Apparent responses of stomata to transpiration and humidity in a hybrid poplar canopy

It has been proposed that the stomatal response to humidity relies on sensing of the transpiration rate itself rather than relative humidity or the saturation deficit per se. We used independent measurements of stomatal conductance (g_s), transpiration (E), and leaf-to-air vapour pressure difference (V) in a hybrid poplar canopy to evaluate relationships between g_s and E and between g_s and V. Relationships between E, V and total vapour phase conductance or crown conductance (g_c) were also assessed. Conductance measurements were made on exposed and partially shaded branches over a wide range of incident solar radiation. In exposed branches, g_s appeared to decline linearly with increasing E and increasing V at both high and low irradiance. However, in a partially shaded branch, a bimodal relationship between g_s and E was observed in which g_s continued to decrease after E had reached a maximum value and begun to decrease. The relationship between g_s and V for this branch was linear. Plots of g_c against E always yielded bimodal or somewhat variable relationships, whereas plots of g_c against V were invariably linear. It was not possible to derive a unique relationship between conductance and E or V because prevailing radiation partially determined the operating range for conductance. Normalization of data by radiation served to linearize responses observed within the same day or type of day, but even after normalization, data collected on partly cloudy days could not be used to predict stomatal behaviour on clear days and vice versa. An additional unidentified factor was thus also involved in determining operating ranges of conductance on days with different overall radiation regimes. We suggest that the simplest mechanism to account for the observed humidity responses is stomatal sensing of the epidermal or cuticular transpiration rate rather than the bulk leaf or stomatal transpiration rate.     

Effects of low level O3 exposure on leaf diffusive conductance and water-use efficiency in hybrid poplar

Abstract Young, amphistomatous hybrid poplar (Populus deltoides x trichocarpa) plants were exposed daily to either background (0.025 cm³ m^-3) or elevated (0.125 cm³ m^-3) concentrations of O₃. Levels of abaxial and adaxial leaf conductance were affected interactively by pollutant treatment, leaf age, and photon fluence rate. Consequently, conductance in O₃-treated leaves was sometimes higher and sometimes lower than in comparable control leaves, depending on leaf age or level of photon fluence rate. For example, at low photon fluence rate or in the dark, conductance was greater in O₃-treated than in control plants, while at high photon fluence rate that relationship was reversed. Exposure to O₃ also reduced the water-use efficiency and range of leaf conductance of individual leaves, and altered the relationship between the conductances of the two leaf surfaces (the ratio of abaxial to adaxial leaf conductance was increased). Furthermore, O₃ treatment resulted in diminished stomatal control of water loss; excised O₃-treated leaves had higher conductances and wilted sooner than excised control leaves of identical ages. Overall, the data indicate that exposure to O₃ resulted in impaired stomatal function.     

Vectors for RNAi technology in poplar

Abstract: The potential of double-stranded RNA interference (RNAi) technology was studied for down-regulation of gene expression in poplar. A set of vectors was constructed generating RNAs capable of duplex formation of sequences specific for the β-glucuronidase (GUS) reporter gene system. These gene cassettes are driven by the CaMV-35S promoter. To address the question of gene silencing, we tested the functionality of these vectors, both in transient assays by transforming protoplasts with the RNAi constructs, and in stably transformed GUSexpressing poplar plants. Agrobacterium -mediated transformation of those GUS-expressing plants with a GUS-specific RNAi construct showed a strong down-regulation of the reporter gene. From these results we conclude that RNAi is also functional in poplar.     

Systemic induction of a potato pin2 promoter by wounding,methyl iasmonate,and abscisic acid in transgenic rice plants

To address the question whether common signal(s) and transduction pathways are used to mediate a systemic wound response in monocot and dicot plants, a fusion of the potato proteinase inhibitor II gene (pin2) promoter and the bacterial -glucuronidase gene (Gus)-coding region was introduced into rice. In transgenic rice plants, the expression of the pin2-Gus fusion gene displays a systemic wound response, although the expression level is relatively low. Incorporation of the first intron from the rice actin 1 gene (Act1) into the 5-untranslated region of the pin2-Gus construct results in high-level, systemically wound-inducible expression of the modified construct in transgenic rice plants. Histochemical analysis shows that this high-level, wound-inducible expression is associated with the vascular tissue in both leaves and roots. Furthermore, the expression of the pin2-Act1 intron-Gus fusion gene in transgenic rice plants can be systemically induced by both methyl jasmonate (MJ) and the phytohormone abscisic acid (ABA). These results suggest that the signal(s) mediating the observed systemic wound response and certain steps of the transduction pathways are conserved between dicot and monocot plants. Transient expression assays show that the pin2-Act1 intron-Gus construct is also actively expressed in transformed cells and tissues of several other monocot plants. Thus, the wound-inducible pin2 promoter in combination with the rice Act1 intron 1 might be used as an efficient regulator for foreign gene expression in transgenic monocot plants.     

毛果杨 NLP 基因家族生物信息学分析与鉴定简

NLP基因家族是一类特殊的转录因子,豆科植物根瘤的形成依赖于该基因家族的存在,在非豆科植物中具有调节植物硝酸盐吸收以及同化的功能。通过对毛果杨（Populus trichocarpa）基因组的生物信息学分析,共鉴定出14个毛果杨NLP基因家族成员,这些成员具有低亲水性的特点,基因结构保守,都含有RWP-RK以及PB1两个保守结构域。通过细胞定位预测,所有成员都定位在细胞核中。直系同源与旁系同源进化分析显示,NLP基因家族成员在漫长的进化过程中经历了严格的选择。染色体定位分析表明,毛果杨NLP基因家族成员坐落在毛果杨9条染色体之上,成员数量的扩增来自于杨柳科染色体自身的扩增事件。芯片数据分析结果显示,NLP基因家族成员在嫩叶,根和雄花中表达,部分基因在木质部以及种子萌发过程之中表达,但所有成员均不在成熟叶片中表达。     

Recovery of transgenic trees after electroporation of poplar protoplasts

Protoplasts from leaflets ofin vitro cuttings were electroporated in osmotically adjusted and buffered solutions containing plasmid DNA: pABD1, carrying thenptII gene for resistance to neomycin; pGH1, carrying a mutant acetolactate synthase gene,als, for resistance to sulfonylurea; and pGSFR781A, carrying a synthetic phosphinothricin acetyltransferase (pat) for resistance to phosphinothricin (Basta). Gene transfer was repeatedly efficient, without use of carrier DNA, in the range of one transformant for 10⁵ to 10⁴ protoplast-derived cell colonies. This was probably due to the high plating efficiency (30%) of protoplasts in our culture process. Selection for expression of foreign genes was applied in liquid medium and repeatedly achieved with 30 M paromomycin for NPTII, 200 nM chlorsulfuron for the mutant ALS ofArabidopsis and 25 M phosphinothricin for PAT expression. Integration of foreign genes into genomic DNA of resistant poplar trees was demonstrated by Southern blot hybridizations, which revealed that for some transformants practically no other part of the vector plasmid than the selected gene was integrated.Effective processes for protoplast culture, efficient selection at the cell colony stage and gene transfer will provide new possibilities in poplar breeding.     

Betaines and free amino acids in salt stressed vitroplants and winter resting buds of Populus trichocarpa X deltoides

Organic solutes, in particular glycine betaine and proline, have been detected as osmoprotective compounds in many microorganisms and herbaceous species. However, for woody plants, very little information is available on mechanisms of adaptation to salt stress. In the present study, effects of NaCI treatment on betaine and free amino acid contents in a clone of Populus trichocarpa X deltoides micropropagated vitroplants were analyzed using HPLC and silicate plate chromatography. The application during 12 days of 50 to 200 m M NaCI to viroplants cultured on Murashige and Skoog medium (Murashige and Skoog 1962. Physiol. Plant. 15: 473–497) led to a progressive decrease of growth, leaf senescence, abscission, and apical necrosis. Populus trichocarpa X deltoides was characterized by the absence of glycine betainc and/or proline accumulation. However, trigonelline was found to increase in vitroplants subjected to 100 m M NaCI. Trigonelline was also present in dormant buds harvested in natural conditions and missing in active buds. In vitroplants, the content of some amino acids was strongly modified by salt stress. A progressive accumulation of alanine and γ-amino butyrate was particularly significant in roots, whereas the relative concentration of glutamine was strongly enhanced in leaves. Leaf content of glutamate and ornithine attained maximum in the presence of 100 and 150 m M NaCI concentrations, and then decreased. Arginine and serine pools were not significantly modified by salt treatment. The variations in vitroplants were of small amplitude compared to those observed in mature poplars where winter rest was associated with a very high arginine level and with a disappearance of nearly all other free amino acids. The results are discussed in relation to previous data obtained with herbaceous species and in relation to some metabolic pathways in poplars where trigonelline could be considered as a sensitive stress indicator.     

全基因组分析杨树WOX基因家族在茎部发育中的作用

WUSCHEL-related homeobox(WOX)家族是植物特有的转录因子家族,参与分生组织细胞分裂分化、初生和次生物质代谢及植物激素信号转导等多个发育过程,目前尚未有从全基因组分析该基因家族参与杨树茎部发育的相关研究。本项研究旨在对杨树WOX基因家族进行鉴定,在杨树基因中发现18个WOX候选基因,将这些候选基因分为三组,同一分组的大多数WOX家族成员具有相似的基因结构和保守的基序。根据不同发育阶段茎部转录组数据,系统分析了WOX家族成员在茎部不同发育阶段的特异表达情况,并采用qRT-PCR对上述结果进行了验证。结果表明,杨树WOX基因家族在茎部不同发育阶段表现出不同的表达模式,为毛果杨WOX家族的功能研究与利用奠定基础。     

Distinct responses to ozone of abaxial and adaxial stomata in three Euramerican poplar genotypes

Ozone induces stomatal sluggishness, which impacts photosynthesis and transpiration. Stomatal responses to variation of environmental parameters are slowed and reduced by ozone and may be linked to difference of ozone sensitivity. Here we determine the ozone effects on stomatal conductance of each leaf surface. Potential causes of this sluggish movement, such as ultrastructural or ionic fluxes modification, were studied independently on both leaf surfaces of three Euramerican poplar genotypes differing in ozone sensitivity and in stomatal behaviour. The element contents in guard cells were linked to the gene expression of ion channels and transporters involved in stomatal movements, directly in microdissected stomata. In response to ozone, we found a decrease in the stomatal conductance of the leaf adaxial surface correlated with high calcium content in guard cells compared with a slight decrease on the abaxial surface. No ultrastructural modifications of stomata were shown except an increase in the number of mitochondria. The expression of vacuolar H⁺/Ca²⁺‐antiports (CAX1 and CAX3 homologs), β‐carbonic anhydrases (βCA1 and βCA4) and proton H⁺‐ATPase (AHA11) genes was strongly decreased under ozone treatment. The sensitive genotype characterized by constitutive slow stomatal response was also characterized by constitutive low expression of genes encoding vacuolar H⁺/Ca²⁺‐antiports.     

Fluorescent proteins in poplar: a useful tool to study promoter function and protein localization

The jellyfish (Aequorea victoria) green fluorescent protein (GFP) and its variants (CFP [cyan] and YFP [yellow]) were successfully used as a vital marker system for the transformation of hybrid poplar (Populus tremula x P. alba). Our results show that, in this woody plant, fluorescent proteins can be expressed: (i) transiently in protoplasts after PEG-mediated transformation, as well as in leaf cells after particle bombardment, and (ii) stably in callus cells and plants after Agrobacterium-mediated transformation. For these studies, we constructed vectors permitting easy recloning of any promoter fragments of interest. Confocal laser scanning microscopy was used both for visualization and differentiation between the different colours of the GFP variants and autofluorescence of chlorophyll and lignified xylem vessels. Peroxisomes were chosen as target organelles for GFP translocation via the peroxisomal targeting sequence PTS1 because this allowed us to concentrate the fluorochrome in the small volume of a few peroxisomes, giving a bright fluorescence over background noise.     

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.     

毛果杨固有无序蛋白质基因克隆及胁迫响应分析

为了解毛果杨中固有无序蛋白PtrIDP1（Potri.010G161200.1）基因的相关信息，探究该基因在毛果杨的不同组织、不同逆境胁迫下的表达特性，本研究根据Phytozome数据库中得到的基因全长序列设计引物，克隆得到该基因的目的片段。该基因完整的CDs区序列长度为423 bp，共编码140个氨基酸。构建亚细胞定位表达载体，瞬时转化洋葱表皮细胞，在激光共聚焦显微镜下观察显示，PtrIDP1定位在细胞核内。利用实时定量RT-PCR技术分析了PtrIDP1基因在毛果杨不同组织中的表达特异性和应对非生物胁迫的表达特性。结果表明：PtrIDP1基因在毛果杨的根、茎、叶中均有表达，其中在根部表达量最低，在茎和叶中相对表达量较高。PtrIDP1基因在高盐和干旱胁迫诱导时其表达量的变化模式不同，初步分析认为PtrIDP1基因参与了毛果杨非生物逆境胁迫的响应过程。     

转双抗虫基因741杨节肢动物群落营养结构及生态位变化

转双抗虫基因741杨（简称转基因741杨）节肢动物群落中,基位物种的植食性昆虫丰富度显著降低,但中性节肢动物丰富度却明显增加.高抗和中抗的节肢动物群落中位物种和顶位物种较之对照有所增多.转基因741杨节肢动物群落的害虫功能团,其优势状况,与对照相比有所改变：天敌优势度高于对照,中性节肢动物丰富度增加,并在天敌-害虫的营养链中起着重要的调控作用.鳞翅目害虫的空间生态位宽度最小,其它各功能类群的生态位宽度较大;捕食性和寄生性天敌与鳞翅目害虫的生态位重叠均较小,而与腐生和游逛种类的生态位重叠较大;各类害虫之间、捕食性天敌与寄生性天敌之间亦存在激烈竞争.转基因741杨对寄生性天敌和捕食性天敌在利用时间资源上有正作用.各种功能类群的时-空二维生态位宽度和生态位重叠均不如单维生态位宽度和生态位重叠值大,但抗性株系天敌类群对环境的适应性优于对照.     

Unique occurrence of pectin-like fibrillar cell wall deposits in xylem fibres of poplar

Using light and electron microscopic techniques, we studied the unique occurrence of fibrillar cell wall deposits in mature xylem fibres from poplar. These cell wall deposits lined the lumen-facing side of the wall, mainly in fibres next to vessel elements. Different lines of evidence point to the pectin-like nature of these fibrillar cell wall deposits. First, specific staining by Alcian Blue 8GX, a dye with high affinity for pectic substances. Second, the strongly reduced staining of the cell wall deposits in microscopic sections treated with pectolytic enzyme. Third, concomitant staining of pits, which are known to consist mainly of pectic substances. Given the pectin-like nature of the fibrillar cell wall deposits as well as their preferred occurrence in fibres neighbouring water-conducting vessel elements, a function for the fibrillar cell wall deposits in lateral water diffusion and stem water storage is hypothesised. The hypothesis is supported by the increased abundance of these cell wall deposits in wood tissue of a drought-sensitive poplar species.     

Life history alterations upon oral and hemocoelic bacterial exposure in the butterfly Melitaea cinxia

Life history strategies often shape biological interactions by specifying the parameters for possible encounters, such as the timing, frequency, or way of exposure to parasites. Consequentially, alterations in life‐history strategies are closely intertwined with such interaction processes. Understanding the connection between life‐history alterations and host–parasite interactions can therefore be important to unveil potential links between adaptation to environmental change and changes in interaction processes. Here, we studied how two different host–parasite interaction processes, oral and hemocoelic exposure to bacteria, affect various life histories of the Glanville fritillary butterfly Melitaea cinxia. We either fed or injected adult butterflies with the bacterium Micrococcus luteus and observed for differences in immune defenses, reproductive life histories, and longevity, compared to control exposures. Our results indicate differences in how female butterflies adapt to the two exposure types. Orally infected females showed a reduction in clutch size and an earlier onset of reproduction, whereas a reduction in egg weight was observed for hemocoelically exposed females. Both exposure types also led to shorter intervals between clutches and a reduced life span. These results indicate a relationship between host–parasite interactions and changes in life‐history strategies. This relationship could cast restrictions on the ability to adapt to new environments and consequentially influence the population dynamics of a species in changing environmental conditions.     

A preliminary investigation of the role of auxin and cytokinin in sylleptic branching of three hybrid poplar clones exhibiting contrasting degrees of sylleptic branching

Sylleptic branches grow out from lateral buds during the same growing season in which the buds are formed. This type of branching is present in poplar and in many tropical species. It results in the production of more branches, more leaves and expanded photosynthetic capacity and is thought to assist in increasing the overall growth and biomass of the tree at a young age. However, very little is known about the physiology of sylleptic branching in poplar, which is an extremely important source of fibre and fuel. In the present study of three hybrid poplar clones (11-11, 47-174 and 49-177) of Populus trichocarpa x P. deltoides exhibiting contrasting degrees of sylleptic branching, an analysis was carried out on parent shoot elongation and sylleptic branching, together with a preliminary comparison of the parent shoots' sensitivity to auxin (naphthaleneacetic acid) as a repressor of lateral bud outgrowth, and cytokinin (benzyladenine) as a promoter. Suggestive evidence was found for an inverse correlation between parent shoot sensitivity to auxin and the degree of sylleptic branching, as well as a partially positive correlation with respect to sensitivity to cytokinin. The present data are consistent with the hypothesis that auxin and cytokinin may play repressive and promotive roles, respectively, in the sylleptic branching of hybrid poplar.