Clelia De-la-Peña

Root-Microbe Communication through Protein Secretion

Biotic interactions in the rhizosphere are biologically important, and although many of those interactions have been well studied, the role of secreted proteins in the cross-talk between microbes and roots has not been investigated. Here, protein secretion was studied during the communication between the roots of two plants (Medicago sativa and Arabidopsis thaliana) and the bacterial symbiont of one of these species (Sinorhizobium meliloti strain Rm1021) and an opportunistic bacterial pathogen of A. thaliana (Pseudomonas syringae pv. tomato DC3000) using a proteomic approach. It was found that protein exudation in the M. sativa-S. meliloti interaction caused an increase in the secretion of seven plant proteins, such as hydrolases, peptidases, and peroxidases among others in two or more time points compared with the plant control. In addition, four proteins, all of bacterial origin, were increased 1.5-fold more in this interaction compared with S. meliloti alone. However, these proteins were not induced when M. sativa was inoculated with P. syringae DC3000. The interaction between A. thaliana and P. syringae DC3000 highly induced the secretion of several plant proteins related to defense soon after initial contact with P. syringae, but these proteins were not secreted in the incompatible interaction with S. meliloti. The results of this study reveal a specific, protein level cross-talk between roots and microbes. These results suggest that secreted proteins may be a critical component in the process of signaling and recognition that occurs between compatible and incompatible interactions.

Altered Profile of Secondary Metabolites in the Root Exudates of Arabidopsis ATP-Binding Cassette Transporter Mutants

Following recent indirect evidence suggesting a role for ATP-binding cassette (ABC) transporters in root exudation of phytochemicals, we identified 25 ABC transporter genes highly expressed in the root cells most likely to be involved in secretion processes. Of these 25 genes, we also selected six full-length ABC transporters and a half-size transporter for in-depth molecular and biochemical analyses. We compared the exuded root phytochemical profiles of these seven ABC transporter mutants to those of the wild type. There were three nonpolar phytochemicals missing in various ABC transporter mutants compared to the wild type when the samples were analyzed by high-performance liquid chromatography-mass spectrometry. These data suggest that more than one ABC transporter can be involved in the secretion of a given phytochemical and that a transporter can be involved in the secretion of more than one secondary metabolite. The primary and secondary metabolites present in the root exudates of the mutants were also analyzed by gas chromatography-mass spectrometry, which allowed for the identification of groups of compounds differentially found in some of the mutants compared to the wild type. For instance, the mutant Atpdr6 secreted a lower level of organic acids and Atmrp2 secreted a higher level of amino acids as compared to the wild type. We conclude that the release of phytochemicals by roots is partially controlled by ABC transporters.

Plant neighbor identity influences plant biochemistry and physiology related to defense

BackgroundChemical and biological processes dictate an individual organisms ability to recognize and respond to other organisms. A small but growing body of evidence suggests that plants may be capable of recognizing and responding to neighboring plants in a species specific fashion. Here we tested whether or not individuals of the invasive exotic weed, Centaurea maculosa, would modulate their defensive strategy in response to different plant neighbors.ResultsIn the greenhouse, C. maculosa individuals were paired with either conspecific (C. maculosa) or heterospecific (Festuca idahoensis) plant neighbors and elicited with the plant defense signaling molecule methyl jasmonate to mimic insect herbivory. We found that elicited C. maculosa plants grown with conspecific neighbors exhibited increased levels of total phenolics, whereas those grown with heterospecific neighbors allocated more resources towards growth. To further investigate these results in the field, we conducted a metabolomics analysis to explore chemical differences between individuals of C. maculosa growing in naturally occurring conspecific and heterospecific field stands. Similar to the greenhouse results, C. maculosa individuals accumulated higher levels of defense-related secondary metabolites and lower levels of primary metabolites when growing in conspecific versus heterospecific field stands. Leaf herbivory was similar in both stand types; however, a separate field study positively correlated specialist herbivore load with higher densities of C. maculosa conspecifics.ConclusionsOur results suggest that an individual C. maculosa plant can change its defensive strategy based on the identity of its plant neighbors. This is likely to have important consequences for individual and community success.

Root secretion of defense-related proteins is development-dependent and correlated with flowering time.

Proteins found in the root exudates are thought to play a role in the interactions between plants and soil organisms. To gain a better understanding of protein secretion by roots, we conducted a systematic proteomic analysis of the root exudates of Arabidopsis thaliana at different plant developmental stages. In total, we identified 111 proteins secreted by roots, the majority of which were exuded constitutively during all stages of development. However, defense-related proteins such as chitinases, glucanases, myrosinases, and others showed enhanced secretion during flowering. Defense-impaired mutants npr1-1 and NahG showed lower levels of secretion of defense proteins at flowering compared with the wild type. The flowering-defective mutants fca-1, stm-4, and co-1 showed almost undetectable levels of defense proteins in their root exudates at similar time points. In contrast, root secretions of defense-enhanced cpr5-2 mutants showed higher levels of defense proteins. The proteomics data were positively correlated with enzymatic activity assays for defense proteins and with in silico gene expression analysis of genes specifically expressed in roots of Arabidopsis. In conclusion, our results show a clear correlation between defense-related proteins secreted by roots and flowering time.

New insights into somatic embryogenesis: leafy cotyledon1, baby boom1 and WUSCHEL-related homeobox4 are epigenetically regulated in Coffea canephora.

Plant cells have the capacity to generate a new plant without egg fertilization by a process known as somatic embryogenesis (SE), in which differentiated somatic cells can form somatic embryos able to generate a functional plant. Although there have been advances in understanding the genetic basis of SE, the epigenetic mechanism that regulates this process is still unknown. Here, we show that the embryogenic development of Coffea canephora proceeds through a crosstalk between DNA methylation and histone modifications during the earliest embryogenic stages of SE. We found that low levels of DNA methylation, histone H3 lysine 9 dimethylation (H3K9me2) and H3K27me3 change according to embryo development. Moreover, the expression of LEAFY COTYLEDON1 (LEC1) and BABY BOOM1 (BBM1) are only observed after SE induction, whereas WUSCHEL-RELATED HOMEOBOX4 (WOX4) decreases its expression during embryo maturation. Using a pharmacological approach, it was found that 5-Azacytidine strongly inhibits the embryogenic response by decreasing both DNA methylation and gene expression of LEC1 and BBM1. Therefore, in order to know whether these genes were epigenetically regulated, we used Chromatin Immunoprecipitation (ChIP) assays. It was found that WOX4 is regulated by the repressive mark H3K9me2, while LEC1 and BBM1 are epigenetically regulated by H3K27me3. We conclude that epigenetic regulation plays an important role during somatic embryogenic development, and a molecular mechanism for SE is proposed.

Biotic interactions in the rhizosphere: a diverse cooperative enterprise for plant productivity

Roots and microbes secrete organic compounds into the rhizosphere that influence plant productivity. Microbes and plants have evolved biochemical mechanisms to communicate with each other. The molecules responsible for such communication are secreted during beneficial or harmful interactions. Hundreds of these molecules secreted into the rhizosphere have been identified, and their functions are being studied in order to understand the mechanisms of interaction and communication among the different members of the rhizosphere community. The importance of root and microbe secretion to the underground habitat in improving crop productivity is increasingly recognized, with the discovery and characterization of new secreting compounds found in the rhizosphere. Different omic approaches, such as genomics, transcriptomics, proteomics, and metabolomics, have expanded our understanding of the first signals between microbes and plants. In this review, we highlight the more recent discoveries related to molecules secreted into the rhizosphere and how they affect plant productivity, either negatively or positively. In addition, we include a survey of novel approaches to studying the rhizosphere and emerging opportunities to direct future studies.

The role of chromatin modifications in somatic embryogenesis in plants.

Somatic embryogenesis (SE) is a powerful tool for plant genetic improvement when used in combination with traditional agricultural techniques, and it is also an important technique to understand the different processes that occur during the development of plant embryogenesis. SE onset depends on a complex network of interactions among plant growth regulators, mainly auxins and cytokinins, during the proembryogenic early stages, and ethylene and gibberellic and abscisic acids later in the development of the somatic embryos. These growth regulators control spatial and temporal regulation of multiple genes in order to initiate change in the genetic program of somatic cells, as well as moderating the transition between embryo developmental stages. In recent years, epigenetic mechanisms have emerged as critical factors during SE. Some early reports indicate that auxins and in vitro conditions modify the levels of DNA methylation in embryogenic cells. The changes in DNA methylation patterns are associated with the regulation of several genes involved in SE, such as WUS, BBM1, LEC, and several others. In this review, we highlight the more recent discoveries in the understanding of the role of epigenetic regulation of SE. In addition, we include a survey of different approaches to the study of SE, and new opportunities to focus SE studies.

Root Secreted Metabolites and Proteins Are Involved in the Early Events of Plant-Plant Recognition Prior to Competition

The mechanism whereby organisms interact and differentiate between others has been at the forefront of scientific inquiry, particularly in humans and certain animals. It is widely accepted that plants also interact, but the degree of this interaction has been constricted to competition for space, nutrients, water and light. Here, we analyzed the root secreted metabolites and proteins involved in early plant neighbor recognition by using Arabidopsis thaliana Col-0 ecotype (Col) as our focal plant co-cultured in vitro with different neighbors [A. thaliana Ler ecotype (Ler) or Capsella rubella (Cap)]. Principal component and cluster analyses revealed that both root secreted secondary metabolites and proteins clustered separately between the plants grown individually (Col-0, Ler and Cap grown alone) and the plants co-cultured with two homozygous individuals (Col-Col, Ler-Ler and Cap-Cap) or with different individuals (Col-Ler and Col-Cap). In particularly, we observed that a greater number of defense- and stress- related proteins were secreted when our control plant, Col, was grown alone as compared to when it was co-cultured with another homozygous individual (Col-Col) or with a different individual (Col-Ler and Col-Cap). However, the total amount of defense proteins in the exudates of the co-cultures was higher than in the plant alone. The opposite pattern of expression was identified for stress-related proteins. These data suggest that plants can sense and respond to the presence of different plant neighbors and that the level of relatedness is perceived upon initial interaction. Furthermore, the role of secondary metabolites and defense- and stress-related proteins widely involved in plant-microbe associations and abiotic responses warrants reassessment for plant-plant interactions.

KNOX1 is expressed and epigenetically regulated during in vitro conditions in Agave spp

BackgroundThe micropropagation is a powerful tool to scale up plants of economical and agronomical importance, enhancing crop productivity. However, a small but growing body of evidence suggests that epigenetic mechanisms, such as DNA methylation and histone modifications, can be affected under the in vitro conditions characteristic of micropropagation. Here, we tested whether the adaptation to different in vitro systems (Magenta boxes and Bioreactors) modified epigenetically different clones of Agave fourcroydes and A. angustifolia. Furthermore, we assessed whether these epigenetic changes affect the regulatory expression of KNOTTED1-like HOMEOBOX (KNOX) transcription factors.ResultsTo gain a better understanding of epigenetic changes during in vitro and ex vitro conditions in Agave fourcroydes and A. angustifolia, we analyzed global DNA methylation, as well as different histone modification marks, in two different systems: semisolid in Magenta boxes (M) and temporary immersion in modular Bioreactors (B). No significant difference was found in DNA methylation in A. fourcroydes grown in either M or B. However, when A. fourcroydes was compared with A. angustifolia, there was a two-fold difference in DNA methylation between the species, independent of the in vitro system used. Furthermore, we detected an absence or a low amount of the repressive mark H3K9me2 in ex vitro conditions in plants that were cultured earlier either in M or B. Moreover, the expression of AtqKNOX1 and AtqKNOX2, on A. fourcroydes and A. angustifolia clones, is affected during in vitro conditions. Therefore, we used Chromatin ImmunoPrecipitation (ChIP) to know whether these genes were epigenetically regulated. In the case of AtqKNOX1, the H3K4me3 and H3K9me2 were affected during in vitro conditions in comparison with AtqKNOX2.ConclusionsAgave clones plants with higher DNA methylation during in vitro conditions were better adapted to ex vitro conditions. In addition, A. fourcroydes and A. angustifolia clones displayed differential expression of the KNOX1 gene during in vitro conditions, which is epigenetically regulated by the H3K4me3 and H3K9me2 marks. The finding of an epigenetic regulation in key developmental genes will make it important in future studies to identify factors that help to find climate-resistant micropropagated plants.

Methyl Jasmonate Induces ATP Biosynthesis Deficiency and Accumulation of Proteins Related to Secondary Metabolism in Catharanthus roseus (L.) G. Hairy Roots

Jasmonates are specific signal molecules in plants that are involved in a diverse set of physiological and developmental processes. However, methyl jasmonate (MeJA) has been shown to have a negative effect on root growth and, so far, the biochemical mechanism for this is unknown. Using Catharanthus roseus hairy roots, we were able to observe the effect of MeJA on growth inhibition, cell disorganization and cell death of the root cap. Hairy roots treated with MeJA induced the perturbation of mitochondrial membrane integrity and a diminution in ATP biosynthesis. Furthermore, several proteins were identified that were involved in energy and secondary metabolism; the changes in accumulation of these proteins were observed with 100 μM MeJA. In conclusion, our results suggest that a switch of the metabolic fate of hairy roots in response to MeJA could cause an increase in the accumulation of secondary metabolites. This is likely to have important consequences in the production of specific alkaloids important for the pharmaceutical industry.