Biosynthesis and distribution of chlorophyll among the photosystems during recovery of the green alga Dunaliella salina from irradiance stress

Abstract

To elucidate the mechanism of an irradiance-dependent adjustment in the chlorophyll (Chl) antenna size of Dunaliella salina, we investigated the regulation of expression of the Chl a oxygenase (CAO) and light-harvesting complex b (Lhcb) genes as a function of Chl availability in the photosynthetic apparatus. After a high-light to low-light shift of the cultures, levels of both CAO and Lhcb transcripts were rapidly induced by about 6-fold and reached a high steady-state level within 1.5 h of the shift. This was accompanied by repair of photodamaged photosystem II (PSII) reaction centers, accumulation of Chl a and Chl b (4:1 ratio), photosystem I (PSI), light-harvesting complex, and by enlargement of the Chl antenna size of both photosystems. In gabaculine-treated cells, induction of CAO and Lhcb transcripts was not affected despite substantial inhibition in de novo Chl biosynthesis. However, cells were able to synthesize and accumulate some Chl a and Chl b (1:1 ratio), resulting in a marked lowering of the Chl a to Chl b ratio in the presence of this inhibitor. Assembly incorporation of light-harvesting complex and a corresponding Chl antenna size increase, mostly for the existing photosystems, was noted in the presence of gabaculine. Repair of photodamaged PSII was not affected by gabaculine. However, assembly accumulation of new PSI was limited under such conditions. These results suggest a coordinate regulation of CAO and Lhcb gene transcription by irradiance, independent of Chl availability. The results are discussed in terms of different signal transduction pathways for the regulation of the photosynthetic apparatus organization by irradiance.

Figure 1

Effect of gabaculine on Chl accumulation and cell growth after an HL → LL shift of D. salina cultures. A, Chl accumulation in the culture. B, Cell density increase. C, Cellular Chl content. D, The Chl a to Chl b ratio. Control (●) and gabaculine-treated cells (○) were shifted from HL to LL growth irradiance at zero time. Data are means from two independent experiments with n = 3 to 5. Error bars represent sd.

Figure 2

A, Effects of gabaculine on carotenoid content in D. salina after an HL → LL shift. B, Effect of gabaculine on cellular de-epoxidation state of xanthophyll pools after an HL → LL shift. The molar ratio of zeaxanthin (Z) + antheraxanthin (A) per Z + A + violaxanthin (V) was calculated after HPLC analysis. Control (●) and gabaculine-treated cells (○) were shifted from HL to LL growth irradiance at zero time. Data are means from two independent experiments with n = 3. Error bars represent sd.

Figure 3

Effects of gabaculine on recovery of the in vivo F_v/F_m Chl fluorescence ratio after an HL → LL shift. Control (●) and gabaculine-treated cells (○) were shifted from HL to LL growth irradiance at zero time. Data are representative of three independent measurements.

Figure 4

Effect of gabaculine on the induction of Lhcb, CAO and CHLG mRNA after an HL → LL shift. Treatment with 1 mm gabaculine and the shift in growth irradiance occurred at zero time. Samples were harvested at the indicated times during incubation. A, Autoradiogram of northern blots and ethidium bromide staining of rRNA. Histogram of the quantitative analysis of Lhcb (B), CAO (C), and CHLG (D) mRNA normalized to the concentration of rRNA in control (black bars) and gabaculine-treated cells (white bars).

Figure 5

Effects of gabaculine on the accumulation of key thylakoid membrane proteins after an HL → LL shift. A, Coomassie Brilliant Blue-stained SDS-PAGE gel. Molecular mass markers (kD) are indicated on the left. Western blot analysis of the LHC-II apoproteins (B), the PsaA/PsaB PSI reaction center proteins (C), and the D1/32-kD reaction center protein (D). The nitrocellulose filters were probed with specific polyclonal antibodies. Cross-reactions were quantitated by densitometric scan (block diagrams in B, C, and D). Lanes for the western blotting of LHC-II proteins were loaded with solubilized thylakoid membranes corresponding to 1.3 × 10⁶ cells. All other runs were loaded with thylakoid membranes equivalent to 5 × 10⁶ cells per lane. Lane 1, HL-grown cells. Lane 2, HL-grown cells after a 24-h incubation under LL conditions in the absence of gabaculine (control). Lane 3, HL-grown cells after a 24-h incubation under LL conditions in the presence of gabaculine.

Figure 6

The light-saturation curve of photosynthesis in control and gabaculine-treated D. salina. A, Rates of oxygen evolution on a per Chl basis were measured as a function of incident light intensity. B, Rates of oxygen evolution on a per cell basis were measured as a function of incident intensity. HL-grown cells were incubated under LL growth conditions for 24 h, either in the absence (control; ●) or in the presence (○) of 1 mm gabaculine. Data are means from two independent experiments with n = 3. Error bars represent sd.