Proteomic analysis of Populus × euramericana (clone I-214) roots to identify key factors involved in zinc stress response

Contamination of soil and water by heavy metals has become a widespread problem; environmental pollution by high zinc (Zn) concentration occurs frequently. Although poplar (Populus spp.) has been identified as suitable for phytoremediation approaches, its response to high Zn concentrations are still not clearly understood. For this reason, we investigated the effects of Zn in Populus × euramericana clone I-214 roots by proteomic analysis. Comparative experiments were conducted on rooted woody cuttings grown in nutrient solutions containing 1 mM (treatment) or 1 μM (control) Zn concentrations. A gel-based proteomic approach coupled with morphological and chemical analysis was used to identify differentially represented proteins in treated roots and to investigate the effect of Zn treatment on the poplar root system. Data shows that Zn was accumulated preferentially in roots, that the antioxidant system, the carbohydrate/energy and amino acid metabolisms were the main pathways modulated by Zn excess, and that mitochondria and vacuoles were the cellular organelles predominately affected by Zn stress. A coordination between cell death and proliferation/growth seems to occur under this condition to counteract the Zn-induced damage.     

Responses of the Populus × euramericana clone I-214 to excess zinc: Carbon assimilation,structural modifications,metal distribution and cellular localization

Poplar (Populus), the model system in tree research, is a fast-growing and high biomass plant which is promising for energy, paper and pulp production, and for growth in soils contaminated with metals. Contamination of soils and water with heavy metals has become a widespread problem; environmental pollution by excess zinc (Zn), one of the more important contaminants, occurs frequently and yet the responses of Populus to high Zn concentrations are still not clearly understood.We investigated the effects of Zn on the functional and structural parameters in the Populus × euramericana clone I-214 by Zn localization in frozen-hydrated leaves and roots by cryo-scanning electron microscopy (cryo-SEM)/energy-dispersive X-ray microanalysis (EDXMA). The experiment was conducted on cuttings grown in nutrient solutions with an increasing Zn concentration gradient (0.001–10 mM).Biomass partitioning and Zn uptake were affected by the metal treatments, showing organ- and tissue-dependent responses. In particular, Zn accumulated in old leaves and moved from shoot to root as the Zn concentration in the growth medium increased. At the highest treatment concentration (10 mM), Zn was preferentially localized in photosynthetic tissues of shoots, and in epidermis and cortex tissues of roots. Gas exchange and chlorophyll measurements showed impairments in leaf biochemistry rather than in stomatal function. Modifications in foliage area, stomatal density and leaf layer thickness were investigated to reduce and/or compensate the negative effects of excess Zn on CO₂ assimilation.To counteract Zn toxicity, clone I-214 adopted different defense/tolerance mechanisms involving complex structural, physiological and biochemical processes, attributed to both Zn excluders and accumulators. This study demonstrates the advantages of combining cryo-SEM/EDXMA, gas exchange and chemical analyses for studying metal localization and structural as well as physiological responses in plants.     

Early responses to cadmium of two poplar clones that differ in stress tolerance

Soil cadmium (Cd) contamination is becoming a matter of great global concern. The identification of plants differentially sensitive to Cd excess is of interest for the selection of genotype adaptive to grow and develop in polluted areas and capable of ameliorating or reducing the negative environmental effects of this toxic metal. The two poplar clones I-214 (Populus × canadensis) and Eridano (Populus deltoides × maximowiczii) are, respectively, tolerant and sensitive to ozone (O₃) exposure. Because stress tolerance is mediated by an array of overlapping defence mechanisms, we tested the hypothesis that these two clones differently sensitive to O₃ stress factor also exhibit different tolerance to Cd. With this purpose, an outdoor pot experiment was designed to study the responses of I-214 and Eridano to the distribution of different Cd solutions enriched with CdCl₂ (0, 50 and 150 μM) for 35 days. Changes in leaf area, biomass allocation and Cd uptake, photosynthesis, chlorophyll fluorescence, leaf concentration of nutrients and pigments, hydrogen peroxide (H₂O₂) and nitric oxide (NO) production and thiol compounds were investigated. The two poplar clones showed similar sensitivity to excess Cd in terms of biomass production, photosynthesis activity and Cd accumulation, though physiological and biochemical traits revealed different defence strategies. In particular, Eridano maintained in any Cd treatment the number of its constitutively wider blade leaves, while the number of I-214 leaves (with lower size) was reduced. H₂O₂ increased 4.5- and 13-fold in I-214 leaves after the lowest (L) and highest (H) Cd treatments, respectively, revealing the induction of oxidative burst. NO, constitutively higher in I-214 than Eridano, progressively increased in both clones with the enhancement of Cd concentration in the substrate. I-214 showed a more elevated antioxidative capacity (GSH/GSSG) and higher photochemical efficiency of PSII (F_v/F_m) and de-epoxidation degree of xantophylls-cycle (DEPS). The glutathione pool was not affected by Cd treatment in both clones, while non-protein thiols and phytochelatins were reduced at L Cd treatment in I-214. Overall, these two clones presented high adaptability to Cd stress and are both suitable to develop and growth in environments contaminated with this metal, thus being promising for their potential use in phytoremediation programmes.     

Leaf structural modifications in Populus × euramericana subjected to Zn excess

In previous experiments elevated but sub-symptomatic applications of Zn (0.1 mM and 1 mM) caused impairments in growth parameters and photosynthetic performance of Populus × euramericana (Dode) Guinier clone I-214. The aim of this work was to evaluate leaf morphological and anatomical traits in this clone in response to the same Zn concentrations. The results showed that Zn treatments induced variations in leaf dry mass, area, mesophyll thickness, intercellular spaces, stomatal density and size. Stronger modifications, especially concerning stomata characteristics induced by 1 mM Zn, were consistent with physiological impairments while those induced by 0.1 mM Zn suggested a compensatory strategy for maintaining functional integrity.     

Responses of two poplar species (Populus alba and Populus x canadensis) to high copper concentrations

With the aim to examine their potentials as renewable resources to decontaminate polluted soils, growth, photosynthesis and nitrogen balance were analyzed in two poplar species (Populus x canadensis, Adda clone and Populus alba, Villafranca clone) to investigate the tolerance to high copper (Cu) concentrations. The two clones showed different responses to Cu in terms of tolerance and metal allocation: P. x canadensis accumulated Cu in roots, displaying features sought in plants suitable for phytostabilization, while P. alba accumulated the metal in leaves, like an indicator species.     

Cloning and expression analysis of Chitinase genes from Populus canadensis

Plant chitinases play a key role in conferring resistance to environmental stresses, including attack by fungal pathogens. In the present study, we employed rapid amplification of cDNA ends (RACE) to identify five chitinase genes in Populus canadensis Moench. Sequence alignment revealed that these genes belong to five subfamilies of chitinase genes. The full-length cDNAs of these genes ranged in size from 991 to 1358 bp and encoded proteins with mol wts from 29.5 to 40.3 kD. Five genes were grouped into three major clades based on amino acid sequences of encoded proteins. Exon-intron gene structure and protein domain analysis further supported the designation. A three-dimensional structure comparison showed the high similarity between five P. canadensis chitinases and three well-studied chitinases from other species. The expression levels of all five genes were up-regulated during Populus infection with the pathogenic fungus Marssonina brunnea, and four of them were highly induced by salt and drought stresses. Furthermore, such factors as elicitors, wounding, and low temperature also elevated the expression of these chitinase genes to varying extents. We postulated that these chitinase genes may be involved in pathways of the defense against fungal infection and function in response to various abiotic stresses.     

Anatomical differences of poplar (<Emphasis Type="Italic">Populus</Emphasis> × <Emphasis Type="Italic">euramericana</Emphasis> clone I-214) roots exposed to zinc excess

Poplar is one of the suitable candidates for phytoremediation due to extensive root system, fast growth rate, easy propagation and high biomass production. Zinc (Zn) is an essential element, but at high concentration becomes toxic to plants, similarly like cadmium (Cd). In order to evaluate the effect of Zn on root tissue development we conducted experiments with poplar (Populus × euramericana clone I-214) grown in hydroponics. Plants were treated with low (control) and excess level of Zn (1 mM). Changes in the development of apoplasmic barriers — Casparian bands and suberin lamellae in endodermis, as well as lignification of xylem vessels have been investigated. We found that both apoplasmic barriers developed closer to the root apex in higher Zn-treated root when compared with control root. Similar changes were observed in lignification of xylem vessels. For localization of Zn within root tissues, cryo-SEM/EDXMA analyses were used. Most of Zn was localized in the cortical tissues and four-time less Zn was determined in the inner part of the root below the endodermis. This indicates that endodermis serves as efficient barrier of apoplasmic Zn transport across the poplar root.     

Over-expression of AQUA1 in <Emphasis Type="Italic">Populus alba</Emphasis> Villafranca clone increases relative growth rate and water use efficiency,under Zn excess condition

Key message

Transgenic Populus alba over-expressing a TIP aquaporin ( aqua1) showed a higher growth rate under Zn excess, suggesting that aqua1 could be involved in water homeostasis, rather than in Zn homeostasis.

Abstract

Populus is the internationally accepted model for physiological and developmental studies of tree traits under stress. In plants, aquaporins facilitate and regulate the diffusion of water, however, few poplar aquaporins have been characterized to date. In this study, we reported for the first time an in vivo characterization of Populus alba clone Villafranca transgenic plants over-expressing a TIP aquaporin (aqua1) of P. x euramericana clone I-214. An AQUA1:GFP chimeric construct, over-expressed in P. alba Villafranca clones, shows a cytoplasmic localization in roots, and it localizes in guard cells in leaves. When over-expressed in transgenic plants, aqua1 confers a higher growth rate compared to wild-type (wt) plants, without affecting chlorophyll accumulation, relative water content (RWC), and fluorescence performances, but increasing the intrinsic Transpiration Efficiency. In response to Zn (1 mM), transgenic lines did not show a significant increase in Zn accumulation as compared to wt plants, even though the over-expression of this gene confers higher tolerance in root tissues. These results suggest that, in poplar plants, this gene could be principally involved in regulation of water homeostasis and biomass production, rather than in Zn homeostasis.

     

Occurrence of different inter-varietal and inter-organ defence strategies towards supra-optimal zinc concentrations in two cultivars of Triticum aestivum L.

Two bread wheat (Triticum aestivum L.) cultivars (Albimonte, traditional cultivar very important in Italy since long time; and Manital, more recent, evincing better productive performances) were grown for 10 d in presence of 0.7 (control), 70 or 350 μM ZnSO₄, to verify whether Zn excess was differently managed at inter-varietal and at inter-organ level. Roots were found to be the main site of Zn accumulation, although a moderate metal translocation to leaves occurred in both cultivars. Despite only slight differences in internal Zn concentrations between cultivars, Albimonte seemed to be more sensitive to Zn excess in terms of growth reduction and H₂O₂ accumulation, suggesting that the diversities in responses to Zn stress should be ascribed here to inter-varietal metabolic differences. In both cultivars, increased NAD(P)H oxidation rate by pH-dependent peroxidases, and reduced detoxification activity by catalase and peroxidases, were responsible for Zn-induced H₂O₂ accumulation, while total superoxide dismutase content and activity seemed in general to not change or even depress. Moreover, differences in the content of thiol-peptide compounds (glutathione and phytochelatins) were detected, suggesting indeed the setting up of differential response mechanisms to Zn excess at an inter-varietal and inter-organ level.     

Comparison of cadmium-induced iron-deficiency responses and genuine iron-deficiency responses in Malus xiaojinensis

The effects of the heavy metal Cd in Malus xiaojinensis were investigated using hydroponic cultures. Chlorophyll and Fe concentrations in young leaves were markedly decreased by Cd treatment, although Fe concentration was significantly enhanced in the roots. A comparative examination of the Fe-deficiency responses due to Fe deficiency and Cd treatment was also performed. Both Fe deficiency and Cd treatment induced responses similar to those of Fe-deficiency in M. xiaojinensis, including acidification of the rhizosphere, enhanced Fe(III) chelate reductase activity, and upregulation of the Fe-deficiency-responsive genes MxIRT1 and MxFRO2-Like. However, the Fe-deficiency responses induced by Cd treatment were different in intensity and timing from those induced by Fe deficiency.     

Consortium of Higher Aquatic Plants and Microalgae Designed to Purify Sewage of Heavy Metal Ions

We selected higher aquatic plants (HAP) and microalgae possessing a high sorption capacity in respect to heavy metals to form a consortium designed to purify contaminated aquatic ecosystems. Accumulation of heavy metals Cd²⁺, Cu²⁺, Pb²⁺, and Zn²⁺ was investigated in plants Pistia stratiotes, Elodea canadensis, and Lemna minor and green microalgae Chlorella vulgaris ВВ-2, Ankistrodesmus sp. ВI-1, Chlamydomonas reinhardtii В-4, and Scеnеdеsmus quadricauda В-1. It was found that intense accumulation of metals occurs in cultures of HAP Pistia stratiotes and Elodea canadensis. These plants are macroconcentrators of zinc, lead, and copper and microconcentrators of cadmium. Out of the examined cultures of microalgae, effective bioaccumulators of heavy metals were C. vulgaris ВВ-2 and Ankistrodesmus sp. ВI-1. It was shown that heavy metals are selectively taken up from the medium in the series Zn²⁺ > Cu²⁺ > Cd²⁺ > Pb²⁺. In order to produce a consortium of higher aquatic plants and microalgae for purification of polluted aquatic ecosystems, we investigated interaction of HAP P. stratiotes and E. canadensis with microalgae C. vulgaris ВВ-2 and Ankistrodesmus sp. ВI-1 in the course of their cocultivation. Neutral relations were detected between the cells of microalgae C. vulgaris ВВ-2 and Ankistrodesmus sp. ВI-1 and HAP E. canadensis. At the same time, the cells of Ankistrodesmus sp. ВI-1 and HAP P. stratiotes formed a symbiosis. Microscopic examination showed numerous points where the cells of microalgae Ankistrodesmus sp. ВI-1 were attached to the roots of P. stratiotes plants. We tested an opportunity to employ the association between P. stratiotes and Ankistrodesmus sp. ВI-1 for purification of simulated wastewater polluted with heavy metal ions. This consortium proved to be capable of eliminating contaminants from the sewage, reducing their level in the sewage to standard values, and active accumulation of heavy metal ions.     

Transient expression for functional gene analysis using Populus protoplasts

Despite the availability of the Populus genome sequence and the development of genetic, genomic, and transgenic approaches for its improvement, the lengthy life span of Populus and the cumbersome process required for its transformation have impeded rapid characterization of gene functions in Populus. Protoplasts provide a versatile and physiologically relevant cell system for high-throughput analysis and functional characterization of plant genes. Here, a highly efficient transient expression system using Populus mesophyll protoplasts was developed based on the following three steps. The first step involved formulating a new enzyme cocktail containing 2 % Cellulase C2605 and 0.5 % Pectinase P2611, which was shown to enable efficient large-scale isolation of homogenous Populus mesophyll protoplasts. The second step involved optimization of transfection conditions, such as the polyethylene glycol concentration and amount of plasmid DNA to ensure a >80 % transfection efficiency for Populus protoplasts. The third step involved using the Populus protoplast transient expression system to successfully determine the subcellular localizations of proteins, emulate signaling events during pathogen infection, and prepare protein extracts for Western blotting and protein–protein interaction assays. This rapid and highly efficient transient gene expression system in Populus mesophyll protoplasts will facilitate the rapid identification of gene functions and elucidation of signaling pathways in Populus.     

Copper and zinc in Elodea canadensis from rivers with various pollution levels

The anthropogenic impact of xenobiotics contributes to environmental risk for the aquatic environment and thus, must be controlled. Elodea canadensis, a cosmopolitan aquatic macrophyte with an important role in the ecology of many littoral zones, may provide an integrated record of pollution. Therefore, it was interesting to investigate the accumulation of Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn in this species and in water and bottom sediments collected from rivers with various levels of contamination. Of these rivers one control and one polluted was selected for the collection of E. canadensis for an experiment to compare the ability of this species to accumulate Cu and Zn. These elements were supplemented at concentrations (mg L⁻¹) of 0.01, 0.02, 0.03, 0.05, 0.08 and 0.14 as CuSO₄·5H₂O, and 0.4, 0.6, 0.9, 1.4, 2.03 and 3.04 as ZnSO₄·7H₂O and in a mixture containing (mg L⁻¹) 0.01Cu + 0.4Zn, 0.02Cu + 0.6Zn, 0.03Cu + 0.9Zn, 0.05Cu + 1.4Zn, 0.08Cu + 2.03Zn and 0.14Cu + 3.04Zn. After the experiment, E. canadensis from the polluted river contained significantly higher Cu and Zn concentrations when applied separately and also significantly higher Cu and Zn concentrations when applied as a mixture compared to the control river. These higher concentrations in E. canadensis from the polluted river were found in all combinations in the experiment. Thus, E. canadensis habituated in polluted sites to the exposure, and long-term influence of elevated metal levels appeared to be better adapted, and it also exhibited a higher increase in biomass than plants from the control river in all the experimental Cu and Zn solutions. Younger leaves of E. canadensis were more resistant to the effects of Cu and Zn than older leaves. Both Cu and Zn negatively affected the cell structure of older leaves, although the influence of Cu on plasma membrane integrity and chloroplast distribution was stronger than that of Zn. The influence of the Cu + Zn mixture on E. canadensis resulted in less pronounced cell disintegration than the influence of Cu added separately.The explanation of differences in the E. canadensis biomass increase and metal concentrations under the binary Cu and Zn impact needs further examination.     

Induced somatic sector analysis of cellulose synthase (CesA) promoter regions in woody stem tissues

The increasing focus on plantation forestry as a renewable source of cellulosic biomass has emphasized the need for tools to study the unique biology of woody genera such as Eucalyptus, Populus and Pinus. The domestication of these woody crops is hampered by long generation times, and breeders are now looking to molecular approaches such as marker-assisted breeding and genetic modification to accelerate tree improvement. Much of what is known about genes involved in the growth and development of plants has come from studies of herbaceous models such as Arabidopsis and rice. However, transferring this information to woody plants often proves difficult, especially for genes expressed in woody stems. Here we report the use of induced somatic sector analysis (ISSA) for characterization of promoter expression patterns directly in the stems of Populus and Eucalyptus trees. As a case study, we used previously characterized primary and secondary cell wall-related cellulose synthase (CesA) promoters cloned from Eucalyptus grandis. We show that ISSA can be used to elucidate the phloem and xylem expression patterns of the CesA genes in Eucalyptus and Populus stems and also show that the staining patterns differ in Eucalyptus and Populus stems. These findings show that ISSA is an efficient approach to investigate promoter function in the developmental context of woody plant tissues and raise questions about the suitability of heterologous promoters for genetic manipulation in plant species.     

Nitric Oxide Is Associated with Long-Term Zinc Tolerance in Solanum nigrum

Nitric oxide (NO) has been identified as a signal molecule that interplays with reactive oxygen species in response to heavy metal stresses. Roles of NO in regulating cadmium toxicity and iron deficiency have been proposed; however, the function of NO in zinc (Zn) tolerance in plants remains unclear. Here, we investigated NO accumulation and its role in plant Zn tolerance. Zn-induced NO production promoted an increase in reactive oxygen species accumulation in Solanum nigrum roots by modulating the expression and activity of antioxidative enzymes. Subsequently, programmed cell death (PCD) was observed in primary root tips. Inhibiting NO accumulation by 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (a specific NO scavenger) or N^G-nitro-l-arginine-methyl ester (a NO synthase inhibitor) prevented the increase of superoxide radical and hydrogen peroxide as well as the subsequent cell death in the root tips, supporting the role of NO in Zn-induced PCD in the root tips. Zn-induced NO production affected the length of primary roots, the number of lateral roots, and root hair growth and thereby modulated root system architecture and activity. Investigation of metal contents in Zn-treated roots suggests that NO is required for metal (especially iron) uptake and homeostasis in plants exposed to excess Zn. Taken together, our results indicate that NO production and the subsequent PCD in root tips exposed to excess Zn are favorable for the S. nigrum seedling response to long-term Zn toxicity by modulating root system architecture and subsequent adaptation to Zn stress.Heavy metal contamination is a serious problem for the environment. Some metallic elements, such as zinc (Zn), are essential micronutrients and play a role as enzyme cofactors in many metabolic reactions. However, uptake of high concentrations of Zn is found to be toxic to plant growth and development. High concentrations of Zn (260–16,000 mg kg⁻¹) have been found in the soil near smelting sites (Bi et al., 2006), and Zn contamination has been of increasing concern in these regions due to its threat to agriculture and human health (Bi et al., 2006).Zn homeostasis is a tightly regulated process because Zn can be both essential and deleterious to plants depending on its concentration. The effects of Zn on plants have been widely reported (Broadley et al., 2007; Wang et al., 2009), including tolerance to Zn accumulation and Zn deficiency as well as the protective effects of Zn in plants. Zn is closely involved in protein synthesis and nitrogen metabolism; the growth of Zn-deficient plants is markedly inhibited. Zn is also a constituent of copper/zinc superoxide dismutase (Cu/Zn SOD). Zn deficiency reduces antioxidative enzyme activity and thereby results in reactive oxygen species (ROS) accumulation and oxidative damage (Sharma et al., 2004). Tolerance to Zn accumulation in plants is a complex phenomenon. Aside from Zn deficiency, excess Zn can also inhibit plant growth and development by disequilibrating the uptake and redistribution of mineral nutrition and by disturbing the antioxidant defense system and metabolic processes such as photosynthesis, transpiration, and antioxidative enzyme activity. Recent studies have shown that Zn toxicity affects the activity of antioxidative enzymes, such as SOD, catalase (CAT), and ascorbate peroxidase (APX), in plants (Wójcik et al., 2006; Tewari et al., 2008). The mechanisms of Zn toxicity are not fully understood; however, they may involve competition for catalytic sites or for transporter proteins (González-Guerrero et al., 2005). Zn toxicity also inhibits the uptake of other nutrient elements, such as iron (Fe). Deficiency of these elements can lead to ROS accumulation and oxidative stress (Bonnet et al., 2000). Excess Zn may bind to proteins and lead to the displacement of other ions, such as Fe²⁺, from protein-binding sites. Plants exposed to excess Zn become Fe deficient (Wintz et al., 2003). However, the effects of Zn stress on root system development have not been elucidated.Nitric oxide (NO) is a free radical gas that has emerged as an important signaling molecule in plants (Neill et al., 2002). NO accumulation in roots mediates auxin-induced lateral root formation (Correa-Aragunde et al., 2004), adventitious root growth (Tewari et al., 2008), and root hair development (Lombardo et al., 2006). Graziano and Lamattina (2007) have reported that root hair proliferation induced by Fe deficiency is involved in NO accumulation in tobacco (Nicotiana tabacum). It has also been indicated that NO protects plant cells against oxidative stress by reducing ROS accumulation (Wink and Mitchell, 1998; Xu et al., 2010). NO enhances the tolerance of plants to heavy metal stresses (Yang et al., 2006; Sun et al., 2007; Zhang et al., 2008; Xu et al., 2009). However, very little is known about the level of Zn-mediated NO accumulation in plants and the physiological and molecular mechanisms of the effects of NO on tolerance to Zn toxicity.Programmed cell death (PCD) is an active process of cellular suicide that is essential for development and stress responses in plants. Diverse abiotic stresses, such as salt, drought, nutrient deficiency, and cadmium (Cd) toxicity, can induce PCD in plants. Salt stress has been reported to induce PCD in root apical meristem cells (Huh et al., 2002). Subbaiah and Sachs (2003) have demonstrated that flooding stress induces PCD-like root tip death in maize (Zea mays) and pea (Pisum sativum) plants. Duan et al. (2010) have shown that drought induces PCD in Arabidopsis (Arabidopsis thaliana) root tips. De Michele et al. (2009) have reported that Arabidopsis cell suspension cultures undergo PCD when exposed to Cd stress and that NO is involved in this process. Many studies have indicated that PCD plays a role in the developmental plasticity of plant architecture (Duan et al., 2010). Excess Zn-induced growth inhibition and root death have been found in various plant species (Lingua et al., 2008; Ozdener and Aydin, 2010). However, whether Zn-induced root death occurs through PCD and its underlying mechanisms are poorly understood.In this study, we used a Zn-hyperaccumulator, Solanum nigrum, to study the effects of excess Zn on root system architecture and the roles of NO and ROS in these effects. S. nigrum is known to hyperaccumulate Zn and Cd in natural soil or in soils contaminated with Zn or Cd. In recent years, physiological characteristics of S. nigrum under Zn or Cd stress have been reported (Wei et al., 2004; Sun et al., 2007; Marques et al., 2008; Xu et al., 2009). The aim of this work was to study plant tolerance to excess Zn and the function of NO produced in Zn-treated plants. Our findings support the model in which NO contributes to rapid ROS accumulation and subsequent PCD in root tips in response to heavy metal stresses; our results also indicate that NO is an important regulator of Zn-modulated root system architecture. Potential mechanisms involved in this process are discussed.