Three Different Methods for Measuring Xylem Cavitation and Embolism: A Comparison

Three different methods for measuring xylem embolism due towater cavitation were compared—the acoustic method, thehydraulic method and the anatomical method. Young plants ofCeratonia siliqua L. were water stressed for 9, 16 and 23 d. Xylem cavitation was detected by counting ultrasound (100–300kHz) acoustic emissions (AE) from 1-year-old twigs (acousticmethod). Xylem embolism was detected by measuring the loss ofhydraulic conductivity of twigs of the same age (hydraulic method).The blockage of single xylem conduits was detected by perfusingSafranin into the xylem of 1-year-old twigs of stressed plantsand measuring the number and the diameters of non-conductingxylem conduits, under the microscope (anatomical method). It was noted that: (a) the number of AE and the loss of conductivityincreased with the water stress applied; (b) a linear relationseemed to exist between the number of AE and the loss of conductivity,suggesting that the AE counted could be only (or mainly) producedin the xylem conduits; (c) the vulnerability of the xylem conduitsto embolism was a direct function of their diameter; and (d)the measured loss of conductivity was of the same order of magnitudeas the theoretical one. The three methods gave fairly similar results. Nonetheless,they are not alternative to one another in that: (a) the acousticmethod allows continuous recordings to be made but does notprovide information about the actual damage suffered by plants;(b) the hydraulic method is very informative but destructive;and (c) the anatomical method is very useful both in phytogeographicaland in genetic improvement studies. Ceratonia siliqua L, Carob tree, water stress, xylem embolism, acoustic method, hydraulic method, anatomical method     

Three Different Methods for Measuring Xylem Cavitation and Embolism: A Comparison

Three different methods for measuring xylem embolism due towater cavitation were compared—the acoustic method, thehydraulic method and the anatomical method. Young plants ofCeratonia siliqua L. were water stressed for 9, 16 and 23 d. Xylem cavitation was detected by counting ultrasound (100–300kHz) acoustic emissions (AE) from 1-year-old twigs (acousticmethod). Xylem embolism was detected by measuring the loss ofhydraulic conductivity of twigs of the same age (hydraulic method).The blockage of single xylem conduits was detected by perfusingSafranin into the xylem of 1-year-old twigs of stressed plantsand measuring the number and the diameters of non-conductingxylem conduits, under the microscope (anatomical method). It was noted that: (a) the number of AE and the loss of conductivityincreased with the water stress applied; (b) a linear relationseemed to exist between the number of AE and the loss of conductivity,suggesting that the AE counted could be only (or mainly) producedin the xylem conduits; (c) the vulnerability of the xylem conduitsto embolism was a direct function of their diameter; and (d)the measured loss of conductivity was of the same order of magnitudeas the theoretical one. The three methods gave fairly similar results. Nonetheless,they are not alternative to one another in that: (a) the acousticmethod allows continuous recordings to be made but does notprovide information about the actual damage suffered by plants;(b) the hydraulic method is very informative but destructive;and (c) the anatomical method is very useful both in phytogcographicaland in genetic improvement studies. Ceratonia siliqua L., Carob tree, water stress, xylem embolism, acoustic method, hydraulic method, anatomical method     

Xylem Cavitation in Nodes and Internodes of Whole Chorisia insignis H.B. et K. Plants Subjected to Water Stress: Relations Between Xylem Conduit Size and Cavitation

Measurements of cavitation occurring in xylem conduits of differentstem parts in whole Chorisia insignis H.B. et. K. plants subjectedto water stress are reported. Pre-stressed plants were shownto undergo cavitation over 10 times greater than watered ones.The most vulnerable parts of plants were one-year-old twigswhere cavitation reached a peak of over 50 acoustic emissions(AE) min^–1 while in two-year-old twigs AE min^–1were about one half this value. Stem zones were found wherecavitation was typically very low even during water stress:these were one-year-old nodes and junctions where branches meet.Measurements of the inside diameters of xylem conduits and distributionof conduit ends in stem parts where AE were detected, showedthat nodes have a significantly larger percentage of narrowxylem conduits than internodes. Similar ‘constricted zones’were found injunctions with respect to two-year-old twigs. Hereabout 50 per cent of the xylem conduits were as narrow as 20to 50 µm in diameter. The distribution of xylem conduitends show about 3 per cent of them ending in the nodes and 1per cent in the internodes of one-year-old twigs. About 11.6per cent of xylem conduits end in the junctions and about ahalf in two-year-old internodes. Our data would give furtherexperimental evidence to the functional concept of ‘plantsegmentation’ into zones (internodes) more efficient inwater conduction, i.e. with wider xylem conduits but more vulnerableto cavitation and others (nodes and junctions) with oppositecharacteristics. Chorisia insignis, acoustic emissions, water stress, nodes, internodes, xylem conduit size, vessel ends     

Wood anatomy of some trees with diffuse- and ring-porous wood: Some functional and ecological interpretations

Abstract

The xylem conduit dimensions (i.e. their width and length) have been measured in 1-year-old internodes, nodes and node-to-petiole (N-P) junctions of three species with diffuse-porous wood, namely Ceratonia siliqua L., Laurus nobilis L. and Olea europaea L. as well as of three species with ring-porous wood, namely Quercus ilex L., Q. suber L. and Q. pubescens Willd‥ The xylem conduit diameter and length distributions have been related to the drought resistance strategies adopted by the six species. C. siliqua and Q. ilex (drought avoiding water spenders) showed the widest xylem conduits (each species within its characteristic pattern of wood anatomy). This is consistent with their high demand of efficient water transport to leaves. L. nobilis (drought avoiding water saver) showed relatively narrow xylem conduits, efficient enough, however, to assure water supply to leaves at the reduced transpiration rate exhibited by the species. O. europaea, Q. suber and Q. pubescens (drought tolerants) showed the narrowest xylem conduits but also the longest ones. The xylem system of C. siliqua and Q. ilex represented a good compromise between efficiency and safety of the water transport, the former as due to wide xylem conduits, the latter to the reduced xylem conduit length as well as to the strong «hydraulic constrictions» at their nodes and N-P junctions. The ecological interpretation of such hydraulic architecture is discussed.     

Stomata on the Primary Root of Ceratonia siliqua

Anomocytic stomatal complexes observed with light and scanningelectron microscopes on the primary roots of Ceratonia siliqua(carob tree) are described. Stomata are randomly distributedthroughout the surface of the root zones which possess maturevascular tissues, i.e. from the zone of root hairs to the transitionzone. Stomatal orientation does not follow that of the rootepidermal cells whilst their distribution does not seem to forma regular pattern. Ceratonia siliqua, primary root, stomata     

Relations between vulnerability to xylem embolism and xylem conduit dimensions in young trees of Quercus corris

Measurements of xylem conduit length and width and the distribution of xylem conduit ends were made in inter-nodes (I), nodes (N) and twig junctions (J) of 1-, 2- and 3-year-old twigs of plants of Quercus cerris L. Parallel measurements were also made of the loss of hydraulic conductivity of twigs subjected to pressure differentials across conduit pit membranes, equalling the leaf water potential at the turgor loss point. The loss of theoretical hydraulic conductivity was calculated as the ratio of i esivr⁴ (where r is the conduit radius) of the non-conducting conduits to that of all the conduits in the outermost wood ring of I, N and J. Stem zones such as 1-year-old nodes and junctions were localized with narrower and shorter xylem conduits and with higher percentages of conduit ends than internodes. Such ‘constricted zonesrsquo; were less vulnerable to embolism than internodes. Latewood conduits were consistently narrower, shorter and less vulnerable to embolism than earlywood ones. A positive relation therefore existed between conduit diameter and length and vulnerability to embolism. The overall vulnerability to embolism of Q. cerris plants is discussed in terms of xylem conduit width and length and of the distribution of conduit ends.     

Xylem conduction and cavitation in Hevea brasiliensis

Clones of Hevea were studied in an attempt to discover the reasonsfor differences in the hydraulic performance of xylem. Differencesbetween clones were determined, including hydraulic conductivityand conduit width and length distributions. However, it hasproved difficult to reconcile anatomical differences with physiologicalperformance for use in future plant breeding programmes. When leaf relative water content (RWC) had been reduced fromabout 95% to 85%, the hydraulic conductivity of petioles decreasedsharply to about 40% of the initial value. This value correspondedwith xylem sap tensions of 1.8–2.0 MPa. Acoustic detectionexperiments revealed that this reduction in hydraulic conductivitycoincided with the greatest occurrence of cavitation. It seemsinescapable that the reduction in hydraulic conductivity wascaused by embolization; thereafter gas bubbles blocked the flowof water inside many of the conduits. There was some indicationthat eventually such bubbles might be dissolved, because thehydraulic conductivity increased again if specimens were fullyrehydrated. Apparently, the incidence of cavitation coincides with the entryof gas bubbles via ultramicroscopic pores into the conduitsthrough the walls according to the air-seeding hypothesis. Whena petiolate leaf is tested in a pressure chamber it is impossibleto make satisfactory measurements of a balancing pressure beyondc. 1.8–2.0 MPa, because air bubbles, mixed with sap andescaping from the conduits, form a persistent froth. Xylem transportin Hevea seems to be disrupted relatively easily under waterstress which is a feature of other tropical species adaptedto rainforest–type environments Key words: Hevea, xylem, cavitation, conduit, hydraulic conductivity     

Vulnerability to cavitation of leaf minor veins: any impact on leaf gas exchange?

Vulnerability to cavitation of leaf minor veins and stems of Laurus nobilis L. was quantified together with that of leaflets, rachides and stems of Ceratonia siliqua L. during air‐dehydration of 3‐year‐old branches. Embolism was estimated by counting ultrasound acoustic emissions (UAE) and relating them to leaf water potential (Ψ_L). The threshold Ψ_L for cavitation was less negative in L. nobilis than in C. siliqua according to the known higher drought resistance of the latter species. Leaf minor vein cavitation was also quantified by infiltrating leaves with fluorescein at different dehydration levels and observing them under microscope. Distinct decreases in the functional integrity of minor veins were observed during leaf dehydration, with high correlation between the two variables. The relationship between leaf conductance to water vapour (g_L) and Ψ_L showed that stomata of L. nobilis closed in response to stem and not to leaf cavitation. However, in C. siliqua, g_L decreased in coincidence to the leaf cavitation threshold, which was, nevertheless, very close to that of the stem. The hypothesis that stem cavitation acts as a signal for stomatal closure was confirmed, while the same role for leaf cavitation remains an open problem.     

Use of centrifugal force in the study of xylem cavitation

Two methods were evaluated for using centrifugal force to measurethe occurrence of cavitation as a function of negative pressuresin xylem. The general protocol was to measure the hydraulicconductivity of xylem segments (stem or root pieces) beforeand after centring them on a centrifuge rotor and spinning themabout their long axis to generate negative xylem pressure. Thepercentage decrease in conductivity from the initial to finalmeasurement was used to quantify the embolism resulting fromcavitation during spinning. In one approach, segments were spunwith their ends exposed to air. This method could only be usedwhen xylem conduits were much shorter than the segment. Resultsfrom an angiosperm (Betula occidentalis) and a gymnosperm (AblesIasiocarpa) corresponded to previous observations of embolismcaused by air dehydration where negative pressure was measuredwith the pressure chamber. Results also agreed with embolismcaused by injection of air into the xylem, in support of theair-seeding hypothesis for cavitation. In a second approach,segments were spun in a rotor designed to keep the segment endsimmersed in water during spinning. This gave the same resultsas for non-immersed segments. Immersing the segment ends allowedmeasurements on any material, regardless of conduit length,as demonstrated for roots of B. occidentalis. The chief advantageof the centrifugal force method is the rapidity and precisionwith which any desired xylem pressure can be imposed. Key words: Cavitation, embolism, drought stress, water relations, water transport     

不同冲洗液对毛白杨和油松枝条水力导度和抵抗空穴化能力测定值的影响

植物通过木质部管道系统进行水分运输, 木质部的水分运输效率和抗空穴化能力等水力结构特征对于植物物种的分布、抗逆能力等方面起关键性作用。目前, 国内外学者一般采用“冲洗法”进行木质部水力结构研究, 然而在该方法中使用的不同冲洗溶质可能对植物木质部水力结构等产生较大影响, 因此该文研究了3种溶质的冲洗溶液对毛白杨(Populus tomentosa)和油松(Pinus tabulaeformis)枝条的水力导度和抵抗空穴化能力的影响。实验结果表明: 相对于去离子水, 用0.01 mol·L^-1的草酸和0.03 mol·L^-1KCl溶液作为冲洗溶液, 均导致毛白杨木质部导管和油松管胞的水力导度测定值的增大。KCl导致毛白杨和油松木质部抵抗空穴化能力测定值的提高, 草酸导致杨树抵抗空穴化能力测定值增强, 但导致油松抗空穴化能力显著(p<0.01)减弱。小枝水平上, 毛白杨和油松的水分运输效率和抗空穴化能力之间没有显著相关性。另外, 在截枝实验中发现, 毛白杨小枝木质部水力导度随长度增加变化不大, 而油松枝条的木质部水力导度有逐渐增大的趋势。以上的实验结果表明不同溶质下毛白杨和油松枝条的木质部水力导度和抵抗空穴化能力不同, 草酸和KCl可能对木质部管道系统及纹孔处的果胶等产生作用, 从而使毛白杨和油松的水力结构发生变化。毛白杨与油松水力结构在去离子水、草酸和KCl的作用下的不同结果及两物种截枝试验下水力导度的不同变化趋势表明, 导管运输系统和管胞运输系统可能具有不同的水分运输影响因素。     

Water storage in the wood and xylem cavitation in 1-year-old twigs of Populus deltoides Bartr.

The possible role of water expelled from cavitated xylem conduits in the rehydration of water-stressed leaves has been studied in one-year-old twigs of populus deltoides Bartr. Twigs were dehydrated in air. At desired values of leaf water potential (Ψ_l) (between near full turgor and -1.62 MPa), twigs were placed in black plastic bags for 1–2h. Leaf water content was measured every 3–5 min before bagging and every 10 min in the dark. Hydraulic conductivity and xylem cavitation were measured both in the open and in the dark. Cavitation was monitored as ultrasound acoustic emissions (AE). A critical Ψ_l value of -0.96 MPa was found, at which AE increased significantly while the leaf water deficit decreased by gain of water. Since the twigs were no longer attached to roots, it was concluded that water expelled from cavitated xylem conduits was transported to the leaves, thus contributing to their rehydration. Xylem cavitation is discussed in terms of a ‘leaf water deficit buffer mechanism’, under not very severe water stress conditions.     

A phi Layer in Roots of Ceratonia siliqua L

The central cylinder of the primary root of the carob tree (Ceratonia siliqua) is encircled by a layer of cells with wall thickenings, known as a phi (φ) cell layer. The development of the φ layer and the chemical composition of the cell wall thickenings have been studied in roots of C. siliqua. The results reveal the presence of condensed tannins in the mature phi thickenings and that the development of the φ layer is asynchronous: at 0–1 cm from the root tip φ thickenings appear before endodermis differentiation at the sites opposite phloem, at 1–4 cm new φ thickenings are developed at the sites opposite xylem, at 4–7 cm the φ layer consists of two layers of cells and it completely encloses the central cylinder.     

Xylem Hydraulic Characteristics, Water Relations and Wood Anatomy of the Resurrection PlantMyrothamnus flabellifoliusWelw.

Myrothamnus flabellifoliusWelw. is a desiccation-tolerant (‘resurrection’)plant with a woody stem. Xylem vessels are narrow (14 µmmean diameter) and perforation plates are reticulate. This leadsto specific and leaf specific hydraulic conductivities thatare amongst the lowest recorded for angiosperms (k_s0.87 kg m^-1MPa^-1s^-1;k_l3.28x10^-5kg m^-1MPa^-1s^-1, stem diameter 3 mm). Hydraulic conductivitiesdecrease with increasing pressure gradient. Transpiration ratesin well watered plants were moderate to low, generating xylemwater potentials of -1 to -2 MPa. Acoustic emissions indicatedextensive cavitation events that were initiated at xylem waterpotentials of -2 to -3 MPa. The desiccation-tolerant natureof the tissue permits this species to survive this interruptionof the water supply. On rewatering the roots pressures thatwere developed were low (2.4 kPa). However capillary forceswere demonstrated to be adequate to account for the refillingof xylem vessels and re-establishment of hydraulic continuityeven when water was under a tension of -8 kPa. During dehydrationand rehydration cycles stems showed considerable shrinking andswelling. Unusual knob-like structures of unknown chemical compositionwere observed on the outer surface of xylem vessels. These maybe related to the ability of the stem to withstand the mechanicalstresses associated with this shrinkage and swelling.Copyright1998 Annals of Botany Company cavitation, desiccation, hydraulic conductivity, refilling, resurrection plant, root pressure, xylem anatomy,Myrothamnus flabellifolius     

The relationship between xylem conduit diameter and cavitation caused by freezing

The centrifuge method for measuring the resistance of xylem to cavitation by water stress was modified to also account for any additional cavitation that might occur from a freeze-thaw cycle. A strong correlation was found between cavitation by freezing and mean conduit diameter. On the one extreme, a tracheid-bearing conifer and diffuse-porous angiosperms with small-diameter vessels (mean diameter <30 μm) showed no freezing-induced cavitation under modest water stress (xylem pressure = −0.5 MPa), whereas species with larger diameter vessels (mean >40 μm) were nearly completely cavitated under the same conditions. Species with intermediate mean diameters (30–40 μm) showed partial cavitation by freezing. These results are consistent with a critical diameter of 44 μm at or above which cavitation would occur by a freeze–thaw cycle at −0.5 MPa. As expected, vulnerability to cavitation by freezing was correlated with the hydraulic conductivity per stem transverse area. The results confirm and extend previous reports that small-diameter conduits are relatively resistant to cavitation by freezing. It appears that the centrifuge method, modified to include freeze–thaw cycles, may be useful in separating the interactive effects of xylem pressure and freezing on cavitation.     

The Vascular System of Maize Stems Revisited: Implications for Water Transport and Xylem Safety

The plexus of vascular bundles in the nodes of grasses is notoriouslycomplex, where long axial bundles pass through a network oftransverse bundles. The xylem pathways for water in maize stemshave been investigated anatomically and with dye and particulatetracers, revealing some of the details of this complexity. Onlyapprox. 3% of axial vessels pass through nodes without beinginterrupted by end walls. Axial bundles at nodes differ fromthose in internodes in having the metaxylem and protoxylem vesselsconnected by small tracheary elements. So it is only at nodesthat exchange of sap occurs between the large vessels withina bundle. End walls, acting as filters for particles and gasbubbles, always separate axial vessels from vessels in transversebundles. The high redundancy of bundle connections in the nodalplexus is interpreted as providing alternative water pathwaysto bypass embolisms and damaged or diseased sections of thexylem. The pores in the filters at the base of nodes and betweenaxial and transverse vessels within nodes are <20 nm in diameter.Where axial vessels connect to transverse vessels, a varietyof unusual shapes of vessel elements mediate two- and three-wayconnections within the plexus.Copyright 2000 Annals of BotanyCompany Zea mays, cryoSEM, maize, node, pits, pit membranes, vessel ends, vessels, xylem embolism, xylem pathogens     

Acclimation to Drought in Acer pseudoplatanus L. (Sycamore) Seedlings

A glasshouse experiment was conducted with well-watered andwater-stressed seedlings of sycamore (Acer pseudoplatanus L.)grown in soil columns. Water was withheld when the seedlingswere 82-d-old. Effects of soil drying on stomatal behaviour,water relations, xylem cavitation, and growth of leaves androots were evaluated. Stomatal conductance declined well before any observable changein bulk leaf water potentials, and was correlated with soilwater status. At seven weeks, osmotic potential had declinedby 0·51 MPa and 0·44 MPa at full and zero turgor,respectively. Drought significantly increased both bulk elasticmodulus and leaf dry weight to turgid weight ratio of water-stressedplants. Drought had no effect on relative water content at zeroturgor. Water cavitation in the xylem was detected as ultrasonic acousticemissions (AE). Water-stressed plants displayed significantlyhigher rates of AE than well-watered plants. Maximum rate ofAE coincided with the minimum level of stomatal conductanceand apparent rehydration of the leaves. Drought caused changes in the root distribution profile andit increased the root weight. The increase in root weight wasmainly due to a substantial shift in assimilates allocated infavour of roots with total biomass being unaffected. Leaf growthwas maintained for six weeks without any significant declinein expansion rate. However, the development of severe waterstress reduced both leaf production and expansion.     

Vulnerability of xylem to embolism in relation to leaf water potential and stomatal conductance in Fagus sylvatica f. purpurea and Populus balsamifera

The vulnerability of xylem vessels to water stress-induced cavitationwas studied by measuring hydraulic conductivity and ultrasoundacoustic emissions (AEs) in Fagus sylvatica L. f. purpurea (Ait.)Schneid. and Populus balsamifera L. The occurrence of xylemembolism in summer was investigated in relation to leaf waterpotential and stomatal conductance. Populus was extremely vulnerableto cavitation, losing functional vessels due to embolism atwater potentials lower than –0.7 MPa. Fagus experiencedembolism when water potential fell below –1.9 MPa. Middaywater potentials often approached these threshold values. Whenevaporative demand increased rapidly on sunny days, water lossbecame limited by low stomatal conductance. Thus water potentialsfell only slightly below the threshold values inducing cavitation.Despite the differences in vulnerability, both species tolerateda similar embolism rate of about 10% in the summer. There wasno embolism reversal during prolonged periods of rain. AEs werepredictive of loss in hydraulic conductivity, indicating thatAEs were mainly confined to vessels. Finally, vessel lengthdistribution, vessel diameter (tangential axis), vessel density,and vessel wall thickness had been determined for both speciesinvestigated. Populus had longer and wider vessels than Fagus,whereas vessel wall thickness was similar in both species. Key words: Acoustic emissions, Fagus, Populus, stomatal closure, xylem embolism     

Lenticel hypertrophy in shoot cultures of Ceratonia siliqua L.

Numerous white surface proliferations appeared in cultures of Ceratonia siliqua L. grown three to four weeks on medium containing 0.5 mg l^-1 BA and 0.1 mg l^-1 IBA. It was histologically confirmed that these proliferations were hypertrophied lenticels. Proliferations appeared first at the basal shoot internode and gradually spread acropetally, covering eventually the whole shoot except the uppermost internodes. Increase of BA concentration in the medium increased both the number of hypertrophied lenticels per shoot and the shoot multiplication index.Abbreviations BA 6-benzylamino-purine - IBA indole-3-butyric acid Dubravka Bojovi-Cveti deceased July 8, 1991.     

Structure-function constraints of tracheid-based xylem: a comparison of conifers and ferns

The ferns comprise one of the most ancient tracheophytic plant lineages, and occupy habitats ranging from tundra to deserts and the equatorial tropics. Like their nearest relatives the conifers, modern ferns possess tracheid-based xylem but the structure-function relationships of fern xylem are poorly understood. Here, we sampled the fronds (megaphylls) of 16 species across the fern phylogeny, and examined the relationships among hydraulic transport, drought-induced cavitation resistance, the xylem anatomy of the stipe, and the gas-exchange response of the pinnae. For comparison, the results are presented alongside a similar suite of conifer data. Fern xylem is as resistant to cavitation as conifer xylem, but exhibits none of the hydraulic or structural trade-offs associated with resistance to cavitation. On a conduit diameter basis, fern xylem can exhibit greater hydraulic efficiency than conifer and angiosperm xylem. In ferns, wide and long tracheids compensate in part for the lack of secondary xylem and allow ferns to exhibit transport rates on a par with those of conifers. We suspect that it is the arrangement of the primary xylem, in addition to the intrinsic traits of the conduits themselves, that may help explain the broad range of cavitation resistance in ferns.     

Production and secretion of {alpha}-galactosidase and endo-{beta}-mannanase by carob (Ceratonia siliqua L.) endosperm protoplasts

Viable protoplasts were isolated for the first time from maturecarob (Ceratonia siliqua L.) endosperm tissue. After 5 d ofincubation 75% of the protoplasts were viable. During incubationthey underwent vacuolation and produced the carob endospermhydrolases, agalactosidase and endo-ß-mannanase, whichwere secreted in the incubation medium. The secretion of bothenzymes were under Ca²⁺ control. Many characteristics of -galactosidaseand endo-ß-mannanase production by protoplasts werethe same as those of whole endosperms: their production didnot require any hormonal signal and was inhibited in the presenceof ABA or the leachate from the carob endosperm/seed coat. Moderatewater stress (—2.0 MPa) neither affected the activityof these hydrolases nor their secretion by endosperm protoplast.However, when the osmoticum of protoplast incubation mediumwas higher, the production and secretion of both hydrolaseswere reduced. Comparison of the hydrolases activities in theincubation media of leached carob endosperms, which were incubatedunder normal and water stress (—1.5 MPa) conditions, withthe activities of the protoplast-secreted hydrolases indicatedthat (i) carob endosperm cell wall acts as a barrier for thesecreted enzymes and (ii) that water stress reduces the cellwall porosity of the carob endosperm cells, and thus the releaseof the secreted -galactosidase and endo-ß-mannanaseis inhibited. The isolation of carob endosperm protoplasts offersa potent experimental system for the study of aspects of endospermcell physiology, such as enzyme secretion Key words: Abscisic acid, carob endosperm, Ceratonia siliqua L, endo-ß-mannanase, -galactosidase, leachate, protoplasts, water stress