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Glutaredoxin-dependent peroxiredoxin from poplar: protein-protein interaction and catalytic mechanism.

Recently, a poplar phloem peroxiredoxin (Prx) was found to accept both glutaredoxin (Grx) and thioredoxin (Trx) as proton donors. To investigate the catalytic mechanism of the Grx-dependent reduction of hydroperoxides catalyzed by Prx, a series of cysteinic mutants was constructed. Mutation of the most N-terminal conserved cysteine of Prx (Cys-51) demonstrates that it is the catalytic one. The second cysteine (Cys-76) is not essential for peroxiredoxin activity because the C76A mutant retained approximately 25% of the wild type Prx activity. Only one cysteine of the Grx active site (Cys-27) is essential for peroxiredoxin catalysis, indicating that Grx can act in this reaction either via a dithiol or a monothiol pathway. The creation of covalent heterodimers between Prx and Grx mutants confirms that Prx Cys-51 and Grx Cys-27 are the two residues involved in the catalytic mechanism. The integration of a third cysteine in position 152 of the Prx, making it similar in sequence to the Trx-dependent human Prx V, resulted in a protein that had no detectable activity with Grx but kept activity with Trx. Based on these experimental results, a catalytic mechanism is proposed to explain the Grx- and Trx-dependent activities of poplar Prx.

Amino Acid Sequence↗

Functional and structural aspects of poplar cytosolic and plastidial type a methionine sulfoxide reductases.

The genome of Populus trichocarpa contains five methionine sulfoxide reductase A genes. Here, both cytosolic (cMsrA) and plastidial (pMsrA) poplar MsrAs were analyzed. The two recombinant enzymes are active in the reduction of methionine sulfoxide with either dithiothreitol or poplar thioredoxin as a reductant. In both enzymes, five cysteines, at positions 46, 81, 100, 196, and 202, are conserved. Biochemical and enzymatic analyses of the cysteine-mutated MsrAs support a catalytic mechanism involving three cysteines at positions 46, 196, and 202. Cys(46) is the catalytic cysteine, and the two C-terminal cysteines, Cys(196) and Cys(202), are implicated in the thioredoxin-dependent recycling mechanism. Inspection of the pMsrA x-ray three-dimensional structure, which has been determined in this study, strongly suggests that contrary to bacterial and Bos taurus MsrAs, which also contain three essential Cys, the last C-terminal Cys(202), but not Cys(196), is the first recycling cysteine that forms a disulfide bond with the catalytic Cys(46). Then Cys(202) forms a disulfide bond with the second recycling cysteine Cys(196) that is preferentially reduced by thioredoxin. In agreement with this assumption, Cys(202) is located closer to Cys(46) compared with Cys(196) and is included in a (202)CYG(204) signature specific for most plant MsrAs. The tyrosine residue corresponds to the one described to be involved in substrate binding in bacterial and B. taurus MsrAs. In these MsrAs, the tyrosine residue belongs to a similar signature as found in plant MsrAs but with the first C-terminal cysteine instead of the last C-terminal cysteine.

Cloning, Molecular↗

Leaching of contaminated leaves following uptake and phytoremediation of RDX, HMX, and TNT by poplar.

The uptake and fate of 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) by hybrid poplars in hydroponic systems were compared and exposed leaves were leached with water to simulate potential exposure pathways from groundwater in the field. TNT was removed from solution more quickly than nitramine explosives. Most of radioactivity remained in root tissues for 14C-TNT, but in leaves for 14C-RDX and 14C-HMX. Radiolabel recovery for TNT and HMX was over 94%, but that of RDX decreased over time, suggesting a loss of volatile products. A considerable fraction (45.5%) of radioactivity taken up by whole plants exposed to 14C-HMX was released into deionized water, mostly as parent compound after 5 d of leaching. About a quarter (24.0%) and 1.2% were leached for RDX and TNT, respectively, mostly as transformed products. Leached radioactivity from roots was insignificant in all cases (< 2%). This is the first report in which small amounts of transformation products of RDX leach from dried leaves following uptake by poplars. Such behavior for HMX was reported earlier and is reconfirmed here. All three compounds differ substantially in their fate and transport during the leaching process.

Azocines↗

Stimulating natural defenses in poplar clones (OP-367) increases plant metabolism of carbon tetrachloride.

Groundwater contamination by carbon tetrachloride (CCl4) presents a health risk as a potential carcinogen and pollutant that is capable of depleting the ozone layer. Although use of poplar trees in a phytoremediation capacity has proven to be cost effective for cleaning contaminated sites, minimizing leaf emission of volatile contaminants remains a pressing issue. We hypothesized that recently fixed carbon plays a key role in CCl4 metabolism in planta yielding nonvolatile trichloroacetic acid (TCA) and that the extent of this metabolism can be altered by heightening plant defenses. Labeling intact leaves with (11)CO2 (t 1/2 20.4 m) can test this hypothesis, because the extremely short half-life of the tracer reflects only those processes involving recently fixed carbon. Using radio-HPLC analysis, we observed [(11)C]TCA from leaf extract from poplar clones (OP-367) whose roots were exposed to a saturated solution of CCl4 (520 ppm). Autoradiography of [(11)C]photosynthate showed increased leaf export and partitioning to the apex within 24 h of CCl4 exposure, suggesting that changes in plant metabolism and partitioning of recently fixed carbon occur rapidly. Additionally, leaf CCl4 emissions were highest in the morning, when carbon pools are low, suggesting a link between contaminant metabolism and leaf carbon utilization. Further, treatment with methyljasmonate, a plant hormone implicated in defense signal transduction, reduced leaf CCl4 emissions two-fold due to the increased formation of TCA.

Acetates↗

Regulation of sulphate assimilation by glutathione in poplars (Populus tremula x P. alba) of wild type and overexpressing gamma-glutamylcysteine synthetase in the cytosol.

Glutathione (GSH) is the major low molecular weight thiol in plants with different functions in stress defence and the transport and storage of sulphur. Its synthesis is dependent on the supply of its constituent amino acids cysteine, glutamate, and glycine. GSH is a feedback inhibitor of the sulphate assimilation pathway, the primary source of cysteine synthesis. Sulphate assimilation has been analysed in transgenic poplars (Populus tremula x P. alba) overexpressing gamma-glutamylcysteine synthetase, the key enzyme of GSH synthesis, and the results compared with the effects of exogenously added GSH. Although foliar GSH levels were 3-4-fold increased in the transgenic plants, the activities of enzymes of sulphate assimilation, namely ATP sulphurylase, adenosine 5'-phosphosulphate reductase (APR), sulphite reductase, serine acetyltransferase, and O-acetylserine (thiol)lyase were not affected in three transgenic lines compared with the wild type. Also the mRNA levels of these enzymes were not altered by the increased GSH levels. By contrast, an increase in GSH content due to exogenously supplied GSH resulted in a strong reduction in APR activity and mRNA accumulation. This feedback regulation was reverted by simultaneous addition of O-acetylserine (OAS). However, OAS measurements revealed that OAS cannot be the only signal responsible for the lack of feedback regulation of APR by GSH in the transgenic poplars.

Cloning, Molecular↗

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↗

An interregional validation of ECOPHYS, a growth process model of juvenile poplar clones.

Field data from poplar plantations in Michigan, Washington, and Wisconsin were used to validate ECOPHYS, a whole-tree growth process model for juvenile poplar. Five clones representing a range of morphological, phenological, and physiological characteristics were planted on the same date at the three sites. Height and diameter measurements were made monthly on 20 trees per clone, and intensive morphological measurements were made every two weeks on two trees per clone. Hourly solar radiation and temperature data were recorded at each site over the growing season. The model was run for each clone x site combination with the weather data and clonal parameters as inputs. A repeated measures ANOVA indicated that there were significant differences in height growth patterns among both clones and sites, as well as significant clone x site interactions. The model generally predicted height growth within a standard deviation of the field plantation means; three of the 15 clone x site simulations were significantly different from the plantation means. The median error between predicted and observed values was 5%. Evaluation of the clonal parameters showed that differences in photosynthetic rates, morphological attributes such as specific leaf area, and timing of budset are primary factors leading to differences in growth.

Journal Article↗

Nutrition, xylem cavitation and drought resistance in hybrid poplar.

Effects of mineral nutrition on susceptibility to cavitation were examined in four hybrid poplar clones. Two drought-sensitive and two drought-resistant hybrid clones of black cottonwood (Populus trichocarpa Torr. & Gray) and eastern cottonwood (P. deltoides Bartr.) were grown at three concentrations of nitrogen (N) applied factorially with two concentrations of phosphorus (P) in a greenhouse, and subjected to varying degrees of drought stress before measurement of cavitation and of anatomical features that might affect cavitation. Mean vessel pit pore diameters were 0.132 micro m at low P, and 0.074 micro m at high P, but no other significant effects of mineral nutrition on vessel dimensions were observed. Vessel diameter and specific conductivity were greater in the drought-resistant clones than in the drought-susceptible clones. Drought-resistant clones did not reach such low water potentials as drought-sensitive clones during the cavitation induction experiments, suggesting better stomatal and cuticular control of water loss. Scanning electron microscope observations showed less damage to pit membranes, also suggesting greater membrane strength in drought-resistant clones than in drought-sensitive clones. High concentrations of N increased cavitation, whereas high concentrations of P decreased cavitation as measured by both hydraulic flow apparatus and dye perfusion techniques. For one test, cavitation was 48% at high N and low P, but only 28% at high N and high P. We consider that N fertilization may make poplars more susceptible to cavitation on dry sites, but P fertilization may reduce this effect.

Journal Article↗

Genotypic variation in drought tolerance of poplar in relation to abscisic acid.

We investigated effects of water stress and external abscisic acid (ABA) supply on shoot growth, stomatal conductance and water status in 1-year-old cuttings of a drought-sensitive poplar genotype Populus x euramericana cv. I-214 (Italica) and a drought-tolerant genotype P. 'popularis 35-44' (popularis). Populus popularis was more productive and maintained higher leaf water potentials throughout the drought treatment than cv. Italica. Supply of ABA to the xylem sap caused a greater decline in growth and more leaf abscission in shoots of cv. Italica than in shoots of P. popularis. Immediately after initiation of the drought treatment in P. popularis, the ABA concentration ([ABA]) of the xylem increased rapidly and stomatal conductance declined; however, stomatal conductance had returned to control values by the third day of the drought treatment, coincident with a gradual decline in xylem [ABA]. In contrast, xylem [ABA] of cv. Italica initially increased more slowly than that of P. popularis in response to the drought treatment, but the increase continued for 3 days at which time a tenfold increase in xylem [ABA] was observed that was followed by abscission of more than 40% of the leaves. We conclude that sensitivity of poplar roots to variation in soil water content varies by clone and that a rapid short-term accumulation of ABA in shoots in response to water stress may contribute to drought tolerance.

Journal Article↗

Ozone-induced changes in biosynthesis of Rubisco and associated compensation to stress in foliage of hybrid poplar.

Experiments were conducted during the growing seasons of 1993-1995 to determine whether exposure to ozone (O(3)) affected the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in hybrid poplar, Populus maximowizii A. Henry x trichocarpa Torr. & A. Gray, Clone 245. As the canopy aged, the concentration of Rubisco decreased at a more rapid rate in lower leaves of hyrid poplar ramets subjected to chronic O(3) exposure in open-top chambers than in comparable foliage of plants grown in charcoal-filtered air. There was no difference in rate of synthesis of Rubisco between treatments, suggesting that loss of this protein in O(3)-treated leaves was caused by an accelerated rate of proteolysis. In foliage higher in the canopy, both concentration and rate of synthesis of Rubisco were stimulated by O(3) for a brief period when the leaves were young. Quantification of mRNA for the small (rbcS) and large (rbcL) transcripts of Rubisco did not reveal changes that were likely to reflect altered synthesis of Rubisco as a prime response to O(3). Analyses of Rubisco concentration and rate of Rubisco synthesis in foliage connected by vascular traces within the canopy indicated that loss of Rubisco in older leaves was associated with an increase in this protein in younger leaves higher in the canopy. These data support the notion that accelerated senescence may provide some compensatory benefit to the plant. In 1995, the rate of synthesis of Rubisco was almost always higher in O(3)-treated foliage than in nontreated foliage, even when the concentration of Rubisco was adversely affected by the O(3) treatment. Because accelerated foliar abscission in response to O(3) was minimal in 1995 compared to other years, we speculate that, when abscission is delayed, Rubisco synthesis and concentration become uncoupled.

Journal Article↗

Transpiration and water relations of poplar trees growing close to the water table.

Sap flow was measured on five branches of two poplar (Populus trichocarpa Torr. & A. Gray x P. tacamahaca L.) trees from June to September 1994 in the south of England with stem-surface, heat balance gauges, and was scaled up to estimate transpiration from single trees on the basis of leaf area. On six days, stomatal conductance and plant water potential were measured simultaneously with a porometer and pressure chamber, respectively. The effects of solar radiation (S), vapor pressure deficit (D) and stomatal conductance on transpiration were evaluated. Sap flow per unit leaf area (F(a)) was closely related to the time course of demand attributable to S and D throughout the season, and only slightly affected by the water content of the top 120 cm of soil. Although F(a) increased linearly at low values of D, it showed a plateau with increases in D above 1.2 kPa. The canopy coupling coefficient (1 - Omega) ranged from 0.48 to 0.78 with a mean of 0.65 +/- 0.01, indicating that transpiration was controlled more by stomatal conductance than by incident radiation. The seasonal pattern of tree water loss followed potential evaporation with a peak in late June or early July. On bright days, daily transpiration over the projected crown area was 3.6 mm early in the season, 3.8 mm in mid-season, and 2.7 mm late in the season. The water balance of the system indicated that poplar trees took 15-60% of water transpired from groundwater, with the proportion increasing as the soil in the unsaturated zone dried out. Access to the water table resulted in high predawn water potentials throughout the season. Estimated hydraulic resistance to water flow in the soil-tree system was in the range of 1.5 to 1.93 x 10(6) MPa s m(-3).

Journal Article↗

Ecotypic and genetic variation in poplar bark storage protein gene expression and accumulation.

Bark storage proteins (BSP) store nitrogen (N) translocated from senescing leaves in autumn, and supply reduced N for spring growth. Expression of bsp and BSP accumulation are associated with short day photoperiod. To determine if photoperiod-associated bsp expression varies among poplars native to different latitudes, Populus deltoides Bartr. clones originating from six latitudes were grown under natural conditions at a common location. Relative amounts of BSP mRNA in these clones were measured at 2-week intervals from August 7 to October 16. The date of maximum BSP mRNA accumulation was correlated with latitude of origin, and maximum accumulation of BSP mRNA occurred earlier in clones native to northern latitudes than in clones native to southern latitudes. This pattern of variation is consistent with photoperiodic responses of plants native to temperate climates. Genotypic variations in BSP accumulation, bark protein concentration and bark N concentration were compared among clones of six hybrid poplar (Populus trichocarpa Torr. and Gray x P. deltoides) full-sib families (three F(2) families, two F(1) families and one BC(1) family) after 6 weeks in a short day photoperiod and at midwinter. Significant differences in BSP accumulation occurred among clones within four of the six full-sib families after 6 weeks in a short day photoperiod and also at midwinter for outdoor-grown plants. Bark protein and bark N concentrations also varied significantly among clones within certain families. In general, the greatest variation was found in F(2) and BC(1) families. Within several families, relative BSP amounts were positively correlated with bark protein concentration and total bark N concentration. These results indicate a role of photoperiod in regulating bsp expression and demonstrate a genetic component underlying seasonal BSP accumulation. The results could have significance in selecting for clones with improved N storage capacity and N-use efficiency.

Gene Expression Regulation, Plant↗

Partitioning of nitrate assimilation among leaves, stems and roots of poplar.

Plants differ in tissue localization of nitrate reduction and assimilation. Some species reduce nitrate primarily in the leaves, whereas other species localize nitrate reduction and assimilation in the roots. We determined how nitrate assimilation is partitioned among leaves, stems and roots of poplar (Populus tremula L. x P. alba L.) by comparing tissue differences in in vivo nitrate reductase activity (NRA), nitrate reductase abundance and tissue nitrate concentration. Compared with stems or roots, NRA was greater in leaves, and the highest leaf NRA was found in young leaves. Leaf and root NRA increased with increasing nitrate supply, whereas stem NRA remained constant. Leaf NRA was at least 10-fold greater than root NRA at all external nitrate concentrations. Nitrate reductase abundance increased in all tissues with increasing nitrate availability, and nitrate reductase abundance was at least 10-fold greater in leaves than in stems or roots at all nitrate availabilities. Tissue nitrate concentration increased with increasing nitrate supply and was greater in roots than in stems and leaves. Photoperiod influenced NRA, with leaf NRA declining in nitrate-fertilized plants with short daily photoperiods (8-h). We conclude that different tissues of poplar vary in nitrate assimilation with little nitrate assimilation occurring in roots and the most nitrate assimilation taking place in leaves.

Nitrate Reductase↗

Carbohydrates in individual poplar fine roots: effects of root age and defoliation.

Late-summer starch accumulation in fine roots of poplars (Populus x canadensis Moench.) defoliated by gypsy moth (Lymantria dispar L.) lagged behind that in fine roots of undefoliated trees. If starch concentration declines with age, defoliation-induced changes in root system age structure could be partly responsible for this difference. To test this hypothesis, we measured fine-root starch and soluble sugar concentrations in roots of known age from trees in defoliated and undefoliated plots. There was a significant interaction between the effects of defoliation and root type (white, brown, or dead) on fine root soluble sugar concentration because of the high concentration of soluble sugars in white roots from trees in undefoliated plots. Both root starch and soluble sugar concentrations were variable among individuals of each age class. The population frequency distributions for starch and soluble sugar concentrations were both right-skewed, and fit by exponential functions. These data are most consistent with direct defoliation effects on a labile and dynamic pool of carbohydrates in poplar fine roots, rather than indirect defoliation effects on root system age structure.

Carbohydrates↗

A method for describing the canopy architecture of coppice poplar with allometric relationships.

A multi-scale biometric methodology for describing the architecture of fast-growing short-rotation woody crops is used to describe 2-year-old poplar clones during the second rotation. To allow for expressions of genetic variability observed within this species (i.e., growth potential, leaf morphology, coppice and canopy structure), the method has been applied to two clones: Ghoy (Gho) (Populus deltoides Bartr. ex Marsh. x Populus nigra L.) and Trichobel (Tri) (Populus trichocarpa Torr. & A. Gray x Populus trichocarpa). The method operates at the stool level and describes the plant as a collection of components (shoots and branches) described as a collection of metameric elements, themselves defined as a collection of elementary units (internode, petiole, leaf blade). Branching and connection between the plant units (i.e., plant topology) and their spatial location, orientation, size and shape (i.e., plant geometry) describe the plant architecture. The methodology has been used to describe the plant architecture of 15 selected stools per clone over a 5-month period. On individual stools, shoots have been selected from three classes (small, medium and large) spanning the diameter distribution range. Using a multi-scale approach, empirical allometric relationships were used to parameterize elementary units of the plant, topological relationships and geometry (e.g., distribution of shoot diameters on stool, shoot attributes from shoot diameter). The empirical functions form the basis of the 3-D Coppice Poplar Canopy Architecture model (3-D CPCA), which recreates the architecture and canopy structure of fast-growing coppice crops at the plot scale. Model outputs are assessed through visual and quantitative comparisons between actual photographs of the coppice canopy and simulated images. Overall, results indicate a good predictive ability of the 3-D CPCA model.

Imaging, Three-Dimensional↗

Petiole length and biomass investment in support modify light interception efficiency in dense poplar plantations.

Leaf architecture, stand leaf area index and canopy light interception were studied in 13 poplar clones growing in a second rotation of a coppice plantation, to determine the role of leaf architectural attributes on canopy light-harvesting efficiency and to assess biomass investment in leaf support tissue. Stand leaf area index (L) varied from 2.89 to 6.99, but L was only weakly associated with canopy transmittance (TC). The weak relationship between TC and L was a result of a higher degree of foliage aggregation at larger values of L, leading to lower light-interception efficiency in stands with greater total leaf area. We observed a strong increase in leaf aggregation and a decrease in light-harvesting efficiency with decreasing mean leaf petiole length (PL) but not with leaf size, possibly because, in cordate or deltoid poplar leaves, most of the leaf area is located close to the petiole attachment to the lamina. Although PL was the key leaf characteristic of light-harvesting efficiency, clones with longer petioles had larger biomass investments in petioles, and there was a negative relationship between PL and L, demonstrating that enhanced light harvesting may lead to an overall decline in photosynthesizing leaf surface. Upper-canopy leaves were generally larger and had greater dry mass (MA) and nitrogen per unit area (NA) than lower-canopy leaves. Canopy plasticity in MA and NA was higher in clones with higher foliar biomass investment in midrib, and lower in clones with relatively longer petioles. These relationships suggest that there is a trade-off between photosynthetic plasticity and biomass investment in support, and also that high light-harvesting efficiency may be associated with lower photosynthetic plasticity. Our results demonstrate important clonal differences in leaf aggregation that are linked to leaf structure and biomass allocation patterns within the leaf.

Biomass↗

Effects of two iron sources on iron and cadmium allocation in poplar (Populus alba) plants exposed to cadmium.

Effects of 10 microM cadmium (supplied as Cd nitrate) on the utilization and allocation of iron (Fe) were investigated in poplar (Populus alba L.) plants grown in nutrient solution with Fe(III)-EDTA or Fe(III)-citrate as the Fe source. The effects of Cd were also compared with those of Fe deprivation. The accumulation of Fe in roots was 10-fold higher in plants grown with Fe-citrate than with Fe-EDTA. Cadmium decreased leaf chlorophyll concentrations and photosynthetic rates, and these decreases were more marked in plants grown with Fe-citrate than with Fe-EDTA. In both Fe treatments, addition of Cd caused large increases in root and shoot apoplasmic and non-apoplasmic Cd contents and increases in root Fe content; however, Cd decreased shoot Fe content, especially in plants grown with Fe-citrate. New leaves of plants grown with Fe-citrate had small cellular (non-apoplasmic) Fe pools, whereas these pools were large in new leaves of plants grown with Fe-EDTA. Non-apoplasmic Cd pools in new leaves were smaller in plants grown with Fe-citrate than with Fe-EDTA, indicating that inactivation of non-apoplasmic Cd pools is facilitated more by Fe-EDTA than by Fe-citrate. In the presence of Cd, Fe-EDTA was also superior to Fe-citrate in maintaining an adequate Fe supply to poplar shoots. Differences in plant responses to Fe-EDTA and Fe-citrate may reflect differences in long-distance transport of Fe rather than in acquisition of Fe by roots.

Cadmium↗

Transgenic sterility in Populus: expression properties of the poplar PTLF, Agrobacterium NOS and two minimal 35S promoters in vegetative tissues.

Transgenic sterility is a desirable trait for containment of many kinds of transgenes and exotic species. Genetically engineered floral sterility can be imparted by expression of a cytotoxin under the control of a predominantly floral-tissue-specific promoter. However, many otherwise desirable floral promoters impart substantial non-floral expression, which can impair plant health or make it impossible to regenerate transgenic plants. We are therefore developing a floral sterility system that is capable of attenuating undesired background vegetative expression. As a first step towards this goal, we compared the vegetative expression properties of the promoter of the poplar (Populus trichocarpa Torr. & Gray) homolog of the floral homeotic gene LEAFY (PTLF), which could be used to impart male and female flower sterility, to that of three candidate attenuator-gene promoters: the cauliflower mosaic virus (CaMV) 35S basal promoter, the CaMV 35S basal promoter fused to the TMV omega element and the nopaline synthase (NOS) promoter. The promoters were evaluated via promoter::GUS gene fusions in a transgenic poplar hybrid (Populus tremula L. x P. alba L.) by both histochemical and fluorometric GUS assays. In leaves, the NOS promoter conveyed the highest activity and had a mean expression level 5-fold higher than PTLF, whereas the CaMV 35S basal promoter fused to the omega element and the CaMV 35S basal promoter alone directed mean expression levels that were 0.5x and 0.35x that of PTLF, respectively. Differential expression in shoots, leaves, stems and roots was observed only for the NOS and PTLF promoters. Strongest expression was observed in roots for the NOS promoter, whereas the PTLF promoter directed highest expression in shoots. The NOS promoter appears best suited to counteract vegetative expression of a cytotoxin driven by the PTLF promoter where 1:1 toxin:attenuator expression is required.

Amino Acid Oxidoreductases↗