Comparative approach for the isolation of genes involved in the osmotolerance of wheat

Improving drought tolerance of wheat is of great agronomical importance. Gene isolation techniques based on expression properties may provide new tools for breeders both in early characterization of new cultivars and in improving drought tolerance via molecular breeding. The main aim of our project is to isolate new drought activated genes which may serve both purposes. As a first step, a subtracted cDNA library was prepared, which represents the difference in the mRNA populations of wheat plantlets grown under 400 mOsm polyethylene-glycol derived osmotic stress versus plantlets grown in optimal conditions. By applying the subtraction approach, the resulted cDNA library becomes enriched in clones of differentially expressed genes including the ones of rare messages as well. This allows us to clone these genes, sequence them and, later, perform in silico analysis. Our first results indicate that the resulted library is enriched in clones coding for membrane associated channel proteins as well as abscisic acid and stress responsive ones

Overexpression of the NMig1 gene encoding a NudC domain protein enhances root growth and abiotic stress tolerance in Arabidopsis thaliana

The family of NudC proteins has representatives in all eukaryotes and plays essential evolutionarily conserved roles in many aspects of organismal development and stress response, including nuclear migration, cell division, folding and stabilization of other proteins. This study investigates an undescribed Arabidopsis homolog of the Aspergillus nidulans NudC gene, named NMig1 (for Nuclear Migration 1), which shares high sequence similarity to other plant and mammalian NudC-like genes. Expression of NMig1 was highly upregulated in response to several abiotic stress factors, such as heat shock, drought and high salinity. Constitutive overexpression of NMig1 led to enhanced root growth and lateral root development under optimal and stress conditions. Exposure to abiotic stress resulted in relatively weaker inhibition of root length and branching in NMig1-overexpressing plants, compared to the wild-type Col-0. The expression level of antioxidant enzyme-encoding genes and other stress-associated genes was considerably induced in the transgenic plants. The increased expression of the major antioxidant enzymes and greater antioxidant potential correlated well with the lower levels of reactive oxygen species (ROS) and lower lipid peroxidation. In addition, the overexpression of NMig1 was associated with strong upregulation of genes encoding heat shock proteins and abiotic stress-associated genes. Therefore, our data demonstrate that the NudC homolog NMig1 could be considered as a potentially important target gene for further use, including breeding more resilient crops with improved root architecture under abiotic stress

Brassinosteroids control meristem size by promoting cell cycle progression in Arabidopsis roots

Brassinosteroids (BRs) play crucial roles in plant growth and development. Previous studies have shown that BRs promote cell elongation in vegetative organs in several plant species, but their contribution to meristem homeostasis remains unexplored. Our analyses report that both loss- and gain-of-function BR-related mutants in Arabidopsis thaliana have reduced meristem size, indicating that balanced BR signalling is needed for the optimal root growth. In the BR-insensitive bri1-116 mutant, the expression pattern of the cell division markers CYCB1;1, ICK2/KRP2 and KNOLLE revealed that a decreased mitotic activity accounts for the reduced meristem size; accordingly, this defect could be overcome by the overexpression of CYCD3;1. The activity of the quiescent centre (QC) was low in the short roots of bri1-116, as reported by cell type-specific markers and differentiation phenotypes of distal stem cells. Conversely, plants treated with the most active BR, brassinolide, or mutants with enhanced BR signalling, such as bes1-D, show a premature cell cycle exit that results in early differentiation of meristematic cells, which also negatively influence meristem size and overall root growth. In the stem cell niche, BRs promote the QC renewal and differentiation of distal stem cells. Together, our results provide evidence that BRs play a regulatory role in the control of cell-cycle progression and differentiation in the Arabidopsis root meristem.Fil: González García, Mary Paz. Centre for Research in Agricultural Genomics. Molecular Genetics Department; EspañaFil: Vilarrasa Blasi, Josep. Centre for Research in Agricultural Genomics. Molecular Genetics Department; EspañaFil: Zhiponova, Miroslava. Ghent University. Department of Plant Biotechnology and Genetics; Bélgica. Vlaams Instituut voor Biotechnologie; BélgicaFil: Divol, Fanchon. Centre for Research in Agricultural Genomics. Molecular Genetics Department; EspañaFil: Mora Garcia, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina. Centre for Research in Agricultural Genomics. Molecular Genetics Department; EspañaFil: Russinova, Eugenia. Ghent University. Department of Plant Biotechnology and Genetics; Bélgica. Vlaams Instituut voor Biotechnologie; BélgicaFil: Caño Delgado, Ana I. Centre for Research in Agricultural Genomics. Molecular Genetics Department; Españ

COI1-dependent jasmonate signalling affects growth, metabolites production and cell wall protein composition in Arabidopsis

Background and Aims: Cultured cell suspensions have been the preferred model to study the apoplast as well as to monitor metabolic and cell cycle-related changes. Previous work showed that methyl jasmonate (MeJA) inhibits leaf growth in a CORONATINE INSENSITIVE 1 (COI1)-dependent manner, with COI1 being the jasmonate (JA) receptor. Here, the effect of COI1 overexpression on the growth of stably transformed arabidopsis cell cultures is described. Methods: Time-course experiments were carried out to analyse gene expression, and protein and metabolite levels. Key Results: Both MeJA treatment and the overexpression of COI1 modify growth, by altering cell proliferation and expansion. DNA content as well as transcript patterns of cell cycle and cell wall remodelling markers were altered. COI1 overexpression also increases the protein levels of OLIGOGALACTURONIDE OXIDASE 1, BETA-GLUCOSIDASE/ENDOGLUCANASES and POLYGALACTURONASE INHIBITING PROTEIN2, reinforcing the role of COI1 in mediating defence responses and highlighting a link between cell wall loosening and growth regulation. Moreover, changes in the levels of the primary metabolites alanine, serine and succinic acid of MeJA-treated Arabidopsis cell cultures were observed. In addition, COI1 overexpression positively affects the availability of metabolites such as β-alanine, threonic acid, putrescine, glucose and myo-inositol, thereby providing a connection between JA-inhibited growth and stress responses. Conclusions: This study contributes to the understanding of the regulation of growth and the production of metabolic resources by JAs and COI1. This will have important implications in dissecting the complex relationships between hormonal and cell wall signalling in plants. The work also provides tools to uncover novel mechanisms co-ordinating cell division and post-mitotic cell expansion in the absence of organ developmental control

Phylogenetic identification of Balkan endemic Stachys species and genomic stability during ex vitro conservation

The genus Stachys is one of the largest in the Lamiaceae family. Representatives of the genus are among the most ancient medicinal plants used in the ethnomedicine. The Balkan endemic species S. thracica, S. bulgarica and S. scardica are included in The Red Data Book of Bulgaria and due to their endangered status are scarcely studied. The aim of the present work was to examine the genetic status of these three endemic Stachys species during the process of their ex situ conservation. To gain information about their taxonomic position in the genus Stachys, we applied the DNA barcoding approach. Nuclear (ITS) and plastid (rbcL, matK and trnH‑psbA) DNA barcodes were generated and aligned with accessions available in the data base. In the constructed phylogenetic trees S. thracica was placed in a cluster together with S. alpina, S. germanica and S. cretica, while S. bulgarica and S. scardica were clustered with S. officinalis. The ex situ conservation was achieved by the initiation of in vitro shoot cultures and their subsequent adaptation in ex vitro conditions. To check the genomic stability of the plants during the acclimatisation from in vitro conditions to ex vitro, analysis by sequence-related amplified polymorphism (SRAP) markers was performed. No difference was detected between the SRAP profiles of in vitro cultivated and ex vitro adapted S. thracica and S. scardica plants. In S. bulgarica, only 0.4% fragment difference was detected. The obtained results indicated that the three Stachys species preserved their genetic stability during the process of in vitro multiplication, which is a prerequisite for conserved bioactive capacity

Catmint (Nepeta nuda L.) phylogenetics defined by nuclear and chloroplast DNA barcodes

Nepeta nuda L. (Lamiaceae) is a medicinal plant with a wide distribution in Europa and Asia. In Bulgaria, N. nuda is also known as “naked (or hairless) catmint”, which likely refers to the naked or sparse short hairy stem and leaves. This study aims to generate DNA barcodes for precise genetic discrimination and determination of phylogenetic position of the plant among other Nepeta species. To achieve this goal, we applied the DNA barcoding technique based on conserved nuclear (internal transcribed spacer/ITS) and chloroplast (rbcL, matK, trnH) DNA regions. The generated DNA barcode sequences were submitted to the Barcode of Life Data System (BOLD) database (https://www.boldsystems.org; accession number BUL002-22). The obtained N. nuda DNA barcodes and corresponding sequences of other Nepeta species available in the BOLD and NCBI database were utilized for taxonomic classification. ITS-derived sequences for Nepeta species were the most enriched in the database, and DNA fragments matching 47 Nepeta species were selected for construction of phylogenetic tree. The results show that N. nuda is clustered in a subclade together with N. sheilae, N. deflersiana, N. isaurica, N. congesta, N. heliotropifolia, N. schiraziana and N. cataria. The information in BOLD was also retrieved for rbcL and matK, and corresponded to 15 and 10 different Nepeta species, respectively. For trnH, only NCBI sequences corresponding to 6 different Nepeta species were found. Тhe three chloroplast markers highlighted the close relation of N. nuda to N. italica, N. tuberosa, N. cataria, N. grandiflora and N. hemsleyana. In conclusion, we suggest a DNA barcode system for genetic discrimination of N. nuda, which could assist its accurate taxonomic characterization