TGTCACA Motif Is a Novel cis-Regulatory Enhancer Element Involved in Fruit-specific Expression of the cucumisin Gene

Abstract

Cucumisin, a subtilisin-like serine protease, is expressed at high levels in the fruit of melon (Cucumis melo L.) and accumulates in the juice. We investigated roles of the promoter regions and DNA-protein interactions in fruit-specific expression of the cucumisin gene. In transient expression analysis, a chimeric gene construct containing a 1.2-kb cucumisin promoter fused to a ␤-glucuronidase (GUS) reporter gene was expressed in fruit tissues at high levels, but the promoter activities in leaves and stems were very low. Deletion analysis indicated that a positive regulatory region is located between nucleotides ؊234 and ؊214 relative to the transcriptional initiation site. Gain-of-function experiments revealed that this 20-bp sequence conferred fruit specificity and contained a regulatory enhancer. Gel mobility shift experiments demonstrated the presence of fruit nuclear factors that interact with the cucumisin promoter. A typical G-box (GACACGTGTC) present in the 20-bp sequence did not bind fruit protein, but two possible cis-elements, an I-box-like sequence (AGATAT-GATAAAA) and an odd base palindromic TGTCACA motif, were identified in the promoter region between positions ؊254 and ؊215. The I-box-like sequence bound more tightly to fruit nuclear protein than the TGTCACA motif. The I-box-like sequence functions as a negative regulatory element, and the TGTCACA motif is a novel enhancer element necessary for fruit-specific expression of the cucumisin gene. Specific nucleotides responsible for the binding of fruit nuclear protein in these two elements were also determined.Timing and levels of gene expression are critical to the proper development of eukaryotic organisms. Regulation of the expression pattern of a particular gene can involve the specific binding of trans-acting factors to the cognate cis-elements, constituting a crucial step in transcriptional initiation and, in turn, on the spatial and temporal expression of genes. Plant genes that show tissue specificity, developmental specificity, and a wide range of expression levels have been characterized, whereas their expression patterns are also influenced by environmental stimuli. A family of genes for fruit proteins provide a model system for the study of the regulatory mechanisms of plant genes, since their expression is restricted to a specific tissue and stage during fruit development (1). A number of fruit-specific genes that are activated during ripening have been isolated from tomato and other fruits, and genes responding to ethylene and nonethylene signals have been identified (1, 2). The promoters of fruit-specific genes would also be of great interest for use in strategies to manipulate fruit metabolism and produce valuable proteins such as antibody, biopharmaceuticals, and edible vaccines through methods of genetic engineering (3-5). However, the detailed mechanisms by which the expression of fruit protein genes are regulated are poorly understood, as many of the essential cis-elements have not been identified.Melon cucumisin, an extracellular...

Functional Analysis of the Cucumisin Propeptide as a Potent Inhibitor of Its Mature Enzyme

“…It comprises more than 10% of the total juice protein and is synthesized in the central parts of the fruits (3). Cucumisin is synthesized and accumulated only in melon fruits, and a cis-regulatory enhancer element in the cucumisin promoter regulates fruit-specific expression of the cucumisin gene (4). We have determined the complete nucleotide sequence of a cucumisin cDNA, the first sequenced plant serine protease, and found that cucumisin is a member of the subtilisin (EC 3.4.21.62) superfamily characterized by a catalytic triad of three amino acids: Asp, His, and Ser (5).…”

Screening of Tissue-Specific Genes and Promoters in Tomato by Comparing Genome Wide Expression Profiles of Arabidopsis Orthologues

“…In the cases of E254367 (flower-and fruit-specific) and E541096 (flower specific) promoters, there were several flower-and pollen-related regulatory elements (VOZATVPP, Mitsuda et al, 2004;POLLEN1LELAT52, Bate and Twell, 1998;AGAMOUSATCONSENSUS, Shiraishi et al, 1993). In particular, we found that E254367 contains fruitspecific cis-elements (TGTCACACUCUMSIN, Yamagata et al, 2002;Fig. 3A).…”

Cyclic GMP acts as a common regulator for the transcriptional activation of the flavonoid biosynthetic pathway in soybean

“…Various lengths of soybean CHR promoter, Arabidopsis CHS promoter, and tomato CAB7 promoter (Bowler et al 1994a) were amplified by PCR using two primers containing XbaI and BamHI sites at their upstream-and downstream-ends, respectively, and introduced into XbaIBamHI sites of pSKGUS3C (Yamagata et al 2002). Two synthesized oligonucleotides for both strands of CaMV 35S minimal promoter (-46 to ?8), 35S(-46) ?…”

“…For the gain-of-function analysis, the upstream region of the CHR promoter between nucleotides -600 to -186 relative to the transcription start site was amplified by PCR using the primers, GmchrproA-Fw1; 5 0 -GGGGTCTAGATA CCATATTTGGATTTG-3 0 and GmchrproA-Rv4; 5 0 -GGGGTCTAGAACAAATGTTTTCAACATT-3 0 with pGmCHRproA as a template, and the product was subcloned into the XbaI site of p35S (-46) GUS3C, after which the direction of the promoter was verified by sequencing. pBIXFF (Yamagata et al 2002) harboring the firefly luciferase (LUC) reporter gene under the control of the CaMV 35S promoter was used as an internal control. Gold particles (1.0 lm in diameter) were coated with 2.5 lg of pBIXFF and an equimolar amount of each promoter-GUS construct by ethanol precipitation according to the manufacturer's protocol (Bio-Rad Laboratories Inc., Hercules, CA, USA).…”

“…Soybean genomic DNA (1 lg) prepared from SB-P cells using cetyltrimethylammonium bromide as described previously (Yamagata et al 2002) was digested with Nco I, and the DNA fragments were self-ligated. This circularized DNA was used as a template for first PCR using a forward primer (Gmchr1, 5 0 -AGCTCTCAAGGAAGCTATCCATCTTGG-3 0 ) and a reverse primer (Gmchr2, 5 0 -ATTCCAACCACTGGCA TCCTCTGTTGG-3 0 ).…”