Mining microsatellite markers from public expressed sequence tags databases for the study of threatened plants

Background Simple Sequence Repeats (SSRs) are widely used in population genetic studies but their classical development is costly and time-consuming. The ever-increasing available DNA datasets generated by high-throughput techniques offer an inexpensive alternative for SSRs discovery. Expressed Sequence Tags (ESTs) have been widely used as SSR source for plants of economic relevance but their application to non-model species is still modest. Methods Here, we explored the use of publicly available ESTs (GenBank at the National Center for Biotechnology Information-NCBI) for SSRs development in non-model plants, focusing on genera listed by the International Union for the Conservation of Nature (IUCN). We also search two model genera with fully annotated genomes for EST-SSRs, Arabidopsis and Oryza, and used them as controls for genome distribution analyses. Overall, we downloaded 16 031 555 sequences for 258 plant genera which were mined for SSRsand their primers with the help of QDD1. Genome distribution analyses in Oryza and Arabidopsis were done by blasting the sequences with SSR against the Oryza sativa and Arabidopsis thaliana reference genomes implemented in the Basal Local Alignment Tool (BLAST) of the NCBI website. Finally, we performed an empirical test to determine the performance of our EST-SSRs in a few individuals from four species of two eudicot genera, Trifolium and Centaurea. Results We explored a total of 14 498 726 EST sequences from the dbEST database (NCBI) in 257 plant genera from the IUCN Red List. We identify a very large number (17 102) of ready-to-test EST-SSRs in most plant genera (193) at no cost. Overall, dinucleotide and trinucleotide repeats were the prevalent types but the abundance of the various types of repeat differed between taxonomic groups. Control genomes revealed that trinucleotide repeats were mostly located in coding regions while dinucleotide repeats were largely associated with untranslated regions. Our results from the empirical test revealed considerable amplification success and transferability between congenerics. Conclusions The present work represents the first large-scale study developing SSRs by utilizing publicly accessible EST databases in threatened plants. Here we provide a very large number of ready-to-test EST-SSR (17 102) for 193 genera. The cross-species transferability suggests that the number of possible target species would be large. Since trinucleotide repeats are abundant and mainly linked to exons they might be useful in evolutionary and conservation studies. Altogether, our study highly supports the use of EST databases as an extremely affordable and fast alternative for SSR developing in threatened plants.

Heavy Metals in Crop Plants: Transport and Redistribution Processes on the Whole Plant Level

Copper, zinc, manganese, iron, nickel and molybdenum are essential micronutrients for plants. However, when present in excess they may damage the plant or decrease the quality of harvested plant products. Some other heavy metals such as cadmium, lead or mercury are not needed by plants and represent pollutants. The uptake into the roots, the loading into the xylem, the acropetal transport to the shoot with the transpiration stream and the further redistribution in the phloem are crucial for the distribution in aerial plant parts. This review is focused on long-distance transport of heavy metals via xylem and phloem and on interactions between the two transport systems. Phloem transport is the basis for the redistribution within the shoot and for the accumulation in fruits and seeds. Solutes may be transferred from the xylem to the phloem (e.g., in the small bundles in stems of cereals, in minor leaf veins). Nickel is highly phloem-mobile and directed to expanding plant parts. Zinc and to a lesser degree also cadmium are also mobile in the phloem and accumulate in meristems (root tips, shoot apex, axillary buds). Iron and manganese are characterized by poor phloem mobility and are retained in older leaves.

Selective Transport of Zinc, Manganese, Nickel, Cobalt and Cadmium in the Root System and Transfer to the Leaves in Young Wheat Plants

• Background and Aims The uptake, translocation and redistribution of the heavy metals zinc, manganese, nickel, cobalt and cadmium are relevant for plant nutrition as well as for the quality of harvested plant products. The long-distance transport of these heavy metals within the root system and the release to the shoot in young wheat (Triticum aestivum ‘Arina’) plants were investigated. • Methods After the application of 65Zn, 54Mn, 63Ni, 57Co and 109Cd for 24 h to one seminal root (the other seminal roots being excised) of 54-h-old wheat seedlings, the labelled plants were incubated for several days in hydroponic culture on a medium without radionuclides. • Key Results The content of 65Zn decreased quickly in the labelled part of the root. After the transfer of 65Zn from the roots to the shoot, a further redistribution in the phloem from older to younger leaves was observed. In contrast to 65Zn, 109Cd was released more slowly from the roots to the leaves and was subsequently redistributed in the phloem to the youngest leaves only at trace levels. The content of 63Ni decreased quickly in the labelled part of the root, moving to the newly formed parts of the root system and also accumulating transiently in the expanding leaves. The 54Mn content decreased quickly in the labelled part of the root and increased simultaneously in leaf 1. A strong retention in the labelled part of the root was observed after supplying 57Co. • Conclusions The dynamics of redistribution of 65Zn, 54Mn, 63Ni, 57Co and 109Cd differed considerably. The rapid redistribution of 63Ni from older to younger leaves throughout the experiment indicated a high mobility in the phloem, while 54Mn was mobile only in the xylem and 57Co was retained in the labelled root without being loaded into the xylem.

Dynamics of Sodium‐22 and Cesium‐134 in Young Wheat Plants

The dynamics of the two alkali metals sodium and cesium in crop plants are relevant in an ecological context. Redistribution processes for these elements in young wheat plants were investigated in the work reported here. Two days old wheat plants (Triticum aestivum L. cv. Arina) were fed for 24 h with sodium‐22 (22Na) and cesium‐134 (134Cs) via the main root and incubated afterwards in a culture room. Cesium‐134 accumulated in newly formed parts of the main root and in the expanding leaves during the first 20 days after labeling, while 22Na accumulated transiently in these plant parts, reached a peak and declined after a few days. A high percentage of 22Na was released from the roots to the medium. Total Na in leaves also accumulated transiently, but its highest accumulation appeared later than the peak of 22Na. Therefore, the distribution and retranslocation processes differ considerably for sodium and cesium in wheat plants. Such differences must be considered for the evaluation of environmental effects (e.g., release of pollutants into the environment) on the quality of harvested cereal products.

Biochemical changes in barley plants after excessive supply of copper and manganese

The present study was undertaken to identify changes in some important proteins involved in CO2 fixation (Rubisco, Rubisco activase (RA), Rubisco binding protein (RBP)), NH4+ assimilation (glutamine synthetase (GS) and glutamate synthase (GOGAT)), using immunoblotting, and in the antioxidative defense as a result of Cu or Mn excess in barley leaves (Hordeum vulgare L. cv. Obzor). Activities and isoenzyme patterns of superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and catalase (CAT), as well as the levels of ascorbate (ASC), non-protein sulfhydryl groups, hydrogen peroxide and oxidative damage to proteins were determined. Data were correlated to the accumulation of Cu or Mn in the leaves after 5 days supply of heavy metal (HM) excess in the nutrient solution. In the highest Cu excess (1500 μM), Rubisco LS and SS were reduced considerably whereas under the highest Mn concentrations (18,300 μM) only minor changes in Rubisco subunits were detected. The RBP was diminished under the highest concentrations of both Cu or Mn. The bands of RA changed differently comparing Cu and Mn toxicity. GS decreased and GOGAT was absent under the highest concentration of Cu. At Mn excess Fd-GOGAT diminished whereas GS was not apparently changed. The development of toxicity symptoms corresponded to an accumulation of Cu or Mn in the leaves and to a gradual increase in protein carbonylation, a lower SOD activity and elevated CAT and GPX activities. APX activity was diminished under Mn toxicity and was not changed under Cu excess. Generally, changes in the isoenzyme profiles were similar under both toxicities. An accumulation of H2O2 was observed only at Mn excess. Contrasting changes in the low-molecular antioxidants were detected when comparing both toxicities. Cu excess affected mainly the non-protein SH groups, while Mn influenced the ASC content. Oxidative stress under Cu or Mn toxicity was most probably the consequence of depletion in low-molecular antioxidants as a result of their involvement in detoxification processes and disbalance in antioxidative enzymes. The link between heavy metal accumulation in leaves, leading to different display of oxidative stress, and changes in individual chloroplast proteins is discussed in the article.

Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and glutathione

The coding sequence of the wild-type, cys-sensitive, cysE gene from Escherichia coli, which encodes an enzyme of the cysteine biosynthetic pathway, namely serine acetyltransferase (SAT, EC 2.3.1.30), was introduced into the genome of potato plants under the control of the cauliflower mosaic virus 35S promoter. In order to target the protein into the chloroplast, cysE was translationally fused to the 5′-signal sequence of rbcS from Arabidopsis thaliana. Transgenic plants showed a high accumulation of the cysE mRNA. The chloroplastic localisation of the E. coli SAT protein was demonstrated by determination of enzymatic activities in enriched organelle fractions. Crude leaf extracts of these plants exhibited up to 20-fold higher SAT activity than those prepared from wild-type plants. The transgenic potato plants expressing the E. coli gene showed not only increased levels of enzyme activity but also exhibited elevated levels of cysteine and glutathione in leaves. Both were up to twofold higher than in control plants. However, the thiol content in tubers of transgenic lines was unaffected. The alterations observed in leaf tissue had no effect on the expression of O-acetylserine(thiol)-lyase, the enzyme which converts O-acetylserine, the product of SAT, to cysteine. Only a minor effect on its enzymatic activity was observed. In conclusion, the results presented here demonstrate the importance of SAT in plant cysteine biosynthesis and show that production of cysteine and related sulfur-containing compounds can be enhanced by metabolic engineering.

Effect of mannitol and cold treatments on phosphate uptake and protein phosphorylation in Lemna minor (L.) plants

This report is aimed at elucidating the effect of mannitol and cold treatments on P uptake and protein phosphorylation in Lemna minor plants. Duckweed p lants were incu bated in the presence of [32P]or [33P]Pi in half-strength phosphate deprived E-medium under constant light regime for 1.5 h. Total plant protein extracts (pellet and supernatant) were then prepared and subjected to IEF x SDS-PAGE. To analyse the effect of the stresses on P uptake and protein labelling, Lemna minor plants were preincubated with 0.1, 0.5 mol · L-1 mannitol and at 4°C respectively, for 4 hours, before adding labelled orthophosphate. The results show that the general protein phosphorylation (including LHCII) is related to the level of P uptake. Radioactive phosphate incorporation is stimulated by a low concentration of mannitol (0.1 mol · L-1) but reduced by 0.5 mol · L-1 mannitol and cold stress in planta. The labelling into proteins is affected neither when stresses were applied to the plants after incubation with labelled orthophosphate, nor after in vitro protein phosphorylation. This indicates that general protein kinase activities in vivo are strictly limited by P uptake. A marked accumulation of soluble hexoses (mainly sucrose, glucose, and fructose) is observed under imposed stress, suggesting that the inhibition of P uptake in response to hyperosmotic and cold stresses is mediated by sugar accumulation in situ. However, metabolisable sugars like glucose did not alter the entry of phosphate at concentrations of 0.5 mol · L-1, showing that the chemical nature of the osmoticum influences P uptake.

Sulfate assimilation in higher plants. Characterization of a stable intermediate in the adenosine 5′-phosphosulfate reductase reaction

The enzyme catalysing the reduction of adenosine 5′-phosphosulfate (AdoPS) to sulfite in higher plants, AdoPS reductase, is considered to be the key enzyme of assimilatory sulfate reduction. In order to address its reaction mechanism, the APR2 isoform of this enzyme from Arabidopsis thaliana was overexpressed in Escherichia coli and purified to homogeneity. Incubation of the enzyme with [35S]AdoPS at 4 °C resulted in radioactive labelling of the protein. Analysis of APR2 tryptic peptides revealed 35SO2–3 bound to Cys248, the only Cys conserved between AdoPS and prokaryotic phosphoadenosine 5′-phosphosulfate reductases. Consistent with this result, radioactivity could be released from the protein by incubation with thiols, inorganic sulfide and sulfite. The intermediate remained stable, however, after incubation with sulfate, oxidized glutathione or AdoPS. Because truncated APR2, missing the thioredoxin-like C-terminal part, could be labelled even at 37 °C, and because this intermediate was more stable than the complete protein, we conclude that the thioredoxin-like domain was required to release the bound SO2–3 from the intermediate. Taken together, these results demonstrate for the first time the binding of 35SO2–3 from [35S]AdoPS to AdoPS reductase and its subsequent release, and thus contribute to our understanding of the molecular mechanism of AdoPS reduction in plants.

A fungal endophyte helps plants to tolerate root herbivory through changes in gibberellin and jasmonate signaling

Plant–microbe mutualisms can improve plant defense, but the impact of root endophytes on below-ground herbivore interactions remains unknown. We investigated the effects of the root endophyte Piriformospora indica on interactions between rice (Oryza sativa) plants and its root herbivore rice water weevil (RWW; Lissorhoptrus oryzophilus), and how plant jasmonic acid (JA) and GA regulate this tripartite interaction. Glasshouse experiments with wild-type rice and coi1-18 and Eui1-OX mutants combined with nutrient, jasmonate and gene expression analyses were used to test: whether RWW adult herbivory above ground influences subsequent damage caused by larval herbivory below ground; whether P. indica protects plants against RWW; and whether GA and JA signaling mediate these interactions. The endophyte induced plant tolerance to root herbivory. RWW adults and larvae acted synergistically via JA signaling to reduce root growth, while endophyte-elicited GA biosynthesis suppressed the herbivore-induced JA in roots and recovered plant growth. Our study shows for the first time the impact of a root endophyte on plant defense against below-ground herbivores, adds to growing evidence that induced tolerance may be an important root defense, and implicates GA as a signal component of inducible plant tolerance against biotic stress.

Taking Transgenic Plants with a Pinch of Salt

Making plants resistant to salty environments would be a boon for developing countries where poor land management has rendered large areas of arable land unfit for crop production. In a Perspective, Frommer and colleagues discuss how genetic engineering can be used to confer salt tolerance on plants ( see Apse et al.) and explore the implications of this feat for improving crop production in developing countries.

Genetic diversity and differentiation follow secondary succession in a multi-species study on woody plants from subtropical China

Aims: Species diversity and genetic diversity may be affected in parallel by similar environmental drivers. However, genetic diversity may also be affected independently by habitat characteristics. We aim at disentangling relationships between genetic diversity, species diversity and habitat characteristics of woody species in subtropical forest. Methods: We studied 11 dominant tree and shrub species in 27 plots in Gutianshan, China, and assessed their genetic diversity (Ar) and population differentiation (F’ST) with microsatellite markers. We tested if Ar and population specific F’ST were correlated to local species diversity and plot characteristics. Multi-model inference and model averaging were used to determine the relative importance of each predictor. Additionally we tested for isolation-by-distance and isolation-by-elevation by regressing pairwise F’ST against pairwise spatial and elevational distances. Important findings: Genetic diversity was not related to species diversity for any of the study species. Thus, our results do not support joint effects of habitat characteristics on these two levels of biodiversity. Instead, genetic diversity in two understory shrubs, Rhododendron simsii and Vaccinium carlesii, was affected by plot age with decreasing genetic diversity in successionally older plots. Population differentiation increased with plot age in Rhododendron simsii and Lithocarpus glaber. This shows that succession can reduce genetic diversity within, and increase genetic diversity between populations. Furthermore, we found four cases of isolation-by-distance and two cases of isolation-by-elevation. The former indicates inefficient pollen and seed dispersal by animals whereas the latter might be due to phenological asynchronies. These patterns indicate that succession can affect genetic diversity without parallel effects on species diversity and that gene flow in a continuous subtropical forest can be restricted even at a local scale.

Amino acid transport in plants

Amino acids are transported between different organs through both xylem and phloem. This redistribution of nitrogen and carbon requires the activity of amino acid transporters in the plasma membrane. In addition, amino acids can be taken up directly by the roots. Amino acid transport has been well characterized in the yeast Saccharomyces cerevisiae, and functional complementation has served as an excellent tool for identifying and characterizing amino acid transporters from plants. The transporters from yeast and plants are related and can be grouped into two large superfamilies. Based on substrate specificity and affinity, as well as expression patterns in plants, different functions have been assigned to some of the individual transporters. Plant mutants for amino acid transporter genes are now being used to study the physiological functions of many of the cloned genes.

Medicinal plants - prophylactic and therapeutic options for gastrointestinal and respiratory diseases in calves and piglets? A systematic review.

BACKGROUND Gastrointestinal and respiratory diseases in calves and piglets lead to significant economic losses in livestock husbandry. A high morbidity has been reported for diarrhea (calves ≤ 35 %; piglets ≤ 50 %) and for respiratory diseases (calves ≤ 80 %; piglets ≤ 40 %). Despite a highly diverse etiology and pathophysiology of these diseases, treatment with antimicrobials is often the first-line therapy. Multi-antimicrobial resistance in pathogens results in international accordance to strengthen the research in novel treatment options. Medicinal plants bear a potential as alternative or additional treatment. Based on the versatile effects of their plant specific multi-component-compositions, medicinal plants can potentially act as 'multi-target drugs'. Regarding the plurality of medicinal plants, the aim of this systematic review was to identify potential medicinal plant species for prevention and treatment of gastrointestinal and respiratory diseases and for modulation of the immune system and inflammation in calves and piglets. RESULTS Based on nine initial sources including standard textbooks and European ethnoveterinary studies, a total of 223 medicinal plant species related to the treatment of gastrointestinal and respiratory diseases was identified. A defined search strategy was established using the PRISMA statement to evaluate 30 medicinal plant species starting from 20'000 peer-reviewed articles published in the last 20 years (1994-2014). This strategy led to 418 references (257 in vitro, 84 in vivo and 77 clinical trials, thereof 48 clinical trials in veterinary medicine) to evaluate effects of medicinal plants and their efficacy in detail. The findings indicate that the most promising candidates for gastrointestinal diseases are Allium sativum L., Mentha x piperita L. and Salvia officinalis L.; for diseases of the respiratory tract Echinacea purpurea (L.) MOENCH, Thymus vulgaris L. and Althea officinalis L. were found most promising, and Echinacea purpurea (L.) MOENCH, Camellia sinensis (L.) KUNTZE, Glycyrrhiza glabra L. and Origanum vulgare L. were identified as best candidates for modulation of the immune system and inflammation. CONCLUSIONS Several medicinal plants bear a potential for novel treatment strategies for young livestock. There is a need for further research focused on gastrointestinal and respiratory diseases in calves and piglets, and the findings of this review provide a basis on plant selection for future studies.