OeBAS and CYP716C67 catalyze the biosynthesis of health-beneficial triterpenoids in olive (Olea europaea) fruits.

The bioactive properties of olive (Olea europaea) fruits and olive oil are largely attributed to terpenoid compounds, including diverse triterpenoids such as oleanolic, maslinic and ursolic acids, erythrodiol, and uvaol. They have applications in the agri-food, cosmetics, and pharmaceutical industries. Some key steps involved in the biosynthesis of these compounds are still unknown. Genome mining, biochemical analysis, and trait association studies have been used to identify major gene candidates controlling triterpenoid content of olive fruits. Here, we identify and functionally characterize an oxidosqualene cyclase (OeBAS) required for the production of the major triterpene scaffold ß-amyrin, the precursor of erythrodiol, oleanolic and maslinic acids, and a cytochrome P450 (CYP716C67) that mediates 2α oxidation of the oleanane- and ursane-type triterpene scaffolds to produce maslinic and corosolic acids, respectively. To confirm the enzymatic functions of the entire pathway, we have reconstituted the olive biosynthetic pathway for oleanane- and ursane-type triterpenoids in the heterologous host, Nicotiana benthamiana. Finally, we have identified genetic markers associated with oleanolic and maslinic acid fruit content on the chromosomes carrying the OeBAS and CYP716C67 genes. Our results shed light on the biosynthesis of olive triterpenoids and provide new gene targets for germplasm screening and breeding for high triterpenoid content.

Two bi-functional cytochrome P450 CYP72 enzymes from olive (Olea europaea) catalyze the oxidative C-C bond cleavage in the biosynthesis of secoxy-iridoids - flavor and quality determinants in olive oil.

Olive (Olea europaea) is an important crop in Europe, with high cultural, economic and nutritional significance. Olive oil flavor and quality depend on phenolic secoiridoids, but the biosynthetic pathway of these iridoids remains largely uncharacterized. We discovered two bifunctional cytochrome P450 enzymes, catalyzing the rare oxidative C-C bond cleavage of 7-epi-loganin to produce oleoside methyl ester (OeOMES) and secoxyloganin (OeSXS), both through a ketologanin intermediary. Although these enzymes are homologous to the previously reported Catharanthus roseus secologanin synthase (CrSLS), the substrate and product profiles differ. Biochemical assays provided mechanistic insights into the two-step OeOMES and CrSLS reactions. Model-guided mutations of OeOMES changed the product profile in a predictable manner, revealing insights into the molecular basis for this change in product specificity. Our results suggest that, in contrast to published hypotheses, in planta production of secoxy-iridoids is secologanin-independent. Notably, sequence data of cultivated and wild olives point to a relation between domestication and OeOMES expression. Thus, the discovery of this key biosynthetic gene suggests a link between domestication and secondary metabolism, and could potentially be used as a genetic marker to guide next-generation breeding programs.

Metabolic and transcriptional changes associated with the use of Ascophyllum nodosum extracts as tools to improve the quality of wine grapes (Vitis vinifera cv. Sangiovese) and their tolerance to biotic stress.

BACKGROUND: Recent studies report that Ascophyllum nodosum extracts, once applied on the canopy of different crops, deliver positive effects, increasing yield, inducing tolerance to biotic stress, and improving the quality of products. However, the mechanisms of action are still unclear. In this research, vines subjected to multiple foliar applications of an A. nodosum extract (ANE) at label doses were compared with untreated vines (NTV) in accordance with a comparative approach. The investigation coupled a field experiment with a second trial conducted under semi-controlled conditions, to clarify the mechanisms of action involved. RESULTS: The biostimulant did not affect soluble solids or the acidity of grapes; instead, it improved their anthocyanin and phenolic concentrations and the respective profiles. At the time of harvest, anthocyanin, and phenolic concentration were increased by 10.4% and 14.5%, respectively, when compared to the NTV. These effects correlated with a specific modulation of genes involved in the flavonoid metabolic pathways. Moreover, grapes from ANE vines witnessed a significant reduction in the spreading of gray mold when they were either assessed in field conditions or in vitro, compared to the grapes of NTV vines. This was related to a significant upregulation of the defense-related genes of the plant. CONCLUSIONS: Overall, the results showed that A. nodosum extracts can be valuable tools in viticulture considering the emergence of challenging environmental conditions; hence, the regulation of specific metabolic pathways is the mechanism of action that leads to an increased tolerance of biotic stress and of changes in the content of grape metabolites. © 2019 Society of Chemical Industry.

An apomixis-linked ORC3-like pseudogene is associated with silencing of its functional homolog in apomictic Paspalum simplex.

Apomixis in plants consists of asexual reproduction by seeds. Here we characterized at structural and functional levels an apomixis-linked sequence of Paspalum simplex homologous to subunit 3 of the ORIGIN RECOGNITION COMPLEX (ORC3). ORC is a multiprotein complex which controls DNA replication and cell differentiation in eukaryotes. Three PsORC3 copies were identified, each one characterized by a specific expression profile. Of these, PsORC3a, specific for apomictic genotypes, is a pseudogene that was poorly and constitutively expressed in all developmental stages of apomictic flowers, whereas PsORC3b, the putative functional gene in sexual flowers, showed a precise time-related regulation. Sense transcripts of PsORC3 were expressed in the female cell lineage of both apomictic and sexual reproductive phenotypes, and in aposporous initials. Although strong expression was detected in sexual early endosperm, no expression was present in the apomictic endosperm. Antisense PsORC3 transcripts were revealed exclusively in apomictic germ cell lineages. Defective orc3 mutants of rice and Arabidopsis showed normal female gametophytes although the embryo and endosperm were arrested at early phases of development. We hypothesize that PsORC3a is associated with the down-regulation of its functional homolog and with the development of apomictic endosperm which deviates from the canonical 2(maternal):1(paternal) genome ratio.

Medicago truncatula CYP716A12 is a multifunctional oxidase involved in the biosynthesis of hemolytic saponins.

Saponins, a group of glycosidic compounds present in several plant species, have aglycone moieties that are formed using triterpenoid or steroidal skeletons. In spite of their importance as antimicrobial compounds and their possible benefits for human health, knowledge of the genetic control of saponin biosynthesis is still poorly understood. In the Medicago genus, the hemolytic activity of saponins is related to the nature of their aglycone moieties. We have identified a cytochrome P450 gene (CYP716A12) involved in saponin synthesis in Medicago truncatula using a combined genetic and biochemical approach. Genetic loss-of-function analysis and complementation studies showed that CYP716A12 is responsible for an early step in the saponin biosynthetic pathway. Mutants in CYP716A12 were unable to produce hemolytic saponins and only synthetized soyasaponins, and were thus named lacking hemolytic activity (lha). In vitro enzymatic activity assays indicate that CYP716A12 catalyzes the oxidation of ß-amyrin and erythrodiol at the C-28 position, yielding oleanolic acid. Transcriptome changes in the lha mutant showed a modulation in the main steps of triterpenic saponin biosynthetic pathway: squalene cyclization, ß-amyrin oxidation, and glycosylation. The analysis of CYP716A12 expression in planta is reported together with the sapogenin content in different tissues and stages. This article provides evidence for CYP716A12 being a key gene in hemolytic saponin biosynthesis.

Genome-Wide Identification of Histone Modification Gene Families in the Model Legume Medicago truncatula and Their Expression Analysis in Nodules.

Histone methylation and acetylation are key processes in the epigenetic regulation of plant growth, development, and responses to environmental stimuli. The genes encoding for the enzymes that are responsible for these chromatin post-translational modifications, referred to as histone modification genes (HMGs), have been poorly investigated in Leguminosae species, despite their importance for establishment and activity of nitrogen-fixing nodules. In silico analysis of Medicago truncatula HMGs identified 81 histone methyltransferases, 46 histone demethylases, 64 histone acetyltransferases, and 15 histone deacetylases. MtHMGs were analyzed for their structure and domain composition, and some combinations that were not yet reported in other plant species were identified. Genes have been retrieved from M. truncatula A17 and R108 genotypes as well as M. sativa CADL and Zhongmu No.1; the gene number and distribution were compared with Arabidopsis thaliana. Furthermore, by analyzing the expression data that were obtained at various developmental stages and in different zones of nitrogen-fixing nodules, we identified MtHMG loci that could be involved in nodule development and function. This work sets a reference for HMG genomic organization in legumes which will be useful for functional investigation that is aimed at elucidating HMGs involvement in nodule development and symbiotic nitrogen fixation.

Functional Characterization of MtrGSTF7, a Glutathione S-Transferase Essential for Anthocyanin Accumulation in Medicago truncatula.

Flavonoids are essential compounds widespread in plants and exert many functions such as defence, definition of organ colour and protection against stresses. In Medicago truncatula, flavonoid biosynthesis and accumulation is finely regulated in terms of tissue specificity and induction by external factors, such as cold and other stresses. Among flavonoids, anthocyanin precursors are synthesised in the cytoplasm, transported to the tonoplast, then imported into the vacuole for further modifications and storage. In the present work, we functionally characterised MtrGSTF7, a phi-class glutathione S-transferase involved in anthocyanin transport to the tonoplast. The mtrgstf7 mutant completely lost the ability to accumulate anthocyanins in leaves both under control and anthocyanin inductive conditions. On the contrary, this mutant showed an increase in the levels of soluble proanthocyanidins (Pas) in their seeds with respect to the wild type. By complementation and expression data analysis, we showed that, differently from A. thaliana and similarly to V. vinifera, transport of anthocyanin and proanthocyanidins is likely carried out by different GSTs belonging to the phi-class. Such functional diversification likely results from the plant need to finely tune the accumulation of diverse classes of flavonoids according to the target organs and developmental stages.

Specific expression of apomixis-linked alleles revealed by comparative transcriptomic analysis of sexual and apomictic Paspalum simplex Morong flowers.

Apomixis is defined as clonal reproduction by seed. A comparative transcriptomic analysis was undertaken between apomictic and sexual genotypes of Paspalum simplex Morong to identify apomixis-related polymorphisms at the level of mRNA. cDNA-AFLP (amplified fragment length polymorphism) profiling of apomictic and sexual flowers at several stages of development yielded 202 amplicons that showed several kinds of expression specificities. Among these, the large majority consisted of amplicons that were present only in specific stages of development of the apomictic flowers. Ten percent of polymorphic amplicons were present with almost identical intensity in all stages of the apomictic flowers and never in the sexual flowers. Reverse transcription-PCR (RT-PCR) and Southern analyses of these amplicons showed that they belong to constitutively expressed alleles that are specifically present on the apomixis-controlling locus of P. simplex. The most frequent biological functions inferred from the sequence homology of the apomixis-linked alleles were related to signal transduction and nucleic acid/protein-binding activities. Most of these apomixis-linked alleles showed nonsense and frameshift mutations, revealing their probable pseudogene nature. None of the amplicons that were present only in specific stages of development of the apomictic flowers co-segregated with apomixis, indicating they did not originate from additional apomictic alleles but more probably from differential regulation of the same allele in apomictic and sexual flowers. The molecular functions inferred from sequence analysis of these latter amplicons were related to seed storage protein and regulatory genes of various types. The results are discussed regarding the possible role in apomictic reproduction of the differentially expressed genes in relation to their specificity of expression and inferred molecular functions.

T-DNA Insertional Mutagenesis and Activation Tagging in Medicago truncatula.

The development of plant genetic transformation techniques has greatly enhanced our capacity to investigate and understand gene function. Since T-DNA constructs insert randomly in genomes, in principle, it is possible to construct a population of individuals harboring one or more T-DNA inserted in any region of the genome. Such populations can be screened following two approaches: (1) given a mutant phenotype, one could find the gene subtending the phenotypic alteration (forward approach), or (2) given a gene of interest, one could identify the phenotypic effect of its expression perturbation (reverse approach).Activation tagging is an application of T-DNA mutagenesis aimed at obtaining gain-of-function mutations. This can be achieved by introducing enhancer sequences randomly in the target genome via a T-DNA shuttle and then analyzing the genomic regions flanking the insertion sites in individuals showing phenotypic alterations. In this chapter, we describe the detailed procedure to obtain and screen an activation-tagged population in Medicago truncatula.

A topoisomerase II-dependent checkpoint in G2-phase plant cells can be bypassed by ectopic expression of mitotic cyclin B2.

DNA topoisomerase II is required for mitotic chromosome condensation and segregation. Here we characterize the effects of inhibiting DNA topoisomerase II activity in plant cells using the non-DNA damaging topoisomerase II inhibitor ICRF-193. We report that ICRF-193 abrogated chromosome condensation in cultured alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.) mitoses and led to bridged chromosomes at anaphase. Moreover, ICRF-193 treatment delayed entry into mitosis, increasing the frequency of cells having a pre-prophase band of microtubules, a marker of late G2 and prophase, and delaying the activation of cyclin-dependent kinase. These data suggest the existence of a late G2 checkpoint in plant cells that is activated in the absence of topoisomerase II activity. To determine whether the checkpoint-induced delay was a result of reduced cyclindependent kinase activity, mitotic cyclin B2 was ectopically expressed. Cyclin B2 bypassed the ICRF-193-induced delay before mitosis, and correspondingly, reduced the frequency of interphase cells with a pre-prophase band. These data provide evidence that plant cells possess a topoisomerase II-dependent G2 cell cycle checkpoint that transiently inhibits mitotic CDK activation and entry into mitosis, and that is overridden by raising the level of CDK activity through the ectopic expression of a plant mitotic cyclin.

CYP72A67 Catalyzes a Key Oxidative Step in Medicago truncatula Hemolytic Saponin Biosynthesis.

In the Medicago genus, triterpenic saponins are bioactive secondary metabolites constitutively synthesized in the aerial and subterranean parts of plants via the isoprenoid pathway. Exploitation of saponins as pharmaceutics, agrochemicals and in the food and cosmetic industries has raised interest in identifying the enzymes involved in their synthesis. We have identified a cytochrome P450 (CYP72A67) involved in hemolytic sapogenin biosynthesis by a reverse genetic TILLING approach in a Medicago truncatula ethylmethanesulfonate (EMS) mutagenized collection. Genetic and biochemical analyses, mutant complementation, and expression of the gene in a microsome yeast system showed that CYP72A67 is responsible for hydroxylation at the C-2 position downstream of oleanolic acid synthesis. The affinity of CYP72A67 for substrates with different substitutions at multiple carbon positions was investigated in the same in vitro yeast system, and in relation to two other CYP450s (CYP72A68) responsible for the production of medicagenic acid, the main sapogenin in M. truncatula leaves and roots. Full sib mutant and wild-type plants were compared for their sapogenin profile, expression patterns of the genes involved in sapogenin synthesis, and response to inoculation with Sinorhizobium meliloti. The results obtained allowed us to revise the hemolytic sapogenin pathway in M. truncatula and contribute to highlighting the tissue specificities (leaves/roots) of sapogenin synthesis.

Reverse genetics in Medicago truncatula using a TILLING mutant collection.

Medicago truncatula is one of the model species for legume molecular genetics. In the last decade different types of mutant populations have been created in this species that can be screened by forward and reverse-genetic approaches to identify and functionally characterize genes of interest. TILLING is a reverse-genetic method combining random chemical mutagenesis and a PCR-based screen to identify point mutations in regions of interest. The different steps of the TILLING analysis are described in a mutant collection of ~2,300 M2 individuals for which genomic DNA and M3 seed were obtained. A two-dimensional DNA pooling strategy was adopted to reduce the number of PCR reactions necessary to screen the collection and to unambigously identify the individual M2 plant carrying the mutation. The genotypic and phenotypic analyses of the mutant M3 progeny provide the possibility to study the gene function. In spite of its reduced size, this mutant collection has proved valid in the study of the biosynthetic pathway of a class of secondary metabolites present in the genus Medicago, the triterpenic saponins.

The Arabidopsis thaliana cysteine-rich receptor-like kinase CRK20 modulates host responses to Pseudomonas syringae pv. tomato DC3000 infection.

In plants, the cysteine-rich repeat kinases (CRKs) are a sub-family of receptor-like protein kinases that contain the DUF26 motif in their extracellular domains. It has been shown that in Arabidopsis thaliana, CRK20 is transcriptionally induced by pathogens, salicylic acid and ozone (O(3)). However, its role in responses to biotic and abiotic stress remains to be elucidated. To determine the function of CRK20 in such responses, two CRK20 loss-of-function mutants, crk20-1 and crk20-2, were isolated from public collections of Arabidopsis T-DNA tagged lines and examined for responses to O(3) and Pseudomonas syringae pv. tomato (Pst) DC3000. crk20-1 and crk20-2 showed similar O(3) sensitivities and no differences in the expression of defense genes when compared with the wild-type. However, pathogen growth was significantly reduced, while there were no differences in the induction of salicylic acid related defense genes or salicylic acid accumulation. Furthermore, correlation analysis of CRK20 gene expression suggests that it has a role in the control of H(2)O and/or nutrient transport. We therefore propose that CRK20 promotes conditions that are favorable for Pst DC3000 growth in Arabidopsis, possibly through the regulation of apoplastic homeostasis, and consequently, of the environment of this biotrophic pathogen.

Do pathogen-specific defense mechanisms contribute to wound-induced resistance in tomato?

A network of shared intermediates/components and/or common molecular outputs in biotic and abiotic stress signaling has long been known, but the possibility of effective influence between differently triggered stresses (co-protection) is less studied. Recent observations show that wounding induces transient protection in tomato (Solanum lycopersicum L.) to four pathogens with a range of lifestyles, locally and systemically. The contribution of ethylene (ET) in basal but also in wound-induced resistance to each pathogen, although dispensable, is demonstrated to be positive (Botrytis cinerea, Phytophthora capsici) or negative (Fusarium oxysporum, Pseudomonas syringae pv. tomato). Furthermore, the expression of several defense markers is influenced locally and/or systemically by wounding and ET, and might be part of that core of conserved molecular responses whereby an abiotic stress such as wounding imparts co-resistance to biotic stress. In this addendum, we speculate on some of the physiological responses to wounding that might contribute to the modulation of resistance in a more pathogen-specific manner.

An Italian functional genomic resource for Medicago truncatula.

BACKGROUND: Medicago truncatula is a model species for legumes. Its functional genomics have been considerably boosted in recent years due to initiatives based both in Europe and US. Collections of mutants are becoming increasingly available and this will help unravel the genetic control of important traits for many species of legumes. FINDINGS: Our report is on the production of three complementary mutant collections of the model species Medicago truncatula produced in Italy in the frame of a national genomic initiative. Well established strategies were used: Tnt1 mutagenesis, TILLING and activation tagging. Both forward and reverse genetics screenings proved the efficiency of the mutagenesis approaches adopted, enabling the isolation of interesting mutants which are in course of characterization. We anticipate that the reported collections will be complementary to the recently established functional genomics tools developed for Medicago truncatula both in Europe and in the United States.

Wounding induces resistance to pathogens with different lifestyles in tomato: role of ethylene in cross-protection.

Many reports point to the existence of a network of regulatory signalling occurring in plants during the interaction with micro-organisms (biotic stress) and abiotic stresses such as wounding. However, the focus is on shared intermediates/components and/or common molecular outputs in differently triggered signalling pathways, and not on the degree and modes of effective influence between abiotic and biotic stresses nor the range of true plant-pathogen interactions open to such influence. We report on local and systemic wound-induced protection in tomato (Solanum lycopersicum L.) to four pathogens with a range of lifestyles (Botrytis cinerea, Fusarium oxysporum f.sp. lycopersici, Phytophthora capsici and Pseudomonas syringae pv. tomato). The role of ethylene (ET) in the phenomenon and in the induction by wounding of several markers of defense was investigated by using the never-ripe tomato mutant plants impaired in ET perception. We showed that PINIIb, PR1b, PR5, PR7 and peroxidase (POD) are influenced locally and/or systemically by wounding and, with the exception of POD activity, by ET perception. We also demonstrated that ET, although not essential, is positively (B. cinerea, P. capsici) or negatively (F. oxysporum, P. syringae pv. tomato) involved not only in basal but also in wound-induced resistance to each pathogen.

Delay of leaf senescence in Medicago sativa transformed with the ipt gene controlled by the senescence-specific promoter SAG12.

We report the successfull delay of leaf senescence in Medicago sativa. A highly regenerable clone of alfalfa was transformed with the construct SAG12-IPT, an approach that has already proved efficient in other crops. Several independent transformants were obtained as determined by Southern analysis and all the transformants expressed the transgene as measured by RT-PCR. In vitro and in vivo analyses showed that SAG12-IPT plants exhibited a stay-green phenotype that has the potential to greatly improve the quantity and quality of alfalfa forage.

Molecular cytogenetics and DNA sequence analysis of an apomixis-linked BAC in Paspalum simplex reveal a non pericentromere location and partial microcolinearity with rice.

Apomixis in plants is a form of clonal reproduction through seeds. A BAC clone linked to apomictic reproduction in Paspalum simplex was used to locate the apomixis locus on meiotic chromosome preparations. Fluorescent in situ hybridisation revealed the existence of a single locus embedded in a heterochromatin-poor region not adjacent to the centromere. We report here for the first time information regarding the sequencing of a large DNA clone from the apomixis locus. The presence of two genes whose rice homologs were mapped on the telomeric part of the long arm of rice chromosome 12 confirmed the strong synteny between the apomixis locus of P. simplex with the related area of the rice genome at the map level. Comparative analysis of this region with rice as representative of a sexual species revealed large-scale rearrangements due to transposable elements and small-scale rearrangements due to deletions and single point mutations. Both types of rearrangements induced the loss of coding capacity of large portions of the "apomictic" genes compared to their rice homologs. Our results are discussed in relation to the use of rice genome data for positional cloning of apomixis genes and to the possible role of rearranged supernumerary genes in the apomictic process of P. simplex.