Studies on leaf spot disease of <Emphasis Type="Italic">Withania somnifera</Emphasis> and its impact on secondary metabolites

During an investigation of the disease profile of Withania somnifera, it was observed that leaf spot is the most prevalent disease. Repeated isolations from infected leaf tissues and pathogenicity tests showed the association of fungal pathogen identified as Alternaria alternata (Fr.) Keissler. Scanning electron microscopy showed various histological changes in the leaf tissues of infected plants. A decrease in total content of reducing sugars (20%) and chlorophyll (26.5%) was observed in diseased leaves whereas an increase was noticed in proline (25%), free amino acids (3%) and proteins (74.3%). High performance thin layer chromatography (HPTLC) analysis of secondary metabolites viz. withanolides, withaferin-A and total alkaloids of the diseased leaves vis-à-vis control revealed reduction in withaferin-A and withanolides contents by 15.4% and 76.3% respectively, in contrast to an increase in total alkaloids by 49.3%, information hitherto unreported in W. somnifera.     

Withanolide production by <Emphasis Type="Italic">in vitro</Emphasis> cultures of <Emphasis Type="Italic">Withania somnifera</Emphasis> and its association with differentiation

Withanolides-steroidal lactones, isolated from various Solanaceous plants have received considerable attention due to their potential biological activities. Five selected withanolides (withanone, withaferin A, withanolide A, withanolide B, withanolide E) were identified by HPLC-UV (DAD) — positive ion electrospray ionization mass spectroscopy in Withania somnifera (L.) Dunal cv. WSR plants and tissues cultured in vitro at different developmental phases. Cultures were established from five explants on Murashige and Skoog’s medium supplemented with different plant growth regulators. Results suggest that production of withanolides is closely associated with morphological differentiation.     

Qualitative and quantitative variations in withanolides and expression of some pathway genes during different stages of morphogenesis in Withania somnifera Dunal

Withania somnifera Dunal is an important and extensively studied medicinal plant; however, there is no report available that relates withanolide content and its profile in relation to the expression of pathway genes during different morphogenic stages. In this study, withanolide A, withaferin A, and withanone, the major withanolides of W. somnifera, were measured in different in vitro stages during organogenesis, viz., shoot to root (direct rhizogenesis)/root to shoot (indirect via callus phase) transition vis-à-vis expression levels of key pathway genes involved in withanolide biosynthetic pathways. The morphogenic transitions were found to be tightly linked to the pattern of accumulation of withanolides. The high expression levels of most of the pathway genes in in vitro shoots in comparison to in vitro root and callus tissues exhibited a direct co-relation with the maximum withanolide content (>2.7 mg/gDW). The biogenesis of withaferin A, a major constituent of the leaves, was however found to be tightly linked to shoots/green tissue. In addition, we were also able to establish an efficient regeneration system from roots for their further utilization in biotechnological applications.     

高盐生境中乌拉尔甘草盐离子分泌与积存格局的研究

采用植物水培方法,以乌拉尔甘草为研究材料,用不同浓度(0、80、160、320mmol·L~(-1))NaCl溶液胁迫处理乌拉尔甘草幼苗3周后,分析其叶片表面盐离子(K~+、Ca~(2+)、Na+)分泌速率的差异,并采集盐化低地草甸重盐土生境中2年生乌拉尔甘草植株,应用ICP-AES测定其不同部位(根、根状茎、茎、老叶和幼叶)中的盐离子(K~+、Na~+、Ga~(2+)、Mg~(2+))含量,探究盐离子在乌拉尔甘草叶片上的分泌格局以及盐离子在植株体内的积存格局,为完善甘草耐盐机理的研究提供依据。结果显示:(1)随着盐胁迫浓度的升高,乌拉尔甘草叶片上K~+、Ca~(2+)、Na+的分泌速率均呈增加趋势,且Na~+的分泌速率远远大于Ca~(2+)和K+的分泌速率。(2)在乌拉尔甘草各部位中,K+的积存量从大到小依次为:幼叶根根状茎茎老叶;Na~+在各个部位的积存量都十分有限,且无论地下部分还是地上部分,差异均不大;Ca~(2+)积存量由大到小依次为:老叶幼叶茎根状茎根,且老叶中Ca~(2+)的积存量显著高于其它部位。研究认为,在重盐碱地生境中,K+主要积存在幼叶中,Ga~(2+)主要积存在老叶中,植株体内各个部位Na~+的积存量很低,乌拉尔甘草表现出明显的拒Na现象;叶片分泌的主要盐离子为Na~+;乌拉尔甘草通过泌盐的方式将Na+排出体外,从而有效降低Na~+在体内的积存,这是其能够在重盐碱地生存生长的重要原因。     

PEX11β induces peroxisomal gene expression and alters peroxisome number during early Xenopus laevis development

Background  

Peroxisomes are organelles whose roles in fatty acid metabolism and reactive oxygen species elimination have contributed much attention in understanding their origin and biogenesis. Many studies have shown that de novo peroxisome biogenesis is an important regulatory process, while yeast studies suggest that total peroxisome numbers are in part regulated by proteins such as Pex11, which can facilitate the division of existing peroxisomes. Although de novo biogenesis and divisions are likely important mechanisms, the regulation of peroxisome numbers during embryonic development is poorly understood. Peroxisome number and function are particularly crucial in oviparous animals such as frogs where large embryonic yolk and fatty acid stores must be quickly metabolized, and resulting reactive oxygen species eliminated. Here we elucidate the role of Pex11β in regulating peroxisomal gene expression and number in Xenopus laevis embryogenesis.     

A loop‐to‐base processing mechanism underlies the biogenesis of plant microRNAs miR319 and miR159

The first step in microRNA (miRNA) biogenesis usually involves cleavage at the base of its fold‐back precursor. Here, we describe a non‐canonical processing mechanism for miRNAs miR319 and miR159 in Arabidopsis thaliana. We found that their biogenesis begins with the cleavage of the loop, instead of the usual cut at the base of the stem–loop structure. DICER‐LIKE 1 (DCL1) proceeds then with three additional cuts until the mature miRNA is released. We further show that the conserved upper stem of the miR319 precursor is essential to organize its biogenesis, whereas sequences below the miRNA/miRNA^* region are dispensable. In addition, the bulges present in the fold‐back structure reduce the accumulation of small RNAs other than the miRNA. The biogenesis of miR319 is conserved in the moss Physcomitrella patens, showing that this processing mechanism is ancient. These results provide new insights into the plasticity of small‐RNA pathways.     

BSD2 is a Rubisco‐specific assembly chaperone,forms intermediary hetero‐oligomeric complexes,and is nonlimiting to growth in tobacco

The folding and assembly of Rubisco large and small subunits into L₈S₈ holoenzyme in chloroplasts involves many auxiliary factors, including the chaperone BSD2. Here we identify apparent intermediary Rubisco‐BSD2 assembly complexes in the model C₃ plant tobacco. We show BSD2 and Rubisco content decrease in tandem with leaf age with approximately half of the BSD2 in young leaves (~70 nmol BSD2 protomer.m²) stably integrated in putative intermediary Rubisco complexes that account for <0.2% of the L₈S₈ pool. RNAi‐silencing BSD2 production in transplastomic tobacco producing bacterial L₂ Rubisco had no effect on leaf photosynthesis, cell ultrastructure, or plant growth. Genetic crossing the same RNAi‐bsd2 alleles into wild‐type tobacco however impaired L₈S₈ Rubisco production and plant growth, indicating the only critical function of BSD2 is in Rubisco biogenesis. Agrobacterium mediated transient expression of tobacco, Arabidopsis, or maize BSD2 reinstated Rubisco biogenesis in BSD2‐silenced tobacco. Overexpressing BSD2 in tobacco chloroplasts however did not alter Rubisco content, activation status, leaf photosynthesis rate, or plant growth in the field or in the glasshouse at 20°C or 35°C. Our findings indicate BSD2 functions exclusively in Rubisco biogenesis, can efficiently facilitate heterologous plant Rubisco assembly, and is produced in amounts nonlimiting to tobacco growth.     

Magnesium and iron deficiencies alter Cd accumulation in Salix viminalis L.

Evidence exists that Cd and certain nutrient elements, such as Fe and Mg, could share similar mechanisms of plant uptake and accumulation. Here we report that Mg and Fe deficiency in mature plants of Salix viminalis, grown in hydroponic solutions containing 5 µg ml^?1 of Cd, caused a significant increase in Cd accumulation in roots, stems and leaves. Cd (µg g^?1 dry weight) was determined following three treatments: 1) Cd treatment in complete nutrient solution; 2) Cd treatment with Fe deficiency; and 3) Cd treatment with Mg deficiency, yielding, respectively: in young leaves (65.3, 76.1, and 92.2), mature leaves (51.5 to 76.3 and 87.1), upper stems (80.6, 116.8, and 130.6) lower stems (67.2, 119, and 102.3), roots (377.1, 744.8, and 442,5). Our results suggest that Cd utilizes the same uptake and transport pathways as Mg and Fe. Evidence exists that Mg and Fe uptake and translocation could be further facilitated by plants as an adaptive response to deficiency of these elements. Such physiological reaction could additionally stimulate Cd accumulation. Although Cd uptake was mostly confined in roots, high Cd content in aerial plant parts (51.5–130.6 µg g^?1) indicates that the analysed Salix viminalis genotype is suitable for phytoextraction.     

Early seedling growth and morphogenesis in the <Emphasis Type="Italic">xantha1</Emphasis> mutant of sunflower with alteration of chloroplast biogenesis

In the xantha1 (xan1) mutant of sunflower (Helianthus annuus L.), the effects on organ anatomy and seedling growth did correlate to the alteration of chloroplast biogenesis. The xan1 seedlings grown under 165 μmol(photon) m⁻² s⁻¹ revealed a severely altered chloroplast ultrastructure in cotyledons and leaves. Cross-sections or clarified tissues of the xan1 cotyledons did not show evident alterations with respect to normal cotyledons suggesting that the impairment of chloroplast biogenesis has negligible consequences on embryonic leaves. By contrast, the analysis of xan1 leaves showed that the defects in chloroplast biogenesis were correlated to a drastic reduction of organ size and to a clear enhancement of the trichome growth. The differentiation of palisade and spongy parenchyma in cotyledons and leaves of the xan1 mutant was normal but both organs displayed a drastic reduction in the plastid number with respect to wild type. In addition, xan1 hypocotyls showed a reduced development of the main vascular bundles in comparison with normal seedlings and an undersized central cylinder of the primary root. The exogenous supply of sucrose was not sufficient to revert in vitro the deficit of xan1 growth and the constraints in morphogenetic processes.     

Characterization of pigment apparatus in winter-green and summer-green leaves of a shade-tolerant plant <Emphasis Type="Italic">Ajuga reptans</Emphasis>

Comparative study was performed to assess the content and proportions of photosynthetic pigments and the violaxanthin cycle (VXC) activity in winter-green and summer-green leaves of bugleweed (Ajuga reptans L.) plants grown in shaded (photosynthetically active radiation, PAR 150 μmol/(m² s)) and sunny (PAR 1200 μmol/(m² s)) habitats in the Botanic Garden of Jagiellonian University (Krakow, Poland). In overwintered and newly formed leaves of shade plants, the content of green and yellow pigments was two times higher than in leaves of sun plants. The shade plants were distinguished by accumulation of β-carotene, while lutein was predominant in leaves of sun plants. Under the action of strong light (2000 μmol/(m²s)), the level of violaxanthin deepoxidation in winter-green leaves of shade and sun plants increased five- to sixfold, whereas it changed insignificantly in summer-green leaves of shade plants. It is concluded that, in a shadetolerant species A. reptans, the photosynthetic apparatus of winter-green leaves in sun and shade plants and of summer-green leaves in sun plants is protected against excess insolation by high activity of VXC. The carotenoids of summer-green leaves in shade plants are supposed to function mainly as light-harvesting pigments.     

<Emphasis Type="Italic">Sonic hedgehog</Emphasis> mutations identified in holoprosencephaly patients can act in a dominant negative manner

Sonic hedgehog (SHH) plays an important instructional role in vertebrate development, as exemplified by the numerous developmental disorders that occur when the SHH pathway is disrupted. Mutations in the SHH gene are the most common cause of sporadic and inherited holoprosencephaly (HPE), a developmental disorder that is characterized by defective prosencephalon development. SHH HPE mutations provide a unique opportunity to better understand SHH biogenesis and signaling, and to decipher its role in the development of HPE. Here, we analyzed a panel of SHH HPE missense mutations that encode changes in the amino-terminal active domain of SHH. Our results show that SHH HPE mutations affect SHH biogenesis and signaling at multiple steps, which broadly results in low levels of protein expression, defective processing of SHH into its active form and protein with reduced activity. Additionally, we found that some inactive SHH proteins were able to modulate the activity of wt SHH in a dominant negative manner, both in vitro and in vivo. These findings show for the first time the susceptibility of SHH driven developmental processes to perturbations by low-activity forms of SHH. In conclusion, we demonstrate that SHH mutations found in HPE patients affect distinct steps of SHH biogenesis to attenuate SHH activity to different levels, and suggest that these variable levels of SHH activity might contribute to some of the phenotypic variation found in HPE patients. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. S. Singh, R. Tokhunts and V. Baubet contributed equally to this work.     

Reduced naïve CD8+ T‐cell priming efficacy in elderly adults

Aging is associated with impaired vaccine efficacy and increased susceptibility to infectious and malignant diseases. CD8⁺ T‐cells are key players in the immune response against pathogens and tumors. In aged mice, the dwindling naïve CD8⁺ T‐cell compartment is thought to compromise the induction of de novo immune responses, but no experimental evidence is yet available in humans. Here, we used an original in vitro assay based on an accelerated dendritic cell coculture system in unfractioned peripheral blood mononuclear cells to examine CD8⁺ T‐cell priming efficacy in human volunteers. Using this approach, we report that old individuals consistently mount quantitatively and qualitatively impaired de novo CD8⁺ T‐cell responses specific for a model antigen. Reduced CD8⁺ T‐cell priming capacity in vitro was further associated with poor primary immune responsiveness in vivo. This immune deficit likely arises as a consequence of intrinsic cellular defects and a reduction in the size of the naïve CD8⁺ T‐cell pool. Collectively, these findings provide new insights into the cellular immune insufficiencies that accompany human aging.     

Cadmium Accumulation in Relation to Organic Acids and Nonprotein Thiols in Leaves of the Recently Found Cd Hyperaccumulator <Emphasis Type="Italic">Rorippa globosa</Emphasis> and the Cd-accumulating Plant <Emphasis Type="Italic">Rorippa islandica</Emphasis>

It has been proposed that organic acid and nonprotein thiol are involved in the hyperaccumulation of metals. In this study, Cd accumulation, organic acid, and nonprotein thiol production and their relationships in the leaves of Cd-hyperaccumulator Rorippa globosa were examined and compared with a closely related species, Rorippa islandica. The results showed that there was no reduction in biomass of R. globosa when treated with 25 μg Cd g⁻¹ (T₂), despite Cd accumulation in the leaves was up to 158.2 μg g⁻¹ DW. On the other hand, the growth of Cd-treated R. islandica was obviously inhibited as it accumulated more than 100 μg g⁻¹ DW in the leaves. Therefore, R. islandica behaved as a Cd-accumulating plant. The Cd treatments could significantly induce the synthesis of acetic acid in both species, suggesting that acetic acid, as the most abundant organic acid, might be related to the Cd accumulation. Significant positive correlations between Cd concentrations and both tartaric and malic acid concentrations in the leaves of R. globosa were observed. There was a significant positive correlation between Cd concentrations and acetic acid concentrations in the leaves of R. islandica. This trend of tartaric and malic acids in the leaves of R. globosa and acetic acid in the leaves of R. islandica might be related to Cd accumulation. In addition, a quadratic relationship was obviously observed for NP-SH contents and total Cd concentrations in the leaves of R. globosa, indicating that NP-SH was significantly related to Cd accumulation and tolerance.     

Fate of 13C in metabolic pathways and effects of high CO2 on the alteration of metabolites in Rumex obtusifolius L.

In present study, in vivo turn-over of ¹³CO₂ to organic acids such as oxalate and citrate in Rumex obtusifolius L. was explored. Conversion of fixed carbon to oxalate was studies using “new leaves”, i.e., leaves removed from 2-month-old-plants grown under different environmental conditions. Collected new leaves and stems were subjected to metabolomic analyses using capillary electrophoresis mass spectrometry. The results showed the mobilization of metabolites from stems to new leaves, where active TCA cycle and oxalate pathways occurred. The ¹³C labeling experiments also indicated that these pathways are active in new leaves. Subsequently, we compared the effects of high carbon dioxide level (1000 ppm) and nutrients (Hoagland’s formulation) on the metabolite accumulation in R. obtusifolius. Data analysed by both principal component and hierarchical clustering analyses revealed significant changes in metabolite accumulation. The accumulation of most abundant metabolite oxalate in leaves was affected by both high CO₂ as the carbon source and nutrients. We suggest that the common weed R. obtusifolius may proliferate in cultivated lands under high CO₂ level, a potential cause of global warming.     

Growth and ion uptake in <Emphasis Type="Italic">Annona muricata</Emphasis> and <Emphasis Type="Italic">A. squamosa</Emphasis> subjected to salt stress

The effects of treatment with NaCl (3, 100 and 300 mM) for 1, 2, 3 and 7 d on plant growth and ion accumulation were analyzed in 2-week and 8-week-old Annona muricata and A. squamosa plants. Fresh mass and root growth inhibition were directly related to the increase in salinity, particularly for A. squamosa. Two-weeks old seedlings were sensitive to 100 and 300 mM NaCl particularly after 7 d, whereas 8-week-old plants were shown to be more resistant to NaCl even at 300 mM NaCl. Na⁺ and Cl^– mostly accumulated in young leaves. Our results suggest that A. squamosa is more sensitive than A. muricata to salt stress and that older seedlings of both species are more tolerant than younger seedlings.     

Differential usage of photoreceptors for chalcone synthase gene expression during plant development

Photoregulation of chalcone synthase (CHS) mRNA accumulation was analysed in parsley (Petroselinum crispum) and mustard (Sinapis alba) plants at different developmental stages. In both species, CHS mRNA accumulation in young etiolated seedlings was primarily under phytochrome control. In leaves of adult re-etiolated plants, a UV-B photoreceptor was predominantly involved in photocontrol. The reduced red light control in mature leaves was not due to the absence of immunoreactive phytochrome. The apparent dependence of photoreceptor usage on the developmental state of the cell or organism was in accordance with observations on the photoregulation of fusion constructs between CHS promoters from parsley or mustard and the β-glucuronidase reporter gene (GUS). When tested in the parsley protoplast transient expression system, both constructs yielded the same type of photoregulation as observed for the endogenous CHS gene.