Differential regulation of cytokinin oxidase genes and cytokinin-induced auxin biosynthesis by cellular cytokinin level in transgenic poplars.

KEY MESSAGE: The present work with transgenic poplar lines producing varying levels of trans -zeatin suggests the existence of a switching threshold for triggering ckx gene expression or suppressing cytokinin-induced auxin. Cytokinins have an important role in growth and developmental processes of plants. Transgenic plants with varying levels of cellular cytokinin are convenient tools for studying its role in morphogenetic as well as molecular responses. In this work, the transgenic lines producing either high level of cellular trans-zeatin (HX lines) or moderate level (MX lines) were compared with regard to their cytokinin oxidase activities and cellular auxin content. The HX lines showed typical cytokinin phenotypes including leafy shoots and spontaneous shoot formation on hormone free medium. In contrast, the MX lines did not show any striking phenotypes. However, in leaf disk culture on hormone free medium, they regenerated roots and subsequently formed shoots from the roots. Determination of cellular IAA content revealed a significant increase in the level in MX lines but not in HX lines. Of nine cytokinin oxidase genes (ckx) examined by qPCR, five were activated in HX lines but not in MX lines. Among them, ckx4 appeared to play a key role in maintaining cellular cytokinin level since it showed more than 1,000-fold increase in HX lines and in the leaf disks of untransformed control exposed to exogenous cytokinins. Although low level of cellular cytokinin did not induce the expression of ckx genes, it appeared to trigger cellular IAA biosynthesis.

Evaluation of a transgenic poplar as a potential biomass crop for biofuel production.

A transgenic poplar, in which the RabG3bCA gene from Arabidopsis was overexpressed, was analyzed for its biomass composition and enzymatic digestibility after chemical pretreatment. In comparison with a wild-type poplar (WT), the transgenic poplar (OX8) showed 9.8% higher glucan content. The levels of other biomass components did not differ greatly between WT and OX8. When WT and OX8 samples were pretreated by sulfuric acid (1%, w/v at 190 °C), sodium hydroxide (1%, w/v at 190 °C), or ammonia (14%, w/w at 80 °C), the washed pretreated solids of OX8 exhibited a higher enzymatic digestibility than those of WT in each chemical pretreatment. The sodium hydroxide pretreatment was the most effective among the three pretreatment processes, showing 58.7% and 69.4% of theoretical glucose yield from the saccharification of pretreated OX8 and WT, respectively. The transgenic poplar, growing faster and taller, was found to contain more glucan and have a higher enzymatic digestibility than WT.

Transgenic poplar trees expressing yeast cadmium factor 1 exhibit the characteristics necessary for the phytoremediation of mine tailing soil.

Genetic engineering of plants for phytoremediation is thought to be possible based on results using model plants expressing genes involved in heavy metal resistance, which improve the plant's tolerance of heavy metals and accumulation capacity. The next step of progress in this technology requires the genetic engineering of plants that produce large amounts of biomass and the testing of these transgenic plants in contaminated soils. Thus, we transformed a sterile line of poplar Populus alba X P. tremula var. glandulosa with a heavy metal resistance gene, ScYCF1 (yeast cadmium factor 1), which encodes a transporter that sequesters toxic metal(loid)s into the vacuoles of budding yeast, and tested these transgenic plants in soil taken from a closed mine site contaminated with multiple toxic metal(loid)s under greenhouse and field conditions. The YCF1-expressing transgenic poplar plants exhibited enhanced growth, reduced toxicity symptoms, and increased Cd content in the aerial tissue compared to the non-transgenic plants. Furthermore, the plants accumulated increased amounts of Cd, Zn, and Pb in the root, because they could establish an extensive root system in mine tailing soil. These results suggest that the generation of YCF1-expressing transgenic poplar represents the first step towards producing plants for phytoremediation. The YCF1-expressing poplar may be useful for phytostabilization and phytoattenuation, especially in highly contaminated regions, where wild-type plants cannot survive.

Microarray and suppression subtractive hybridization analyses of gene expression in hybrid poplar (Populus alba × Populus tremula var. glandulosa) cell suspension cultures after exposure to NaCl.

The gene expression profiles of hybrid poplar (Populus alba × Populus tremula var. glandulosa) cells in suspension culture after exposure to salinity (NaCl) induced stress were examined by constructing two suppression subtractive hybridization (SSH) libraries. cDNA from non-treated cells was used as a driver and cDNA samples from cell suspension cultures exposed to 150 mM NaCl for 2 or 10 h were used as testers. Randomly selected clones from each SSH library were sequenced and 727 high-quality expressed sequence tags (ESTs) were obtained and analyzed. Four novel ESTs were identified. Between the two libraries, 542 unique SSH clones were selected for placement on a cDNA microarray. In total, 18 differentially expressed genes were identified with 4 and 12 genes being significantly differentially expressed 2 and 10 h after the treatment, respectively. Genes related to metabolism and protein synthesis and several genes whose protein products are implicated in salt or other abiotic stress-related responses were expressed in the salt-stressed cells.

Overexpression of constitutively active Arabidopsis RabG3b promotes xylem development in transgenic poplars.

An Arabidopsis small GTPase, RabG3b, was previously characterized as a component of autophagy and as a positive regulator for xylem development in Arabidopsis. In this work, we assessed whether RabG3b modulates xylem-associated traits in poplar in a similar way as in Arabidopsis. We generated transgenic poplars (Populus alba × Populus tremula var. glandulosa) overexpressing a constitutively active form of RabG3b (RabG3bCA) and performed a range of morphological, histochemical and molecular analyses to examine xylogenesis. RabG3bCA transgenic poplars showed increased stem growth due to enhanced xylem development. Autophagic structures were observed in differentiating xyelm cells undergoing programmed cell death (PCD) in wild-type poplar, and were more abundant in RabG3bCA transgenic poplar plants and cultured cells. Xylogenic activation was also accompanied by the expression of secondary wall-, PCD- and autophagy-related genes. Collectively, our results suggest that Arabidopsis RabG3b functions to regulate xylem growth through the activation of autophagy during wood formation in Populus, as does the same in Arabidopsis.

Drought, salt and wounding stress induce the expression of the plasma membrane intrinsic protein 1 gene in poplar (Populus alba×P. tremula var. glandulosa).

Water uptake across cell membranes is a principal requirement for plant growth at both the cellular and whole-plant levels; water movement through plant membranes is regulated by aquaporins (AQPs) or major intrinsic proteins (MIPs). We examined the expression characteristics of the poplar plasma membrane intrinsic protein 1 gene (PatPIP1), a type of MIP, which was isolated from a suspension cell cDNA library of Populus alba×P. tremula var. glandulosa. Examination of protoplasts expressing the p35S-PatPIP1::sGFP fusion protein revealed that the protein was localized in the plasma membrane. Northern blot analysis revealed that the gene was strongly expressed in poplar roots and leaves. Gene expression was inducible by abiotic factors including drought, salinity, cold temperatures and wounding, and also by plant hormones including gibberellic acid, jasmonic acid and salicylic acid. Since we found that the PatPIP1 gene was strongly expressed in response to mannitol, NaCl, jasmonic acid and wounding, we propose that PatPIP1 plays an essential role in the defense of plants against water stress.

EST analysis of functional genes associated with cell wall biosynthesis and modification in the secondary xylem of the yellow poplar (Liriodendron tulipifera) stem during early stage of tension wood formation.

A cDNA library was constructed from secondary xylem in the stem of a 2-year-old yellow poplar after being bent for 6 h with a 45° configuration to isolate genes related to cell wall modification during the early stages of tension wood formation. A total of 6,141 ESTs were sequenced to generate a database of 5,982 high-quality expressed sequence tags (ESTs). These sequences were clustered into 1,733 unigenes, including 822 contigs and 911 singletons. Homologs of the genes regulate many aspects of secondary xylem development, including those for primary and secondary metabolism, plant growth hormones, transcription factors, cell wall biosynthesis and modification, and stress responses. Although there were only 1,733 annotated ESTs (28.9%), the annotated ESTs obtained in this study provided sequences for a broad array of transcripts expressed in the stem upon mechanical bending, and the majority of them were the first representatives of their respective gene families in Liriodendron tulipifera. In the case of lignin, xylem-specific COMTs were identified and their expressions were significantly downregulated in the tension wood-forming tissues. Additionally, the majority of the auxin- and BR-related genes were downregulated significantly in response to mechanical bending treatment. Despite the small number of ESTs sequenced in this study, many genes that are relevant to cell wall biosynthesis and modification have been isolated. Expression analysis of selected genes allow us to identify the regulatory genes that may perform essential functions during the early stages of tension wood formation and associated cell wall modification.

Transgenic poplar expressing Arabidopsis NDPK2 enhances growth as well as oxidative stress tolerance.

Nucleoside diphosphate kinase 2 (NDPK2) is known to regulate the expression of antioxidant genes in plants. Previously, we reported that overexpression of Arabidopsis NDPK2 (AtNDPK2) under the control of an oxidative stress-inducible SWPA2 promoter in transgenic potato and sweetpotato plants enhanced tolerance to various abiotic stresses. In this study, transgenic poplar (Populus alba × Poplus glandulosa) expressing the AtNDPK2 gene under the control of a SWPA2 promoter (referred to as SN) was generated to develop plants with enhanced tolerance to oxidative stress. The level of AtNDPK2 expression and NDPK activity in SN plants following methyl viologen (MV) treatment was positively correlated with the plant's tolerance to MV-mediated oxidative stress. We also observed that antioxidant enzyme activities such as ascorbate peroxidase, catalase and peroxidase were increased in MV-treated leaf discs of SN plants. The growth of SN plants was substantially increased under field conditions including increased branch number and stem diameter. SN plants exhibited higher transcript levels of the auxin-response genes IAA2 and IAA5. These results suggest that enhanced AtNDPK2 expression affects oxidative stress tolerance leading to improved plant growth in transgenic poplar.

Arabidopsis PCR2 is a zinc exporter involved in both zinc extrusion and long-distance zinc transport.

Plants strictly regulate the uptake and distribution of Zn, which is essential for plant growth and development. Here, we show that Arabidopsis thaliana PCR2 is essential for Zn redistribution and Zn detoxification. The pcr2 loss-of-function mutant was compromised in growth, both in Zn-excessive and -deficient conditions. The roots of pcr2 accumulated more Zn than did control plants, whereas the roots of plants overexpressing PCR2 contained less Zn, indicating that PCR2 removes Zn from the roots. Consistent with a role for PCR2 as a Zn-efflux transporter, PCR2 reduced the intracellular concentration of Zn when expressed in yeast cells. PCR2 is located mainly in epidermal cells and in the xylem of young roots, while it is expressed in epidermal cells in fully developed roots. Zn accumulated in the epidermis of the roots of pcr2 grown under Zn-limiting conditions, whereas it was found in the stele of wild-type roots. The transport pathway mediated by PCR2 does not seem to overlap with that mediated by the described Zn translocators (HMA2 and HMA4) since the growth of pcr2 hma4 double and pcr2 hma2 hma4 triple loss-of-function mutants was more severely inhibited than the individual single knockout mutants, both under conditions of excess or deficient Zn. We propose that PCR2 functions as a Zn transporter essential for maintaining an optimal Zn level in Arabidopsis.

Isolation and characterization of osmotic stress-induced genes in poplar cells by suppression subtractive hybridization and cDNA microarray analysis.

Osmotic stress induces changes in the expression of various genes including those associated with drought tolerance, cell wall metabolism and defense. We isolated 852 cDNA clones, the expression of which is induced by osmotic stress, from cells of a hybrid poplar (Populus alba x Populus tremula var. glandulosa) by suppression subtractive hybridization after mannitol treatment. We examined how stress affected their expression using cDNA microarray analysis, which identified 104 genes significantly up-regulated by osmotic stress. These include genes with functions related to transcription, signal transduction, cell wall metabolism and defense. Other gene transcripts encoding cysteine protease and aquaporin are also up-regulated during osmotic stress. The function of about one-third of the genes in poplar cells that were significantly up-regulated by stress is not known, suggesting that the cell suspension may offer an opportunity of finding novel genes otherwise never expressed and that we still need more information at the molecular level.

Differential expression of a poplar SK2-type dehydrin gene in response to various stresses.

Dehydrins are group II, late embryogenesis abundant proteins that act putatively as chaperones in stressed plants. To elucidate the function of dehydrins in poplar, we isolated the SK(2)-type dehydrin gene Podhn from Populus alba x P. tremula var. glandulosa suspension cells and analyzed its expression following treatments of abiotic stress, wounding and plant growth regulator. Sequence homology and phylogenetic analyses indicate Podhn encodes an acidic dehydrin (pI 5.14, 277 amino acids, predicted size 25.6 kDa) containing two lysine-rich "K-segments" and a 7-serine residue "S-segment", both characteristic of SK(2)-type dehydrins. Southern blots show Podhn genes form a small gene family in poplar. Podhn was expressed in all tissues examined under unstressed conditions, but most strongly in cell suspensions (especially in the stationary phase). Drought, salt, cold and exogenous abscisic acid (ABA) treatments enhanced Podhn expression, while wounding and jasmonic acid caused its reduction. Therefore, Podhn might be involved in ABA or stress response.

Identification of genes upregulated by pinewood nematode inoculation in Japanese red pine.

Pine wilt disease caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, has destroyed huge areas of pine forest in East Asia, including Japan, China and Korea. No protection against PWN has been developed, and the responses of pine trees at the molecular level are unrecorded. We isolated and analyzed upregulated or newly induced genes from PWN-inoculated Japanese red pine (Pinus densiflora Sieb. et Zucc.) by using an annealing control primer system and suppression subtractive hybridization. Significant changes occurred in the transcript abundance of genes with functions related to defense, secondary metabolism and transcription, as the disease progressed. Other gene transcripts encoding pathogenesis-related proteins, pinosylvin synthases and metallothioneins were also more abundant in PWN-inoculated trees than in non-inoculated trees. Our report provides fundamental information on the molecular mechanisms controlling the biochemical and physiological responses of Japanese red pine trees to PWN invasion.

Growth-phase-dependent gene expression profiling of poplar (Populus alba x Populus tremula var. glandulosa) suspension cells.

Complex sequences of morphological and biochemical changes occur during the developmental course of a batch plant cell culture. However, little information is available about the changes in gene expression that could explain these changes, because of the difficulties involved in isolating specific cellular events or developmental phases in the overlapping phases of cell growth. In an attempt to obtain such information we have examined the global growth phase-dependent gene expression of poplar cells in suspension cultures by cDNA microarray analysis. Our results reveal that significant changes occur in the expression of genes with functions related to protein synthesis, cell cycling, hormonal responses and cell wall biosynthesis, as cultures progress from initiation to senescence, that are highly correlated with observed developmental and physiological changes in the cells. Genes encoding protein kinases, calmodulin and proteins involved in both ascorbate metabolism and water-limited stress responses also showed strong stage-specific expression patterns. Our report provides fundamental information on molecular mechanisms that control cellular changes throughout the developmental course of poplar cell cultures.

Molecular cloning of a peroxidase gene from poplar and its expression in response to stress.

To elucidate the precise functions of peroxidase in poplar (Populus alba x P. tremula var. glandulosa), we cloned a peroxidase gene (PoPOD1) from poplar suspension culture cells and examined its expression pattern in response to various stresses. PoPOD1 showed the highest homology with a bacterial-induced peroxidase gene from cotton (Gossypium hirsutum L.). The PoPOD1 gene encodes a putative 316 amino acid protein with an N-terminal signal peptide of 23 residues. The DNA blot analysis indicated that PoPOD1 is a single copy gene in the poplar genome. The RNA blot analyses indicated that PoPOD1 shows cell-culture-specific expression. Expression of PoPOD1 is down-regulated by various treatments including treatment with some metals, NaCl, methyl viologen and polyethylene glycol, and by the plant growth regulators, jasmonic acid (JA) and gibberellic acid (GA(3)). The gene is significantly up-regulated by the bacterial-elicitor laminarin and by wounding. Thus, PoPOD1 gene expression is sensitively and specifically regulated at the transcription level. Because both JA and GA3 appear to be involved in the regulation of PoPOD1 expression in poplar cells, we postulate that the peroxidase encoded by PoPOD1 plays a pivotal role in defense against pathogen invasion, possibly through the formation of a cell wall barrier over the wound.

AtATM3 is involved in heavy metal resistance in Arabidopsis.

AtATM3, an ATP-binding cassette transporter of Arabidopsis (Arabidopsis thaliana), is a mitochondrial protein involved in the biogenesis of iron-sulfur clusters and iron homeostasis in plants. Our gene expression analysis showed that AtATM3 is up-regulated in roots of plants treated with cadmium [Cd(II)] or lead (II); hence, we investigated whether this gene is involved in heavy metal tolerance. We found that AtATM3-overexpressing plants were enhanced in resistance to Cd, whereas atatm3 mutant plants were more sensitive to Cd than their wild-type controls. Moreover, atatm3 mutant plants expressing 35S promoter-driven AtATM3 were more resistant to Cd than wild-type plants. Since previous reports often showed that the cytosolic glutathione level is positively correlated with heavy metal resistance, we measured nonprotein thiols (NPSH) in these mutant plants. Surprisingly, we found that atatm3 contained more NPSH than the wild type under normal conditions. AtATM3-overexpressing plants did not differ under normal conditions, but contained less NPSH than wild-type plants when exposed to Cd(II). These results suggest a role for AtATM3 in regulating cellular NPSH level, a hypothesis that was further supported by our gene expression study. Genetic or pharmacological inhibition of glutathione biosynthesis led to the elevated expression of AtATM3, whereas expression of the glutathione synthase gene GSH1 was increased under Cd(II) stress and in the atatm3 mutant. Because the closest homolog of AtATM3 in fission yeast (Schizosaccharomyces pombe), HMT1, is a vacuolar membrane-localized phytochelatin-Cd transporter, it is tempting to speculate that glutathione-Cd(II) complexes formed in the mitochondria are exported by AtATM3. In conclusion, our data show that AtATM3 contributes to Cd resistance and suggest that it may mediate transport of glutamine synthetase-conjugated Cd(II) across the mitochondrial membrane.

AtPDR12 contributes to lead resistance in Arabidopsis.

Arabidopsis (Arabidopsis thaliana) contains about 130 ATP-binding cassette (ABC) proteins, which are likely to contribute to the transport of diverse materials, including toxic substances. However, the substrates of ABC transporters remain unknown in most cases. We tested which ABC transporter is involved in detoxification of lead [Pb(II)]. Among the many tested, we found that the message level of only AtPDR12 increased in both shoots and roots of Pb(II)-treated Arabidopsis, suggesting that it may be involved in the detoxification of Pb(II). AtPDR12-knockout plants (atpdr12) were used to further test this possibility. In Pb(II)-containing medium, atpdr12 plants grew less well and had higher Pb contents than those of wild-type plants. In contrast, AtPDR12-overexpressing Arabidopsis plants were more resistant to Pb(II) and had lower Pb contents than wild-type plants. The mutant phenotypes and their Pb contents, as well as the localization of the GFP:AtPDR12 fusion protein at the plasma membrane, suggest that AtPDR12 functions as a pump to exclude Pb(II) and/or Pb(II)-containing toxic compounds from the cytoplasm. Inhibition of glutathione synthesis by addition of buthionine sulfoximine to the growth medium exacerbated the Pb(II)-sensitive phenotype of atpdr12 plants, consistent with a glutathione-dependent detoxification mechanism operating in parallel with an AtPDR12-dependent mechanism. Thus, we propose that AtPDR12 is an ABC transporter that contributes to Pb(II) resistance in Arabidopsis.

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

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