Light stress-regulated two-helix proteins in Arabidopsis thaliana related to the chlorophyll a/b-binding gene family

Abstract

The chlorophyll a/b, chlorophyll a/c, and chlorophyll a/a light-harvesting proteins are part of an extended gene family that also includes the transiently expressed stress proteins, the Elips (early light-induced proteins). Four Elip homologue proteins, encoded by single-copy nuclear genes, have been identified in the Arabidopsis thaliana database. These proteins were divided into two groups according to the expression pattern under light-stress conditions and the predicted secondary structure. Group one included two members of the Elip family with three predicted transmembrane helices and a gene expression strictly related to light stress. Group two included two proteins, the Seps (stress-enhanced proteins), which possessed two predicted transmembrane segments. The transcripts of Sep1 and Sep2 were present under low light conditions, but their level increased 4- to 10-fold during illumination of plants with high-intensity light. Preliminary data indicated that the induced transcripts were translated in vivo. Other physiological stress conditions, such as cold, heat, desiccation, salt, wounding, or oxidative stress, did not significantly influence the expression of Sep genes. In vitro import of radioactively labeled precursors of Seps into isolated chloroplasts confirmed the thylakoid membrane localization of these proteins. Considering the predicted protein structure and homology to other pigment-antenna proteins, the two-helix Seps might represent an evolutionary missing link between the one- and three-helix antenna proteins present in pro- and eukaryota.

Figure 1

Amino acid sequence alignment of Elips and Seps. The sequences were aligned manually with the assistance of the multiple alignment program clustalw. Identical amino acids are shown on a gray background. Gaps introduced for the alignment are indicated by dashes.

Figure 2

Predicted secondary structure of Elips and Seps. (A) Hydropathy plots of the translated cDNA sequences of Elips and Seps; (B) schematic representation of the predicted secondary structure of Elips and Seps. The predicted length of transmembrane domains, stromal and luminal connectors, and transit peptides are shown. Numbers indicate the amount of amino acids (aa) in each domain.

Figure 3

Thylakoid membrane localization of Elips and Seps. In vitro-transcribed and -translated Elip and Sep precursors (lane 1) were imported into intact pea chloroplasts before their separation into the thylakoid membranes (lane 2) and the soluble stromal fraction (lane 3). Autoradiogram of proteins from both chloroplast fractions separated by SDS/PAGE is shown. pElip and pSep, precursors of Elip and Sep.

Figure 4

Comparison of the transmembrane domains of Elips, Seps and Cab proteins from Arabidopsis. Highly conserved amino acid residues common for the Elips, Seps, and Cabs are marked in red; additional residues with important structural functions discussed in text are marked in blue. The sequences of Cab proteins from Arabidopsis are according to ref. .

Figure 5

Genomic DNA gel blot analysis of Elips and Seps. Five micrograms of Arabidopsis genomic DNA were digested with the restriction enzymes: (1) BamHI, (2) EcoRI, or (3) HindIII, and the blot was hybridized with the complete Elip1, Elip2, Sep2, and Sep2 coding regions derived from the EST cDNA clones. The sizes of the fragments are marked.

Figure 6

Regulation of Elip and Sep gene expression by stress. (A) Arabidopsis leaves were either directly used for isolation of total RNA (C, control) or exposed to light stress for 3 hr before RNA extraction (HL, high light). Northern blot hybridization was performed by using ³²P-labeled cDNA fragments of the Elip1, Elip2, Sep1, Sep2 clones and selected members of the Cab gene family, such as Lhca1, Lhcb3, and Lhcb6. The α-tubulin gene (clone ID 32C11T7) was hybridized as an internal control. (B) Arabidopsis leaves were exposed for 2 hr to various stress conditions: (1) control, (2) cold stress, (3) heat shock, (4) wounding, (5) salt stress, (6) desiccation, (7) oxidative stress, and (8) UV-A illumination. Isolated total RNA was used for Northern blot hybridization with the radioactively labeled inserts of the Elip and Sep cDNA clones. The rRNA pattern in the gel, visualized by staining with ethidium bromide, is shown in A (Right) and B (Lower).