Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry

ABSTRACT: Understanding protein and gene function requires identifying interaction partners usingbiochemical, molecular or genetic tools. In plants, searching for novel protein-proteininteractions is limited to protein purification assays, heterologous in vivo systems such as the yeast-two-hybrid or mutant screens. Ideally one would be able to search for novel proteinpartners in living plant cells. We demonstrate that it is possible to screen for novel proteinproteininteractions from a random library in protoplasted Arabidopsis plant cells and recoversome of the interacting partners. Our screen is based on capturing the bi-molecularcomplementation of mYFP between an YN-bait fusion partner and a completely random preyYC-cDNA library with FACS. The candidate interactions were confirmed using in plantaBiFC assays and in planta FRET-FLIM assays. From this work, we show that the wellcharacterized protein Calcium Dependent Protein Kinase 3 (CPK3) interacts with APX3,HMGB5, ORP2A and a ricin B-related lectin domain containing protein At2g39050. This isone of the first random in planta screens to be successfully employed.     

硫酸铜,硝酸银和脱落酸对草地早熟禾胚性愈伤诱导的影响

试验以草地早熟禾4个品种的成熟种子为外植体,研究了CuSO4、AgNO3、脱落酸(ABA)对草地早熟禾胚性愈伤诱导的影响,结果表明,K3诱导培养基中添加CuSO4对草地早熟禾胚性愈伤组织的形成有促进作用,除DP192-10之外的3个品种都发生了明显的形态上的变化,DP-LF-300和2W42-117在添加5μmol/LCuSO4的培养基上诱导出了乳白色、致密的胚性愈伤组织,2W42-117的胚性愈伤率高达40%。在MS基本培养基中添加AgNO3、ABA对草地早熟禾4个品种的胚性愈伤组织的诱导没有作用。     

Development of an express method for measuring soil nitrate,phosphate, potassium,and pH for future in-field application

Background

In practical farming, there is often a need for short-term availability of information on the soil nutrient status.

Aims

To develop a new express method for the extraction of major plant-available nutrients and measurement of soil nutrients. In future, this method shall serve for in-field measurements of soil samples with an ion-sensitive field-effect transistor (ISFET).

Methods

Various extraction conditions such as type of extractant, soil-to-solution ratio, time, and intensity were investigated on a broad selection of dried soil samples in the laboratory. Based on 83 field-moist soil samples with varying clay contents, these conditions were compared to standard laboratory methods.

Results

With increasing extraction time, the nutrient concentrations increased. When the soil-to-solution ratio was reduced, a greater share of nutrients was extracted, independent of soil type. H₂O and 0.01 M CaCl₂ and standard calcium-acetate-lactate (CAL) solution proved to be too weak in the short period to reach the ISFET sensor measurement range. Higher concentrated CAL solutions performed much better. Finally, a 5-min CaCl₂ extraction followed by the removal of an aliquot for the determination of soil pH and NO₃⁻ was found to be effective. The remaining solution was then mixed with 0.20 M CAL solution for the analysis of H₂PO₄⁻ and K⁺ at 10 min of extra extraction time. This extraction method showed very good correlations with the values based on the German laboratory reference methods for pH (R² = 0.91) and for nitrate (R² = 0.95). For phosphorus and potassium, we obtained an R² of 0.70 and 0.81, respectively, for all soils. When soils were grouped according to clay content higher correlations were found.

Conclusions

A new express method based on a wet-chemical approach with a soil preparation procedure was successfully developed and validated. This seems to be a valuable basis for future in-field measurements via ISFET.     

Effects of Carbohydrate-free Diets on the Performance of Lactating Mink (Mustela vison) and the Growth Performance of Suckling Kits

The effects of carbohydrate-free diets on body weight changes, feed consumption, and plasma concentrations of nutrients, metabolites, and hormones in lactating mink and on growth performance in kits were investigated in a total of 108 litters during the lactation period of seven weeks. The litters were distributed in three experimental groups fed different ratios of metabolisable energy (ME) derived from protein and fat (61:38; 47:52; 33:66). The females and kits were weighed at parturition and 2, 3, 4, and 7 weeks after parturition. Blood samples were collected from females in the second, fourth, and sixth week of the lactation period. Carbohydrate-free diets fed to lactating mink can in principle support normal performance, health of the females, and growth performance of the kits. However, mink need a sufficient supply of available gluconeogenic precursors in the form of amino acids. In the present experiment, the diet with 33% of ME from protein contained insufficient amounts of gluconeogenic precursors for lactating mink, and three weeks post partum this group was excluded from the experiment. The differences in dietary protein level (61% and 47% of ME) and fat level (38% and 52% of ME) influenced the plasma concentrations of free fatty acids (FFA) and urea, but had minimal influence on plasma concentrations of other nutrients, metabolites, and hormones.     

Physical carbon‐sequestration mechanisms under special consideration of soil wettability

The protective impact of aggregation on microbial degradation through separation has been described frequently, especially for biotically formed aggregates. However, to date little information exists on the effects of organic‐matter (OM) quantity and OM quality on physical protection, i.e., reduced degradability by microorganisms caused by physical factors. In the present paper, we hypothesize that soil wettability, which is significantly influenced by OM, may act as a key factor for OM stabilization as it controls the microbial accessibility for water, nutrients, and oxygen in three‐phase systems like soil. Based on this hypothesis, the first objective is to evaluate new findings on the organization of organo‐mineral complexes at the nanoscale as one of the processes creating water‐repellent coatings on mineral surfaces. The second objective is to quantify the degree of alteration of coated surfaces with regard to water repellence. We introduce a recently developed trial that combines FTIR spectra with contact‐angle data as the link between chemical composition of OM and the physical wetting behavior of soil particles. In addition to characterizing the wetting properties of OM coatings, we discuss the implications of water‐repellent surfaces for different physical protection mechanisms of OM. For typical minerals, the OM loading on mineral surfaces is patchy, whereas OM forms nanoscaled micro‐aggregates together with metal oxides and hydroxides and with layered clay minerals. Such small aggregates may efficiently stabilize OM against microbial decomposition. However, despite the patchy structure of OM coating, we observed a relation between the chemical composition of OM and wettability. A higher hydrophobicity of the OM appears to stabilize the organic C in soil, either caused by a specific reduced biodegradability of OM or indirectly caused by increased aggregate stability. In partly saturated nonaggregated soil, the specific distribution of the pore water appears to further affect the mineralization of OM as a function of wettability. We conclude that the wettability of OM, quantified by the contact angle, links the chemical structure of OM with a bundle of physical soil properties and that reduced wettability results in the stabilization of OM in soils.     

Improved RP‐HPLC and anion‐exchange chromatography methods for the determination of amino acids and carbohydrates in soil solutions

In spite of their low concentrations in soil solutions, low–molecular weight organic substances (LMWOS) such as amino acids, sugars, and uronic acids play a major role in the cycles of C and N in soil. With respect to their low concentrations and to possible matrix interferences, their analysis in soil leachates is a challenging task. We established two HPLC (high‐performance liquid chromatography) methods for the parallel determination of amino acids and carbohydrates in soil leachates. The pre‐column derivatization of amino acids with an o‐phthaldialdehyde (OPA) mercaptoethanol solution yields quantitation limits between 0.03 and 0.44 µmol L^–1 and S_D values of <8.3% (n = 9). High‐performance anion‐exchange chromatography (HPAEC) on a Dionex CarboPac PA 20 column with a NaOH acetate gradient combined with pulsed amperometric detection (PAD) was used for the determination of carbohydrates. The calibration curves obtained for 11 carbohydrates showed excellent linearity over the concentration range from 0.02 to 50.0 mg L^–1. Recovery studies revealed good results for all analytes (89%–108%). Interferences from Hg(II) salts and chloroform used for stabilization of the leachates did not occur with both chromatographic methods. The optimized method was successfully used for quantitative determinations of amino acids and carbohydrates in soil leachates.     

核苷酸营养(一):核苷酸在动物饲料中的使用

从本期起我们将陆续向读者推出核苷酸的营养作用。本期首先介绍核苷酸是怎样促进年轻动物的生长发育的。