Andreas Blöchl

Stoichiometric controls of nitrogen and phosphorus cycling in decomposing beech leaf litter

Resource stoichiometry (C:N:P) is an important determinant of litter decomposition. However, the effect of elemental stoichiometry on the gross rates of microbial N and P cycling processes during litter decomposition is unknown. In a mesocosm experiment, beech (Fagus sylvatica L.) litter with natural differences in elemental stoichiometry (C:N:P) was incubated under constant environmental conditions. After three and six months, we measured various aspects of nitrogen and phosphorus cycling. We found that gross protein depolymerization, N mineralization (ammonification), and nitrification rates were negatively related to litter C:N. Rates of P mineralization were negatively correlated with litter C:P. The negative correlations with litter C:N were stronger for inorganic N cycling processes than for gross protein depolymerization, indicating that the effect of resource stoichiometry on intracellular processes was stronger than on processes catalyzed by extracellular enzymes. Consistent with this, extracellular protein depolymerization was mainly limited by substrate availability and less so by the amount of protease. Strong positive correlations between the interconnected N and P pools and the respective production and consumption processes pointed to feed-forward control of microbial litter N and P cycling. A negative relationship between litter C:N and phosphatase activity (and between litter C:P and protease activity) demonstrated that microbes tended to allocate carbon and nutrients in ample supply into the production of extracellular enzymes to mine for the nutrient that is more limiting. Overall, the study demonstrated a strong effect of litter stoichiometry (C:N:P) on gross processes of microbial N and P cycling in decomposing litter; mineralization of N and P were tightly coupled to assist in maintaining cellular homeostasis of litter microbial communities.

Preparation of starch and soluble sugars of plant material for the analysis of carbon isotope composition: a comparison of methods

Starch and soluble sugars are the major photosynthetic products, and their carbon isotope signatures reflect external versus internal limitations of CO(2) fixation. There has been recent renewed interest in the isotope composition of carbohydrates, mainly for use in CO(2) flux partitioning studies at the ecosystem level. The major obstacle to the use of carbohydrates in such studies has been the lack of an acknowledged method to isolate starch and soluble sugars for isotopic measurements. We here report on the comparison and evaluation of existing methods (acid and enzymatic hydrolysis for starch; ion-exchange purification and compound-specific analysis for sugars). The selectivity and reproducibility of the methods were tested using three approaches: (i) an artificial leaf composed of a mixture of isotopically defined compounds, (ii) a C(4) leaf spiked with C(3) starch, and (iii) two natural plant samples (root, leaf). Starch preparation methods based on enzymatic or acid hydrolysis did not yield similar results and exhibited contaminations by non-starch compounds. The specificity of the acidic hydrolysis method was especially low, and we therefore suggest terming these preparations as HCl-hydrolysable carbon, rather than starch. Despite being more specific, enzyme-based methods to isolate starch also need to be further optimized to increase specificity. The analysis of sugars by ion-exchange methods (bulk preparations) was fast but produced more variable isotope compositions than compound-specific methods. Compound-specific approaches did not in all cases correctly reproduce the target values, mainly due to unsatisfactory separation of sugars and background contamination. Our study demonstrates that, despite their wide application, methods for the preparation of starch and soluble sugars for the analysis of carbon isotope composition are not (yet) reliable enough to be routinely applied and further research is urgently needed to resolve the identified problems.

Short-term dynamics of nonstructural carbohydrates and hemicelluloses in young branches of temperate forest trees during bud break

Nonstructural carbohydrates (NSC) are the most important C reserves in the tissues of deciduous and evergreen tree species. Besides NSC, cell-wall hemicelluloses as the second most abundant polysaccharides in plants have often been discussed to serve as additional mobile carbon (C) reserves during periods of enhanced carbon-sink activities. To assess the significance of hemicelluloses as mobile carbon reserves, branches of two deciduous (Carpinus betulus L. and Fagus sylvatica L.) and two evergreen (Picea abies L. and Pinus sylvestris L.) tree species were sampled in a mature mixed forest stand in short intervals before and during bud break to assess NSC and hemicellulose concentrations in response to the increased carbon demand during bud break. Starch concentrations in branch sapwood of deciduous trees strongly decreased immediately before bud break and increased after bud break. In both evergreen species, only small changes of NSC were found in branch sapwood. However, 1-year-old needles exhibited a significant increase in starch concentration shortly before bud break which declined again after flushing. Hemicellulose concentrations (on an NSC-free dry matter basis) in branch sapwood of Carpinus decreased significantly shortly before bud break, but increased again after bud break. Contrarily, in Fagus branch sapwood, hemicellulose concentrations remained constant during bud break. Moderate increases of total hemicellulose concentrations before bud break were found in 1-year-old needles of both conifers, which could be explained by an accumulation of glucose units within the hemicellulose fraction. Overall, cell-wall hemicelluloses appeared to respond in a species-specific manner to the enhanced carbon demand during bud break. Hemicelluloses in branch sapwood of Carpinus and in 1-year-old needles of conifers likely act as additional carbon reserves similar to starch.

Starch Serves as Carbohydrate Storage in Nematode-Induced Syncytia

The plant parasitic nematode Heterodera schachtii induces specific syncytial feeding sites in the roots of Arabidopsis thaliana from where it withdraws all required nutrients. Therefore, syncytia have to be well supplied with assimilates and generate strong sinks in the host plants transport system. Import mechanisms and consequent accumulation of sucrose in syncytia were described recently. In this work, we studied the starch metabolism of syncytia. Using high-performance liquid chromatography and microscopic analyses, we demonstrated that syncytia store carbohydrates by starch accumulation. Further, we monitored the expression of genes involved in the starch metabolic pathway by gene chip analysis and quantitative reverse transcription-PCR. Finally, we provide functional proof of the importance of starch synthesis for nematode development using T-DNA insertion lines. We conclude that syncytia accumulate starch as a carbohydrate buffer to compensate for changing solute uptake by the nematode and as long-term storage during juvenile development.

Enzymatic breakdown of raffinose oligosaccharides in pea seeds

Both alkaline and acidic α-galactosidases (α-d-galactoside galactohydrolases, E.C.3.2.1.22) isolated from various plant species have been described, although little is known about their co-occurrence and functions in germinating seeds. Here, we report on the isolation of two cDNAs, encoding for α-galactosidases from maturing and germinating seeds of Pisum sativum. One was identified as a member of the acidic α-galactosidase of the family 27 glycosyl hydrolase cluster and the other as a member of the family of alkaline α-galactosidases, which are highly homologous to seed imbibition proteins (SIPs). PsGAL1 transcripts, encoding for the ACIDIC α-GALACTOSIDASE, were predominately expressed during seed maturation and acidic enzyme activities were already present in dry seeds, showing little changes during seed germination. Compartmentation studies revealed that acidic α-galactosidases were located in protein storage vacuoles (PSVs). PsAGA1, encoding for the ALKALINE α-GALACTOSIDASE, was only expressed after radicle protrusion, when about 50% of RFOs have already been broken down. RFO breakdown was markedly decreased when the translation of the alkaline enzyme was inhibited, providing evidence that PsAGA1 indeed functioned in RFO degradation. Based on these data, we present an integrated model of RFO breakdown by two sequentially active α-galactosidases in pea seeds.