Quantitative analysis of cell division in leaves: methods,developmental patterns and effects of environmental conditions

In planta quantitative studies of cell cycle are necessary for examining the role of cell division in the response of plants to environmental conditions and to analyse the behaviour of transformed plants in this context. We present and discuss non-intrusive kinematic methods which allow estimating the duration of cell cycle with a high spatial resolution in the leaf. Different methods are proposed and discussed for monocotyledons and dicotyledons, and compared with methods involving the use of chemicals. In monocotyledon leaves, cell division is restricted to a limited zone near the leaf insertion point, twice as long in the mesophyll as in the epidermis. In dicotyledons, cell division occurs in the whole leaf with a uniform and constant cell cycle duration for a determinate number of cell cycles, representing about half of leaf development. Over several experiments, this number is well conserved in a given leaf zone in the absence of stresses, but larger near the leaf base than near the leaf tip. After that, cell cycle duration increases because cells are progressively blocked in G1 while the durations of S-G2-M phases do not change with time. Leaf temperature affects neither the distribution of nuclei in each phase of the cycle nor the number of cell cycles in a leaf. Water or light deficits both cause a partial blockage of nuclei in G1 during the stress only, thereby increasing cell cycle duration and decreasing final cell number. These results suggest that a strong developmental programme drives cell division in leaves, so a simple framework allows analysis of temporal patterns, of spatial gradients and of the effect of environmental conditions.     

The emergy of metabolism in different ecosystems under the same environmental conditions in the agro-pastoral ecotone of northern China

The sustainability of ecosystem productivity and rules governing ecosystem development are important topics of scientific research. The emergy approach is an effective method for investigating these topics, especially when used to evaluate systems that have developed under the same environmental conditions, such as climate and soil. In this paper, emergy differences between terrestrial ecosystems were studied in Guyuan County, a region representative of the agro-pastoral ecotone in Hebei Province, China. A combination of field tests and a questionnaire survey were carried out between June and August 2015. The ecosystems studied included natural grassland, artificial grassland, field crops and commercial crops. These four ecosystems were further subdivided into a total of ten ecosystems. Natural grassland was divided into free-grazing and mowed ecosystems; artificial grassland consisted of oat, Chinese leymus and corn silage; field crops included naked oats, flax and wheat; and commercial crops consisted of cabbage and potatoes. The results showed that the rain input of 4.78 × 10¹⁴ seJ/ha/yr constituted the highest renewable natural resource emergy and that the purchased emergy inputs of the ten ecosystems ranged from 3.53 to 147.67 × 10¹⁴ seJ/ha/yr. Natural resource emergy input was the basic power to maintain the ecosystem, and purchased emergy input was the direct cause of the development of the ecosystems. Groundwater was the most important non-renewable purchased energy for the production of economic crops. The emergy investment ratios (EIR) for potatoes (27.81) and cabbage (19.03) were higher than those of the other ecosystems, but mowed and artificial Chinese leymus grassland had the higher emergy self-sufficiency rates (ESR). Natural grassland, artificial Chinese leymus grassland and traditional grain crops had a low environmental load and high sustainability, whereas potatoes and cabbage had a high environmental load and low sustainability. Overall, rain-fed artificial grassland has a high development potential from the perspective of environment and productivity.     

烤烟硝酸盐含量与土壤养分的关系

采用多元统计分析方法研究了湖南烤烟叶片硝酸盐、亚硝酸盐含量与土壤养分之间的关系,结果表明：（1）典型相关分析证实烟叶硝酸盐、亚硝酸盐含量与土壤主要含氮养分（土壤全氮、碱解氮、铵态氮）及有机质含量的关系密切,与土壤其他养分含量的相关性较小,反映出在一定范围内,随着土壤含氮养分及有机质含量的增加,烟叶（亚）硝酸盐含量呈现增加的趋势,且土壤含氮养分及有机质含量与烟叶硝酸盐含量关系的密切程度高于与亚硝酸盐含量的相关;（2）根据烟叶硝酸盐、亚硝酸盐含量及其相应的植烟土壤养分（有机质、全氮、碱解氮、铵态氮）含量的大小,通过聚类分析把同一等级的烟叶样品分为高、中、低3类,不同类别相比较,有机质含量越高的土壤,其土壤全氮、碱解氮、铵态氮含量以及相应的烟叶硝酸盐、亚硝酸盐含量也越高,说明土壤有机质的高低,直接影响了土壤氮素的供应状况,进而影响了烟叶硝态氮含量的积累;（3）根据聚类分析结果建立了不同等级烟叶硝酸盐、亚硝酸盐含量高低的判别函数,可作为烟叶硝态氮含量在不同土壤肥力条件下判别归类的参考依据。     

Comparison of allometric relationships and morphological differences in Tilia cordata Mill. outer leaves exposed to different environmental conditions

The study is based on four leaf parameters: leaf width (LW), lobe length (LL), leaf size (LS) and leaf shape which is calculated as LW to leaf length (LW/LL) ratio. Under different environmental conditions, LL is an isometric character, LW shows positive allometry, whereas LW/LL shows negative allometry. Regression analysis results indicated that there is no significant difference either in slopes or in regression coefficients between investigated sites. Thus, in this study, we found that allometric relationships between leaf parameters and LS are character specific and that they tended not to differ significantly between Tilia cordata Mill. outer leaves exposed to different environmental conditions. Also, there are no significant interpopulation differences for both principal component PC1 and PC2 scores. The stepwise discriminant functional analysis results allowed us to identify a set of two leaf parameters (LS and LL) with a moderate discriminating ability (59.8%). T. cordata outer leaves are significantly larger and broader in the reference area (R-leaves) than leaves from polluted (P-leaves) site. The data also indicated that there is a relatively larger petiole size in R-leaves than in P-leaves. We found that in P-leaves, LW increased faster with increasing LS than in R-leaves.     

Inheritance of body size in the Barnacle Goose under different environmental conditions

Heritabilities, genetic variances and covariances for body size traits, i.e. tarsus length, head length and body mass, were estimated under different environmental conditions in a Barnacle Goose (Branta leucopsis) population. Under poor growth conditions, that is, when average body size of fully grown offspring in a given cohort was small, the offspring-parent regressions and full-sib analyses yielded heritability estimates not significantly different from zero. By contrast, when growth conditions were normal or good the heritability estimates were generally significantly positive. Comparisons of genetic covariance estimates indicated that they also differed across the analysed environmental conditions. This result, together with similar results obtained in studies of passerine birds, suggests that genotype-environment interactions might be frequent within the range of environments normally encountered by birds in natural populations. If general, such results might question the validity of assuming approximate constancy of additive genetic variances and covariances over time and environments in evolutionary models.     

Variation of the essential oil content and composition in leaves from cultivated plants of Hypericum androsaemum L

The amount and composition of the essential oil from leaves of Hypericum androsaemum L. cultivated in Arouca (Portugal) were determined in six samples harvested during 1 year at intervals of 2 months. The seasonally dependent essential oil content ranged from 0.7 mg/g biomass dry weight in September to 3.4 mg/g in February. The oil contained more than 80 compounds, 70 of which (constituting 88-93% of the total oil) were identified by GC and GC-MS. An approximation of the absolute quantification of each compound and compound class was performed using a GC method with an internal standard. The relative and the absolute content of each compound and compound class changed during the year. At the end of the winter and in the spring, the essential oil was dominated by sesquiterpene hydrocarbons and accumulated a high number of intermediate to long chain n-alkanes and 1-alkenes. In September, the essential oil contained the lowest levels of sesquiterpene hydrocarbons (43%) and the highest levels of 1-octene and 2-hexenal (38%). In February, the essential oil had the highest level of sesquiterpene hydrocarbons (73%) and the highest diversity of intermediate to long chain n-alkanes and 1-alkenes.     

In vivo cytochrome and alternative pathway respiration in leaves of Arabidopsis thaliana plants with altered alternative oxidase under different light conditions

The in vivo activity of the alternative pathway (ν(alt)) has been studied using the oxygen isotope fractionation method in leaves of Arabidopsis thaliana modified for the expression of the AtAOX1a gene by anti-sense (AS-12) or overexpression (XX-2). Under non-stressful conditions, ν(alt) was similar in all plant lines regardless of its different alternative pathway capacities (V(alt)). Total leaf respiration (V(t)) and V(alt) were directly related to growth light conditions while electron partitioning between the cytochrome pathway (CP) and alternative pathway (AP) was unchanged by light levels. Interestingly, the AP functioned at full capacity in anti-sense plants under both growth light conditions. The role of the AP in response to a high light stress induced by short-term high light treatment (HLT) was also studied. In wild type and XX-2, both CP and AP rates increased proportionally after HLT while in AS-12, where the AP was unable to increase its rate, the CP accommodated all the increase in respiration. The results obtained under high light stress suggest that flexibility in the response of the mitochondrial electron transport chain is involved in sustaining photosynthetic rates in response to this stress while the saturated AP in AS-12 plants may contribute to the observed increase in photoinhibition.     

A possible influence of extracellular polysaccharides on the analysis of intracellular metabolites from Trichoderma harzianum grown under carbon-limited conditions

Intracellular metabolites were evaluated during the continuous growth of Trichoderma harzianum P49P11 under carbon-limited conditions. Four different conditions in duplicate were investigated (10 and 20 g/L of glucose, 5.26/5.26 g/L of fructose/glucose and 10 g/L of sucrose in the feed). Differences in the values of some specific concentrations of intracellular metabolites were observed at steady-state for the duplicates. The presence of extracellular polysaccharide was confirmed in the supernatant of all conditions based on FT-IR and proton NMR. Fragments of polysaccharides from the cell wall could be released due to the shear stress and since the cells can consume them under carbon-limited conditions, this could create an unpredictable carbon flow rate into the cells. According to the values of the metabolite concentrations, it was considered that the consumption of those fragments was interfering with the analysis.     

Quantitative analysis of photosynthetic carbon metabolism in protoplasts and intact leaves of barley. Determination of carbon fluxes and pool sizes of metabolites in different cellular compartments

Rates of carbon fluxes and pool sizes of photosynthetic metabolites in different cellular compartments of barley protoplasts were calculated from the time curves of their labeling in the medium of ¹⁴CO₂. Using membrane filtration procedure, kinetics of ¹⁴C incorporation into the products of steady-state photosynthesis was determined separately in chloroplasts, mitochondria and cytosol of barley protoplasts illuminated for different periods in the air containing ¹⁴CO₂. To extract the quantitative information, analytical labeling functions P(t) describing the dependence of ¹⁴C content in the primary, intermediate and end products of a linear reaction chain upon the duration of tracer feeding have been derived. The parameters of these functions represent pool sizes of metabolites and rates of carbon fluxes. The values of these parameters were determined by fitting the experimental labeling curves to the functions P(t) by means of non-linear regression procedure. To elucidate the possible effects of fractionation on the photosynthetic carbon metabolism, the parameters of protoplasts were compared with corresponding values in intact leaves of barley.     

Expression analysis of the two ferrochelatase genes in Arabidopsis in different tissues and under stress conditions reveals their different roles in haem biosynthesis

The Arabidopsis thaliana genome has two genes (AtFC-I and AtFC-II), encoding ferrochelatase, the terminal enzyme of haem biosynthesis. The roles of the two enzymes in the synthesis of haem for different haemoproteins was investigated using reporter gene analysis. A 1.41 kb fragment from the 5' upstream region of the AtFC-II gene was fused to the luciferase gene, and then introduced into tobacco plants, followed by luciferase activity measurements. AtFC-II-LUCwas expressed in all aerial parts of the plant, and was highest in flowers, but it was not expressed in roots. It was unaffected by viral infection, and considerably reduced by wounding or oxidative stress. Similarly, a 1.76 kb region of the AtFC-I promoter was fused to the uidAgene encoding -glucuronidase. AtFC-I-GUS was expressed in all tissues of the plant, but was higher in roots and flowers than in leaves or stems. It was induced by sucrose, wounding and oxidative stress and, most markedly, by plants undergoing the hypersensitive response to TMV infection. Levels of endogenous ferrochelatase activity were increased in pea chloroplasts isolated from wounded leaves, indicating that the induction in promoter activity is likely to result in increased haem biosynthetic potential. Salicylic acid, but not methyl-jasmonate was able to replace the stress treatment in induction of AtFC-I expression, suggesting that the requirement for haem synthesis is part of the defence response. The implications of the results for the different roles of the two ferrochelatases in haem biosynthesis are discussed.     

Metabolome analysis of photosynthesis and the related primary metabolites in the leaves of transgenic rice plants with increased or decreased Rubisco content

Because the comprehensive effects on metabolism by genetic manipulation of leaf Rubisco content are unknown, metabolome analysis was carried out on transgenic rice plants with increased or decreased Rubisco content using the capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) technique. In RBCS-sense plants, an increase in Rubisco content did not improve light-saturated photosynthesis. Glyceraldehyde 3-phosphate and sedoheputulose 7-phosphate levels increased, but ribulose bisphosphate (RuBP), ATP and ADP levels were not affected. It is considered from these results that RuBP regeneration independent of ATP supply became a bottleneck for photosynthesis. In RBCS-antisense plants, a decline in Rubisco content decreased photosynthesis with a substantial accumulation of RuBP. ATP and ADP levels also increased and were associated with increases in the diphosphate and triphosphate compounds of other nucleosides. These results imply that a decline in Rubisco content slowed down the Calvin cycle and that the resultant excess energy of ATP was transferred to other nucleoside diphosphates and triphosphates. The levels of amino acids tended to decline in RBCS-sense plants and increase in RBCS-antisense plants, probably reflecting the demand for Rubisco synthesis. Starch and carbohydrate levels decreased only in RBCS-antisense plants. Thus, genetic manipulation of Rubisco contents widely affected C and N metabolism in rice.     

Source metabolism dominates the control of source to sink carbon flux in tuberizing potato plants throughout the diurnal cycle and under a range of environmental conditions

The aim of this work was to investigate the effect of environmental conditions on the control of source to sink carbon flux in potato during tuberization. Top down metabolic control analysis was used to quantitatively determine the extent to which source and sink reactions control the flux from CO₂ to the tuber. Under all conditions investigated (beginning of the dark period, low nitrogen availability, variation in temperature) source metabolism accounts for at least 80% of the control. In plants grown with limiting nitrogen, or when measurements were made at low temperature the extent to which source metabolism controls flux increased to 90%.     

不同种源麻栎种子和苗木性状地理变异趋势面分析

麻栎(Quercus acutissima Carr.)是优良的能源和用材树种,在我国分布广泛并已有悠久栽培利用的历史,研究其种子和苗木性状地理变异规律对选择优良种源和适生区十分必要.研究收集了13个省(区)36个种源的麻栎种子,在测定麻栎种子性状的基础上,在安徽省滁州市红琊山林场进行了苗期试验和苗木性状测定,并采用趋势面分析方法对这些性状在经纬2维方向的地理变异模式进行了剖析.结果表明:1)麻栎种源间种子长度、宽度、百粒重和营养内含物存在极显著差异.种子百粒重、长度和宽度总体表现双向渐变趋势,随经度增高而增大,随纬度增高而减小,主要受到经度的控制;以西南到东北为中间地带,可溶性糖含量向东南表现先下降后上升的趋势,向西北则相反;淀粉含量从西北到东南呈逐渐减小的趋势.2)麻栎种源苗高、地径、生物量、热值和木材化学组分存在极显著差异.苗高、地径和生物量均呈双向渐变,经正向变异且变化幅度较大,纬负向变异且变化幅度较小,经度影响大于纬度;热值拟合回归方程不显著,方程无意义.木质素含量北部大于南部,但北部以西北部最高,南部以东南部最高.3)研究显示,麻栎种子和苗木性状多数存在显著的地理变异模式,这也是麻栎在长期进化过程中为适应复杂多变的环境而产生与之相适应的遗传变异结果.     

同一居群韭莲不同植株减数分裂行为差异的遗传分析

对韭莲(2n=48)小孢子母细胞减数分裂及小孢子发育进行研究。结果显示同一居群植株的减数分裂行为存在明显差异。多数韭莲植株小孢子母细胞减数分裂存在少量落后染色体、微核等现象,平均每株中具有异常分离行为的母细胞占14.02%,小孢子发育正常,但花粉无活力。并首次从减数分裂后期Ⅰ的特殊的细胞学形态证明韭莲是臂内倒位杂合体。而少数植株韭莲的小孢子母细胞减数分裂极其紊乱,后期Ⅰ出现多极分离、大量落后染色体,小孢子母细胞减数分裂总异常分离高达94.3%。四分孢子期多分孢子体高达73.4%。分析认为:前者减数分裂行为异常的原因主要由染色体结构变异所致,而后者的原因除染色体结构变异外,还可能与控制纺锤体形成的基因突变有关。     

微生物生态研究中基于BIOLOG方法的数据分析

BIOLOG微平板法作为一种方便快速的微生物检验技术,已广泛应用于环境微生物检测,微生物生态研究等方面,发挥着越来越重要的作用。该方法可以获得关于微生物群落碳源利用能力的大量数据,反映出关于微生物活性的丰富信息。然而大量的数据也对解释和分析提出了挑战,分析了应用于BIOLOG产生数据的统计分析方法,对常用的AWCD值计算,多样性指数计算,主成分分析(PCA),聚类分析,相关、回归等方法深入探讨,阐述各自的功能、不足以及在应用中容易出现的问题。另外也对一些不常见的方法,如非参数多元分析(Non-Parametric version of MANOVA/Permutation version of MANOVA)、动力学参数分析、多元回归树、典范对应分析等也进行了讨论。通过对不同方法应用目标和原理的分析论述了各自优缺点,对微生物研究中基于BIOLOG方法数据分析的选择应用提供参考。     

Craniometric analysis in groups from tierra del Fuego/Patagonia and the peopling of the south extreme of the Americas

Five large craniometric samples from extinct tribes (Selk’nam, Kawéskar and Yámana) from Tierra del Fuego and from Patagonia have been analyzed through multivariate techniques. The purpose was to test the hypothesis of one or two different migration waves in the peopling of the south extreme of South America. A cluster analysis has been made, using the squared Euclidean distance as a measure of proximity, and the UPGMA and neighbor joining algorithms as a tree building method. The robustness of the branches has been assessed with bootstrap analysis through 100 random iterations of the original data set. Results show that, despite their cultural differences, the three hunter-gatherer groups. from Tierra del Fuego tend to cluster together, indicating a similar morphological pattern. This suggests that geographic distance (in latitudinal sense) is the main factor that influenced the differentiation of the human groups from Tierra del Fuego and Patagonia, from a single ancestral population.