Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles. Identification of the crucial role of a threonine residue for enzyme activity

Volatile esters, a major class of compounds contributing to the aroma of many fruit, are synthesized by alcohol acyl-transferases (AAT). We demonstrate here that, in Charentais melon (Cucumis melo var. cantalupensis), AAT are encoded by a gene family of at least four members with amino acid identity ranging from 84% (Cm-AAT1/Cm-AAT2) and 58% (Cm-AAT1/Cm-AAT3) to only 22% (Cm-AAT1/Cm-AAT4). All encoded proteins, except Cm-AAT2, were enzymatically active upon expression in yeast and show differential substrate preferences. Cm-AAT1 protein produces a wide range of short and long-chain acyl esters but has strong preference for the formation of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a wide range of substrates but with very strong preference for producing benzyl acetate. Cm-AAT4 is almost exclusively devoted to the formation of acetates, with strong preference for cinnamoyl acetate. Site directed mutagenesis demonstrated that the failure of Cm-AAT2 to produce volatile esters is related to the presence of a 268-alanine residue instead of threonine as in all active AAT proteins. Mutating 268-A into 268-T of Cm-AAT2 restored enzyme activity, while mutating 268-T into 268-A abolished activity of Cm-AAT1. Activities of all three proteins measured with the prefered substrates sharply increase during fruit ripening. The expression of all Cm-AAT genes is up-regulated during ripening and inhibited in antisense ACC oxidase melons and in fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon

Sequence of two tomato nuclear genes encoding chlorophyll a/b -binding proteins of CP24, a PSII antenna component

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43424/1/11103_2004_Article_BF00017734.pd

Chloroplast DNA sequences integrated into an intron of a tomato nuclear gene

DNA sequences capable of hybridizing with chloroplast DNA have previously been reported to exist in the nuclear genome of higher plants. Here we show that the third intron of the cultivated tomato ( Lycopersicon esculentum ) nuclear gene Cab -7, which resides on chromosome 10 and which we recently cloned and sequenced, contains two DNA fragments derived from the coding region of the chloroplast gene psb G. The first fragment, 133 bp long, is located at a site 63 bp from the 3′ end of the 833 bp intron. The exact sequence of the 11 nucleotides at the 3′ end of the inserting chloroplast sequence is also found at the 5′ border of the insertion. A small (107 bp) chloroplast DNA fragment is inserted near the middle of the intron, again with the 3′ end of the inserting element (6 bp) duplicated at the 5′ border of the insertion. The second insert is a subfragment of the first insert, and is most likely directly derived from it. The psb G insertion sequence was found to be present in the Cab -7 gene of all tomato species examined but not in species from related genera (e.g. Solanum, Petunia, Nicotiana ), suggesting that the original transposition event (chloroplast to nucleus) occurred relatively recently-since the divergence of the genus Lycopersicon from other genera in the family Solanaceae, but before radiation of species in that genus.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47568/1/438_2004_Article_BF00331304.pd

Tomato: a crop species amenable to improvement by cellular and molecular methods

Tomato is a crop plant with a relatively small DNA content per haploid genome and a well developed genetics. Plant regeneration from explants and protoplasts is feasable which led to the development of efficient transformation procedures. In view of the current data, the isolation of useful mutants at the cellular level probably will be of limited value in the genetic improvement of tomato. Protoplast fusion may lead to novel combinations of organelle and nuclear DNA (cybrids), whereas this technique also provides a means of introducing genetic information from alien species into tomato. Important developments have come from molecular approaches. Following the construction of an RFLP map, these RFLP markers can be used in tomato to tag quantitative traits bred in from related species. Both RFLP's and transposons are in the process of being used to clone desired genes for which no gene products are known. Cloned genes can be introduced and potentially improve specific properties of tomato especially those controlled by single genes. Recent results suggest that, in principle, phenotypic mutants can be created for cloned and characterized genes and will prove their value in further improving the cultivated tomato.

Nucleotide sequence and chromosomal location of Cab -7, the tomato gene encoding the type II chlorophyll a/b-binding polypeptide of photosystem I

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43420/1/11103_2004_Article_BF00016015.pd

Determination of steady-state mRNA levels of individual chlorophyll a/b binding protein genes of the tomato cab gene family

The steady-state levels of mRNA produced by 14 genes encoding members of the tomtato chlorophyll a/b binding protein family were quantified. All genes were found to be expressed in leaf tissue, but the mRNAs accumulated to significantly different levels. The transcripts of cab 1A, cab 1B, cab 3A and cab 3B, encoding the Type I LHC proteins of photosystem II, are abundant, while low levels were measured for mRNAs encoding the Type II LHC II and the LHC I proteins. Sequences from the 5′ upstream regions (−400 to translational start) of some cab genes were determined in this study, and a total of 16 tomato cab gene promoters for which sequences are now available were analyzed. Significant sequence conservation was found for those genes which are tandemly linked on the chromosome. However, the level of sequence conservation is different for the different cab subfamilies, e.g. 85% similarity between cab 1A and cab 1D vs. 45% sequence similarity between cab 3A and cab 3C upstream sequences. Characteristic GATA repeats with a conserved spacing were found in 5′ upstream sequences of cab 1AD, cab 3 A-C, cab 11 and cab 12. The consensus sequence CCTTATCAT, which is believed to mediate light responsiveness, was found at different locations in the upstream sequences of cab 6B, cab 7, cab 8, cab 9, cab 10A, cab 10B and cab 11. In 11 out of 15 genes the transcription initiation site was found to center on the triplet TCA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47581/1/438_2004_Article_BF00280298.pd

Species-Specific Expansion and Molecular Evolution of the 3-hydroxy-3-methylglutaryl Coenzyme A Reductase (HMGR) Gene Family in Plants

Kazakh dandelion (Taraxacum kok-saghyz, Tk) is a rubber-producing plant currently being investigated as a source of natural rubber for industrial applications. Like many other isoprenoids, rubber is a downstream product of the mevalonate pathway. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the conversion of 3-hydroxy-3-methylglutaryl-CoA to mevalonic acid, a key regulatory step in the MVA pathway. Such regulated steps provide targets for increases in isoprenoid and rubber contents via genetic engineering to increase enzyme activities. In this study, we identify a TkHMGR1 gene that is highly expressed in the roots of Kazakh dandelion, the main tissue where rubber is synthesized and stored. This finding paves the way for further molecular and genetic studies of the TkHMGR1 gene, and its role in rubber biosynthesis in Tk and other rubber-producing plants

Isolation and characterization of tomato cDNA and genomic clones encoding the ubiquitin gene ubi3

We report here the isolation and nucleotide sequence of tomato cDNA and genomic clones encoding a ubiquitin extension protein homologous to the yeast gene ubi3 . Sites similar to upstream activating sites commonly found in the promoters of yeast ribosomal genes were observed in the tomato promoter. The tomato ubi3 promoter also contained elements found in the rbcS promoter from pea. The transcription initiation site was determined to occur 66 bp upstream of the initiating Met. RFLP mapping revealed that the gene was located on chromosome 1, 23 cM from marker TG301. A ubi3 gene-specific probe hybridized to a single 800 nt transcript. Expression was reduced in heat-shocked plants and plants kept in the dark. Expression was highest in young leaves and immature green fruit and lowest in mature leaves and petals. We isolated the original cDNA clone using an antibody prepared against chloroplast polypeptides. Immunological studies did not detect ubiquitin or ubiquitin extension proteins in the chloroplast. However, higher-molecular-weight chloroplast proteins were detected with ubiquitin antisera suggesting that ubiquitin conjugates are transported into the chloroplast.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43430/1/11103_2004_Article_BF00028735.pd