Plant metabolite 5-pentadecyl resorcinol is produced by the Amazonian fungus Penicillium sclerotiorum LM 5679

Since the classic studies of Alexander Flemming, Penicillium strains have been known as a rich source of antimicrobial substances. Recent studies have identified novel metabolites produced by Penicillium sclerotiorum that have antibacterial, antifouling and pharmaceutical activities. Here, we report the isolation of a P. sclerotiorum (LM 5679) from Amazonian soil and carry out a culture-based study to determine whether it can produce any novel secondary metabolite(s) that are not thus-far reported for this genus. Using a submerged culture system, secondary metabolites were recovered by solvent extract followed by thin-layer chromatography, nuclear magnetic resonance, and mass spectroscopy. One novel secondary metabolite was isolated from P. sclerotiorum (LM 5679); the phenolic compound 5-pentadecyl resorcinol widely known as an antifungal, that is produced by diverse plant species. This metabolite was not reported previously in any Penicillium species and was only found once before in fungi (that time, in a Fusarium). Here, we discuss the known activities of 5-pentadecyl resorcinol in the context of its mode-of-action as a hydrophobic (chaotropicity-mediated) stressor.

A Versatile and Efficient Plant Protoplast Platform for Genome Editing by Cas9 RNPs

The ultimate goal of technology development in genome editing is to enable precisely targeted genomic changes in any cells or organisms. Here we describe protoplast systems for precise and efficient DNA sequence changes with preassembled Cas9 ribonucleoprotein (RNP) complexes in Arabidopsis thaliana, Nicotiana benthamiana, Brassica rapa, and Camelina sativa. Cas9 RNP-mediated gene disruption with dual gRNAs could reach ∼90% indels in Arabidopsis protoplasts. To facilitate facile testing of any Cas9 RNP designs, we developed two GFP reporter genes, which led to sensitive detection of nonhomologous end joining (NHEJ) and homology-directed repair (HDR), with editing efficiency up to 85 and 50%, respectively. When co-transfected with an optimal single-stranded oligodeoxynucleotide (ssODN) donor, precise editing of the AtALS gene via HDR reached 7% by RNPs. Significantly, precise mutagenesis mediated by preassembled primer editor (PE) RNPs led to 50% GFP reporter gene recovery in protoplasts and up to 4.6% editing frequency for the specific AtPDS mutation in the genome. The rapid, versatile and efficient gene editing by CRISPR RNP variants in protoplasts provides a valuable platform for development, evaluation and optimization of new designs and tools in gene and genomic manipulation and is applicable in diverse plant species.

Inoculation of Herbaspirillum Seropedicae Increases Biomass in Maize Roots in the Early Stages of Plant Development

Herbaspirillum seropedicae is a plant growth-promoting bacteria isolated from diverse plant species. In this work, the main objective was to investigate the efficiency of H. seropedicae strain SmR1 in colonizing and increasing maize growth in the early stages of development under greenhouse conditions. Inoculation with H. seropedicae resulted in 10.51 and 19.43% in mean of increase of root biomass concerning non-inoculated controls, mainly in the initial stages of plant development, at 21 days after emergence (DAE). Quantification of H. seropedicae in roots and leaves was performed by quantitative PCR.. H. seropedicae was detected only in maize inoculated roots by qPCR, and a slight decrease in DNA copy number g−1 of fresh root weight was observed from 7 to 21 DAE, suggesting that there was initial effective colonization on maize plants. H. seropedicae strain SmR1 efficiently increased maize root biomass exhibiting its potencial to be used as inoculant in agricultures systems.

Comparative Review of Plant Transformation Techniques

Plant transformation is now an important biotechnological tool in plant biology and a practical tool for transgenic plant development. There are many verified methods for stable introduction of novel genes into the nuclear genomes of diverse plant species. As a result, gene transfer and regeneration of transgenic plants are no longer the factors limiting the development and application of practical transformation systems for many plant species. However, the desire for higher transformation efficiency has stimulated work on not only improving various existing methods but also in inventing novel methods. Different methods of transferring the gene into plant cells have been developed and continuous efforts have been made to increase its efficiency. Both direct and indirect methods of gene transfer have their own merits and demerits. Efforts have been made continuously to eliminate drawbacks and to develop an easy and eco-friendly method to transfer foreign genes. Many methods of genetic transformation have been proposed and tried in the laboratories, but most of them result to transient expressions. However, transformation work based on particle bombardment with DNA coated micro projectiles and Agrobacterium mediated transformation have proved to be promising in producing stable transgenic plants from a range of plant species.

Insights into Metabolic Engineering of the Biosynthesis of Glycine Betaine and Melatonin to Improve Plant Abiotic Stress Tolerance

Metabolic engineering in plant can be describe as a tool using molecular biological technologies which promotes enzymatic reactions that can enhance the biosynthesis of existing compounds such as glycine betaine (GB) in plant species that are able to accumulate GB, or produce news compounds like GB in non-accumulators plants. Moreover we can include to these definition, the mediation in the degradation of diverse compounds in plant organism. For decades, one of the most popular ideas in metabolic engineering literature is the idea that the improvement of gly betaine or melatonin accumulation in plant under environmental stress can be the main window to ameliorate stress tolerance in diverse plant species. A challenging problem in this domain is the integration of different molecular technologies like transgenesis, enzyme kinetics, promoter analysis, biochemistry and genetics, protein sorting, cloning or comparative physiology to reach that objective. A large number of approaches have been developed over the last few decades in metabolic engineering to overcome this problem. Therefore, we examine some previous work and propose some understanding about the use of metabolic engineering in plant stress tolerance. Moreover, this chapter will focus on melatonin (Hormone) and gly betaine (Osmolyte) biosynthesis pathways in engineering stress resistance.

Enzyme Complexes of Ptr4CL and PtrHCT Modulate Co-enzyme A Ligation of Hydroxycinnamic Acids for Monolignol Biosynthesis in Populus trichocarpa

Co-enzyme A (CoA) ligation of hydroxycinnamic acids by 4-coumaric acid:CoA ligase (4CL) is a critical step in the biosynthesis of monolignols. Perturbation of 4CL activity significantly impacts the lignin content of diverse plant species. In Populus trichocarpa, two well-studied xylem-specific Ptr4CLs (Ptr4CL3 and Ptr4CL5) catalyze the CoA ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. Subsequently, two 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) mediate the conversion of 4-coumaroyl-CoA to caffeoyl-CoA. Here, we show that the CoA ligation of 4-coumaric and caffeic acids is modulated by Ptr4CL/PtrHCT protein complexes. Downregulation of PtrHCTs reduced Ptr4CL activities in the stem-differentiating xylem (SDX) of transgenic P. trichocarpa. The Ptr4CL/PtrHCT interactions were then validated in vivo using biomolecular fluorescence complementation (BiFC) and protein pull-down assays in P. trichocarpa SDX extracts. Enzyme activity assays using recombinant proteins of Ptr4CL and PtrHCT showed elevated CoA ligation activity for Ptr4CL when supplemented with PtrHCT. Numerical analyses based on an evolutionary computation of the CoA ligation activity estimated the stoichiometry of the protein complex to consist of one Ptr4CL and two PtrHCTs, which was experimentally confirmed by chemical cross-linking using SDX plant protein extracts and recombinant proteins. Based on these results, we propose that Ptr4CL/PtrHCT complexes modulate the metabolic flux of CoA ligation for monolignol biosynthesis during wood formation in P. trichocarpa.

Identifying synergies and antagonisms between plant defense traits in Asclepias syriaca

As a general rule, plants defend against herbivores with multiple traits. The defense synergy hypothesis posits that some traits are more potent when co-expressed with others, compared to their independent potency. However, this hypothesis has rarely been tested outside of synergies within a class of particular phytochemicals, and seldom under field conditions. We tested for synergies between multiple defense traits of common milkweed (Asclepias syriaca) by assaying the performance of two specialist herbivores on plants in natural populations. We employed both standard regression and exploratory analysis using a novel application of Random Forests that allowed us to detect synergies between defense traits. In hypothesis testing, we found the first empirical evidence for a previously hypothesized synergy between one pair of co-expressed defense traits (latex and cardenolides), but not another (latex and trichomes). When exploring all potential interactions between pairs of traits we found eight synergies and five antagonisms in predicting herbivore performance. Half of the identified synergies involved carbon, which is the basis of several defenses including chemical and physical barriers to feeding, and also essential nutrients. Our findings suggest that defense synergies could explain co-expression of latex and cardenolides in milkweeds. This synergy may be common among the diverse plant species that employ latex as a defense. Future studies should test carbon-based synergies, which our work suggests are prevalent, as well as the other synergies identified in our exploratory analysis. Our analytic approach provides a general, flexible framework for more broadly discovering and predicting the coexpression of traits through their synergistic function.

Characterization of Erwinia gerundensis A4, an Almond-Derived Plant Growth-Promoting Endophyte

The rapidly increasing global population and anthropogenic climate change have created intense pressure on agricultural systems to produce increasingly more food under steadily challenging environmental conditions. Simultaneously, industrial agriculture is negatively affecting natural and agricultural ecosystems because of intensive irrigation and fertilization to fully utilize the potential of high-yielding cultivars. Growth-promoting microbes that increase stress tolerance and crop yield could be a useful tool for helping mitigate these problems. We investigated if commercially grown almonds might be a resource for plant colonizing bacteria with growth promotional traits that could be used to foster more productive and sustainable agricultural ecosystems. We isolated an endophytic bacterium from almond leaves that promotes growth of the model plant Arabidopsis thaliana. Genome sequencing revealed a novel Erwinia gerundensis strain (A4) that exhibits the ability to increase access to plant nutrients and to produce the stress-mitigating polyamine spermidine. Because E. gerundensis is known to be able to colonize diverse plant species including cereals and fruit trees, A4 may have the potential to be applied to a wide variety of crop systems.

Transcriptional Landscape and Splicing Efficiency in Arabidopsis Mitochondria

Plant mitochondrial transcription is initiated from multiple promoters without an apparent motif, which precludes their identification in other species based on sequence comparisons. Even though coding regions take up only a small fraction of plant mitochondrial genomes, deep RNAseq studies uncovered that these genomes are fully or nearly fully transcribed with significantly different RNA read depth across the genome. Transcriptomic analysis can be a powerful tool to understand the transcription process in diverse angiosperms, including the identification of potential promoters and co-transcribed genes or to study the efficiency of intron splicing. In this work, we analyzed the transcriptional landscape of the Arabidopsis mitochondrial genome (mtDNA) based on large-scale RNA sequencing data to evaluate the use of RNAseq to study those aspects of the transcription process. We found that about 98% of the Arabidopsis mtDNA is transcribed with highly different RNA read depth, which was elevated in known genes. The location of a sharp increase in RNA read depth upstream of genes matched the experimentally identified promoters. The continuously high RNA read depth across two adjacent genes agreed with the known co-transcribed units in Arabidopsis mitochondria. Most intron-containing genes showed a high splicing efficiency with no differences between cis and trans-spliced introns or between genes with distinct splicing mechanisms. Deep RNAseq analyses of diverse plant species will be valuable to recognize general and lineage-specific characteristics related to the mitochondrial transcription process.