Greening under high light or cold temperature affects the level of xanthophyll-cycle pigments, early light-inducible proteins, and light-harvesting polypeptides in wild-type barley and the chlorina f2 mutant

Abstract

Etiolated seedlings of wild type and the chlorina f2 mutant of barley (Hordeum vulgare) were exposed to greening at either 5 degrees C or 20 degrees C and continuous illumination varying from 50 to 800 &mgr;mol m-2 s-1. Exposure to either moderate temperature and high light or low temperature and moderate light inhibited chlorophyll a and b accumulation in the wild type and in the f2 mutant. Continuous illumination under these greening conditions resulted in transient accumulations of zeaxanthin, concomitant transient decreases in violaxanthin, and fluctuations in the epoxidation state of the xanthophyll pool. Photoinhibition-induced xanthophyll-cycle activity was detectable after only 3 h of greening at 20 degrees C and 250 &mgr;mol m-2 s-1. Immunoblot analyses of the accumulation of the 14-kD early light-inducible protein but not the major (Lhcb2) or minor (Lhcb5) light-harvesting polypeptides demonstrated transient kinetics similar to those observed for zeaxanthin accumulation during greening at either 5 degrees C or 20 degrees C for both the wild type and the f2 mutant. Furthermore, greening of the f2 mutant at either 5 degrees C or 20 degrees C indicated that Lhcb2 is not essential for the regulation of the xanthophyll cycle in barley. These results are consistent with the thesis that early light-inducible proteins may bind zeaxanthin as well as other xanthophylls and dissipate excess light energy to protect the developing photosynthetic apparatus from excess excitation. We discuss the role of energy balance and photosystem II excitation pressure in the regulation of the xanthophyll cycle during chloroplast biogenesis in wild-type barley and the f2 mutant.

Figure 1

Effects of growth regime on the kinetics of Chl accumulation during greening of etiolated wild-type barley and the f2 mutant. A, 20/250; B, 20/800; C, 5/50; D, 5/250. All data are means ± sd of three to five replicate plants in one experiment. The experiment was repeated at least once. ▪, Wild-type Chl a; •, wild-type Chl b; □, f2 Chl a. FW, Fresh weight.

Figure 2

Dynamics of the accumulation of xanthophyll-cycle intermediates during greening of etiolated wild-type barley and the f2 mutant. A, Wild type at 20/250; B, wild type at 20/800; C, f2 at 20/250; D, f2 at 20/800. •, Violaxanthin; ▵, antheraxanthin; ○, zeaxanthin. All data are expressed as a percentages of the total xanthophyll pool (V+A+Z) and are the averages of three to five replicate plants. For clarity of presentation, the error bars were omitted. The errors averaged less than 10%.

Figure 3

Dynamics of the accumulation of xanthophyll-cycle intermediates during greening of etiolated wild-type barley and the f2 mutant. A, Wild type at 5/50; B, wild type at 5/250; C, f2 at 5/50; D, f2 at 5/250. •, Violaxanthin; ▵, antheraxanthin; ○, zeaxanthin. All data are expressed as a percentages of the total xanthophyll pool (V+A+Z) and are the averages of three to five replicate plants. For clarity of presentation, the error bars were omitted. The errors averaged less than 10%.

Figure 4

Fluctuations in epoxidation state (EPS) during greening of wild-type barley and the f2 mutant. A, Wild-type barley at 20/250 (•) and 20/800 (▪). B, f2 mutant at 20/250 (○) and 20/800 (□). Data were calculated from data in Figure 2.

Figure 5

Chromatograms of HPLC pigment separations illustrating the effects of exposure to low-temperature photoinhibition on the conversion of violaxanthin to zeaxanthin at various times during greening (GR) of etiolated wild-type barley. Photoinhibition conditions consisted of exposure to 1600 μmol m⁻² s⁻¹ at 5°C (high light [HL]) for 2 h. A, Wild-type barley controls before exposure to photoinhibition exposed to greening at 20/250. B, Wild-type barley after exposure to photoinhibition. MP, Mature wild-type barley plants after greening had been completed at 20/250. The data are the results of a single experiment.

Figure 6

The effects of greening on the accumulation of ELIPs, Lhcb2, and Lhcb5. Thylakoids were isolated at various times (3, 6, 12, 24, and 48 h) from wild-type (WT) barley and the f2 mutant exposed to greening at either 20/250 (A and C) or 20/800 (B and D). Similarly, thylakoids were isolated at various times (24, 48, 72, and 100 h) from wild-type barley and the f2 mutant exposed to greening at either 5/250 (E and G) or 5/50 (F and H). Immunoblots from SDS-PAGE were probed with polyclonal antibodies raised against ELIP (14 kD), Lhcb2 (27 kD), and Lhcb5 (29 kD).

Figure 7

Correlation between zeaxanthin and relative ELIP abundance. A, Wild-type barley at 5/50 (□) and 5/250 (▪). B, f2 mutant at 20/250 (○) and 20/800 (•). ELIP abundance was estimated from immunoblots using a specific probe for the 14-kD ELIP, as described in Methods. FW, Fresh weight.

Figure 8

Correlation between qN and 1 − qP (A) and qN and the total xanthophyll-pool size (V+A+Z) (B) in wild-type barley (closed symbols) and the f2 mutant (open symbols) developed under various growth regimes. ▪, □, 20/50; •, ○, 20/250; ▴, ▵, 20/800; ▾, ▿, 5/50; ♦, ⋄, 5/250. All values of qN and 1 − qP were obtained under growth conditions and represent means ± se from three independent experiments. FW, Fresh weight.