Guralnick, Lonnie J.

Hanscom, Zac

Rorabaugh, Patricia A.

English

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Guralnick LJ, PA Rorabaugh, Z Hanscom (1984) Influence of photoperiod and leaf age on Crassulacean acid metabolism in Portulacaria afra (L.) Jacq.

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Roger Williams UniversityDOCS@RWUFeinstein College of Arts & Sciences FacultyPublications Feinstein College of Arts and Sciences1984Guralnick LJ, PA Rorabaugh, Z Hanscom (1984)Influence of photoperiod and leaf age onCrassulacean acid metabolism in Portulacaria afra(L.) Jacq.Lonnie J. GuralnickRoger Williams University, lguralnick@rwu.eduPatricia A. RorabaughZac HanscomFollow this and additional works at: http://docs.rwu.edu/fcas_fpPart of the Plant Sciences CommonsThis Article is brought to you for free and open access by the Feinstein College of Arts and Sciences at DOCS@RWU. It has been accepted forinclusion in Feinstein College of Arts & Sciences Faculty Publications by an authorized administrator of DOCS@RWU. For more information, pleasecontact mwu@rwu.edu.Recommended CitationGuralnick LJ, Patricia A. Rorabaugh, Zac Hanscom. 1984. "fluence of photoperiod and leaf age on Crassulacean acid metabolism inPortulacaria afra (L.) Jacq." Plant Physiology 74:454-457Plant Physiol. (1984) 75, 4544570032-0889/84/75/0454/04/$01.00/0Influence of Photoperiod and Leaf Age on Crassulacean AcidMetabolism in Portulacaria afra (L.) Jacq.'Received for publication September 13, 1983 and in revised form January 15, 1984LONNIE J. GURALNICK, PATRICIA A. RORABAUGH, AND ZAC HANSCOM 1II*Department ofBotany and Plant Sciences, University ofCalifornia, Riverside, Califomia 92551 (L. J. G.);Plant Science Department, Utah State University, Logan, Utah 84322 (P. A. R); and Department ofBiology, San Diego State University, San Diego, California 92182 (Z. H.)ABSTRACTThe possibility that ssace ad metabolism (CAM) is subjectto long day pbotoperiodic control in Portudacaria afia (L.) Jacq., afaculative CAM plat, was studed. Periodic measurements of 14CO2uptake, stomatal resist e, and titratable acidity were made on plantsexposed to long and short day photoperiods. Results indictes that water-strsed P. 4J1 had primly notura CO2 uptake, daytime stomatalclosre, and a brge diurnal acid fluctatiod in either photoperiod. Maturelaf tssue from nwessed pats der klog days exhibited a moderatediunl acid fintion ad midday stomatal closure. Under short days,there was a reduced diunl acd fltation in mature leaf tissue. Youngleaftsme taken from essed pints did not utilize theCAM pathwayunder either photoperiod as indicated by daytine CO2 uptalke lack ofdiWnal acid fl an, d incomplete daytie stomatal closure.The induction of CAM in P. afra appears to be related to the waterstatus of the plat and the age of the laf tissue. The photsyntheticmetabolism of mature leaves may be partly under the contol of waterstress and of photoperiod, where CAM is favored under long days.Photoperiodic induction ofCAM was first reported for Kalan-choe blossfeldiana (3), which under a LD photoperiod exhibiteda C3 mode of photosynthesis. With an increasing number of SD,there was increased nocturnal CO2 uptake. Subsequent studiesof K. blossfeldiana showed that malic acid and the enzymesassociated with CAM increased in activity after a switch to a SDphotoperiod (1, 9). K blossfeldiana undergoes a winter droughtin its natural habitat; thus, it is hypothesized that induction ofCAM by SD constitutes preparation of a metabolic mechanismto withstand this drought (2). There is still very little informationon the influence ofphotoperiod in other facultative CAM plants.Portulacaria afra is a faculative CAM species which respondsto water stress by switching from C3 photosynthesis to CAM (5,1 1). Irrigated plants of P. afra exhibit daytime CO2 uptake andlittle diurnal organic acid fluctutation. Nighttime CO2 uptakeoccurs in conjunction with a large diurnal fluctuation of organicacids only when the plants are water- or salt-strsed. P. afra isendemic to South Africa in an area which is characterized by adistinct summer drought (Richard Cowling, personal commu-nication). Based on preliminary findings, we suggest that CAMis photoperiodically triggered in P. afra by LD. We report here aset of experiments testing the hypothesis that CAM may beinduced in P. afra by LD and short nights.' Supported by the Departments of Biology and Botany and theSystems Ecology Research Group at San Diego State UniversityMATERIAIS AND METHODSPlants. Portulacaria afra (L.) Jacq. clones were propagatedfrom a large potted plant growing in the San Diego State Uni-versity greenhouse. The shrub (2.5-3 m in height) was wateredbiweekly and fertilized monthly. Cuttings from the parent plantwere rooted in U.C.-2 potting mix and irrigted biweekly withdeionizedH20. After rooting, six nonstessed plants were wateredevery 3rd d to maintain high tissue water potentials, and sixstsed plants were watered every 6th d with one-halfthe amountofwater applied to the nonsressed group. Full-strength Hoaglandsolution was applied to all plants biweekly. Plants were trimmedto induce new seasonal growth at the beginning of the growingseason.Growth Chamber Experiments. To study the effect of photo-period, 12 randomly selected plants were transferred to a growthchamber and were allowed to adjust to a LD (15 h light/9 hdark) photoperiod for 2 months. Light was provided by fluores-cent (Sylvania F48T12-CV-VHO) and incandescent (100 w)lamps with a photosynthetic photon flux density, measuredbelow the top ofthe canopy, of 500 ,umol m-2 sr' (Li-Cor LI-185quantum meter). After analyses, the same plants were allowedto adjust to a SD (9 h light/ 15 h dark) photoperiod for 2 months.Daytime temperatures during each photoperiod regime weremaintained at 30°C and nighttime temperatures at 15°C. TheRH fluctuated between 65% during the day and 75% at night.Add Titrations. Six leaves per treatment were collected ran-domly, weighed, and placed on dry ice. The samples were lyoph-ilized and stored in a desiccator until assayed. The dried sampleswere ground to a fine powder with a mortar and pestle, and ahomogenate was made with 20 ml glass-distilled H20. Thehomogenate was titrated with 0.01 N KOH to a pH 7.0 endpoint.In addition, morning and evening organic acid levels of matureleaves from a large potted shrub growing in the San Diego StateUniversity greenhouse were assayed for a year.Gas Exchange Measurements. Stomatal reistance to watervapor was measured with an autoporometer (Li-Cor, Li-65). Theporometer was calibrated at several temperatures with knownresistances to obtain regression lines of reistance versus time.Values exceeding 90 s cm'i were assumed to indicate stomatalclosure (5). The sensor was placed on the abaxial surface ofattached leaves.'4C02 uptake was measured using the system of Oechel andMustafa (8). A single detached leafwas enclosed in a cuvette andexposed to 14CO2 for 45 s in the growth chamber. Exposed plantmaterial (0.168 cm2 leaf punch) was immediately harvested anddropped into a 1:1 mixture of cold phenethylamine:methanol.After 48 h, the plant sample was washed with 1 ml of ethyleneglycol monoethyl ether (cellosolve) and then dried for 24 h at80°C, weighed, and combusted. Radioactivity of the phenethyl-454 www.plantphysiol.orgon October 26, 2017 - Published by Downloaded from Copyright © 1984 American Society of Plant Biologists. All rights reserved.PHOTOPERIOD AND LEAF AGE IN PORTULACARIA4(2(-l(JUNE SEPT. DEC.1980MONTH OF YEARMAR.1981FIG. 1. Accumulation of titratable acidity for P. afra from June 1980to June 1981. Each datum was calculated from a minimum of 12 aciditydeterminations, six each at sunrise and sunset.3003Ui.I-oW0ZLJ;: 200-JUI-j< 100!-SD'L_ -_ _6 12 18 24 6HOUR OF DAYFIG. 2. Diurnal variation in titratable acidity of mature, nonstesedP. afra leaves grown under a LD (@ -) or SD (O---O) photoperiod.Night period indicated (LD) or (SD). Each point represents themean of six determinations ± SE.amine:methanol, cellosolve, and combusted plant tissue wasmeasured with liquid scintillation methods, pooled, and used toderive '4CO2 rates for one side of leaf tissue (8).RESULTSCAM is usually characterized by a minimum of 100 &eq acidfluctuation from morning to evening (7). Thus, a rate of 12.5teq h-' (for an 8-h d) would be an indicator of CAM activity.The change in titratable acidity for P. afra decreased from over40 in late July to below 10 peq h-I in December, and remainedlow throughout much of the following spring (Fig. 1). On thebasis ofthese results, we attempted to determine if the change incarbon metabolism was related to photoperiod.Mature, Nonstressed Leaves. Irrigated plants which weregrown under a LD (15 h light/9 h dark) photoperiod for 2months exhibited a moderate 100 Neq g-' FW2 fluctuation oforganic acids (Fig. 2). Minimum stomatal reistance values on2Abbreviation: FW, fresh weight.the order of 2 to 10 s cm-' were noted at the end of the darkperiod and beginning of the light period (Fig. 3). Completestomatal closure was observed through the middle of the lightperiod. Stomatal resistance decreased during the latter part ofthe light period. Maximum 'CO2 uptake in nonstesd P. afraoccurred at the begnning of the light period and at the end ofthe dark period (Fig. 4). Low levels of '4CO uptake were seenduring the remainder of the dark period and again at the end ofthe light period.Aftergrowth for 30 d undera 9-h light/15-h dark photoperiod,there was a reduced organic acid fluctuation ('50 jseq g-' FW)from that observed under LD (Fig. 2). Stomatal resistance wasminimal at the beginning of the light period and at the end ofthe dark period (Fig 3). In addition, stomatal resistance increasedthroughout most of the light period. The stomatal openingobserved during the dark period was not accompanied by appre-ciable '4C02 uptake (Fig. 4). The major peaks of '4CO2 uptakein the SD plants occurred during the early portion of the lightperiod and at the very end ofthe 15-h dark period.Maetur, Strssed Leaves. Titratable acidity of water-stressedP. afra grown under a LD photoperiod underwent a large 250peq g7' FW fluctuation (Fig. 5). Stomatal reistance was high6 12 18 24 6HOUR OF DAYFIG. 3. Diurnal variation in stomatal resistance of mature, non-strssed P. afra leaves grown under a LD (@-) or SD (O---O)photoperiod. Each point repesents the mean of three determinations +SE.c3-ELDLi2I-e ;D~~~~S6 12 18 24 6HOUR OF DAYFIG. 4. Time course of '4C02 uptake of mature, nonstrssed P. afraleaves grown under a LD (@-*) or SD (O---O) photoperiod. Eachpoint repesents the mean of six determinations SE.1.I0crm4w49I I * I I -1 I00* 0. 00~~~~~~~~~~~0..* 0~~~0- .0o 0s | | ^ | ffi |0 00 000 0O 0455I I I I www.plantphysiol.orgon October 26, 2017 - Published by Downloaded from Copyright © 1984 American Society of Plant Biologists. All rights reserved.Plant Physiol. Vol. 75, 1984300R.,T>rIaLJ-icoHr0oo[loo0SDt LD-6 12 18 24 6HOUR OF DAYFIG. 5. Diurnal variation in titratable acidity of mature, water-sressed P. afra leaves grown under a LD (@-4) or SD (O---O)photoperiod. Each period represents the mean of six determinations ±SE.S9O-0-____LDEw I~~~~~SL60z I30-0SDt- --LD*6 1 2 18 246HOUR OF DAYFIG. 6. Diurnal variation in stomatal resistance of mature, water-stressed P. afra leaves grown under a LD ( -)or SD (O---O)photoperiod. Each point represents the mean of three determinations±SE.during the light period, with a slight decrease at the beginningand the end of the light period (Fig. 6). Stomatal resistancegradually decreased during the dark period with minimum valuesoccurring near the end ofthe dark period. I4C02 uptake occurredat the beginning of the light period, was low during the middleof the day, and began to rise during the latter part of the day(Fig. 7). Low rates of 4C02 uptake were observed throughoutthe dark period.After growth for 30 d under a SD photoperiod, the acidityresults were only slightly different from those of plants grownunder a LD photoperiod (Fig. 5). Stomatal resistance patternswere only slightly different under the SD photoperiod relative tothat observed under LD (Fig. 6). Low levels of '4C02 uptakeoccurred during the light period and increased to moderate levelsthroughout the dark period (Fig. 7).Young, Nonstressed Leaves. Fluctuations in titratable acidityof young, nonstressed P. afra leaves were markedly differentfrom those seen in mature leaves (Fig. 8). Acid levels remainedquite high throughout the day and night, and the fluctuationobserved was minor. Stomatal resistances were moderately lowN3-EELJ2 %LLSDL* LD ,6 12 HOUR OF 24 6FIG. 7. Time course of '4CO2 uptake ofmature, water-stressed P. afraleaves grown under a LD ( -) or SD (O---O) photoperiod. Eachpoint represents the mean of six deteminations ± SE.300_LDLA-2100EH,6 12 18 24 6HOUR OF DAYFIG. 8. Diurnal variation in titratable acidity of young P. afra leavesgrown under a ID (@-) or SD (O-O) photoperiod. Each pointrepresents the mean of six determinations + SE.throughout the 24-h cycle, with minimal levels observed at thebeginning ofthe light period and near the end ofthe dark period(Fig. 9). Low rates of 4C02 uptake occurred throughout the 24-h cycle, with peaks at the beginning of the light period, near theend ofthe light period, and near the end ofthe dark period (Fig.10). Titratable acidity fluctuation and stomatal rsstances inyoung nonstressed leaves, after 66 d of growth under a SDphotoperiod, were similar to those of plants grown under a LDphotoperiod (Figs. 8 and 9). After 66 SD, daytime 14C02 uptakewas still greater than nighttime uptake, though low levels ofuptake occurred throughout the dark period (Fig. 10).DISCUSSIONNonstressed P. afra showed low organic acid fluctuation whencompared to strssed plants (Figs. 2 and 5). These results aresimilar to those reported earlier for P. afra (11). Nonstressedplants underwent a moderate acid fluctuation during LD pho-toperiods, indicating some CAM activity. With an increasingnumber of SD, there is a reduction in the magnitude ofthe acidfluctuation. Nocturnal stomatal opening occurs during bothphotoperiod regimes, but there is a slight reduction of nocturnal'4C02 uptake during SD, an indication ofreduced CAM activity.Ii456 GURALNICK ET AL. www.plantphysiol.orgon October 26, 2017 - Published by Downloaded from Copyright © 1984 American Society of Plant Biologists. All rights reserved.PHOTOPERIOD AND LEAF AGE IN PORTUL,ACARIA)90ELiiZ 60nLii-JI-< 3006SDL- - DL- i_24 6DAYFIG. 9. Diurnal variation in stomatal reisstance of young P. afraleaves grown under a LD (-4*) or SD (O--- ) photoperiod. Eachpoint represents the mean of three determinations ± SE.E0'Y. 2LIJ~ ~ ~ ~6 12 18 24 6HOUR OF DAYFIG. 10. Time course of 14CO2 uptake of young P. afra leaves grownunder a LD (e-@) or SD (O---O) photoperiod. Each point repre-sents the mean of six determinations ± SE.Differences between LD and SD plants are even less pro-nounced in young leaves. The magnitude of the acid fluctuationwas not sufficiently large to consider the leaves to have switchedto CAM. The gas exchange data collected from young plants areindicative of C3 mode of photosynthesis, with '4CO2 uptakeoccurring primarily during the day. One notable difference be-tween young and mature leaves is that stomatal closure in youngleaves is not as complete as in mature leaves. Similar results werereported by Jones (6) whose results indicated that gas exchangepatterns in young Bryophyllumfedtschenkoi leaves differed fromolder leaves. This pattern of low daytime stomatal resistance hasalso been observed in young Opuntia basilaris stems (4). It thusappears that young tissues in general, when not stressed, exhibitlower stomatal resistance than do mature tissues.Data reported here support the contention that baseline acidlevels are related to leaf age. Young leaves maintain high acidlevels when not fluctuating, whereas older leaves maintain rela-tively lower acid levels when not fluctuating. P. afra maintainshigh organic acid levels even when the C3 photosynthetic pathwayis operating (10). Other facultative CAM plants do not share thisfeature (7). We assume that acid fluctuations commence in youngleaves when daytime stomatal closure occurs. The hypothesisthat daytime stomatal closure is a first response during CAMinduction is supported by Ting (10).Stressed plants had a gas exchange and acid fluctuation patterntypical ofCAM regardless of the photoperiod under which theywere grown. Plants which were not water-stressed and were grownunder a LD/short-night photoperiod had some characteristics ofCAM, including some nocturnal CO2 uptake and a moderatefluctuation of organic acids. Young leaf tissue did not displayany characteristics of CAM in either photoperiod. Our dataindicate that water stress overrides any effect of photoperiod inP. afra, while in well-watered plants the induction of CAM is amaturation process where CAM is favored under LD. In sum-mary, it appears that CAM in P. afra is induced by a combinationoffactors which include water stress, photoperiod, and leafaging.Thus, the original hypothesis which suggests that CAM is inducedin P. afra by a LD/short night photoperiod was only partiallysupported by our data.Acknowledgment-We would like to extend our thanks to Dr. Irwin P. Ting forhis help during the initial stages of the research and to Drs. Ken Johnson, JochenKummerow, and Michael Donoghue for their advice and input on the manuscript.Jim Malusa helped by doing numerous acid titrations, and Jan Lippert did thegraphics.LITERATURE CrITD1. BRULFERT J, D GUERRIER, 0 QUEIROZ 1975 Photoperiodism and enzymerhythms: kinetic characteistics of the photoperiodic induction of Crassula-cean acid metabolism. Planta 125: 33-442. BRULFERT J, D GUERRIER, 0 QUEIROZ 1982 Photoperiodism and Crassulaceanacid metabolism. II. Relations with leaf aging and photoperiod in Crassula-cean acid metabolism induction. Planta 154: 332-3383. GREGORY FG, I SPEAR, KV THIMANN 1954 The interrelation between CO2metabolism and photoperiodism in Kalanchoe. Plant Physiol 29: 220-2294. HANSCOM Z, IP TING 1977 Physiological responses to irrigation in Opuntiabasilaris Engelm & Bigel. Bot Gaz 138:159-1675. HANSCOM Z, IP TING 1978 Responses ofsucculents to plant water stress. PlantPhysiol 61: 327-3306. JONES MB 1975 The effect of leaf age on leaf resstance and CO2 exchange ofthe CAM plant Bryophyllumfedtschenkoi. Planta 123: 91-967. KLUGE M, IP TING 1978 Crassuln Acid Metabolism: Analysis of anEcological Adaptation. Ecological Studies, Vol 3. Springer-Verlang, NewYork8. OECHEL WC, J MUSTAFA 1979 Energy utilization and carbon metabolism inMediterranean scrub vegetation ofChile and California. H. The relationshipbetween photosynthesis and cover in chaparral evergreen shrubs. Oecologia41: 305-3159. QUEIROZ0 1965 Sur le metabolisme acide des Crassulacoes Action along termde la temperature de nuit sur la synthese d'acide malique par Kalanchoeblossfeldiana "Tom Thumb" place enjours courts. Physiol Veg 3: 203-21310. TING IP 1981 Effects ofabscisic acid on CAM in Portulacaria afra. PhotosynthRes 2:39-481 1. Ting IP, Z Hanscom 1977 Induction of acid metabolism in Ponulacaria afra.Plant Physiol 59: 511-514457TSD'-~1,A- www.plantphysiol.orgon October 26, 2017 - Published by Downloaded from Copyright © 1984 American Society of Plant Biologists. All rights reserved.

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Guralnick LJ, PA Rorabaugh, Z Hanscom (1984) Influence of photoperiod and leaf age on Crassulacean acid metabolism in Portulacaria afra (L.) Jacq.

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