Fluorescence microscopy: a powerful technique to detect low GUS activity in vascular tissues

We have previously shown that the Eucalyptus gunnii EgCAD2 promoter was preferentially expressed in vascular tissues in different transgenic plants (poplar, tobacco, Arabidopsis and grapevine). In order to delineate the cis elements governing this vascular expression pattern, promoter deletion analysis was performed allowing us to identify the proximal region [-340/-124] as essential for vascular cambium/xylem-specific expression. In plants transformed with the smallest promoter region [-124/+117], the GUS activity was difficult to detect using conventional bright field microscopy. To overcome this problem, we used fluorescence microscopy, enabling us to show that the [-124/+117] region contained cis-elements driving activity in phloem fibres but not in secondary xylem. The technical improvement of the histochemical detection of GUS activity using fluorescence microscopy enables accurate investigation of low GUS activity in phenol-rich tissues.     

Wound-induced and developmental activation of a poplar tree chitinase gene promoter in transgenic tobacco

Wounding hybrid poplar (Populus trichocarpa × P. deltoides) trees results in the expression of novel wound-inducible (win) mRNAs thought to encode proteins involved in defense against pests and pathogens. Members of thewin6 gene family encode acidic multi-domain chitinases, with combined structure and charge characteristics that differ from previously described chitinases.Win6 expression has been shown to occur in pooled unwounded leaves of a wounded (on multiple leaves) poplar plant. Here we demonstrate that wounding a single leaf induceswin6 expression locally, in the wounded leaf, and remotely, in specific unwounded leaves with strong vascular connections to the wounded leaf. We also demonstrate that awin6 promoter--glucuronidase (GUS) gene fusion (win6-GUS) responds to wounding locally and remotely in transgenic tobacco. These data indicate that the poplarwin6 promoter has regulatory elements that are responsive to wound signals in the heterologous host. In addition,win6-GUS is developmentally activated in unwounded young leaves and floral tissues of transgenic tobacco. Similar developmental expression patterns are found to occur forwin6 in poplar trees, demonstrating that a herbaceous plant can serve as a host for woody tree transgene analysis and can accurately predict expression patterns in tree tissues (e.g. flowers) that would be difficult to study in free-living trees.     

Anaerobic induction of the maize<Emphasis Type="Italic"> GapC4</Emphasis> promoter in poplar leaves requires light and high CO<Subscript>2</Subscript>

The maize (Zea mays L.) glyceraldehyde-3-phosphate dehydrogenase gene 4 (GapC4) promoter confers anaerobic gene expression in tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.) and Arabidopsis thaliana (L.) Heynh. Here we have investigated its expression in hybrid poplar (Populus tremula × P. alba). Our results show that the promoter is not expressed in leaves and stems under normoxic conditions while anaerobiosis induces reporter gene expression in leaves up to a level observed for the STLS-1 promoter from potato that is shown to confer leaf-specific gene expression in transgenic poplar. Anaerobic induction is cell autonomous and requires a CO₂ atmosphere and light. As in tobacco, the GapC4 promoter in poplar is wound inducible. The induction by CO₂ and light may reflect a natural situation because flooding, a natural cause of anaerobiosis, is often accompanied by high CO₂ concentrations in the floodwater. Our results show that the GapC4 promoter is suitable as an anaerobic reporter and as an inducible gene expression system in poplar.Abbreviations CaMV cauliflower mosaic virus - GapC4 glyceraldehyde-3-phosphate dehydrogenase gene 4 - GUS -glucuronidase - 4-MU methylumbelliferone - STLS-1 stem- and leaf-specific promoter 1     

The Polyadenylation of RNA in Plants

Stem sections from poplar that were stably transformed with a eucalypt cinnamyl alcohol dehydrogenase promoter-[beta]-glucuronidase construct were prepared by using either a technique routinely used in herbaceous species or a technique designed to take into account the particular anatomy of woody plants. Although both preparation techniques confirmed the pattern of expression previously observed (C. Feuillet, V. Lauvergeat, C. Deswarte, G. Pilate, A. Boudet and J. Grima-Pettenati [1995] Plant Mol Biol 27: 651-657), the latter technique also allowed the detection of other sites of promoter activity not revealed by the first technique. In situ hybridization confirmed the expression pattern obtained with the second sample preparation technique.     

History and progress of the genomics studies in the model system of perennial plant species

Strong justifications have been developed for why woody plants should be viewed as mod-el systems in plant biology. The genus Populus possesses many characteristics that are conductive to functional genomic studies,and therefore leads to its emergence as a model system in extrapolating findings in perennial plant species that are different from annual herbaceous plants. With the pro-ceeding of the whole genome sequencing,poplars will be act as a wide reference for functional ge-nomics studies in perennial plant species and will also contribute towards answering some fundamen-tal scientific questions. This paper reviewed the history and progress of the poplar genome studies and the potential keen topics in the future. The contents mainly address on: （1）the somatic genet-ics studies in Populus;（2）the genomics studies carried out in Populus,including genetic map-ping,genome sequ encing,physical map construction,microarray analysis and linkage disequilibri-um analysis;（3）the potential application of the genome information of Populus for facilitating our understanding of some basic scientific questions.     

多年生植物模式物种基因组研究的历史及进展

木本植物有许多不同于一年生草本植物的生物学特性,生物学家提出将木本植物 作为研究多年生植物的模式体系。杨属Populus树种由于研究基础较好且基因组较小,目前已 被广泛地接受作为多年生植物基因组研究的模式物种。随着杨属树种全基因组序列的测定, 杨属树种在多年生植物的功能基因组研究及一些基础科学问题的研究中将发挥重要作用。本 文综述了杨属树种基因组研究的历史、进展及将来的研究热点,旨在为我国多年生植物基因 组研究提供参考和借鉴。本文主要论述了以下几个方面的内容:（1）对杨属树种开展的细胞 遗传学研究;（2）在分子水平上对杨属树种进行的基因组研究,内容包括遗传作图、基因组 测序、物理图谱构建、基因芯片及连锁不平衡分析;（3）杨属树种基因组信息在探讨一些基 础科学问题中的潜在应用。     

Transgenic analysis of a hybrid poplar wound-inducible promoter reveals developmental patterns of expression similar to that of storage protein genes.

The wound-inducible win3 multigene family from hybrid poplars (Populus trichocarpa x Populus deltoides) encodes proteins with structural similarities with Kunitz-type protease inhibitors (H.D. Bradshaw Jr., J.B. Hollick, T.J. Parsons, H.R.G. Clarke, M.P. Gordon [1990] Plant Mol Biol 14: 51-59), and at least one member, win3.12, is transcribed de novo in the injured and uninjured leaves of wounded trees (J.B. Hollick, M.P. Gordon [1993] Plant Mol Biol 22: 561-572). A previous study demonstrated that 1352 bp of 5' flanking DNA from the win3.12 gene confers local wound-regulated expression of the beta-glucuronidase gene in transgenic tobacco (Nicotiana tabacum cv Xanthi n.c.) (J.B. Hollick, M.P. Gordon [1993] Plant Mol Biol 22: 561-572). We extend this transgenic analysis here by examining the developmental regulation and systemic wound induction conferred by the same transgene construct in tobacco. Biochemical and histochemical surveys of beta-glucuronidase activity are described for four, independent transgenic lines. The observed spatial and temporal expression patterns coincide with dormant storage tissues and with previously described expression patterns for both seed and vegetative storage protein genes. Developmental northern blot analysis of win3 RNA levels in poplar seeds confirms that proper temporal expression of the reporter gene is maintained during tobacco seed maturation. These results demonstrate that a putative Kunitz-type protease inhibitor can be wound inducible in addition to being expressed in developing seeds.     

Biochemical characterization of two differentially expressed polyphenol oxidases from hybrid poplar

Two polyphenol oxidase isoforms with distinct expression patterns were identified in hybrid poplar (Populus trichocarpaxP. deltoides). PPO-1, corresponding to the previously cloned PtdPPO (Constabel et al., Plant Physiol. 124: 285-295) was primarily leaf tissue-specific and detected only after wounding. PPO-2 was expressed constitutively in all tissue types tested except mature leaves, with highest expression in very young leaves and conducting tissues such as roots, stems and petioles. These two PPO isoforms were partially purified from hybrid poplar by ammonium sulfate fractionation followed by hydrophobic interaction chromatography. They were found to differ in stability, pH optimum, and activation by SDS. Tests with common phenolic substrates showed that PPO-1 had a broader substrate specificity than PPO-2. The distinct enzymatic properties and expression patterns of these two PPO isoforms suggest that they may have different physiological functions in hybrid poplar.     

Seasonal patterns of dehydrins and 70-kDa heat-shock proteins in bark tissues of eight species of woody plants

Although considerable effort has been directed at identifying and understanding the function and regulation of stress-induced proteins in herbaceous plants, reports concerning woody plants are limited. Studies with herbaceous crops have revealed similarities in the types of proteins that accumulate in response to a wide array of abiotic stresses and hormonal cues such as the accumulation of abscisic acid. Many of the identified proteins appear to be related to dehydrins (the D-11 subgroup of late-embryogenesis-abundant proteins). The objective of the present study was to determine if seasonal induction of dehydrins is a common feature in woody plants and to see if seasonal patterns existed for other stress-induced proteins. Bark tissues from eight species of woody plants were collected monthly for a period of 1.5 years. The species included: peach (Prunus persica) cv. Loring; apple (Malus domestica) cv. Golden Delicious; thornless blackberry (Rubus sp.) cv. Chester; hybrid poplar (Populus nigra); weeping willow (Salix babylonica); flowering dogwood (Cornus florida); sassafras (Sassafras albidum); and black locust (Robinia pseudo-acacia). Immunoblots of bark proteins were probed with a polyclonal antibody recognizing a conserved region of dehydrin proteins, and monoclonal antibodies directed against members of the HS70 family of heat-shock proteins. Some proteins, immunologically related to dehydrins, appeared to be constitutive; however, distinct seasonal patterns associated with winter acclimation were also observed in all species. The molecular masses of these proteins varied widely, although similarities were observed in related species (willow and poplar). Identification of proteins using the monoclonal antibodies (HSP70, HSC70, BiP) was more definitive because of their inherent specificity, but seasonal patterns were more variable among the eight species examined. This study represents only a precursory examination of several proteins reported to be stress related in herbaceous plants, but the results indicate that these proteins are also common to woody plants and that further research to characterize their regulation and function in relation to stress adaptation and the perennial life cycle of woody plants is warranted.     

Rapid alkalinization factors in poplar cell cultures. Peptide isolation,cDNA cloning,and differential expression in leaves and methyl jasmonate-treated cells

A family of peptides inducing rapid pH alkalinization in hybrid poplar (Populus trichocarpa x Populus deltoides) cell culture medium was isolated from hybrid poplar leaves. Five related approximately 5-kD peptides were purified by high-performance liquid chromatography and analyzed by matrix-assisted laser desorption ionization-mass spectrometry. The N-terminal sequence of one of the isolated peptides was very similar to a previously characterized peptide from tobacco (Nicotiana tabacum), rapid alkalinization factor (RALF), which causes a rapid increase in culture medium pH when added to tobacco cell cultures (G. Pearce, D.S. Moura, J. Stratmann, C.A. Ryan [2001] Proc Natl Acad Sci USA 98: 12843-12847). Two unique poplar RALF cDNAs (PtdRALF1 and PtdRALF2) were isolated from a poplar cDNA library and used to study RALF expression in poplar saplings and cultured poplar cells. Both genes were found to be expressed constitutively in poplar saplings and cultured cells. However, PtdRALF2 was expressed in leaves at very low levels, and its expression in suspension culture cells was transiently suppressed by methyl jasmonate (MeJa). Although the function of these novel peptides remains enigmatic, our experiments suggest their role may be developmental rather than stress related. Overall, our study confirms the presence of active RALF peptides in other plants, and provides new data on the complexity of the RALF gene family in poplar.     

Chitinase accumulates systemically in wounded poplar trees

Young leaves of poplar ( Populus spp.) trees accumulate novel messenger RNAs shortly after the mature leaves have been mechanically wounded. These systemically wound‐induced ( win ) mRNAs are thought to encode proteins involved in plant defense. In the present paper, transgenic tobacco plants that ectopically expressed a win6 cDNA contained a novel chitinase activity that was not present in normal tobacco. This demonstrated Win6 was a chitinase. Win6 and a related protein Win8 accumulated in wounded poplars. Win6 and Win8 had low isoelectric points (ca 4) as predicted from their nucleotide sequence. The wound‐inducible increase in Win6 and Win8 was correlated with an increase in chitinase (EC 3.2.1.14) activity in poplar leaf extracts. We conclude that mechanical wounding induces chitinase in poplar trees, and speculate that the induced chitinase activity could act to increase the tolerance of poplars to opportunistic wound pathogens.