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 fingerlike 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.
Somatic embryogenic cultures of white spruce (Picea glauca) represent a valuable system to study molecular mechanisms regulating embryo development because many embryos of defined developmental stages can be generated. The inclusion of polyethylene glycol (PEG) in the maturation medium can improve the number and quality of embryos produced. To learn more about the mechanism of action of PEG, we analyzed transcript profiles of stage-specific embryos matured without (control) or with (PEG treated) PEG. RNA extracted from maturing spruce embryos was analyzed on DNA microarrays containing 2,178 cDNAs from loblolly pine (Pinus taeda). The efficiency of heterologous hybridization between spruce and pine species on microarrays has been documented previously (L. van Zyl, S. von Arnold, P. Bozhkov, Y. Chen, U. Egertsdotter, J. MacKay, R. Sederoff, J. Shen, L. Zelena, D. Clapham [2002] Comp Funct Genomics 3: 306-318). Several pine genes, including the apparent homologs to the Arabidopsis genes ZWILLE, FIDDLEHEAD, FUSCA, and SCARECROW, increased in expression after PEG treatments. These genes are known to be involved in the formation of the embryo body plan and in the control of the shoot and root apical meristems. The increased transcript levels of these genes in immature PEG-treated embryos suggest that PEG may improve the quality of spruce somatic embryos by promoting normal differentiation of the embryonic shoot and root. Changes in the transcript levels of many genes involved in sucrose catabolism and nitrogen assimilation and utilization were also observed between control and PEG-treated embryos.Embryogenesis is a critical stage of the plant life cycle because it establishes the basic body plan. Little is known about the molecular mechanisms that regulate the process (Harada, 1999). This paucity of information is partially because of the location of the embryos, which are embedded within the maternal tissue and are difficult to dissect. The generation of embryos in culture through somatic embryogenesis has become a model system for investigating factors that affect embryo growth. Somatic embryogenesis provides a large number of embryos at defined stages of development, and allows alterations of the embryonic environment through manipulations of the culture conditions. White spruce (Picea glauca) is an economically important species in North America, utilized for pulpwood and lumber production (Hosie, 1979). Regeneration of this species via somatic embryogenesis (Hakman and Fowke, 1987;Lu and Thorpe, 1987) has represented a means of propagation and a model system for conducting physiological and biochemical studies (for review, see Stasolla et al., 2002). Generation of white spruce somatic embryos is commonly achieved by transferring embryogenic tissue onto an abscisic acid (ABA)-containing maturation medium (Lu and Thorpe, 1987). Although such embryos may appear "morphologically" mature, they do not perform well during postembryonic growth without the imposition of a drying period. Improvement of embryo quality can ...
Somatic embryogenesis of Norway spruce (Picea abies L.) is a versatile model system to study molecular mechanisms regulating embryo development because it proceeds through defined developmental stages corresponding to specific culture treatments. Normal embryonic development involves early differentiation of proembryogenic masses (PEMs) into somatic embryos, followed by early and late embryogeny leading to the formation of mature cotyledonary embryos. In some cell lines there is a developmental arrest at the PEM-somatic embryo transition. To learn more about the molecular mechanisms regulating embryogenesis, we compared the transcript profiles of two normal lines and one developmentally arrested line. Ribonucleic acid, extracted from these cell lines at successive developmental stages, was analyzed on DNA microarrays containing 2178 expressed sequence tags (ESTs) (corresponding to 2110 unique cDNAs) from loblolly pine (Pinus taeda L.). Hybridization between spruce and pine species on microarrays has been shown to be effective (van Zyl et al. 2002, Stasolla et al. 2003). In contrast to the developmentally arrested line, the early phases of normal embryo development are characterized by a precise pattern of gene expression, i.e., repression followed by induction. Comparison of transcript levels between successive stages of embryogenesis allowed us to identify several genes that showed unique expression responses during normal development. Several of these genes encode proteins involved in detoxification processes, methionine synthesis and utilization, and carbohydrate metabolism. The potential role of these genes in embryo development is discussed.
The mechanisms underlying defence reactions to a pathogen attack, though well studied in crop plants, are poorly understood in conifers. To analyze changes in gene transcript abundance in Pinus sylvestris L. root tissues infected by Heterobasidion annosum (Fr.) Bref. s.l., a cDNA microarray containing 2109 ESTs from P. taeda L. was used. Mixed model statistical analysis identified 179 expressed sequence tags differentially expressed at 1, 5 or 15 days post inoculation. In general, the total number of genes differentially expressed during the infection increased over time. The most abundant group of genes up-regulated upon infection coded for enzymes involved in metabolism (phenylpropanoid pathway) and defence-related proteins with antimicrobial properties. A class III peroxidase responsible for lignin biosynthesis and cell wall thickening had increased transcript abundance at all measurement times. Real-time RT-PCR verified the microarray results with high reproducibility. The similarity of the expression profiling pattern observed in this pathosystem to those documented in crop pathology suggests that angiosperms and gymnosperms use similar genetic programs in responding to invasive growth by microbial pathogens.
Inclusions of reduced glutathione (GSH) in the maturation medium increased the conversion frequency of white spruce somatic embryos without the need of a partial drying treatment (PDT). This beneficial effect was the result of major alterations in morphology and gene expression during the maturation period. Compared with control embryos, GSH-treated embryos showed a differential accumulation of storage products, i.e. preferential deposition of starch, the reduced formation of protein bodies, and increased vacuolation of cells. These morphological changes correlated with extensive alterations of gene expression occurring throughout the maturation period. The transcript profiles of stage-specific embryos matured with or without GSH were analysed using a DNA microarray containing 2 178 cDNAs from loblolly pine (Pinus taeda). The efficiency of heterologous hybridization between spruce and pine species on microarrays has previously been documented. The results indicate that several genes involved in a variety of signal regulatory pathways were differentially expressed in developing GSH- treated embryos. The transcript levels of many genes involved in carbohydrate metabolism and protein synthesis were altered by the presence of GSH and denoted differences in physiology between treatments. Extensive changes in the expression of genes participating in hormone synthesis, nucleotide metabolism, and meristem formation were also observed and related to the post-embryonic performance of the embryos.
Background: The need to perform microarray experiments with small amounts of tissue has led to the development of several protocols for amplifying the target transcripts. The use of different amplification protocols could affect the comparability of microarray experiments.
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