Energy transfer for low temperature fluorescence in PS II mutant thylakoids

Photosynthesis Research 44: 117-125, 1995. © 1995 KluwerAcademic Publishers. Printed in the Netherlands.

Regular paper

Energy transfer for low temperature fluorescence in PS II mutant thylakoids

B r e n t W. K r u g h & D o n a l d Mi le s Division of Biological Sciences, University of Missouri-Columbia, 108 Tucker Hall, Columbia, MO 65211, USA

Received 17 November 1994; accepted in revised form 21 January 1995

Key words: chlorophyll fluorescence, chlorophyll-protein complex, maize mutant, photosynthesis, thylakoid membrane

Abstract

The Chl-protein complexes of three maize (Zea mays L.) mutants and one barley (Hordeum vulgare L.) mutant were analyzed using low temperature Chl fluorescence emissions spectroscopy and LDS-polyacrylamide gel electrophoresis. The maize mutants hcf-3, hcf-19, and hcf-114 all exhibited a high Chl fluorescence (hc3') phenotype indicating a disruption of the energy transfer within the photosynthetic apparatus. The mutations in each of these maize mutants affects Photosystem II. The barley mutant analyzed was the well characterized Chl b-less mutant chlorina-f2, which did not exhibit the hcfphenotype. Chlorina-f2 was used because no complete Chl b-less mutant of maize is available. Analysis of hcf-3, hcf-19, and hcf-114 revealed that in the absence of CP43, LHC II can still transfer excitation energy to CP47. These results suggest that in mutant membranes LHC II can interact with CP47 as well as CP43. This functional interaction of LHC II with CP47 may only occur in the absence of CP43, however, it is possible that LHC II is positioned in the thylakoid membranes in a manner which allows association with both CP43 and CP47.

Abbreviations: h c f - high chlorophyll fluorescence; LDS - lithium dodecyl sulfate; LHC II - light-harvesting complex of Photosystem II; LHC I - light-harvesting complex of Photosystem I; CPIa - chlorophyll-protein complex consisting of LHC I and the PSI core complex; CPI - chlorophyll-protein complex consisting of the PS I core complex; CP47 - 47 kDa chlorophyll-protein of the Photosystem II core; CP43 - 43 kDa chlorophyll-protein of the Photosystem II core; CP29 - 29 kDa chlorophyll-protein of Photosystem II; CP26 - 26 kDa chlorophyll-protein of Photosystem II; CP24 - 24 kDa chlorophyll-protein of Photosystem II; fp - free pigments

Introduction

All of the Chl in plants is complexed with several membrane polypeptides (Thornber 1975; Markwell et al. 1979). The nature of these polypeptides and their interaction with the Chl molecules imparts unique energy absorption and transfer characteristics to each structure. These Chl-protein complexes are assembled in the thylakoid membranes forming the multicompo- nent pigment-protein complexes termed PSI and PS II (Thornber 1986).

PSII can be composed of at least seven Chl-protein complexes; LHC IIb (LHC II), LHC IIa (CP29), LHC IIc/c' (CP26), LHC IId (CP24), LHC IIe, CP47, CP43

(Bassi et al. 1987b; Green 1988; Dreyfuss and Thorn- ber 1994a). LHC IIb is the major light harvesting Chl- protein complex of PSII and is the complex which is most often referred to as LHC II. LHC IIb binds both Chl a and b in an a/b ratio of approximately 1.3 and exists as trimeric pigment-protein units (Bassi et al. 1987b; Green 1988; Dreyfuss and Thornber 1994a). LHC IIa (CP29), LHC IIc/c' (CP26), and LHC IId (CP24), and LHC IIe are all nuclear gene encoded proteins of minor Chl a/b peripheral antenna complexes of PSII and have approximate Chl a/b ratios of 2.2, 2.0, 0.9 and 1.4, respectively (Peter and Thorn- ber 1991; Dreyfuss and Thornber 1994a). LHC IIa is composed of a single apoprotein resolving at 31 kDa

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under denaturing conditions which shows immuno- logical cross-reactivity with a portion of LHC I (the peripheral light harvesting antenna of PSI) (Bassi et al. 1987b; Peter and Thornber 1991). LHC IIc/c' (CP26) has two apoproteins of 26.5 and 29kDa which are not immunologically related to CP29 or LHC I (Bassi et al. 1987b; Peter and Thornber 1991; Dreyfuss and Thornber 1994a). LHC IId (CP24) consists of a 2 lkDa apoprotein, has been isolated from both PSI and PSII, may be identical to the portion of LHC I designated as LHCI-680, and may have a role in energy 'spillover' between the two photosystems (Dunahay and Staehelin 1986; Bassi and Simpson 1987a; Green 1988). LHC IIe was recently discovered and contains an apoprotein of 13kDa (Peter and Thornber 1991; Dreyfuss and Thornber 1994a).

CP47 and CP43 are Chl a core antennas each consisting of one polypeptide ranging from 45-51 kDa and 40-45 kDa, respectively, depending on the electrophoresis system and the degree of denaturation. Both are encoded by the chloroplast genome; the psbB and psbC genes, respectively, and serve as intermedi- ate transducers of excitation energy from the Chl a/b peripheral antenna (LHC II) to the PSII reaction center Chl (P680) (Bricker 1990). Because CP43 is much easier to remove from the PSII core complex (by treat- ment with chaotropic agents or detergents) than CP47, CP47 may be more closely associated with the PSII reaction center than CP43.

PSI can be composed of at least five Chl-protein complexes; LHC Ia (LHC 1-680), LHC Ib (LHC 1- 730), LHC Ic, PSI-A, and PSI-B (Bassi and Simpson 1987a; Green 1988; Goldbeck and Bryant 1991; Zil- ber and Malkin 1992; Dreyfuss and Thornber 1994b). LHC Ia (LHC 1-680) and LHC Ib (LHC 1-730) are Chl a/b peripheral antenna complexes which are often distinguished by their respective low temperature fluorescence emission maxima and which have Chl a/b ratios of 2.0-3.1 and 2.2-4.4 in barley, respectively (Ikeuchi et al. 1991; Dreyfuss and Thornber 1994b). PSI-A and PSI-B are the core polypeptides containing the Chl a PSI reaction center (P700). PSI-A, PSI-B, and P700 make up the PSI reaction center complex designated as CPI when resolved on SDS-polyacrylamide gels (Green 1988).

Evidence is accumulating for the assignment of low temperature Chl fluorescence emission bands from thylakoid membranes to specific Chl-protein membrane complexes. Krause and Weis (1991) presented the fol- lowing assignments of low temperature Chl emission bands to some of the Chl-protein complexes: LHC

II(F680), CP43(F685), CP47(F695), PSI core(F720), and LHC I(F735). These assignments have been based on fluorescence analysis of isolated membrane complexes and Chl-protein bands excised from polyacrylamide gels as well as studies of a limited number of photosynthetic mutants.

We analyzed three maize (Zea mays L.) mutants and one barley (Hordeum vulgare L.) mutant using octyl glucoside solubilization, LDS-PAGE ('Green gels'), and low temperature (77 K) Chl fluorescence emissions. The maize mutants (hcf-3, hcf-19, hcf-114) had previously been shown to lack one or more components of the photosynthetic apparatus and to exhibit an high Chl fluorescence (hc~ phenotype. The hcfphenotype indicates that their respective mutations resulted in some disruption of energy transfer within the photosynthetic apparatus. The barley mutant used was a Chl b-less barley which exhibits a yellow-green phenotype but not the hcfphenotype. Our LDS-PAGE system did not resolve all of the Chl-protein complexes discussed above but did resolve the Chl-proteins in a similar fash- ion as reported in Camm and Green (1980), Bassi et al. (1987b), and Green (1988). We used LDS-PAGE and low temperature Chl fluorescence emissions to determine if Chl-protein bands on polyacrylamide gels could be correlated with low temperature Chl fluorescence emissions bands, and to study the energy transfer characteristics of the Chl-protein complexes.

Materials and methods

Plant material

The maize (Zea mays L.) mutants used in this study were: hcf-3, hcf-19 (Miles 1980), and hcf-ll4 (Cook and Miles 1992). The control maize referred to as 'normal maize' was the inbred line, B-73, from Pioneer Seed Company.

The barley (Hordeum vulgare L.) used in this study was the Chl b-less barley mutant chlorina-f2 (Robert- son 1937; Highkin 1950; Thornber and Highkin 1974; Machold et al. 1979).

Seedlings were grown in a growth chamber for 10 days under 300 #mol m -2 s -1 of light 12 h daily. After emergence they were supplemented with a (15- 30-15) nutrient solution, 'Miracle Grow' at concentrations recommended by the manufacturer. The light temperature was 29 °C and the dark temperature was 26 °C.

119

t..) i -

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o~ f_

g r-4 la_

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J / [ I I 650 700 750 800

'Nave lengt.h (rim) Fig. 1. Low temperature fluorescence emissions spectra from normal maize: A) thylakoid membranes solubilized with octyl glucoside and B) native thylakoid membranes.

hcf mutants were visually screened and selected with long wave UV light (Miles 1982).

Thylakoid isolation and solubilization

The leaves of the maize and barley seedlings were collected and rinsed in reverse osmosis purified water. The thylakoid membranes were isolated as described by Carom and Green (1980). Chl determinations of the thylakoid suspensions were as described in Arnon (1949). Aliquots of the thylakoid suspensions were extracted with n-octyl t , D-glucopyranoside (octyl glucoside) and incubated at room temperature, in the dark, for 1 h then centrifuged at 20,000 x g for 20 min to remove undissolved material (Camm and Green 1980). The detergent to Chl ratios were 40:1 for normal maize and 50:1 for all mutants. Since the mutant plants showed a decrease in Chl compared to normal maize, a higher detergent to Chl ratio was used with the mutant thylakoids. The green supernatant fractions from the centrifuged octyl glucoside extracted sam- pies were collected for LDS-PAGE and low temperature fluorescence emissions analysis. During these procedures, the thylakoid membrane suspensions were maintained at 4 °C and in the dark.

Electrophoresis

Polyacrylamide gel electrophoresis (PAGE) was car- ried out using discontinuous gels as reported in Laemmli (1970). The gels were 1.5 mm thick 18 cm wide, and 16 cm long with acrylamide concentrations

Fig. 2. Chlorophyll-protein complexes of normal maize, chlorina-f2, hcf-3, hcf-114, and hcf-19 separated by LDS-PAGE.

of 5% in the stacking gel and 12.5% in the resolving gel. The electrophoresis buffer was 192 mM glycine and 25 mM Tris (pH 8.3) (Laemmli 1970). Lithium dodecyl sulfate (LDS) was added to 0.2% (w/v) (Camm and Green 1980) and EDTA was added to 1 mM (Dele- pelaire and Chua 1979) to the upper buffer reservoir. In order to achieve clear resolution of the Chl-protein bands, it was necessary to pre-run the gels (without sample proteins in the wells) for at least 3 h at 5 watts constant power. Thylakoid samples of various Chl concentrations (5-50 #g) were used, however, best resolution occurred with 10-15 #g of Chl. The samples were electrophoresed at 4 °C under non-denaturing conditions at 2.5 watts constant power until samples entered the resolving gel and then 5 watts constant power until bands were sufficiently separated (approximately 2.0 h).

Fluorescence

Low temperature Chl fluorescence emissions analysis was performed on thylakoid samples solubilized in octyl glucoside and thylakoid samples not treated with detergent (native thylakoids) using a Spex fluorolog F-112. Samples of thylakoids containing 6/ lg of Chl were diluted to 150 ~1 in 2 mM Tris-maleate pH 7.0 and then to 300/~1 in glycerin. Each sample was placed in a quartz tube yielding a sample path length of approximately 2 mm and cooled to 77 K in liquid nitrogen. The excitation wavelength was 470 nm and the excitation monochrometer had a band pass of 4.7 nm. At 50% of the bandpass, the excitation light intensity was 100 #mol m 2- s -1. The fluorescence emission was

120 A

B A

L L

I I I

B50 700 750 BOO

Wavelength (nm) Fig. & Low temperature fluorescence emissions spectra from octyl glucoside solubilized thylakoid membranes of A) normal maize and B) chlorina-f2.

t ' -

O.} C.)

0

,--i h

A

A

sso 700 7s0 a00 Wavelength (nm)

Fig. 4. Low temperature fluorescence emissions spectra from native thylakoid membranes of A) normal maize and B) chlorina-f2.

recorded over the range of 650-800 nm with a double grating monochrometer having a band pass of 0.43 nm and a photon counting circuit.

R e s u l t s

Normal maize

In normal maize, we observed the characteristic low temperature Chl fluorescence emission bands from chloroplasts as reported by Murata and Satoh (1986); Siefermann-Harms (1988) and Krause and Weis (1991). Octyl glucoside solubilized thylakoids showed low temperature emission peaks at 680 nm and

(,o

,r o -1

,--i LL

B

B A

650 700 750 BOO

Wavelength (nm)

Fig. 5. Low temperature fluorescence emissions spectra from native thylakoid membranes of A) normal maize and B) hcf-3.

735 nm (Fig. 1) but native thylakoid samples showed bands at 685, 695, and 735 nm (Fig. 1). The 680 nm, 685 nm, 695 nm, and 735 nm emissions (F680, F685, F695, F735) are generally accepted as emanating from LHC II, CP43, CP47, and LHC I, respectively. Although there was some variation in the intensity of the long wavelength peak (F735) between the solubilized and native samples, there was no indication that solubilization shifted the emission wavelength of this

peak. Our LDS-PAGE system did not resolve all of the

described LHC II complexes. Normal maize thylakoids which were solubilized with octyl glucoside and separated by LDS-PAGE resolved into the six previously reported bands; CPIa, CPI, LHC II oligomer, CP47, CP43, and LHC II monomer (Camm and Green 1980; Bassi et al. 1987b; Green 1988) (Fig. 2). CPIa, the slowest migrating band, contains the PSI core complex (PSI-A, PSI-B, and P700) and LHC I. This band probably contains LHC Ia, LHC Ib, and LHC Ic but our method does not resolve the LHC I Chl a/b complexes. CPI, migrates slightly faster than CPIa and contains the PSI core complex (PSI-A, PSI-B, and P700) without LHC I. The next lower band on the gel is the LHC II oligomer band that likely consists of the LHC IIb oligomeric complex. The two less distinct bands migrating below the LHC II oligomer band are CP47 and CP43, respectively. The fastest migrating Chl- protein band may contain a number of components. It is termed 'LHC II monomer' because it contains monomeric forms of the minor LHC II Chl a/b antenna complexes (Camm and Green 1980; Bassi et al.

121

A

C QJ LJ tO oJ f.-

t.L.

I I I 6 5 0 700 750 800

WavelengLh (nm) Fig. 6. Low temperature fluorescence emissions spectra from native thylakoid membranes of A) normal maize and B) hcf-ll4.

8

LL

B

I I I 650 700 750 fl00

Wavelength (nm)

Fig. 7. Low temperature fluorescence emissions spectra from native thylakoid membranes of A) normal maize and B) hcf-19.

1987b; Green 1988). This band may also contain LHC Ia (LHCI-680) (Bassi et al. 1987b) or any combina- tion of the minor LHC II chlorophyll a/b complexes, although our data does not distinguish these components. Free pigments (fp) form a diffuse band running ahead of the Chl-protein bands.

Chlorina-f2

Barley mutant chlorina-f2 yielded low temperature Chl fluorescence emissions peaks at 685 nm and 720 nm in solubilized thylakoids (Fig. 3) and 685 nm, 695 nm, and 720 nm for native thylakoids (Fig. 4). LDS-PAGE gels revealed normal amounts of the CPI, CP47, and CP43 bands but an absence of the CPIa and LHC II oligomer and monomer bands. These results agree with the previous data and therefore give us confidence in the effectiveness of our experimental techniques.

hcf-3

The low temperature Chl fluorescence emissions spectrum of maize mutant hcf-3 showed bands at 680 nm, 695 nm, and 735 nm in native thylakoids (Fig. 5). Due to the separation of complexes and the overlapping of fluorescence bands, the low temperature fluorescence emissions of solubilized hcf-3 thylakoids showed no significant difference from that of solubilized normal maize except for an overall increase in emissions intensity. Native hcf-3 thylakoids showed the loss of F685 and the appearance of F680. The 680 nm peak is not present with non-detergent treated normal maize thy-

lakoids and its appearance with hcf-3 reflect the lack of energy transfer from LHC II (F680) to CP43 (F685) in this mutant. These data suggest maize mutant hcf-3 is lacking CP43. Low temperature fluorescence emissions analysis of hcf-3 also revealed a 3 nm shift in the F695 emission band to a peak emission at the 698 nm position. Separation of the Chl-protein complexes of hcf-3 by LDS-PAGE indicated normal amounts of the CPIa, CPI, LHCII oligomer, and LHC II monomer bands but there was a decrease in the CP47 and CP43 bands (Fig. 2). This agrees with the detailed study of Coomassie Brilliant Blue stained polypeptides reported by Metz and Miles (1982).

hcf-114

Maize mutant hcf-ll4 yielded low temperature Chl fluorescence emissions bands at 685 nm, 698 nm, and 735 nm in native thylakoids (Fig. 6). Solubi- lized thylakoids of hcf-114 show the same emission bands as normal maize thylakoids but hcf-114 exhibited an overall greater fluorescence yield compared to normal maize. Although, the same number of peaks that are observed in native normal maize thylakoids are present in hcf-114, the native hcf-114 thylakoids show a decrease in F685 and a shift in a peak from the 695 nm position to the 698 nm position. LDS-PAGE of this PSII mutant resolved normal amounts of CPIa, CPI, CP47, CP43, and LHC II monomer bands but decreased amounts (approximately 50%) of the total LHC II oligomer band compared to normal maize (Fig. 2).

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hcf-19

Maize mutant hcf-19 showed low temperature Chl fluorescence emissions bands at 680 nm, 698 nm, and 735 nm in native thylakoids (Fig. 7). The low temperature Chl fluorescence emissions spectrum of solubilized thylakoids of hcf-19 was virtually identical to that of normal maize. The native sample of hcf-19 differed from normal maize in that hcf-19 exhibited a loss of F685, the appearance of F680, and also a shift in an emission peak from 695 nm to the 698 nm position. LDS-PAGE revealed normal amounts of the CPIa, CPI, LHC II oligomer, and LHC II monomer bands but there was a decrease in the CP47 and CP43 bands (Fig. 2).

Discussion

The low temperature Chl fluorescence emissions characteristics of normal maize thylakoid membranes depended on whether the samples were native or solubilized with detergent. Solubilized thylakoids yielded a narrow fluorescence peak at 680 nm and a broad peak at 735 nm with the 680 nm peak being the most intense. Previous reports suggest that within the 735 nm peak there is a smaller 720 nm peak emanating from the PSI reaction center complex (Murata and Satoh 1986; Krause and Weis 1991). Native thylakoid low temperature fluorescence emissions yielded a narrow peak at 685 nm, a shoulder at 695 nm, and a broad more intense peak at 735 nm (also thought to include the 720 nm peak). The appearance of the 680 nm peak and the disappearance of the 685 nm and 695 nm peaks in the octyl glucoside solubilized sample is due to the disruption of the thylakoid membrane resulting in the separation of the PSII Chl a/b peripheral antenna LHC IIb from the PSII Chl a core antennas CP43 and CP47. This separation prevents the transfer of energy from LHC IIb to CP43 and CP47, therefore excess energy is emitted as F680, the characteristic low temperature emission band of LHC IIb (Murata and Satoh 1986; Krause and Weis 1991). In the absence of octyl glucoside, the thylakoid complexes are more intact, thus allowing energy transfer from LHC IIb to CP43 and CP47 where the excess energy is dissipated as F685 and F695, respectively, and not as F680. This reflects the efficiency of energy transfer from LHC IIb to the Chl a core antennas since there is no significant 680 nm emission in intact thylakoids.

The barley mutant, chlorina-f2, contains no Chl b and therefore is missing functional LHC I and LHC II (Highkin 1950; Thornber and Highkin 1974; Machold et al. 1979). Our results confirm these findings with both LDS-PAGE and low temperature Chl fluorescence emissions analysis (Figs. 2A, 2B, 3). Due to the efficient transfer of energy to CP43 and CP47 in native thylakoids, the loss of LHC lib (F680) was only apparent with octyl glucoside solubilized thylakoid membranes. Chlorina-f2 differed from normal maize by a drastic decrease in the F680 and F735 emission bands making it possible to observe the emission peaks at 685 nm and 720 nm (Fig. 2A). The absence of F735, and therefore LHC Ib, was also observed in native thylakoid membranes (Fig. 3). The PAGE data for this mutant shows the loss of LHC I by the absence of the CPIa band and the loss of LHC II by the absence of the LHC II oligomer and monomer bands (Fig. 2B). These data agree with previous reports on chlorina-f2 (Highkin 1950; Thornber and Highkin 1974; Machold et al. 1979) and appear to support the assignments of F680 to LHC II and F735 to LHC I (Murata and Satoh 1986; Krause and Weis 1991). However, the data does not distinguish which Chl-protein complex (LHC I or LHC II) is responsible for each emission bands (F680 or F735) or if one or both of the Chl-protein complexes emits fluorescence at both 680 and 735 nm.

The PAGE data presented above for the maize mutant hcf-3 revealed decreases in both CP47 and CP43 but, this was not necessarily reflected in the low temperature Chl fluorescence emissions. The low temperature Chl fluorescence emissions spectrum of native hcf-3 thylakoids showed a loss of F685 and the appearance of F680. These results indicate a decrease in CP43 resulting in the absence of an energy acceptor subsequent to LHC IIb and therefore the appearance of F680. In contrast, there was not only an increase in F695 but also a shift in this peak to the 698 nm position. The presence of CP47 and the absence of CP43 was only evident with the native thylakoids where the F685 peak was not obscured among the strong emission of LHC IIb at 680 nm as occurs with the solubilized membranes. Native thylakoids of normal maize have low temperature Chl fluorescence emission peaks at 685 nm, 695 nm, and 735 nm. However, native hcf-3 thylakoids had peak emissions at 680 nm, 698 nm (increased compared to F695 of normal maize), and 735 nm. The decrease in F685 correlates with the decrease in the CP43 band on polyacrylamide gel, thus supporting previous reports based on detailed analysis of stained polypeptides suggesting a loss of CP43 in

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this mutant (Leto and Miles 1980; Leto and Arntzen 1981; Metz and Miles 1982). The appearance of F680 results from the absence of CP43 and therefore the loss of a site for LHC IIb to transfer absorbed energy. Keeping in mind the previous assignment of the 695 nm emission band to CP47 (Murata and Satoh 1986; Krause and Weis 1991), it may seem peculiar that our data shows an increase in 698 nm emission band (assuming that this band results from the same source as F695) along with a decrease in the CP47 Chl- protein band on polyacrylamide gels. However, these results may be explained by referring to the model of energy transfer through the Chl-protein complexes of PSII proposed by Bassi et al. (1987b). This model suggests that CP43 is positioned such that LHC II, CP24, CP26, CP29, and CP47 surround it. This model also predicts that energy transfer within PSII occurs either from LHC II through CP43 to CP47 and then to the PSII reaction center or from CP29 and CP26 to CP43 to CP47 and then to the PSII reaction center. In both pathways depicted in this model, energy is transferred from the antenna complexes through CP43 to CP47, however, our data suggests that CP43 may not be necessary for energy transfer to CP47. If CP43 was necessary for energy transfer to CP47 one would expect to observe a decrease in the CP47 low temperature emission (F695) whenever a decrease in CP43 is observed. In contrast, our data shows an increase in the CP47 fluorescence emission band (although shifted 3 nm) despite the apparent decrease in this component reflected in the PAGE data. These results indicate that in mutant membranes LHC IIb may be able to transfer energy directly to CP47 and that CP47 would receive energy from both LHC IIb and the portion of CP43 which is still present. If this occurs, one would expect an increase in the CP47 associated fluorescence emissions (F695) to occur when less CP43 is present. The transfer of energy from LHC II to CP47 may only occur when CP43 is absent allowing a closer association of LHC II and CP47. However, it is possible that the in vivo association of these complexes is such that transfer of energy can occur either through CP43 to CP47 (possibly the most favorable route) or directly from LHC II to CP47 but this is not distinguished in our data. The shift in the low temperature Chl fluorescence emissions peak from the 695 nm position to the 698 nm position which also occurs in two other maize mutants (hcf-114 and hcf-19) that show disruption in CP43 content, may arise from altered energetic characteristics of the energy transfer system due to the absence of CP43 and/or the direct transfer of energy from LHC II to

CP47. For example, if the LHC II to CP47 energy transfer route is less efficient than the CP43 to CP47 route, more energy may be lost through other process- es and therefore a slightly longer wavelength emission may occur.

Maize mutant hcf-114 showed an approximate 50% decrease in the LHC II oligomer band when compared to normal maize on polyacrylamide gels. Immuunolog- ical studies of PSII polypeptides and stained bands for CP43 and CP47 are generally supported by the present work though the slight decrease in CP43 and CP47 that was reported for this mutant (Cook and Miles 1992) were not always evident in our studies. This may have resulted from comigration of other complexes with CP43 and CP47 or since the gels were loaded on an equal Chl basis, the diminished LHC IIb content may have caused the sample to become some- what enriched in CP43 and CP47. Although there was an overall increase in the low temperature Chl fluorescence of solubilized hcf-114 thylakoids, the Chl fluorescence characteristics were similar to that of normal maize. Native thylakoids of hcf-114, however, showed a decrease in F685, a slight increase in F695, and a shift of F695 to the 698 nm position. These data are consis- tent with the data obtained from hcf-3 except that the appearance of F680 does not occur with hcf-114. This is likely due to the substantial decrease in LHC IIb. The decrease ofLHC IIb can also explain why hcf-114 exhibited a smaller increase in F695 compared to hcf- 3. This lack of LHC IIb causes less light energy to be initially absorbed and therefore less energy ultimately arrives at CP47. These data also support the model that LHC IIb may be able to transfer light energy directly to CP47 in these mutant thylakoids.

hcf-19 shows even less CP47 and CP43 than observed in hcf-3. The low temperature Chl fluorescence emissions spectrum for solubilized hcf-19 thylakoids is nearly identical to that of normal maize but native thylakoids show a peak at 680 nm instead of 685 nm and a shift of the 695 nm peak to the 698 nm position. In contrast to hcf-3, the intensity of the 698 nm peak emission in hcf-19 remains the same as F695 of normal maize. The appearance of the 680 nm peak and the subsequent loss of the 685 nm peak as occurred with hcf-3 suggests a decrease in CP43 in hcf-19. The fact that the F698 emission band did not increase as in hcf-3 and hcf-114 may be due to the greater loss of CP47 in hcf-19.

There are two possibilities for the shift of F695 to the 698 nm position. A decrease in CP43 could slightly alter the energetic characteristics of the system causing

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a shift in the emission band for CP47. However, one would expect only changes in fluorescence emission intensity if the same energy transfer course was being utilized in the mutants as in the normal plants. The most likely explanation is that in the absence of CP43, LHC II can transfer energy directly to CP47 which results in altered energetic properties of the system and therefore a shift in the CP47 emission band. This may occur as a result of novel assembly of these components within the thylakoid membrane in the absence of CP43. However, it is also conceivable that this route of energy transfer is always possible but is less favorable than transfer through CP43.

The origin of F695 and assignment to CP47 alone may be questioned though a thylakoid preperation containing only CP47 was shown to have a peak fluorescence at 693 nm (Siefermann-Harms 1988). The four mutants reported here all have some amount of CP47 and all retain F695. In hcf3 in the absences of an assembled reaction center the fluorescence remains at F695. In hcf114 the reaction center is assembled with CP47, but these is no functional electron transport. In other mutants the PSII reaction center is assembled with reduced electron transport and all retain F695. It appears that if CP47 is present, assembled into an active reaction center or not, F695 remains (though it may be shifted to 698 nm).

In summary, previous reports have suggested that the low temperature Chl fluorescence emissions bands F680, F685, F695, F720, and F735 emanate from the Chl-protein complexes LHC II, CP43, CP47, the PSI core complex, and LHC I, respectively (Murata and Satoh 1986; Krause and Weis 1991). The data presented in this report are the results from LDS-PAGE and low temperature Chl fluorescence emissions analysis of three maize and one barley mutant in comparison to normal maize. This data appears to support the previous assignments ofF680 to LHC II, F685 to CP43, and F695 to CP47 but may suggest an alternative energy transfer route within the intact PSII complex that was reported by Bassi et al. (1987b). The alternative route would involve direct energy transfer from LHC II to CP47 instead of transfer from LHC II to CP43 and then to CP47. This alternative energy transfer route may only occur when CP43 is absent due to reassem- bly of the Chl-protein complexes within the thylakoid membranes. However, it is possible that the normal arrangement of these complexes in the thylakoid membranes may allow for such transfer under certain cir- cumstances.

Acknowledgements

This research was supported by USDA Grant 90- 37280-5459 and by the Interdisciplinary Plant Group at the University of Missouri-Columbia.

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Energy transfer for low temperature fluorescence in PS II mutant thylakoids