ABSTRACT
We investigated changes in gene expression in Iris hollandica flowers by microarray technology. Flag tepals were sampled daily, from three days prior to flower opening to the onset of visible senescence symptoms. Gene expression profiles were compared with biochemical data including lipid and protein degradation and DNA coiling, and with morphological data. Plasmodesmata of mesophyll cells closed about two days before flower opening, while in the epidermis they closed concomitant with opening. Similarly, the onset of visible senescence in the epidermis cells occurred about two days later than in the mesophyll. About 1400 PCR-amplified clones, derived from a subtractive cDNA library enriched for tepal-specific genes, were spotted and about 240 clones, including 200 that were expressed most differentially, were sequenced. The expression patterns showed three main clusters. One exhibited high expression during tepal growth (cluster A). These genes were putatively associated with pigmentation, cell wall synthesis and metabolism of lipids and proteins. The second cluster (B) was highly expressed during flower opening. The third cluster (C) related to the final stages of senescence, with genes putatively involved in signal transduction, and the remobilization of phospholipids, proteins, and cell wall compounds. Throughout the sampling period, numerous plant defence genes were highly expressed. We identified an ion channel protein putatively involved in senescence, and some putative regulators of transcription and translation, including a MADS-domain factor.
Subject(s)
Flowers/genetics , Gene Expression Profiling , Magnoliopsida/genetics , Blotting, Northern , Cluster Analysis , DNA, Complementary/chemistry , DNA, Complementary/genetics , Flowers/growth & development , Flowers/ultrastructure , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Gene Library , Magnoliopsida/growth & development , Microscopy, Electron, Scanning , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis/methods , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, DNAABSTRACT
Fruit flavor is a result of a complex mixture of numerous compounds. The formation of these compounds is closely correlated with the metabolic changes occurring during fruit maturation. Here, we describe the use of DNA microarrays and appropriate statistical analyses to dissect a complex developmental process. In doing so, we have identified a novel strawberry alcohol acyltransferase (SAAT) gene that plays a crucial role in flavor biogenesis in ripening fruit. Volatile esters are quantitatively and qualitatively the most important compounds providing fruity odors. Biochemical evidence for involvement of the SAAT gene in formation of fruity esters is provided by characterizing the recombinant protein expressed in Escherichia coli. The SAAT enzyme showed maximum activity with aliphatic medium-chain alcohols, whose corresponding esters are major components of strawberry volatiles. The enzyme was capable of utilizing short- and medium-chain, branched, and aromatic acyl-CoA molecules as cosubstrates. The results suggest that the formation of volatile esters in fruit is subject to the availability of acyl-CoA molecules and alcohol substrates and is dictated by the temporal expression pattern of the SAAT gene(s) and substrate specificity of the SAAT enzyme(s).
Subject(s)
Acyltransferases/genetics , Fruit/enzymology , Acyltransferases/chemistry , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA Primers , DNA, Complementary , Escherichia coli/genetics , Fruit/genetics , Genes, Plant , Molecular Sequence Data , Plant Proteins , Sequence Homology, Amino AcidABSTRACT
DNA microarray technology is a new and powerful technology that will substantially increase the speed of molecular biological research. This paper gives a survey of DNA microarray technology and its use in gene expression studies. The technical aspects and their potential improvements are discussed. These comprise array manufacturing and design, array hybridisation, scanning, and data handling. Furthermore, it is discussed how DNA microarrays can be applied in the working fields of: safety, functionality and health of food and gene discovery and pathway engineering in plants.
