Photosynthetic acclimation to light gradients

in Typha latifolia

Emil Arboe Jespersen – 20105855

60 ECTS speciale

Vejleder: Brian K. Sorrell

Institut for Bioscience Aarhus Universitet

Marts 2016

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Indholdsfortegnelse:

Forord................................................................................................................................... s. 3

Abstract................................................................................................................................ s. 4

Resumé.................................................................................................................................. s. 5

Introduktion

- 1.1 Lys........................................................................................................................ s. 6

- 1.2 Lysresponskurven og akklimering....................................................................... s. 7

Bladakklimering

- 2.1 Kutikulaen........................................................................................................... s 10

- 2.2 Trikomere........................................................................................................... s. 10

- 2.3 Stomatadensitet.................................................................................................. s. 11

- 2.4 Bladvinkel og størrelse...................................................................................... s. 12

- 2.5 Bladtykkelse....................................................................................................... s. 13

- 2.6 Allokering på bladniveau................................................................................... s. 16

Kloroplastakklimering

- 3.1 Kloroplasten...................................................................................................... s. 17

- 3.2 Fotosystem 1 og 2.............................................................................................. s. 18

- 3.3 Xantofylcyklussen.............................................................................................. s. 19

- 3.4 Elektrontransport og Rubisco............................................................................ s. 20

- 3.5 Allokering på kloroplastniveau.......................................................................... s. 21

Artikelmanuskript

- Introduction............................................................................................................. s. 23

- Method and materials.............................................................................................. s. 24

- Results….................................................................................................................. s. 29

- Discussion................................................................................................................ s. 32

References........................................................................................................................... s. 35

Figures and Tables............................................................................................................. s. 43

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Forord

Specialet vil være inddelt i to overordnede afsnit, hvor det første afsnit er et litteraturstudie af

planters akklimering til lys. Dette afsnit er skrevet på dansk, mens det efterfølgende afsnit er

et artikelmanuskript til ”Functional plant biology”, og derfor på engelsk. Artikelmanuskriptet

er skrevet efter de normer der gælder for det pågældende tidsskrift.

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Abstract

Light acclimation has been widely studied in many different kinds of plants, especially in

broad leaved and herbaceous species. Plants respond to increased light by altering their

morphology; the cuticle becomes thicker, trichome and stomatal density increases, leaf size

decreases while its thickness increases and leaf angle compared to incident light changes.

Biochemical responses are also seen, as the pigment content is altered together with key

enzymes responsible for processing excitations energy. Finally, allocation patterns change at

the leaf and chloroplast level. The genus Typha poses a special case of light acclimation, as it

is native to freshwater wetlands, which are often nutrient rich and highly productive. It grows

from a basal meristem with erect leaves that reduce light attenuation in monospecific stands

compared to plants with horizontal leaves. The growth form and habitat therefor distinguishes

Typha from many of the broad leafed and herbaceous species. The objectives of this study

were therefore to investigate how Typha latifolia L. acclimates its photosynthetic metabolism

and pigment composition to the light availability in its canopy. This was done by growing T.

latifolia under two light intensities under controlled laboratory conditions and in a field study,

where two stands differing in light attenuation were compared. Comparisons were based on

gas exchange parameters, chlorophyll (chl) florescence, pigment content and morphology.

The light environment affected gas exchange, pigment content and morphology. The

laboratory study showed that T. latifolia could acclimate leaf morphology and photosynthesis

to high and low light with a “sun-shade” response similar to those seen in many other species.

In both studies, photosynthesis rates for T. latifolia in high light were very high for a C3 plant

(> 40 µmolCO2 m-2

s-1

). High photosynthesis rates and high light saturation requirements for

photosynthesis were maintained throughout the canopy in the field, consistent with relatively

low light attenuation by the linear-leaf growth form of this species. The xanthophyll (VAZ)

pool, which protects leaves against excess light, was extremely high in all T. latifolia in this

study, and as predicted was lower in plants subject to lower light intensities.. These data

confirm that in field conditions, T. latifolia is able to maintain high photosynthetic activity

throughout its canopy and avoid photoinhibition under high light conditions through high

investment in the VAZ pool. This pattern of acclimation is likely adaptive in the field as T.

latifolia is native to high light habitats, and explains why this species is so successful and

dominant in high-light, high-nutrient wetlands.

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Resumé

Lysakklimering har været undersøgt i mange forskellige slags planter, specielt i bredbladet

træer og urteagtige planter. Planter responderer på øget lys intensitet ved at ændre deres

morfologi; kutikulaen bliver tykkere, trikomer- og stomatadensiteten stiger, bladets areal

bliver større mens tykkelsen aftager og bladets vinkel i forhold til det indfaldne lys ændres.

Planter responderer også biokemisk ved at ændre pigmentindholdet samt mængden af

nøgleenzymer involveret i omsætningen af eksitationsenergi. Allokeringsmønsteret ændres

også både på blad og kloroplastniveau. Slægten Typha udgør et særtilfælde i forhold til

lysakklimering da den er hjemmehørende i ferskvandsvådområder, som oftest er næringsrige

og meget produktive. Slægten vokser via et basalt meristem og har oprejste blade, der giver

en lav lyssvækkelse i monospecifikke stande i forhold til planter med horisontale blade.

Vækstformen og habitattypen adskiller Typha fra mange bredbladet træer og urteagtige

planter. Formålet med dette studie var derfor at undersøge hvordan Typha latifolia L.

akklimerer dens fotosyntetiske metabolisme og pigmentsammensætning i forhold til den

tilgængelige mængde lys. Dette blev gjort ved at dyrke T. latifolia under kontrollerede

laboratorieforhold ved to lysintensiteter og derudover i et feltforsøg, hvor to stande med

forskellige lyssvækkelse blev sammenlignet. Sammenligningen var baseret på målinger af

gasudveksling, klorofylfluorescens, pigmentindhold og morfologi. Lysmiljøet påvirkede

gasudvekslingen, pigmentindholdet og morfologien. Laboratoriestudiet viste at T. latifolia

kunne akklimere bladmorfologi og fotosyntese (A) til høj og lav lysintensitet via en ”sol-

skygge” respons tilsvarende til, hvad man ser i mange andre arter. I begge studier var A

meget høj for en C3 plante (> 40 µmolCO2 m-2

s-1

). En høj A og et højt lysmætningspunkt (Ik)

blev opretholdt ned igennem løvtaget i felten, hvilket er i overenstemmelse med den lave

lyssvækkelse lineære blade skaber. Xantofylpuljen (VAZ puljen), som kan beskytte blade når

der er for meget lys, var ekstremt høj i alle T. latifolia i dette studie, og var som forventet

lavere i planter udsat for lavere lysintensiteter. Data bekræfter, at under feltforhold er T.

latifolia i stand til at opretholde en høj A ned igennem løvtaget og undgår fotoinhibering

under høj lysintensitet ved at investere i VAZ puljen. Dette akklimeringsmønster er

formodentligt adaptivt i felten da T. latifolia er hjemmehørende i habitater med høj

lysintensitet og forklarer, hvorfor denne art er så succesfuld og dominant i

ferskvandsvådområder med høj lysintensitet og næringsindhold.

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Introduktion

1.1 Lys

Af de mange faktorer der påvirker en organismes evne til at øge sin biomasse, har lys en

vigtig betydning for de fotoautotrofe organismer. For fotoautotrofe organismer, der omfatter

planter, alger og bakterier, er lys den primære energikilde når CO2 skal omdannes til

biomasse. Intensiteten af lys har en effekt på fotosynteseraten (A), hvilket ses ud fra en

lysresponskurve. I takt med at lysintensiteten stiger stiger A også. Ved lav lysintensitet er der

et lineært sammenhæng mellem lysintensitet og A. Dette sammenhæng aftager og flader ud,

hvis lysintensiteten foresætter med med at stige, og A kan til sidst begynde at falde, hvis

lysintesiteten forsat stiger. A er kun et øjebliksbillede af plantens CO2 optagelse og siger ikke

noget om tidslig variation hen over døgnet (Givnish 1988). Lysintensiteten et blad udsættes

for i løbet af dagen kan variere meget. Især lys med en høj intensitet over en kortvarig

periode skabt af bevægelser i blade over det pågældende blad bidrager til dette fænomen

(solpletter). Den tidslige variation kan kontrolleres ved brug af et klimakammer, hvor lysflux

og antal lystimer kan kontrolleres. Alternativt kan den totale daglige lysflux over dagen

måles, og her ud fra kan den totale A for perioden beregnes. A kan udtrykkes på baggrund af

forskellige parametre. Tre af disse er bladets areal(Aareal), bladets masse(Amasse) eller plantens

masse(Aplant). Alle tre parametre korrelerer med den relative vækstrate (RGR), dog findes den

bedste korrelation mellem Aplante og RGR (r2=0,93) (Kruger and Volin 2006). Andre studier

har fundet samme korrelation for Aareal og Amasse dog ikke for skyggeakklimerede planter. For

skyggeakklimerede planter korrelerede RGR bedst med en kombination af Amasse og bladets

vægtratio(LWR g blad g plante-1

) (Walters et al. 1993). LWR er tilsvarende bladets

masseratio (LMR). LMR vil blive brugt fremover. Kombinationen af Amasse og LMR svarer

til Aplante, dog afhænger dette af om ikke-fotosyntetiserende vævs respiration er medtaget.

Metoden brugt i Kruger and Volin (2006) og Walters et al. (1993) er ikke ens. Kruger and

Volin (2006) måler lysfluxen over døgnet og kombinerer dette med anden data, som er

indsamlet fra andre artikler. Walters et al. (1993) benytter sig af en konstant lysflux og antal

lystimer. Aplante påvirkes af forskellige faktorer. Da Aplante omfatter hele planten, vil forskellig

grad af allokering til forskellige organer have en effekt på Aplante. En høj grad af allokering til

fotosyntetiserende væv (eksempelvis blade) fremfor andet ikke fotosyntetiserende

(eksempelvis rødder), vil øge Aplante, mens en mindre grad af allokering vil have den

omvendte effekt. Udover øget allokering til blade vil ændringer i respirationsrater påvirke

Aplante.

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1.2 Lysresponskurven og akklimering

Når næringssalte ikke er en begrænsende faktor, kan en variabels effekt på RGR analyseres

via følgende formel

(Lambers et al. 2008) s. 322

Hvor Aa er fotosynteseraten per areal, SLA er det specifikke bladareal, LMR, SMR, RMR er

masseratioen af hhv. blade, stamme og rødder, LRm, SR og RR er massebaseret blad-,

stamme- og rodrespiration. Når en plante oplever at lysintensiteten øges eller sænkes, og

lysintensiteten bliver på det nye niveau over en længerevarende periode, initieres en

akklimeringsprocess. Denne proces har til formål at udnytte den givne mængde lys på en

sådan måde, at det hverken er skadeligt for planten, eller at fotoner ikke udnyttes optimalt

indenfor den pågældende genetisk isoleret populations rammer for akklimering (Bjorkman

and Holmgren 1963; Bjorkman 1968b). Om denne proces nødvendigvis er en fordel kan i

nogle situationer være uklar, afhængigt af hvordan parametrerne udtrykkes (Givnish 1988).

Bjorkman and Holmgren (1963); Bjorkman (1968b) brugte bla. en lysresponskurve til at

vurdere effekten af akklimering på det fotosyntetiske apparart. Han sammenlignede quantum

yield (Φ) og brutto A ved lysmætning (ABmax) i Solidago virgauera fra hhv. to skyggede og to

soleksponerede lokaliteter dyrket ved forskellige lysintensiteter.

Φ er den initiale hældning på en lysresponskurve og angiver, hvor effektivt det fotosyntetiske

apparart er til at udnytte eksitationsenergi fra fotonerne til optage CO2. Φ angives med

enheden µmol CO2 µmol fotoner-1

. Amax angives normalt som en nettorate (ANmax), men kan

også angives som en bruttorate (ABmax). Amax beskriver den lysmættede fotosyntetiske rate

bladet kan opnå når lys ikke længere begrænser fotosyntesen. Værdien angives normalt med

enheden µmol CO2 m-2

s-1

(Amax-area), men er også blevet angivet med andre enheder end m2.

Et eksempel er massebaseret A, hvor enheden bliver µmol CO2 g-1

s-1

(Amax-mass) . Måden Amax

bestemmes på er ikke endtydig, hvilket bla. skyldes at lysresponskurver tilpasses de

eksperimentielt bestemte målepunkter med forskellige ligninger (Lobo et al. 2013). En ofte

anvendt formel er

√( )

(Prioul and Chartier 1977)

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Fig 1: Angiver punkter eller regioner for de parameter som kan aflæses eller beregnes ud fra

lysreponskurven.

Udover Φ og Amax findes der andre parametre på en lysresponskurve, der beskriver AN som

funktion af lysintensiteten, som kan bruges til at vurdere akklimeringsgraden til forskellige

lysintensiteter. Ved en lysintensitet på 0 finder man på en lysresponskurve mørkerespiration

(Rd). Denne værdi er kun gældende for bladets respiration. En beregning af hele plantens

respiration, på baggrund af denne værdi, vil føre til en underestimering, da Rd i det ikke-

fotosyntetiserende væv ofte er højere end i bladet. Ved en lysintensitet, der tillader en høj nok

A til at modsvare Rd, findes lyskompensationspunktet (Ic). Ic findes ikke på en

lysresponskurve, der beskriver AB som funktion af lysintensitet. Konveksiteten (θ) beskriver

overgang fra den lysbegrænsede del af kurven, som er karakteriseret ved en stejl hældning, til

den CO2/Rubisco begrænsede del af lysresponskurven. I den CO2/Rubisco begrænsede del af

lysresponskurven er hældning aftagende. Efter denne overgang stabiliserer A sig og

førnævnte Amax nåes. Til at beskrive ved hvilken lysintensitet overgangen mellem den

lysbegrænsende og CO2/Rubisco begræsende del findes, bruges lysmætningspunktet (Isat eller

Isat(n%)). Dette punkt er skæringen mellem følgende to ligninger og

, men kan også angives som den mængde lys, der skal til at mætte fotosyntesen med n % i

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forhold til Amax (Lobo et al. 2013). Et alternativ til dette er at definere Ik som den værdi, hvor

A ikke stiger længere (Hopkins and Huner 2009) s. 216. Ik bestemt ved sidste defination vil i

forhold til den første metode overestimere værdien. Det er derfor vigtigt at vide hvilken

metode, der er brugt til bestemmelse af Ik, hvis en sammenligning på tværs af studier udføres.

Størrelsen og effekten af akklimering blandt genetisk isoleret populationer kan variere meget.

Denne forskel vurderes bla. ud fra forskelle på lysresponskurver og

kloroplastsammensætning mm. for en genetisk isoleret population dyrket under forskellige

lysintensiteter. Kapaciteten (forskellen mellem sol- og skygge akklimeret individer) til at

akklimere mentes at afspejle om den genetisk isoleret population bestod af planter adapteret

til høj eller lav lysintensitet (solplanter med høj kapacitet, skyggeplanter med lav kapacitet)

(Bjorkman 1968a; Boardman 1977). Det har sidenhen vist sig, at akklimeringskapaciteten i

højere grad afspejler variationer i lysmiljøet i populationens naturlige habitat sådan at

intermediære populationer, der både lever i sol og skygge, har den størst kapacitet til at

akklimere (Murchie and Horton 1997; Balaguer et al. 2001). Termerne sol- og skyggeplanter

bruges stadig om genetisk isoleret populationer adapteret til høj eller lav lysintensitet, men

også om individer akklimerer til høj og lav lysintensitet, hvorved der kan opstå tvetydighed.

Termerne solblade og skyggeblade bruges derimod kun om blade der er akklimeret til høj og

lav lysintensitet (Lambers et al. 2008) s. 26. Fremover vil termerne skygge- og solplanter

henvise til planter, der er er akklimeret til høj eller lav lysintensitet. Dette har dog ikke ændret

på at lysresponskurver er et vigtigt værktøj når akklimeringskapacitet og mønster skal

vurderes. Hvor god en isoleret genetisk population er til at akklimere er genetisk bestemt, og

der findes derfor en grænse i disse træk. Der findes f. eks. en nedre grænse for tykkelsen af

blade (Niinemets 2007). Afhængig af den genetisk isoleret population vil bladets tykkelse

kun i mindre grad ændres, når først det er færdigudviklet, modsat mængden af enzymer og

pigmenter som løbende vil kunne op og ned reguleres i det pågældende blad (Sims and

Pearcy 1992; Oguchi et al. 2003). I de efterfølgende afsnit vil forskellige

akklimeringensresponser i forhold til lysintensitet blive gennemgået. Først et afsnit

omhandlende aklimering på bladniveau og derefter et andet afsnit på kloroplastnievau. Fælles

for begge overordnede afsnit(blad- og kloroplastnieveau) er et underafsnit omhandlende

allokering til forskllige væv med fokus på fordelingen af nitrogen (N). N-allokering

behandlet i afsnittet ”Allokering på bladniveau” vil blive gennemgået i forhold til hele

planten.

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Bladakklimering

2.1 Kutikulaen

Kutikulaen hos planter har flere funktioner. Den er med til at mindske vandtabet fra bladet til

atmosfæren, men kutikulaen kan også mindske mængden af lys via sine reflektive

egenskaber. Dette gør at det kan være svært at adskille effekten på kutikulaen fra forskellige

abiotiske stressfaktorer. Reed and Tukey (1982) fandt at kutikulaen ændrer sig afhængigt af

både temperatur og lysintensitet, hvilket understreger kompleksiteten. Derudover er lys,

temperatur og fugtighed i en vis grad sammenhængdende, hvilket yderlige komplicerer dette.

Effekten af lysintensitet på kutikulaen vil blive gennemgået heri. Der henvises til Shepherd

and Griffiths (2006) for en mere detaljeret gennegang af effekten af temperatur og fugtighed.

Lys kan være skadeligt i for store mængder(Bjorkman 1981) og i særdeleshed kan UV være

skadeligt for planter, især UVB (Holmes and Keiller 2002). Ved at fjerne kutikulaen viste

Cameron (1970) at reflektansen faldt og A efterfølgende steg, da mere lys blev tilgængeligt.

At reflektansen stiger, når kutikulaens tykkelse øges, er dokumenteret af andre (Baltzer and

Thomas 2005). På baggrundt af dette kunne man forestille sig, at planter akklimeret til lysrige

habitater vil have en kutikula, der er tykkere og af en sådan sammensætning, at den

reflekterer mere lys for at undgå fotoinhibering. Flere studier har vist at kutikulaens tykkelse

øges i takt med stigende lysintensiteter(Osborn and Taylor 1990; Ashton and Berlyn 1994;

Baltzer and Thomas 2005; Shepherd and Griffiths 2006), mens Baker (1974) og Reed and

Tukey (1982) påviste en effekt i form af øget tykkelse men også ændret vokslag. I habitater

med begrænset lys kan kutikulaen reflektere lys og dermed begrænse mængden af lys, der er

tilgængelig for fotosyntesen, og reducere plantens vækstpotentiale. Under sådanne forhold vil

en relativ stor andel voks, i forhold til kutikulaens tykkelse, kombineret med en tynd cutin

(cuticular proper) være fordelagtigt. Årsagen er, at en tyk kutikula reflekterer mere lys

(Baltzer and Thomas 2005), mens vokslaget primært reflekterer UV lys (Holmes and Keiller

2002). Studier der undsøger kombinationen af lysintensitet og kvalitet i forhold til variation i

kutikulaen er sparsomme.

2.2 Trikomere

På kutikulaen findes der forskellige former for trikomere, som kan variere i densitet. Her gør

problematikken fra før sig igen gældende, da trikomerne har flere forskellige funktioner,

hvilket besværligegøre en klar og entydig fortolkning af deres effekt og funktion. En generel

observation er at densiteten af trikomere stiger i takt med stigende lysintensitet (Filella and

Penuelas 1999; Klich 2000; Tattini et al. 2000; Camarero et al. 2012; Laureau et al. 2015),

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dog med en enkelt undtagelse, hvor der ikke var nogen signifikant forskel (Pereira et al.

2009). Der er dog et problem med disse undersøgelse, hvilket er, at de er udført på forskellige

lokaliteter eller, at blade er taget fra toppen (solblade) og andre fra lavere højder

(skyggeblade) af samme plante. Dette skaber den mulighed, at andet end lysintensiten kan

variere og dermed være årsag til ændringer i trikomerdensiteten. Et studie har begrænset dette

problem og kommer stadig frem til, at trikomerdensiteten stiger med stigende lysintensitet

(Perez-Estrada et al. 2000). Førnævnte studier påviser også, at andre abiotiske faktorer (vand)

kan påvirke trikomerdensiteten, hvilket understreger problemet med førnævnte studier.

Effekten af trikomere er, at de reflekterer lys (Holmes and Keiller 2002). Under forhold hvor

der er overskydende lys i forhold til, hvad der bruges i fotosyntesen, kan trikomere bridrage

til at undgå fotoinhibering. Trikomere reflekterer lys i hele PAR området (Holmes and Keiller

2002), men de kan også absorbere UV, hvis de indeholder flavonoider (Liakopoulos et al.

2006). At blade er i stand til at reflektere lys og dermed undgå fotoinhibering er ikke den

eneste gavnlige effekt af trikomerne eller kutikulaen (afsnit 2.1). Temperaturen i aktivt

fotosyntetiserende blade kan være højere end den omgivende temperatur grundet

absorptionen af lys og den efterfølgende uundgåelige varmefrigivelse. Ved at reflektere lyset

mindskes varmebelastningen på vævet, hvilket kan nedsætte temperaturen og have en gavnlig

effekt på plantens vandbalance (Vogelmann 1993).

2.3 Stomata

På kutikulaen findes stomata hvorigennem hovedparten af gasudvekslingen foregår.

Åbningsgraden af stomata er under stærk kontrol for at undgå et unødvendigt stort vandtab

men stadig tillade at nok CO2 optages, sådan at CO2 og ribulose 1,5 bisphosphate (RuBP) co-

begrænser A (Farquhar and Sharkey 1982). Dette tyder på at stomatadensiteten og størrelsen

kan påvirkes af flere forskellige faktorer. Fokus her vil ligge på responsen på ændret

lysintensitet. I takt med stigende lysintensitet skal mere eksitationsenergi fra fotonerne

omsættes. Dette kan ske via flere forskellige mekanismer. Hvis eksitationsenergien fra

fotonerne anvendes til at reducere CO2, vil en højere lysintensitet kræve en højere influx af

CO2. Dette afhjælpes til dels af en større koncentrationsforskel i CO2 mellem bladet og

atmosfæren, og at stomata åbnes mere. Flere stomata per areal samt større stomata tillader en

større gasudveksling og kan dermed mindske den potentielle CO2 begræsning. Flere studier

viser at stomatadensiteten stiger i takt med stigende lysintensitet (Lichtenthaler et al. 1981;

Abrams and Kubiske 1990; Lee et al. 1996; Cao 2000; Mendes et al. 2001; Holscher 2004;

Matos et al. 2009; Brodribb and Jordan 2011; Carrion-Tacuri et al. 2011; Carins Murphy et

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al. 2012). Disse studier er dog feltforsøg, hvor de tidligere problemer i forbindelse med

kutikulaen og trikomere igen kan påvirke stomatadensiteten, dog viser et enkelt

labaratoriestudie tilsvarende resultater (Valladares et al. 2002). Andre studier har kun kunne

påvisse en tendens og ikke en signifikant forskel (Nardini et al. 2012). Den generelle respons

er altså at stomatadensiteten stiger i takt med stigende lysintensitet, selvom få studier har vist,

at det omvendte kan gøre sig gældende (James and Bell 2000). Årsagen til den omvendte

sammenhæng mellem lysintensitet og stomatadensitet kan evt. være forsaget af et stærkt

sammenhæng mellem vandtab og vandforsyning (Brodribb and Jordan 2011), eller at faldet i

densitet er blevet kompenseret for af større stomata, hvilket understreger kompleksiteten i

stomataplasticiteten og akklimeringen. Influxen af CO2 er også afhængig af størrelsen på

stomata, da større stomata kan åbnes mere og dermed tillade en højere flux. Ved at undersøge

densitet og størrelse interspecifikt fandt Hetherington and Woodward (2003) ud af, at

størrelsen på stomata faldt i takt med stigende densitet. Om dette også gør sig gældende

intraspecifikt i form af akklimering er mere uklart. Mindre stomata kan være fordelagtigt

under høj lysintensitet, da relativt små stomata teoretiske tillader højere influx af CO2 per

areal (Bidwell 1974) i (Abrams and Kubiske 1990). Små stomata giver derudover en

hurtigere stomatarespons og dermed bedre kontrol over gasudvekslingen (Drake et al. 2013).

Der er ikke noget entydigt billed på, at stomata bliver større eller mindre afhængig af

lysintensistet (Abrams and Kubiske 1990; Cao 2000; Valladares et al. 2002). Manglen på et

endtydigt billed kan skyldes, at andre faktorer har påvirket størrelsen af stomata i højere grad

end lysintensitet.

2.4 Bladets vinkel og størrelse

Bladets vinkel i forhold til det indfaldne lys har betydning for, hvordan lysintensiteten aftager

ned igennem et løvtag (Terashima and Hikosaka 1995). Lyset aftager i hht.

( ), hvor Ix er lysintensiteten i en højde svarende til et sammenlagt blad areal

indeks (LAI) på x, I0 er lysintensiteten af det indfaldne lys, K er ekstinktionskoefficienten og

Fx er summeret bladareal indtil en højde x. I takt med at bladet orienteres parallelt i forhold til

det indfaldne lys bliver ekstinktionskoefficienten lavere (lyset absorberes i mindre grad ned

igennem løvtaget) mens den for blade parallelt i forhold til det indfaldne går mod 1 (lyset

absorberes i højere grad ned igennem løvtaget) (Monsi and Saeki 2005). Dette skyldes at hvis

bladet er parallelt med det indfaldne lys, får bladet et lille projekteret areal, hvilket betyder at

en mindre mængde lys kan absorberes i forhold til, hvis bladet var parallelt og havde et stort

projekteret areal (Falster and Westoby 2003). Dette er kun gældende for løvtag med ens LAI,

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da LAI også påvirker lysabsorptionen ned igennem et løvtag. Bladvinklen kan også påvirke

lysabsorptionen i løbet af dagen. Om morgenen og aftenen, hvor solen står lavt på himlen, vil

oprejste blade (græsser f. eks) absorbere mere lys end et tilsvarende blad, der er parallelt med

jorden. Midt på dagen vil dette forhold være omvendt (Falster and Westoby 2003). Ud fra

dette opstår der to scenarier hvor oprejste blade kan øge den daglige mængde optaget CO2.

Enten ved at øge lysabsorptionen om morgenen og aftenen under lysbægrænsende forhold

eller ved at mindske fotoinhiberingen midt på dagen. Via en model viste Falster and Westoby

(2003) at oprejste blade havde den største gavnlige effekt af mindsket fotoinhibering midt på

dagen baseret på daglig CO2 assimilation. Ud fra dette er det ikke nogen overraskelse, at flere

studier har dokumenteret at bladets vinkel udviser stor plastictet i forhold til lysintensitet

(Mcmillen and Mcclendon 1979; He et al. 1996; Valladares and Pearcy 1998; Balaguer et al.

2001; Larbi et al. 2015).

Andre studier har udover bladets vinkel også målt bladets totale areal (He et al. 1996;

Valladares and Pearcy 1998; Markesteijn et al. 2007; Carins Murphy et al. 2012; Larbi et al.

2015). Det generelle billede er, at bladet udviklet i sol får et mindre totalt areal. Der er

fremsat forskellige bud på hvorfor bladene bliver mindre med stigende lysintensitet. Et af de

tidlige bud var, at solblade skulle være bedre til at skaffe sig af med varme grundet det

mindre totale areal men også grundet ændringer i kutikulaen og trikomere (Vogel 1968).

Udover at have en possitiv effekt på plantens varmebalance er det også blevet foreslået, at de

små blade i solplanter reducerer grænselaget og dermed øger CO2 influxen (Parkhurst and

Loucks 1972). Falster and Westoby (2003) viste via deres model, at små blade var med til at

skabe mere selvskygning, og at dette havde større betydning for den daglige carbon

assimilation end bladets vinkel. At selvskygningen havde denne effekt kan også forklare,

hvorfor nogle arter udviser plastictet i deres fyllotaxis (Valladares and Pearcy 1998; Galvez

and Pearcy 2003).

2.5 Bladtykkelse

Udover bladets totale størrelse ses der også variation i bladet tykkelse blandt forskellige arter.

Bladtykkelse viser også stor plastisitet i forhold til lysintensiteten under opvæksten(Nobel et

al. 1975; Evans and Poorter 2001). Bladets tykkelse kan udtrykkes på flere forskellige måder.

Bladets totale tykkelse kan måles direkte, tykkelsen kan estimeres ud fra bladets specifikke

bladareal (SLA), ud fra bladets friskvægt i forhold til bladets areal (LMA) eller en

kombination af SLA og vægten af tørstof i forhold til vådvægt (LDMC) (Vile et al. 2005).

Ved at sammenligne absorptionsspektre fra esktraherede pigmenter i en opløsning med

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absorptionspektret for pigmenter i et blad, viste Ruhle and Wild (1979) at pigmenter i et blad,

er arrangeret på en sådan måde i forhold til pigmenter i en opløsning, at absorptionen øges.

Dette indikerer, at den indre tredimensionelle opbygning af bladet og pigmenternes position

heri har betydning for absorptionskarakteristika. Bladets tykkelse er plastisk, hvilket primært

skyldes ændringer i tykkelsen af mesofylvævet. Mesofylvævet kan opdeles i to lag hhv.

palisadevæv og svamepvæv. Hvordan påvirker plasticiteten i bladtykkelsen absorption af lys

og CO2 optaget? Under forhold med høj lysintensitet får nogle arter flere lag palisadevæv

eller et tykkere lag palisadevæv (Bjorkman 1981; Cao 2000; Markesteijn et al. 2007). Et

tykkere lag svampevæv medvirker også til et tykkere blad under høj lysintensitet, dog ikke i

så høj grad som palisadevævet. I direkte sollys er langt størstedelen af det indfaldne lys i

form af direkte lys, mens en mindre del består af diffust lys (Bird and Riordan 1986). Dette

har implikationer for absorbtionen af lys i hhv. svampe og palisadevæv. Palisadevæv

faciliterer i højere grad end svampevæv direkte lys ned igennem det underliggende væv

(Vogelmann and Martin 1993; Vogelmann et al. 1996), hvilket kan forklare, hvorfor der er

størst variation i palisadevævets tykkelse fremfor svampevævet. Epidermiscellernes form gør,

at de kan fokusere direkte lys (Brodersen and Vogelmann 2007). Fokuspunkteter afhænger

bla. af vinklen på det indfaldne lys, hvor lys vinkelret på bladet har et dybere fokuspunkt end

mere parallelt lys(Brodersen and Vogelmann 2010). Fokuseringen og faciliteringen af lys

giver en mere jævn fordeling af lys igennem bladet, hvilket kan give en større A for hele

bladet, da flere kloroplaster fotosyntetiserer omkring det punkt, hvor de lysmættes

(Terashima and Saeki 1985). I forhold til lysabsorbtion er det tykkere blad fordelagtigt, men i

forhold til CO2 diffusion kan et tykt blad udgøre en begrænsning, da CO2 har en længere

diffusionsvej i et tykt kontra tyndt blad. Tidligere mente man, at den største begrænsning var

fra atmosfæren og ind igennem stomata til det intracellulære rum i mesofylet (Farquhar and

Sharkey 1982). Dette understøttes af et tidligere afsnit (afsnit 2.3), hvor man så at

stomatadensiteten steg med stigende lysintensitet, hvilket tyder på, at diffusion fra den

omkringværende luft og ind i bladet udgjorde en begrænsning. Denne begrænsning blev

reduceret af den lavere modstand, som flere stomata giver. Derfor burde den længere vej ikke

udgøre nogen stor begrænsning, da begrænsingen fra stomata er meget større. Det tyder dog

på, at modstanden fra atmosfæren og ind til det intracellulære rum ikke er så stor i forhold til

andre modstande undervejs (Terashima et al. 2006). Det tykkere blad kan faktisk vise sig at

være en fordel under høj lysintensitet, da diffusionsbegrænsningen ser ud til at være når CO2

skal fra det ekstracellulære rum og ind i mesofylcellen og videre til kloroplasten, hvor

Rubisco befinder sig, og ikke fra luften omkring bladet og ind i bladet via stomata

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(Terashima et al. 2006). Bladtykkelsen korrelerer med andelen af mesofylcellernes areal i

forhold til bladets areal (Ames/A) (Nobel et al. 1975; Terashima et al. 2006), hvilket viser at

mere areal til CO2 optagelse er til rådighed i et tykt blad. Arealet af kloroplaster, der er

eksponeret mod mesofylcellens kant (Sc), er også af betydning for diffusionen af CO2 ind til

rubisco i kloroplasten (Evans et al. 1994). Denne stiger, ligesom Ames/A gjorde, i forhold til

bladtykkelsen og korrelerer desuden med den interne konduktans (gi, ledningsevnen mellem

det substomatale rum og kloroplastens stroma) som igen korrelerer med A (Terashima et al.

2006). I forhold til CO2 diffusion fra den omgivende atmosfære og ind til kloroplasten er et

tykkere blad altså fordelagtigt, mere kloroplastareal er eksponeret til CO2 optag.

Omkostningerne i form af biomasse til at danne et tykt blad er større end for et tyndt blad

(Poorter et al. 2006). Dette kan begrænse tykkelsen af bladet især under lysbegrænsende

forhold. Ovenstående kan også forklare hvorfor man i nogle tilfælde ser varierende grad af

succesfuld akklimation af i forevejen færdigudviklede blade (Oguchi et al. 2003, 2005). Hvis

mesofylcellerne har kloroplaster langs hele kanten, ses der en ringe grad af akklimering i

færdiguviklede blade. Det er altså i høj grad andelen af ikke optaget plads i mesofylcellerne

som gør at nogle arter er i stand til at akklimere færdigudviklede blade i større grad end

andre.

Udover at være billigere i konstruktionsomkostninger grundet deres ringe tykkelse tillader

den ringe tykkelse et større total areal af bladet, hvilket forbedre plantens lysabsoprtion mere

end hvis den dobbelt mængde N var investeret i det halve bladareal (Bjorkman 1981;

Niinemets 2007). Andelen af absorberet lys reduceres relativt mere i et skyggeblad i forhold

til et solblad når mesofylvævet fyldes med oile (DeLucia et al. 1996). Når lys rammer en

grænseflade, der er i stand til at sprede lyset via reflektering, fungerer dette som en lysfælde

(Vogelmann 1993). Grænsefladen mellem mesofylcellerne og luften inde i svampevævet har

disse karakteristika. Bladet virker derfor som en lysfælde, og absorbtionen øges med

efterfølgende gavnlige effekt på den daglige carbon balance. Ved at fylde bladets svampevæv

med olie ændres grænsefladen og dermed også dens karakteristika, sådan at lyset ikke

længere fanges i samme grad. Denne effekt er selvfølgelig målbar i både et sol- og

skyggeblad, men andelen absorptionen sænkes med er relativt størst for skyggebladet.

Epidermiscellernes evne til at fokusere lys har også givet ophav til spekulationer om, hvordan

dette er fordelagtigt under lav lysintensitet (Vogelmann 1993). Selvom nogle studier har vist,

at løvtagets samlede fotosynteserate er højere under diffust lys kontra direkte lys (Roderick et

al. 2001; Farquhar and Roderick 2003), tyder det ikke på, at dette skyldes epidermiscellernes

evne til at fokusere lys(Brodersen and Vogelmann 2007; Brodersen et al. 2008).

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Skyggebladet ser altså ud til at absorbere en så stor andel af lyset som muligt, mens solbladet

via palisadecellernes lysfacilitering og epidermiscellens fokusering tillader at lyset trænger

dybt ned i bladet, sådan at så meget muligt kloroplastareal kan eksponeres.

2.6 Allokering på bladniveau

Alokkering er indirekte blevet berørt i forgående afsnit, da en tykkere kutikula eller blad

nøvendigvis må kræve mere biomasse, som ellers kunne have været brugt et andet sted i

planten. Dette afsnit vil forsøge at besvare, hvordan fordelingen af biomasse varierer i

forhold til mængden af lys planten modtager. Brouwer (1983) fremsatte en hypotese, som

siger at fordeling af biomasse ændres afhængigt af miljøpåvirkningen, sådan størst mulig

optag af den begrænsende faktor opnåes. Hvis lys begrænser væksten, vil mere biomassen i

højere grad allokeres til de organger, som står for at absorbere lys. Under lysbegrænsede

forhold vil der altså investeres mere biomasse i blade. Under forhold hvor lys ikke begrænser,

allokeres der mere biomasse til rødder, hvis næring eller vand er begrænsende for plantens

vækst. I forbindelse med fortolkningen af forsøg, der undersøger allokeringsmønsteret, kan

der opstå nogle problemer, da allokeringsmønsteret skifter i forhold til plantens alder. En

nyspirret frøplante vil fra start have en høj andel af rødder i forhold til overjordisk biomasse

(R:S ratio). I takt med at planten vokser dannes der flere blade og R:S ratioen falder. Dette

skift skyldes ikke miljøpåvirkninger men udelukkende ontogenetisk skift i allokeringen,

planten følger sin allometriske bane (Weiner 2004). I tillæg til dette korrelerer alder og

størrelse ikke i nær samme grad som hos. f. eks. dyr. For nærmere diskusion af denne

problematik henvises der til Weiner (2004), da dette er et for omfattende emne her. Poorter

and Nagel (2000) argumenterede for, at den klassiske opdeling af over- og

undergrundsbiomasse blev yderligt opdelt, sådan at overgrundsbiomasse opdeles i fraktionen

udgjort af hhv. blade (LMF) og stamme (SMF). Via en metaanalyse viste de, at LMF faldt

med stigende lysintensitet. Udover en mindre LMF faldt SMF også. Fraktionen af rødder

(RMF) steg til gengæld med stigende lysintensitet, hvilket indikerer, at biomassen fra bladene

og stammen i stedet blev brugt til at danne rødder. Dette er i overenstemmelse med Brouwers

hypotese. Undersøges de individuelle studier, som metaanalysen bestod af, finder man

studier, der ikke understøtter Brouwers hypotese. Andre studier udgivet efter metaanalysen

støtter umiddelbart heller ikke Brouwers hypotese i forhold til LMF (Poorter 2001). Årsagen

til dette kan være, at studierne ikke har taget bladets levealder i betragtning, da sol og

skyggeblade er forskellige i både levetid og tilbagebetalingsomkostninger (Poorter et al.

2006).

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N allokering til forskellige blade afhænger af lysintensiteten(Hirose and Werger 1987a,

1987b). For bladet gjorde det sig gældende at når hver kloroplast fotosyntetiserer omkring sit

lysmætningspunkt opnåes den højeste A for hele bladet (Terashima and Saeki 1985). Et

tilsvarende scenarie gør sig gældende for bladene og hele planten, da planten som helhed

opnår den højeste A, hvis hvert enkelt blad fotosyntetiserer omkring sit lysmætningspunkt.

Dette kan ske ved at sende mere lys ned igennem løvtaget eller ved en skæv fordeling af N

mellem de øverste og nederste blade, sådan at N afspejler lysgradienten ned igennem

løvtaget. Via deres model viste Hirose and Werger (1987a) at den observerede fordeling af N

var mere ensartede, end hvad modellens optimum angav. Dog gav den observerede fordeling

stadig ophav til højere A end for en helt ensartet N-fordeling.

Kloroplastakklimering

3.1 Kloroplasten

Antallet af kloroplaster per areal stiger i takt med stigende lysintensitet, hvilket skyldes flere

celler per areal (Chow et al. 1988) grundet det tykkere blad (se afsnit 2.5). Udover at der

bliver færre kloroplastere under lav lysintensitet, bliver selve kloroplasten større og i højere

grad fyldt ud med thylakoidmembran, samtidig med at granumdannelsen øges (Anderson et

al. 1988). Det større antal kloroplaster ved høje lysintensiteter giver mulighed for et større

kloroplastvolumen per bladareal til fotosyntesens enzymer, som befinder sig inde i

kloroplasten (Oguchi et al. 2006). Den store kloroplast med meget thylakoidmembran gør, at

bladet kan have et højt klorofylindhold per bladareal på trods af det mindre antal kloroplaster

per areal (Bjorkman 1981; Anderson et al. 1988). Thylakoidmembranen udgører en stor del

af kloroplasten, og i denne membran finder lysabsorptionen sted. Dette sker via de to

fotosystemer (PS1 og 2), hvor hvert PS består af et reaktionscenter (RC) forsynet af op til

flere lyshøstningskomplekser (LHC). Fælles for RC og LHC er at de består af

lysabsorberende pigmenter indlejret i et proteinskellet, mens kun RC har redoxkomponenter

tilknyttet (Croce and van Amerongen 2014). I RC i PS2 findes P680, mens P700 findes i PS1.

De to RC har forskellige absorbstionspektre (Wientjes et al. 2013). PS1 og 2 er knyttet til

hver deres særskilte region af thylakoidmembranen (Anderson 1986). PS1 er knyttet til den

eksponerede region forstået på den måde, at den eksponeres imod stroma, mens PS2 er

knyttet til den sammentrykte region og er eksponeret mod en anden thylakoidmembran.

Forholdet imellem PS1 og 2 varierer i forhold til lysintensiteten, hvilket andelen af

eksponeret og sammentrykt membran også gør (Anderson et al. 1988). Udover PS findes

ATP syntasen også i membranen og er ligesom PS1 tilknyttet den eksponerede del af

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membranen (Miller and Staehelin 1976). I membranen findes der også et tredje kompleks

kaldet cytochrome b/f kompekset (cyt b/f), hvilket er involveret i protonpumpning fra

thylakoidlumen og ud i kloroplastens stroma. Dette kompleks modtager elektronener fra PS2

via plastoquinon (PQ), elektronen transporteres videre til PS1 via plastocyanin (PC), og i

sidste ende reduceres NADP til NADPH, som efterfølgende forbruges af Rubisco under CO2

reduktionen (Lambers et al. 2008) s. 11-15. Hvert trin undervejs fra PS til CO2 udgør et

muligt reguleringspunkt, sådan en optimal balance imellem de forskellige trin opnåes. De

efterfølgende afsnit vil beskæftige sig med nogle af disse trin, og hvordan de reguleres sådan

en mere favorabel A opnåes i forhold til ingen regulering.

3.2 Fotosystem 1 og 2

Forholdet imellem PS1 og 2 varierede i forhold til lysintensiteten, men tidlige forsøg har

givet resultater af tvetydig karakter (Anderson et al. 1988). Årsagen til dette kan skyldes, at

både intensiteten og kvaliteten af lyset påvirker forholdet imellem PS1 og 2 med størst effekt

af kvaliteten (Chow et al. 1990) og at responsen er artsafhængig (Murchie and Horton 1998).

Ændringer i mængden af PS2 for hhv kvalitet og kvantitet er i hver sin retning (Anderson et

al. 1995). Ændringerne i forholdet skyldes ikke ændringer i mængden af PS1 da denne er

relativt konstant, men derimod ændringer i mængden af PS2 (Rochaix 2014). Udover at

ændre forholdet imellem PS1 og PS2 kan der ske ændringer i mængden af LCH2, der

forsyner PS2 men overraskende nok kan LCH2 også forsyne PS1, mens forholdet imellem

PS1 og LHC1 er konstant (Ballottari et al. 2007). Under forhold med lav lysintensitet

opreguleres mængden af LHC2 for at kompensere for den mindre mængde lys og omvendt

under forhold med høj lysintensitet, sådan overeksitering og efterfølgende skader undgåes.

Ændringer i kvaliteten af lyset forsager at LHC2 migrerer imellem PS1 og 2 sådan en balance

imellem de to systemer opnåes, og overeksitering undgåes (Rochaix 2014). Lyskvaliteten

under et løvtag er beriget i langbølget rødt lys (Smith 1982). Denne type lys absorberes

primært af PS1, sådan at PS1 overeksiteres i forhold til PS2 i skygge. Ved at LHC2 migrerer

mellem PS1 og 2, sikres det at PS1 og 2 eksiteres i lige høj grad. For en mere detaljeret og

molekylær forklaring af reguleringmekanismerne bag migrationen imellem de to PS’er og op

og nedreguleringen af LHC2 henvises der til Rochaix (2014) og Eberhard et al. (2008).

Ratioen mellem klorofyl a og b (chl a/b ratio) mentes at afspejle forholdet mellem PS og

LHC, da PS kun indeholder klorofyl a mens LHC indeholder klorofyl a og b (Anderson et al.

1988). Dette har været brugt til at vurdere akklimeringen. En lav chl a/b ratio afspejlede en

større investering i LHC da klorofyl b ikke findes i PSerne. Sidenhen er der sket større

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fremskridt indenfor, hvordan PS og LHC er opbygget samt deres indbyrdes forhold. Dette er

sket i forbindelse med mere avancerede tekniker især indenfor molekylærbiologien. På trods

af at den oprindelig model for LHC og PS har ændret sig, har validiteten af klorofyl a og b

ratioen, som målestok for om akklimering har ført til en større effektivitet i

lyshøstningen(flere LHC) eller en større kapacitet for omsætningen(flere PS) af fotoner, ikke

ændret sig (Rochaix 2014).

3.3 Xantofylcyklussen

Udover klorofyl a og b findes der andre typer af pigmenter i thylakoidmembranen i

forbindelse med PSerne. Tre af disse pigmenter er violaxanthin (V), antherxanthin (A) og

zeaxanthin (Z), som tilsammen udgøre xantofylcyklussen (VAZ) (DemmigAdams and

Adams 1996). De tre pigmenter adskiller sig fra hinanden ved antallet af epoxidgrupper.

Epoxidgrupperne har betydning for antallet af dobbeltbindinger og den steriske

konformation. Man har været bekendt med VAZ og at planter er i stand til at skaffe sig af

med overskudsenergi fra lys via en pH afhængig mekanisme i længere tid (DemmigAdams

and Adams 1996). Forbindelsen imellem disse to processer blev først opdaget via indirekte

evidens (Demmig et al. 1987) men sidenhen er der kommet mere direkte evidens (Bilger and

Bjorkman 1990). Mekanismen bag denne regulering imellem de tre pigmenter er forslået at

ske på følgende måde (DemmigAdams and Adams 1996; Lambers et al. 2008) s. 11-15. Når

lys absorberes af PS2 eksiteres en elektron fra H2O som efterfølgende henfalder i

energiniveau via en række kontrolerede rekationer. Denne proces sker via

elektrontransportkæden i thylakoidmembranen. Energien fra disse henfald pumper protoner

ind i thylakoidlumen, sådan pH-værdien sænkes, og elektronen reducerer til sidst NADP til

NADPH. pH forskellen imellem thylakoidlumen og stroma kobles til ATP dannelse via ATP

syntasen. Hvis ATP syntasen ikke forbruger protongradienten med samme hastighed som den

opretholdes, forsurres lumen mere og mere. Deepoxideringen imellem V, A og Z er pH

afhængig. En lav pH favoriserer omdannelsen af V og A til A og Z, mens høj pH favoriserer

omdannelsen af Z og A til A og V. Z påvirker LHC2 via konformationsændringer sådan at

nogle af fotonerne omsættes til varme i stedet for at blive brugt i fotosyntesen. Derudover har

Z yderlig en rolle i form af antioxidant (Jahns and Holzwarth 2012). En forudsigelse baseret

på denne mekanisme vil være at solplanter vil have en højere andel af Z i forhold til en

skyggeplante. Denne forudsigelse er understøttet af flere forsøg, hvor man kan se at andelen

af Z i forhold til A og V stiger med stigende lysintensitet (DemmigAdams and Adams 1996;

Grace and Logan 1996). Udover at andelen af Z stiger, stiger størrelsen af hele VAZ puljen i

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forhold til bladareal eller klorofylindhold også (DemmigAdams and Adams 1996; Grace and

Logan 1996; Niinemets et al. 2003). Det har også vidst sig at konverteringen af Z tilbage til

A og V er langsommere når planten har vokset ved høj kontra lav lysintensitet, hvilket svarer

til en form for hukommelse, der kan ”huske” det tidligere lysmiljø (Jahns and Holzwarth

2012).

3.4 Elektrontransport og Rubisco

Tre overordnede processer er involveret fra absoprtionen af fotonen til reduktionen af CO2.

De tre processer er lysabsorptionen, den efterfølgende elektrontransport frit men også

tilknyttet thylakoidmembranen og PS1 og 2, og til sidst selve reduktionen. Disse tre processer

har ikke ens rekationshastigheder (Lambers et al. 2008) s. 14. Fotokemien er hurtigst

efterfulgt af elektrontransporten og langsomst er reduktionen af CO2. Dette kan have

implikationer for mængden i hver af de tre processer. Elektron transport i PS1 og 2 er blevet

behandlet tidligere, hvor man så hvordan PS1 og 2 varierede i forhold til lysintensiteten for at

balancere absorsption og efterfølgende elektrontransport i PS komplekserne (afsnit 3.2). I

forlængelse af dette kunne man forestille sig en regulering af elektrontransportkapaciteten

imellem PS1 og 2, og tilsvarende en regulering i raten af NADPH forbruget, når

lysintensiteten variede. Cyt b/f, PQ og PC som alle er involveret i elektrontransporten mellem

de to PS’er stiger med stigende lysintensitet (Leong and Anderson 1984; Anderson et al.

1988; Schottler and Toth 2014).

Reduktionen af CO2 sker via flere enzymer, men da Rubisco er det enzym som udgøre den

største andel af enzymer i bladet (Hopkins and Huner 2009) s. 88, vil fokus være på dette

enzym, selvom andre enzymer i forbindelse med reduktionen også må forventes at blive

reguleret. Aktiviteten af Rubisco kan reguleres på kort sigt via aktiveringen af Rubisco (Parry

et al. 2008), men også på længere sigt, hvis mængden af Rubisco ændres. Sidstnævnte vil

blive behandlet her, for gennemgang af korttidsregulering henvises der til Parry et al. (2008).

Ud fra rent morfologiske betragtninger vil man forvente et højere Rubisco indhold i

kloroplasten når lysintensiteten stiger. Årsagen til dette er at solplanter har mindre

thylakoidmembran i kloroplasten (Anderson et al. 1988), hvilket gør, at de potentielt kan

have mere Rubisco, som befinder sig i kloroplastens stroma. På basis af bladareal vil Rubisco

indholdet også stige i en solplante, da bladet indeholder flere celler per areal og hver celler

indeholder flere kloroplastere (Lambers et al. 2008) s. 34. Dette vil også give mening ud fra

et fysiologisk perspektiv. Elektrontransporten udviste større kapacitet med stigende

lysintensitet, og disse elektroner skal derfor også omsættes i højere grad (Evans and Poorter

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2001). Der bruges ikke NADPH af Rubisco, men det gør de efterfølgende trin i reduktionen

af CO2, hvorfor det kunne forventes at Rubisco indholdet ville øges. Hvis mængden af

Rubisco ikke er begrænsende for Amax, eller at NADPH i høj grad kan forbruges andre steder,

vil man ikke forvente at se en korrelation mellem Rubisco og Amax. Bjorkman (1981) viste at

der var en stærk korrelation (r2 = 0,96) imellem Amax og Rubisco aktiviteten. Dette gjorde han

via indsamlede data fra 11 forskellige arter, som var blevet dyrket ved forskellige

lysintensiteter. Nogle arter var repræsenteret af både soladapterede og skyggeadapterede

genetisk isolerede populationer.

3.5 Allokering på kloroplastniveau

Mængden af N per bladareal varierer mellem sol og skyggeplanter, med mest N per areal i

solplanter (Lambers et al. 2008) s. 34. Andelen af N allokeret til proteiner i

thylkaoidmembranen varierer afhænigt af den undersøgte art. Den generelle respons menes at

være, at andelen af N allokeret til enzymer i forbindelse med Calvin-cyklussen stiger med

stigende lysintensitet. Dette sker på bekostningen af mindre N til thylakoidmembranen

(Evans 1989). Hvis kun den andel af N, der allokeres til thylakoidmembranen betragtes, ser

man også variationer i denne i forhold til lysintensiteten. Allokering i thylakoid membranen

af N kan enten ske til lyshøstning eller til elektrontransport. Fordelingen er sålede at ved høj

lysintensitet investeres der en større andel N i elektrontransport på bekostning af N til

lyshøstningen og omvendt under lav lysintensitet. (Evans 1987; Lambers et al. 2008) s. 34.

Dette kunne forståes ud fra eksitationspresset på de forskellige komplekser (se afsnit 3.2).

Her vil det blive betragtet ud fra et N økonomisk perspektiv. LHC består af en høj andel

pigment per protein i forhold til PS (Evans 1987; Croce and van Amerongen 2014). Årsagen

til dette er at der findes en større andel protein i PS’erne, som har til formål at orientere

pigmenterne. I forlængelse af dette indeholder PS’erne forskellige redoxkomponenter som

ikke findes i LHC. Dette gør at en opregulering i LHC fremfor PS, giver en mere effektiv

lyshøstning i forhold til investeringen af N i proteiner. N allokering varierer ikke kun med

lysintensiteten men også med mængden af N, samt interaktion imellem N og lysintensitet.

Ovenstående er derfor baseret på at planterne har adgang til rigeligt N, og ikke er begrænset

af N. Der henvises til Hikosaka and Terashima (1995) for en model der beskriver effekten af

både lysintensiteten og adgang til N.

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Light attenuation and photosynthetic

acclimation in Typha latifolia, a productive

wetland species with erect linear leaves

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Introduction

Light can be strongly attenuated through plant canopies, due to light absorption by plant

tissue, which creates a gradient in the light environment experienced by individual leaves.

The magnitude of the gradient depends on several factors, such as structural and chemical

properties of the leaf itself but also the arrangement of leaves in relation to each other in the

canopy (Hirose 2005). Leaves are plastic in response to their light environment (Bjorkman

1981) and will acclimate to the light intensity at a given height in the canopy,ensuring that

photosynthesis can optimally exploit incident radiation to maximize canopy photosynthesis

(Hirose and Werger 1987a). Higher overall canopy photosynthesis also depends on the leaf

angle compared to the incident light, as vertically inclined leaves, such as those of grasses,

should have a higher leaf area index (LAI) compared to more horizontal leaves, such as those

of broad leaved species, to maximize overall canopy photosynthesis in full light conditions

(Hirose 2005). The mechanism behind the higher overall canopy photosynthesis is an even

distribution of photons between the leaves in the canopy, and acclimation of individual

leaves, and even individual chloroplasts, such that no individual leaf is operating above its

light saturation point (Ik), and thereby wasting excitation energy (Terashima et al. 2006).

Freshwater marshes and swamps are known to possess some of the highest net

primary production (NPP) of any ecosystem on earth (Westlake 1963; Whittaker 1975;

Wetzel 1992; Rocha and Goulden 2009), and this may be in part due to the widespread

dominance of tall graminoids with such vertically aligned leaves, together with their high

nutrient and water availability. Nitrogen (N) is known to scale positively with light saturated

photosynthesis (Amax) (Evans 1989) and the high NPP can be related to high nutrient supply,

and also to the ease of access to water (Keefe 1972). Water is lost as an inevitable

consequence of CO2 assimilation, but as water is plentiful and unlimiting in a freshwater

wetland habitat, few of the negative consequences normally associated with water stress and

desiccation at high transpiration (T) rates would be expected. This can allow very high

stomatal conductances (gs) in wetland plants, and could potentially uncouple the general

close relationship between gs and Amax in plants (Wong et al. 1979). Others have ascribed the

high productivity to high carbon use efficiency (CUE), as respiratory requirements would be

lower when N is supplied in the form of ammonium instead of nitrate and high allocation to

leaves rather than to stems and roots (Rocha and Goulden 2009).

The possible importance of the linear leaf growth form for maximising light

penetration and maintaining extreme high photosynthesis rates and productivity in freshwater

wetland plants in nature has not been widely studied. The prevalence of basal meristems in

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such plants also allows additional plasticity in response to light. Leaves produced by species

with apical meristems will with time experience increased self-shading at the lower leaves,

because new leaves are produced above the older leaf. As the leaf ages it will at some point

be shed when light intensity becomes low enough (Ackerly 1999). The situation is different

in species with basal or intermediate meristems, such as those that frequently dominate

nutrient-rich wetlands. Leaves will still be shed at some point, but the oldest part of the leaf

will be at the leaf tip. The leaf will emerge from the leaf sheath and over time is pushed

upwards, by further cell division and expansion in the growth zone.

One genus featuring species with particularly high productivity, basal growth and

capabilities to make dense monospecific stands in wetlands is Typha (Grace and Harrison

1986; Miao et al. 2000). The Typha growth form allows a higher proportion of light to

penetrate down to the lower layers, i.e. less light attenuation, compared to other species from

the same habitat, but with less vertically inclined leaves (Hirtreiter and Potts 2012). The high

productivity, basal growth, and low light attenuation make this genus an interesting study

subject in regards to light acclimation, which seldom has been studied in these kinds of

plants.

The main purpose of this study was therefore to investigate how the Typha growth

form, featuring erect linear leaves and potentially lower light attenuation could affect light

acclimation in Typha latifolia L. when grown under optimal water and nutrients supply, both

in laboratory and field conditions. Specifically we aimed to test two hypotheses regarding

photosynthesis in high light conditions. First, that the linear growth form minimizes self-

shading and hence allows T. latifolia to maintain high photosynthetic rates and sun adapted

photosynthetic light responses throughout its canopy. Second, that the pigment content and

composition will reflect the amount of light experienced at different heights in the canopy.

Materials and methods

Laboratory study: Plant material and growth conditions

Seeds of T. latifolia were collected at Femmøller Beach, 50 km north of Aarhus, Denmark, in

February 2015. Seeds were planted in peat soil and later transferred to beach sand. Plants of

similar size and in good conditions were selected and transplanted from sand to individual

indoor 1.4 L hydroponic glass jars darkened with black plastic in April 2015. Plants were

mounted in the jars with polystyrene lids. The lids had a hole in the middle for the plant and

great care was taken to ensure that the stem was not mounted to tight. With time the stem

expanded and the hole was subsequently made bigger. The nutrition solution consisted of

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0.01% (w/w) “Grøn Pioner NPK 19-2-15 + Mg” and 0.001‰ (w/w) “Mikro plus with iron”

(Brøste, Lungby, Denmark).

The plants were distributed evenly in a growth chamber (Bio 2000S, Weiss

Umwelttechnik GmbH, Lindenstruth, Germany) programmed with a day:night cycle of 16 h

light:8 h dark, 22 oC:18

oC, 70% RH:80% RH and a light intensity of 600 µmol m

-2 s

-1 PAR.

After eight days’ pretreatment under these conditions, a high (HL) and low light (LL)

treatment were made by shading 12 of the 24 plants with neutral density shading cloth. Light

intensities were measured with a Li-COR LI-250 light meter and HL had a light intensity at

the top of the plants of 700 µmol m-2

s-1

PAR whereas LL had 200 µmol m-2

s-1

PAR. One of

the HL plants died and reduced the number of replicates for this treatment to 11. Water levels

were checked three times per week. Water was alternately added or completely changed with

fresh nutrient solution in the cultures. After 35 days under HL or LL, measurements were

started.

Field study: Study site and plant selection

The site was located north of Aarhus and east of Egå Engsø in east Jutland (56.2175N

10.23448E), a restored wetland, which was reflooded in 2006 and colonized naturally by T.

latifolia. Egå Engsø is fed with water from Egå Stream and Ellebækken creek and discharges

into Aarhus Bay through the town of Egå. The sediment consists of a 30 cm deep layer of

organic matter and the nutrient content of the sediment is… East Jutland has an average

rainfall of 722 mm year-1

. Average mean temperature during the year is 7.7oC with an

average night and day temperature of 3.8oC and 11.4

oC (www.dmi.dk). A total of 10 plots in

the T. latifolia vegetation were chosen for the experimental procedure, five in which

individual shoots were not shaded by other vegetation (Open stand, O) versus five in the

middle of a dense monospecific stand where there was intense self-shading (Dense stand, D).

Plants were not selected at randomly but instead the most densely shaded plants in D and

least shaded plants in O were chosen.

Light environment

Light profiles were made to characterize the light environment in both the laboratory and

field experiments. Light intensities were measured with two Li-COR LI-250 light meters in

the field. One was situated above the canopy to account for variation in incident radiation

while the other was used to measure light intensities at a given height. Profiles were made in

the field for both within the monospecific D stand and the other in the O area. For the O-area

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light intensities was measured 0, 40, 80, 120 and 160 cm above ground and in the D-plot 20,

60, 100, 140 and 180 cm above ground. Light intensity was measured five times at each

height to produce an average.

In the laboratory experiment a light profile was made for both the HL and LL

treatment. In the HL treatment light intensities were measured 21, 41 and 61 cm from the top

of the chamber and in the LL treatment 51, 71, 91, 111 and 130 cm from the top. For both

treatments measurements were made in each corner and in the middle of the rectangle

produced by the corner measurements.

Gas-exchange measurements

For both the field and laboratory experiment, gas-exchange measurements were made with a

portable LI-6400 XT infrared gas analyser (IRGA), (LI-COR Biosciences, Inc., Lincoln, NE,

USA). Light response curves were measured at 2500, 2000, 1500, 1000, 500, 250, 120, 60,

30, 15 and 0 µmol m-2

s-1

PAR with a minimum waiting time of 90 s and a maximum of 180 s

per light intensity. Measurements were made in the order of high to low light to avoid CO2

limitation of photosynthesis due to a sluggish stomata response. Light was supplied from the

built in red-blue 6400-02B LED array. The leaf chamber was air conditioned at 20oC in the

field and at 22oC in the laboratory experiment. CO2 was set to 400 µmol L

-1 and the air flow

500 µmol s-1

. The humidity in the incoming air ranged from 10-50% RH during

measurements in the laboratory and 45-80% RH in the field. For both experiments a fully

developed and mature set of leaves were selected. All measurements were conducted between

09.00 and 15.00 h. In the growth chamber experiment measurements were made on a

segment of the leaf on the HL treated plants at a height corresponding to a light intensity of

700 µmol m-2

s-1

. On the LL treated plants measurements were made on two sections of the

same leaf. The first measurement was at a height corresponding to a light intensity of 150

µmol m-2

s-1

(LLB), and the second corresponding to 250 µmol m-2

s-1

(LLT). These were at

different heights due to heterogeneity in the light environment in the growth chamber. The

leaf segments used for gas-exchange measurements were excised and divided into two

sections. One section was stored at -80oC for later pigment analysis. The other section was

used for specific leaf area measurements.

In the field experiment gas-exchange measurements were made on two segments of

the same leaf. These two segments were respectively 15cm (T) and 70cm (B) from the tip of

the leaf and were made on plants from the O-stand and D-stand. After gas-exchange

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measurements the segments were divided into two sections. One was stored at -80oC for later

pigments analysis. The other was used for specific leaf area measurements.

To obtain parameters from the light response curve, data was fitted according to the curve

described by Prioul and Chartier (1977)with the program Photosynthesis Assistant software

(Version 1.1, Dundee Scientific, Dundee, UK).

√( )

Where An is the photosynthetic rate, Φ is the quantum yield, Amax is the light saturated

photosynthetic rate, I is the intensity of the incoming light, θ is the convexity of the light

response curve and Rd is dark respiration.

Fluorescence measurements

Fluorescence measurements were made with a portable fluorometer (PAM-2000, Walz Mess-

und Regeltechnik, Germany). The effective quantum yield of photosystem II (Fv/Fm ratio)

was measured after dark incubating the plants for 1 h. Measurements were conducted in very

dim ambient light. Fluorescence measurements were only made on plants from the growth

chamber experiment, and were made on the leaf from the selected mature leaf pair that was

not used for gas-exchange measurements. Great care was taken to ensure measurements were

at the same height as gas-exchange measurements were made. After the fluorescence

measurements, the segment measured was removed and divided into two sections. One

section was used for porosity measurements, and the other for stomatal density.

Morphological measurements

For both the field experiment and growth chamber experiment, specific leaf area and stomatal

density were measured, while leaf porosity, total leaf area and total shoot biomass were also

measured for the growth chamber experiment. For specific leaf area the area of the sections

was obtained with a Li-COR LI-3100 area meter in the growth chamber experiment and with

a ruler for the field experiment. For both experiments, fresh weights of the section were

determined (precision 0.1 mg). After drying at 70 oC for a minimum of 24 hours, sections

were re-weighed for dry weight.

To obtain the stomatal density a negative imprint of the fresh leaf was made with dentist

impression gum (elite HD+ light fast vinylpolysiloxane (addition silicone) impression

material) and returned to the laboratory. In both experiments measurements were done on the

second leaf of the pair selected, i.e. the one not used for gas-exchange measurements. Great

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care was taken to ensure measurements were done at the same height as gas-exchange

measurements were made. In the laboratory a positive imprint was made with nail polish

(GOSH clear nail polish) and transferred to a slide and number of stomata was obtained from

a picture taken with a light microscope (Leica DM4000B microscope and Leica application

suite 3.8.0).

Porosity was measured with a pycnometer (Burdick 1989). First fresh weight of the

leaf was measured (FW) and then the pycnometer filled with water (Wwater). Then the weight

of the pycnometer filled with water and the leaf section (Wwater+leaf). After that the leaf section

was homogenised and more water added to the pycnometer it was then weighed again

(Whomo). Finally the porosity was calculated.

Great care was taken to ensure that the temperature of the water was constant during

measurements.

Ten days after measurements were started, all plants were harvested and total leaf area

was measured with a Li-COR LI-3100 area meter before drying at 70oC for a minimum of 24

hours and for dry weight.

Pigment analysis

Analysis of pigments by HPLC was conducted on plants from both the field experiment and

the growth chamber experiment. Extraction was done according to (Thayer and Bjorkman

1990) with some modifications. The frozen leaf was ground in liquid nitrogen and

approximately 40 mg was added to 5 mL cold acetone in a Tenbroeck tissue grinder

(Wheaton). The sample was then ground and sparged with N2 for 5 min. and centrifuged for 5

min at 5000 rpms at 5oC. The supernatant was removed and extraction procedure was

repeated. The two extractions were pooled and filtered through a 0.45 µm PTFE filter.

Samples were kept cold during the extraction and performed in dim light.

The extract was then analysed using a high-performance liquid chromatography

(HPLC) Thermo Scientific Ultimate 3000 equipment provided with a with a diode array

detector (DAD), automatic sampler and column oven. The analysis was done according to

(Hou et al. 2011) with some modifications. The samples were separated on a Kinetex 2.6μ C8

100 Å column (100 mm × 3.0 mm ID) using methanol:1M ammonium acetate (70:30) as

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mobile phase A and 100% methanol as mobile phase B. Mobile phase gradient started with

80% of A (20% of B), linearly decreased to 5% of A (95% of B) within 10 min and finally

returned to the initial conditions in 2 min and hold for extra 2min. A flow rate of 0.5 mL min-

1 was held for the 14 min run, while the column oven was set to 50°C. The sample injection

volume was set at 20 μL and the detector signal was monitored at λ = 450 nm.

Pigment standards were obtained from DHI (Hørsholm, Denmark), and samples were

analysed for the following pigments: Antheraxanthin (A), zeaxanthin (Z), violaxanthin (V),

chlorophyll a and b (chl a and chl b) and β-carotene. Limits of determination (LOD) and

quantifications (LOQ) are as following A (5/15 mg L-1

), Z (7/21 mg L-1

), V (6/19 mg L-1

), chl

a (15/44 mg L-1

) and b (12/36 mg L-1

) and β-carotene (7/22 mg L-1

).

Statistical analysis

Parameters measured are reported as mean ± sd and were compared between treatments at the

0.05 significance level using a one-way ANOVA and Tukeys HSD test with the software

JMP 11.1.1 (SAS Institute, Cary, North Carolina, USA). Data was tested for unequal variance

with Levene’s test, and log, x2 or square root transformed when they did not satisfy variance

homogeneity. For the laboratory study light saturated photosynthesis on an area or mass basis

(Amax-area or Amax-mass), gs, internal CO2 concentration (Ci), T, intrinsic water use efficiency

(WUE) and total leaf area per plant were not successfully transformed, but were still

included. Factors used in the comparison were HL, LLT, LLB for the laboratory experiment

and OT, OB, DT, DB for the field experiment.

Results

Light environment

The light environment in the growth chamber was heterogeneous (Fig 1). Light intensity was

ca. 1000 µmol photons m-2

s-1

at the leaf tips and ca. 600 µmol photons m-2

s-1

at the bottom

in the HL treatment. For the LL treatment light intensity was 300 and 150 µmol photons m-2

s-1

. I both treatments light intensity followed an exponential decay.

In the field, light attenuation by T. latifolia biomass was lower in the O-plot than the

D-plot (Fig 2). The lower light attenuation allowed an unknown grass species to form a

ground layer in the O-plot. The grass was not present in the D-plot, probably due to the low

light intensity at the bottom of the D-plot.

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Laboratory study: Morphology

The morphological responses in the laboratory experiment are shown in Table 1. Specific leaf

area (SLA) decreased significantly with increasing light intensity, while the LL plants had

higher SLA with increasing leaf height (see Fig. 4 for SLA). Stomatal density increased

significantly with increasing light intensity, but only in the LLT treatment. For the LLB

treatment there was no difference compared to the HL treatment. Stomatal ratio between the

adaxial and abaxial side of the leaf differed between treatments. The LLB treatment had the

highest ratio, and was greater than 1 with most stomata on the adaxial side. The LLT

treatment showed the lowest ratio, due to having most stomata on the abaxial side. The HL

treatment was not different from the LLT or the LLB treatments and had equal stomatal

numbers on the adaxial and abaxial sides of the leaf. The porosity measurement revealed no

difference in the fractional air volume between the LLT and HL treatments. Leaves from the

LLB treatment differed from the LLT and HL treatments in having higher fractional air

volume. Even though the HL treatment resulted in significantly more biomass per plant than

the LL treatment there was no difference in total leaf are per plant.

Laboratory study: Gas exchange

Amax-area increased significantly from the bottom to the top of the LL treated plants, and was

highest for the HL treatment (Table 2). These differences disappeared when Amax was

expressed on a mass basis; the LLB treatment became lower than the HL and LLT treatments.

Rd and Ic followed the same trend, and was significantly higher for the HL treatment

compared to the LLT treatment, with the LLB treatment intermediate, but not different from

the two other treatments. No differences were observed in Φ for any of the treatments. θ was

generally low and was lowest for the HL treatment. Ik was higher for the HL treatment

compared to the LL treatment, with no differences between the top and bottom

measurements. There was a significantly higher gs in the HL treatment compared to the LLT

and LLB treatments. Ci was significantly higher in the HL treatment compared to the LLB

and LLT top treatment. This was not expected based on the higher Amax-area in the HL

treatment, but is consistent with the high gs. T and WUE followed a reverse pattern, with HL

having the highest T and lowest WUE compared to the LLB and LLT treatment.

Laboratory study: Pigment content and chlorophyll fluorescence

The only significant difference in pigment content on a leaf area basis was for chl b, where

the HL treatment resulted in less chl b compared to the LLT and LLB treatments (Table 3).

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The lower content of chl b in the HL treatment resulted in a higher ratio of chl a relative to

chl b (chl a/b ratio). This changed when pigment content was assessed on a mass basis

instead. Both chl a and b were lowest in the HL treatment and highest in the LLB treatment

with the LLT treatment intermediate. Consequently chl a+b followed the same pattern. The

xanthophyll (VAZ) pool size and β-carotene content was highest in the LLT treatment with

no difference between the HL and LLB treatment. The percentage of Z in the VAZ pool was

lowest for the HL treatment with the equal amounts in the LLB and LLT treatments. The

reverse was true for A, with the HL treatment having the highest percentage of A, and no

difference between the LLT and LLB treatment. Even though there were differences in the

amount of Z and A, this did not result in a difference in the deepoxidation state between the

treatments. When the size of the VAZ pool was expressed relative to chl the HL treatment

had the biggest VAZ pool with no difference between the LLB and LLT treatment. Finally

there was a significant but small difference in Fv/Fm, with the HL treatment being higher than

the LLT and LLB treatments.

Field study: Gas exchange and morphology

The parameters that showed significant differences were Amax-area, Rd, Ik, Amax-mass and SLA

(Table 4). For all other measurements there was no significant difference between plots or

height. Amax-area and Amax-mass was high in the bottom part and increased with height in both

plots. When compared at the same height there was no difference between the plots. Ik

reflected Amax, but there was only a tendency for higher values with increasing height, not a

significant difference. Rd was highest in OB and no difference was seen between the three

others. SLA decreased from top to bottom in both stands, and tended to be higher in the C

area when comparisons were made at the same height.

Field study: Pigment content

Chlorophyll a/b ratio, VAZ/chl ratio, chl a and chl b on mass basis, chl a+b also on mass

basis and the size of the VAZ pool on a mass basis were the only parameters that differed

significantly (Table 5). The chl a/b ratio was highest at the top on both the O and D stand. At

the same height the D stand had a lower chl a/b ratio compared to the O stand. OB showed

the lowest chl b content per mass compared to the other three, of which none differed from

each other. The combined amount of chl a and b followed the same pattern as β-carotene. The

trend was that DT had the highest chlorophyll content, but did not differ significantly from

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OT. There was also a difference from top to bottom, with the values for the top being highest.

There was no difference between measurements from the bottom on the D and O stand.

Discussion

Consistent with our first hypothesis, T. latifolia was indeed able to maintain higher

photosynthetic rates and remained sun acclimated throughout the canopy in the field. Higher

irradiance increased Amax-area which would be expected to be followed by an increase in the

proportion of mesophyll area that is exposed to the intercellular airspace per leaf area

(Ames/A) so that adequate CO2 is supplied (Terashima et al. 2006). Greater porosity would

also increase mesophyll conductance and hence diffusion rates (Terashima et al. 2001), but

the HL and LLT treatment had no effect on porosity. Even though porosity did not respond to

changes in irradiance, it was very high compared to a maximum value of 36% reported in a

compiled literature study of 597 non-wetland species (Niinemets 1999). High porosity can

increase internal conductance if the airspace is associated with the spongy tissue, but in T.

latifolia the airspaces responsible for the high porosity are likely predominantly associated

with the aerenchyma involved in convective gas flow (Sorrell and Brix 2003). The increase in

CO2 demand per area in response to higher Amax-area reduced SLA to values well within the

limits reported for trees and herbs in plasticity but also absolute values of SLA (Meziane and

Shipley 1999; Rozendaal et al. 2006). This is in contrast to other studies on T. latifolia where

no differences in SLA to changes in irradiance were observed (Ojanguren and Goulden

2013). They argued that the lack of response was because T. latifolia was adapted to a high

light environment, but this could also be explained due to changes in SLA after maturation of

leaves (Oguchi et al. 2005) in combination with their transfer study. Other studies on

Chenopodium album and several deciduous trees have reported that acclimation to light

intensity after leaf development was due to chloroplasts filling unoccupied mesophyll cell

border (Oguchi et al. 2003, 2005). T. latifolia could then be adapted to the light intensities

experienced in its natural habitat by having a large proportion of unfilled mesophyll border.

As the leaf elongates and is pushed up through the canopy more and more unoccupied

mesophyll cell border is occupied. Oguchi et al. (2005) argued that if mesophyll border did

not become occupied it would render an extra cost, but as T. latifolia is native to high light

habitats, close to complete occupation would be expected and no biomass would therefore be

wasted. The difference in SLA reported here could be due to that the measurements were

made at different relative heights on the leaf, as SLA scales with relative height (Knapp and

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Yavitt 1995), and in Fig. 4 the SLA values for HL are all below the regression line, indicating

in fact that there is a difference in SLA due to light the environment.

The decrease in SLA allows a higher area-based activity of the enzymes, in the

Calvin-cycle, but also other enzymes, which is known to scale positively with Amax-area

(Walters 2005). Another consequence of the difference in SLA was that even though higher

irradiance resulted in more biomass it did not translate into more leaf area. This indicates that

the LL treatment resulted in an allocation pattern where light harvesting is maximised, and

this is supported by the higher chl a+b content per mass. The HL treatment increased the

numbers of stomata present on both sides of the leaf. The increase in stomatal density and gs

allowed a high Ci which is in contrast to other reports on T. latifolia, where no effects on Ci

were observed (Ojanguren and Goulden 2013). An unavoidable consequence of taking up

CO2 is the concomitant loss of water. Since T. latifolia is associated with waterlogged soil,

loss of water should not impose a restriction on growth as much as for terrestrial plants. In

fact the high transpiration could allow a high nutrient uptake, to match the high Amax-area,

especially in the HL treatment, resulting in the high productivity reported for Typha sp.

(Westlake 1963; Whittaker 1975; Wetzel 1992; Rocha and Goulden 2009).

The pigment composition of T. latifolia in this study was consistent with our second

hypothesis, reflecting the light gradient in the canopy. Biochemical changes could also be

responsible for the increase in Amax-area, as chl b decreased with increasing irradiance in the

laboratory study. This affected the chl a/b ratio, because no changes were observed in chl a.

The chl a/b ratio is thought to reflect the investment between light harvesting and electron

capacity, because chl b is only present in the antenna complex (Walters 2005). With

increasing irradiance the capacity of the photosystems is increased while the amount of

antenna complexes is downregulated, so the amount of excitation energy absorbed is matched

with the capacity to process electrons. This is in agreement with previously mentioned

correlation between Amax-area and enzyme capacity, as these enzymes are involved in further

processing of the excitation energy. The chl a/b ratio reported for T. latifolia does not deviate

substantially from the chl a/b ratio reported in other species and is not particularly plastic

(Murchie and Horton 1997; Esteban et al. 2015)

The VAZ pool size increased with increasing irradiance, but only when expressed

relative to the amount of chl. This increase reflects a higher level of possible photoprotection

and is very high in T. latifolia compared to other species (Demmigadams and Adams 1992;

Esteban et al. 2015). In fact the size of the VAZ pool for T. latifolia in this study is one of the

largest ever reported, lying within the top 0.5% of all plant species as reviewed by Esteban et

34/51

al. (2015) This high level of protection could offer a competitive advantage, because T.

latifolia is found growing in habitats that are exposed to full sunlight most of the day. Inter

conversion between the three pools of V, A and Z determines the actual protection, and the

size of the three pools differed in response to irradiance. When comparing the treatments the

LLT and LLB treatment had the highest amount converted to Z, while the opposite was true

for A. This could seem contradictory at first, but as the inter conversion reflects the actual

protection, i.e. just before the segment was frozen, it can be explained as that during gas

exchange measurements both treatments received the same amount of light, but because the

HL treated plants had a higher Amax-area a higher proportion of excitation energy was

processed through the Calvin cycle resulting in a lower pH gradient over the thylakoid

membrane. The pH gradient is known to exert control of the inter conversion between V, A

and Z (Gilmore 1997). In the LLT treatment the pH gradient would have been higher and

thereby explaining the higher proportion of Z in the VAZ cycle for the LLT and LLB

treatments. In the field study this artefact from measuring was not evident, possibly due to the

much higher light intensity experienced by field plants. Another measure of the protection

level is the deepoxidation state, but due to the opposite trend for A and Z between treatments

in the laboratory study or lack of response in the field no differences in the deepoxidation

state was observed. In general there was a substantial amount (ca. one fifth to one third) of V

in the VAZ pool, indicating that the high irradiance experienced in the field was below

harmful levels in this species. The same conclusion can be drawn from the Fv/Fm ratio even

though it differs between treatments. The Fv/Fm ratio in green healthy non photo inhibited

leaves is ≈0.83 (Demmigadams and Adams 1992). This value is not substantially different

from the measured value reported here, so even though there is a significant difference

between treatments, this difference is likely too small to be of any biological relevance to

plant performance.

Ontogenetic effects are evident from the measurements on individual plants at

different heights. Because the meristematic zone in T. latifolia is positioned at the base of the

leaf, cell division and cell expansion push the leaf upwards. Growth by this mechanism

results in the leaf tip being the oldest part of the leaf with decreasing age down to the leaf

base. Changes in SLA can mediate responses to light acclimation, and explain differences

along the leaf, but changes along the leaf are not mediated through SLA in T. latifolia, which

is apparent from the difference in Amax-mass along the leaf. The changes along the leaf could be

caused by ontogenetic effects, with the lower part of the leaf not possessing fully developed

photosynthetic machinery. This would explain why pigment content on mass basis is low in

35/51

the bottom part of the leaf and why Rd is comparable or even higher than in the top part of the

leaf. The lower part of the leaf is still synthesising pigments which raises the respiratory

demands. Higher porosity in the lower part also indicates that all cells are not yet fully

developed, yet again increasing the respiratory demands in the lower part. This agrees with

the previously mentioned acclimation strategy where mesophyll cell border was left

unoccupied. Some of the differences along the leaf are possibly not due to ontogenetic

effects. This is seen in the chl a/b ratio, as it increase with higher leaf position. The same is

true for VAZ when expressed relative to the amount of chl. From VAZ/chl it can also be seen

that the field plants actually acclimated to their light environment even though it did not lead

to differences in Amax-area. This is so because the lower part in the O-stand received more light

than in the D-stand, which was processed through the relatively larger VAZ pool.

In conclusion, T. laifolia appears to acclimate to light by modulating SLA and

chl a/b ratio in response to its light environment, but these changes are not substantial

compared to other species. Instead the VAZ pool size is very high, and is likely adaptive in T.

latifolia’s native habitat, where conditions with full sunlight are frequent. High porosity and

gs were also high; allowing high rates of CO2 diffusion to meet the demand required for high

Amax-area in this species. Due to its basal growth ontogenetic effects were apparent in the lower

part of the leaf, but were still capable of fairly high Amax-area. Further studies could address the

importance of filling unoccupied mesophyll cell border with chloroplasts in response to light

acclimation.

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Figures and Tables

Fig 1: Light attentuation as a function of the height in the growth chamber. A height of 0 m

corresponds to the leaf tip. LL = low light, HL = high light = HL, LR and RR = left or right

rear, LF and RF = left or right front, M = middle, and corresponds to where in the chamber

the measurement were made at. LL regressions: LR Δ y = 325e-1.029x R² = 0.97; RR □ y =

369e-1.174x R² = 0.99; LF ▲ y = 284e-1.039x R² = 0.96; RF ♦ y = 305e-1.013x R² = 0.97;

M ○ y = 305e-1.013x R² = 0.97. HL regressions: LR Δ y = 1036e-0.855x R² = 0.98; RR □ y =

980e-1.059x R² = 0.99; LF ▲ y = 1005e-0.915x R² = 0.99; RF ♦ y = 958e-1.17x R² = 0.98; M

○ y = 1039e-0.895x R² = 0.97.

HL

550

650

750

850

950

1050

0 0,2 0,4

Lig

ht in

tensity (

µm

ol m

-2 s

-1)

Height (m)

LL

110

160

210

260

310

360

0 0,2 0,4 0,6 0,8

Lig

ht in

tensity (

µm

ol m

-2 s

-1)

Height (m)

44/51

Fig 2: Light attentuation as a function of the height in the canopy in the field study. The

measurment at the greatest height is immediately above the the leaf tips. The green line

represents the top of the grass understory. C = closed stand, O = open stand. Values are mean

±sd (n=5) for each profile.

C y = 0.0275e2.042x

R² = 0.99

0

0,2

0,4

0,6

0,8

1

0 0,5 1 1,5 2Rela

tive lig

ht in

tensity

Height (m)

O y = 0.2875e0.786x

R² = 0.96

0

0,2

0,4

0,6

0,8

1

0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8Rela

tive lig

ht in

ten

sity

Height (m)

45/51

Fig 3: A: Light response curves from the laboratory study. LLT = low light top, HL = high

light, LLB = low light bottom. B: Light response curves from the field study. DB = dense

stand bottom, DT = dense stand top, OB = open stand bottom, OT = open stand top. Values

are mean ± sd. For the laboratory study n=11-12, for the field study n=4-5.

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Table 1: Morphological responses to the three irradiance treatments in the growth cabinet

during the laboratory study. LLT = Low light top, LLB = low light bottom, HL = high light.

Values are mean±sd, (n = 11-12). Small letters in superscript indicates significant difference

between treatments at the 0.05 level for Tukeys HSD test

Parameter

LLT LLB HL

Stomatal density (mm-2

)B

314±44a 335±31

ab 373±40

b

Stomatal ad:ab ratioA

0.93±0.09*a

1.08±0.15*b 1.03±0.12

ab

Porosity (%) 72±7a 78±4

b 70±3

a

Leaf area (m2 plant

-1) 0.10±0.04 0.11±0.01

Above ground biomass (gDW plant-1

) 4.4±1.8a 6.5±0.8

b

A Comparison within treatment for stomata ad:ab ratio was made with a t-test to see if the

ratio differed from one. * indicates significant difference at the 0.05 level for the t-test.

B Stomatal density is the average of the adaxial (ad) and abaxial (ab) side.

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Figure 4: Correlation between relative height measurements were made at and SLA. □ is HL

= high light, ▲ is LLT = low light top and ● is LLB = low light bottom. The regression line

is based exclusively on LLB and LLT (▲ and ●). LLT 24.5±5 m2 kgDW

-1 was significant

higher than LLB 17.8±3 m2 kgDW

-1 and HL 16.5±2 m

2 kgDW

-1 at the 0.05 level for Tukeys

HSD test. Values are mean±sd, (n=11-12).

y = 19.1x + 11.2 R² = 0.47 p = 0.002

0

5

10

15

20

25

30

35

0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0

SLA

(m

-2 k

gD

W-1

)

Relative height

48/51

Table 2: Responses of gas exchange parameters to the three irradiance treatments in

laboratory study. LLT = low light top, LLB = low light bottom, HL = high light. Values are

mean±sd, (n = 11-12). Small letters in superscript indicates significant difference between

treatments at the 0.05 level for Tukeys HSD test

ParametersA

LLT LLB HL

Amax-area (µmolCO2 m-2

s-1

) 22.6±7.5a 16.6±4.4

b 41.5±3.5

c

Rd (µmolCO2 m-2

s-1

) 0.75±0.2a 0.85±0.3

ab 1.00±0.2

b

Φ (µmolCO2 µmol photons-1

) 0.06±0.01 0.06±0.01 0.06±0.01

θ 0.32±0.3a 0.48±0.2

a 0.09±0.2

b

Isat (µmol photons m-2

s-1

) 415±160a 305±97

a 763±112

b

Icomp (µmol photons m-2

s-1

) 13±4a 15±5

ab 18±2

b

gs (molH2O m-2

s-1

) 0.48±0.4a 0.22±0.1

a 1.13±0.2

b

Ci (ppmCO2) 269±48a 261±27

a 313±13

b

T (mmolH2O m-2

s-1

) 6.8±4.2a 4.2±1.8

a 13±1.1

b

WUE (µmolCO2 molH2O-1

) 63±33a 71±18

a 30±8

b

Amax-mass (µmolCO2 gDW-1

s-1

) 0.57±0.3a 0.30±0.1

b 0.68±0.1

a

Relative height of measurements 0.69±0.09a

0.36±0.03b

0.51±0.05c

A Amax-area = Area based light saturated photosynthesis, Rd = area based dark respiration, Φ =

quantum yield, θ = convexity, Ik = light saturation point, Ic = light compensation point, gs =

stomata conductance, T = transpiration, WUE = instantaneous water use efficiency, Amax-mass

= mass based light saturated photosynthesis.

49/51

Table 3: Comparison of pigment content and chlorophyll fluorescence to the three irradiance

treatments for the laboratory study. LLT = low light top, LLB = low light bottom, HL = high

light. Values are mean±sd, (n = 10-11). Small letters in superscript indicates significant

difference between treatments at the 0.05 level for Tukeys HSD test

ParameterA

LLT LLB HL

Chl a (µmol m-2

) 258±60 264±56 206±66

Chl b (µmol m-2

) 67±17a 68±16

a 49±16

b

Chl a+b (µmol m-2

) 324±77 333±72 254±83

VAZ (µmol m-2

) 63±15 62±13 70±21

β-carotene (µmol m-2

) 35±9 36±7 31±9

Chl a/b ratio 3.9±0.2a 3.9±0.2

a 4.2±0.1

b

VAZ/chl ratio 0.19±0.02a 0.19±0.01

a 0.28±0.03

b

V/VAZ ratio 0.31±0.03 0.32±0.05 0.28±0.04

A/VAZ ratio 0.04±0.02a

0.03±0.03a

0.12±0.04b

Z/VAZ ratio 0.65±0.03a 0.65±0.03

a 0.59±0.03

b

Deepoxidation state 1.3±0.1 1.3±0.1 1.3±0.1

Chl a (µmol g-1

DW). 6.2±1.2a 4.7±1.1

b 3.3±0.9

c

Chl b (µmol g-1

DW) 1.6±0.3a 1.2±0.3

b 0.79±0.2

c

Chl a+b (µmol g-1

DW) 7.8±1.5a 5.9±1.4

b 4.1±1.1

c

VAZ (µmol g-1

DW) 1.50±0.4a 1.10±0.3

b 1.14±0.3

b

β-carotene (µmol g-1

DW) 0.84±0.2a 0.63±0.2

b 0.50±0.1

b

Fv/Fm 0.83±0.02a 0.84±0.01

a 0.81±0.01

b

A chl a = chlorophyll a, chl b = chlorophyll b, V = violaxanthin, A = antheraxanthin, Z =

zeaxanthin, chl a/b ratio = chlorophyll a relative to b, Fv/Fm = effective quantum yield of

PSII.

50/51

Table 4: Responses of gas exchange parameters and morphology to irradiance and leaf height

for the field study. CT = closed stand top, CB closed stand bottom, OT = open stand top, OB

open stand bottom. Values are mean±sd, (n = 4-5). Small letters in superscript indicates

significant difference between treatments at the 0.05 level for Tukeys HSD test

ParameterA CT CB OT OB

Amax-area (µmolCO2 m-2

s-1

) 40.5±5.3a 26.7±2.9

b 40.2±6.5

a 27.8±2.8

b

Rd area (µmolCO2 m-2

s-1

) 1.1±0.25a

0.93±0.24a

0.98±0.20a

1.54±0.21b

Φ (µmolCO2 µmol photons-1

) 0.07±0.02 0.06±0.01 0.06±0.01 0.06±0.01

θ 0.02±0.03 0.02±0.03 0.03±0.05 0.10±0.09

Ik (µmol photons m-2

s-1

) 644±129ab

470±79b 751±171

a 490±79

ab

Ic (µmol photons m-2

s-1

) 17±6 16±4 18±5 25±3

gs (molH2O m-2

s-1

) 0.91±0.37 0.37±0.15 0.68±0.14 0.60±0.26

Ci (ppmCO2) 283±39 285±14 277±17 290±25

T (mmolH2O m-2

s-1

) 6.5±1.8 4.7±0.6 4.5±0.6 4.4±2.2

WUE (µmolCO2 molH2O-1

) 41±21 46±11 46±9 42±17

Amax-mass (µmolCO2 gDW-1

s-1

) 0.57±0.08a 0.27±0.04

b 0.51±0.08

a 0.23±0.03

b

SLA (m2 kgDW

-1) 14.0±2

a 10.3±2

bc 12.4±1

ac 8.1±1

b

Stomatal density (mm-2

)B

366±63 401±82 356±20 415±19

A Amax-area = Area based light saturated photosynthesis, Rd = area based dark respiration, Φ =

quantum yield, θ = convexity, Ik = light saturation point, Ic = light compensation point, gs =

stomata conductance, T = transpiration, WUE = instantaneous water use efficiency, Amax-mass

= mass based light saturated photosynthesis, SLA = specific leaf area. B Stomatal density is

the average of the adaxial (ad) and abaxial (ab) side.

51/51

Table 5: Differences in pigment content to irradiance and effects of leaf height for the field

study. CT = closed stand top, CB closed stand bottom, OT = open stand top, OB open stand

bottom. Values are mean±sd. (n = 4-5). Small letters in superscript indicates significant

difference between treatments at the 0.05 level for Tukeys HSD test

ParameterA

CT CB OT OB

Chl a (µmol m-2

) 297±46 297±60 297±47 280±81

Chl b (µmol m-2

) 80±12 88±17 75±13 77±22

Chl a+b (µmol m-2

) 377±58 385±77 371±60 357±103

VAZ (µmol m-2

) 0.21±0.06 0.23±0.06 0.21±0.04 0.18±0.05

β-carotene (µmol m-2

) 36±7 36±6 38±6 35±6

Chl a/b ratio 3.7±0.1a 3.4±0.1

b 4.0±0.1

c 3.7±0.1

a

VAZ/chl ratio 0.24±0.01ab

0.21±0.02b 0.25±0.02

a 0.25±0.03

a

V/VAZ ratio 0.21±0.06 0.23±0.06 0.21±0.04 0.18±0.05

A/VAZ ratio 0.15±0.03 0.12±0.02 0.16±0.03 0.15±0.02

Z/VAZ ratio 0.64±0.05 0.65±0.05 0.63±0.05 0.67±0.05

Deepoxidation state 1.4±0.1 1.4±0.1 1.4±0.1 1.5±0.1

Chl a (µmol g-1

) 4.1±0.6a 3.0±0.7

bc 3.7±0.4

ac 2.3±0.6

b

Chl b (µmol g-1

) 1.1±0.2b 0.90±0.2

b 0.92±0.1

b 0.62±0.2

a

Chl a+b (µmol g-1

) 5.3±0.8a 3.9±0.9

bc 4.6±0.5

ac 2.9±0.7

b

VAZ (µmol g-1

) 1.27±0.22a 0.80±0.13b 1.16±0.13a 0.71±0.12

b

β-carotene (µmol g-1

) 0.50±0.1a 0.37±0.1

bc 0.47±0.1

ac 0.28±0.0

b

A chl a = chlorophyll a, chl b = chlorophyll b, V = violaxanthin, A = antheraxanthin, Z =

zeaxanthin, chl a/b ratio = chlorophyll a relative to b.