White light promotes the formation of diferulic acid in maize coleoptile cell walls by enhancing PAL activity

To elucidate the mechanism by which white fluorescent light (5 W m^-2) stimulates the formation of diferulic acid (DFA) in cell walls, the effect of light on phenylalanine-and tyrosine-ammonia-lyase (PAL, EC 4.3.1.5 and TAL, EC 4.3.1.5) and peroxidase activities was studied using coleoptiles of maize ( Zea mays L. cv. Cross Bantam T51). Growth rate of dark-grown coleoptiles was highest at the basal zone and decreased towards the tip, while continuous irradiation caused an inhibition of growth, especially at the basal zone. Light decreased the cell wall extensibility in all zones of the coleoptile. The amounts of DFA, ferulic acid (FA) and p -coumaric acid ( p -CA) increased by severalfold in cell walls of light-grown maize coleoptiles as compared with those grown in the dark. Strong correlations were observed between the increase in the contents of either DFA, FA or p -CA and the decrease in cell wall extensibility. Light decreased the wall-bound peroxidase activity. No correlation was found between DFA content and peroxidase activity. The activities of PAL and TAL were enhanced upon white light irradiation. The increment in either DFA, FA or p -CA content was correlated with an increase in PAL activity, but not with that in TAL activity. White light may promote DFA formation in the cell walls of maize coleoptiles by enhancing PAL activity.     

White light‐induced sugar distribution controls growth and osmotic properties in the coleoptile and the first leaf in Zea mays seedlings

The correlation of white light‐induced changes in osmotic concentration in the coleoptile and the first leaf and the growth rate of these organs in maize seedlings, was examined in relation to sugar distribution and invertase activity. One hour irradiation with white light decreased the osmotic concentration in basal zones of the coleoptile and increased it in the apical zones of the first leaf. The change in the osmotic concentration was positively correlated with the growth rate of both organs. The amount of total osmotic solutes in each zone was highly correlated with that of soluble sugars. Light decreased the activity of wall‐bound invertase in the coleoptile, but increased it in the first leaf. A high correlation existed between the content of soluble sugars and invertase activity in both organs. During 1 h incubation in the light, ca 2 µmol of soluble sugars per seedling was lost from the coleoptile and gained in the first leaf. Light promoted sugar exudation from the excised coleoptile, but the amount of soluble sugar exuded represented 5% of sugar loss from the coleoptile in intact seedlings.
These results indicate that in maize seedlings white light controls the growth rate of the coleoptile and the first leaf through the osmotic concentration. Light may have an osmoregulatory function in the control of sugar distribution between the coleoptile and the first leaf by regulating the activity of wall‐bound invertase.     

Diferulic and ferulic acid in the cell wall of Avena coleoptiles—Their relationships to mechanical properties of the cell wall

Alkaline hydrolysis liberated ferulic and diferulic acid from polysaccharides of the Avena coleoptile ( Avena sativa L. cv. Victory I) cell walls. The amount of the two phenolic acids bound to cell walls increased substantially at day 4–5 after sowing, when the growth rate of the coleoptile started to decrease. The level of these acids was almost constant from the tip to base in 3-day-old coleoptiles, but increased toward the basal zone in 4- and 5-day-old ones. The ratio of diferulic acid to ferulic acid was almost constant irrespective of coleoptile age and zone. An increase in the amount of ferulic and diferulic acids bound to cell wall polysaccharides correlated with a decrease in extensibility and with an increase in minimum stress-relaxation time and relaxation rate of the cell wall. The level of lignin in the cellulose fraction increased as coleoptiles aged, but this increase did not correlate with changes in mechanical properties of the cell walls. These results suggest that ferulic acid, ester-linked to cell wall polysaccharides, is oxidized to give diferulic acid, which makes the cell wall mechanically rigid by cross-linking matrix polysaccharides and results in limited cell extension growth. In addition, it is probable that the step of feruloylation of cell wall polysaccharides is rate-limiting in the formation of in-termolecular bridges by diferulic acid in Avena coleoptile cell walls.     

The biophysical basis of cell elongation and organ maturation in coleoptiles of rye seedlings: implications for shoot development

The relationships between changes in irreversible and reversible organ length, turgor (P), osmotic pressure (pi), and metabolic activity of the cells were investigated in intact coleoptiles of rye seedlings ( Secale cereale L.) that were either grown in darkness or irradiated with continuous white light. Cessation of growth at day 4 after sowing was associated with an apparent mechanical stiffening of the cell walls. Turgor pressure was measured in epidermal and mesophyll cells with a miniaturized pressure probe. No gradient of turgor was found between the peripheral and internal cells. In juvenile (growing) coleoptiles, average turgor was 0.60 MPa and a negative water potential (P - pi) was established in these cells. Upon emergence of the primary leaf, turgor declined, but P was maintained at values of 0.43 and 0.52 MPa in 7-day-old light- and dark-grown coleoptiles, respectively. Water potential in non-growing cells approached zero. The rate of dark respiration and elongation growth were not correlated. Surgical removal of the mature coleoptile revealed that the erect position of the 7-day-old shoot was dependent on the presence of this sturdy, turgid organ sheath. It is concluded that, during the first week of seedling development, the pierced, metabolically active coleoptile fulfills an essential function as an elastic basal tube for the juvenile shoot.     

Suppression of cell wall stiffening along coleoptiles of wheat (Triticum aestivum L.) seedlings grown under osmotic stress conditions

Effects of polyethylene glycol (PEG)-induced osmotic stress on the mechanical properties of cell walls and the levels of their components were investigated along intact wheat (Triticum aestivum L.) coleoptiles. Stress-relaxation analysis showed that the cell walls of stressed coleoptiles were loosened as compared with those of unstressed ones not only in the apical but in the basal regions. The amounts of wall-bound ferulic acid (FA) and diferulic acid (DFA) of stressed coleoptiles were substantially lower than those of unstressed ones in all regions. The cellulose and hemicellulose contents increased toward the coleoptile base. Osmotic stress reduced the cellulose content in the basal region but it slightly affected the hemicellulose content. The molecular weight of hemicellulose in the apical region of stressed coleoptiles was higher than that of unstressed ones, while that in the basal region was almost the same in both coleoptiles. FA, DFA and cellulose contents correlated with the cell wall mechanical property. The amount and molecular weight of hemicellulose, however, did not correlate. These results suggest that the reduced levels of FA and DFA in all regions and cellulose in the basal region of wheat coleoptiles are involved in maintaining the cell wall extensibility under osmotic stress.     

Automorphosis of maize shoots under simulated microgravity on a three-dimensional clinostat

Seedlings of maize ( Zea mays L. cv. Golden Cross Bantam T-51) were grown under microgravity conditions simulated by a three-dimensional clinostat. On the clinostat, maize shoots exhibited curvatures in three different portions: (1) the basal transition zone connecting roots and mesocotyls, (2) the coleoptile node located between mesocotyls and coleoptiles, and (3) the elongating region of the coleoptiles. Even non-clinostatted control shoots showed some degree of curvature away from the caryopsis in the transition zone and bending toward the caryopsis in the coleoptile node. Clinostat rotation greatly stimulated these curvatures. Control coleoptiles elongated almost straightly, whereas coleoptiles on the clinostat bent either away from or toward the caryopsis depending on the timing of rotation. The curvature in all three portions became larger with time, both in control and clinostatted seedlings. There was no difference in the osmotic concentration of the cell sap between the convex and the concave halves of any portion. However, in coleoptile nodes and coleoptiles, the faster-expanding convex side exhibited a higher extensibility of the cell wall than the opposite side, and this appears to be a cause of the curvature. Thus, changes in the cell wall metabolism may be involved in automorphosis, which governs the life cycle of plants under a microgravity environment.     

Changes in osmotic pressure and cell wall properties during auxin- and ethylene-induced growth of intact coleoptiles of rice

Indole-3-acetic acid and 1-aminocyclopropane-1-carboxylic acid, the precursor of ethylene, stimulated elongation of coleoptiles of seedlings of intact rice ( Oryza sativa L. cv. Sasanishiki) submerged in buffer solution with constant air-bubbling. The osmotic pressure of the cell sap decreased during elongation of coleoptiles. In the presence of 30 μ M aminooxyacetic acid, an inhibitor of ethylene biosynthesis, in-dole-3-acetic acid at 30 μ M accelerated the decrease in the osmotic pressure in the early stage of growth. 1-Aminocyclopropane-1-carboxylic acid at 30 μ M did not influence the decrease in the osmotic pressure.
Both indole-3-acetic acid and 1-aminocyclopropane-1-carboxyIic acid decreased the minimum stress-relaxation time and the relaxation rate of the cell wall, suggesting that both auxin and ethylene induce elongation of rice coleoptiles by stimulating cell wall loosening. These growth regulators caused an increase in the level of glucose in hemicelluloses in the early stage of growth and a decrease in the level in the subsequent last growth phase. Indole-3-acetic acid decreased the hydroxyproline and glucosamine levels per unit dry weight of the cell wall. These changes in the level of cell wall components may be associated with the changes in the mechanical properties of the cell walls caused by auxin and ethylene.     

Viscoelastic versus plastic cell wall extensibility in growing seedling organs: a contribution to avoid some misconceptions

In a recent publication (Kutschera, 1996), it was reported thatthe cell walls of growing rye coleoptiles exhibit irreversible(plastic) extensibility in a rheological extension test. Basicallysimilar measurements with cell walls of maize coleoptiles hadpreviously shown that the apparent plastic extensibility determinedin this material is in reality due to the slowly reversible(viscoelastic) extensibility of the walls. A recent reinvestigationof this discrepancy showed that rye coleoptile walls also behaveas a perfectly viscoelastic material if precautions are takento prevent measuring artefacts. Similar results were obtainedwith cell walls from the growing zone of various other seedlingorgans (maize mesocotyl, maize root, cucumber hypocotyl). Itis concluded that plastic extensibility has not yet been convincinglydemonstrated by rheological tests that determine the intrinsicmaterial properties of cell walls. Reported changes in mechanicalmaterial properties of cell walls produced by growth-controllingfactors such as auxin or light may generally be attributed tochanges in viscoelasticity which are not directly related tothe chemo-rheological processes controlling wall extension ofgrowing cells. Key words: Cell wall extensibility, extension growth, plastic cell wall extensibility, viscoelastic cell wall extensibility     

Involvement of Cell Wall-Bound Diferulic Acid in Light-Induced Decrease in Growth Rate and Cell Wall Extensibility of Oryza Coleoptiles

Irradiation of white fluorescent light (5 W m^–2) inhibitedthe growth of Oryza coleoptiles. Light irradiation increasedstress-relaxation parameters of coleoptile cell walls, minimumstressrelaxationtime and relaxation rate, and decreased cellwall extensibility (strain/load). Under light conditions, thecontents of ferulic and diferulic acids ester-linked to thehemicellulosic arabinose residue in cell walls increased andcorrelated with the modification of the cell wall mechanicalproperties. These results suggest that light irradiation enhancesthe formation of diferulic acid bridges in hemicelluloses, makingcell walls mechanically rigid and thus inhibits cell elongationin rice coleoptiles. Also, irrespective of coleoptile age orthe presence of light, the ratio of diferulic acid to ferulicacid was almost constant, suggesting that the rate limitingstep in the formation of diferulic acid bridges in Oryza cellwalls is in the step of feruloylation. (Received September 24, 1991; Accepted December 3, 1991)     

Effect of anti-wall protein antibodies on auxin-induced elongation, cell wall loosening, and β-D-glucan degradation in maize coleoptile segments

Antiserum raised against the LiCl extract of maize shoot cell walls suppresses auxin-induced elongation of maize coleoptile segments. A series of polyclonal antibodies were raised against protein fractions separated from the LiCl extract of maize ( Zea mays L. cv. B73 x Mo17) coleoptiles by SP-Sephadex and Bio-Gel P-150 chromatography. To understand the role of cell wall proteins in growth regulation, the effect of these antibodies on auxin-induced elongation and changes in the cell walls of maize coleoptiles was examined. Four of the fractions prepared reacted with the antiserum raised against the total LiCl extract and effectively suppressed its growth-inhibiting activity. Only these fractions contained the proteins responsible for eliciting growthinhibiting antibodies. The antibodies capable of growth inhibition of auxin-induced elongation of segments also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time of the cell walls) of segments. The antibodies raised against one of the protein fractions separated by SP-Sephadex inhibited the autolytic reactions of isolated cell walls and the auxin-induced decrease in (1→3), (1→4)-β-D-glucans in the cell walls. Thus, the degradation of β-D-glucans by cell wall enzymes may be associated with the cell wall loosening that is responsible for cell elongation. Because the other antibodies did not influence the auxin-induced degradation of (1→3), (1→4)-β-D-glucanses, β-D-glucanases and other cell wall enzymes may cooperate in regulation of cell elongation in maize coleoptiles.     

Inhibition of the Coleoptile Growth in Avena sativa by Endosperm Removal--Changes in Auxin, Osmotica and Cell Wall

Removal of the endosperm from 84-h-old etiolated oat seedlingsstrongly retarded the subsequent growth of coleoptiles. Thecontribution of the endosperm to coleoptile growth was studied.Endosperm removal was found to: (1) decrease the endogenouslevel of indole-3-acetic acid (IAA) in the coleoptile tip. IAAapplied to the coleoptile tip stimulated coleoptile growth inseedlings with and without the endosperm. The sensitivity ofthe coleoptile to a suboptimal concentration of IAA was higherin seedlings without the endosperm than in intact ones. At theoptimal concentration of IAA, however, the final length of thecoleoptile was larger in intact seedlings than in those withoutthe endosperm. (2) decrease the concentration of the solublesugars and amino acids in the cell sap. (3) retard the increasein the amount of polysaccharides in the cell wall of the coleoptile,particularly noncellulosic ones. (4) make the cell wall mechanicallyrigid according to stress-relaxation analysis of the cell wall.(5) induce an increase in the osmotic potential of the coleoptilecell sap. From these results, it was concluded that the endosperm suppliesthe coleoptile with IAA, sugars and amino acids, thus promotingcoleoptile growth. (Received September 24, 1987; Accepted February 3, 1988)     

Galactose inhibition of auxin-induced cell elongation in oat coleoptile segments

Auxin-induced cell elongation in oat coleoptile segments was inhibited by galactose; removal of galactose restored growth. Galactose did not appear to affect the following factors which modify cell elongation: auxin uptake, auxin metabolism, osmotic concentration of cell sap, uptake of tritium-labeled water, auxin-induced wall loosening as measured by a decrease in the minimum stress-relaxation time and auxininduced glucan degradation. Galactose markedly prevented incorporation of [¹⁴C]-glucose into cellulosic and non-cellulosic fractions of the cell wall. It was concluded that galactose inhibited auxin-induced long-term elongation of oat coleoptile segments by interfering with cell wall synthesis.     

Role of Expansin in Cell Enlargement of Oat Coleoptiles (Analysis of Developmental Gradients and Photocontrol)

Expansins are wall proteins that mediate a type of acid-induced extension in isolated plant cell walls (S. McQueen-Mason, D.M. Durachko, D.J. Cosgrove [1992] Plant Cell 4: 1425-1433). To assess the role of these proteins in the process of cell enlargement in living tissues, we compared the spatial and temporal growth patterns of oat (Avena sativa L.) coleoptiles with four wall properties related to expansin action. These properties were (a) the ability of isolated walls and living segments to extend in acidic buffer, (b) the ability of heat-inactivated walls to extend upon application of expansins, (c) the amount of immunologically detectable expansin in wall protein extracts, and (d) the extractable expansin activity of walls. Growth rate was maximal in the apical half of dark-grown coleoptiles and negligible in the basal region. This growth pattern correlated with properties a and b; in contrast, the amount and activity of extractable expansin (properties c and d) were reduced only in the most basal region. Upon exposure to white light, coleoptiles abruptly ceased elongation at 8 to 10 h after start of irradiation, and this cessation correlated with reductions in properties a to c. The growth cessation at 8 to 10 h also coincided with the loss of growth response to exogenous auxin and fusicoccin in excised coleoptile segments. These results lend correlative support to the hypothesis that expansin action is important for growth responses of living oat coleoptiles (e.g. responses to acidic buffers, auxin, fusicoccin, aging, and light). Our results suggest that changes in the susceptibility of the wall to expansin action, rather than changes in expansin activity, may be a key determinant of the growth patterns in oat coleoptiles.     

Light-induced increase in the contents of ferulic and diferulic acids in cell walls of Avena coleoptiles: its relationship to growth inhibition by light

White fluorescent light (5 W m⁻²) inhibited Avena coleoptile growth. Light caused in increase in minimum stress relaxation time and a decrease in extensibility (strain/load) of coleoptile cell walls. Light increased the contents of ferulic acid (FA) and diferulic acid (DFA) ester-linked to the hemicellulose I in cell walls. These changes in the phenolic contents correlated with those of the mechanical properties of cell walls, suggesting that light stimulates the formation of DFA in hemicellulose I, making cell walls rigid, and thus results in growth inhibition. The ratio of DFA to FA was almost constant in the dark, but decreased in light, although it was almost constant in Oryza coleoptiles either in the dark or in light (Tan et al. 1992). From this fact, it is speculated that in the light condition, the formation of DFA in cell walls is limited in the step of the peroxidase catalyzed coupling reaction to produce DFA, while in the dark it is limited in the step of the feruloylation of hemicellulose I.     

Correlation between cell wall extensibility and the content of diferulic and ferulic acids in cell walls of Oryza sativa coleoptiles grown under water and in air

Rice ( Oryza sativa L. cv. Sasanishiki) coleoptiles grown under water achieved greater length than those grown either in air or under water with constant air bubbling. The extensibility of cell walls in coleoptiles grown under water was larger than that in the other treatments. Per unit length of the coleoptile, the content of ferulic and diferulic acids ester-linked to hemicelluloses was higher in air and bubbling type coleoptiles than in water type ones. The extensibility of the coleoptile cell walls correlated with the content of diferulic acids per unit length and per hemicellulose, suggesting that the enhancement of the formation of diferulic acid bridges in hemicelluloses in air or under water with air bubbling makes the cell walls mechanically rigid; thereby inhibiting cell elongation in rice coleoptiles. In addition, the ratio of diferulic acid to ferulic acid was almost constant irrespective of coleoptile age, zone and growth conditions, suggesting that the feruloylation of hemicelluloses is rate-limiting in the formation of diferulic acid bridges in the cell walls of rice coleoptiles.     

The effect of auxin on stress relaxation in isolated Avena coleoptiles

In order to characterize further the mechanical properties of coleoptile cell walls, stress relaxation measurements were made on methanol-boiled sections of Avena coleoptiles. Relaxation was measured both in mechanically conditioned specimens and in specimens which had not been previously extended. In both cases the relaxation was proportional to log time. Mechanical conditioning increased the relaxation modules and decreased the relative rate of relaxation. In contrast, pretreatment of the live coleoptiles with indoleacetic acid reduced the relaxation modulus and the absolute rate of relaxation but did not affect the relative rate of relaxation. Essentially similar pictures of the mechanical properties of coleoptile walls are obtained from stress relaxation and creep tests; the wall behaves as a nonlinear viscoelastic material.     

Increased molecular mass of hemicellulosic polysaccharides is involved in growth inhibition of maize coleoptiles and mesocotyls under hypergravity conditions.

Elongation growth of dark grown maize (Zea mays L cv. Cross Bantam T51) coleoptiles and mesocotyls was suppressed by hypergravity at 30 g and above. Acceleration at 300 g significantly decreased the mechanical extensibility of cell walls of both organs. Hypergravity increased the amounts of hemicellulose and cellulose per unit length in mesocotyl walls, but not in coleoptile walls. The weight average molecular masses of hemicellulosic polysaccharides were also increased by hypergravity in both organs. On the other hand, the activities of beta-glucanases extracted from coleoptile and mesocotyl cell walls were decreased by hypergravity. These results suggest that the decreased activities of beta-glucanases by hypergravity cause an increase in the molecular mass of hemicellulosic polysaccharides of both organs. The upshift of molecular mass of hemicellulosic polysaccharides as well as the thickening of cell walls under hypergravity conditions seems to be involved in making the cell wall mechanically rigid, thereby inhibiting elongation growth of maize coleoptiles and mesocotyls.     

Osmoregulation in the Avena coleoptile in relation to auxin and growth

A study has been made of the effects of auxin and growth on the ability of Avena coleoptile sections to osmoregulate, i.e. to take up solutes so as to maintain their osmotic concentration, turgor pressure, and growth rate. The high auxin-induced growth rate of Avena coleoptiles is maintained when cells are provided sucrose, glucose, NaCl, or KCl as a source of absorbable solutes, but not when 2-deoxy-d-glucose or 3-O-methyl-d-glucose is used. In the absence of auxin, cells take up solutes from a 2% sucrose solution and the osmotic concentration increases. The rate of solute uptake is even greater in the presence of auxin or fusicoccin, but the osmotic concentration rises only slightly because of the water taken up during growth. Solute uptake is not stimulated by auxin when growth is inhibited osmotically or by calcium ions. Solute uptake appears to have two components: a basal rate, independent of auxin or growth, and an additional uptake which is proportional to growth. Osmoregulation of sections may be limited by the rate of entry of solutes into the tissue rather than by their rate of uptake into the cells.     

Inhibition of auxin-induced cell elongation of maize coleoptiles by antibodies specific for cell wall glucanases

Polyclonal antibodies were raised in rabbits in response to the administration of purified exo- and endoglucanases extracted from cell walls of maize (Zea mays L. B37 × Mo17) coleoptiles. Since the antibodies formed specific conjugates when challenged with the glucanase antigens in immunoblot assays they were employed to evaluate the participation of glucanases in tissue growth. Indole-3-acetic acid induced cell elongation of abraded coleoptile segments was inhibited when the antibodies were supplied as a short term pretreatment (25-200 microgram/milliliter of serum protein). The extent of inhibition of IAA induced cell elongation was additive when endo- and exoglucanase antibodies were applied together. The results suggest that both enzymes have a role in mediating IAA-induced cell elongation. Pretreatment with exo- and endoglucanases antibodies also inhibited IAA induced degradation of noncellulosic β-d-glucans and the increased level of cellulosic polymers in maize coleoptiles. Antibodies also inhibited the expression of the autohydrolytic degradation of glucans in isolated cell walls. The extent of inhibition was dependent on the antibody concentration applied. The results support the contention that enzymatic processes mediated by exo- and endoglucanases are responsible for cell wall autolytic reactions and that these reactions are linked to the mechanism for expressing auxin induced cell elongation in maize coleoptiles.     

Growth and cell wall changes in rice coleoptiles

A fractionation of non-cellulosic sugars of Oryza sativa L. coleoptile cell walls was carried out and the composition of each fraction was studied during coleoptile growth.Percentages of fractions extracted with boiling water and with oxalate (pectic substances) were almost constant throughout development. An increase in the K II hemicellulosic fraction (extracted with 24% KOH) content, and a decrease in the K I hemicellulosic fraction (extracted with 10% KOH) were detected, when coleoptile growth finished.The percentage of glucose content in the K I hemicellulosic fraction was highest in young coleoptiles and lowest in old ones. Furthermore, a highly significant linear relationship between amounts of glucose and growth rate was obtained, while a inverse relationship between the amount of xylose and arabinose and growth rate was attained.Abreviations GLC gas liquid chromatography - IAA indole-3-acetic-acid - TFA trifluoroacetic acid - T_o minimum stress-relaxation time