Isolation and Functional Characterization of a Lycopene β-cyclase Gene Promoter from Citrus

Abstract

Lycopene β-cyclases are key enzymes located at the branch point of the carotenoid biosynthesis pathway. However, the transcriptional regulatory mechanisms of LCYb1 in citrus with abundant carotenoid accumulation are still unclear. To understand the molecular basis of CsLCYb1 expression, we isolated and functionally characterized the 5' upstream sequences of CsLCYb1 from citrus. The full-length CsLCYb1 promoter and a series of its 5' deletions were fused to the β-glucuronidase (GUS) reporter gene and transferred into different plants (tomato, Arabidopsis and citrus callus) to test the promoter activities. The results of all transgenic species showed that the 1584 bp upstream region from the translational start site displayed maximal promoter activity, and the minimal promoter containing 746 bp upstream sequences was sufficient for strong basal promoter activity. Furthermore, the CsLCYb1 promoter activity was developmentally and tissue-specially regulated in transgenic Arabidopsis, and it was affected by multiple hormones and environmental cues in transgenic citrus callus under various treatments. Finer deletion analysis identified an enhancer element existing as a tandem repeat in the promoter region between -574 to -513 bp and conferring strong promoter activity. The copy numbers of the enhancer element differed among various citrus species, leading to the development of a derived simple sequence repeat marker to distinguish different species. In conclusion, this study elucidates the expression characteristics of the LCYb1 promoter from citrus and further identifies a novel enhancer element required for the promoter activity. The characterized promoter fragment would be an ideal candidate for genetic engineering and seeking of upstream trans-acting elements.

Keywords: carotenoid; cis-element; citrus; enhancer; lycopene β-cyclase; promoter.

FIGURE 1

The 5′ upstream promoter sequences of the CsLCYb1 gene. Numbers indicate the positions relative to the ATG start codon (0). The putative TATA-box (double underlined), CAAT-box (double underlined), transcriptional start site (TSS, highlighted), and some cis-elements (highlighted) are labeled under the sequences. Primers for amplifying a series of 5′ truncated fragments are also underlined and labeled. The pair of reverse complementary sequences are italic in blue color. The 20 bp tandem repeat sequences are dot outlined.

FIGURE 2

Schematic representation of the CsLCYb1 promoter::GUS vectors construction. These constructs are based on the pCAMBIA1301 vector. LB, left border; 35S PolyA, Cauliflower Mosaic Virus 35S terminator; Hyg, hygromycin resistance gene; 35S P, Cauliflower Mosaic Virus 35S promoter; GUS, β-glucuronidase reporter gene; Nos PolyA, nopaline synthase terminator; RB, right border. Hollow arrows indicate the positions of the promoter insertion in the vectors. The promoters contain the full-length sequence (LP) and its five 5′ truncated fragments (LP1, LP2, LP3, LP4, and LP5). Numbers indicate the sequence length from the first base of the ATG.

FIGURE 3

Histochemical GUS staining of tomato green fruit. Transgenic lines carrying the GUS reporter gene under the control of the CaMV35S promoter were used as the positive control (35S) and untransformed tomato was used as the negative control (-CK). LP, LP1, LP2, LP3, LP4, and LP5 represent transgenic lines under the control of the full-length CsLCYb1 promoter and its five 5′ truncated fragments, respectively. Bars, 1 cm.

FIGURE 4

GUS assays of transgenic Arabidopsis plants. (Up) Qualitative GUS staining. Tissues (root, leave, flower, stem, and fruit) from each construct were separately subjected to histochemical GUS staining. Transgenic lines carrying the GUS reporter gene under the control of the CaMV35S promoter were used as the positive control (35S) and untransformed Arabidopsis were used as the negative control (WT). Bars, 2 mm. (Down) Quantitative GUS assays of transgenic Arabidopsis seedlings carrying the full-length promoter construct (LP) during seedling development (at bottom left) and transgenic Arabidopsis seedlings carrying different promoter constructs (at bottom right). Leaves were harvested on day 24 after seeding. Data are means ± SD of three independent experiments. Lowercase letters indicate significant differences at P < 0.05. Uppercase letters indicate significant differences at P < 0.01.

FIGURE 5

GUS assays of transgenic citrus callus. (Up) Qualitative GUS staining. Citrus callus carrying different promoter constructs were separately subjected to histochemical GUS staining. Transgenic lines carrying the GUS reporter gene under the control of the CaMV35S promoter were used as the positive control (35S) and untransformed callus was used as the negative control (WT). (Down) Quantitative GUS assays of different promoter deletions in stably transformed citrus callus under various treatments, including abscisic acid (ABA), auxin (IAA), gibberellin (GA), salicylic acid (SA), Methyl Jasmonate (JA), Kinetin (KT), sucrose (Suc), glucose (Glu), and NaCl. Data are means ± SD of three independent experiments. Significant differences between values are indicated by asterisk (^∗P < 0.05, ^∗∗P < 0.01).

FIGURE 6

Finer deletion analysis of the 20 bp fragment. (A) Schematic representation of the internal deletion promoter constructs. Numbers indicate the sequence length from the first base of the ATG. (B) Quantitative GUS assays of different constructs in stably transformed citrus callus.

FIGURE 7

Analysis of LCYb1 promoters from various citrus species. (A) Schematic representation of promoter structure of LCYb1 from four citrus species. Green lines represent the coding sequences of LCYb1 genes. Red lines represent the promoter sequences of LCYb1. Gray lines represent the inserted large fragment. The inserted position and fragment size are indicated. Yellow rhombuses represent the 20 bp enhancer elements. (B) SSR screening of different LCYb1 promoters from various citrus varieties. Numbers on the left denote the three electrophoretic bands. Four citrus species including pummelo, grapefruit, sweet orange and mandarin were detected. W, White-flesh Guanxi pummelo; R, Red-flesh Guanxi pummelo; Ht, Huanong red pummelo; H, HB pummelo; S, Star Ruby grapefruit; M, Marsh grapefruit; F, Flame grapefruit; Hq, Washington navel orange; Ca, Cara Cara navel orange; AL, Anliu sweet orange; HAL, HongAnliu sweet orange; B, Bendizao mandarin; I, Qingjiang ponkan; MJ, Mangshan wild tangerine.