MtNODULE ROOT1 and MtNODULE ROOT2 Are Essential for Indeterminate Nodule Identity.

Symbiotic interactions between legume plants and rhizobia result in the formation of nitrogen-fixing nodules, but the molecular actors and the mechanisms allowing for the maintenance of nodule identity are poorly understood. Medicago truncatula NODULE ROOT1 (MtNOOT1), Pisum sativum COCHLEATA1 (PsCOCH1), and Lotus japonicus NOOT-BOP-COCH-LIKE1 (LjNBCL1) are orthologs of Arabidopsis (Arabidopsis thaliana) AtBLADE-ON-PETIOLE1/2 and are members of the NBCL gene family, which has conserved roles in plant development and is essential for indeterminate and determinate nodule identity in legumes. The loss of function of MtNOOT1, PsCOCH1, and LjNBCL1 triggers a partial loss of nodule identity characterized by the development of ectopic roots arising from nodule vascular meristems. Here, we report the identification and characterization of a second gene involved in regulating indeterminate nodule identity in M. truncatula, MtNOOT2MtNOOT2 is the paralog of MtNOOT1 and belongs to a second legume-specific NBCL subclade, the NBCL2 clade. MtNOOT2 expression was induced during early nodule formation, and it was expressed primarily in the nodule central meristem. Mtnoot2 mutants did not present any particular symbiotic phenotype; however, the loss of function of both MtNOOT1 and MtNOOT2 resulted in the complete loss of nodule identity and was accompanied by drastic changes in the expression of symbiotic, defense, and root apical meristem marker genes. Mtnoot1 noot2 double mutants developed only nonfixing root-like structures that were no longer able to host symbiotic rhizobia. This study provides original insights into the molecular basis underlying nodule identity in legumes forming indeterminate nodules.

The Arabidopsis thaliana checkpoint kinase WEE1 protects against premature vascular differentiation during replication stress.

A sessile lifestyle forces plants to respond promptly to factors that affect their genomic integrity. Therefore, plants have developed checkpoint mechanisms to arrest cell cycle progression upon the occurrence of DNA stress, allowing the DNA to be repaired before onset of division. Previously, the WEE1 kinase had been demonstrated to be essential for delaying progression through the cell cycle in the presence of replication-inhibitory drugs, such as hydroxyurea. To understand the severe growth arrest of WEE1-deficient plants treated with hydroxyurea, a transcriptomics analysis was performed, indicating prolonged S-phase duration. A role for WEE1 during S phase was substantiated by its specific accumulation in replicating nuclei that suffered from DNA stress. Besides an extended replication phase, WEE1 knockout plants accumulated dead cells that were associated with premature vascular differentiation. Correspondingly, plants without functional WEE1 ectopically expressed the vascular differentiation marker VND7, and their vascular development was aberrant. We conclude that the growth arrest of WEE1-deficient plants is due to an extended cell cycle duration in combination with a premature onset of vascular cell differentiation. The latter implies that the plant WEE1 kinase acquired an indirect developmental function that is important for meristem maintenance upon replication stress.

Histone acetyltransferases in plant development and plasticity.

In eukaryotes, transcriptional regulation is determined by dynamic and reversible chromatin modifications, such as acetylation, methylation, phosphorylation, ubiquitination, glycosylation, that are essential for the processes of DNA replication, DNA-repair, recombination and gene transcription. The reversible and rapid changes in histone acetylation induce genome-wide and specific alterations in gene expression and play a key role in chromatin modification. Because of their sessile lifestyle, plants cannot escape environmental stress, and hence have evolved a number of adaptations to survive in stress surroundings. Chromatin modifications play a major role in regulating plant gene expression following abiotic and biotic stress. Plants are also able to respond to signals that affect the maintaince of genome integrity. All these factors are associated with changes in gene expression levels through modification of histone acetylation. This review focuses on the major types of genes encoding for histone acetyltransferases, their structure, function, interaction with other genes, and participation in plant responses to environmental stimuli, as well as their role in cell cycle progression. We also bring together the most recent findings on the study of the histone acetyltransferase HAC1 in the model legumes Medicago truncatula and Lotus japonicus.

Transcriptional and Metabolic Profiling of Arabidopsis thaliana Transgenic Plants Expressing Histone Acetyltransferase HAC1 upon the Application of Abiotic Stress-Salt and Low Temperature.

Augmented knowledge of plant responses upon application of stress could help improve our understanding of plant tolerance under abiotic stress conditions. Histone acetylation plays an important role in gene expression regulation during plant growth and development and in the response of plants to abiotic stress. The current study examines the level of transcripts and free metabolite content in transgenic Arabidopsis thaliana plants expressing a gene encoding histone acetyltransferase from Medicago truncatula (MtHAC1) after its heterologous expression. Stable transgenic plants with HAC1 gain and loss of function were constructed, and their T5 generation was used. Transgenic lines with HAC1-modified expression showed a deviation in root growth dynamics and leaf area compared to the wild-type control. Transcriptional profiles were evaluated after the application of salinity stress caused by 150 mM NaCl at four different time points (0, 24, 48, and 72 h) in treated and non-treated transgenic and control plants. The content and quantity of free metabolites-amino acids, mono- and dicarbohydrates, organic acids, and fatty acids-were assessed at time points 0 h and 72 h in treated and non-treated transgenic and control plants. The obtained transcript profiles of HAC1 in transgenic plants with modified expression and control were assessed after application of cold stress (low temperature, 4 °C).

A common F-box gene regulates the leucine homeostasis of Medicago truncatula and Arabidopsis thaliana.

The F-box domain is a conserved structural protein motif that most frequently interacts with the SKP1 protein, the core of the SCFs (SKP1-CULLIN-F-box protein ligase) E3 ubiquitin protein ligases. As part of the SCF complexes, the various F-box proteins recruit substrates for degradation through ubiquitination. In this study, we functionally characterized an F-box gene (MtF-box) identified earlier in a population of Tnt1 retrotransposon-tagged mutants of Medicago truncatula and its Arabidopsis thaliana homolog (AtF-box) using gain- and loss-of-function plants. We highlighted the importance of MtF-box in leaf development of M. truncatula. Protein-protein interaction analyses revealed the 2-isopropylmalate synthase (IPMS) protein as a common interactor partner of MtF-box and AtF-box, being a key enzyme in the biosynthesis pathway of the branched-chain amino acid leucine. For further detailed analysis, we focused on AtF-box and its role during the cell division cycle. Based on this work, we suggest a mechanism for the role of the studied F-box gene in regulation of leucine homeostasis, which is important for growth.

A replication stress-induced synchronization method for Arabidopsis thaliana root meristems.

Synchronized cell cultures are an indispensable tool for the identification and understanding of key regulators of the cell cycle. Nevertheless, the use of cell cultures has its disadvantages, because it represents an artificial system that does not completely mimic the endogenous conditions that occur in organized meristems. Here, we present a new and easy method for Arabidopsis thaliana root tip synchronization by hydroxyurea treatment. A major advantage of the method is the possibility of investigating available Arabidopsis cell-cycle mutants without the need to generate cell cultures. As a proof of concept, the effects of over-expression of a dominant negative allele of the B-type cyclin-dependent kinase CDKB1;1 gene on cell-cycle progression were tested. The previously observed prolonged G2 phase was confirmed, but was found to be compensated for by a reduced G1 phase. Furthermore, altered S-phase kinetics indicated a functional role for CDKB1;1 during the replication process.

Expanding the Benefits of Tnt1 for the Identification of Dominant Mutations in Polyploid Crops: A Single Allelic Mutation in the MsNAC39 Gene Produces Multifoliated Alfalfa.

Most major crops are polyploid species and the production of genetically engineered cultivars normally requires the introgression of transgenic or gene-edited traits into elite germplasm. Thus, a main goal of plant research is the search of systems to identify dominant mutations. In this article, we show that the Tnt1 element can be used to identify dominant mutations in allogamous tetraploid cultivated alfalfa. Specifically, we show that a single allelic mutation in the MsNAC39 gene produces multifoliate leaves (mfl) alfalfa plants, a pivot trait of breeding programs of this forage species. Finally, we discuss the potential application of a combination of preliminary screening of beneficial dominant mutants using Tnt1 mutant libraries and genome editing via the CRISPR/Cas9 system to identify target genes and to rapidly improve both autogamous and allogamous polyploid crops.

MERE1, a low-copy-number copia-type retroelement in Medicago truncatula active during tissue culture.

We have identified an active Medicago truncatula copia-like retroelement called Medicago RetroElement1-1 (MERE1-1) as an insertion in the symbiotic NSP2 gene. MERE1-1 belongs to a low-copy-number family in the sequenced Medicago genome. These copies are highly related, but only three of them have a complete coding region and polymorphism exists between the long terminal repeats of these different copies. This retroelement family is present in all M. truncatula ecotypes tested but also in other legume species like Lotus japonicus. It is active only during tissue culture in both R108 and Jemalong Medicago accessions and inserts preferentially in genes.

Osmotic shock improves Tnt1 transposition frequency in Medicago truncatula cv Jemalong during in vitro regeneration.

Insertion mutant collections are powerful tools for genetic studies in plants. Although large-scale insertional mutagenesis using T-DNA is not feasible in legumes, the Tnt1 tobacco retrotransposon can be used as a very efficient mutagen in the Medicago truncatula R108 genotype. In this article, we show that Tnt1 can also be exploited to create insertional mutants via transformation and/or regeneration in the reference cultivar Jemalong. Tnt1 insertional mutagenesis in Jemalong following Agrobacterium tumefaciens-mediated transformation was found to be very efficient, with an average of greater than 15 insertions/line. In contrast, regeneration using low-copy transgenic starter lines resulted in a highly variable rate of new Tnt1 insertions. With the goal of increasing the number of additional Tnt1 insertions during regeneration of starter lines, we have compared the insertion frequencies for a number of different regeneration protocols. In addition, we have been able to show that sucrose-mediated osmotic shock preceding regeneration significantly increases the transposition frequency. Under optimal conditions, 95% of the regenerated Jemalong plants possess new insertions.

Different functions of the histone acetyltransferase HAC1 gene traced in the model species Medicago truncatula, Lotus japonicus and Arabidopsis thaliana.

In eukaryotes, histone acetyltransferases regulate the acetylation of histones and transcription factors, affecting chromatin structural organization, transcriptional regulation, and gene activation. To assess the role of HAC1, a gene encoding for a histone acetyltransferase in Medicago truncatula, stable transgenic lines with modified HAC1 expression in the model plants M. truncatula, Lotus japonicus, and Arabidopsis thaliana were generated by Agrobacterium-mediated transformation and used for functional analyses. Histochemical, transcriptional, flow cytometric, and morphological analyses demonstrated the involvement of HAC1 in plant growth and development, responses to internal stimuli, and cell cycle progression. Expression patterns of a reporter gene encoding beta-glucuronidase (GUS) fused to the HAC1 promoter sequence were associated with young tissues comprised of actively dividing cells in different plant organs. The green fluorescent protein (GFP) signal, driven by the HAC1 promoter, was detected in the nuclei and cytoplasm of root cells. Transgenic lines with HAC1 overexpression and knockdown showed a wide range of phenotypic deviations and developmental abnormalities, which provided lines of evidence for the role of HAC1 in plant development. Synchronization of A. thaliana root tips in a line with HAC1 knockdown showed the involvement of this gene in the acetylation of two core histones during S phase of the plant cell cycle.

Transformation of leguminous plants to study symbiotic interactions.

Legume plants are important in agriculture because they represent an important source of protein for human and animal consumption. This high protein content results from their capacity to use atmospheric nitrogen for their nutrition as a consequence of their symbiotic interaction with rhizobia. Understanding this interaction at the molecular level is a prerequisite for its better use in agriculture and for the long term objective of its transfer to other crops. Agrobacterium-mediated transformation is a tool of choice for studying this interaction and for unraveling the function of the different genes discovered through classical genetic approaches. However, legume plants are often recalcitrant to regeneration and transformation. This paper describes the technology developments (regeneration, transformation, insertion mutagenesis) related to Agrobacterium transformations that were established in the legume plants, as well as different examples of the technology developments or gene discoveries resulting from these studies.

Recent Progress in Development of Tnt1 Functional Genomics Platform for Medicago truncatula and Lotus japonicus in Bulgaria.

Legumes, as protein-rich crops, are widely used for human food, animal feed and vegetable oil production. Over the past decade, two legume species, Medicago truncatula and Lotus japonicus, have been adopted as model legumes for genomics and physiological studies. The tobacco transposable element, Tnt1, is a powerful tool for insertional mutagenesis and gene inactivation in plants. A large collection of Tnt1-tagged lines of M. truncatula cv. Jemalong was generated during the course of the project 'GLIP': Grain Legumes Integrated Project, funded by the European Union (www.eugrainlegumes.org). In the project 'IFCOSMO': Integrated Functional and COmparative genomics Studies on the MOdel Legumes Medicago truncatula and Lotus japonicus, supported by a grant from the Ministry of Education, Youth and Science, Bulgaria, these lines are used for development of functional genomics platform of legumes in Bulgaria. This review presents recent advances in the evaluation of the M. truncatula Tnt1 mutant collection and outlines the steps that are taken in using the Tnt1-tagging for generation of a mutant collection of the second model legume L. japonicus. Both collections will provide a number of legume-specific mutants and serve as a resource for functional and comparative genomics research on legumes. Genomics technologies are expected to advance genetics and breeding of important legume crops (pea, faba bean, alfalfa and clover) in Bulgaria and worldwide.