Effects of calcium and kinetin on growth and cell wall composition of pea epicotyls

Kinetin and CaCl₂, in the presence of indoleacetic acid, promoted lateral expansion of epicotyls of decapitated and derooted Alaska pea seedlings (Pisum sativum L.) and inhibited their elongation. This growth response was correlated with the development of cell walls unusually rich in pectic uronic acids. Epicotyls in calcium-auxin solutions continued to enlarge and to add new wall material long after tissues in auxin only had stopped. Longitudinal enlargement, associated with the development of walls poor in pectic uronic acids, was favored by KCl, MgCl₂, and ethylenediaminetetraacetate. The last of these agents promoted the loss of ⁴⁵Ca from the epicotyls. Seedings grown in vermiculite moistened with CaCl₂, KCl, or MgCl₂ solutions did not differ in appearance or in the composition of their walls. They responded similarly to experimental treatment except that the decapitated epicotyls of the MgCl₂-grown plants suffered an absolute loss of pectic uronate when incubated in that salt.     

Effects of gibberellic Acid, calcium, kinetic, and ethylene on growth and cell wall composition of pea epicotyls

The influence of gibberellic acid (GA), calcium, kinetin, and ethylene on growth and cell-wall composition of decapitated pea epicotyls (Pisum sativum L. var. Alaska) was investigated. Calcium, kinetin, and ethylene each caused an inhibition of GA-induced elongation of pea stems. Gibberellic acid did not reverse the induction of swelling by Ca²⁺, kinetin, or ethylene. Both Ca²⁺ and ethylene significantly inhibited the stimulatory effects of GA on the formation of residual wall material. Although GA promoted the development of walls relatively low in pectic substances and pectic uronic acid, Ca²⁺, kinetin, and ethylene favored the formation of walls rich in these constituents. Calcium, kinetin, and GA, alone or in combination, had no effect on the production of ethylene by pea epicotyls.     

Effect of red radiation, kinetin and linuron on growth and ethylene production in Chlorella

Both short-term and continuous red radiation stimulated while far red radiation inhibited growth and ethylene production in Chlorella pyrenoidosa. Kinetin and linuron also affected culture density and ethylene production, depending on their concentration. Phytochrome might participated in the regulation of growth and ethylene production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interaction of kinetin and calcium in relation to their effect on stimulation of ethylene production

Application of kinetin and Ca²⁺ caused a striking synergistic increase in ethylene production by mung bean (Phaseolus aureus Roxb) hypocotyl segments. The effect of kinetin on Ca²⁺ uptake and of Ca²⁺ on the uptake and metabolism of kinetin in relation to their effect on ethylene production was studied. Tracer experiments showed that kinetin greatly increased the uptake of ⁴⁵Ca²⁺ after 6 hours of incubation. Reciprocally, Ca²⁺ stimulated the uptake of kinetin-8-¹⁴C and remarkably enhanced the metabolism of kinetin-8-¹⁴C into several polar metabolites. Consequently, the quantity of free kinetin-8-¹⁴C remaining in Ca²⁺-treated segments was much less than in control segments. A possible mechanism accounting for the synergism between kinetin and calcium on ethylene production is discussed.     

Hydroxyproline-rich cell wall protein (extensin): Biosynthesis and accumulation in growing pea epicotyls

Plant cell walls contain a glycoprotein component rich in the otherwise rare amino acid hydroxyproline. We examined the synthesis and accumulation of wall hydroxyproline during different states of elongation growth in pea epicotyls. Light-grown peas contained more wall hydroxyproline than their taller, dark-grown counterparts. When elongation was studied by marking growing stems in situ, there was a marked accumulation of wall hydroxyproline coincident with the cessation of elongation. Dividing and elongating regions of the epicotyl showed less wall hydroxyproline than did regions where elongation was no longer occurring.Hydroxyproline biosynthesis was examined by incubation of excised sections of tissues in various growth states in ¹⁴C-proline. The extent of conversion of these residues to ¹⁴C-hydroxyproline served as a measure of the rate of hydroxyproline synthesis. This rate was highest in tissues which had ceased elongation. The low rate of hydroxyproline synthesis in dividing and elongating cells was probably not due to the inability to hydroxylate peptidyl proline or to secrete proteins.These data show a positive correlation between the synthesis and accumulation of cell wall hydroxyproline and the cessation of cell elongation in pea epicotyls.     

Effect of osmotic stress on the growth of epicotyls of Cicer arietinum in relation to changes in cell wall composition

The inhibition of growth by polyethlene glycol (PEG)-induced osmotic stress led to modifications in the changes taking place in cell wall composition during normal growth of epicotyls of Cicer arietinum L. cv. Castellana. Epicotyls growing under normal conditions showed a decrease in the amount of pectic fractions and an increase in the hemicellulosic fractions and α-cellulose that led to an increase in the rigidity and a loss in growth capacity. Among the hemicellulosic fractions, the KI-2 fraction (insoluble fraction of 10% KOH-extracted hemicelluloses) seemed to be the only one related to the elongation process and subsequent rigidity. During normal growth a decrease was observed in the total amount of galactose, mainly from the pectic fractions. The inhibition of elongation led to an increase in the amount of the cell walls, due to inhibition of cellular elongation. PEG prevented the increase in the hemicelluloses and the α-cellulose, indicating that these changes were related to elongation. Thus, during the inhibition of elongation there is probably an inhibition of new synthesis that prevents cell wall rigidity and maintains cell wall growth capacity. Changes in the pectic fractions during growth were not affected by the inhibition of elongation, showing that these fractions are related to cell wall loosening rather than to elongation. Study of the cell wall composition confirms the idea that the autolytic process is regulated by changes in the cell wall structure during epicotyl growth     

The Casparian strip in pea epicotyls: Effects of light on its development

The Casparian strip, which is specific to roots, was studied in the epicotyls of dark-grown seedlings of pea (Pisum sativum L.) where it was found to have the same morphology and properties as the strip in roots. In dark-grown seedlings, the distance between the upper-most position of the Casparian strip and the bending point of the hook (about 37 mm) did not change during growth of the seedlings. In the uppermost 0.5-mm region of the region in which the Casparian strip could be detected by fluorescence microscopy, the plasma membrane was not firmly attached to the cell wall. The development of the Casparian strip continued for about 42 h after dark-grown seedlings were transferred to the light, indicating that (i) the cells that have been determined to form the Casparian strip in darkness form the strip in the light, and that (ii) it takes about 42 h for the cells to complete formation of the strip. Cells in the hook of dark-grown seedlings did not form a Casparian strip when such seedlings were transferred to the light. The Casparian strip was formed in rapidly elongating internodes of light-grown seedlings when the seedlings were transferred to darkness. Light did not control the formation of the Casparian strip in roots.Abbreviation PBS phosphate-buffered saline     

Effects of brefeldin A on the development of the Casparian strip in pea epicotyls

Summary The Casparian strip, a structure that is present in roots, is also present in epicotyls of dark-grown pea seedlings. In a dark-grown epicotyl, the cells in each stage of the development of the Casparian strip have been suggested to be lined up basipetally in the region 3 to 37 mm below the bending point of the hook, in order of the developmental stage. Brefeldin A (BFA), a specific inhibitor of secretory transport, was administrated at 200 M. to dark-grown pea epicotyls for 2 h via a thread passed through the epicotyl 40 mm below the bending point. The basipetal sequence of development of the modification of the cell wall at the Casparian strip, as judged by fluorescence microscopy, stopped 5 h after the start of 2 h treatment with BFA and resumed after 30 h. This basipetal sequence of development did not stop in control seedlings. Electron micrographs of endodermal cells in epicotyls treated with BFA showed striking morphological changes in the Golgi stacks and the ER. Histological examination made 20 h after the start of the experiment revealed that the basipetal sequence of development of the cell wall modification stopped at a point which was present at 25.2 ± 1.6 mm (mean with SD, n=5) from the bending point of the hook at the start while the basipetal sequence of development of the tight adhesion of the plasma membrane to the cell wall at the Casparian strip stopped 0.9 ± 0.5 mm (mean with SD, n=5) below this point. These results indicate the involvement of secretory transport not only in the introduction of the modification of the cell wall but also in the completion of the tight adhesion of the plasma membrane.Abbreviations BFA brefeldin A - PBS phosphate-buffered saline - ER endoplasmic reticulum     

Amyloplast development in etiolated and ethylene-treated pea epicotyls

The amyloplasts found in the apical hook cells of etiolated pea (Pisum sativum L.) epicotyls were randomly distributed. Sedimentation of endodermal amyloplasts in the direction of gravity became apparent in the transition from the hook to the top of the main axis of the epicotyl. Cortical amyloplasts in this region were not, however, sedimented. These patterns of sedimentation could not be related to changes in amyloplast size, and it is proposed that cytoplasmic properties determine amyloplast behaviour.The differentiation of plastids in the hook differed between the amyloplast-containing endodermal cells and the cortical cells, in which amoeboid plastids predominated over amyloplasts. Amyloplasts disappeared from the cortical cells in the main axis of the epicotyl, but in the endodermal cells sedimented amyloplasts were found throughout the upper epicotyl.Etiolated epicotyls induced to grow horizontally by treatment with ethylene had a normal content of amyloplasts, sedimented in the direction of gravity.     

Stimulation of ethylene production in the mung bean hypocotyls by cupric ion, calcium ion, and kinetin

The synergistic stimulation of ethylene production by kinetin and Ca²⁺ in hypocotyl segments of mung bean (Phaseolus aureus Roxb.) seedling was further studied. The requirement for Ca²⁺ in this system was specific. Except for Sr²⁺, which mimicked the effect of Ca²⁺, none of the following divalent cations, including Ba²⁺, Mg⁶⁺, Cu²⁺, Hg²⁺, Co²⁺, Ni²⁺, Sn²⁺, and Zn²⁺, showed synergism with kinetin on ethylene production. Fe²⁺, however, showed a slight synergism with kinetin. Some of them (Hg²⁺, Co²⁺, and Ni²⁺) had a strong inhibitory effect, while others (Zn²⁺, Mg²⁺, Sn²⁺, and Ba²⁺) had a slight or no inhibitory effect on ethylene production in the absence or presence of kinetin.     

Effect of red light on geotropism in pea epicotyls

Dose response curves were determined for phytochrome phototransformation and for a phytochrome-controlled decrease in geotropic curvature in epicotyls of dark-grown Pisum sativum L. cv. Alaska. Ten times as much light was required to produce a spectrophotometrically detectable transformation of phytochrome as was required to produce a significant change in the geotropic response. The red light energy required for a 50% phytochrome transformation caused a 90% change in the physiological response.     

Autolysis of cell walls in pea epicotyls during growth. Enzymatic activities involved

Pisum sativum L. (cv. Lincoln) epicotyl cell walls show autohydrolysis and release into the incubation medium up to 120 μg of sugar per mg of cell wall dry weight in 30 h. Cell walls from younger epicotyls with high growth capacity showed higher auto-lytic capacity than older epicotyls. This suggests that both processes, growth and au-tolysis, are related. The proteins responsible for autolysis were extracted from the wall fraction with high saline solution (3 M LiCl) and enzymatic activities associated with the proteins were studied. The highest activity corresponded to α-galactosidase; lower activities were found for β-galactosidase, a-arabinosidase and exoglucanase. Changes in enzymatic activities and changes in the proportion of sugars released in autolysis by cell walls during the growth of epicotyls support the notion that α-galac-tosidase is one of the enzymes involved in the process of autolysis, and that the liberation of arabinose and galactose in this process occurs as arabinogalactan.     

Synergistic effect of kinetin on IAA-induced ethylene production

Kinetin has a significant synergistic effect on IAA-inducedehtylene production in hypocotyl sections of eitolated mungbean(Phaseolus mungo L.) seedlings when it is administered immediatelyafter cutting. If the addition of kinetin was delayed, it becameless effective. Kinetin-pretreated sections followed by incubationeither in IAA or in IAA plus kinetin produced ehtylene essentiallyat an identical rate to the one treated with IAA plus kinetinat zero time. However, the buffer-preincubated sections followedby incubation in IAA or in IAA plus kinetin shows a much reducedrate of ehtylene production as compared with the one treatedwith IAA or with IAA plus kinetin at zero time, respectively.These results suggest that kinetin is required for the maximalstimulation of ethylene production only during the early incubationperiod while IAA is required continuously throughout the incubation;the removal of IAA from the IAA-treated tissues causes an immediatecessation of ethylene production. ¹Present address: Mann Laboratory, Department of Vegetable Crops,University of California, Davis, California 95616, U.S.A. (Received May 10, 1973; )     

Galactoglucomannan oligosaccharides inhibition of elongation growth is in pea epicotyls coupled with peroxidase activity

The effect of galactoglucomannan oligosaccharides — GGMOs, GGMOs-r (GGMOs with reduced reducing ends), and GGMOs-g (GGMOs with reduced number of d-galactose units) on peroxidase activity was determined in pea epicotyls. GGMOs didn’t significantly modify the activity of soluble peroxidases. However, cell wall-associated peroxidases activity increased after GGMOs and GGMOs-r treatment, while in the presence of GGMOs-g this activity was significantly lower. These results are inversely related to the GGMOs, GGMOs-r, and GGMOs-g effect on elongation growth induced by 2,4-D (2,4-dichlorophenoxyacetic acid) in pea epicotyls. It can be concluded that GGMOs evoked inhibition of the elongation growth induced by auxin is probably associated with cell wall modifications catalysed by peroxidase.     

Ethylene as a factor regulating the growth of pea epicotyls subjected to physical stress

Pea epicotyls (Pisum sativum, cv. Alaska) were enclosed in chambers in which their elongation was restricted by means of a foam neoprene stopper or by a medium of glass beads. These treatments increased evolution of ethylene and resulted in reduced length and increased diameter of both the internodes and the cells of the internodes. These responses increased with increasing degrees of restriction. A time-sequence study of the emergence of epicotyls through 90 mm of glass beads showed that an accelerated evolution of ethylene preceded a reduction in elongation. As the epicotyls elongated through the glass bead medium and less resistance was encountered, evolution of ethylene declined and rapid elongation was resumed. The morphological and anatomical effects of a 120-mm column of glass beads were duplicated by applied ethylene concentrations of 0.2 ppm or less. Evolution of CO₂ was inhibited slightly by the ethylene treatments. The data indicate that production of ethylene by pea epicotyls is increased by nonwounding physical stress, and that the ethylene acts as an endogenous growth regulator, decreasing elongation and increasing diameter in response to increasing increments of stress.     

On the role of calcium in indole-3-acetic acid movement and graviresponse in etiolated pea epicotyls

To determine whether Ca²⁺ plays a special role in the early graviresponse of shoots, as has been reported for roots, we treated etiolated pea epicotyls with substances known to antagonize Ca²⁺ (La³⁺), to remove Ca²⁺ from the wall (spermidine, EGTA), to inhibit calmodulin mediated reactions (chlorpromazine), or to inhibit IAA transport (TIBA). We studied the effect of these substances on IAA and Ca²⁺ uptake into 7 mm long subapical 3rd internode etiolated pea epicotyl sections and pea leaf protoplasts, on pea epicotyl growth, and graviresponse and on lateral IAA redistribution during gravistimulation.Our results support the view that adequate Ca²⁺ in the apoplast is required for normal IAA uptake, transport and graviresponse. Experiments with protoplasts indicate that Ca²⁺ may be controlling a labile membrane porter, possibly located on the external surface of cell membrane, while inhibitor experiments suggest that calmodulin is also implicated in both the movement of IAA and graviresponse. Since a major transfer of Ca²⁺ through free space during graviresponse has not yet been demonstrated, and since inhibition of calcium channels does not affect IAA redistribution (Migliaccio and Galston, 1987, Plant Physiology 85:542), we conclude that no clear evidence links prior Ca²⁺ movement with IAA redistribution during graviresponse in stems.Abbreviations IAA indole-3-acetic acid - CPZ chlorpromazine - EGTA ethylene glycol bis-(aminoethyl ether) N, N, N¹, N¹-tetracetic acid - G C gravicurvature The research was supported by NASA grant NSG-7290 to AWG.     

Changes in composition of the outer epidermal cell wall of pea stems during auxin-induced growth

The gross composition of the outer epidermal cell wall from third internodes of Pisum sativum L. cv. Alaska grown in dim red light, and the effect of auxin on that composition, was investigated using interference microscopy. Pea outer epidermal walls contain as much cellulose as typical secondary walls, but the proportion of pectin to hemicellulose resembles that found in primary walls. The pectin and hemicellulose fractions from epidermal peels, which are enriched for outer epidermal wall but contain internal tissue as well, are composed of a much higher percentage of glucose and glucose-related sugars than has been found previously for pea primary walls, similar to non-cellulosic carbohydrate fractions of secondary walls. The epidermal outer wall thus has a composition rather like that of secondary walls, while still being capable of elongation. Auxin induces a massive breakdown of hemicellulose in the outer epidermal wall; nearly half the hemicellulose present is lost during 4 h of growth in the absence of exogenous sugar. The percentage breakdown is much greater than has been seen previously for whole pea stems. It has been proposed that a breakdown of xyloglucan could be the basis for the mechanical loosening of the outer wall. This study provides the first evidence that such a breakdown could be occurring in the outer wall.M.S. Bret-Harte would like to thank Dr. Peter M. Ray, of Stanford University, for helpful discussions and for technical and editorial assistance, Dr. Winslow R. Briggs, of the Camegie Institude of Washington, for the use of experimental facilities and for helpful discussions, Dr. Wendy K. Silk, of the University of California, Davis, for helpful discussions and financial support, Dr. Paul B. Green for financial support, and Drs. John M. Labavitch and L.C. Greve, of the University of California, Davis, for performing the -cellulose analysis on short notice, in response to a request by an anonymous reviewer. This work was supported by a National Science Foundation Graduate Fellowship to M.S. B.-H., National Science Foundation Grant DCB8801493 to Paul B. Green, and the generosity of Wendy K. Silk (Department of Land, Air, and Water Resources, University of California, Davis) during the final writing.     

Purification and properties of diamine oxidase from pea epicotyls

Diamine oxidase (DAO) (EC 1.4.3.6) was purified from pea epicotyls to homogeneity by the criterion of polyacrylamide gel electrophoresis (PAGE). The pu