Cadmium in white lupin nodules: Impact on nitrogen and carbon metabolism

The aims of this work were to investigate the microlocalisation of cadmium (Cd) in Lupinus albus L. cv. Multolupa nodules, and to determine its effects on carbon and nitrogen metabolism. Nodulated white lupin plants were grown in a growth chamber with or without Cd (150 μM). Energy-dispersive X-ray microanalysis showed the walls of the outer nodule cortex cells to be the main area of Cd retention, helping to reduce the harmful effect Cd might have on the amount of N₂ fixed by the bacteroids. Sucrose synthase activity declined by 33% in the nodules of the Cd-treated plants, and smaller reductions were recorded in glutamine synthetase, aspartate aminotransferase, alkaline invertase and NADP-dependent isocitrate dehydrogenase activities. The Cd treatment also sharply reduced nodule concentrations of malate, succinate and citrate, while that of starch doubled, but that of sucrose experienced no significant change. In summary, the present results show that white lupins accumulate significant amounts of Cd in their root nodules. However, the activity of some enzymes involved in ammonium assimilation did decline, promoting a reduction in the plant N content. The downregulation of sucrose synthase limits the availability of carbon to the bacteroids, which might interfere with their respiration. Carbon metabolism therefore plays a primary role in the impaired function of the white lupin root nodule caused by Cd, while N metabolism appears to have a more secondary involvement.     

Changes in chloroplast morphology of different parenchyma cells in leaves of Haberlea rhodopensis Friv. during desiccation and following rehydration

The size, shape, and number of chloroplasts in the palisade and spongy parenchyma layers of Haberlea rhodopensis leaves changed significantly during desiccation and following rehydration. The chloroplasts became smaller and more rounded during desiccation, and aggregated in the middle of the cell. The size and number of chloroplasts in the palisade parenchyma cells were higher than in spongy parenchyma. The good correlation observed between the size or number of chloroplasts and the cross-sectional area of mesophyll cells, the cross-sectional width of the leaf and its water content suggested that the palisade cells were more responsive to water availability than the spongy cells. Changes in chloroplast number during desiccation and rehydration process are characteristic features for desiccation-tolerant plants (especially in homoiochlorophyllous strategy).     

Response to oxidative stress induced by cadmium and copper in tobacco plants (Nicotiana tabacum) engineered with the trehalose-6-phosphate synthase gene (AtTPS1)

The response of tobacco plants genetically engineered with the AtTPS1 gene to stress induced by excess Cu and Cd was evaluated in hydroponic solution (100 and 400 μM Cu and 50 and 200 μM Cd) after a 48 h exposure. Two transgenic lines, transformed with the AtTPS1 (trehalose-6-phosphate synthase) gene from Arabidopsis, with different levels of trehalose-6-phosphate synthase expression (B5H, higher and B1F, lower), and a wild type (WT) were investigated. Protein content, antioxidative enzymes (CAT, POD, SOD, and APX), glucose, fructose, lipid peroxidation, hydrogen peroxide and Cd and Cu contents were determined in leaves. The two transgenic lines were differently influenced by Cd and Cu exposure as they induced a different antioxidant enzymatic defense response. B1F and B5H plants showed a better acclimation to Cd and excess Cu compared to WT. Furthermore B1F was more tolerant than B5H to Cd and excess Cu. B1F accumulated less Cd and Cu in leaves, probably due to a more efficient exclusion mechanism. Catalase was shown to be the most important enzyme in the antioxidative system of these plants.     

Cadmium-stress in white lupin: effects on nodule structure and functioning

Cadmium effects on nodule structure and changes in organic and amino acids, proteins, nutrients and some stress indicators were studied in nodules of white lupin (Lupinus albus L., cv. Multolupa). Plants were grown hydroponically on perlite for 49 d with (18 μM) or without Cd in the nutrient solution. Cadmium-treated plants showed decreases in leaf chlorophyll and shoot sucrose concentrations, but sucrose did not change in nodules. Cadmium application produced alterations in nodule cortex and infected zone structure. Furthermore, Cd supply caused a marked decrease in P, K, leghemoglobin, N–amino compounds, malate, succinate and soluble protein in the nodules. Conversely, the levels of lipid peroxidation and total thiols increased strongly. Results obtained suggest that white lupin nodules are Cd sensitive, in spite of Cd sequestering by cell walls and thiols. The main phytotoxic effects of Cd on nodule structure and function were the occlusion with glycoprotein of intracellular spaces of nodule cortex, alterations in symbiosomes, enrichment in Cd of cell walls and oxidative stress. Glycoprotein accumulation and leghemoglobin depletion may be considered useful indicators of Cd stress in white lupin nodules.     

Leaf anatomy of highbush blueberry grownin vitro and during acclimatization toex vitro conditions

Leaves of micropropagated highbush blueberry (Vaccinium corymbosum) cv. ‘Bluetta’ have been observed during the acclimatization phase. In vitro-developed leaf cells were circular and small, the spongy parenchyma was discontinuous and disorganized and formed by 1–2 layers of cells with large intercellular spaces and the palisade to spongy mesophyll thickness ratio was 1:1.5. After rooting ex vitro, the first leaves formed under natural conditions showed substantial changes in the anatomical characteristics. After 6 months, the plants produced leaves similar to those in field-grown plants. The palisade cells were rectangular, the spongy parenchyma was formed by 3–4 layers of cells and the intercellulars were around the stomata. Leaves from field-grown plants lost 24 % of water during 150 min after excision while leaves from in vitro shoots lost about 50 % of water in the same time. Leaves from in vitro shoots showed a higher number of smaller stomata (361 per mm²), with the guard cells forming a circular ring; the stomata frequency in field-grown leaves was 241 per mm² and the guard-cells were elliptical.     

接骨草的显微结构及其绿原酸组织化学定位研究

运用石蜡切片法和荧光显微镜观察法研究了3个不同接骨草(Sambucus chinensis Lindl.)居群营养器官的显微结构及其绿原酸的分布规律。结果表明:(1)接骨草地上茎厚角组织明显,髓部由大小不等的两类薄壁细胞组成,且有单宁细胞分布;地下根状茎厚角组织细胞小,髓部薄壁细胞大小差异不明显,皮层及髓中有油细胞分布。(2)叶片为异面叶,栅栏组织细胞为短柱状,油细胞不明显。(3)绿原酸分布在根状茎皮层部分细胞、茎厚角组织部分细胞及叶片的海绵组织中,以海绵组织中含量最高。研究认为,髓部薄壁细胞大小的差异可作为接骨草的一个鉴别特征;荧光显微镜观察法可迅速准确显示绿原酸的分布;在所研究的3个接骨草居群中,怀化居群的绿原酸含量最高,若以绿原酸为有效成分来采收接骨草,可以只采收叶。     

Active chitinases in the apoplastic fluids of healthy white lupin (Lupinus albus L.) plants

Acidic exocellular class III chitinase (EC 3.2.1.14) was previously identified in healthy white lupin (Lupinus albus L.) plants and suspension-cultured cells by N-terminal microse-quencing. In this study, the detection of chitinase activity with Remazol Brilliant Violet 5R (RBV)-labelled chitin derivatives is described. Chitinase activity was observed in protein fractions of cytoplasmic or exocellular origin from roots, hypocotyls, cotyledons, and leaves of healthy white lupin plants. Using isoelectrofocusing followed by a new overlay technique with carboxymethyl chitin-RBV conjugate-containing gel, up to six different chitinase isoforms were visualised. Their activity was distributed fairly evenly within a plant with acidic isoforms predominating in cell walls and basic (or neutral) ones found intracellularly. Exocellular location of some chitinase isoforms were also confirmed by detection of their activities in intercellular washing fluids from white lupin tissues. Chitinase activity was demonstrated in culture filtrates and cell walls of suspension-cultured white lupin cells.     

中国假鹰爪属和皂帽花属植物叶的形态结构及其分类学意义

利用扫描电镜技术、叶片离析法和石蜡切片法研究了假鹰爪属Desmos 4种植物和皂帽花属Dasy-maschalon 3种植物叶片的形态结构。结果表明：假鹰爪属植物叶片近轴面表皮具大型球状含晶簇细胞和不含晶簇的表皮细胞两种类型,远轴面表皮细胞均具一较小的晶簇;叶肉组织明显分化为栅栏组织细胞和海绵组织细胞,油细胞分布于第2层的栅栏组织和海绵组织内,单位毫米叶宽油细胞数为4～6个;主脉维管组织被薄壁细胞分隔成束状。皂帽花属植物叶片近轴面表皮细胞形状相同,均具一晶簇,远轴面表皮细胞的晶簇和近轴面表皮细胞的晶簇相似;靠近上、下表皮的叶肉组织均分化为栅栏组织细胞,在两层栅栏组织细胞之间分化为一至几层海绵组织细胞,油细胞分布于海绵组织内,单位毫米叶宽油细胞数为2～3个;主脉维管组织形成连续的环状。由此可见两属叶的结构具有明显的差异,因而支持假鹰爪属和皂帽花属为两个独立属的观点。     

石蒜属12 种植物叶片比较解剖学研究

 对石蒜属( Lycoris Herb. ) 12 种植物叶片的比较解剖学研究表明: (1) 石蒜属植物叶片横切面的端部、中部及基部的轮廓基本呈浅“W”或“V”字型, 有些种的表皮细胞上具有明显的尖刺状乳突; ( 2)石蒜属植物均为异面叶, 叶肉组织有一定的栅栏组织和海绵组织分化, 但二者的厚度、叶肉中所占比例及栅栏组织的细胞层数在种间有一定的差异; (3) 海绵组织发达、具有大而明显的薄壁细胞或细胞裂溶后形成空腔(分泌腔或气腔); (4) 叶片中维管束数目大多为奇数, 叶脉维管束鞘由薄壁细胞组成; (5) 石蒜属植物横切面上叶缘的形状分为圆弧形和楔形两种类型。石蒜属植物叶的解剖结构具有许多相似特征; 同时又具有一定的种间差异, 可为石蒜属植物的种间关系和开发利用提供有价值的信息。     

Leaf anatomy during leaf development of photoautotrophically <Emphasis Type="Italic">in vitro</Emphasis>-grown tobacco plants as affected by growth irradiance

Tobacco (Nicotiana tabacum L.) plants were cultured in vitro photoautotrophically at three levels of irradiance (PAR 400–700 nm): low (LI, 60 μmol m⁻² s⁻¹), middle (MI, 180 μmol m⁻² s⁻¹) and high (HI, 270 μmol m⁻² s⁻¹). Anatomy of the fourth leaf from bottom was followed during leaf development. In HI and MI plants, leaf area expansion started earlier as compared to LI plants, and both HI and MI plants developed some adaptations of sun species: leaves were thicker with higher proportion of palisade parenchyma to spongy parenchyma tissue. Furthermore, in HI and MI plants palisade and spongy parenchyma cells were larger and relative abundance of chloroplasts in parenchyma cells measured as chloroplasts cross-sectional area in the cell was lower than in LI plants. During leaf growth, chloroplasts crosssectional area in both palisade and spongy parenchyma cells in all treatments considerably decreased and finally it occupied only about 5 to 8 % of the cell cross-sectional area. Thus, leaf anatomy of photoautotrophically in vitro cultured plants showed a similar response to growth irradiance as in vivo grown plants, however, the formation of chloroplasts and therefore of photosynthetic apparatus was strongly impaired.     

Leaf anatomy of Desmos and Dasymaschalon (Annonaceae) from China in relation to taxonomic significance

Leaf morphology in four species of Desmos and three species of Dasymaschalon was comparatively studied using scanning electron microscopy (SEM) together with epidermal maceration and paraffin methods. The results showed that there were some remarkable foliar anatomical differences between Desmos and Dasymaschalon. In leaves of Desmos, some of the adaxial epidermal cells were enlarged into globose cells each containing one large cluster crystal, while other epidermal cells were normal without any crystal, and in abaxial epidermis each cell contained one smaller cluster crystal. The leaf structure was typically bifacial, and the mesophyll cells were differentiated into palisade tissue and spongy tissue. Oil cells were distributed in the second layer of palisade and the whole spongy tissue, and the number of oil cells per mm leaf width ranged from 4 to 6. The vascular tissue in the midrib was separated into bundles by parenchyma cells. In leaves of Dasymaschalon, all the adaxial epidermal cells contained one cluster crystal, and the crystal size was similar to that of thecrystals in abaxial epidermal cells. The leaf structure was more or less isobilateral. Oil cells were distributed only in the spongy tissue between the two layers of the palisade, and the number of oil cells per mm leaf width ranged from 2 to 3. The vascular tissue in the midrib formed a continuous circle. It is clear that the anatomical differences between Desmos and Dasymaschalon are remarkable, supporting the treatment of Desmos and Dasymaschalon as two independent genera.     

Effects of nitrogen deprivation on cell division and expansion in leaves of Ricinus communis L.

The effects of nitrogen deprivation on leaf extension, cell numbers and epidermal cell size were followed in leaves of Ricinus communis L. The extent to which reductions in final cell number or final epidermal cell size contributed to the reduction in final leaf size depended on the developmental stage of the leaf at the time of N deprivation. In leaves which already had their full complement of cells (leaf 2), the reduction in final leaf size following nitrogen deprivation was associated with a reduction in final cell size. In leaves that were at earlier stages of development at the onset of N deprivation (leaves 3 and 4), the reduction in final leaf size was greater than in leaf 2. In these younger leaves, the final cell size was even smaller than in leaf 2, but the greatest contribution to reduced final leaf size was a reduction in the number of cells produced. This accounted for approximately 80% of the reduction in final leaf size in leaf 4. During leaf development, the contribution from different tissue layers to the total cell number changed. In the smallest leaf sizes, the contribution from upper and lower epidermis and spongy parenchyma was greater than that from palisade parenchyma. As the leaf size increased, cells in the palisade parenchyma continued to divide for longer than in the other layers. At final leaf size, the contribution from the different tissue layers to total cell number was the same for leaves 2, 3 and 4, irrespective of N treatment. In these final leaf structures, palisade parenchyma contributed 60% of the total cell number. Thus, although nitrogen deprivation affected leaf size variously through cell division and cell expansion, depending on leaf developmental stage at the time of nitrogen deprivation, the ratio of cell numbers and sizes in different tissue layers, at final leaf size, was unaffected.     

地黄叶和茎的解剖学及组织化学研究

采用解剖学和组织化学方法对地黄叶和茎的显微结构以及梓醇、多糖的分布进行观察研究,以明确梓醇和多糖在地黄叶和茎中的分布特征。结果显示:(1)地黄叶的上、下表皮均分布有腺毛和非腺毛,腺毛都属于头状腺毛,包括长柄和短柄的头状腺毛,两类腺毛的分泌物化学成分主要是黄酮和多糖;叶的上、下表皮上都分布有无规则型气孔,下表皮的气孔密度比上表皮的大,但气孔指数相差不大;栅栏组织由2～3层薄壁细胞构成,排列紧密,海绵组织薄壁细胞形状无规则,细胞间隙大。(2)组织化学研究表明,海绵组织中黄斑样的薄壁细胞是梓醇和多糖的贮存场所,这类薄壁细胞在叶片边缘的齿末端处最为集中,茎的皮层、韧皮部和木质部的薄壁细胞也都是梓醇和多糖的贮存场所。     

The spatial distribution of chloroplast water in Acer platanoides sun and shade leaves

We evaluated a new, two-dimensional (2-D) nuclear magnetic resonance (NMR) imaging technique as a method for measuring the distribution of chloroplasts in leaves. NMR images that showed the distribution of chloroplast water and of total water as a function of depth into Acer platanoides sun and shade leaves were compared with the distribution of chlorophyll in the same leaf types (as measured by fluorescence microscopy), with the cellular structure (by scanning electron microscopy), and with published information. Results showed that the volume fraction of chloroplast water was much larger in shade than in sun leaves, and that it averaged about one-third larger in the palisade than in the spongy parenchyma region of both leaf types. Chlorophyll fluorescence was more intense in shade than in sun leaves. In sun leaves, fluorescence was maximal in the palisade region near the junction with the spongy parenchyma, while in shade leaves, fluorescence was maximal in the upper part of the spongy layer. We concluded that 2-D NMR imaging reliably indicates the location of chloroplast water.     

Kinetics of mercury uptake by oilseed rape and white lupin: influence of Mn and Cu

Mercury influx in oilseed rape and white lupin was studied using short time influx experiments. The effect of Cu and Mn in Hg influx was also tested. Plants were grown for 2 weeks and then roots were incubated with increasing Hg concentrations (0–50 μM HgCl₂), both at 20 °C and ice-cold temperature. An active, saturable component in Hg uptake was found in oilseed rape and white lupin, with K _m and V _max values in the range of low affinity transporters for essential micronutrients. A reduction in Hg uptake was observed in the presence of Mn for oilseed rape, suggesting that Hg influx is mediated by a Mn transporter. No effects of Cu on Hg influx were observed for any of the two plant species, suggesting a different transport system for Hg and Cu in roots of oilseed rape and white lupin.     

Excess copper predisposes photosystem II to photoinhibition in vivo by outcompeting iron and causing decrease in leaf chlorophyll

Photoinhibition of photosystem II was studied in vivo with bean (Phaseolus vulgaris) plants grown in the presence of 0.3 (control), 4, or 15 microM Cu(2+). Although photoinhibition, measured in the presence of lincomycin to block concurrent recovery, is faster in leaves of Cu(2+)-treated plants than in control leaves, thylakoids isolated from Cu-treated plants did not show high sensitivity to photoinhibition. Direct effects of excess Cu(2+) on chloroplast metabolism are actually unlikely, because the Cu concentration of chloroplasts of Cu-treated plants was lower than that of their leaves. Excess Cu in the growth medium did not cause severe oxidative stress, collapse of antioxidative defenses, or loss of photoprotection. Thus, these hypothetical effects can be eliminated as causes for Cu-enhanced photoinhibition in intact leaves. However, Cu treatment lowered the leaf chlorophyll (Chl) concentration and reduced the thylakoid membrane network. The loss of Chl and sensitivity to photoinhibition could be overcome by adding excess Fe together with excess Cu to the growth medium. The addition of Fe lowered the Cu(2+) concentration of the leaves, suggesting that Cu outcompetes Fe in Fe uptake. We suggest that the reduction of leaf Chl concentration, caused by the Cu-induced iron deficiency, causes the high photosensitivity of photosystem II in Cu(2+)-treated plants. A causal relationship between the susceptibility to photoinhibition and the leaf optical density was established in several plant species. Plant species adapted to high-light habitats apparently benefit from thick leaves because the rate of photoinhibition is directly proportional to light intensity, but photosynthesis becomes saturated by moderate light.     

龙苍沟国家森林公园7种花楸属植物的叶解剖特征及其环境适应性

采用石蜡切片法对四川省龙苍沟国家森林公园内7种花楸属（Sorbus）植物的叶解剖特征进行研究,探究其结构特征与生境的相关性。结果显示：7种植物的叶片均为典型的背腹叶;叶片厚度介于108.16~208.21 μm,种间差异极显著（P<0.01）;上表皮厚度均大于下表皮厚度,且复叶物种的下表皮细胞均有乳突;栅栏组织由1~2层细胞构成,仅多对西康花楸（S. prattii var. aestivalis）的栅海比（栅栏组织与海绵组织的厚度比）为1.93,其余6种植物的栅海比均小于1;中脉维管束均呈心型,为典型的外韧型维管束,种间中脉突起度存在极显著差异（P<0.01）。各解剖结构中,上、下表皮可塑性最大,在生境中具有较强的潜在适应能力;中脉可塑性最小,整体结构较为稳定。栅栏组织、海绵组织和中脉组织是7种植物中种间差异最大的解剖结构。叶解剖结构与生境因子的相关性分析表明,栅栏组织厚度、栅海比和紧密度与年降水量、最暖季降水和海拔正相关（P<0.05）,与季节性温差负相关（P<0.05）;中脉直径和突起度与季节性温差呈正相关（P<0.01）,与年降水量、最暖季降水和海拔正相关负相关（P<0.05）。叶解剖结构性状的适应性变化,体现了7种花楸属植物在龙苍沟国家森林公园的生存策略。     

Leaf responses to iron nutrition and low cadmium in peanut: anatomical properties in relation to gas exchange

Aims

This study evaluated how iron nutrition affect leaf anatomical and photosynthetic responses to low cadmium and its accumulation in peanut plants.

Methods

Seedlings were treated with Cd (0 and 0.2 μM CdCl₂) and Fe (0, 10, 25, 50 or 100 μM EDTA-Na₂Fe) in hydroponic culture.

Results

Cadmium accumulation is highest in Fe-deficient plants, and dramatically decreased with increasing Fe supply. The biomass, gas exchange, and reflectance indices were highest at 25 μM Fe²⁺ treatments, indicating the concentration is favorable for the growth of peanut plants. Both Fe deficiency and Cd exposure impair photosynthesis and reduce reflectance indices. However, they show different effects on leaf anatomical traits. Fe deficiency induces more and smaller stomata in the leaf surface, but does not affect the inner structure. Low Cd results in a thicker lamina with smaller stomata, thicker palisade and spongy tissues, and lower palisade to spongy thickness ratio. The stomatal length and length/width ratio in the upper epidermis, spongy tissue thickness, and palisade to spongy thickness ratio were closely correlated with net photosynthetic rate, stomatal conductance, and transpiration rate.

Conclusions

Cd accumulation rather than Fe deficiency alters leaf anatomy that may increase water use efficiency but inhibit photosynthesis.     

Effects of high zinc concentration on poplar leaves: A morphological and biochemical study

Plants growing under zinc excess store it in leaves, the organs in charge with carbon assimilation. Therefore, leaf modifications can help to understand the plant responses to environmental stresses. In this study, we tested the hypothesis that high zinc concentration can affect leaf anatomy and physiology of Populus alba cv. Villafranca, a tree species with interesting perspectives of application in phytoremediation.Plants were supplemented or not with 300 mg zinc per kg of soil and grown for six months. Green and macroscopically symptom-less leaves from poplars of each treatment were analyzed by light and electron microscopy (TEM, SEM-EDS) and by Raman spectroscopy. LHCII and D1-D2 protein expression were considered to evaluate PSII functioning. Zinc was histochemically detected in the leaves.Zinc strongly altered leaf morphology and ultrastructure, and negatively affected PSII activity decreasing D1 and D2, but not LHCII, protein expression. Zinc was mostly detected in the cell walls of the xylem and of the parenchyma cells surrounding the bundles of zinc treated plants, but not in the controls. A significant increase in the number of calcium oxalate crystals was observed in the leaves of zinc treated plants, but the latter metal was not incorporated in crystals, suggesting an increase of free calcium following zinc accumulation in the cell walls.     

In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves

Accurate localization of phytoalexins is a key for better understanding their role. This work aims to localize stilbenes, the main phytoalexins of grapevine. The cellular localization of stilbene fluorescence induced by Plasmopara viticola, the agent of downy mildew, was determined in grapevine leaves of very susceptible, susceptible, and partially resistant genotypes during infection. Laser scanning confocal microscopy and microspectrofluorimetry were used to acquire UV-excited autofluorescence three-dimensional images and spectra of grapevine leaves 5-6 days after inoculation. This noninvasive technique of investigation in vivo was completed with in vitro spectrofluorimetric studies on pure stilbenes as their fluorescence is largely affected by the physicochemical environment in various leaf compartments. Viscosity was the major physicochemical factor influencing stilbene fluorescence intensity, modifying fluorescence yield by more than two orders of magnitude. Striking differences in the localization of stilbene fluorescence induced by P. viticola were observed between the different genotypes. All inoculated genotypes displayed stilbene fluorescence in cell walls of guard cells and periclinal cell walls of epidermal cells. Higher fluorescence intensity was observed in guard-cell walls than in any other compartment due to increased local viscosity. In addition stilbene fluorescence was found in epidermal cell vacuoles of the susceptible genotype and in the infected spongy parenchyma of the partially resistant genotype. The very susceptible genotype was devoid of fluorescence both in the epidermal vacuoles and the mesophyll. This strongly suggests that the resistance of grapevine leaves to P. viticola is correlated with the pattern of localization of induced stilbenes in host tissues.