Sustainable bioenergy for climate mitigation: developing drought-tolerant trees and grasses.

BACKGROUND AND AIMS: Bioenergy crops are central to climate mitigation strategies that utilize biogenic carbon, such as BECCS (bioenergy with carbon capture and storage), alongside the use of biomass for heat, power, liquid fuels and, in the future, biorefining to chemicals. Several promising lignocellulosic crops are emerging that have no food role - fast-growing trees and grasses - but are well suited as bioenergy feedstocks, including Populus, Salix, Arundo, Miscanthus, Panicum and Sorghum. SCOPE: These promising crops remain largely undomesticated and, until recently, have had limited germplasm resources. In order to avoid competition with food crops for land and nature conservation, it is likely that future bioenergy crops will be grown on marginal land that is not needed for food production and is of poor quality and subject to drought stress. Thus, here we define an ideotype for drought tolerance that will enable biomass production to be maintained in the face of moderate drought stress. This includes traits that can readily be measured in wide populations of several hundred unique genotypes for genome-wide association studies, alongside traits that are informative but can only easily be assessed in limited numbers or training populations that may be more suitable for genomic selection. Phenotyping, not genotyping, is now the major bottleneck for progress, since in all lignocellulosic crops studied extensive use has been made of next-generation sequencing such that several thousand markers are now available and populations are emerging that will enable rapid progress for drought-tolerance breeding. The emergence of novel technologies for targeted genotyping by sequencing are particularly welcome. Genome editing has already been demonstrated for Populus and offers significant potential for rapid deployment of drought-tolerant crops through manipulation of ABA receptors, as demonstrated in Arabidopsis, with other gene targets yet to be tested. CONCLUSIONS: Bioenergy is predicted to be the fastest-developing renewable energy over the coming decade and significant investment over the past decade has been made in developing genomic resources and in collecting wild germplasm from within the natural ranges of several tree and grass crops. Harnessing these resources for climate-resilient crops for the future remains a challenge but one that is likely to be successful.

Poplar genetic engineering: promoting desirable wood characteristics and pest resistance.

Worldwide biomass demand for industrial applications, especially for production of biofuels, is increasing. Extended cultivation of fast growing trees such as poplars may contribute to satisfy the need for renewable resources. However, lignin, which constitutes about 20-30% of woody biomass, renders poplar wood recalcitrant to saccharification. Genetic engineering of the enzymes of the lignification pathway has resulted in drastic decreases in lignin and greatly improved the carbohydrate yield for ethanol fermentation. While uncovering key enzymes for lignification facilitated rapid biotechnological progress, knowledge on field performance of low-lignin poplars is still lagging behind. The major biotic damage is caused by poplar rust fungi (Melampsora larici-populina), whose defense responses involve lignification and production of phenolic compounds. Therefore, manipulation of the phenylpropanoid pathway may be critical and should be tightly linked with new strategies for improved poplar rust tolerance. Emerging novel concepts for wood improvement are discussed.

Isoprene function in two contrasting poplars under salt and sunflecks.

In the present study, biogenic volatile organic compound (BVOC) emissions and photosynthetic gas exchange of salt-sensitive (Populus x canescens (Aiton) Sm.) and salt-tolerant (Populus euphratica Oliv.) isoprene-emitting and non-isoprene-emitting poplars were examined under controlled high-salinity and high-temperature and -light episode ('sunfleck') treatments. Combined treatment with salt and sunflecks led to an increased isoprene emission capacity in both poplar species, although the photosynthetic performance of P. × canescens was reduced. Indeed, different allocations of isoprene precursors between the cytosol and the chloroplast in the two species were uncovered by means of (13)CO2 labeling. Populus × canescens leaves, moreover, increased their use of 'alternative' carbon (C) sources in comparison with recently fixed C for isoprene biosynthesis under salinity. Our studies show, however, that isoprene itself does not have a function in poplar survival under salt stress: the non-isoprene-emitting leaves showed only a slightly decreased photosynthetic performance compared with wild type under salt treatment. Lipid composition analysis revealed differences in the double bond index between the isoprene-emitting and non-isoprene-emitting poplars. Four clear metabolomics patterns were recognized, reflecting systemic changes in flavonoids, sterols and C fixation metabolites due to the lack/presence of isoprene and the absence/presence of salt stress. The studies were complemented by long-term temperature stress experiments, which revealed the thermotolerance role of isoprene as the non-isoprene-emitting leaves collapsed under high temperature, releasing a burst of BVOCs. Engineered plants with a low isoprene emission potential might therefore not be capable of resisting high-temperature episodes.

Fungal soil communities in a young transgenic poplar plantation form a rich reservoir for fungal root communities.

Fungal communities play a key role in ecosystem functioning. However, only little is known about their composition in plant roots and the soil of biomass plantations. The goal of this study was to analyze fungal biodiversity in their belowground habitats and to gain information on the strategies by which ectomycorrhizal (ECM) fungi form colonies. In a 2-year-old plantation, fungal communities in the soil and roots of three different poplar genotypes (Populus × canescens, wildtype and two transgenic lines with suppressed cinnamyl alcohol dehydrogenase activity) were analyzed by 454 pyrosequencing targeting the rDNA internal transcribed spacer 1 (ITS) region. The results were compared with the dynamics of the root-associated ECM community studied by morphotyping/Sanger sequencing in two subsequent years. Fungal species and family richness in the soil were surprisingly high in this simple plantation ecosystem, with 5944 operational taxonomic units (OTUs) and 186 described fungal families. These findings indicate the importance that fungal species are already available for colonization of plant roots (2399 OTUs and 115 families). The transgenic modification of poplar plants had no influence on fungal root or soil communities. Fungal families and OTUs were more evenly distributed in the soil than in roots, probably as a result of soil plowing before the establishment of the plantation. Saprophytic, pathogenic, and endophytic fungi were the dominating groups in soil, whereas ECMs were dominant in roots (87%). Arbuscular mycorrhizal diversity was higher in soil than in roots. Species richness of the root-associated ECM community, which was low compared with ECM fungi detected by 454 analyses, increased after 1 year. This increase was mainly caused by ECM fungal species already traced in the preceding year in roots. This result supports the priority concept that ECMs present on roots have a competitive advantage over soil-localized ECM fungi.

The role of abscisic acid and auxin in the response of poplar to abiotic stress.

The plant hormones auxin and abscisic acid may at first sight appear to be a conflicting pair of plant regulators. Abscisic acid content increases during stress and protects plant water status. The content of free auxin in the developing xylem of poplar declines during stress, while auxin conjugates increase. This indicates that specific down-regulation of a signal transduction chain is important in plant adaptation to stress. Diminished auxin content may be a factor that adapts growth and wood development of poplar during adverse environmental conditions. To allow integration of environmental signals, abscisic acid and auxin must interact. Data are accumulating that abscisic acid-auxin cross-talk exists in plants. However, knowledge of the role of plant hormones in the response of trees to stress is scarce. Our data show that differences in the localisation of ABA synthesis exist between the annual, herbaceous plant Arabidopsis and the perennial woody species, poplar.

Salinity tolerance of Populus.

The genus Populus has a wide distribution in different climatic zones. Besides its economic and ecological relevance, Populus also serves as a model for elucidating physiological and molecular mechanisms of stress tolerance in tree species. In this review, adaptation strategies of poplars to excess soil salinity are addressed at different scales, from the cellular to the whole-plant level. Striking differences in salt tolerance exist among different poplar species and ecotypes, with Populus euphratica being outstanding in this respect. Key mechanisms identified in this species to mediate salt tolerance are compartmentalisation of Cl(-) in the vacuoles of the root cortex cells, diminished xylem loading of NaCl, activation of Na(+) extrusion into the soil solution under stress, together with simultaneously avoiding excessive K(+) loss by regulation of depolarisation-activated cation channels. This leads to improved maintenance of the K(+)/Na(+) balance, a crucial precondition for survival under salt stress. Leaf cells of this species are able to compartmentalise Na(+) preferentially in the apoplast, whereas in susceptible poplar species, as well as in crop plants, vacuolar Na(+) deposition precedes apoplastic transport. ABA, Ca(2+)and ROS are involved in stress sensing, with higher or faster activation of defences in tolerant than in susceptible poplar species. P. euphratica develops leaf succulence after prolonged salt exposure as a plastic morphological adaptation that leads to salt dilution. Transgenic approaches to improve salt tolerance by transformation of candidate genes have had limited success, since salt tolerance is a multigenic trait. In future attempts towards increased salt resistance, barriers between different poplar sections must be overcome and application of novel biotechnological tools, such as gene stacking, are recommended.

VOC emissions of Grey poplar leaves as affected by salt stress and different N sources.

Nitrogen nutrition and salt stress experiments were performed in a greenhouse with hydroponic-cultured, salt-sensitive Grey poplar (Populus x canescens) plants to study the combined influence of different N sources (either 1 mm NO(3) (-) or NH(4)(+)) and salt (up to 75 mm NaCl) on leaf gas exchange, isoprene biosynthesis and VOC emissions. Net assimilation and transpiration proved to be highly sensitive to salt stress and were reduced by approximately 90% at leaf sodium concentrations higher than 1,800 microg Na g dry weight (dw)(-1). In contrast, emissions of isoprene and oxygenated VOC (i.e. acetaldehyde, formaldehyde and acetone) were unaffected. There was no significant effect of combinations of salt stress and N source, and neither NO(3)(-) or NH(4)(+) influenced the salt stress response in the Grey poplar leaves. Also, transcript levels of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (PcDXR) and isoprene synthase (PcISPS) did not respond to the different N sources and only responded slightly to salt application, although isoprene synthase (PcISPS) activity was negatively affected at least in one of two experiments, despite high isoprene emission rates. A significant salt effect was the strong reduction of leaf dimethylallyl diphosphate (DMADP) content, probably due to restricted availability of photosynthates for DMADP biosynthesis. Further consequences of reduced photosynthetic gas exchange and maintaining VOC emissions are a very high C loss, up to 50%, from VOC emissions related to net CO(2) uptake and a strong increase in leaf internal isoprene concentrations, with maximum mean values up to 6.6 microl x l(-1). Why poplar leaves maintain VOC biosynthesis and emission under salt stress conditions, despite impaired photosynthetic CO(2) fixation, is discussed.

Interaction of nitrogen nutrition and salinity in Grey poplar (Populus tremula x alba).

Salinity represents an increasing environmental problem in managed ecosystems. Populus spp. is widely used for wood production by short-rotation forestry in fertilized plantations and can be grown on saline soil. Because N fertilization plays an important role in salt tolerance, we analysed Grey poplar (Populus tremula x alba, syn. Populus canescens) grown with either 1 mM nitrate or ammonium subjected to moderate 75 mM NaCl. The impact of N nutrition on amelioration of salt tolerance was analysed on different levels of N metabolism such as N uptake, assimilation and N (total N, proteins and amino compounds) accumulation. Na concentration increased in all tissues over time of salt exposure. The N nutrition-dependent effects of salt exposure were more intensive in roots than in leaves. Application of salt reduced root increment as well as stem height increase and, at the same time, increased the concentration of total amino compounds more intensively in roots of ammonium-fed plants. In leaves, salt treatment increased concentrations of total N more intensively in nitrate-fed plants and concentrations of amino compounds independently of N nutrition. The major changes in N metabolism of Grey poplar exposed to moderate salt concentrations were detected in the significant increase of amino acid concentrations. The present results indicate that N metabolism of Grey poplar exposed to salt performed better when the plants were fed with nitrate instead of ammonium as sole N source. Therefore, nitrate fertilization of poplar plantations grown on saline soil should be preferred.

Warm and cold parental reproductive environments affect seed properties, fitness, and cold responsiveness in Arabidopsis thaliana progenies.

Conditions in the parental environment during reproduction can affect the performance of the progenies. The goals of this study were to investigate whether warm or cold temperatures in the parental environment during flowering and seed development affect Arabidopsis thaliana seed properties, growth performance, reproduction and stress tolerance of the progenies, and to find candidate genes for progeny-related differences in stress responsiveness. Parental plants were raised at 20 degrees C and maintained from bolting to seed maturity at warm (25 degrees C) or cold (15 degrees C) temperatures. Analysis of seed properties revealed significant increases in nitrogen in seeds from warm temperature and significant increases in lipids and in the ratio of alpha-linolenic to oleic acid in seeds from the cold parental environment. Progenies of the warm parental environment showed faster germination rates, faster root elongation growth, higher leaf biomass and increased seed production at various temperatures compared with those from the cold parental environment. This indicates that under stable environmental conditions, progenies from warm parental environments had a clear adaptive advantage over those from cold parental environments. This parental effect was presumably transmitted by the higher nitrogen content of the seeds developed in warm conditions. When offspring from parents grown at different temperatures were exposed to chilling or freezing stress, photosynthetic yield recovered faster in progenies originating from cold parental environments. Cold acclimation involved up-regulation of transcripts of flavanone 3-hydroxylase (F3H) and pseudo response regulator 9 (PRR9) and down-regulation of growth-associated transcription factors (TFs) NAP and AP2domain containing RAP2.3. NAP, a regulator of senescence, and PRR9, a temperature-sensitive modulator of the circadian clock, were probably involved in mediating parent-of-origin effects, because they showed progeny-related expression differences under chilling. Because low temperatures also delay senescence, cold responsiveness of NAP suggests that this factor is linked with the regulatory network that is important for environmental acclimation of plants.

Paxillus involutus mycorrhiza attenuate NaCl-stress responses in the salt-sensitive hybrid poplar Populusxcanescens.

In order to characterise the effect of ectomycorrhiza on Na+-responses of the salt-sensitive poplar hybrid Populus x canescens, growth and stress responses of Paxillus involutus (strain MAJ) were tested in liquid cultures in the presence of 20 to 500 mM NaCl, and the effects of mycorrhization on mineral nutrient accumulation and oxidative stress were characterised in mycorrhizal and non-mycorrhizal poplar seedlings exposed to 150 mM NaCl. Paxillus involutus was salt tolerant, showing biomass increases in media containing up to 500 mM NaCl after 4 weeks growth. Mycorrhizal mantle formation on poplar roots was not affected by 150 mM NaCl. Whole plant performance was positively affected by the fungus because total biomass was greater and leaves accumulated less Na+ than non-mycorrhizal plants. Energy dispersive X-ray microanalysis using transmission electron microscopy analysis of the influence of mycorrhization on the subcellular localisation of Na+ and Cl- in roots showed that the hyphal mantle did not diminish salt accumulation in root cell walls, indicating that mycorrhization did not provide a physical barrier against excess salinity. In the absence of salt stress, mycorrhizal poplar roots contained higher Na+ and Cl- concentrations than non-mycorrhizal poplar roots. Paxillus involutus hyphae produced H2O2 in the mantle but not in the Hartig net or in pure culture. Salt exposure resulted in H2O2 formation in cortical cells of both non-mycorrhizal and mycorrhizal poplar and stimulated peroxidase but not superoxide dismutase activities. This shows that mature ectomycorrhiza was unable to suppress salt-induced oxidative stress. Element analyses suggest that improved performance of mycorrhizal poplar under salt stress may result from diminished xylem loading of Na+ and increased supply with K+.

Adaptation to high salinity in poplar involves changes in xylem anatomy and auxin physiology.

To investigate the physiological basis of salt adaptation in poplar, we compared the effect of salt stress on wood anatomy and auxin physiology of the salt-resistant Populus euphratica and salt-sensitive Populus x canescens. Both poplar species showed decreases in vessel lumina associated with increases in wall strength in response to salt, however, in P. euphratica at three-fold higher salt concentrations than in P. x canescens. The predicted hydraulic conductivity of the wood formed under salt stress decreased in P. x canescens, while in P. euphratica, no significant effects of salt on conductivity and transpiration were observed. The concentration of free indole-3-acetic acid (IAA) decreased under salt stress in the xylem of both poplar species, but to a larger extent in P. x canescens than in P. euphratica. Only salt-treated P. euphratica exhibited an increase in IAA-conjugates in the xylem. Genes homologous to the auxin-amidohydrolase ILL3 were isolated from the xylems of P. euphratica and P. x canescens. For functional analysis, the auxin-amidohydrolase from P. x canescens was overexpressed in Arabidopsis. Transgenic Arabidopsis plants were more resistant to salt stress than the wild-type plants. Increased sensitivity of the transgenic Arabidopsis to IAA-Leu showed that the encoded hydrolase used IAA-Leu as a substrate. These results suggest that poplar can use IAA-amidoconjugates in the stem as a source of auxin to balance the effects of salt stress on auxin physiology.

Physiological responses of forest trees to heat and drought.

The heat wave of summer 2003 was the largest and the most persistent ever experienced in Central Europe and has fuelled concern about the effects of climate change on European ecosystems. Since forests constitute the most important European ecosystems, in this review article we assess current knowledge on the effects of heat and drought on key metabolic processes for growth and productivity of forest trees. In particular, the general consequences of heat and drought on (1) photosynthesis and respiration at the cellular and community level, and (2) on nutrient uptake, partitioning and competition for nutrients are summarized. The latter are a major sink for photosynthetic energy and, therefore, are indirectly but strongly connected to the performance of photosynthesis. In addition, the interaction of heat and drought with stress compensation mechanisms and emission of biogenic volatile organic compounds (BVOC) are discussed, since these processes are directly connected to carbon metabolism. Effects on the emission of BVOC are also included because they constitute an important feedback mechanism on ozone formation and, thus, on atmospheric pollution. As far as available, data collected during the 2003 heat wave are included and discussed.

Net carbon storage in a poplar plantation (POPFACE) after three years of free-air CO2 enrichment.

A high-density plantation of three genotypes of Populus was exposed to an elevated concentration of carbon dioxide ([CO(2)]; 550 micromol mol(-1)) from planting through canopy closure using a free-air CO(2) enrichment (FACE) technique. The FACE treatment stimulated gross primary productivity by 22 and 11% in the second and third years, respectively. Partitioning of extra carbon (C) among C pools of different turnover rates is of critical interest; thus, we calculated net ecosystem productivity (NEP) to determine whether elevated atmospheric [CO(2)] will enhance net plantation C storage capacity. Free-air CO(2) enrichment increased net primary productivity (NPP) of all genotypes by 21% in the second year and by 26% in the third year, mainly because of an increase in the size of C pools with relatively slow turnover rates (i.e., wood). In all genotypes in the FACE treatment, more new soil C was added to the total soil C pool compared with the control treatment. However, more old soil C loss was observed in the FACE treatment compared with the control treatment, possibly due to a priming effect from newly incorporated root litter. FACE did not significantly increase NEP, probably as a result of this priming effect.

Effect of auxin transport inhibitors and ethylene on the wood anatomy of poplar.

The influence of the auxin transport inhibitors naphthylphthalamic acid (NPA) and methyl-2-chloro-9-hydroxyflurene-9-carboxylate (CF), as well as the gaseous hormone ethylene on cambial differentiation of poplar was determined. NPA treatment induced clustering of vessels and increased vessel length. CF caused a synchronized differentiation of cambial cells into either vessel elements or fibres. The vessels in CF-treated wood were significantly smaller and fibre area was increased compared with controls. Under the influence of ethylene, the cambium produced more parenchyma, shorter fibres and shorter vessels than in controls. Since poplar is the model tree for molecular biology of wood formation, the modulation of the cambial differentiation of poplar towards specific cell types opens an avenue to study genes important for the development of vessels or fibres.

Compatible and incompetent Paxillus involutus isolates for ectomycorrhiza formation in vitro with poplar (Populus x canescens) differ in H2O2 production.

Isolates of Paxillus involutus (Batsch) Fr. collected from different hosts and environmental conditions were screened for their ability to form ectomycorrhizal symbiosis with hybrid poplar P. x canescens (= Populus tremula L. x P. alba) in vitro. The ability to form ectomycorrhiza varied between the fungal isolates and was not correlated with the growth rate of the fungi on agar-based medium. The isolate MAJ, which was capable of mycorrhiza synthesis under axenic conditions, and the incompetent isolate NAU were characterized morphologically and anatomically. MAJ formed a typical hyphal mantle and a Hartig net, whereas NAU was not able to penetrate the host cell walls and caused thickenings of the outer cell walls of the host. MAJ, but not NAU, displayed strong H2O2 accumulation in the outer hyphal mantle. Increases in H2O2 in the outer epidermal walls and adjacent hyphae of the incompetent isolate were moderate. No increases of H2O2 in response to the mycobionts were found inside roots. Suggested functions of H2O2 production in the outer hyphal mantle of the compatible interaction are: growth regulation of the host's roots, defence against other invading microbes, or increasing plant-innate immunity. The system established here for P. x canescens compatible and incompetent fungal associations will be useful to take advantage of genomic information now available for poplar to study tree-fungal interactions at the molecular and physiological level.

Activating effect of Tanacetum vulgare L. pectin polysaccharide on ionic channels of neuronal membrane.

The membranotropic effects of TVF tanacetan pectin polysaccharide derived from Tanacetum vulgare L. was studied by the voltage-clamp method on isolated neurons of Lymnaea stagnalis mollusk. TVF in concentrations of 0.1-10.0 microg/ml nonselectively activated the outward potassium and total inward (sodium and calcium) ionic currents (slightly dose-dependently and reversibly increased their amplitude by 5-10%) and decreased nonspecific leakage current.

Lignification in beech (Fagus sylvatica) grown at elevated CO2 concentrations: interaction with nutrient availability and leaf maturation.

Beech (Fagus sylvatica L.) seedlings were grown in an ambient or elevated CO2 concentration ([CO2]) either in small stands in microcosms for three to four seasons or individually in pots fertilized at different nutrient supply rates. Leaves at different stages of development, as well as stems and roots at the end of the growing season, were used for analysis of structural biomass and lignin. In elevated [CO2], lignification of leaves was slightly retarded compared with structural biomass production and showed a strong correlation with the activities of ionically, cell-wall-bound peroxidases but not with total soluble peroxidases or covalently wall-bound peroxidases. The effect of elevated [CO2] on lignin concentration of mature tissues was dependent on nutrient supply rate. In leaves and roots, elevated [CO2] increased the lignin concentration in dry mass in N-limited plants. In seedlings grown with high nutrient supply, the lignin concentration in dry mass was unaffected or diminished by elevated [CO2]. Because elevated [CO2] enhanced seedling growth in the high nutrient supply treatments, the total amount of lignin produced per seedling was higher in these treatments. We predict that long-term sequestration of carbon will increase as long as biomass production is stimulated by elevated [CO2] and that tissue quality will change depending on developmental stage and nutrient availability.

Structure of tanacetan, a pectic polysaccharide from tansy Tanacetum vulgare L.

Tanacetan TVF was found to have a branched structure with a backbone of linear alpha-1,4-D-galacturonan. The ramified regions consist of linear alpha-1,2-L-rhamno-alpha-1,4-D-galacturonan as the core. The side chains appear to attach to the 4-position of the L-rhamnopyranose residues. They are present as single beta-galactopyranose residues or a branching beta-1,4-galactopyranan bearing 4,6-substituted beta-D-galactopyranose residues as branched points. In addition, the ramified regions contain side chains of a branched alpha-1,5-arabinofuranan possessing 2,5- and 3,5-substituted alpha-L-arabinofuranose residues as branching points. Some side chains of rhamnogalacturonan appear to be arabinogalactan which contains branched sugar chains of alpha-1,5-arabinofuranan attached to the linear chains of beta-1,4-galactopyranan by 1,3- and 1,6-linkages. The residues of alpha-L-arabinofuranose seem to occupy the terminal positions of the arabinogalactan side chains.

Cadmium-induced changes in antioxidative systems, hydrogen peroxide content, and differentiation in Scots pine roots.

To investigate whether Cd induces common plant defense pathways or unspecific necrosis, the temporal sequence of physiological reactions, including hydrogen peroxide (H(2)O(2)) production, changes in ascorbate-glutathione-related antioxidant systems, secondary metabolism (peroxidases, phenolics, and lignification), and developmental changes, was characterized in roots of hydroponically grown Scots pine (Pinus sylvestris) seedlings. Cd (50 microM, 6 h) initially increased superoxide dismutase, inhibited the systems involved in H(2)O(2) removal (glutathione/glutathione reductase, catalase [CAT], and ascorbate peroxidase [APX]), and caused H(2)O(2) accumulation. Elongation of the roots was completely inhibited within 12 h. After 24 h, glutathione reductase activities recovered to control levels; APX and CAT were stimulated by factors of 5.5 and 1.5. Cell death was increased. After 48 h, nonspecific peroxidases and lignification were increased, and APX and CAT activities were decreased. Histochemical analysis showed that soluble phenolics accumulated in the cytosol of Cd-treated roots but lignification was confined to newly formed protoxylem elements, which were found in the region of the root tip that normally constitutes the elongation zone. Roots exposed to 5 microM Cd showed less pronounced responses and only a small decrease in the elongation rate. These results suggest that in cells challenged by Cd at concentrations exceeding the detoxification capacity, H(2)O(2) accumulated because of an imbalance of redox systems. This, in turn, may have triggered the developmental program leading to xylogenesis. In conclusion, Cd did not cause necrotic injury in root tips but appeared to expedite differentiation, thus leading to accelerated aging.