The authors thank Susan Mathes for outstanding technical assist- ance in preparing ... Griffiths WT, Oliver RP (1984) Protochlorophyllide reductase- structure ...
Plant Physiol. (1989) 91, 163-169 0032-0889/89/91/01 63/07/$01 .00/0
Received for publication September 12, 1988 and in revised form April 14, 1989
Light Quality and Irradiance Level Interaction in the Control of Expression of Light-Harvesting Complex of Photosystem 11 Pigments, Pigment-Proteins, and mRNA Accumulation Kenneth Eskins*, Peter Westhoff', Phillip D. Beremand Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1815 N. University St., Peoria, Illinois 61604 ABSTRACT
development, where the work of Jenkins and Smith (16) and our own work ( 11) has shown that during long-term irradiation, variation of Pfr/Ptot2 from 0.37 to 0.78 had no effect on the steady state levels of mRNA for LHCPII and small subunits of RUBPCO and variations of Pfr/Ptot from 0.05 to 0.8 had no effect on accumulation of pigments or pigmentproteins associated with LHCPII. At least two other receptors operate in conjunction with phytochrome during early development: protochlorophyllide and the blue-light receptor. Protochlorophyllide is necessary for Chl pigment synthesis and may control development via photosynthetic and pigment-protein assembly processes. Protochlorophyllide is synthesized in the dark and is present in the proplastid and in the etioplast. Its conversion to chlorophyllide is possible under both red and blue light and is also affected by irradiance level (14). Interaction of phytochrome and protochlorophyllide occurs during early events since the synthesis of protochlorophyllide, itself, is controlled by phytochrome at the level of 6-aminolevulinic acid (23). In addition, there is an inverse relationship between phytochrome induction and the synthesis oftranscript for protochlorophyllide reductase (2). It is not known what interaction, if any, occurs during long-term growth or whether protochlorophyllide has any effect other than that on pigment accumulation. The action of blue-light receptor in higher plants is not well characterized. It appears to be necessary for the synthesis of anthocyanins (29), but this synthesis is also affected by phytochrome (8). Recent evidence suggests that the blue-light receptor may be more important in responses that occur in mature green tissue than in etiolated tissue (13). Since chloroplast development proceeds differently under natural conditions than during greening of etiolated tissue (31), photoreceptor action must be studied in plants grown under light/dark cycles for extended periods of time with controlled light qualities and irradiance levels. In such studies, plastids reach an equilibrium state consistent with the type and irradiance level of light, and experiments must accommodate the fact that developmental effects of early switching
Effects of red and blue light at irradiances from 1.6 to 28.3 micromolar per square meter per second on chloroplast pigments, light-harvesting pigment-proteins associated with photosystem 11, and the corresponding mRNA were evaluated in maize (Zea mays L.) plants (OP Golden Bantum) grown for 14 days under 14 hours light/10 hours dark cycles. Accumulation of pigments, pigment-proteins, and mRNA was less in blue than in red light of equal irradiance. The difference between blue and red light, however, varied as a function of irradiance level, and the pattem of this variation suggests irradiance-controlled activation/deactivation (switching) of blue-light receptor. The maximum reduction in blue light of mRNA and proteins associated with light-harvesting complex occurs at lower irradiance levels than the maximum reduction of chlorophylls a and b.
Function of individual photoreceptors and interactions between photoreceptors are strongly influenced by light quality, irradiance level, and the stage of plant and leaf development (4, 6, 27). In addition, the regulation of plastid and nuclear gene expression may change during thylakoid membrane biogenesis (25). Thus, in very early stages of plastid development, the conversion of proplastids or etioplasts to chloroplasts requires the conversion of phytochrome to Pfr by a short red-light signal (3). This change results in the synthesis of numerous enzymes and other constituents necessary for
chloroplast development (26). During this first stage of development, phytochrome appears to operate as a light quality-controlled switching device initiated by red light and reversed by far-red light (23). This switching is initiated at very low irradiance levels, and genes coding for light-harvesting complexes and ribulose bisphosphate carboxylase apparently have different levels of very low fluence activation (17). However, the action of phytochrome during early development may be different from that found during long-term growth because most reactions escape from phytochrome reversibility after 24 to 48 h of continuous irradiation (15, 24). This is especially true in chloroplast
2Abbreviations: LHCPII, the light-harvesting complex of PSII; Ptot, total phytochrome; psbA, gene coding for 32 kD reaction center protein of PSII.
' Present address: University of Dusseldorf, Dusseldorf, FRG.
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by phytochrome are strongly supplemented by the action of blue/UV and protochlorophyllide receptors (1 1). These studies should also attempt to understand how blue and red quality effects are dependent on species, developmental state and irradiance level. We have attempted to further characterize the interaction of light quality and irradiance level with stage of development during long-term growth by measuring accumulated components associated with LHCPII, i.e. pigments, LHCPII proteins, and mRNA for the LHCPII proteins. Assembly of this complex can be regulated at the transcriptional, translational, and pigment-insertion stages. Our data suggest that amounts of this complex in plants developing under blue or red light are controlled by a mechanism involving quantum counting and an interaction between at least two photoreceptor systems.
MATERIALS AND METHODS Growth Conditions
Corn seeds (Zea mays L. cv OP Golden Bantum) were planted in plastic trays in a mixture of vermiculite and top soil (2:1), watered, and placed in growth chambers maintained at 50% humidity and 25°C. Plants were grown for 14 d under 14 h light/10 h dark cycles at irradiances from 1.6 to 28.3 MAmol m-2s-' of red or blue light. The first and second leaves from plants grown under red light, and from plants grown under blue light, were sampled between the fourth and fifth hour of light on the 14th day. Samples from the first emergent leaves were approximately 10 d old and were designated as red old (RO) and blue old (BO). Samples from the second emergent leaves were approximately 5 d old and were designated red young (RY) and blue young (BY). Samples for pigment analysis were taken from the middle section of the first and second leaf. Thylakoids and total mRNA were extracted from whole first and second leaves. Light Sources Growth chambers were fitted with three banks of lights, each controlled separately. Each bank consisted of three double rows of 4 ft, 40 W fluorescent bulbs, controlled by a Lutron dimming system for variable fluorescent levels. Red light was from General Electric fluorescent tubes (GE F40R Red). Blue light was from General Electric fluorescent tubes (GE F40B Blue) plus a blue plastic filter (Roscolux No. 83 Medium Blue). Spectra of red and blue light sources are shown in Figure 1. Spectra and irradiance levels were measured at regular intervals during growth by a spectroradiometer (LICOR LI-1800). Pigment Analysis
Chloroplast pigments from total leaf pigment extracts were analyzed by a reverse phase HPLC procedure (10). One-cm strips of at least six leaves taken from six separate plantings were cut into pieces and pooled for sampling. Triplicate samples taken from this pool were averaged, and standard deviations were 10% or less.
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Thylakoid Proteins Chloroplast membranes were isolated from mesophyll cells of the first and second leaves of 14-d-old maize plants as previously described (1 1), and stored at -80°C until analysis. Gel electrophoresis was by the method of Delepelaire and Chua (7), which preserves some pigment-proteins during separation. Gels were stained with Coomassie brilliant blue G250, scanned by a Zeineh soft laser scanning densitometer (Biomed Instruments, Inc., Fullerton, CA), and integrated by computer software. Since our gel system is not capable of separating all individual apoproteins of the LHCPII family, LHCPII proteins are reported as a summation of 31 to 27 kD proteins and as a percent of total proteins. Results reported are an average of three to four separate gels run on protein isolates. To ascertain a measure of reproducibility, 12 samples (one-half of the total number of samples) were isolated from repeat light-growth experiments and analyzed. mRNA for LHCPII
Total RNA was isolated from first and second leaves of 14d-old plants by a procedure utilizing 5 M guanidine-HCl (5). Equal amounts (20 ,ug) of total RNA from each treatment were separated on formaldehyde gels (18), transferred to Magnagraph nylon (Fisher Biotechnology), and probed with a fulllength 32P-labeled single-stranded pSP64/65 recombinant containing a spinach LHCPII fragment (M Wedel, R Herrmann, unpublished data). Labeled RNA obtained by runoff transcription with SP6 polymerase and the Riboprobe transcription system was hybridized to nylon membrane by using published procedures (22). For quantitation, autoradiograms were used to mark radiolabeled bands on the nylon membrane which were cut out and counted in a scintillation counter. Results are averages of data from three separate gels. Samples from irradiance levels of 1.6, 8.0, and 25 Mmol m-2s-' were duplicated to assess experimental variability. The time involved in growing and processing these long-term growth experiments did not allow us to reproduce all data, but the representative repeats varied by approximately 15 to 20%, while the difference between red and blue samples at 8.0 ,umol m-2Is- varied from 50 to 70%. Typical Northern analysis showed reproducibility at 8.0 ,umol m-2s-'.
LIGHT-QUALITY/IRRADIANCE INTERACTION
RESULTS
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carotene increased 15%. Reduced pigmentation under blue
Chi a and Chi b Accumulation Chloroplast pigments generally increased over the irradiance range of 1.6 to 28.3 ,umol m-2s-'. This accumulation of pigments with increasing irradiance level, however, was not linear and depended more on light quality than on the 5-d difference in leaf age (Figs. 2 and 3). Chl a and Chl b both accumulated to a greater extent in older leaves (RO and BO) under irradiance levels from 1.6 to 10 ,umol m-2s-'. Above 10 ,umol m-2s-', pigment accumulation was greater in young leaves (RY and BY). In all cases, blue light-grown leaves accumulated less Chl a and Chl b than red light-grown leaves and this reduced pigmentation under blue light was more apparent in the older leaf than in the younger.
Carotenoids Over the irradiance range of 1.6 to 28.3 ,umol m-2s', carotenoids behaved in a manner similar to Chl a and Chl b. Neoxanthin increased in RY by 152%, in BY by 126%, and in RO by 110%, but in BO it only increased 53%. This most closely parallels the behavior of Chl b. Violaxanthin increased as well, but the magnitude of change was smaller; approximately 50% for RY and RO but only 22% for BY and 15% for BO. Lutein and ,B-carotene increased by approximately 100% in RY and RO, but the increase in BY (lutein) was 47% and in BY (carotene) 78%, while in BO both lutein and zuuu RY
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The ratio of Chl a to Chl b, which is an indicator of lightharvesting complex assembly, first decreased with increasing irradiance level, then increased again at higher irradiance levels (Fig. 4). mRNA for LHCPII
Equal amounts of total RNA from plants grown at each irradiance level were probed with a labeled LHCPII fragment. Normalized values (Fig. 5) showed that the pool of LHCPII mRNA present is less in old than in young leaves and is also less in blue than in red light-grown plants. As with pigments, the differences between young and old leaves and between blue and red light depended on irradiance level. Proteins of LHCPII Amounts of light-harvesting proteins associated with PSII (Fig. 6) increase with increasing irradiance, reach a maximum at 14 ,mol m-2s-', decline to a minimum at 25 umol m-2s-', and then begin to increase again at 28.3 ,umol m-2s-'. Protein levels are higher in old (RO, BO) than in young (RY, BY) leaves from 1.6 to 14 ,umol m-2s-' but are nearly equal from 14 to 28.3 jAmol m-2s-'. At almost all irradiance levels, LHCPII proteins are lower in blue-grown leaves than in redgrown leaves. Differences between blue and red, however, are more prominent in old than in young leaves. The effect of age on amount of LHCPII protein is opposite to that on the amount of mRNA, but the effects of blue light irradiance are the same. That is, both proteins and mRNA are most often strongly reduced by blue light in old leaves at irradiance levels below 14 and at about 25 Umol m-2s'1.
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Blue Light Effects The effects of blue light on Chl a, Chl b, proteins, and mRNA for LHCPII as compared to those due to red light of equal irradiance are shown for young leaves in Figure 7 and for older leaves in Figure 8. In young leaves, amounts of Chl A A
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Plant Physiol. Vol. 91, 1989
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Figure 5. (a) Northern analysis of maize mesophyll total RNA isolated from red or blue grown plants. RY, RO (red grown leaves 5 and 10 d old) BY, BO (blue grown leaves 5 and 10 d old). Irradiance levels of 1.6, 2.2, 8.0 #1, 8.0 #2, 25 smol m-2s-' probed with full length LHCPII clone from spinach. (b) Irradiance-response curve for LHCPII mRNA from mesophyll cells of 14-d-old plants. RY, RO (5- and 1 0-dold leaves grown under red light); BY, BO (5- and 1 0-d-old leaves grown under blue light). Normalized responses are counts from a radiolabeled ([32P]UTP) LHCPII probe hybridized to total mRNA.
and Chl b are lowered by blue light at 1.6 ,umol m-2s-t. This effect is abated at 2.2 ,mol m-2st', especially for Chl a. Blue reduction then increases again to a maximum at 14 gmol m-2rs', followed by a steady decline. The behavior of LHCPII protein and mRNA is nearly opposite that of the pigments. While amounts of Chl a and Chl b are lower in blue light than in red light at 1.6 MAmol m-2S-', both protein and message show no difference. At 2.2 ,umol m-2S-', as Chl a and Chl b are suppressed less, blue light reduction of proteins and message is increasing. At 8 ,umol m-2s-t, mRNA is strongly reduced in blue light. This effect is less at 14-25 MAmol m-2s-' then increases again at 28.3 gmol m-2S-'. Overall, blue reduction of protein and mRNA follows similar patterns, which are quite dissimilar to that followed by Chl a and Chl b. In old leaves, blue reduction of pigments does not show a minimum at 2.2 gAmol m-2S-' as in young leaves. Instead, it increases steadily to a maximum beginning at 14 Mumol m W2S-'.
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