12 research outputs found
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
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
(r2 = 0.78 ā 0.86), and somewhat lower for embryogenic cultures (r2 = 0.68 ā 0.83).
The correlation of gene expression for tobacco leaves and needles of each of the three
conifer species was lower but sufficiently high (r2 = 0.52 ā 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
Studying the Functional Genomics of Stress Responses in Loblolly Pine With the Expresso Microarray Experiment Management System
Conception, design, and implementation of cDNA microarray experiments present a
variety of bioinformatics challenges for biologists and computational scientists. The multiple
stages of data acquisition and analysis have motivated the design of Expresso, a
system for microarray experiment management. Salient aspects of Expresso include
support for clone replication and randomized placement; automatic gridding, extraction of
expression data from each spot, and quality monitoring; flexible methods of combining
data from individual spots into information about clones and functional categories; and the
use of inductive logic programming for higher-level data analysis and mining. The
development of Expresso is occurring in parallel with several generations of microarray
experiments aimed at elucidating genomic responses to drought stress in loblolly pine
seedlings. The current experimental design incorporates 384 pine cDNAs replicated and
randomly placed in two specific microarray layouts. We describe the design of Expresso as
well as results of analysis with Expresso that suggest the importance of molecular
chaperones and membrane transport proteins in mechanisms conferring successful
adaptation to long-term drought stress
Microarray Analyses of Gene Expression during Adventitious Root Development in Pinus contorta
In order to investigate the gene expression pattern during adventitious root development, RNA of Pinus contorta hypocotyls, pulse-treated with the auxin indole-3-butyric acid and harvested at distinct developmental time points of root development, was hybridized to microarrays containing 2,178 cDNAs from Pinus taeda. Over the period of observation of root development, the transcript levels of 220 genes changed significantly. During the root initiation phase, genes involved in cell replication and cell wall weakening and a transcript encoding a PINHEAD/ZWILLE-like protein were up-regulated, while genes related to auxin transport, photosynthesis, and cell wall synthesis were down-regulated. In addition, there were changes in transcript abundance of genes related to water stress. During the root meristem formation phase the transcript abundances of genes involved in auxin transport, auxin responsive transcription, and cell wall synthesis, and of a gene encoding a B-box zinc finger-like protein, increased, while those encoding proteins involved in cell wall weakening decreased. Changes of transcript abundance of genes related to water stress during the root meristem formation and root formation phase indicate that the plant roots had become functional in water transport. Simultaneously, genes involved in auxin transport were up-regulated, while genes related to cell wall modification were down-regulated. Finally, during the root elongation phase down-regulation of transcripts encoding proteins involved in cell replication and stress occurred. Based on the observed changes in transcript abundances, we suggest hypotheses about the relative importance of various physiological processes during the auxin-induced development of roots in P. contorta