Differential Effect of Auxin on Molecular Weight Distributions of Xyloglucans in Cell Walls of Outer and Inner Tissues from Segments of Dark Grown Squash (Cucurbita maxima Duch.) Hypocotyls

Effects of indole-3-acetic acid (IAA) on the mechanical properties of cell walls and structures of cell wall polysaccharides in outer and inner tissues of segments of dark grown squash (Cucurbita maxima Duch.) hypocotyls were investigated. IAA induced the elongation of unpeeled, intact segments, but had no effect on the elongation of peeled segments. IAA induced the cell wall loosening in outer tissues as studied by the stress-relaxation analysis but not in inner tissues. IAA-induced changes in the net sugar content of cell wall fractions in outer and inner tissues were very small. Extracted hemicellulosic xyloglucans derived from outer tissues had a molecular weight about two times as large as in inner tissues, and the molecular weight of xyloglucans in both outer and inner tissues decreased during incubation. IAA substantially accelerated the depolymerization of xyloglucans in outer tissues, while it prevented that in inner tissues. These results suggest that IAA-induced growth in intact segments is due to the cell wall loosening in outer tissues, and that IAA-accelerated depolymerization of hemicellulosic xyloglucans in outer tissues is involved in the cell wall loosening processes.     

Stress relaxation properties of the cell wall of growing intact plants

The cell wall of dark-grown Avena coleoptiles and the epidermisof light-grown mungbean hypocotyls was subjected to stress-relaxationanalysis and the following results were obtained. 1. Actively growing apical regions of the organs, either coleoptilesor hypocotyls, had certain threshold values of minimum stress-relaxationtime, T_O, 0.04 sec for coleoptile cell wall and 0.03 sec forthe epidermal cell wall of hypocotyls. The cell wall of thebasal region of the organs, which were mature and not growing,had a higher value of To. 2. When the apical regions of the organs, either coleoptilesor hypocotyls, ceased to grow, their cell walls showed T_O valuesabove these thresholds. 3. The relaxation rate, b, was small in the cell wall of activelygrowing regions of the organs, compared with that of non-growingregions. 4. The maximum relaxation time, T_m, was variable and no significantrelationship with growth capacity was found. 5. The extensibility, mm/gr, was large not only in activelygrowing regions of the organs but also in fully grown regions,suggesting that the value represents complex properties of thecell wall including the history of cell wall extension. From these results, we concluded that biochemical modificationsoccur in the cell wall matrix of actively growing organs ofeither monocots or dicots, and these are the bases of the capacityof the cell wall to extend and are represented chiefly by T_oand possibly by b. (Received August 12, 1974; )     

Silicon-induced changes in viscoelastic properties of sorghum root cell walls

Silicon is deposited in the endodermal tissue in sorghum (Sorghum bicolor L. Moench) roots. Its deposition is thought to protect vascular tissues in the stele against invasion by parasites and drying soil via hardening of endodermal cells. We studied the silicon-induced changes in mechanical properties of cell walls to clarify the role of silicon in sorghum root. Sorghum seedlings were grown in nutrient solution with or without silicon. The mechanical properties of cell walls were measured in three separated root zones: basal, apical and subapical. Silicon treatment decreased cell-wall extensibility in the basal zone of isolated stele tissues covered by endodermal inner tangential walls. The silicon-induced hardening of cell walls was also measured with increases in elastic moduli (E) and viscosity coefficients (eta). These results provided new evidence that silicon deposition might protect the stele as a mechanical barrier by hardening the cell walls of stele and endodermal tissues. In contrast to the basal zone, silicon treatment increased cell-wall extensibility in the apical and subapical zones with concomitant decrease in E and eta. Simultaneously, silicon promoted root elongation. When root elongation is promoted by silicon, one of the causal factors maybe the silicon-enhanced extensibility of cell walls in the growing zone.     

Purification of Xyloglucan Hydrolase/Endotransferase from Cell Walls of Azuki Bean Epicotyls

An enzyme involved in the breakdown of xyloglucans was purifiedfrom an extract of cell walls of azuki bean epicotyls obtainedwith 1 M NaCl and purified by column chromatography on severaldifferent resins. The purified enzyme gave a single band ofa protein with a molecular mass of about 32 kDa after SDS-PAGE.The enzyme hydrolyzed the xyloglucans of high molecular massfrom azuki cell walls to yield fragments of about 50 kDa withoutproduction of any oligo- or monosaccharides. Moreover, the enzymehad hardly any effect on xyloglucans of less than 60 kDa. Theenzyme also hydrolyzed xyloglucans from tamarind, but it didnot react with cellulose derivatives. In the presence of pyridylamino-labeledxyloglucan oligosac-charides as acceptor substrates, the enzymecatalyzed the transfer of 50-kDa products to the oligosaccharides.The K_m value of the enzyme for xyloglucans of 540 kDa was similarin the presence and in the absence of xyloglucan oligosaccharidesas acceptors: 1.0 mg ml^–1. These results suggest thatthe enzyme was an endotransferase but had unusual acceptor specificity,preferring smaller acceptors such as water. (Received September 9, 1996; Accepted March 16, 1997)     

Turgor and Longitudinal Tissue 12Helianthus annuus

The quantitative relationship between turgor and the pressureexerted by the inner tissues (cortex, vascular tissue, and pith)on the peripheral cell walls (longitudinal tissue pressure)was investigated in hypocotyls of sunflower seedlings (Helianthusannuus L.) In etiolated hypocotyls cell turgor pressures, asmeasured with the pressure probe, were in the range 0·38to 0·55 MPa with an average of 0·48 MPa. In irradiatedhypocotyls turgor pressures varied from 0·40 to 0·57MPa with a, mean at 0·49 MPa. The pressure exerted bythe inner tissues on the outer walls was estimated by incubatingpeeled sections in a series of osmotic test solutions (polyethyleneglycol 8000). The length change was measured with a transducer.In both etiolated and irradiated hypocotyls an external osmoticpressure of 0·5 MPa was required to inhibit elongationof the inner tissues, i.e. the average cell turgor and the longitudinaltissue pressure are very similar quantities. The results indicatethat the turgor of the inner tissues is displaced to and borneby the thick, growth-limiting peripheral cell walls of the hypocotyl. Key words: Helianthus annuus, hypocotyl growth, tissue pressure, turgor pressure, wall stress     

Isolation of xyloglucans from etiolated Glycine max and Vigna sesquipedalis hypocotyls

Xyloglucans isolated from cell walls of etiolated Glycine maxand Vigna sesquipedalis hypocotyls were subjected to fragmentationanalysis with cellulase for structural comparison with thosederived from Phaseolus aureus hypocotyls. The xyloglucans fromG. max and V. sesquipedalis had glucose, xylose, galactose andfucose in the approximate molar ratio of 10:6:4:1 and 10:7:3:1,respectively. However, the results of cellulase fragmentationanalysis of xyloglucans from the three species suggested thatthe basic structure of the xyloglucans in the cell walls ofthese bean-hypocotyls is almost the same; the structure is basedon two repeating oligosaccharide units, one of which consistsof glucose and xylose and the other of glucose, xylose, galactoseand fucose. ¹ Present address: Toppan Printing Co., Ltd., Okaji, Sendai980, Japan. (Received February 3, 1977; )     

Distribution of xyloglucan in Phaseolus aureus seeds

Phaseolus aureus seeds were separated into three parts: hulls,cotyledons and hypocotyls. After the polysaccharides in eachpart had been fractionated by successive extraction with water,0.25% ammonium oxalate-oxalic acid, 4% KOH and 24% KOH, thexyloglucan content in each fraction was determined by Kooiman'smethod for quantitative analysis of amyloid coupled with cellulasetreatment. In each part, most of the xyloglucans were localizedin the 24% KOH extracts and moreover, these xyloglucans fromthe three seed parts had almost identical properties, whichagreed well with those of xyloglucans from the cell walls ofetiolated bean hypocotyls. (Received April 18, 1977; )     

Inhibition of the Breakdown of Xyloglucans in Azuki Bean Epicotyls by Concanavalin A

Indole-3-acetic acid at 10 µM caused a 30% decrease inthe weight-average molecular mass of xyloglucans extracted with24% KOH from the cell walls of epicotyl segments of azuki bean(Vigna angularis Ohwi et Ohashi cv. Takara). Concanavalin A(Con A) at 2 g liter^–1 completely inhibited the IAA-inducedchange in the molecular mass of the xyloglucans. Con A alsosuppressed the autolysis of pectin-depleted cell walls, as wellas the breakdown of xyloglucans by a protein fraction that hadbeen extracted with 1 M NaCl from the cell walls of azuki beanepicotyls. These results indicate that Con A is a potent inhibitorof the breakdown of xyloglucans both in vivo and in vitro. Mostof the activity responsible for the decrease in staining byiodine and the increase in reducing power of solution of xyloglucansin the protein fraction from cell walls bound to a column ofCon A-Sepharose and was eluted by the specific hapten, methyl     

Changes in Organelle Movement in the Nuclear Region during the Cell Cycle of Adiantum Protonema

Movements of organelles in the nuclear region as the cell cycleprogresses in single-celled protonemata of Adiantum capillus-veneriswere examined by digital image processing techniques and microscopyof particle movement. Organelles in the nuclear region werenot very crowded and moving directionally along the longitudinalaxis of the filamentous cell in the G₁ and S phases. They beganto gather and accumulate in the nuclear region in early G₂ phase,after which directional movement changed to undirectional Brownianmotion-like movement in late G₂ phase. Movement of organelleslocated on the lateral surface of the nucleus slowed after premitoticpositioning of nucleus and lasted until the nucleolus disappeared.Movement of organelles in the cytoplasm surrounding the nucleoplasmresumed just after the nucleolus disappeared, whereas organelleslocated in the outer regions of the apical and basal surfacesof the nucleus moved rapidly during prophase but did not moveduring metaphase, movement being resumed after chromosome separation.Thus, organelle movement in the nuclear region showed temporaland spatial change during the cell cycle. (Received August 24, 1983; Accepted December 28, 1983)     

Growth and cell wall changes in azuki bean epicotyls I. Changes in wall polysaccharides during intact growth

Measurement of endogenous growth rates and the mechanical propertyof the cell wall in various regions of light-grown azuki beanepicotyls revealed diat the minimum stress-relaxation time (T_o)was the shortest in the upper region (0–30 mm below theapex) of the epicotyl, where vigorous endogenous growth tookplace, and became longer toward the basal region, which wasmature and not growing. In the upper region of the epicotyl, a lower percentage of a-celluloseand a higher percentage of pectic substances than in the lowerregion were found. The percentage of hemicellulose content wasalmost constant over the whole epicotyl. Major components ofnoncellulosic neutral sugars in the cell wall were galactoseand xylose. The percentage of the galactose content to the noncellulosicpolysaccharide was highest in the upper region and lowest inthe basal region of the epicotyl, and a clearly negative correlationbetween the galactose composition and the T_o value was obtained.On the contrary, the percentage of die xylose content was highestin the basal region and lowest in die upper region, and a clearlypositive correlation between die xylose composition and theT_o value was obtained. During die endogenous growth of die intactepicotyl, all die neutral sugars, particularly galactose, increasedin die upper region, whereas in die middle and basal regions,only xylose increased. Similar changes in sugar compositionswere observed during IAA-induced elongation of die segment excisedfrom various regions of die epicotyl. (Received July 27, 1978; )     

Cessation of cell elongation in rye coleoptiles is accompanied by a loss of cell-wall plasticity

The mechanism by which endogenous cessation of coleoptile elongationafter emergence of the primary leaf is brought about was investigatedin rye seedlings (Secale cereale L.) that were either grownin darkness or irradiated with continuous white light. In 3-d-oldetiolated (growing) coleoptiles a turgor pressure of 0.59 MPawas measured. In 6-d-old coleoptiles, which had ceased to elongate,cell turgor was 0.51 MPa and thus only 13% lower than in therapidly growing organ. Hence, the driving force for growth (turgor)is largely maintained. Cell-wall plasticity (E_pl) and elasticity(E_Ql were determined with a constant load extensiometer bothin vivo (turgid coleoptile segments) and in vitro (frozen-thawedsamples). Cessation of coleoptile elongation was correlatedwith a 95% reduction in E_pl9 whereas E_Ql was only slightly affected.Extension kinetics were measured with living and frozen-thawedsegments cut from growing and non-growing coleoptiles. The correspondingstress-strain (load-extension) curves indicate that the cellwall of the growing coleoptile behaves like an elastic-plasticmaterial whereas that of the non-growing organ shows the behaviourof an elastic solid. These data demonstate that E_pl representsa true plastic (irreversible) deformation of the cell wall.It is concluded that cessation of coleoptile growth after emergenceof the primary leaf is attributable to a loss of cell-wall plasticity.Hence, a mechanical stiffening of the cell wall and not a lossof turgor pressure may be responsible for the deceleration ofcell elongation in the rye coleoptile. Key words: Extension growth, rye coleoptile, cell-wall extensibility, turgor pressure     

Effect of Hydrogen Peroxide on Degradation of Cell Wall Associated Proteins in Growing Bean Hypocotyls

The possible involvement of active oxygen species and an apoplasticendopeptidase (EP) in the digestion of cell wall proteins wasstudied in extracellular fluid (EF) from hypocotyls of Phaseolusvulgaris at different stages of elongation. EF proteins underwentsignificant changes in polypeptide pattern during hypocotylgrowth, which were characterized by increases in 35, 39, 40and 50 kDa peptides and appearance of 61, 70 and 75 kDa peptidesat the exponential growth phase. EFs also contain endopeptidase[Gómez et al. (1994) Agriscientia 11:3]. Autolysis experimentswithout or with purified EP revealed that many cell wall polypeptidesare liable to degradation by the protease. Besides, EF polypeptidesincreased their susceptibility to EP during hypocotyl elongation.The 50 and 40 kDa polypeptydes were poorly degraded when extractedfrom hypocotyls in active growth, but greatly hydrolyzed whenextracted from fully elongated tissues, suggesting that in thecourse of growth proteins underwent modifications that renderedthem more prone to proteolytic attack. These modifications seemedto involve active oxygen species, as indicated by: (a) H₂O₂level rised when protein susceptibility to EP increased; and(b) EF proteins from growing hypocotyls (comparatively lesssusceptible to EP) treated with H₂O₂ were rapidly degraded bythe protease. (Received April 27, 1995; Accepted July 31, 1995)     

The Relationship Between the Distribution of Periclinal Cell Divisions in the Shoot Apex and Leaf Initiation

Periclinal cell divisions in vegetative shoot apices of Pisumand Silene were recorded from serial thin sections by mappingall the periclinal cell walls formed less than one cell cyclepreviously. The distribution of periclinal divisions in theapical domes corresponded to the distributions subsequentlyoccurring in the apices when the young leaf primordia were forming.In Pisum, periclinal divisions were almost entirely absent fromthe I₁ region of the apical dome for half a plastochron justafter the formation of a leaf primordium and appeared, simultaneouslyover the whole of the next potential leaf site, about half aplastochron before the primordium formed. In Silene periclinaldivisions seemed to always present in the apical dome at thepotential leaf sites and also round the sides of the dome wherethe ensheathing leaf bases were to form. Periclinal divisionstherefore anticipated the formation of leaf primordia by occuring,in Pisum about one cell cycle and in Silene two or more cellcycles, before the change in the direction of growth or deformationof the surface associated with primordial initiation. Pisum, Silene, planes of cell division, orientation of cell walls, leaf primordia, shoot apical meristem, plastochron     

Changes in Wall Polysaccharides of Squash (Cucurbita maxima Duch.) Hypocotyls under Water Stress Condition: II. Composition of Pectic and Hemicellulosic Polysaccharides

Hypocotyl growth of dark-grown squash (Cucurbita maxima Duch.)seedlings was greatly reduced by the addition of polyethyleneglycol (60 mM) to the hydroponic solution through inhibitionof cell elongation. Measurement of the mechanical propertiesof the cell walls revealed that the cell wall of stressed hypocotylswas loosened as much as that of the unstressed hypocotyls, suggestingthat the stressed hypocotyl could not elongate even though thecell wall loosened. Galactose and arabinose in the pectic fraction,which are probably attached to high mol wt rhamnogalacturonans,increased under stressed as well as under unstressed condition.Other polysaccharides including pectic low mol wt galacturonans,hemicellulosic xyloglucans, galactoglucomannans, arabinans,and glucuronoarabinoxylans increased more under unstressed condition.The mol wt of xyloglucans in the hemicellulosic fraction increasedunder unstressed but not under stressed condition. These results suggest that changes in wall structure, such asincreases in high mol wt rhamnogaracturonans rich in arabinoseand galactose residues, and the suppression of polymerizationof xyloglucans are involved in the process of cell wall loosening. (Received December 15, 1986; Accepted June 8, 1987)     

The epidermal-growth-control theory of stem elongation: an old and a new perspective

The botanist G. Kraus postulated in 1867 that the peripheral cell layers determine the rate of organ elongation based on the observation that the separated outer and inner tissues of growing stems spontaneously change their lengths upon isolation from each other. Here, we summarize the modern version of this classical concept, the "epidermal-growth-control" or "tensile skin" theory of stem elongation. First, we present newly acquired data from sunflower hypocotyls, which demonstrate that the expansion of the isolated inner tissues is not an experimental artefact, as recently claimed, but rather the result of metabolism-independent cell elongation caused by the removal of the growth-controlling peripheral walls. Second, we present data showing that auxin-induced elongation of excised stem segments is attributable to the loosening of the thick epidermal walls, which provides additional evidence for the "epidermal-growth-control concept". Third, we show that the cuticle of aerial organs can be thin and mechanically weak in seedlings raised at high humidity, but thick and mechanically important for organs growing under relatively dry air conditions. Finally, we present a modified model of the "tensile skin-theory" that draws attention to the mechanical and physiological roles of (a) the thickened, helicoidal outer cell walls, (b) the mechanical constraint of a cuticle, and (c) the interactions among outer and inner cell layers as growth is coordinated by hormonal signals.     

Cell-wall synthesis and elongation growth in hypocotyls of Helianthus annuus L.

The relationship between growth and increase in cell-wall material (wall synthesis) was investigated in hypocotyls of sunflower seedlings (Helianthus annuus L.) that were either grown in the dark or irradiated with continuous white light (WL). The peripheral three to four cell layers comprised 30–50% of the entire wall material of the hypocotyl. The increase in wall material during growth in the dark and WL, respectively, was larger in the inner tissues than in the peripheral cell layers. The wall mass per length decreased continuously, indicating that wall thinning occurs during growth of the hypocotyl. When dark-grown seedlings were transfered to WL, a 70% inhibition of growth was observed, but the increase in wall mass was unaffected. Likewise, the composition of the cell walls (cellulose, hemicellulose, pectic substances) was not affected by WL irradiation. Upon transfer of dark-grown seedlings into WL a drastic increase in wall thickness and a concomitant decrease in cell-wall plasticity was measured. The results indicate that cell-wall synthesis and cell elongation are independent processes and that, as a result, WL irradiation of etiolated hypocotyls leads to a thickening and mechanical stiffening of the cell walls.     

Monitoring Primary Response to Chilling Stress in Etiolated Vigna radiata and V. mungo Seedlings Using Thermal Hysteresis of Water Proton NMR Relaxation Times

Thermal hysteresis of longitudinal relaxation times (T₁) ofwater protons in hypocotyls of etiolated Vigna radiata and V.mungo seedlings was investigated by pulse nuclear magnetic resonance(NMR) spectroscopy. Various lengths of chilling exposures duringa cool-warm cycle between 20 and 0?C (below 10?C, about 4 h)for the T₁ hysteresis measurement did not cause any visibleinjury symptoms in hypocotyls. However, the profiles of T₁ hysteresisvaried as a result of different chilling exposures. The sumsof the T₁ ratio (for detail see Introduction) reflecting T₁prolongation or shortening upon the warming process were a goodquantitative index for the extent of T₁ hysteresis, and thewide dispersion of this value ranging on the "minus" side (T₁prolongation upon warming) suggested the occurrence of a primaryresponse of cells to chilling stress before obvious visiblesymptoms occur while the T₁ ratio sums on the "plus" side (T₁shortening upon warming) corresponded to a response of seriousvisible injury. Therefore, the sums of the T₁ ratio can be usedas a non-destructive diagnostic tool for monitoring the primaryevent of chilling injury when lacking any visible injury symptoms.The data indicate that the critical temperature for the occurrenceof primary response for chilling stress was around 7.5?C forV. radiata and 12.5?C for V. mungo. (Received February 1, 1988; Accepted June 1, 1988)     

The Oligosaccharide Units of the Xyloglucans in the Cell Walls of Bulbs of Onion, Garlic and their Hybrid

Xyloglucans were isolated from the 24% KOH-soluble fractionof the cell walls of bulbs of onion (Allium cepa), garlic (Alliumsativum) and their hybrid. The polysaccharides yielded singlepeaks upon gel filtration with average moleular weights of 65,000for onion, 55,000 for garlic and 82,000 for the hybrid. Compositionalanalysis of the oligosaccharide units after digestion with anendo-1,4-ß-glucanase from Streptomyces indicated thatthe polysaccharides were constructed of four kinds of repeatingoligosaccharide unit, namely, a decasaccharide (glucose/xylose/galactose/fucose,4 : 3 : 2 : 1), a nonasaccharide (glucose/xylose/galactose/fucose,4 : 3 : 1 : 1), an octasaccharide (glucose/xylose/galactose,4 : 3 : 1 ) , and a heptasaccharide (glucose/xylose, 4 : 3).The xyloglucan from the hybrid contained highly fucosylatedunits that resembled those from onion rather than from garlic.The analysis also revealed that the xyloglucans from Alliumspecies contain highly substituted xylosyl residues with fucosyl-galactosylresidues, suggesting that these monocotyledonous plants resembledicotyledons in the structural features of their xyloglucans. (Received November 1, 1993; Accepted June 16, 1994)     

Stimulation of elongation growth and xyloglucan breakdown in Arabidopsis hypocotyls under microgravity conditions in space

Seedlings of Arabidopsis thaliana (L.) Heynh. (ecotype Columbia and an ethylene-resistant mutant etr1-1) were cultivated for 68.5, 91.5 and 136 h on board during the Space Shuttle STS-95 mission, and changes in the elongation growth and the cell wall properties of hypocotyls were analyzed. Elongation growth of dark-grown hypocotyls of both Columbia and etr1-1 was stimulated under microgravity conditions in space. There were no clear differences in the degree of growth stimulation between Columbia and etr1-1, indicating that the ethylene level was not abnormally high in the cultural environment of this space experiment. Microgravity also increased the mechanical extensibility of cell walls in both cultivars, and such an increase was attributed to the increase in the apparent irreversible extensibility. The levels of cell wall polysaccharides per unit length of hypocotyls decreased in space. Microgravity also reduced the weight-average molecular mass of xyloglucans in the hemicellulose-II fraction. Also, the activity of xyloglucan-degrading enzymes extracted from hypocotyl cell walls increased under microgravity conditions. These results suggest that microgravity reduces the molecular mass of xyloglucans by increasing xyloglucan-degrading activity. Modifications of xyloglucan metabolism as well as the thickness of cell wall polysaccharides seem to be involved in an increase in the cell wall extensibility, leading to growth stimulation of Arabidopsis hypocotyls in space.     

The Role of the Epidermis in the Control of Elongation Growth in Stems and Coleoptiles

The peripheral cell wall(s) of stems and coleoptiles are 6 to 20 times thicker than the walls of the inner tissues. In coleoptiles, the outer wall of the outer epidermis shows a multilayered, helicoidal cellulose architecture, whereas the walls of the parenchyma and the outer wall of the inner epidermis are unilayered. In hypocotyls and epicotyls both the epidermal and some subepidermal walls are multilayered, helicoidal structures. The walls of the internal tissues (inner cortex, pith) are unilayered, with cellulose microfibrils oriented primarily transversely. Peeled inner tissues rapidly extend in water, whereas the outer cell layer(s) contract on isolation. This indicates that the peripheral walls limit elongation of the intact organ. Experiments with the pressure microprobe indicate that the entire organ can be viewed as a giant, turgid cell: the extensible inner tissues exert a pressure (turgor) on the peripheral wall(s), which bear the longitudinal wall stress of the epidermal and internal cells. Numerous studies have shown that auxin induces elongation of isolated, intact sections by loosening of the growth-limiting peripheral cell wall(s). Likewise, the effect of light on reduction of stem elongation and cell wall extensibility in etiolated seedlings is restricted to the peripheral cell layers of the organ. The extensible inner tissues provide the driving force (turgor pressure), whereas the rigid peripheral wall(s) limit, and hence control, the rate of organ elongation.