Heterologous array analysis in Pinaceae: hybridization of Pinus taeda cDNA arrays with cDNA from needles and embryogenic cultures of P. taeda, P. sylvestris or Picea abies

Abstract

Hybridization of labelled cDNA from various cell types with high-density arrays of expressed sequence tags is a powerful technique for investigating gene expression. Few conifer cDNA libraries have been sequenced. Because of the high level of sequence conservation between Pinus and Picea we have investigated the use of arrays from one genus for studies of gene expression in the other. The partial cDNAs from 384 identifiable genes expressed in differentiating xylem of Pinus taeda were printed on nylon membranes in randomized replicates. These were hybridized with labelled cDNA from needles or embryogenic cultures of Pinus taeda, P. sylvestris and Picea abies, and with labelled cDNA from leaves of Nicotiana tabacum. The Spearman correlation of gene expression for pairs of conifer species was high for needles (r 2 = 0.78 − 0.86), and somewhat lower for embryogenic cultures (r 2 = 0.68 − 0.83). The correlation of gene expression for tobacco leaves and needles of each of the three conifer species was lower but sufficiently high (r 2 = 0.52 − 0.63) to suggest that many partial gene sequences are conserved in angiosperms and gymnosperms. Heterologous probing was further used to identify tissue-specific gene expression over species boundaries. To evaluate the significance of differences in gene expression, conventional parametric tests were compared with permutation tests after four methods of normalization. Permutation tests after Z-normalization provide the highest degree of discrimination but may enhance the probability of type I errors. It is concluded that arrays of cDNA from loblolly pine are useful for studies of gene expression in other pines or spruces.

Important processes during differentiation and early development of somatic embryos of Norway spruce as revealed by changes in global gene expression

“…We have previously demonstrated that Norway spruce cDNAs hybridize strongly with arrayed loblolly pine cDNA, thus allowing pine arrays to be used to assay spruce gene expression (Van Zyl et al 2002). To further investigate the hybridization efficiencies of the Norway spruce transcriptome towards the heterologous array, we have compared the sequence identity among Pinus and Picea species for genes used in qRT-PCR to validate the microarray results.…”

Proteomic Analysis of Somatic Embryogenesis in Medicago truncatula. Explant Cultures Grown under 6-Benzylaminopurine and 1-Naphthaleneacetic Acid Treatments

“…Two-dimensional gel electrophoresis-based proteomic approaches have been applied to investigate molecular changes during SE in several species (Choi and Sung, 1984;Gianazza et al, 1992;Reinbothe et al, 1992;Boyer et al, 1993;Pedroso et al, 1995;Yoshida et al, 1995;Dodeman and Ducreux, 1996;Giroux and Pauls, 1996;Dodeman et al, 1998;Sallandrouze et al, 1999) with limited success. Large-scale transcription analyses of embryogenesis have also been reported in several species (Giroux and Pauls, 1997;van Zyl et al, 2002van Zyl et al, , 2003Stasolla et al, 2003;Thibaud-Nissen et al, 2003;Stasolla et al, 2004).…”

Microarray Analyses of Gene Expression during Adventitious Root Development in Pinus contorta

“…Microarray analysis has previously been used for identifying genes involved in cell wall biosynthesis during xylogenesis in P. taeda (Whetten et al, 2001). Recently, we showed that arrays from P. taeda can be used for studying gene expression in Picea abies and Pinus sylvestris (van Zyl et al, 2002a(van Zyl et al, , 2002bStasolla et al, 2003). At present, the possibility of sampling single cells or a small number of cells, and also obtaining synchronized populations of cells, is restricted (Hertzberg et al, 2001;van Zyl et al, 2002a).…”