Downregulation of high-isoelectric-point extracellular superoxide dismutase mediates alterations in the metabolism of reactive oxygen species and developmental disturbances in hybrid aspen

Authors

  • Vaibhav Srivastava,

    Corresponding author
    1. Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 901 83 Umeå, Sweden,
      Joint first authors.
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  • Helga Schinkel,

    Corresponding author
    1. Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstr.6, 52074 Aachen, Germany,
      Joint first authors.
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  • Johanna Witzell,

    1. Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 901 83 Umeå, Sweden,
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  • Magnus Hertzberg,

    1. Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 901 83 Umeå, Sweden,
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  • Mikaela Torp,

    1. Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 901 83 Umeå, Sweden,
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  • Manoj Kumar Srivastava,

    Corresponding author
    1. Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 901 83 Umeå, Sweden,
      Present address: Department of Biochemistry, JC Bose Institute of Life Sciences, Bundelkhand University, Jhansi (UP) 284 128, India.
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  • Barbara Karpinska,

    1. Department of Botany, Stockholm University, Lilla Frescativ. 5, SE-10691 Stockholm, Sweden, and
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  • Michael Melzer,

    1. Department of Molecular Cell Biology, Institute of Plant Genetics and Crop Plant Research, 06466 Gatersleben, Germany
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  • Gunnar Wingsle

    Corresponding author
    1. Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 901 83 Umeå, Sweden,
      (fax +46 90 786 5901; e-mail gunnar.wingsle@genfys.slu.se).
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(fax +46 90 786 5901; e-mail gunnar.wingsle@genfys.slu.se).

Joint first authors.

Present address: Department of Biochemistry, JC Bose Institute of Life Sciences, Bundelkhand University, Jhansi (UP) 284 128, India.

Summary

Transgenic hybrid aspen (Populus tremula L. × P. tremuloides Michx.) plants expressing a high-isoelectric-point superoxide dismutase (hipI-SOD) gene in antisense orientation were generated to investigate its function. Immunolocalization studies showed the enzyme to be localized extracellularly, in the secondary cell wall of xylem vessels and phloem fibers. The antisense lines of hipI-SOD exhibited a distinct phenotype; growth rate was reduced, stems were thinner and leaves smaller than in wild-type (WT) plants. The abundance of hipI-SOD was reduced in the bark and xylem of plants from these antisense lines. The vascular tissue of transgenic lines became lignified earlier than in WT plants and also showed an increased accumulation of reactive oxygen species (ROS). Xylem fibers and vessels were shorter and thinner in the transgenic lines than in WT plants. The total phenolic content was enhanced in the antisense lines. Furthermore, microarray analysis indicated that several enzymes involved in cell signaling, lignin biosynthesis and stress responses were upregulated in apical vascular tissues of transgenic plants. The upregulation of selected genes involved in lignin biosynthesis was also verified by real-time PCR. The results suggest that, in the transgenic plants, a premature transition into maturation occurs and the process is discussed in terms of the effects of increased accumulation of ROS due to reduced expression of hipI-SOD during development and differentiation.

Introduction

In plants, reactive oxygen species (ROS) are continually produced as byproducts of metabolic pathways localized in different cellular compartments (Foyer and Harbinson, 1994). Various plant oxidases and peroxidases also generate ROS in response to certain environmental conditions (Allan and Fluhr, 1997; Bolwell et al., 1998, 2002). Reactive oxygen species cause oxidative stress in plants, leading to oxidative damage to proteins, DNA and lipids. It has been recently shown that, as well as causing oxidative stress, ROS may also function as signaling molecules in plants and are involved in the control and regulation of biological processes such as programmed cell death, hormonal signaling, stress responses and development (Foreman et al., 2003; Karpinski et al., 1999; Kwak et al., 2003; Laloi et al., 2004; Neill et al., 2002; Overmyer et al., 2003).

To prevent the harmful effects of ROS generated by cellular processes or in response to external conditions, plants use a number of defense systems against oxidative stress (Bowler et al., 1992; Larson, 1988). Central components of these defense systems are the superoxide dismutase (SOD; superoxide: superoxide oxidoreductase, EC 1.15.1.1) enzymes, which exists in three different forms (Cu/Zn-SOD, Fe-SOD and Mn-SOD) with metal co-factors (Fridovich, 1986).

Superoxide dismutases catalyze the dismutation of toxic superoxide radicals to molecular oxygen (O2) and hydrogen peroxide (H2O2), thus preventing the oxidation of biological molecules either by the radicals themselves or by their derivatives (Liochev and Fridovich, 1994).

An isoform of CuZn-superoxide dismutase (CuZn-SOD) with an unusually high isoelectric point (>9.5), referred to as hipI-SOD, was recently discovered in both Scots pine (Karpinska et al., 2001) and hybrid aspen (Schinkel et al., 2001). In pine, hipI-SOD has been detected by immunolocalization in the plasma membrane of phloem sieve cells, in the Golgi apparatus of albuminous cells and in secondary walls and intercellular spaces in the xylem (Karpinska et al., 2001). In hybrid aspen, hipI-SOD activity has been found in apical tissues as well as in phloem and xylem (Schinkel et al., 2001). The extracellular localization of hipI-SOD suggests that it has a specific function, such as the regulation of ROS production. Other studies have revealed the involvement of NAD(P)H oxidase in the generation of Oinline image and of CuZn-SOD in the generation of H2O2 in vascular tissue during lignification (Ogawa et al., 1997) and pathogen defense (Desikan et al., 1996; Kliebenstein et al., 1999). Karlsson et al. (2005) have discussed the implications of the expression of hipI-SOD and the presence of H2O2 in the development of secondary cell walls. H2O2 has been shown to be involved in the differentiation of secondary cell walls (Potikha et al., 1999) and in the polymerization of cinnamyl alcohols in the secondary cell walls of lignifying xylem vessels (Barcelo, 2005). These studies, together with its conventional dismutation function, suggest a putative role for hipI-SOD in regulation of ROS and plant development.

In the present study, we examined the function of hipI-SOD by generating transgenic hybrid aspen plants expressing the c-DNA of hipI-SOD in an antisense orientation under the control of the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter. Reduction of gene expression using antisense technology has been identified as a powerful tool for investigating the function of target genes (Bird et al., 1991; Kooter and Mol, 1993). The phenotype of the antisense plants was analyzed and the implications of lowered expression of hipI-SOD are discussed in terms of increased accumulation of ROS and concomitant effects on plant development and maturation.

Results

Identification of expressed sequence tags and gene model of hipI-SOD

Four expressed sequence tags (ESTs; corresponding to two contigs, POPLAR.5824.C1 and POPLAR.5824.C2) encoding hipI-SOD in hybrid aspen were identified from the poplar EST database (PopulusDB; http://www.populus.db.umu.se/). The two closest gene models for hipI-SOD (estExt_Genewise1_v1.C_LG_XIII1983 and grail3.0065004601) were identified from the Populus trichocarpa genome database (http://genome.jgi-psf.org/Poptr1/Poptr1.home.html). All the predicted protein sequences for hipI-SOD were similar (Figure 1a). A putative peroxisomal CuZn-SOD, CSD3 (NP_197311) was found to be the closest homolog of hipI-SOD in Arabidopsis, sharing 71% identity. HipISOD1 (CAC33847) and hipISOD2 (CAC33846) are 98% identical to each other, whereas Ptt5824C2 (POPLAR.5824.C2) is 100% identical to hipI-SOD1. The two predicted gene models (Pttgene1 and Pttgene2) for hipI-SOD in hybrid aspen share 94% similarity. The phylogenetic tree shown in Figure 1(b) suggests that all the predicted hipI-SODs in poplar, CSD3 in Arabidopsis (AtCSD3) and the ice plant SOD (McSOD) can be grouped together.

Figure 1.

 Sequence analysis of hipI-SOD. (a) Sequence comparison of all predicted hipI-SOD proteins in poplar and their homologs from other plants. Conserved residues are shaded in black, dark gray shading indicates similar residues in at least seven out of 10 sequences and light gray shading indicates similar residues in five out of 10 sequences. (b) Phylogenetic relationships among SODs in poplar and other plants. The phylogenetic tree was based on the alignment derived using the clustalw program. Parsimony bootstrap analysis was performed using paup* version 4.0b10 with the default settings selected (with the exception that 1000 replicates were carried out). Bootstrap support values are indicated.

Immunolocalization

Cross-sections of the stem at the fourth internode of wild-type (WT) hybrid aspen plants were used for immunolabeling of hipI-SOD with Alexa 488 (Molecular Probes; Invitrogen, GmbH Karlsruhe, Germany). Analysis of fluorescence and transmission images (taken with a Zeiss LSM 510 META confocal laser scanning microscope) showed significant antibody signals in the lignified secondary cell walls of phloem fibers and xylem vessels (Figure 2).

Figure 2.

 Immunolocalization of hipI-SOD with Alexa 488 in cross-sections of stems of WT hybrid aspen plants. Fluorescence signals showing location of hipI-SOD in secondary wall forming cells (a), and same section showing internal structure (b) in a transverse section from internode 4. Ph, phloem fibers; X, xylem vessels; scale bars represent 20 μm.

Generation of hipI-SOD antisense plants

Twenty independently transformed lines (see Experimental procedures) were regenerated. Southern blot analysis of the transgenic lines indicated that lines 9 and 24 each had a single T-DNA insert (data not shown) and were denoted AS-SOD9 and AS-SOD24, respectively.

Phenotype of hipI-SOD antisense plants

Plants belonging to two lines (AS-SOD9 and AS-SOD24) exhibited clear phenotypic differences from WT plants (Figure 3a). Compared with the WT, they were stunted (Figure 4a), had reduced radial stem growth (Figure 4b) and fewer and shorter internodes (Figure 4c,d). Plants of line AS-SOD9 had a sloping stem habit and developed numerous lateral shoots. The leaves of the antisense plants were smaller and showed developmental abnormalities compared to the WT (Figure 3b). The leaves of both transgenic lines were much smaller than leaves of WT plants. Wild-type leaves first grew longitudinally and then laterally, resulting in an oval leaf shape. In contrast, the lateral growth of leaves of AS-SOD9 plants seemed to be inhibited and the leaves of AS-SOD24 plants grew in both directions simultaneously (Figure 3b). Furthermore, whilst the leaf margins of WT plants remained curled up to the position of leaf 8–11, they uncurled earlier in AS-SOD9 and AS-SOD24 (at leaf positions 3–6 and 4–6, respectively). The younger foliage in line AS-SOD24 was paler green than in WT plants of the same age (Figure 3b).

Figure 3.

 Phenotype of WT and hipI-SOD antisense plants. (a) Phenotypes of the transgenic lines AS-SOD9 and AS-SOD24 and WT hybrid aspen plants (left to right). All plants were 13 weeks old. (b) Leaf development in hybrid aspen plants WT, line AS-SOD9 and line AS-SOD24. From left to right leaf numbers 5, 8, 11, 14, 16, 22, are shown, where leaf 1 is the first leaf longer than 1 cm.

Figure 4.

 Characterization of the stems of WT, AS-SOD9 and AS-SOD24 plants. (a) Growth increment, (b) stem diameter, (c) internode number, (d) internode length. The error bars represent standard deviations for results from three (line AS-SOD9) or four samples (WT and line AS-SOD24).

Expression of hipI-SOD

Expression levels of hipI-SOD protein were compared by western blot analysis of high-isoelectric-point proteins obtained by partial purification of bark and xylem tissue from stem samples. The plants tested from both antisense lines showed a reduced amount of hipI-SOD compared with WT. Furthermore, one band of approximately 18 kDa and two bands of approximately 18 and 20 kDa were found in bark and xylem tissue, respectively (Figure 5).

Figure 5.

 HipI-SOD expression pattern in hybrid aspen by Western blot analysis. Western blot of partially purified protein from bark and xylem tissue of three individual plants (a–c) from the WT, AS-SOD9 and AS-SOD24 lines. The size of the bands on the blot is indicated on the right.

ROS and lignin

Examination of cross sections of phloroglucinol-stained stems indicated only slight lignification of the xylem and no lignification of phloem fibers in the WT plants at internode 4 (counted from the top); whilst the xylem in AS-SOD24, and both the xylem and phloem fibers in AS-SOD9 plants were lignified at this point (Figure 6a–c).

Figure 6.

 Histochemical analysis of vascular tissue. (a–c) Phloroglucinol staining for lignin in the vascular tissues of WT (a), AS-SOD9 (b), and AS-SOD24 (c) plants. (d–f) DAB staining for H2O2 in vascular tissues of WT (d), AS-SOD9 (e) and AS-SOD24 (f) plants. (g–i) NBT staining for superoxide in vascular tissues of WT (g), AS-SOD9 (h) and AS-SOD24 (i) plants. Ca, cambium; Epi, epidermis; P, pith; Ph, phloem; S, sclerenchyma. Scale bars represent 100 μm.

Cross-sections of the stem at internode 4 stained with 3–3′-diaminobenzidine tetrahydrochloride (DAB) showed an increased accumulation of H2O2 in lignified xylem vessels in lines AS-SOD9 and AS-SOD24 compared with WT plants, whereas the concentration of H2O2 in phloem fibers was almost equal in WT and AS-SOD24 plants but higher in those of line AS-SOD9 (Figure 6d–f).

However, in sections of the same region stained with nitro blue tetrazolium (NBT) increased levels of blue formazan precipitate (produced by the reduction of NBT by superoxide) were found in the cambial region, as well as in the xylem vessels of both antisense lines (Figure 6g–i). The superoxide radical concentration was lower in phloem fibers than in xylem vessels in all of the plants examined (Figure 6g–i). Control experiments were performed on WT stem cross-sections for both DAB and NBT staining. Sections were incubated with a well-known ROS scavenger (10 mm reduced glutathione; Karlsson et al., 2005; Larson, 1988), together with DAB and NBT respectively. A clear reduction in precipitate of both types of staining was achieved, indicating the specificity of staining reactions with ROS (Figure S1).

The total lignin content of mature stems (taken from the base of the plant) was determined. Both the hipI-SOD antisense lines exhibited a slight decrease in lignin content compared with WT plants (Figure 7a). The lignin content was found to be approximately 22–26% of the cell wall residue (CWR) on a dry weight basis.

Figure 7.

 Total lignin content and phenolic profiles in stems of WT and transgenic plants. (a) Total lignin content in WT and transgenic plants. Values are given as percentage of CWR and correspond to the mean of three determinations. The vertical bar represents standard deviation. (b) Total concentration of salicylates (salicin, salicortin and tremulacin) and (c) total concentration of phenolic acids (chlorogenic, p-coumaric and cinnamic acid) in both young and old internodes of WT, AS-SOD9 and AS-SOD24 plants. The error bars represent standard error. Levels of significance were calculated from anova data (*P < 0.05).

Histological and morphological analysis

Comparison of cross-sections of the tenth internode of WT and transgenic plants showed histological differences (Figure 8). The epidermis of plants from both antisense lines was discontinuous and ruptured. An interesting observation in antisense plants was the presence of two layers of phloem fibers. In both antisense lines, the cambium was compressed and disorganized. Furthermore, cells were smaller in size and rays were fewer and inconsistent.

Figure 8.

 Light microscopy of transverse sections of stem. Comparable morphological analysis of WT, AS-SOD9 and AS-SOD24: (a–c) epidermis and periderm; (d–f) sclerenchyma, phloem and cambium; (g–i) xylem with rays (arrows). Ca, cambium; Epi, epidermis; Ph, phloem; S, sclerenchyma. Scale bars represent 50 μm.

Downregulation of the hipI-SOD gene in antisense plants affected the length and diameter of both xylem fibers and vessels in fully matured internodes. Fibers in the transgenic lines were approximately 20% shorter and 14% thinner, while vessels were 17% shorter and 36% thinner than WT plants (Figure 9a–f).

Figure 9.

 Fiber and vessel measurements. (a) Xylem fiber length, (b) xylem fiber diameter, (c) xylem vessel total length, (d) xylem vessel length, (e) xylem vessel diameter, (f) individual vessel showing measured parameters. All the measurement values represent means for four independently generated lines. The vertical bar represents standard error.

Analysis of phenolic profiles

The main phenolic compounds found in poplar tissues were hydroxycinnamic acid derivatives (with UV spectra resembling either that of chlorogenic acid or p-coumaric acid) and salicylates. Flavonoids were not detected in quantifiable amounts in stems, although some kaempherol and quercetin derivatives were present in the leaves of the same plants (data not shown). Compared with WT plants, the stems of transgenic plants generally had higher total concentrations of phenolic acids and salicylates (Figure 7b,c).

Microarray and real-time RT-PCR analysis

The expression of genes that were upregulated or downregulated by a factor of at least 1.8 in the transgenic lines AS-SOD9 and AS-SOD24 compared with the WT was regarded as being significantly altered. Genes that fulfilled this criterion in lines AS-SOD9 and AS-SOD24 are listed in Table 1. Some genes involved in lignin biosynthesis (4-coumarate:coA ligase, dehydroquinate dehydratase/shikimate dehydrogenase precursor and basic peroxidase) were upregulated in both antisense lines. A number of genes encoding proteins connected with stress responses were also upregulated, including: the endopeptidase Clp, a drought-induced protein from sunflower, phosphatase 2C, histone H1, the protein ERD 15 and several proteins related to pathogen defense such as phospholipase A2 and a patatin-like protein. We also found genes involved in signal transduction (such as ankyrins, a putative GTPase, serine/threonine protein kinase, the GTPase Rab 11C and the two calmodulins) to be upregulated. A ubiquitin-conjugating enzyme was also upregulated in both antisense lines, and in line AS-SOD9 a clear increase in mRNA encoding pectate lyases was observed. All of the genes discussed above had high scores in blastx searches of GenBank, confirming their assigned functions (Table 1). A few up- and downregulated sequences were also found in antisense plants that coded for hypothetical proteins or showed no similarity to any other sequence in GenBank (data not shown).

Table 1.   Results of the microarray experiment comparing gene expression in transgenic lines AS-SOD9 and AS-SOD24 to WT
Acc. no.AnnotationScoreE24E9
  1. Expression ratio of genes that were up- or downregulated in lines AS-SOD9 and AS-SOD24. Genes that were upregulated ≥1.8-fold or downregulated ≥1.8-fold (ratios 0.55 and less) compared with the WT are displayed. Acc. no stands for GenBank accession number of the gene on the array. blastx score (score) refers to the similarity to the GenBank annotation given. Only the highest score in blastx searches is given except for * which was the second highest score (the first being a hypothetical protein). E9 and E24 show the expression ratios between transgenic lines (AS-SOD9 and AS-SOD24, respectively) and the WT. All annotations refer to Arabidopsis thaliana genes except where another species is given. Put., putative.

AI162626IRE homolog, protein kinase like505.22.4
AI161963Chloroplast ribosomal protein S1220531.1
AI164191Ubiquitin conjugating enzyme-like protein1032.91.6
AI162541Endopeptidase Clp1282.91.7
AI1635944-Coumarate:coA ligase2112.81.4
AI163374Cytoplasmic aconitate hydratase342.61.5
AI162418Drought-induced protein (Helianthus)*932.41.4
AI163572Dehydroquinate dehydratase/Shikimate dehydrogenase precursor862.21.6
AI164389Put. Phospolipase A2 (Dianthus)1572.11.7
AI162596RAS-related protein RAB11C (Lotus)2712.11.6
AI164638Put. ankyrin992.11.5
AI163617Patatin-like protein 1 (Nicotiana)8221.1
AI162640Beta 1,2-xylosyltransferase17621
AI164044Histone H1602.42
AI162170Put. beta-hydroxyacyl ACP dehydratase431.91.4
AI163562Put. GTPase1951.91.3
AI165218Kinase-like protein1811.80.8
AI165265Similarity to protein phosphatase 2C1051.81.6
AI164312Similarity to ‘tub’ protein gp1081.81.4
AI166132Put. pectate lyase1791.42.3
AI165989Put. serine/threonine protein kinase (Mesembryanthemum)1051.22.3
AI162623Chlorophyll A/B-binding protein precursor (Oryza)830.92.2
AI163946Calmodulin-like protein6112
AI162944Basic peroxidase (Vigna)2281.52
AI163950Calmodulin-like protein801.21.9
AI162822Dehydration-induced protein ERD151321.71.9
AI162241Oxygen evolving enhancer protein 1 precursor (Bruguiera)12011.9
AI163014Ribosomal protein S 15A640.90.6
AI164188Ribosomal protein L 29 (Panax)1201.20.5
AI161865GTP-binding protein (Glycine)660.90.5
AI165142GSH-dependent dehydroascorbate reductase1921.10.5
AI165127NADH-cytochrome b5 reductase1040.51
AI165359Methyltransferase (Prunus)970.51.1
AI163311Cytochrome c oxidase III (Trypanosoma)370.40.8

To further validate the microarray results, several genes involved in lignin biosynthesis were selected for real time RT-PCR. The results showed that the expression of hydroxycinnamate: CoA ligase (4CL), caffeic acid/5-hydroxy ferulic acid O-methyl transferase (COMT) and hydroxycinnamyl alcohol dehydrogenase (CAD) was significantly increased in both antisense lines compared with the WT (Figure 10a–c). However, no significant differences were found in the expression level of phenylalanine ammonia lyase (PAL), caffeoyl-CoA O-methyltransferase 6 (CCoAOMT) and cinnamate 4-hydroxylase (C4H) in WT and line AS-SOD9, but levels were higher in line AS-SOD24 (Figure 10d–f).

Figure 10.

 Relative transcript abundance in apical tissues of WT and transgenic hybrid aspen. Gene expression was analyzed using quantitative RT-PCR. Results were normalized to the expression of 18S ribosomal RNA. The vertical bars represent the standard errors of the mean normalized expression value for three separate biological replicates. Relative expression levels of 4CL (a), COMT (b), CAD (c), PAL (d), CCoAOMT (e) and C4H (f) in apical shoot of WT and transgenic plants are shown.

Discussion

Expression and localization studies of a hipI-SOD in both pine and the Zinnia tracheary element (TE) differentiation system (Karlsson et al., 2005; Karpinska et al., 2001), and gene expression data from a high-resolution transcript profile from Populus (Schrader et al., 2004), suggest that the enzyme could be involved in the development and lignification of secondary cell walls. Fluorescence analysis showed specific staining in xylem vessels and phloem fibers, confirming the localization of hipI-SOD in secondary wall-forming cells, in agreement with published transcript profiling data and localization studies (Karlsson et al., 2005; Karpinska et al., 2001; Schrader et al., 2004).

To elucidate the function of hipI-SOD, we generated transgenic antisense plants and confirmed that the two lines showing phenotypic differences from the WT (AS-SOD9 and AS-SOD24) contained reduced quantities of hipI-SOD in their bark and xylem tissues. The Western blot analysis showed bands of different sizes in xylem and phloem tissues than the expected size of hipI-SOD monomers (16 kDa). The discrepancy between theoretical and experimental molecular masses may be due to the presence of two isoforms differing slightly in molecular mass, or the presence of post-translational modifications. Tissue-specific differences in hipI-SOD isozyme pools in hybrid poplar have previously been reported, and it is hypothesized that there are at least two isoforms of hipI-SOD (Schinkel et al., 2001). The Populus genome database (http://genome.jgi-psf.org/Poptr1/Poptr1.home.html) also contains two gene models for hipI-SOD. The adaptive benefits of having a number of hipI-SOD isoforms are not yet clear, but similar isoforms of other SODs have been reported (Schinkel et al., 1998; Streller and Wingsle, 1994; Zhu and Scandalios, 1993), and such redundancy or isovariant dynamics (Meagher et al., 1999) may be needed to ensure the constant presence of active hipI-SOD in different tissues.

The dwarfed phenotype of the transgenic plants is apparently due to reductions in both cell division and expansion, probably caused by a premature transition into maturation, as indicated by the early onset of lignification. The antisense lines had an increased number of lateral branches and AS-SOD9 appeared to have reduced apical dominance. Since the meristem responsible for producing the leaf blade is positioned around the leaf margins (Esau, 1977), the earlier uncurling of the leaf blade suggests that meristematic activity had ceased earlier in the transgenic lines.

In the antisense plants, enhanced accumulation of H2O2 and Oinline image was found in the vascular tissue of the younger internodes. In particular, H2O2 was found in the cell walls of phloem fibers and xylem vessels. Also, in xylem vessels, elevated Oinline image was also localized in the living cells of the cambium region in the transgenic lines. Lignification of phloem fibers and xylem vessels was correlated with the elevated accumulation of H2O2, with earlier initiation in younger parts of the stem in the transgenic lines compared with WT plants.

Xylem fibers and vessels were shorter and thinner in antisense plants than in the WT. As the lignification process is initiated prematurely whilst vessels and fibers are still expanding in antisense plants, their cell walls may become more rigid, thereby limiting the cell expansion. In the transgenic plants, the cells in the cambium region of the stem were compressed and looked ‘disorganized’ and cell division and expansion towards both the phloem and xylem side were impeded, possibly accounting for the stunted phenotype. This may be a direct effect of increased oxidative burst caused by the downregulation of hipI-SOD in these tissues, thus inhibiting cell division as reported earlier (Reichheld et al., 1999).

Analysis of the total lignin content of stem samples taken from the base of fully mature plants gave values in accordance with published data (Baucher et al., 1996). However, a slight difference was found between transgenic plants and the WT, indicating that the downregulation of hipI-SOD in antisense plants affects the initiation of lignification but not the overall lignin content.

Further indications that the transgenic plants are stressed are the elevated levels of phenolics observed in their stems. The phenolics detected in hybrid poplar stems were mainly hydroxycinnamic acid derivatives and salicylates (salicin, salicortin and tremulacin), in agreement with published data on the phenolic chemistry of poplar (Lindroth and Koss, 1996). Accumulation of phenolic acids in transgenic poplar stems might be activating an antioxidative system to diminish the oxidative stress (Sakihama et al., 2002), or may be involved in increased lignification (Robertsen and Svalheim, 1990). Accumulation of phenolics has also been described as a defensive or stress response in plants (Dixon and Paiva, 1995).

Further, the overexpression of genes such as GTPase, IRE homolog, serine/threonine protein kinase, RAS related protein Rab 11C and calmodulins (CAM) in transgenic plants is indicative of the involvement of ROS in plant signaling (Baxter-Burrell et al., 2002; Lamb and Dixon, 1997). Also, several genes encoding proteins associated with stress responses were also found to be upregulated. Meskiene et al. (2003) have proposed that the mitogen activated protein kinase (MAPK)-signal pathway is controlled by the redox-sensitive protein phosphatase 2C (PP2C), which is induced under oxidative conditions. A similar type of protein phosphatase 2C was highly expressed in the antisense plants. The upregulated proteins phospholipase A2 and a patatin-like protein have been shown to be rapidly induced during the hypersensitive response in tobacco (Dhondt et al., 2000).

The upregulation of the xylem-specific 4CL isoform may be helping to direct phenylpropanoids to the lignification pathway in the transgenic plants. An increased expression of COMT and CAD was observed in the apical tissue of antisense plants. The expression of basic peroxidase, which has been described as one of the enzymes responsible for cell wall lignification in Zinnia elegans (Barcelo and Pomar, 2001), was found to be enhanced in antisense plants. In Arabidopsis, most of the discussed genes were upregulated in bolting stems (Ehlting et al., 2005).

Reduced expression of hipI-SOD in the antisense lines leads to an increase in localized accumulation of Oinline image and H2O2. In the absence of SOD, Oinline image has been shown to be dismutated at a non-enzymatic dismutation rate close to 105 m−1 sec−1 (Torres et al., 2002), and may thus result in an enhanced production of H2O2 compared with when it is dismutated in a SOD-dependent manner (Liochev and Fridovich, 1994). The increased accumulation of ROS seems to induce both defense responses during pathogen infections of plants (Kangasjärvi et al., 2005) and signal transduction pathways important for developmental processes. In addition, the increased H2O2 in the transgenic plants may also participate directly in the peroxidase-mediated oxidation of monolignols to monolignol radicals in lignin polymerization (Ogawa et al., 1997). The localization of hipI-SOD in phloem fibers and xylem vessels also suggest a role for the enzyme as a regulator of ROS levels in these cells.

The dwarfed phenotype of the transgenic plants is apparently due to reductions in both cell division and expansion, probably caused by a premature transition into maturation, as indicated by the early onset of lignification. Whether this progression is caused by a change in the concentration of ROS acting as signaling molecules or as oxidation equivalents in the production of monolignol radicals, is not clear. More experimental data are needed to unambiguously define the function of hipI-SOD.

Experimental procedures

Database search and sequence analysis

To identify the number of ESTs and the most accurate gene model encoding hipI-SOD in hybrid aspen, amino acid sequences held in the NCBI database (accession numbers AJ278671 and AJ278670 for hipI-SOD1 and hipI-SOD2, respectively; Schinkel et al., 2001) were used for Blast (Basic Local Alignment Search Tool; Altschul et al., 1997) searches against the Populus EST database (PopulusDB; http://www.populus.db.umu.se/) and the P. trichocarpa genome database (http://genome.jgi-psf.org/Poptr1/Poptr1.home.html), respectively. Proteins with similarity to hipI-SOD were also identified by performing blast analysis (http://www.ncbi.nlm.nih.gov/BLAST/) of the hipI-SOD protein against a translated nucleotide database.

Amino acid sequences of all selected proteins were aligned using clustalw (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html) followed by phylogenetic analysis using the Paup* program (version 4.0b10; Norberg et al., 2005).

Accession numbers

GenBank accession numbers of the sequences analyzed for protein alignment (Figure 1a) and phylogenetic analysis (Figure 1b) were as follows: AtCSD1 (NP_172360), AtCSD2 (NP_565666), AtCSD3 (NP_197311), McSOD (O49044), OsSOD (P28757), PshipISOD (CAC34448), PtcytSOD (AAD01605), PttcpSOD (CAC33844), PttcytSOD (CAC33845), PtthipISOD1 (CAC33847), PtthipISOD2 (CAC33846) and ZmSOD (P11428).

Pttgene1 and Pttgene2 were annotated as estExt_Genewise1_v1.C_LG_XIII1983 and grail3.0065004601, respectively, in the P. trichocarpa genome database, whereas Ptt5824C1 was annotated as POPLAR.5824.C1 and Ptt5824C2 as POPLAR.5824.C2 in the Populus EST database.

Antisense vector construction and plant transformation

The antisense construct used in this study contained the entire hipI-SOD 2 gene (Schinkel et al., 2001), cloned as a SpeI fragment into the binary vector P 11/2 (provided by Dr B. Reiss, MPI, Köln, Germany). The bacterial XL1-blue MRF′ system was used to amplify constructs.

Hybrid aspen (Populus tremula L. × P. tremuloides Michx.) plants were transformed and regenerated as previously described (Nilsson et al., 1992). A total of 19 independent lines were recovered and multiplied by cuttings and rooted. Wild-type plants were multiplied in tissue culture in the same way and served as controls.

Plant growth parameters

Plants from the transgenic lines AS-SOD9 and AS-SOD24, and WT plants of the same age, were measured weekly starting 8 weeks after the plants had been transferred to the greenhouse. Each week, the height and basal diameter of the stem of each plant were measured, and the number of internodes and average internode length were recorded. When the plants had been in the greenhouse for 13 weeks, the maximal width and length of the blades of each of the 20 youngest leaves of plants from all three lines were measured.

Protein extraction and Western blot analysis

Three plants from each of the WT and both antisense lines were selected for protein extraction and Western blot analysis. Tissues were sampled from bark peeled off internodes 5–25 and by scraping off xylem with a sharp blade (Karpinska et al., 2004). Protein was extracted from 0.4 g of fresh tissue sampled from both bark and xylem tissues. The samples were homogenized in extraction buffer [50 mm 2-amino-2(hydroxymethyl)-1,3-propanediol (TRIS)-HCl, pH 8.0, 1 mm EDTA, 1 mm DTT], and centrifuged at 12 000 g for 15 min at 4°C. The supernatant was heated at 70°C for 10 min and centrifuged again at 12 000 g for 15 min at 4°C. The supernatant was purified using an anion exchange column (HiQ XL, GE Healthcare, Uppsala, Sweden) and proteins in the purified fractions were precipitated overnight at −20°C by four volumes of chilled acetone.

Protein preparations were mixed with loading buffer to a final concentration of 1% SDS (w/v) and 50 mm DTT then incubated at 95°C for 5 min prior to loading. Proteins in samples containing approximately 22 μg of protein were electrophoretically separated in a 14% SDS-polyacrylamide gel using a MiniProtean II electrophoresis assembly (Bio-Rad, Hercules, CA, USA) and electroblotted onto nitrocellulose membranes (Hybond®-ECL®, GE Healthcare). Western blot analysis was performed using a specific hipI-SOD antibody (Schinkel et al., 2001) as the primary antibody and horseradish peroxidase-linked anti-rabbit IgG as the secondary antibody (GE Healthcare). Fluorescence signals were generated by incubation with ECL plus Western blotting detection reagents (GE Healthcare). Pre-stained broad range SDS-PAGE standards (Bio-Rad) were used to estimate the size of the protein bands.

Fixation, substitution and embedding for light microscopy

For the primary fixation of samples, cross-sections of stem approximately 1 mm thick were incubated overnight at room temperature (RT, 22°C) in 50 mm cacodylate buffer, pH 7.2, containing 0.5% (v/v) glutaraldehyde and 2.0% (v/v) formaldehyde, followed by one wash with buffer and two washes with distilled water. Samples for immunolabeling studies were dehydrated stepwise by increasing the concentration of ethanol and concomitantly lowering the temperature (method of progressive lowering of temperature, PLT) using an automated freeze substitution unit (AFS, Leica, Benzheim, Germany). The steps used were as follows: 30% (v/v), 40% (v/v) and 50% (v/v) ethanol for 1 h each at 4°C; 60% (v/v) and 75% (v/v) ethanol for 1 h each at −15°C; 90% (v/v) ethanol and 2x 100% (v/v) ethanol for 1 h at −35°C. The samples were subsequently infiltrated with Lowycryl HM20 resin (Plano GmbH, Marburg, Germany) by incubating them in the following mixtures: 33% (v/v), 50% (v/v) and 66% (v/v) HM 20 resin in ethanol for 4 h each and then 100% (v/v) HM 20 overnight. Samples were transferred into gelatin capsules, incubated for 3 h in fresh resin and polymerized at 35°C for 3 days under indirect UV light.

Samples for ultrastructural and light microscopy analyses were transferred after primary fixation into a solution of 1% (w/v) OsO4. After 1 h, samples were washed three times with distilled water. Dehydration at 25°C was carried out by increasing the ethanol concentration in the following steps: 30% (v/v), 50% (v/v), 60% (v/v), 75% (v/v), 90% (v/v) and 2x 100% (v/v) ethanol for 1 h. After additional dehydration with propylene oxide for 1 h, the samples were infiltrated with Spurr's resin (Plano GmbH, Marburg, Germany) as follows: 33% (v/v), 50% (v/v) and 66% (v/v) Spurr's resin in propylene oxide for 4 h each and then 100% (v/v) Spurr's resin overnight. Samples were transferred into embedding molds, incubated for 3 h in fresh resin and polymerized at 70°C for 24 h.

Microscopy

Semi-thin sections, 1 μm thick, were cut with a Histo diamond knife, mounted on glass slides and used for fluorescence labeling. To avoid non-specific antibody binding, sections were blocked at RT for 20 min in PBS buffer containing 3% (w/v) bovine serum albumin and 0.1% Tween. Rabbit polyclonal antibody raised against hipI-SOD and Alexa 488 Fluor anti-rabbit IgG (Invitrogen GmbH, Karlsruhe, Germany) as the secondary antibody were used for immunolabeling. The system used to detect Alexa 488 was a Zeiss Axiovert 200 microscope (Carl Zeiss, Göttigen, Germany) attached to a confocal laser scanning system (Zeiss LSM 510 META). Fluorescence of Alexa 488 was induced by an excitation light at 488 nm produced by an Ar-laser. A 505–535 nm emission filter and emission fingerprinting were used to eliminate remaining autofluorescence signals from other cell components. Control labeling without using primary antibody and Alexa 488, respectively, gave no significant signals (data not shown). Additional images were obtained in transmission mode.

Light microscopical analyses of fresh and fixed stem material from WT and antisense plants were carried out with a CCD camera system (Zeiss AxioCam attached to a Zeiss Axiovert 135M microscope; Carl Zeiss).

Transverse sections of fresh poplar stem were used for histochemical analysis of lignin, H2O2 and superoxide radicals. A Leica Vibratome VT 1000S (Leica Microsystems, Bensheim, Germany) was used to prepare sections with an approximate thickness of 150 μm from different internodes (1, 2, 3,…). To detect the presence of lignin, sections were incubated for 30 min in 1% phloroglucinol in ethanol followed by the addition of 10% HCl (Ogawa et al., 1997). H2O2 was analyzed as described by Holm et al. (2003): stem sections were incubated in 2 mm DAB dissolved in 50 mm TRIS-HCl buffer pH 6.8. To detect superoxide radicals, sections were incubated for 1 h in 0.25 mm NBT in 10 mm sodium phosphate, pH 7.8 (Ogawa et al., 1997).

For histological analysis, semi-thin sections of fixed stem material, approximately 3 μm thick, were prepared using a Leica Ultramicrotome UCT (Leica Microsystems, Bensheim, Germany) and a Histo diamond knife. The sections were mounted on slides and stained for 2 min with 1% (w/v) methylene blue/1% (w/v) Azur II in 1% (w/v) aqueous borax at 60°C prior to light microscopical examination.

Lignin analysis

The lignin content of three individual plants from each of the WT and AS-SOD9 and AS-SOD24 lines were determined by a modified Klason lignin method (Kirk and Obst, 1988). Wood tissue was ball milled to a fine powder which was then subjected to successive extraction with water, ethanol, toluene:ethanol (1:1) and acetone. The resulting cell wall residue was used for lignin determination. Five milliliters of 76% H2SO4 was added to 1 g of cell wall residue and the mixture was incubated for 1 h at 30°C in a water bath. The digestion was terminated by adding 140 ml of water to each sample and placing the sample in an autoclave for 1 h at 120°C. Whilst maintaining the temperature of the solution the lignin was filtered through a pre-weighed glass fiber filter (Whatman GF/A, Maidstone, UK). The residue was thoroughly washed with hot water to completely remove the acid.

Fiber and vessel measurement

Four plants from the WT and each of the antisense lines were chosen for fiber and vessel measurement. Trimmed pieces of outer xylem from the mature part of each plant were prepared, and xylem pieces were macerated as described by Eriksson et al. (2000). After maceration, the fibers and vessels were separated from each other by shaking vigorously in water. The length and diameter of 100 fibers and 50 vessels per sample were measured using a microscope fitted with a camera (Axio vision3, Zeiss, Germany).

Analyses of phenolics

Young and old internodes (taken from the top and bottom of plants, respectively) from plants of lines AS-SOD9, AS-SOD24 and the WT were analyzed for phenolic glucosides (salicin, salicortin, tremulacin) and phenolic acids (p-coumaric acid, cinnamic acid, chlorogenic acid and benzoic acid).

All plant material for the phenolic analyses was air dried at ambient temperature, milled to an homogeneous powder and extracted as described by Witzell et al. (2003). Phenolic metabolites were analyzed using a Merck Hitachi LaChrom HPLC system (L-7100 pump, L-7200 autosampler, L-7360 column oven and L-7455 diode array detector; Darmstadt, Germany). The oven temperature was set to 30°C. The compounds were separated on a reversed-phase 100 × 4.6 mm internal diameter, 3 μm particle size C-18 column (Thermo Quest HyPURITY®, Waltham, MA, USA). The mobile phase consisted of the following complex gradient: 100% A for 0–1 min, declining linearly to 60% A at 6 min, then to 40% at 12 min and 0% A at 28 min, which was maintained until 30 min, before switching to 100% A until 33 min, followed by washing and equilibration to initial conditions where A is H2O (pH adjusted to 3.0 with H2PO4) and the balance was provided by methanol (4:6 v/v). The flow rate was 1 ml min−1 throughout and the injection volume was 15 μl. Individual phenolics were identified by comparing their retention times and UV spectra (200–400 nm) with those of standard compounds (Sigma, St Louis, MO, USA), and their total concentration was calculated. Salicylates (salicin, salicortin and tremulacin) were quantified as salicin (Sigma) equivalents.

Microarray experiments

Poly A+ RNA was prepared from the apical part (top 3 cm) of a primary shoot (with leaves attached) of plants of lines AS-SOD9, AS-SOD24 and the WT, using oligo-dT-coated magnetic beads (DYNAL, Oslo, Norway) according to the manufacturer's recommendations. Four hundred nanograms of mRNA from each sample was labeled using a CyScribe First-Strand cDNA labeling kit (Amersham Pharmacia Biotech, Uppsala, Sweden) and unincorporated nucleotides were removed using an AutoSeq G-50 column (Amersham Pharmacia Biotech). A hybrid aspen cDNA microarray (Hertzberg et al., 2001) was hybridized with the labeled targets using the competitive hybridization technique described in Schena et al. (1995). Hybridization, washing, scanning and data analysis were performed as described by Hertzberg et al. (2001). Annotation of the genes from the array presented in Table 1 was based on the best hit obtained using blastx to search the NCBI non-redundant protein database.

Quantitative real-time PCR

Genes encoding the following phenylpropanoid biosynthesis enzymes were chosen for quantitative real-time PCR analysis: phenylalanine ammonia lyase (PAL, EC 4.2.1.4), cinnamate 4-hydroxylase (C4H, EC1.14.13.11), hydroxycinnamate: CoA ligase (4CL, EC 6.2.1.12), caffeic acid/5-hydroxy ferulic acid O-methyl transferase (COMT, E.C. 2.1.1.68), caffeoyl-CoA O-methyltransferase 6 (CCoAOMT, EC 2.1.1.104) and hydroxycinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195). The primers were chosen from the following Populus tremuloides Michx. cDNA sequences present in the NCBI database: PtPAL2 (AF480620), PtC4H (U47293), Pt4CL1(AF041049), CAD (AF217957) and the predicted coding region of the COMT gene (PTOMT1 gene, U13171). The consensus sequence of the contig POPLAR.912.C2 found in PopulusDB was used to synthesize the primers for amplification of CCoAOMT cDNA. The nucleotide sequence was translated to amino acids and was identical with the sequence of CCoAOMT1 (AJ223621) found in poplar by Chen et al. (2000).

Three plants from each of the WT and AS-SOD9 and AS-SOD24 lines were chosen for quantitative real-time PCR of all the genes mentioned above. Total RNA was extracted from the apical part (top 3 cm) of primary shoots using an Aurum total RNA mini kit (Bio-Rad) according to the manufacturer's instructions. One microgram of RNA was used for cDNA synthesis using an iScript® cDNA Synthesis Kit (Bio-Rad). Quantification was performed as described by Norberg et al. (2005) with an iCycler MyiQ real-time PCR detection system (Bio-Rad) using the Bio-Rad iQ SYBR Green Supermix. Each sample was loaded in triplicate and the results were normalized to the expression of 18S ribosomal RNA.

Statistical analyses

All data from the analysis of phenolics were analyzed using anova (analysis of variance). Differences between samples were considered significant if the probability of the difference arising from random sampling errors was <0.05, expressed as *P < 0.05.

Acknowledgements

We thank Dr B Reiss for the kind donation of vector P11/2 and Kjell Olofsson and Eva Mellerowicz for their valuable suggestions. This research was funded by grants to SUAS from the Swedish Council for FORMAS, the Swedish Research Council (VR), the Swedish Foundation for Strategic Research (SSF) and by a grant from the Kempe foundation. This work was also supported in part by Professor Dr Isabella Prokhorenko by grant 436 RUS 17/87/03 and RUS 17/131/05 from the Deutsche Forschungsgemeinschaft (DFG).

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