Regulation of squalene synthase, a key enzyme of sterol biosynthesis, in tobacco

Abstract

Squalene synthase (SS) represents a putative branch point in the isoprenoid biosynthetic pathway capable of diverting carbon flow specifically to the biosynthesis of sterols and, hence, is considered a potential regulatory point for sterol metabolism. For example, when plant cells grown in suspension culture are challenged with fungal elicitors, suppression of sterol biosynthesis has been correlated with a reduction in SS enzyme activity. The current study sought to correlate changes in SS enzyme activity with changes in the level of the corresponding protein and mRNA. Using an SS-specific antibody, the initial suppression of SS enzyme activity in elicitor-challenged cells was not reflected by changes in the absolute level of the corresponding polypeptide, implicating a post-translational control mechanism for this enzyme activity. In comparison, the absolute level of the SS mRNA did decrease approximately 5-fold in the elicitor-treated cells, which is suggestive of decreased transcription of the SS gene. Study of SS in intact plants was also initiated by measuring the level of SS enzyme activity, the level of the corresponding protein, and the expression of SS gene promoter-reporter gene constructs in transgenic plants. SS enzyme activity, polypeptide level, and gene expression were all localized predominately to the shoot apical meristem, with much lower levels observed in leaves and roots. These later results suggest that sterol biosynthesis is localized to the apical meristems and that apical meristems may be a source of sterols for other plant tissues.

Figure 1

A, Validation of an antibody with immunospecificity for TSS. A carboxy-truncated TSS cDNA (TSS-1.1) was expressed in E. coli and the soluble SS activity used for immunoprecipitation assays. B, Aliquots of the soluble SS were incubated with IgGs isolated from preimmune serum (●) or serum of a rabbit immunized with a 23-kD amino-terminal fragment of the TSS enzyme (▪) and the immunoprecipitable SS activity determined. C, Duplicate samples of 200 ng of the 23-kD TSS peptide and 10 μg of total microsomal protein were separated by 8% to 16% (w/v) SDS-PAGE and transferred to polyvinylidene difluoride membranes, and the membranes probed with either IgGs purified from preimmune serum (lanes 1 and 2) or immune serum (lanes 3 and 4).

Figure 2

Suppression of TSS enzyme activity in elicitor-treated tobacco cell suspension cultures is accompanied by changes in the level of the TSS protein and mRNA. Cells were collected at the indicated time points after addition of 1 μg of P. parasitica elicitin per milliliter of cell suspension culture and kept frozen until analyzed for TSS enzyme activity (A), TSS protein (B), and TSS mRNA (C). Microsomes isolated from the cells were used to measure SS enzyme activity and to quantify TSS protein by chemiluminescent immunodetection, whereas TSS mRNA was detected by RNA-blot hybridization using a full-length cDNA to probe total RNA isolated from aliquots of the collected cells.

Figure 3

A, The intron-exon organization of the two TSS genomic clones is nearly identical. Numbers above exons indicate the number of amino acids encoded by the exonic DNA. Numbers below introns indicate the number of base pairs in an intron. Start and stop codons and polyadenylation sites are noted as such. Also shown for comparison is a full-length TSS cDNA. The DNA sequences for the genes reported here have been deposited in GenBank: cTSS-1, U60057; gTSS-1, U59683; and gTSS-2, AY096801. B, The 5′ regions of gTSS-1 and gTSS-2 corresponding to their promoter regions are aligned and annotated for comparison. The ATG translation start codon is outlined, and the adenosine of the ATG start codon is designated as +1 nucleotide. The transcription start site, determined previously by Devarenne et al. (1998), is in bold and underlined. Identical sequence alignments are noted by an asterisk below the two sequences.

Figure 4

The TSS-1 and TSS-2 promoters direct expression of the GUS reporter gene in transgenic tobacco plants. Full-length promoter and 5′-deletion constructs were prepared by fusing the indicated promoter fragments to the GUS reporter gene in the pBI101 vector and engineering these gene fusions into transgenic tobacco via standard Agrobacterium tumefaciens-mediated transformation. Promoter length designations refer to the 5′-end point of a construct relative to the translation start site at +1. R₁ seeds were germinated in the presence of kanamycin, and only 30-d-old plantlets able to grow on kanamycin for the first 15 d were evaluated quantitatively (A) or qualitatively (B) for GUS activity. The values presented in A represent the averaged data for a minimum of 10 independent transgenic lines for each construct. Representative transgenic plants harboring the 1.4-kb TSS1 or 1.2-kb TSS2 promoter-GUS reporter constructs were stained histochemically for GUS expression for 48 h. Error bars in A represent the sds, whereas the inset bar in B represents 1 cm.

Figure 5

Analysis of 1-month-old wild-type tobacco plant tissues for TSS enzyme activity and protein levels. A 1-month-old wild-type tobacco plant (A) was dissected into its component parts as indicated (B and C), and the level of TSS enzyme activity (D) and enzyme protein (E) was determined. Dashed lines indicate how organs were excised into specific tissues; LB, leaf blade; LM, leaf margin; PT, petiole; AM, apical meristem; ST, stem; and RT, roots. The inset bars in A through C equal 1 cm. Microsomes prepared from each of the tissues were used simultaneously to measure TSS enzyme activity (D) and the level of the TSS protein by chemiluminescent immunodetection (E).