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Potassium-dependent cambial growth in poplar.

To study the involvement of potassium in wood formation, poplar plants ( Populus tremula L. x Populus tremuloides Michx.) were grown over a period of one growing season, under different potassium regimes. Seasonal changes in cambial potassium content, osmotic potential, and cambial activity correlated strongly throughout the season, increasing from spring to summer and decreasing from summer to autumn. Moreover, changing the potassium supply during the growing season affected the seasonal changes of these parameters in a similar way. Low potassium supply markedly reduced cambial activity, the number of expanding cambial cell derivatives, the seasonal rate of radial wood increment, and the vessel frequency. The possible effect of hormones on potassium-dependent cambial growth was investigated and revealed that abscisic acid (ABA) strongly decreased the potassium content within the cambial zone and reduced cambial activity, as well as the number of expanding cambial cell derivatives. In summary, our results indicate a key role for potassium in the regulation of cambial growth and wood formation due to its strong impact on osmoregulation in expanding cambial cells. They also demonstrate involvement of ABA in regulation of potassium-dependent cambial growth.

Abscisic Acid↗

PubMed2004

Genetics of resistance to transgenic Bacillus thuringiensis poplars in Chrysomela tremulae (Coleoptera: Chrysomelidae).

The area under genetically engineered plants producing Bacillus thuringiensis (Bt) toxins is steadily increasing. This increase has magnified the risk of alleles conferring resistance to these toxins being selected in natural populations of target insect pests. The speed at which this selection is likely to occur depends on the genetic characteristics of Bt resistance. We selected a strain of the beetle Chrysomela tremulae Fabricius on a transgenic Bt poplar clone Populus tremula L. x Populus tremuloides Michx producing high levels of B. thuringiensis Cry3Aa toxin. This strain was derived from an isofemale line that generated some F2 offspring that actively fed on this Bt poplar clone. The resistance ratio of the strain was >6400. Susceptibility had decreased to such an extent that the mortality of beetles of the strain fed Bt poplar leaves was similar to that of beetles fed nontransgenic poplar leaves. Genetic crosses between susceptible, resistant, and F1 hybrids showed that resistance to the Cry3Aa toxin was almost completely recessive (D(LC) = 0.07) and conferred by a single autosomal gene. The concentration of Cry3Aa produced in the transgenic Bt poplar used in this study was 6.34 times higher than the LC99 of the F1 hybrids, accounting for the complete recessivity (D(ML) = 0) of survival on Bt poplar leaves. Overall, the genetic characteristics of the resistance of C. tremulae to the Cry3Aa toxin are consistent with the assumptions underlying the high-dose refuge strategy, which aims to decrease the selection of Bt resistance alleles in natural target pest populations.

Animals↗

PubMed2005

Transcriptome profiling of vertical stem segments provides insights into the genetic regulation of secondary growth in hybrid aspen trees.

In order to better understand the genetic regulation of secondary growth in hybrid aspen (Populus tremula L.xP. alba L.), we carried out a series of cDNA-amplified fragment length polymorphism (AFLP)-based transcriptome analyses in vertical stem segments that represent a gradient of developmental stages with regard to secondary growth. This approach allowed us to screen >80% of the transcriptome expressed in six samples and identify genes differentially expressed with the progress of secondary growth, in a tissue-specific manner. Of the 76,800 transcript-derived fragments (TDFs) analyzed, 271 TDFs were selected and sequenced based on their differential expression patterns. Many of the xylem-up-regulated genes were involved in cell wall and lignin biosynthesis, while the bark-up-regulated genes had diverse functional roles. About 25% of the xylem-up-regulated TDFs analyzed were involved in the phenylpropanoid biosynthesis pathway, which produces the cell wall polymer lignin and various wood extractives. In addition, many of the TDFs showing secondary xylem-specific expression were annotated as genes not previously reported in Populus, including novel cell death proteins, cytoskeleton-interacting proteins, transporters and putative transcription factors.

Blotting, Northern↗

PubMed2006

The poplar glycosyltransferase GT47C is functionally conserved with Arabidopsis Fragile fiber8.

Xylan is the major hemicellulose in dicot wood. Unraveling genes involved in the biosynthesis of xylan will be of importance in understanding the process of wood formation. In this report, we investigated the possible role of poplar GT47C, a glycosyltransferase belonging to family GT47, in the biosynthesis of xylan. PoGT47C from the hybrid poplar Populus alba x tremula exhibits 84% sequence similarity to Fragile fiber8 (FRA8), which is involved in the biosynthesis of glucuronoxylan in Arabidopsis. Phylogenetic analysis of glycosyltransferase family GT47 in the Populus trichocarpa genome revealed that GT47C is the only close homolog of FRA8. In situ hybridization showed that the PoGT47C gene was expressed in developing primary xylem, secondary xylem and phloem fibers of stems, and in developing secondary xylem of roots. Sequence analysis suggests that PoGT47C is a type II membrane protein, and study of the subcellular localization demonstrated that fluorescent protein-tagged PoGT47C was located in the Golgi. Immunolocalization with a xylan monoclonal antibody LM10 revealed a nearly complete loss of xylan signals in the secondary walls of fibers and vessels in the Arabidopsis fra8 mutant. Expression of PoGT47C in the fra8 mutant restored the secondary wall thickness and xylan content to the wild-type level. Together, these results suggest that PoGT47C is functionally conserved with FRA8 and it is probably involved in xylan synthesis during wood formation.

Amino Acid Sequence↗

PubMed2002

Genomics and the tree physiologist.

During the last decade, a strong case has been made for viewing trees as model systems in plant biology. More recently, forest biologists have argued for the sequencing of the genome of a forest tree. Now, the United States Department of Energy has announced plans for sequencing the genome of the Populus trichocarpa clone, "Nisqually-1." Thus, forest biology is poised to enter an exciting period of scientific discovery. Embracing new technology and new research paradigms, however, is never easy. It is, therefore, timely to ask how tree physiologists will take advantage of the Populus genome data. We contend that the most attractive opportunities will arise through genome-wide research designed to: (1). examine the differences between trees and herbaceous annuals; (2). explore questions relating to the temporal and spatial scales that characterize the life histories and growth of trees; and (3). investigate cause-and-effect relationships that are intractable to conventional research methodologies. To highlight the potential applications of genomics in tree physiology, we briefly discuss each of these approaches.

Genome, Plant↗

PubMed2003

Free-air CO2 enrichment (FACE) enhances biomass production in a short-rotation poplar plantation.

This paper investigates the possible contribution of Short Rotation Cultures (SRC) to carbon sequestration in both current and elevated atmospheric CO2 concentrations ([CO2]). A dense poplar plantation (1 x 1 m) was exposed to a [CO2] of 550 ppm in Central Italy using the free-air CO2 enrichment (FACE) technique. Three species of Populus were examined, namely P. alba L., P. nigra L. and P. x euramericana Dode (Guinier). Aboveground woody biomass of trees exposed to elevated [CO2] for three growing seasons increased by 15 to 27%, depending on species. As a result, light-use efficiency increased. Aboveground biomass allocation was unaffected, and belowground biomass also increased under elevated [CO2] conditions, by 22 to 38%. Populus nigra, with total biomass equal to 62.02 and 72.03 Mg ha-1 in ambient and elevated [CO2], respectively, was the most productive species, although its productivity was stimulated least by atmospheric CO2 enrichment. There was greater depletion of inorganic nitrogen from the soil after three growing seasons in elevated [CO2], but no effect of [CO2] on stem wood density, which differed significantly only among species.

Biomass↗

PubMed2003

Ecophysiology of riparian cottonwoods: stream flow dependency, water relations and restoration.

Cottonwoods (Populus spp.) are adapted to riparian or floodplain zones throughout the Northern Hemisphere; they are also used as parents for fast-growing hybrid poplars. We review recent ecophysiological studies of the native cottonwoods Populus angustifolia James, P. balsamifera L., P. deltoides Marsh., P. fremontii S. Watson and P. trichocarpa T. & G. in North America, and P. nigra L. in Europe. Variation exists within and across species and hybrids; however, all riparian cottonwoods are dependent on shallow alluvial groundwater that is linked to stream water, particularly in semi-arid regions. This conclusion is based on studies of their natural occurrence, decline following river damming and dewatering (water removal), water relations, isotopic composition of xylem water, and by the establishment of cottonwoods along formerly barren natural channels after flow augmentation in response to the conveyance of irrigation water. When alluvial groundwater is depleted as a result of river dewatering or groundwater pumping, riparian cottonwoods exhibit drought-stress responses including stomatal closure and reduced transpiration and photosynthesis, altered 13C composition, reduced predawn and midday water potentials, and xylem cavitation. These physiological responses are accompanied by morphological responses including reduced shoot growth, altered root growth, branch sacrifice and crown die-back. In severe cases, mortality occurs. For example, severe dewatering of channels of the braided Big Lost River in Idaho led to mortality of the narrowleaf cottonwood, P. angustifolia, and adjacent sandbar willows, Salix exigua Nutt., within 5 years, whereas riparian woodlands thrived along flowing channels nearby. The conservation and restoration of cottonwoods will rely on the provision of river flow regimes that satisfy these ecophysiological requirements for survival, growth and reproduction.

Adaptation, Physiological↗

PubMed2003

Bacterial disease resistance of transgenic hybrid poplar expressing the synthetic antimicrobial peptide D4E1.

Important losses in poplar productivity occur because of susceptibility to microbial pathogens. To enhance disease resistance in susceptible genotypes, the gene coding for D4E1, a synthetic antimicrobial peptide consisting of 17 amino acid residues, was introduced into poplar (Populus tremula L. x Populus alba L.) via Agrobacterium-mediated transformation. Four kanamycin-resistant transformants were selected based on significant accumulation of the D4E1 transcript and confirmed by reverse transcription-polymerase chain reaction and RNA dot-blot analysis. These transgenic poplar lines were tested for resistance to Agrobacterium tumefaciens, Xanthomonas populi pv. populi and Hypoxylon mammatum (Wahl.) Miller. One transgenic poplar line, Tr23, bearing the highest transcript accumulation for the D4E1 gene, showed a significant reduction in symptoms caused by A. tumefaciens and X. populi. However, none of the transgenic poplar lines showed a significant difference in disease response to the fungal pathogen H. mammatum.

Cecropins↗

PubMed2005

Differential expression of a poplar copper chaperone gene in response to various abiotic stresses.

Copper chaperone (CCH) is upregulated during Arabidopsis (Arabidopsis thaliana L. Columbia) leaf senescence, suggesting that it mobilizes certain metal ions in leaves and transports them to other growing parts of the plants. The CCHs are also involved in defense mechanisms against oxidative stress in Arabidopsis and tomato (Lycopersicon esculentum Mill. cv. 'Ailsa Craig'). To elucidate the functions of CCH in poplar, we cloned a CCH gene (PoCCH) from Populus (Populus alba x P. tremula var. glandulosa) suspension cells and tested its expression in response to various treatments including heavy metals, plant growth regulators and abiotic stresses. The PoCCH cDNA is 540 bp in length, including a 55-bp 5' noncoding domain, a 258-bp open reading frame (ORF), and 227-bp 3' termination region. The coding region of PoCCH represents a putative 85-amino-acid protein with a molecular weight of 8.9 kDa. The deduced amino acid sequence of the PoCCH gene product is 87 and 78% identical to those of tomato and Arabidopsis, respectively, with a high degree of conservation in both the metal-binding and lysine-rich regions. However, the PoCCH gene product lacks the C-terminal extension identified in Arabidopsis CCH. Southern blot analysis suggested that the PoCCH gene is present in low copy numbers in poplar. The expression of PoCCH increased under copper deprivation conditions. The expression of PoCCH was down-regulated by high concentrations of copper, whereas some metals, such as aluminum and zinc, markedly induced PoCCH, and others including cadmium, cobalt and lead had little effect on PoCCH expression. The plant growth regulator jasmonic acid caused an increase in PoCCH transcript whereas abscisic acid, salicylic acid and gibberellic acid did not. The gene was highly induced when cells were exposed to physical stress by high-speed agitation on a gyratory shaker. Other effective inducers of PoCCH expression in suspension culture were methyl viologen and NaCl. Thus, PoCCH does not respond to all stresses, but responds specifically to certain metals and abiotic stresses that induce oxidative damage. Our results suggest that JA is involved in regulating PoCCH expression in poplar cells.

Adaptation, Physiological↗

PubMed2006

Growth and physiological responses to supplemental UV-B radiation of two contrasting poplar species.

We compared the growth and physiological responses of cuttings of Populus kangdingensis C. Wang et Tung and P. cathayana Rehder originating from altitudes of 3500 m and 1500 m, respectively, when exposed to three ultraviolet-B (UV-B) radiation regimes: zero UV-B, ambient (4.5 kJ m(-2) day(-1)) and twice-ambient (9 kJ m(-2) day(-1)) biologically effective UV-B radiation for one growing season, to determine if Populus trees that are adapted to contrasting UV-B habitats exhibit different tolerances to enhanced UV-B radiation. Compared with cuttings grown without UV-B radiation, twice-ambient UV-B radiation significantly decreased total biomass, total leaf area and internode length in both species, whereas root/shoot ratio, leaf number, amount of photosynthetic pigments and ascorbate peroxidase activity were unaffected. Differences in responses were also observed between the study species. Cutting height increment, total biomass, total leaf area, free proline concentration and membrane damage assessed by electrolyte leakage were significantly more affected by the twice-ambient UV-B radiation in P. cathayana than in P. kangdingensis. However, specific leaf mass, amount of UV-B absorbing compounds and superoxide dismutase and guaiacol peroxidase activities increased more with increasing UV-B radiation in P. kangdingensis than in P. cathayana, perhaps reflecting important characteristics of species with low to moderate tolerance to UV-B radiation. Overall, the results indicated that P. kangdingensis, which originates from altitudes of 3500 m, has greater tolerance to enhanced UV-B radiation than P. cathayana originating from altitudes of 1500 m.

Adaptation, Physiological↗

PubMed2002

Differential expression of two distinct phenylalanine ammonia-lyase genes in condensed tannin-accumulating and lignifying cells of quaking aspen.

Lignins, along with condensed tannins (CTs) and salicylate-derived phenolic glycosides, constitute potentially large phenylpropanoid carbon sinks in tissues of quaking aspen (Populus tremuloides Michx.). Metabolic commitment to each of these sinks varies during development and adaptation, and depends on L-phenylalanine ammonia-lyase (PAL), an enzyme catalyzing the deamination of L-phenylalanine to initiate phenylpropanoid metabolism. In Populus spp., PAL is encoded by multiple genes whose expression has been associated with lignification in primary and secondary tissues. We now report cloning two differentially expressed PAL cDNAs that exhibit distinct spatial associations with CT and lignin biosynthesis in developing shoot and root tissues of aspen. PtPAL1 was expressed in certain CT-accumulating, non-lignifying cells of stems, leaves, and roots, and the pattern of PtPAL1 expression varied coordinately with that of CT accumulation along the primary to secondary growth transition in stems. PtPAL2 was expressed in heavily lignified structural cells of shoots, but was also expressed in non-lignifying cells of root tips. Evidence of a role for Pt4CL2, encoding 4-coumarate:coenzyme A ligase, in determining CT sink strength was gained from cellular co-expression analysis with PAL1 and CTs, and from experiments in which leaf wounding increased PAL1 and 4CL2 expression as well as the relative allocation of carbon to CT with respect to phenolic glycoside, the dominant phenolic sink in aspen leaves. Leaf wounding also increased PAL2 and lignin pathway gene expression, but to a smaller extent. The absence of PAL2 in most CT-accumulating cells provides in situ support for the idea that PAL isoforms function in specific metabolic milieus.

Arabidopsis Proteins↗

PubMed2002

Cloning, functional expression, and subcellular localization of multiple NADPH-cytochrome P450 reductases from hybrid poplar.

NADPH:cytochrome P450 reductase (CPR) provides reducing equivalents to diverse cytochrome P450 monooxygenases. We isolated cDNAs for three CPR genes (CPR1, CPR2, and CPR3) from hybrid poplar (Populus trichocarpa x Populus deltoides). Deduced CPR2 and CPR3 amino acid sequences were 91% identical, but encoded isoforms divergent from CPR1 (72% identity). CPR1 and CPR2 were co-expressed together with the P450 enzyme cinnamate-4-hydroxylase (C4H) in yeast (Saccharomyces cerevisiae). Microsomes isolated from strains expressing CPR1/C4H or CPR2/C4H enhanced C4H activities approximately 10-fold relative to the C4H-only control strain, and catalyzed NADPH-dependent cytochrome c reduction. The divergent CPR isoforms (CPR1 and CPR2/3) contained entirely different N-terminal sequences, which are conserved in other plant CPRs and are diagnostic for two distinct classes of CPRs within the angiosperms. C-terminal green fluorescent protein fusions to CPR1 and CPR2 were constructed and expressed in both yeast and Arabidopsis. The fusion proteins expressed in yeast retained the ability to support C4H activity and, thus, were catalytically active. Both CPR::green fluorescent protein fusion proteins were strictly localized to the endoplasmic reticulum in transgenic Arabidopsis. The lack of localization of either isoform to chloroplasts, where P450s are known to be present, suggests that an alternative P450 reduction system may be operative in this organelle. Transcripts for the three poplar CPR genes were present ubiquitously in all tissues examined, but CPR2 showed highest expression in young leaf tissue.

Amino Acid Sequence↗

PubMed2003

Gene expression in autumn leaves.

Two cDNA libraries were prepared, one from leaves of a field-grown aspen (Populus tremula) tree, harvested just before any visible sign of leaf senescence in the autumn, and one from young but fully expanded leaves of greenhouse-grown aspen (Populus tremula x tremuloides). Expressed sequence tags (ESTs; 5,128 and 4,841, respectively) were obtained from the two libraries. A semiautomatic method of annotation and functional classification of the ESTs, according to a modified Munich Institute of Protein Sequences classification scheme, was developed, utilizing information from three different databases. The patterns of gene expression in the two libraries were strikingly different. In the autumn leaf library, ESTs encoding metallothionein, early light-inducible proteins, and cysteine proteases were most abundant. Clones encoding other proteases and proteins involved in respiration and breakdown of lipids and pigments, as well as stress-related genes, were also well represented. We identified homologs to many known senescence-associated genes, as well as seven different genes encoding cysteine proteases, two encoding aspartic proteases, five encoding metallothioneins, and 35 additional genes that were up-regulated in autumn leaves. We also indirectly estimated the rate of plastid protein synthesis in the autumn leaves to be less that 10% of that in young leaves.

Arabidopsis Proteins↗

PubMed2003

Rapid alkalinization factors in poplar cell cultures. Peptide isolation, cDNA cloning, and differential expression in leaves and methyl jasmonate-treated cells.

A family of peptides inducing rapid pH alkalinization in hybrid poplar (Populus trichocarpa x Populus deltoides) cell culture medium was isolated from hybrid poplar leaves. Five related approximately 5-kD peptides were purified by high-performance liquid chromatography and analyzed by matrix-assisted laser desorption ionization-mass spectrometry. The N-terminal sequence of one of the isolated peptides was very similar to a previously characterized peptide from tobacco (Nicotiana tabacum), rapid alkalinization factor (RALF), which causes a rapid increase in culture medium pH when added to tobacco cell cultures (G. Pearce, D.S. Moura, J. Stratmann, C.A. Ryan [2001] Proc Natl Acad Sci USA 98: 12843-12847). Two unique poplar RALF cDNAs (PtdRALF1 and PtdRALF2) were isolated from a poplar cDNA library and used to study RALF expression in poplar saplings and cultured poplar cells. Both genes were found to be expressed constitutively in poplar saplings and cultured cells. However, PtdRALF2 was expressed in leaves at very low levels, and its expression in suspension culture cells was transiently suppressed by methyl jasmonate (MeJa). Although the function of these novel peptides remains enigmatic, our experiments suggest their role may be developmental rather than stress related. Overall, our study confirms the presence of active RALF peptides in other plants, and provides new data on the complexity of the RALF gene family in poplar.

Acetates↗

PubMed2003

Ectopic expression of a horseradish peroxidase enhances growth rate and increases oxidative stress resistance in hybrid aspen.

We previously demonstrated that overexpression of the horseradish (Armoracia rusticana) peroxidase prxC1a gene stimulated the growth rate of tobacco (Nicotiana tabacum) plants. Here, the cauliflower mosaic virus 35S::prxC1a construct was introduced into hybrid aspen (Populus sieboldii x Populus grandidentata). The growth rate of these transformed hybrid aspen plants was substantially increased under greenhouse conditions. The average stem length of transformed plants was 25% greater than that of control plants. There was no other obvious phenotypic difference between the transformed and control plants. Fast-growing transformed hybrid aspen showed high levels of expression of prxC1a and had elevated peroxidase activities toward guaiacol and ascorbate. However, there was no increase of the endogenous class I ascorbate peroxidase activities in the transformed plants by separate assay and activity staining of native polyacrylamide gel electrophoresis. Furthermore, calli derived from the transformed hybrid aspen grew faster than those from control plants and were resistant to the oxidative stress imposed by hydrogen peroxide. Therefore, enhanced peroxidase activity affects plant growth rate and oxidative stress resistance.

Gene Expression Regulation, Plant↗

PubMed2003

Activation tagging of a dominant gibberellin catabolism gene (GA 2-oxidase) from poplar that regulates tree stature.

We identified a dwarf transgenic hybrid poplar (Populus tremula x Populus alba) after screening of 627 independent activation-tagged transgenic lines in tissue culture, greenhouse, and field environments. The cause of the phenotype was a hyperactivated gene encoding GA 2-oxidase (GA2ox), the major gibberellin (GA) catabolic enzyme in plants. The mutation resulted from insertion of a strong transcriptional enhancer near the transcription start site. Overexpression of the poplar GA2ox gene (PtaGA2ox1) caused hyperaccumulation of mRNA transcripts, quantitative shifts in the spectrum of GAs, and similarity in phenotype to transgenic poplars that overexpress a bean (Phaseolus coccineus) GA2ox gene. The poplar PtaGA2ox1 sequence was most closely related to PsGA2ox2 from pea (Pisum sativum) and two poorly known GA2oxs from Arabidopsis (AtGA2ox4 and AtGA2ox5). The dwarf phenotype was reversible through gibberellic acid application to the shoot apex. Transgenic approaches to producing semidwarf trees for use in arboriculture, horticulture, and forestry could have significant economic and environmental benefits, including altered fiber and fruit production, greater ease of management, and reduced risk of spread in wild populations.

Amino Acid Sequence↗

PubMed2004

Poplar peroxiredoxin Q. A thioredoxin-linked chloroplast antioxidant functional in pathogen defense.

Peroxiredoxins are ubiquitous thioredoxin- or glutaredoxin-dependent peroxidases, the function of which is to destroy peroxides. Peroxiredoxin Q, one of the four plant subtypes, is a homolog of the bacterial bacterioferritin comigratory proteins. We show here that the poplar (Populus tremula x Populus tremuloides) protein acts as a monomer with an intramolecular disulfide bridge between two conserved cysteines. A wide range of electron donors and substrates was tested. Unlike type II peroxiredoxin, peroxiredoxin Q cannot use the glutaredoxin or cyclophilin isoforms tested, but various cytosolic, chloroplastic, and mitochondrial thioredoxins are efficient electron donors with no marked specificities. The redox midpoint potential of the peroxiredoxin Q catalytic disulfide is -325 mV at pH 7.0, explaining why the wild-type protein is reduced by thioredoxin but not by glutaredoxin. Additional evidence that thioredoxin serves as a donor comes from the formation of heterodimers between peroxiredoxin Q and monocysteinic mutants of spinach (Spinacia oleracea) thioredoxin m. Peroxiredoxin Q can reduce various alkyl hydroperoxides, but with a better efficiency for cumene hydroperoxide than hydrogen peroxide and tertiary butyl hydroperoxide. The use of immunolocalization and of a green fluorescence protein fusion construct indicates that the transit sequence efficiently targets peroxiredoxin Q to the chloroplasts and especially to those of the guard cells. The expression of this protein and of type II peroxiredoxin is modified in response to an infection by two races of Melampsora larici-populina, the causative agent of the poplar rust. In the case of an hypersensitive response, the peroxiredoxin expression increased, whereas it decreased during a compatible interaction.

Amino Acid Sequence↗

PubMed2004

Cloning and overproduction of gibberellin 3-oxidase in hybrid aspen trees. Effects on gibberellin homeostasis and development.

To broaden our understanding of gibberellin (GA) biosynthesis and the mechanism whereby GA homeostasis is maintained in plants, we have investigated the degree to which the enzyme GA 3-oxidase (GA3ox) limits the formation of bioactive GAs in elongating shoots of hybrid aspen (Populus tremula x Populus tremuloides). We describe the cloning of a hybrid aspen GA3ox and its functional characterization, which confirmed that it has 3beta-hydroxylation activity and more efficiently converts GA9 to GA4 than GA20 to GA1. To complement previous studies, in which transgenic GA 20-oxidase (GA20ox) overexpressers were found to produce 20-fold higher bioactive GA levels and subsequently grew faster than wild-type plants, we overexpressed an Arabidopsis GA3ox in hybrid aspen. The generated GA3ox overexpresser lines had increased 3beta-hydroxylation activity but exhibited no major changes in morphology. The nearly unaltered growth pattern was associated with relatively small changes in GA1 and GA4 levels, although tissue-dependent differences were observed. The absence of increases in bioactive GA levels did not appear to be due to feedback or feed-forward regulation of dioxygenase transcripts, according to semiquantitative reverse transcription polymerase chain reaction analysis of PttGA20ox1, PttGA3ox1, and two putative PttGA2ox genes. We conclude that 20-oxidation is the limiting step, rather than 3beta-hydroxylation, in the formation of GA1 and GA4 in elongating shoots of hybrid aspen, and that ectopic GA3ox expression alone cannot increase the flux toward bioactive GAs. Finally, several lines of evidence now suggest that GA4 has a more pivotal role in the tree hybrid aspen than previously believed.

Cloning, Molecular↗