High-throughput seed quality analysis in faba bean: leveraging Near-InfraRed spectroscopy (NIRS) data and statistical methods.

Near-infrared spectroscopy (NIRS) provides a high-throughput phenotyping technique to assist breeding for improved faba bean seed quality. We combined chemical analysis of protein, oil content (and composition) with NIRS through chemometrics, employing Partial Least Squares (PLS), Elastic Net (EN), Memory-based Learning (MBL), and Bayes B (BB) as prediction models. Protein was the most reliably predicted trait (R2 = 0.96-0.98) across field trials, followed by oil (R2 = 0.82-0.86) and oleic acid (R2 = 0.31-0.68). Samples for training the models were selected using K-means clustering. The optimal statistical approach for prediction was compound-specific: PLS for protein (Root Mean Squared Error - RMSE = 0.46), BB for oil (RMSE = 0.067), and EN for oleic acid content (RMSE = 2.83). Reduced training set simulations revealed different effects on prediction accuracy depending on the model and compound. Several NIR regions were pinpointed as highly informative for the compounds, using the shrinkage and variable selection capabilities of EN and BB.

The challenge of breeding for reduced off-flavor in faba bean ingredients.

The growing interest in plant protein sources, such as pulses, is driven by the necessity for sustainable food production and climate change mitigation strategies. Faba bean (Vicia faba L.) is a promising protein crop for temperate climates, owing to its remarkable yield potential (up to 8 tonnes ha-1 in favourable growing conditions) and high protein content (~29% dry matter basis). Nevertheless, the adoption of faba bean protein in plant-based products that aim to resemble animal-derived counterparts is hindered by its distinctive taste and aroma, regarded as "off-flavors". In this review, we propose to introduce off-flavor as a trait in breeding programs by identifying molecules involved in sensory perception and defining key breeding targets. We discuss the role of lipid oxidation in producing volatile and non-volatile compounds responsible for the beany aroma and bitter taste, respectively. We further investigate the contribution of saponin, tannin, and other polyphenols to bitterness and astringency. To develop faba bean varieties with diminished off-flavors, we suggest targeting genes to reduce lipid oxidation, such as lipoxygenases (lox) and fatty acid desaturases (fad), and genes involved in phenylpropanoid and saponin biosynthesis, such as zero-tannin (zt), chalcone isomerase (chi), chalcone synthase (chs), ß-amyrin (bas1). Additionally, we address potential challenges, including the need for high-throughput phenotyping and possible limitations that could arise during the genetic improvement process. The breeding approach can facilitate the use of faba bean protein in plant-based food such as meat and dairy analogues more extensively, fostering a transition toward more sustainable and climate-resilient diets.

Structure of a post-translationally processed heterodimeric double-headed Kunitz-type serine protease inhibitor from potato.

Potato serine protease inhibitor (PSPI) constitutes about 22% of the total amount of proteins in potato tubers (cv. Elkana), making it the most abundant protease inhibitor in the plant. PSPI is a heterodimeric double-headed Kunitz-type serine protease inhibitor that can tightly and simultaneously bind two serine proteases by mimicking the substrate of the enzyme with its reactive-site loops. Here, the crystal structure of PSPI is reported, representing the first heterodimeric double-headed Kunitz-type serine protease inhibitor structure to be determined. PSPI has a ß-trefoil fold and, based on the structure, two reactive-site loops bearing residues Phe75 and Lys95 were identified.

Water Holding as Determinant for the Elastically Stored Energy in Protein-Based Gels.

To evaluate the importance of the water holding capacity for the elastically stored energy of protein gels, a range of gels were created from proteins from different origin (plant: pea and soy proteins, and animal: whey, blood plasma, egg white proteins, and ovalbumin) varying in network morphology set by the protein concentration, pH, ionic strength, or the presence of specific ions. The results showed that the observed positive and linear relation between water holding (WH) and elastically stored energy (RE) is generic for globular protein gels studied. The slopes of this relation are comparable for all globular protein gels (except for soy protein gels) whereas the intercept is close to 0 for most of the systems except for ovalbumin and egg white gels. The slope and intercept obtained allows one to predict the impact of tuning WH, by gel morphology or network stiffness, on the mechanical deformation of the protein-based gel. Addition of charged polysaccharides to a protein system leads to a deviation from the linear relation between WH and RE and this deviation coincides with a change in phase behavior.

Conformational stability of the potato serine protease inhibitor group.

The thermal unfolding of potato serine protease inhibitor (PSPI), the most abundant protease inhibitor group in potato tuber, was measured using far UV CD spectroscopy, fluorescence spectroscopy, and DSC. The results indicate that the thermal as well as the guanidinium-induced unfolding of PSPI occurs via a non-two-state mechanism in which at least one stable intermediate is present. Additionally, the occurrence of aggregation, especially at low scan rates, increases the apparent cooperativity of the unfolding and makes the system kinetically rather than thermodynamically controlled. Aggregate formation seems to occur via a specific mechanism of which PSPI in a tetrameric form is the end product and which may involve disulfide interchanges.

Structure and stability of the potato cysteine protease inhibitor group (cv. Elkana).

The conformational stability of potato cysteine protease inhibitor (PCPI), the second most abundant protease inhibitor group in potato tuber, was investigated at ambient temperature and upon heating using far- and near-UV circular dichroism spectroscopy, fluorescence spectroscopy, and differential scanning calorimetry (DSC). The PCPI isoforms investigated have a highly similar structure at both the secondary and the tertiary level. PCPI isoforms show structural properties similar to those of the potato serine protease inhibitor group and the Kunitz type soybean trypsin inhibitor, a known beta-II protein. Therefore, PCPI isoforms are also classified as members of the beta-II protein subclass. Results show that the thermal unfolding of PCPI isoforms does not follow a two-state mechanism and that at least one intermediate is present. The occurrence of this intermediate is most apparent in the thermal unfolding of PCPI 8.3 as indicated by the presence of two peaks in the DSC thermogram. Additionally, the formation of aggregates (>100 kDa), especially at low scan rates, increases the apparent cooperativity of the unfolding.

Tentative assignment of the potato serine protease inhibitor group as beta-II proteins based on their spectroscopic characteristics.

Potato serine protease inhibitor (PSPI) is the most abundant protease inhibitor group in potato tuber. The investigated PSPI isoforms have a highly similar structure at both the secondary and the tertiary level. From the results described, PSPI is classified as a beta-II protein based on (1) the presence in the near-UV spectra of sharp peaks, indicating a rigid and compact protein; (2) the sharp transition from the native to the unfolded state upon heating (only 6 degrees C) monitored by a circular dichroism signal at 222 nm; and (3) the similarity in secondary structure to soybean trypsin inhibitor, a known beta-II protein, as indicated by a similar far-UV CD spectrum and a similar amide I band in the IR spectrum. The conformation of PSPI was shown also to be stable at ambient temperature in the pH range 4-7.5. Upon lowering the pH to 3.0, some minor changes in the protein core occur, as observed from the increase of the intensity of the phenylalanine peak in the near-UV CD spectrum.

Structural characterization of potato protease inhibitor I (Cv. Bintje) after expression in Pichia pastoris.

In the present study the structural properties of potato protease inhibitor 1 (PI-1) were studied as a function of temperature to elucidate its precipitation mechanism upon heating. A cDNA coding for PI-1 from cv. Bintje was cloned and expressed in Pichia pastoris. Using the recombinant PI-1 it was suggested that PI-1 behaves as a hexameric protein rather than as a pentamer, as previously proposed in the literature. The recombinant protein seems either to have a predominantly unordered structure or to belong to the beta-II proteins. Differential scanning calorimetry analysis of PI-1 revealed that its thermal unfolding occurs via one endothermic transition in which the hexameric PI-1 probably unfolds, having a dimer instead of a monomer as cooperative unit. The transition temperature for the recombinant PI-1 was 88 degrees C. Similar results were obtained for a partially purified pool of native PI-1 from cv. Bintje.

The most abundant protease inhibitor in potato tuber (cv. Elkana) is a serine protease inhibitor from the Kunitz family.

The gene of the most abundant protease inhibitor in potato cv. Elkana was isolated and sequenced. The deduced amino acid sequence of this gene showed 98% identity with potato serine protease inhibitor (PSPI), a member of the Kunitz family. Therefore, the most abundant protease inhibitor was considered to be one of the isoforms of PSPI. The PSPI group represents approximately 22% of the total amount of proteins in potato cv. Elkana and is composed of seven different isoforms that slightly differ in isoelectric point. Antibodies were raised against the two most abundant isoforms of PSPI. The binding of these antibodies to PSPI isoforms and protease inhibitors from different groups of protease inhibitor in potato showed that approximately 70% of the protease inhibitors present in potato juice belong to the Kunitz family.

Chrysosporium lucknowense C1 arabinofuranosidases are selective in releasing arabinose from either single or double substituted xylose residues in arabinoxylans.

Two novel arabinofuranosidases, Abn7 and Abf3 from Chrysosporium lucknowense (C1), belonging to the glycoside hydrolase family 43 and 51 were purified and characterized. Abn7 is exclusively able to hydrolyze arabinofuranosyl residues at position O-3 of double substituted xylosyl residues in arabinoxylan-derived oligosaccharides, an activity rarely found thus far. Abf3 is able to release arabinose from position O-2 or O-3 of single substituted xyloses. Both enzymes performed optimal at pH 5.0 and 40°C. Combining Abn7 and Abf3 resulted in a synergistic increase in arabinose release from arabinoxylans. This synergistic effect is due to the action of Abf3 on the remaining arabinose residues at position O-2 on single substituted xylosyl residues resulting from the action of Abn7 on double substituted xylosyl residues. Arabinose release was further increased when an endo-1,4-ß-xylanase was present during digestion. The efficiency of these arabinohydrolases from C1 on insoluble arabinoxylan substrates is discussed.

Crystallization and preliminary X-ray crystallographic studies on a Kunitz-type potato serine protease inhibitor.

Interest in protease inhibitors has been renewed because of their potent activity in preventing carcinogenesis in a wide variety of in vivo and in vitro model systems. Potato tubers contain a wide range of such protease inhibitors. In cv. Elkana potato tubers, protease inhibitors represent about 50% of the total amount of soluble protein. Potato serine protease inhibitor (PSPI), one of the isoforms of the most abundant group of protease inhibitors, is a dimeric double-headed Kunitz-type inhibitor. No high-resolution structural information on this type of inhibitor has so far been obtained, as all currently known structures are of the monomeric single-headed or monomeric double-headed types. Crystals were grown in 0.1 M HEPES pH 7.5, 10% PEG 8000 and 8% ethylene glycol complemented with 9 mM 1-s-octyl-beta-D-thioglucoside or 0.1 M glycine. Data were collected from a single crystal under cryoconditions to 1.8 A resolution. The protein crystallized in space group P2(1), with unit-cell parameters a = 54.82, b = 93.92, c = 55.44 A, beta = 100.7 degrees; the scaling Rsym is 0.044 for 45,456 unique reflections.

In vivo uniform (15)N-isotope labelling of plants: using the greenhouse for structural proteomics.

Isotope labelling of proteins is important for progress in the field of structural proteomics. It enables the utilisation of the power of nuclear magnetic resonance spectroscopy (NMR) for the characterisation of the three-dimensional structures and corresponding dynamical features of proteins. The usual approach to obtain isotopically labelled protein molecules is by expressing the corresponding gene in bacterial or yeast host organisms, which grow on isotope-enriched media. This method has several drawbacks. Here, we demonstrate that it is possible to fully label a plant with (15)N-isotopes. The advantage of in vivo labelling of higher organisms is that all constituting proteins are labelled and become available as functional, post-translationally modified, correctly folded proteins. A hydroponics set-up was used to create the first example of a uniformly (15)N-labelled (> 98%) plant species, the potato plant (Solanum tuberosum L., cv. Elkana). Two plants were grown at low costs using potassium-[(15)N]-nitrate as the sole nitrogen source. At harvest time, a total of 3.6 kg of potato tubers and 1.6 kg of foliage, stolons and roots were collected, all of which were fully (15)N-labelled. Gram quantities of soluble (15)N-labelled proteins (composed mainly of the glycoprotein patatin and Kunitz-type protease inhibitors) were isolated from the tubers. NMR results on the complete proteome of potato sap and on an isolated protease inhibitor illustrate the success of the labelling procedure. The presented method of isotope labelling is easily modified to label other plants. Its envisioned impact in the field of structural proteomics of plants is discussed.