Designing Novel Strategies for Improving Old Legumes: An Overview from Common Vetch.

Common vetch (Vicia sativa L.) is a grain legume used in animal feeding, rich in protein content, fatty acid, and mineral composition that makes for a very adequate component to enrich feedstuff. In addition, relevant pharmacological properties have been reported in humans. The common vetch, similar to other legumes, can fix atmospheric nitrogen, a crucial feature for sustainable agricultural systems. These properties enhance the use of vetch as a cover crop and its sowing in intercropping systems. Moreover, several studies have recently pointed out the potential of vetch in the phytoremediation of contaminated soils. These characteristics make vetch a relevant crop, which different potential improvements target. Varieties with different yields, flowering times, shattering resistance, nutritional composition, rhizobacteria associations, drought tolerance, nitrogen fixation capacity, and other agronomic-relevant traits have been identified when different vetch accessions are compared. Recently, the analysis of genomic and transcriptomic data has allowed the development of different molecular markers to be used for assisted breeding purposes, promoting crop improvement. Here, we review the potential of using the variability of V. sativa genetic resources and new biotechnological and molecular tools for selecting varieties with improved traits to be used in sustainable agriculture systems.

Common Vetch, Valuable Germplasm for Resilient Agriculture: Genetic Characterization and Spanish Core Collection Development.

Common vetch (Vicia sativa L.) is a legume used for animal feed because of its high protein content and great capacity for nitrogen fixation, making this crop relevant in sustainable agriculture. The Spanish vetch collection, conserved at the Spanish Plant Genetic Resources Center (CRF), is one of the largest collections of this species worldwide, including landraces, wild relatives mainly collected in Spain, and commercial cultivars, but also accessions of international origin. The analysis of the genetic diversity of this material, whose genome has not been sequenced yet, and the assembly of a representative collection could play a pivotal role in conserving and exploiting these genetic resources in breeding programs mainly in those focused on consequences and demands of climate change. In this work, a set of 14 simple sequence repeat (SSR) reference alleles for genetic diversity analysis of the CRF vetch collection has been developed, used for genotyping more than 545 common vetch accessions from all over the world and validated. All the tested markers were polymorphic for the analyzed accessions. Overall, at least 86 different loci were identified with 2-11 alleles per locus with an average of 6.1 alleles per locus. Also, the analyses of the generated SSR database support that most of these SSR markers are transferable across closely related species of Vicia genus. Analysis of molecular variance revealed that wild relatives have a higher genetic diversity than landraces. However, cultivars have similar diversity than landraces, indicating that genetic variability has been barely lost due to the breeding of this legume. Low differences of genetic variations between Spanish and non-Spanish accessions have been observed, suggesting a high degree of diversity within Spanish genotypes, which provide 95% of the total genetic variation, so we have focused our efforts on characterizing genotypes of Spanish origin that were further studied using storage protein profiles. Based on SSR, seed protein profiles, and agromorphological and passport data, a vetch core collection (VCC) containing 47 V. sativa accessions of Spanish origin has been established. In this collection, the characterization has been expanded using ISSR markers, and it has been reevaluated with new agromorphological data, including drought tolerance characters. This VCC presents a minimum loss of genetic diversity concerning the total collection and constitutes an invaluable material that can be used in future breeding programs for direct use in a resilient agricultural system.

Molecular bases for drought tolerance in common vetch: designing new molecular breeding tools.

BACKGROUND: Common vetch (Vicia sativa L.) is a forage grain legume of high protein content and high nitrogen fixation, relevant in sustainable agriculture systems. Drought is the main limiting factor of this crop yield. Genetic resources collections are essential to provide genetic variability for breeding. The analysis of drought associated parameters has allowed us to identify drought tolerant and sensitive ecotypes in a vetch core collection. RESULTS: To understand the mechanisms involved in drought response we analysed transcriptomic differences between tolerant and sensitive accessions. Polymorphic variants (SNPs and SSRs) in these differential expressed genes (DEGs) have also been analysed for the design of drought-associated markers. A total of 1332 transcripts were commonly deregulated in both genotypes under drought. To know the drought adaptive response, we also analysed DEGs between accessions. A total of 2646 transcripts are DEG between sensitive and tolerant ecotypes, in watered and drought conditions, including important genes involved in redox homeostasis, cell wall modifications and stress-response. The integration of this functional and genetic information will contribute to understand the molecular mechanisms of drought response and the adaptive mechanisms of drought tolerance in common vetch. The identification of polymorphic variants in these DEGs has also been screened for the design of drought-associated markers that could be used in future breeding program strategies. CONCLUSIONS: Our studies shed light for the first time in common vetch about the genes and pathways associated with drought tolerance. In addition, we identify over 100 potential drought associated polymorphism, as SNPs or SSRs, which are differently present in drought and tolerant genotypes. The use of these molecular markers for trait prediction would enable the development of genomic tools for future engineering strategies by screening of germplasm crop collections for traits related with crop drought resilience, adaptability or yield in vetch.

The transcription factor OBP4 controls root growth and promotes callus formation.

Plant growth and development require a continuous balance between cell division and differentiation. In root meristems, differentiated cells acquire specialized functions, losing their mitotic potential. Some plant cells, such as pericycle cells, have a remarkable plasticity to regenerate new organs. The molecular mechanisms underlying cell reprogramming are not completely known. In this work, a functional screening of transcription factors identified Arabidopsis OBP4 (OBF Binding Protein 4) as a novel regulator of root growth and cell elongation and differentiation. Overexpression of OBP4 regulates the levels of a large number of transcripts in roots, many involved in hormonal signaling and callus formation. OBP4 controls cell elongation and differentiation in root cells. OBP4 does not induce cell division in the root meristem, but promotes pericycle cell proliferation, forming callus-like structures at the root tip, as shown by the expression of stem cell markers. Callus formation is enhanced by ectopic expression of OBP4 in the wild-type or alf4-1, but is significantly reduced in roots that have lower levels of OBP4. Our data provide molecular insights into how differentiated root cells acquire the potential to generate callus, a pluripotent mass of cells that can regenerate fully functional plant organs.

Whole genome duplications in plants: an overview from Arabidopsis.

Polyploidy is a common event in plants that involves the acquisition of more than two complete sets of chromosomes. Allopolyploidy originates from interspecies hybrids while autopolyploidy originates from intraspecies whole genome duplication (WGD) events. In spite of inconveniences derived from chromosomic rearrangement during polyploidization, natural plant polyploids species often exhibit improved growth vigour and adaptation to adverse environments, conferring evolutionary advantages. These advantages have also been incorporated into crop breeding programmes. Many tetraploid crops show increased stress tolerance, although the molecular mechanisms underlying these different adaptation abilities are poorly known. Understanding the physiological, cellular, and molecular mechanisms coupled to WGD, in both allo- and autopolyploidy, is a major challenge. Over the last few years, several studies, many of them in Arabidopsis, are shedding light on the basis of genetic, genomic, and epigenomic changes linked to WGD. In this review we summarize and discuss the latest advances made in Arabidopsis polyploidy, but also in other agronomic plant species.

Deciphering the molecular bases for drought tolerance in Arabidopsis autotetraploids.

Whole genome duplication (autopolyploidy) is common in many plant species and often leads to better adaptation to adverse environmental conditions. However, little is known about the physiological and molecular mechanisms underlying these adaptations. Drought is one of the major environmental conditions limiting plant growth and development. Here, we report that, in Arabidopsis thaliana, tetraploidy promotes alterations in cell proliferation and organ size in a tissue-dependent manner. Furthermore, it potentiates plant tolerance to salt and drought stresses and decreases transpiration rate, likely through controlling stomata density and closure, abscisic acid (ABA) signalling and reactive oxygen species (ROS) homeostasis. Our transcriptomic analyses revealed that tetraploidy mainly regulates the expression of genes involved in redox homeostasis and ABA and stress response. Taken together, our data have shed light on the molecular basis associated with stress tolerance in autopolyploid plants.

ANTI-SILENCING FUNCTION1 proteins are involved in ultraviolet-induced DNA damage repair and are cell cycle regulated by E2F transcription factors in Arabidopsis.

ANTI-SILENCING FUNCTION1 (ASF1) is a key histone H3/H4 chaperone that participates in a variety of DNA- and chromatin-related processes, including DNA repair, where chromatin assembly and disassembly are of primary relevance. Information concerning the role of ASF1 proteins in the post-ultraviolet (UV) response in higher plants is currently limited. In Arabidopsis (Arabidopsis thaliana), an initial analysis of in vivo localization of ASF1A and ASF1B indicates that both proteins are mainly expressed in proliferative tissues. In silico promoter analysis identified ASF1A and ASF1B as potential targets of E2F corresponds to Adenovirus E2 Binding Factor. [corrected]. These observations were experimentally validated, both in vitro, by electrophoretic mobility shift assays, and in vivo, by chromatin immunoprecipitation assays and expression analysis using transgenic plants with altered levels of different E2F transcription factors. These data suggest that ASF1A and ASF1B are regulated during cell cycle progression through E2F transcription factors. In addition, we found that ASF1A and ASF1B are associated with the UV-B-induced DNA damage response in Arabidopsis. Transcript levels of ASF1A and ASF1B were increased following UV-B treatment. Consistent with a potential role in UV-B response, RNA interference-silenced plants of both genes showed increased sensitivity to UV-B compared with wild-type plants. Finally, by coimmunoprecipitation analysis, we found that ASF1 physically interacts with amino-terminal acetylated histones H3 and H4 and with acetyltransferases of the Histone Acetyl Transferase subfamily, which are known to be involved in cell cycle control and DNA repair, among other functions. Together, we provide evidence that ASF1A and ASF1B are regulated by cell cycle progression and are involved in DNA repair after UV-B irradiation.

Auxin and epigenetic regulation of SKP2B, an F-box that represses lateral root formation.

In plants, lateral roots originate from pericycle founder cells that are specified at regular intervals along the main root. Here, we show that Arabidopsis (Arabidopsis thaliana) SKP2B (for S-Phase Kinase-Associated Protein2B), an F-box protein, negatively regulates cell cycle and lateral root formation as it represses meristematic and founder cell divisions. According to its function, SKP2B is expressed in founder cells, lateral root primordia and the root apical meristem. We identified a novel motif in the SKP2B promoter that is required for its specific root expression and auxin-dependent induction in the pericycle cells. Next to a transcriptional control by auxin, SKP2B expression is regulated by histone H3.1/H3.3 deposition in a CAF-dependent manner. The SKP2B promoter and the 5' end of the transcribed region are enriched in H3.3, which is associated with active chromatin states, over H3.1. Furthermore, the SKP2B promoter is also regulated by H3 acetylation in an auxin- and IAA14-dependent manner, reinforcing the idea that epigenetics represents an important regulatory mechanism during lateral root formation.

Genome-wide analysis of histone H3.1 and H3.3 variants in Arabidopsis thaliana.

Nucleosomes package eukaryotic DNA and are composed of four different histone proteins, designated H3, H4, H2A, and H2B. Histone H3 has two main variants, H3.1 and H3.3, which show different genomic localization patterns in animals. We profiled H3.1 and H3.3 variants in the genome of the plant Arabidopsis thaliana and found that the localization of these variants shows broad similarity in plants and animals, along with some unique features. H3.1 was enriched in silent areas of the genome, including regions containing the repressive chromatin modifications H3 lysine 27 methylation, H3 lysine 9 methylation, and DNA methylation. In contrast, H3.3 was enriched in actively transcribed genes, especially peaking at the 3' end of genes, and correlated with histone modifications associated with gene activation, such as histone H3 lysine 4 methylation and H2B ubiquitylation, as well as RNA Pol II occupancy. Surprisingly, both H3.1 and H3.3 were enriched on defined origins of replication, as was overall nucleosome density, suggesting a novel characteristic of plant origins. Our results are broadly consistent with the hypothesis that H3.1 acts as the canonical histone that is incorporated during DNA replication, whereas H3.3 acts as the replacement histone that can be incorporated outside of S-phase during chromatin-disrupting processes like transcription.

Regulation of plant MSH2 and MSH6 genes in the UV-B-induced DNA damage response.

Deleterious effects of UV-B radiation on DNA include the formation of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). These lesions must be repaired to maintain the integrity of DNA and provide genetic stability. Of the several repair systems involved in the recognition and removal of UV-B-induced lesions in DNA, the focus in the present study was on the mismatch repair system (MMR). The contribution of MutSα (MSH2-MSH6) to UV-induced DNA lesion repair and cell cycle regulation was investigated. MSH2 and MSH6 genes in Arabidopsis and maize are up-regulated by UV-B, indicating that MMR may have a role in UV-B-induced DNA damage responses. Analysis of promoter sequences identified MSH6 as a target of the E2F transcription factors. Using electrophoretic mobility shift assays, MSH6 was experimentally validated as an E2F target gene, suggesting an interaction between MMR genes and the cell cycle control. Mutations in MSH2 or MSH6 caused an increased accumulation of CPDs relative to wild-type plants. In addition, msh2 mutant plants showed a different expression pattern of cell cycle marker genes after the UV-B treatment when compared with wild-type plants. Taken together, these data provide evidence that plant MutSα is involved in a UV-B-induced DNA damage response pathway.

Chromatin dynamics during the plant cell cycle.

Cell cycle progression depends on a highly regulated series of events of which transcriptional control plays a major role. In addition, during the S-phase not only DNA but chromatin as a whole needs to be faithfully duplicated. Therefore, both nucleosome dynamics as well as local changes in chromatin organization, including introduction and/or removal of covalent DNA and histone modifications, at genes with a key role in cell proliferation, are of primary relevance. Chromatin duplication during the S-phase and the chromosome segregation during mitosis are cell cycle stages critical for maintenance of epigenetic marks or for allowing the daughter products to acquire a distinct epigenetic landscape and, consequently, a unique cell fate decision. These aspects of chromatin dynamics together with the strict coupling of cell proliferation, cell differentiation and post-embryonic organogenesis have a profound impact on plant growth, development and response to external signals.

The many faces of chromatin assembly factor 1.

Chromatin organization requires that histones associate with DNA in the form of nucleosomes the position and composition of which is crucial for chromatin dynamics. Histone chaperones help to deliver specific histone proteins to the sites where chromatin is being newly formed or remodeled. Association of H3-H4 during DNA replication depends on the chromatin assembly factor 1. The study of Arabidopsis plants carrying loss-of-function alleles in each of the three chromatin assembly factor 1 subunits has highlighted the links between chromatin assembly in proliferating cells and other cellular processes. These are the G2 DNA damage checkpoint, homologous recombination, endoreplication control and transcriptional regulation of specific gene sets, all contributing to the plasticity of plants in dealing with alterations in DNA replication-associated chromatin assembly.

E2F regulates FASCIATA1, a chromatin assembly gene whose loss switches on the endocycle and activates gene expression by changing the epigenetic status.

Maintenance of genome integrity depends on histone chaperone-mediated chromatin reorganization. DNA replication-associated nucleosome deposition relies on chromatin assembly factor-1 (CAF-1). Depletion of CAF-1 in human cells leads to cell death, whereas in Arabidopsis (Arabidopsis thaliana), where it is involved in heterochromatin compaction and homologous recombination, plants are viable. The mechanism that makes the lack of CAF-1 activity compatible with development is not known. Here, we show that the FASCIATA1 (FAS1) gene, which encodes the CAF-1 large subunit, is a target of E2F transcription factors. Mutational studies demonstrate that one of the two E2F binding sites in its promoter has an activator role, whereas the other has a repressor function. Loss of FAS1 results in reduced type A cyclin-dependent kinase activity, inhibits mitotic progression, and promotes a precocious and systemic switch to the endocycle program. Selective up-regulation of the expression of a subset of genes, including those involved in activation of the G2 DNA damage checkpoint, also occurs upon FAS1 loss. This activation is not the result of a global change in chromatin structure, but depends on selective epigenetic changes in histone acetylation and methylation within a small region in their promoters. This suggests that correct chromatin assembly during the S-phase is required to prevent unscheduled changes in the epigenetic marks of target genes. Interestingly, activation of the endocycle switch as well as introduction of activating histone marks in the same set of G2 checkpoint genes are detected upon treatment of wild-type plants with DNA-damaging treatments. Our results are consistent with a model in which defects in chromatin assembly during the S-phase and DNA damage signaling share part of a pathway, which ultimately leads to mitotic arrest and triggers the endocycle program. Together, this might be a bypass mechanism that makes development compatible with cell division arrest induced by DNA damage stress.

Cell type-specific role of the retinoblastoma/E2F pathway during Arabidopsis leaf development.

Organogenesis in plants is almost entirely a postembryonic process. This unique feature implies a strict coupling of cell proliferation and differentiation, including cell division, arrest, cell cycle reactivation, endoreplication, and differentiation. The plant retinoblastoma-related (RBR) protein modulates the activity of E2F transcription factors to restrict cell proliferation. Arabidopsis contains a single RBR gene, and its loss of function precludes gamete formation and early development. To determine the relevance of the RBR/E2F pathway during organogenesis, outside its involvement in cell division, we have used an inducible system to inactivate RBR function and release E2F activity. Here, we have focused on leaves where cell proliferation and differentiation are temporally and developmentally regulated. Our results reveal that RBR restricts cell division early during leaf development when cell proliferation predominates, while it regulates endocycle occurrence at later stages. Moreover, shortly after leaving the cell cycle, most of leaf epidermal pavement cells retain the ability to reenter the cell cycle and proliferate, but maintain epidermal cell fate. On the contrary, mesophyll cells in the inner layers do not respond in this way to RBR loss of activity. We conclude that there exists a distinct response of different cells to RBR inactivation in terms of maintaining the balance between cell division and endoreplication during Arabidopsis (Arabidopsis thaliana) leaf development.

The genes encoding Arabidopsis ORC subunits are E2F targets and the two ORC1 genes are differently expressed in proliferating and endoreplicating cells.

Initiation of eukaryotic DNA replication depends on the function of pre-replication complexes (pre-RC), one of its key component being the six subunits origin recognition complex (ORC). In spite of a significant degree of conservation among ORC proteins from different eukaryotic sources, the regulation of their availability varies considerably in different model systems and cell types. Here, we show that the six ORC genes of Arabidopsis thaliana are regulated at the transcriptional level during cell cycle and development. We found that Arabidopsis ORC genes, except AtORC5, contain binding sites for the E2F family of transcription factors. Expression of AtORC genes containing E2F binding sites peaks at the G1/S-phase. Analysis of AtORC gene expression in plants with reduced E2F activity, obtained by expressing a dominant negative version of DP, the E2F heterodimerization partner, and with increased E2F activity, obtained by inactivation of the retinoblastoma protein, led us to conclude that all AtORC genes, except AtORC5 are E2F targets. Interestingly, Arabidopsis contains two AtORC1 (a and b) genes, highly conserved at the amino acid level but with unrelated promoter sequences. AtORC1b expression is restricted to proliferating cells. However, AtORC1a is preferentially expressed in endoreplicating cells based on our analysis in endoreplicating tissues and in a mutant with altered endocycle pattern. This suggests a differential expression of the two ORC1 genes in Arabidopsis.

Balance between cell division and differentiation during plant development.

The processes which make possible that a cell gives rise to two daughter cells define the cell division cycle. In individual cells, this is strictly controlled both in time and space. In multicellular organisms extra layers of regulation impinge on the balance between cell proliferation and cell differentiation within particular ontogenic programs. In contrast to animals, organogenesis in plants is a post-embryonic process that requires developmentally programmed reversion of sets of cells from different differentiated states to a pluripotent state followed by regulated proliferation and progression through distinct differentiation patterns. This implies a fine coupling of cell division control, cell cycle arrest and reactivation, endoreplication and differentiation. The emerging view is that cell cycle regulators, in addition to controlling cell division, also function as targets for maintaining cell homeostasis during development. The mechanisms and cross talk among different cell cycle regulatory pathways are discussed here in the context of a developing plant.

Role of an atypical E2F transcription factor in the control of Arabidopsis cell growth and differentiation.

The balance between cell proliferation and differentiation is crucial in multicellular organisms, where it is regulated by complex gene expression networks. This is particularly relevant in plants because organogenesis is a continuous postembryonic process. Here, we investigate the function of Arabidopsis thaliana E2Ff, an atypical member of the E2F family of transcription factors, which acts independently of a dimerization partner. We have focused our analysis on roots and hypocotyls, organs where (1) cell proliferation and differentiation are spatially and/or temporally separated, (2) growth depends on cell expansion in the longitudinal axis, and (3) the AtE2Ff promoter is active. AtE2Ff overexpression produced a reduction in the size of differentiated cells of these organs. Cells of mutant e2ff-1 plants with reduced levels of AtE2Ff mRNA were larger, especially in the hypocotyl, suggesting a role as a growth regulator. These effects of AtE2Ff are not associated with changes in nuclear ploidy levels or in the expression of cell cycle marker genes. However, expression of a subset of cell wall biogenesis genes is misregulated in an AtE2Ff-dependent manner, and based on chromatin immunoprecipitation experiments, they seem to be direct E2F targets. Our results highlight the complex regulatory function exerted by E2F and suggest a possible role of AtE2Ff in repressing cell wall biosynthesis genes during cell elongation in differentiated cells.

Geminivirus DNA replication and cell cycle interactions.

The Geminiviridae family includes a large number of viruses that infect plants and have a unique geminate virion particle, a single-stranded genome of approximately 2.6-3.0 kb, and replicate through a rolling-circle mechanism. Since they encode for just a few proteins (4-6 depending on the members that belong to four different genera), a rich variety of interactions has evolved between viral proteins and host factors to develop the virus replicative cycle. Among them, we have been particularly interested so far: (i). in the interference with cell cycle regulatory proteins of the retinoblastoma-related (RBR)/E2F pathway and (ii). in the interaction with host DNA replication factors necessary for the assembly of a functional replication complex at the viral origin of DNA replication during the rolling-circle stage. Yeast two-hybrid assays revealed that wheat dwarf virus RepA protein, but nor Rep protein, interacts with plant RBR protein. Interestingly, deletion of the C-terminal domain of Rep confers the truncated protein the ability to interact with RBR, suggesting that this domain may hinder the LXCXE RBR-binding motif. Secondary structure predictions support such a possibility.

A genome-wide identification of E2F-regulated genes in Arabidopsis.

The completion of the Arabidopsis genomic sequence offers the possibility to extract global information about regulatory mechanisms. Here, we describe a data mining strategy in combination with gene expression analysis to identify bona fide genes regulated by the E2F transcription factor. Starting with a genome-wide search of chromosomal sites containing E2F-binding sites, we studied in depth two of the most abundant E2F-binding sites within the Arabidopsis genome and identified over 180 potential E2F target genes. Among them and in addition to cell cycle-related genes, we have also identified genes belonging to other functional categories, e.g. transcription, stress and defense or signaling. We have determined the expression levels of genes selected from different categories under two experimental situations. Using cultured cells partially synchronized with aphidicolin, we found that most potential E2F targets identified in silico show a cell cycle-regulated expression pattern with a peak in early/mid S-phase. In addition, we used Arabidopsis transgenic plants expressing a DP gene containing a truncated DNA-binding domain, which likely has a dominant-negative effect on AtE2Fa, b and c (also named AtE2F3, 1 and 2, respectively), which require DP for efficient DNA binding. Contrary to the up-regulation observed in early/mid S-phase-cultured cells, the expression of a large number of potential E2F targets was decreased in the transgenic plants. Our results strongly support that the RBR/E2F pathway plays a crucial role in regulating the expression of the genes identified in this study.