solutions applied directly to the surface of leaf disks in order to produce known doses on a leaf fresh weight basis gave results similar to the systemic treatment for weight gain, leaf area consumed and developmental rate, while mortality was higher in the latter treatment.     

Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves

The quantity and quality of light required for light-stimulated cell expansion in leaves of Phaseolus vulgaris L. have been determined. Seedlings were grown in dim red light (RL; 4 micromoles photons m-2 s-1) until cell division in the primary leaves was completed, then excised discs were incubated in 10 mM sucrose plus 10 mM KCl in a variety of light treatments. The growth response of discs exposed to continuous white light (WL) for 16 h was saturated at 100 micromoles m-2 s-1, and did not show reciprocity. Extensive, but not continuous, illumination was needed for maximal growth. The wavelength dependence of disc expansion was determined from fluence-response curves obtained from 380 to 730 nm provided by the Okazaki Large Spectrograph. Blue (BL; 460 nm) and red light (RL; 660 nm) were most effective in promoting leaf cell growth, both in photosynthetically active and inhibited leaf discs. Far-red light (FR; 730 nm) reduced the effectiveness of RL, but not BL, indicating that phytochrome and a separate blue-light receptor mediate expansion of leaf cells.     

Induction of Phenylalanine Ammonia-lyase in Strawberry Leaf Disks: Action Spectra and Effects of Wounding, Sucrose, and Light

The increase in phenylalanine ammonia-lyase (PAL) activity in strawberry (Fragaria vesca var. WSU-1232) leaf disks required wounding, sucrose, and light and was cycloheximide-sensitive. In injured leaves and in leaf disks, the highest PAL activity was detected nearest the wounded tissues. Without wounding, no increase in activity was observed when leaves were cultured in sucrose and light.     

Effect of cutting on solute uptake by plasma membrane vesicles from sugar beet (Beta vulgaris L.) leaves.

The uptake of sucrose, 3-O-methylglucose (3-O-MeG), and valine were studied in discs and in purified plasma membrane vesicles (PMV) prepared from sugar beet (Beta vulgaris L.) exporting leaves. The uptake capacities of freshly excised leaf discs were compared with the uptake in discs that had been floated for 12 h on a simple medium (aging) and with discs excised from leaves that had been cut from the plant 12 h before the experiments (cutting). After cutting, sucrose uptake amounted to twice the uptake measured in fresh discs, whereas the uptake of 3-O-MeG and valine remained unaffected. In aged leaf discs, there was a general stimulation of uptake, which represented 400, 300, and 400% of the uptake measured in fresh discs for sucrose, 3-O-MeG, and valine, respectively. Sucrose uptake in fresh discs was sensitive to N-ethylmaleimide (NEM), to p-chloromercuribenzenesulfonic acid (PCMBS), and to mersalyl acid (MA). Although cutting induced the appearance of a sucrose uptake system that is poorly sensitive to NEM but sensitive to PCMBS and MA, aging induced the development of an uptake system that is sensitive to NEM but poorly sensitive to PCMBS and MA. Autoradiographs of discs fed with [14C]sucrose show that cutting resulted in an increase of vein labeling with little effect in the mesophyll, whereas aging induced an increase of labeling located mainly in the mesophyll. The data show that cutting is sufficient to induce dramatic and selective changes in the uptake properties of leaf tissues and that the effects of cutting and aging on the uptake of organic solutes are clearly different. Parallel experiments were run with purified PMV prepared from fresh and cut leaves. The uptake of sugars and amino acids was studied after imposition of an artificial proton motive force (pmf). Comparison of the uptake properties of PMV and of leaf tissues indicate that the recovery of the sucrose uptake system in PMV is better than the recovery of the hexose and of the valine uptake systems. As observed with the leaf discs, cutting induced a 2-fold increase of the initial rate of sucrose uptake in PMV but did not affect the uptake of valine and 3-O-MeG. Cutting induced an increase of both Vmax and Km of the sucrose transport system in PMV. Measurements of the pmf imposed on the vesicles indicated that the increase of sucrose uptake induced by cutting was not due to a better integrity of the vesicles. Hexoses did not compete with sucrose for uptake in PMV from fresh and cut leaves, and maltose was a stronger inhibitor of sucrose uptake in PMV from cut leaves than in PMV from fresh leaves. The sensitivity of sucrose uptake to NEM, PCMBS, and MA in PMV from fresh and cut leaves paralleled that described above for the corresponding leaf discs. These data show that (a) the changes induced by cutting on sucrose uptake by leaf discs are due to membrane phenomena and not to the metabolism of sucrose; (b) the study of sucrose uptake with PMB gives a good account of the physiological situation; and (c) the specific effects induced by cutting on the sucrose uptake system are not lost during the preparation of the PMV.     

Invertase and sucrose synthase activity, carbohydrate status and endogenous IAA levels during Citrus leaf development

Levels of activity of the sucrose catabolizing enzymes, acid invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13), were measured during development of new leaves of Citrus sinensis (L.) Osbeck cv. Shamouti. Soluble acid invertase showed a peak activity of 32 nkat (g fresh weight)⁻¹ at ca 60% of full leaf expansion and rapidly declined toward and after full expansion. There was no concomitant increase in an insoluble form of the enzyme. Sucrose synthase activity, measured in the synthesis direction, declined from 33% of full leaf expansion [10 nkat (g fresh weight)⁻¹] 10, and following, full expansion. Highest sucrose synthase activity, measured in the cleavage direction, was 6 nkat (g fresh weight)⁻¹ and showed little change during development. Acid invertase has a K_m of 5 m M for sucrose, while sucrose synthase had a K_m of 118 m M for sucrose. Changes in acid invertase activity correlated with changes in the reducing sugar:sucrose ratio. These results suggest that soluble acid invertase activity is the primary enzyme responsible for sucrose catabolism in the expanding Citrus leaf. Changes in leaf expansion rate and invertase activity did not correlate positively with changes in endogenous free IAA level, as determined by enzyme linked immunoassay.     

The Roles of Cell Division and Cell Expansion in the Growth of Alfalfa Leaf Mesophyll

Leaf mesophyll of Medicago sativa (L.) was investigated to determinethe roles of cell division and cell expansion in tissue growth.Samples of leaf tissue were macerated, stained, and squashed.The slides were studied under a phase microscope to determinethe percentage of recently divided cells and the average celldiameter for leaflets of varying lengths. Cell division wasgreatest in young leaflets and virtually ceased as a leaf lengthof 12 mm was attained. For leaflets less than 12 mm in length,the rate of increase in cell size appeared to be inversely associatedto the degree of cell division. For alfalfa leaflets greaterthan 12 mm in length, the mean cell size increased in proportionto leaf length since cell division had virtually ceased.