Fine-tuning CRISPR/Cas9 gene editing in common bean ( L.) using a hairy root transformation system and prediction models.

The stable transformation of common bean is a challenging and time-consuming process. Although CRISPR/Cas9 has revolutionized gene editing with its high efficiency and specificity, the performance of the system can be affected by multiple factors, such as sgRNA specificity and effectiveness, and the choice of promoter used to drive Cas9 expression. The use of a hairy root transformation system to initially check the efficiency of sgRNAs and the impact of different promoters could speed up this process and increase the chances of success.

Impact of drought and salt stress on galactinol and raffinose family oligosaccharides in common bean ().

Due to climate change, farmers will face more extreme weather conditions and hence will need crops that are better adapted to these challenges. The raffinose family oligosaccharides (RFOs) could play a role in the tolerance of crops towards abiotic stress. To investigate this, we determined for the first time the importance of galactinol and RFOs in the roots and leaves of common bean under drought and salt stress conditions.

Genome-Wide Classification and Phylogenetic Analyses of the GDSL-Type Esterase/Lipase (GELP) Family in Flowering Plants.

GDSL-type esterase/lipase (GELP) enzymes have key functions in plants, such as developmental processes, anther and pollen development, and responses to biotic and abiotic stresses. Genes that encode GELP belong to a complex and large gene family, ranging from tens to more than hundreds of members per plant species. To facilitate functional transfer between them, we conducted a genome-wide classification of GELP in 46 plant species.

The Hidden Potential of High-Throughput RNA-Seq Re-Analysis, a Case Study for DHDPS, Key Enzyme of the Aspartate-Derived Lysine Biosynthesis Pathway and Its Role in Abiotic and Biotic Stress Responses in Soybean.

DHDPS is a key enzyme in the aspartate-derived lysine biosynthesis pathway and an evident object of study for biofortification strategies in plants. DHDPS isoforms with novel regulatory properties in were demonstrated earlier and hypothesized to be involved in abiotic and biotic stress responses. Here, we present a phylogenetic analysis of the gene family in land plants which establishes the existence of a legume-specific class of DHDPS, termed DHDPS B-type, distinguishable from the DHDPS A-type commonly present in all land plants.

Transcriptome-Guided Identification of Pectin Methyl-Esterase-Related Enzymes and Novel Molecular Processes Effectuating the Hard-to-Cook Defect in Common Bean ( L.).

The hard-to-cook defect in common beans is dictated by the ability to achieve cell separation during cooking. Hydrolysis of pectin methyl-esters by the pectin methyl-esterase (PME) enzyme influences cell separation. However, the contributions of the PME enzyme and the cell wall to the hard-to-cook defect have not been studied using molecular tools.

Identification and Expression Analysis of the Genes Involved in the Raffinose Family Oligosaccharides Pathway of and .

Raffinose family oligosaccharides (RFO) play an important role in plants but are also considered to be antinutritional factors. A profound understanding of the galactinol and RFO biosynthetic gene families and the expression patterns of the individual genes is a prerequisite for the sustainable reduction of the RFO content in the seeds, without compromising normal plant development and functioning. In this paper, an overview of the annotation and genetic structure of all galactinol- and RFO biosynthesis genes is given for soybean and common bean.

Dehydrin ERD14 activates glutathione transferase Phi9 in Arabidopsis thaliana under osmotic stress.

Background: Fully intrinsically disordered plant dehydrin ERD14 can protect enzymes via its chaperone-like activity, but it was not formally linked with enzymes of the plant redox system yet. This is of particular interest, as the level of HO in Arabidopsis plants increases during osmotic stress, which can be counteracted by overexpression of ERD14.

Methods: The proteomic mass-spectrometry analysis of stressed plants was performed to find the candidates affected by ERD14.

New Phenotypes of Potato Co-induced by Mismatch Repair Deficiency and Somatic Hybridization.

As plants are sessile they need a very efficient system for repairing damage done by external or internal mutagens to their DNA. Mismatch repair (MMR) is one of the systems that maintain genome integrity and prevent homeologous recombination. In all eukaryotes mismatches are recognized by evolutionary conserved MSH proteins often acting as heterodimers, the constant component of which is MSH2.

Biotechnological Approaches for Biomass and Cardenolide Production in Digitalis purpurea L.

Digitalis purpurea L. is one of the main economically viable sources of cardenolides (cardiac glycosides) for the pharmaceutical industry. Nevertheless, production of cardenolides in plants grown by traditional agriculture is not always an efficient process and can be affected by biotic and abiotic factors.

Optimization of a gas chromatography-mass spectrometry method with methyl chloroformate derivatization for quantification of amino acids in plant tissue.

Rapid, easy and reliable quantification of amino acids is crucial in research on plant amino acid metabolism and nutritional improvement of crops via enrichment of essential amino acids. A recently reported analysis method, based on solid phase extraction (SPE), derivatization with methyl chloroformate and gas chromatography-mass spectrometry was optimized and tested on three-week-old Arabidopsis thaliana leaf tissues. Optimization of the SPE cleanup yielded recovery rates of minimum 95% for all amino acids (except arginine).

CENH3-GFP: a visual marker for gametophytic and somatic ploidy determination in Arabidopsis thaliana.

Background: The in vivo determination of the cell-specific chromosome number provides a valuable tool in several aspects of plant research. However, current techniques to determine the endosystemic ploidy level do not allow non-destructive, cell-specific chromosome quantification. Particularly in the gametophytic cell lineages, which are physically encapsulated in the reproductive organ structures, direct in vivo ploidy determination has been proven very challenging.

From the tumor-inducing principle to plant biotechnology and its importance for society.

This dialogue was held between the Guest Editors of the Special Issue on "Plant Transgenesis" of the Int. J. Dev.

Thirty years of transgenic research in plants.

In 1983, the first transgenic tissues and plants were generated by means of disarmed Agrobacterium tumefaciens strains, in which the oncogenes had been replaced by antibiotic resistance markers. Hence, this Special Issue of The International Journal of Developmental Biology celebrates 30 years of transgenic research in plants! Eminent scientists working in the field of plant transformation or plant biotechnology have contributed to this publication and reviewed the state of the art of their particular subdomain or summarized the importance of transgenic research in the discovery of new mechanisms and the establishment of an entirely new field, such as epigenetics.

T-DNA transfer and T-DNA integration efficiencies upon Arabidopsis thaliana root explant cocultivation and floral dip transformation.

T-DNA transfer and integration frequencies during Agrobacterium-mediated root explant cocultivation and floral dip transformations of Arabidopsis thaliana were analyzed with and without selection for transformation-competent cells. Based on the presence or absence of CRE recombinase activity without or with the CRE T-DNA being integrated, transient expression versus stable transformation was differentiated. During root explant cocultivation, continuous light enhanced the number of plant cells competent for interaction with Agrobacterium and thus the number of transient gene expression events.

Excision of a selectable marker gene in transgenic banana using a Cre/lox system controlled by an embryo specific promoter.

Antibiotic and herbicide resistance genes have been used in transgene technology as powerful selection tools. Nonetheless, once transgenic events have been obtained their presence is no longer needed and can even be undesirable. In this work, we have developed a system to excise the selectable marker and the cre recombinase genes from transgenic banana cv.

Regulatory mechanisms after short- and long-term perturbed lysine biosynthesis in the aspartate pathway: the need for isogenes in Arabidopsis thaliana.

The aspartate-derived amino acid pathway in plants is an intensively studied metabolic pathway, because of the biosynthesis of the four essential amino acids lysine, threonine, isoleucine and methionine. The pathway is mainly controlled by the key regulatory enzymes aspartate kinase (AK; EC 2.7.

Glutamate metabolism in plant disease and defense: friend or foe?

Plant glutamate metabolism (GM) plays a pivotal role in amino acid metabolism and orchestrates crucial metabolic functions, with key roles in plant defense against pathogens. These functions concern three major areas: nitrogen transportation via the glutamine synthetase and glutamine-oxoglutarate aminotransferase cycle, cellular redox regulation, and tricarboxylic acid cycle-dependent energy reprogramming. During interactions with pathogens, the host GM is markedly altered, leading to either a metabolic state, termed "endurance", in which cell viability is maintained, or to an opposite metabolic state, termed "evasion", in which the process of cell death is facilitated.

Medicago truncatula dihydrodipicolinate synthase (DHDPS) enzymes display novel regulatory properties.

Lysine biosynthesis in plants is tightly regulated by feedback inhibition of the end product on the first enzyme of the lysine-specific branch, dihydrodipicolinate synthase (DHDPS). Three complete DHDPS coding sequences and one partial sequence were obtained in Medicago truncatula via inverse PCR. Analysis of the MtDHDPS sequences indicated the presence of isozymes (MtDHDPS2 and MtDHDPS3) with multiple amino acid substitutions on positions previously shown to be involved in feedback inhibition and of residues important for catalytic activity, possibly affecting the enzymatic properties of these isoforms.

Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system.

Selectable marker genes are indispensable for efficient production of transgenic events, but are no longer needed after the selection process and may cause public concern and technological problems. Although several gene excision systems exist, few have been optimized for vegetatively propagated crops. Using a Cre-loxP auto-excision strategy, we obtained transgenic banana plants cv.

Influence of over-expression of cytosolic aspartate aminotransferase on amino acid metabolism and defence responses against Botrytis cinerea infection in Arabidopsis thaliana.

Arabidopsis possesses several genes encoding aspartate aminotransferase, which catalyzes the bidirectional conversion of aspartate into glutamate. These amino acids together with asparagine and glutamine play an important role in N storage and distribution. In addition, they act as precursors for other amino acids.

Evaluation of seven promoters to achieve germline directed Cre-lox recombination in Arabidopsis thaliana.

Site-specific recombination systems, such as Cre-lox from bacteriophage P1, have become very important tools for plant genome engineering. In many cases a constitutive promoter is used to express the recombinase gene. However, for certain research and commercial applications constitutive Cre-mediated recombination may not be desirable.

Marker-free transgenic plants through genetically programmed auto-excision.

We present here a vector system to obtain homozygous marker-free transgenic plants without the need of extra handling and within the same time frame as compared to transformation methods in which the marker is not removed. By introducing a germline-specific auto-excision vector containing a cre recombinase gene under the control of a germline-specific promoter, transgenic plants become genetically programmed to lose the marker when its presence is no longer required (i.e.

Tepary bean (Phaseolus acutifolius).

Phaseolus beans are among the major legumes for food consumption, especially in Latin America, Africa, and Asia. Tepary bean (Phaseolus acutifolius L. Gray) is one of the five cultivated species of the genus Phaseolus.

Light strongly promotes gene transfer from Agrobacterium tumefaciens to plant cells.

Light conditions during Agrobacterium-based plant transformation, the most routinely used method in plant genetic engineering, differ widely and, to our knowledge, have not been studied systematically in relation to transformation efficiency. Here, light effects were examined in two already optimized transformation procedures: coculture of Agrobacterium tumefaciens with callus from two genotypes of the crop plant Phaseolus acutifolius (tepary bean) and coculture of root segments from two ecotypes of Arabidopsis thaliana. Except for the light conditions during coculture, all steps followed established procedures.