Organization of Ripening and Ethylene Regulatory Regions in a Fruit-Specific Promoter from Tomato (Lycopersicon esculentum)

Abstract

Tomato (Lycopersicon esculentum) fruit ripening is initiated by an increase in ethylene hormone concentration. E8 gene transcription is fruit-specific and is activated at the onset of ripening and in unripe fruit treated with exogenous ethylene. To understand how E8 gene transcription is controlled during ripening, we analyzed the effect of deletions of flanking DNA sequences on E8 gene expression in transgenic tomato fruit. We found that a minimum of three 5' and one 3' regions influence E8 gene expression during fruit ripening. DNA sequences that confer responsiveness to exogenous ethylene in unripe fruit are distinct from DNA sequences that are sufficient for expression during fruit ripening.Ethylene is one of five classical plant hormones that control plant growth and development. Ethylene affects shoot and root growth and differentiation, promotes leaf abscission, induces fruit ripening and flower senescence, and is an important part of the plant's response to wounding and pathogen attack (13). Experiments have shown that ethylene exerts its effect on plant growth and development, at least in part, by controlling the transcription of specific genes (1,11,18). In tomato (Lycopersicon esculentum) fruits, ripening is controlled by an increase in ethylene hormone production (6,9,19,23).The E8 predicted polypeptide is a member of a family of dioxygenases found in plants and microorganisms (15) MATERIALS AND METHODS Plant Material and TransformationTomato (Lycopersicon esculentum cv Ailsa Craig) plants were grown under standard greenhouse conditions. Fruit maturity stage was determined as described (10). To treat with ethylene, unripe fruit were placed in a 25-L chamber and exposed for 6 h to 4.5 L/min of ethylene (10 ,L/L) in humidified air. Construction of Chimeric Genes and Plant TransformationThe E8-Tag construct (4) was used to generate a nested set of 5' deletions as described by Henikoff (7). Deletion end points were determined by DNA sequencing. For plant transformation, genes bearing 5' deletions were subcloned into the EcoRI site of shuttle vector pMLJ1 using EcoRI/HindIII adapters (New England Biolabs). The 3' deletion was constructed using a unique HgaI restriction endonuclease site. This deletion removed 337 bp of 3'-flanking DNA and left 185 bp of DNA 3' of the poly(A) addition site. The E8-Tag plasmid was digested with HgaI, and DNA polymerase was used to fill in the single-stranded ends of the HgaI restriction endonuclease site. EcoRI linkers (New England Biolabs) were ligated to the fragments, which were then digested with EcoRI. The resulting 4.2-kb E8-Tag fragment was purified by agarose gel electrophoresis and ligated into the EcoRI site of pMLJ1. Plant transformation was carried out as described (4)

Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis

“…2D). This pattern of pteridine accumulation fits with the expected activity of the E8 promoter that was used to drive GCHI expression (25).…”

“…2 A) and by adjusting the sequence context around the start codon to fit the plant consensus (26). This synthetic gene was placed behind the ripening-specific E8 promoter (25) in an Agrobacterium binary vector and introduced into tomato plants. Screening fruit by Western analysis identified transformants that expressed a protein of the correct size (27 kDa) in a ripening-specific fashion (Fig.…”

“…The pMON10086 vector (24) containing the tomato E8 promoter (25) and pea Rubisco small subunit terminator, and the npt II kanamycin resistance gene was modified by ablating the NotI site and ligating a polylinker (5Ј-GGATCCGCGGCCGCGAATTCAGGCCTG-GTACCGGCGCGCCAGATCT-3Ј) into the unique BamHI site between the E8 promoter and the terminator. A mammalian GCHI cDNA (GenBank accession no.…”

Transient expression of organophosphorus hydrolase to enhance the degrading activity of tomato fruit on coumaphos

“…X13437) described by Deikman et al(1992). The 5′-and 3′-primer sequences were 5′-CCG GGT ACC AAG CTT AGG AAT TTC ACG AAA TCG-3′ and 5′-GCC GTC GAC GGA TCC TCT TTT GCA CTG TGA ATG-3′, respectively.…”

Characteristics of fruit ripening in tomato mutant epi

“…E8, a function unclear gene, expression is regulated during fruit ripening both by ethylene, and by ethylene-independent ripening signals (Deikman et al, 1992). pTOM5 and pTOM6 were confirmed to encode phytoene synthetase and polygalacturonase (PG) respectively, with both being ethylene-regulated (Bird et al, 1991;Grierson et al, 1986).…”