Consequences of chlorophyll deficiency for leaf carotenoid

Journal of Experimental Botany, Vol. 49, No. 320, pp. 535–546, March 1998

Consequences of chlorophyll deficiency for leafcarotenoid composition in tobacco synthesizingglutamate 1-semialdehyde aminotransferase antisenseRNA: dependency on developmental age and growth light

Heiko Hartel1,2,3 and Bernhard Grimm1

1 Institut fur Pflanzengenetik und Kulturpflanzenforschung, Corrensstrasse 3, D-06466 Gatersleben, Germany2 Humboldt Universitat zu Berlin, Institut fur Biologie/Pflanzenphysiologie, Philippstrasse 13, D-10115 Berlin,Germany

Received 26 June 1997; Accepted 5 November 1997

Abstract suggests a co-regulation between carotenoid andchlorophyll synthesis; the second emphasizes the

Transgenic tobacco (Nicotiana tabacum), with aspecial role of carotenoids for protection against

reduced chlorophyll content of up to less than 10% oflight stress.

the wild-type level due to a different expression ofantisense RNA coding for glutamate 1-semialdehyde

Key words: Light acclimation, photoprotection, quantumaminotransferase, were used to study the relationshipyield, lutein, xanthophyll cycle.between chlorophyll accumulation and changes in

carotenoid composition in developing and matureleaves grown either under low (30 mmol photons

Introductionm−2 s−1) or high light (300 mmol photons m−2 s−1).Regardless of the extent to which chlorophyll synthesis Like chlorophylls (Chls), carotenoids of leaves are ubi-was reduced, under low light the ratios of total chloro- quitous structural components of the photosyntheticphyll to carotenoids remained constant. In contrast, apparatus. In higher plants, Chls and carotenoids areunder high light the content of carotenoids was elev- bound to specific proteins to form either reaction centreated relative to chlorophyll and increased further with pigment–protein complexes or light-harvesting pigment–progressive inhibition in chlorophyll synthesis. The protein complexes (LHCs) of photosystem (PS) I andxanthophyll-cycle pigment pool was most strongly PSII (LHCII ). Attempts to ascertain the distribution ofincreased (up to 18-fold) upon supression of chloro- carotenoids within the multiple pigment–protein com-phyll synthesis. Concurrently to the higher pool sizes plexes by detergent fractionation yielded varying resultsa higher extent of violaxanthin was converted into between research groups, probably due to distinct isola-antheraxanthin and zeaxanthin and this was found to tion procedures and differences in both the growth condi-be correlated with a decrease in the quantum yield of tions and plant species. Nevertheless, b-carotene wasphotosystem II photochemistry. While lutein increased consistently found in the reaction centre/core complexes(up to 3-fold) with decreasing chlorophyll contents in of both PSI and PSII, whereas xanthophylls mainly bindhigh light transformants, neoxanthin remained rather to LHCs (Thayer and Bjorkman, 1992; Bassi et al., 1993;constant in all plants analysed. Based on the present Ruban et al., 1994; Lee and Thornber, 1995). Lutein isresults, two different levels for the regulation of carot- normally the predominant xanthophyll, accounting forenoid synthesis are proposed depending on (i) the up to 50% of total carotenoids in most plants. 80% andchlorophyll synthesizing capacity, and (ii) the photo- more of all the lutein and neoxanthin were found to be

associated with the peripheral trimeric LHCII, consistingsynthetic light utilization efficiency. The first point

3 To whom correspondence should be addressed at Gatersleben: Fax: +49 39482 5139. E-mail: [email protected]

© Oxford University Press 1998

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536 Hartel and Grimm

of the major polypeptides Lhcb1 and Lhcb2 (Thayer and molecular structure of the thylakoid membrane, and theirchemical and physical properties are strongly influencedBjorkman, 1992; Bassi et al., 1993; Ruban et al., 1994).

However, low amounts of lutein were frequently found by other molecules in their vicinity, such as proteins andmembrane lipids (Plumley and Schmidt, 1987;to be associated with the reaction centre/core complex as

well (Bassi et al., 1993; Lee and Thornber, 1995). Whilst Kuhlbrandt et al., 1994; for a review see Britton, 1995).Because the pigment concentration of leaves per unit arealutein is considered to bind within the protein backbone

of LHCs, neoxanthin seems to bind along the periphery can vary considerably in wild-type plants dependent onthe growth conditions (Bjorkman, 1981), no consistent(Plumley and Schmidt, 1987; Kuhlbrandt et al., 1994).

Violaxanthin (V ) and its de-epoxidation products anther- correlation between the photosynthetic pigment accumu-lation and the growth light intensity was found until now.axanthin (A) and zeaxanthin (Z) are enriched in the

inner LHCII antenna proteins Lhcb4 to Lhcb6 (Bassi In this respect the recently described glutamate1-semialdehyde aminotransferase (GSA-AT) antisenseet al., 1993).

Carotenoids have important functional roles in the RNA plants of tobacco provide a novel tool. These plantsare characterized by different degrees of lowered GSA-ATphotosynthetic apparatus of higher plants. They serve as

accessory pigments by supplementing Chl in light har- activities leading to a reduced 5-aminolevulinic acid-synthesizing capacity and in consequence to a loweredvesting and thereby enable plants to utilize light efficiently

over a broad wavelength range. Furthermore, carotenoids Chl content relative to the leaf area (Hofgen et al., 1994;Hartel et al., 1997). The plants show a wide variety ofhave long been associated with a pivotal role in the

photoprotection of the photosynthetic apparatus by limit- inheritable Chl variegation pattern in tobacco GSA-ATantisense RNA plants (Hofgen et al., 1994; Hartel et al.,ing the destructive reactions of 1O2 via direct quenching

of 1O2 or of the Chl triplet state that sensitizes formation 1997). The spectrum of Chl abundance ranged from wildtype-like to uniformly bleached yellow leaves. Otherof reactive oxygen species (Cogdell et al., 1992; Telfer

et al., 1994; for reviews see Siefermann-Harms, 1987; enzymes of the tetrapyrrole pathway seem not to beinfluenced by the antisense gene effect, which largely rulesBartley and Scolnik, 1995). Because of their supposed role

in deactivating excess excitation energy of light (Demmig- out the possibility that potentially dangerous tetrapyrroleintermediates accumulate (Hartel et al., 1997). Therefore,Adams et al., 1996), the regulation of the synthesis of the

xanthophyll-cycle pigments has attracted much interest. these GSA-AT antisense plants offer a novel opportunityto gain greater insight into how the carotenoid biosyn-It is thought that the carotenoids are involved in the

structural reorganization of the individual PS components thetic pathway is regulated in plants upon Chl depriva-tion. Since carotenoids are of crucial importance for thein response to varying light conditions. Variations in

carotenoid composition and energy dissipation among photoprotection of the photosynthetic apparatus, studiesof these plants improves the understanding of the strat-different species grown under various field conditions

have been described previously (Thayer and Bjorkman, egies developed to cope with excess light energy. Theresults demonstrate a distinctly different response of indi-1990; Demmig-Adams and Adams, 1992; Johnson et al.,

1993; Brugnoli et al., 1994; Demmig-Adams et al., 1995). vidual carotenoid components under Chl shortage. It issuggested that the carotenoid pattern in leaves dependsMoreover, the abundance of individual carotenoids is

differently affected in mutant plants with altered antenna on both the Chl turnover and the photosynthetic lightutilization efficiency.composition (Hofer et al., 1987; Knoetzel and Simpson,

1991; Schindler et al., 1994; Falbel et al., 1994; Plumleyand Schmidt, 1995; Hartel et al., 1996) and in transgenicplants with a genetically lowered photosynthetic capacity

Materials and methodsas well (Bilger et al., 1995).However, the regulation of carotenoid synthesis and Plant material and growth conditions

the precise function of carotenoids in photosynthesis and Wild-type tobacco (Nicotiana tabacum var. Samsun NN) plantsand GSA-AT antisense RNA transformants were cultivated inthe assembly of the photosynthetic apparatus have notsoil with a 12/12 h light/dark cycle at 65% relative humidity inbeen completely understood. The use of mutants affecteda growth chamber under two different photon flux densitiesin their ability to synthesize specific carotenoids is one(PFD, 400–700 nm) constant throughout the light period: High

important tool to assign their function in vivo (Rock light (300 mmol photons m−2 s−1, HL) and low light (30 mmolet al., 1992; Pogson et al., 1996). Another promising photons m−2 s−1, LL) were both provided by an array of

halogen lamps (SON-T AGRO 400, Phillips, Eindhoven,approach to gain new information on the functional andNetherlands). PFDs incident upon the top of plants wereregulatory significance of carotenoids is the manipulationdetermined using a quantum sensor (LI-189A, Li-Cor, Lincoln,of Chl synthesis. It seems well established that both ChlNebraska, USA). The day/night temperature regime was

and carotenoid biosynthesis exclusively take place in controlled at 25/20 °C. For all measurements, 7–10-week-oldchloroplasts. Carotenoids and Chls are characterized by plants were usually used. The construction of the GSA-AT

antisense gene transformants is described in Hofgen et al. (1994).their well-defined location and orientation within the

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Carotenoids in chlorophyll-deficient tobacco 537

Pigment analysis

Samples were harvested 4–5 h after the beginning of lightexposure. Leaves were rapidly frozen in liquid nitrogen forsubsequent pigment determination. Pigments were extractedand analysed by high-performance liquid chromatographyapplying the method of Thayer and Bjorkman (1990) modifiedas described (Hartel et al., 1996). All data shown represent themean of three to five replicates on different plants of the sameline. The de-epoxidation state of xanthophyll-cycle pigments(DPS) was calculated as DPS=(A+Z)/(V+A+Z).

Chl a fluorescence measurements

Measurements of room temperature Chl a fluorescence wereperformed in the growth chamber with a portable PAM-2000Chl fluorometer in conjunction with a leaf clip holder ( Walz,Effeltrich, Germany) immediately before the same samples wereharvested for carotenoid determinations. The PSII quantumyield (WPSII ) was calculated according to Genty et al. (1989). Inorder to ensure the coverage of representative leaf sections,fluorescence was measured in triplicate for light-exposed plantsat three different locations on the leaf. The same leaf sectionswere used for pigment determinations.

Results

Effect of GSA-AT antisense RNA synthesis on Chlaccumulation in dependence on developmental age andgrowth light

GSA-AT tobacco transformants nos. 57, 25 and 42 were Fig. 1. Typical leaves of wild-type tobacco (SNN) and the GSA-ATselected for these investigations. They have been previ- antisense RNA transformants nos 57, 25 and 42 of plants that were

grown for 7 weeks under HL at a PFD of 300 mmol photons m−2 s−1.ously subjected to an extensive genetical and biochemicalcharacterization (Hofgen et al., 1994; Hartel et al., 1997).These transformants cover a wide range of reduced Chlcontent per leaf tissue, which correspond to the reduction in particular between the wild type and the most chlorotic

transformant no. 42, further increased (Fig. 2B). A minorof GSA-AT activity (Fig. 1). The leaves of the mostchlorotic transformant no. 42 are uniformly bleached, shift in the maximum Chl contents towards leaves 8 and

10 occurred in HL-grown wild type and transformantwhile those of other transformants are slightly variegatedin the pigment pattern. To get further information about no. 57.

The Chl a/b ratios were higher in HL leaves of eachthe mechanism which controls carotenoid accumulation,plants were grown under two distinct different PFDs, line compared to those of LL leaves of the same line,

except for the oldest leaves in transformant no. 4230 mmol m−2 s−1 (LL) and 300 mmol m−2 s−1 (HL). Nosignificant macroscopic differences of the phenotypes were (Fig. 2C, D). The Chl a/b ratio strongly altered through-

out leaf development in transformant no. 42 with higherobserved in plants grown under LL or HL conditions.For pigment determinations either whole leaves ratios in young leaves and lower ratios in old leaves in

comparison to both the HL transformants nos. 57 and(number 2 and 4) or large leaf areas (9 cm2) coveringrepresentative leaf sections were used. Figure 2 depicts 25 and the wild type. Remarkably, in all LL lines, the

Chl a/b ratios were very similar, with the exeption of theChl contents measured in total leaf extracts of wild typeand transformants dependent on leaf age and growth very young leaves of no. 42, despite the fact that the Chl

content was reduced up to a factor of seven in trans-light. In LL-exposed plants of wild type and transformantno. 57, maximal amounts of Chl were determined in formants versus the wild type (Fig. 2A, B). Unchanged

Chl a/b ratios upon Chl-deficiency have not been previ-leaves 6 or 8 numbered from the plant top downwards(Fig. 2A), while in chlorotic transformants nos. 25 and ously reported for pigment mutants ( Knoetzel and

Simpson, 1991; Falbel et al., 1994) or intermittent-light42 the Chl content was only marginally changed through-out the entire leaf development. Chl accumulated during grown plants (Hofer et al., 1987; Hartel et al., 1996),

indicating the distinct different response of the GSA-ATthe development of HL-exposed plants similar as observedfor LL plant, even though the differences in Chl content, transformants to the Chl shortage.

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Fig. 2. Chl content on a unit fresh weight basis (A, B) and Chl a/b ratio (C, D) in tobacco wild type (%) and the GSA-AT transformants no. 57(( ), no. 25 (#) and no. 42 (6) grown either under LL (closed symbols) or HL (open symbols) dependent on developmental leaf age. The standarddeviations were always less than 22% (A, B) and 14% (C, D), respectively.

The carotenoid content and composition of GSA-AT this early stage of leaf development are mainly controlledby endogenous factors associated with chloroplast differ-transformants depends on developmental age and growthentation (e.g. Chl and protein synthesis), whereas changeslightin carotenoids relative to Chl upon further maturation of

Figure 3 shows that the reduction in the total carotenoid leaves are additionally influenced by the light regime (ascontent parallels the deprivation of Chl synthesis in indicated by the increase in the carotenoid to Chl ratiotransformants. Like total Chls, GSA-AT transformants in HL transformants nos. 25 and 42). The higher caroten-grown under LL possessed considerably lower levels of oid to Chl ratio in the premature leaves of transformantcarotenoids per unit fresh weight compared to wild-type no. 42 is likely due to the steadily synthesized low amountplants. As a consequence, except for the very young leaves of carotenoids that is usually found in dark-grown plants2 and 4 of transformant no. 42, the relative proportions independent of the presence of Chl (Frosch and Mohr,between Chls and carotenoids did not vary between wild 1980).type and transformants (Fig. 3A). However, a different During assembly of light-harvesting antenna com-response was observed for plants grown under HL. More plexes, antenna protein and pigment biosynthesis arecarotenoids accumulated in the most Chl-deficient trans- tightly linked regulated processes (Hartel et al., 1997).formants nos. 25 and 42, yielding higher levels of caroten- Chl b is exclusively associated with LHCs, in particularoids per Chl relative to those of wild type (Fig. 3B). In the peripheral LHCII. Hence, the amount of Chl b is aall HL plants maximal carotenoid contents were present rough indicator of the amount of LHCs. Furthermore,in leaf 8. A quantitative inspection of the amounts of lutein and neoxanthin are considered to be predominantlyindividual carotenoids revealed that the higher carotenoid associated with the LHCs. Thus, alterations in thesecontents in HL-grown transformants were mainly due to pigments should be indicative for changes in the light-higher amounts of lutein and the xanthophyll-cycle pig- harvesting antenna organization. In LL plants, thements V+A+Z (Fig. 3H, K ), whereas neoxanthin and neoxanthin/Chl b and lutein/Chl b ratios were indistin-to a lesser extent b-carotene yielded similar ratios to Chl guishable, with the exception of the young leaves 2 andin all plants during leaf development (Fig. 3D, F ). The 4 of no. 42, which contained higher levels of neoxanthindecline in the carotenoid content in older leaves, which and lutein compared to Chl b (Fig. 4A, C). This constantis most prominent in chlorotic transformants nos. 25 and stoichiometry between these carotenoids and Chl b42, may be due to the increasing distance from the light throughout an increase in the Chl b content of nearly onesource and shading from upper leaves. order of magnitude from transformant no. 42 to the wild

Interestingly, in leaf 2 the ratio of all individual caroten- type (Fig. 2A) indicate that the synthesis of these pig-oid components to Chl was very similar when the same ments is closely coupled under LL conditions. On thelines were compared independent of whether plants were other hand, a pronounced increase, in particular in thegrown under LL or HL conditions. This may indicate lutein/Chl b ratio but also the neoxanthin/Chl ratio, was

noticed in the HL-exposed transformants nos 42 and 25that changes in carotenoid content and composition at

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Fig. 3. Contents of total carotenoids (A, B), b-carotene (C, D), neoxanthin (E, F), lutein (G, H ) and xanthophyll-cycle pigments (I, K) relative tothe Chl content in tobacco wild type (%) and the GSA-AT transformants no. 57 (( ), no. 25 (#) and no. 42 (6) grown either under LL (closedsymbols) or HL (open symbols) dependent on developmental leaf age. The standard deviations were always less than 10%. V, A, Z denote thecontents of violaxanthin, antheraxanthin and zeaxanthin, respectively.

in comparison both with their LL counterparts and the between carotenoid and Chl content in this particularstage of leaf development. Figure 5 shows the relationshipHL wild type (Fig. 4D). Virtually no differences werebetween the content of total carotenoids and total Chlsfound in the neoxanthin/Chl b and lutein/Chl b ratiosin leaves of wild type and GSA-AT transformants, eachbetween HL and LL variants of wild type and trans-expressed on a unit area basis. A strict linear relationformant no. 57 (Fig. 4B, D).between both pigment groups was found (r2=0.952, solidline) irrespective of the PFD that plants experiencedThe pigment content and composition is distinctly differentduring growth and the degree of suppression of Chlin mature leaves of tobacco wild type and GSA-ATsynthesis. The correlation improved further (r2 ≥0.99)transformantswhen LL and HL variants were plotted separately (see

Since both carotenoid and Chl maximally accumulated dashed lines). Whilst the LL plot extrapolates to theorigin, the HL plot does not, which is due to higherin leaf 8 further analyses were focused on the relations

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Fig. 4. Ratios of neoxanthin/Chl b (A, B) and lutein/Chl b (C, D) in tobacco wild type (%) and the GSA-AT transformants no. 57 (( ), no. 25(#) and no. 42 (6) grown either under LL (closed symbols) or HL (open symbols) dependent on developmental leaf age. The standard deviationswere always less than 8%.

LL. When HL were compared with LL plants, theV+A+Z pool sizes increased 2.0-fold (wild type),3.0-fold (no. 57), 3.1-fold (no. 25) and 10.4-fold (no. 42),respectively. Due to the different light response of Chlsynthesis (Fig. 2), the differences in V+A+Z contentson a Chl basis between wild type and transformants weremore pronounced under HL exposure yielding 1.4-fold(wild type) to 18.3-fold (no. 42) higher pool sizes than inLL plants (Fig. 6D). On a Chl basis, HL-grown no. 42contained a 12.8-fold higher V+A+Z pool size than theHL-grown wild type. The higher V+A+Z pool sizes didnot simply occur at the expense of b-carotene, which is aprecursor in the synthesis of xanthophyll-cycle pigments.

Fig. 5. Relationship between total leaf carotenoid and Chl content per The b-carotene contents were equal or even higher uponunit area in mature leaves of tobacco wild type (%) and the GSA-AT HL exposure in all lines (Fig. 6A). An intriguing resulttransformants no. 57 (( ), no. 25 (#) and no. 42 (6) grown either

is the dramatic increase in lutein contents throughout leafunder LL (closed symbols) or HL (open symbols). The standarddeviations were always less than 10%. The solid line correspond to the maturation (up to 300%) in HL transformants no. 25 andfirst-order regression for both the LL and HL data. In addition separate 42, while virtually no changes were found in the wild typeregression lines for LL or HL data are fitted (dashed lines).

and transformant no. 57 (Fig. 6B). On the other hand,neoxanthin accumulates independent on changes in Chl

amounts of carotenoids in particular in the chlorotic content and PFD (Fig. 6C).transformants nos 25 and 42.

The Chl content increased per unit area in theThe carotenoid composition correlates with the quantum

HL-grown wild type to 142% of the LL content, and inefficiency of PSII photochemistry

the HL-grown transformant no. 57 to 147%, remainedrather constant in no. 25 (99%) and displayed a decrease Figure 7 illustrates the relationship between the levels of

the various carotenoid components (as a percentage ofin no. 42 (58%) (Table 1). Consequently, the most severeChl-deficient transformant no. 42 contained 16% (LL) total carotenoids) and the relative quantum yield of PSII

photochemistry, WPSII, which reflects the degree ofand 7% (HL) of the respective wild-type Chl content.Neoxanthin and to a lesser extent also b-carotene contents absorbed light utilized in photochemistry under the

respective light-adapted state. In LL plants, virtually nofollowed the same pattern. In contrast, lutein contentincreased 1.5-fold in leaves of HL plants relative to LL differences in the relative distribution were found among

all individual carotenoids. In particular, HL-grown Chl-plants. Of the carotenoids, xanthophyll-cycle pigmentsresponded most strongly to the acclimation to HL versus deficient transformants showed dramatic changes in the

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Table 1. Pigment contents in mature leaves of wild-typpe tobacco (SNN) and the GSA-AT transformants nos 57, 25 and 42 grownunder low light (LL) or high light (HL) conditions on a leaf area basis (in mmol m−2)All values represent the means of at least seven different plants. The standard deviations were always less than 15%.

Lines LL HL

Chl b-carotene V+A+Z Neoxanthin Lutein Chl b-carotene V+A+Z Neoxanthin Lutein

SNN 360.2 32.0 10.2 14.9 42.6 511.4 52.7 20.7 19.8 61.957 263.7 25.2 8.0 11.0 34.5 387.7 40.8 24.1 17.8 50.025 163.3 18.0 6.0 7.3 19.1 161.0 20.4 18.4 6.8 25.542 58.6 4.8 1.7 2.5 6.7 33.9 4.8 17.6 1.6 10.0

Fig. 6. Relationship between individual carotenoids normalized to Chl and the Chl content per unit area in mature leaves of tobacco wild type (%)and the GSA-AT transformants no. 57 (( ), no. 25 (#) and no. 42 (6) grown either under LL (closed symbols) or HL (open symbols). Thestandard deviations were always less than 8%.

carotenoid pattern. The strongest alteration was found excitation pressure on PSII in parallel to the reduction inChl content in HL transformants. A close relationshipfor xanthophyll-cycle pigments, which increased from

about 10% in LL plants to more than 50% in (r2=0.977) was also apparent when the V+A+Z poolsize was plotted against the DPS (Fig. 8B). No differenceHL-transformant no. 42. While the WPSII values were

similar for all LL plants (solid symbols), they showed a was found in the DPS levels in plants grown under LL.Virtually all violaxanthin remained epoxidized. Leaves ofdecrease with reduced Chl content in HL transformants

(open symbols), indicative for a higher extent of excess HL plants did not only reveal higher pool sizes ofxanthophyll-cycle pigments, but also showed a higherlight in HL transformants. There is a positive correlation

between these changes in the WPSII values and the fractions DPS than LL plants. 85% of the total xanthophyll-cyclepigments were present as A and Z in transformant no.of b-carotene (r2=0.896, Fig. 7A), lutein (r2=0.860,

Fig. 7B) and neoxanthin (r2=0.885, Fig. 7C) and a nega- 42 upon HL conditions.The high differences in the pool sizes of V+A+Z (ontive correlation between WPSII and the fraction of xantho-

phyll-cycle pigments (r2=0.935, Fig. 7D). a Chl basis) between the wild type and transformantsunder HL conditions rule out that the PFD to which theDecreases in WPSII were linearly related to increases in

the DPS (Fig. 8A). All values closely fit to the same plants were exposed during growth is the sole determin-ator. In fact, there appears to be a close link between theregression line (r2=0.959), despite the large differences

in both the absolute amounts of A and Z present as well V+A+Z pool size and the depression of WPSII, i.e. theincrease in the excitation energy pressure on PSII. Toas in the Chl content (Fig. 2; Table 1). Since both WPSII

and DPS are mainly controlled by the acidification of the support this idea, the amounts of xanthophyll-cycle pig-ments, DPS and WPSII were correlated in leaves of differentthylakoid lumen space, these results point to an increasing

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Fig. 7. Relationship between b-carotene (A), lutein (B) and neoxanthin (C) and xanthophyll-cycle pigments each expressed as a percentage of totalcarotenoids and WPSII in mature leaves of tobacco wild type (%) and the GSA-AT transformants no. 57 (( ), no. 25 (#) and no. 42 (6) growneither under LL (closed symbols) or HL (open symbols). The standard deviations were always less than 8%. The dashed lines correspond to thefirst-order regression to the data.

Fig. 8. Relationship between the DPS and either WPSII (A) or the pool size of xanthophyll-cycle pigments (B) in mature leaves of tobacco wild type(%) and the GSA-AT transformants no. 57 (( ), no. 25 (#) and no. 42 (6) grown either under LL (closed symbols) or HL (open symbols). Thestandard deviations were always less than 14%. The dashed lines correspond to the first-order regression to the data.

age of transformant no. 42 grown either under LL or HL leaf gradient of both LL and HL-grown transformant no.42 (r2=0.962; Fig. 9C).conditions. No. 42 has the greatest difference in the

V+A+Z pool size between LL and HL-grown plantsand among the leaves of different age of HL plants. DiscussionFigure 9A shows the plot of the DPS versus the corres-ponding V+A+Z pool size. As apparent, a curvilinear The present studies were conducted to understand better

the regulatory mechanisms determining carotenoid syn-relationship was obtained between both parameters. Thisis mainly caused by the fact that the DPS did not respond thesis and accumulation in plants. For this purpose,

transgenic tobacco plants with differently reduced Chlto the altered V+A+Z pool size in LL transformants(see also Fig. 8A), while in HL transformants a strong contents were grown under two different PFDs. The

alterations in the Chl content were achieved by expressinglinear correlation exists. A similar relationship wasobtained when WPSII was plotted against the pool size of antisense RNA for GSA-AT in tobacco plants (Hofgen

et al., 1994; Hartel et al., 1997). GSA-AT catalyses thexanthophyll-cycle pigments (Fig. 9B). However, WPSII lin-early correlated with the DPS along the developmental last step in the formation of 5-aminolevulinic acid, which

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Fig. 9. Relationship between the DPS (A) or WPSII (B) and the pool size of xanthophyll-cycle pigments, and between WPSII and the DPS (C) indeveloping leaves of the GSA-AT transformant no. 42 (6) grown either under LL (closed symbols) or HL (open symbols). Values obtained withleaves 2 to 8 are shown. The standard deviations were always less than 14%. The dashed line in (C) corresponds to the first-order regression to the data.

is rate limiting for the entire Chl synthesis (Beale and this finding suggests that Chl and carotenoid synthesisare co-regulated in plants. In fact, both the Chl andWeinstein, 1990). This approach enables us to follow

changes in the carotenoid content from two different carotenoid pathway share the precursor geranyl geranylpyrophosphate (Beale and Weinstein, 1990; Bartley andpoints of view. First, the large range of Chl reduction of

selected transgenic plants allows a more detailed study Scolnik, 1995). Thus, co-ordination of the synthesis ofChls and carotenoids could take place at the level ofon the interrelations between Chl and carotenoid synthesis

in developing and mature leaves. Second, it was possible geranyl geranyl pyrophosphate availability. A largelyproportional decrease in Chl and carotenoid contents wasto study the effects of light dosis on the carotenoid pattern

in plants of a single species, but with genetically defined recently also reported for spinach leaves that were exposedto nitrogen stress (Verhoeven et al., 1997), which mayreductions in Chl content under highly standardized

growth conditions in the laboratory. So far, most studies corroborate the assumption of a tight control in thesynthesis of both pigment groups.on carotenoid changes under different PFDs were carry

out with plants grown under field conditions, which inhere However, under HL conditions the gradual reductionof total Chl in GSA-AT transformants quite differentlythe difficulties to exclude secondary effects by other

environmental factors. affects the abundance of individual carotenoids. In alllines higher carotenoid contents were found relative toDiminished GSA-AT activity results in dramatic

changes not only in the Chl but also in the carotenoid Chl (Fig. 5). Xanthophyll-cycle pigments responded moststrongly to the change of the light regime. Consistentcontents (Figs 2, 3). Independent of the extent of Chl

deficiency, the proportions between total Chl and caroten- with results obtained with numerous different field-grownplant species (Thayer and Bjorkman, 1990; Demmig-oids appreciably correlated under the LL regime. This

commensurate decline of carotenoids with Chls is cer- Adams and Adams, 1992; Johnson et al., 1993; Brugnoliet al., 1994; Demmig-Adams and Adams, 1996)tainly related to the changes in the amount of individual

pigment-protein complexes which bind carotenoids, but HL-grown plants contained elevated pool sizes of xantho-phyll-cycle pigments compared with LL plants (Figs 3, 6;which obligatorily require Chl for stabilization (Hartel

et al., 1997, and references cited therein). Assuming that Table 1). However, large differences in the enhancementof the V+A+Z pool size between wild type and the palethe carotenoid contents measured mainly reflect rates of

synthesis and are not merely due to higher turnover rates, GSA-AT transformants were found. Depending on

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whether Chl or leaf area was used as reference unit the (1994), leaves of plants grown under HL showed signific-antly higher lutein contents (up to 3-fold in line no. 42;increase varied from 1.4- or 2.0-fold, respectively, in the

wild type to 18.3- or 10.4-fold, respectively, in trans- Figs 3H, 6C) than those grown under LL. On the otherhand, no appreciable differences were found in neoxanthinformant no. 42. This implies that HL exposure to plants

does not lead to a proportionate increase in the pool size contents in LL and HL grown plants (Figs 3F, 6C).Under the assumption that (i) all carotenoids are alsoof xanthophyll-cycle pigments and that a clear trend exists

for the V+A+Z content to increase with decreasing bound to proteins in transgenic lines, and (ii) the indi-vidual pigment-binding proteins possess specific bindingChl content.

One may argue that the lack of a direct correlation sites for the different pigments, one might in fact expect,due to the higher Chl a/b ratios in transformants, abetween total V+A+Z and the PFD incident on trans-

genic plants could simply be explained by the variations decrease in the levels of both neoxanthin and lutein thatare thought to be associated mostly with the peripheralin the amount of Chl, since this was the reference unit

for expressing the pool sizes. First of all, differences in LHCII. While the neoxanthin/Chl b ratios showed, exceptfor the younger leaves of no. 42, minor differencesthe pool sizes were not found in Chl-deficient GSA-AT

transformants grown under LL conditions when expressed (Fig. 4A, B), the higher lutein/Chl b ratios inHL-transformants no. 42 and 25 (Fig. 4D) may indicateon a Chl basis. Apart from this, it has to be emphasized

that in HL plants the amount of xanthophyll-cycle pig- that either the proportion of lutein associated with theinner PSII antenna increased and/or large amounts ofments drastically more increased than does the Chl con-

tent declined (Fig. 2; Table 1). Moreover, the progressive lutein must bind to the reaction centre/core complexesunder Chl-deficiency. In favour of a regulatory responseincrease in V+A+Z contents at consistent low levels of

Chl in expanding leaves of the most chlorotic trans- is the progressive increase in the lutein content throughoutleaf development in HL transformants no. 42 and 25formant no. 42 (Fig. 3K) is clearly indicative of a regu-

latory response. On the other hand, due to the drastic (Fig. 3H), which seems to parallel the changes in theV+A+Z contents (Fig. 3K). This increase of luteindecline of the Chl content in GSA-AT transformants, a

progressive decline in the proportion of incident light that contents would be difficult to understand, if large propor-tions of lutein are present as free (unbound to proteinis absorbed by leaves can be predicted. This implies that

the changes in the V+A+Z pool size under both LL complexes) pigments in transformants.The changes in the amounts of xanthophyll-cycle pig-and HL conditions are not a direct consequence of the

photon receipt by leaves and that another intrinsic factor ments in GSA-AT transformants are fully consistent withtheir proposed role in photoprotection. Evidence has beenmust control the carotenoid synthesis. Owing to the

standardized growth conditions, the possibility can be accumulated that dissipation of potentially harmful excessexcitation energy occurs directly within the LHCs of PSIexcluded that differences in light quality and temperature

may have affected the V+A+Z pool size. Importantly, and PSII and that the xanthophyll cycle mediates thisprocess (for recent reviews see Demmig-Adams et al.,the V+A+Z pool size correlated with the PSII quantum

efficiency. The transformants with the lowest WPSII values, 1996; Gilmore, 1997). Recent work has indicated theimportance of the minor LHCs of PSII in the process ofi.e. with the lowest fraction of absorbed light that is

utilized in PSII photochemistry, had much higher excess excitation energy dissipation. They are enriched inxanthophyll-cycle pigments (Bassi et al., 1993; RubanV+A+Z pool sizes than the wild type and those trans-

formants with higher WPSII values (Figs 8, 9). This suggests et al., 1994) and they seem to be the main site for theformation of the quenching species which mediate heatthat the excitation energy pressure on the photosynthetic

apparatus seems to determine the V+A+Z pool size and dissipation (Crofts and Yerkes, 1994; Hartel and Lokstein,1995; Gilmore, 1997). Since lutein is a structural isomernot the PFD during growth. Given the fact that the

degree to which the xanthophyll cycle was converted to of zeaxanthin, differing only in the location of one doublebound, it is tempting to assume that it also plays a roleA+Z tended to correlate with the degree to which the

quantum yield of PSII photochemistry was depressed in in excess excitation energy dissipation, that could alsoprovide a reasonable explanation for its increase inHL transformants, the lowered WPSII values could be due

a higher extent of non-radiative excitation energy dissipa- HL-exposed transformants. The strong differences in theV+A+Z pool sizes and in lutein contents make thetion in these plants (Demmig-Adams et al., 1995;

Verhoeven et al., 1997). GSA-AT transformants an interesting tool with which tocorrelate the pigment alterations in leaves with theirThe content of the two other xanthophylls, neoxanthin

and lutein, was quite differently affected in transformants capacity to dissipate excess light energy non-radiatively.The Chl a/b ratio is very sensitive to the amounts ofby the different growth PFD. In agreement with previous

reports on several field-growing sun and shade species LHCs per PS unit and, hence, is a good indicator notonly for changes in the antenna organization, but also(Thayer and Bjorkman, 1990; Demmig-Adams and

Adams, 1992, 1996), but in contrast to Brugnoli et al. the PSII antenna size. Under LL conditions the ratio of

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Chl a/b was virtually the same for the wild type and all a high regulatory complexity between Chl synthesis andassembly of photosystems.transformants, indicating that the antenna organization

is not affected by the strongly restricted Chl synthesizing In summary, the commensurate accumulation of carot-enoids with Chls and their dependency on the light regimecapacity. This finding confirms our previous study, that

the Chl deficiency in GSA-AT transformants does not provide evidence for the high flexibility of their synthesisand/or turnover to adapt to the respective environmentalreduce the antenna size in tobacco grown under low

PFDs (Hartel et al., 1997). As expected, HL plants conditions. Based on the results presented at least twofactors seem to control the carotenoid pattern of plants:showed higher Chl a/b ratios than plants grown under

LL conditions, which is consistent with a decrease of the (i) the Chl-synthesizing capacity, and (ii) the photosyn-thetic light utilization efficiency. Although our results doperipheral PSII antenna size relative to the reaction

centre/core complex, and an increase in the proportion not explain the molecular mechanism, they are consistentwith the idea that a co-regulation exists between Chl andof non-appressed thylakoid membranes (Anderson,

1986). The Chl a/b ratio of HL-transformants concomit- carotenoid synthesis, which in turn is governed by aselective accumulation of individual carotenoid compon-antly increased with lowering Chl content (Fig. 2D). This,

together with the changes in carotenoids, points to a ents. It is suggested that the ratio of absorbed to photo-chemically utilized light is an important determiningdifferent adaptive response in antenna organization

during LL/HL acclimation under Chl deficiency. factor in the signalling pathway leading to this selectiveaccumulation. It remains a challenging task to find outPresumably, the acclimation to HL conditions is associ-

ated with a deprivation of the peripheral antenna size. the mechanism for interactive control of Chl and caroten-oid synthesis.However, the distribution of Chls and carotenoids among

the pigment–protein complexes and the amount of thesecomplexes remains to be elucidated.

HL-grown wild type and transformant no. 57 accumu- Acknowledgementslated more Chl than under LL conditions, whereas lineno. 25 accumulated similar amounts and line no. 42 less This work was supported by the Deutsche Forschungs-

gemeinschaft (grants no. Gr 936/4 and 936/5 and Ho 1757/1)Chl (Fig. 2, Table 1). An explanation for the decreasingand by a grant from the Innovationskolleg ‘Zellspezialisierung’Chl content could be an additional inactivation of Chl(Deutsche Forschungsgemeinschaft) to HH Barbara Hickel is

synthesis in HL-grown pale transformants. It is well acknowledged for expert technical assistance.established that synthesis of 5-aminolevulinic acid is con-trolled by light (Kannangara and Gough, 1979). Atpresent, it can not be excluded that, additionally to the

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Consequences of chlorophyll deficiency for leaf carotenoid

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