Light controls phospholipase A2alpha and beta gene expression in Citrus sinensis

Abstract

The low-molecular weight secretory phospholipase A2alpha (CssPLA2alpha) and beta (CsPLA2beta) cloned in this study exhibited diurnal rhythmicity in leaf tissue of Citrus sinensis. Only CssPLA2alpha displayed distinct diurnal patterns in fruit tissues. CssPLA2alpha and CsPLA2beta diurnal expression exhibited periods of approximately 24 h; CssPLA2alpha amplitude averaged 990-fold in the leaf blades from field-grown trees, whereas CsPLA2beta amplitude averaged 6.4-fold. Diurnal oscillation of CssPLA2alpha and CsPLA2beta gene expression in the growth chamber experiments was markedly dampened 24 h after transfer to continuous light or dark conditions. CssPLA2alpha and CsPLA2beta expressions were redundantly mediated by blue, green, red and red/far-red light, but blue light was a major factor affecting CssPLA2alpha and CsPLA2beta expression. Total and low molecular weight CsPLA2 enzyme activity closely followed diurnal changes in CssPLA2alpha transcript expression in leaf blades of seedlings treated with low intensity blue light (24 micromol m(-2) s(-1)). Compared with CssPLA2alpha basal expression, CsPLA2beta expression was at least 10-fold higher. Diurnal fluctuation and light regulation of PLA2 gene expression and enzyme activity in citrus leaf and fruit tissues suggests that accompanying diurnal changes in lipophilic second messengers participate in the regulation of physiological processes associated with phospholipase A2 action.

Fig. 1.

Amino acid sequence alignment of CssPLA₂α and CsPLA₂β. The KxEL endoplasmic reticulum (ER) retention sequence in the 3′-terminus is only present in CsPLA₂β. Eukaryotic signal peptide cleavage site present only in CssPLA₂α is underlined. ‘*’, identical amino acids; ‘:’, conserved substitutions; ‘.’, semi-conserved substitutions; ‘–’, sequence gaps; ↓, conserved Cys residues. Ca²⁺-binding loop, PLA₂ catalytic motifs, and endoplasmic reticulum (ER) retention sequence are enclosed in boxes.

Fig. 2.

Nucleotide sequences of CssPLA₂α (A) and CsPLA₂β (B) promoter regions. One kb 5′ upstream sequences from the transcription initiation start sites (ATG) are listed. The light- and clock-regulated binding elements described in Table 1 are underlined. SRE, shade response element; CCA1B, CCA1 binding element; CBE, clock and blue-light-regulated element; EE, evening element; all other elements presented are listed in Table 1.

Fig. 3.

Diurnal oscillation in CssPLA₂α (upper graph) and CsPLA₂β (lower graph) gene expression under field conditions. (A) Oscillation of gene expression in leaf blades (LB, closed circles), leaf abscission zones (LAZ, open circles), fruit flavedo (FF, closed triangles), and fruit abscission zones (FAZ, open triangles) during natural daylight (white area) and darkness (grey area) over a 48 h period. (B) Enlargement of LAZ, FF, and FAZ gene expression showing diurnal pattern. Actual time of days is indicated on the x-axis. Vertical bars represent standard error of mean (n=4).

Fig. 4.

Diurnal regulation of CssPLA₂α (upper graph) and CsPLA₂β (lower graph) gene expression in leaf blade under light/dark (open triangles), constant dark (closed circles) or constant light (open circles). Subjective light and dark photoperiods are indicated by open and closed boxes, respectively. Double arrow lines indicate the samples taken from the last 10 h (light period) of the 7 d entrainment under light/dark condition. Vertical bars represent standard error of mean (n=4).

Fig. 5.

Diurnal expression of CssPLA₂α (upper graph) and CsPLA₂β (lower graph) under blue and green 12/12 h light/dark cycles. Seedlings were exposed to 12/12 h light/dark cycle of blue (24 μmol m⁻² s⁻¹, closed circles; and 3 μmol m⁻² s⁻¹, open circles), green (3 μmol m⁻² s⁻¹, open triangles) or dark conditions (0 μmol m⁻² s⁻¹ , closed squares). Subjective light and dark photoperiods are indicated by open and closed boxes and white or grey background. Vertical bars represent standard error of mean (n=4).

Fig. 6.

(A) Diurnal CssPLA₂α (open symbols) and CsPLA₂β (closed symbols) expression, and (B) CsPLA₂ total (open symbols) and low molecular weight (sPLA₂) (closed triangles) enzyme activity under 12/12 h blue/dark cycle, or 24 h constant dark. Blue light was applied at 24 μmol m⁻² s⁻¹. Subjective light and dark photoperiods are indicated by open and closed boxes and white or grey background, respectively. Vertical bars represent standard error of mean (n=4).