Summary
I had the fortune to start my scientific carrier during the early stages of the development of plant transformation in one of the leading laboratories in the field. Here, I describe my ...personal experience in the laboratory of Marc van Montagu and Jeff Schell, and some important contributions that the group made to the development of the technology to produce transgenic plants. I also briefly summarize the impact of this technology on the development of modern plant biology and in plant molecular improvement.
Summary
Phosphate (Pi) is a critical macronutrient for the biochemical and molecular functions of cells. Under phosphate limitation, plants manifest adaptative strategies to increase phosphate ...scavenging. However, how low phosphate sensing links to the transcriptional machinery remains unknown.
The role of the MEDIATOR (MED) transcriptional co‐activator, through its MED16 subunit in Arabidopsis root system architecture remodeling in response to phosphate limitation was assessed. Its critical function acting over the SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1)‐ALUMINUM‐ACTIVATED MALATE TRANSPORT1 (ALMT1) signaling module was tested through a combination of genetic, biochemical, and genome‐wide transcriptomic approaches.
Root system configuration in response to phosphate scarcity involved MED16 functioning, which modulates the expression of a large set of low‐phosphate‐induced genes that respond to local and systemic signals in the Arabidopsis root tip, including those directly activated by STOP1. Biomolecular fluorescence complementation analysis suggests that MED16 is required for the transcriptional activation of STOP1 targets, including the membrane permease ALMT1, to increase malate exudation in response to low phosphate.
Our results unveil the function of a critical transcriptional component, MED16, in the root adaptive responses to a scarce plant macronutrient, which helps understanding how plant cells orchestrate root morphogenesis to gene expression with the STOP1‐ALMT1 module.
SUMMARY
Low‐molecular‐weight organic acid (OA) extrusion by plant roots is critical for plant nutrition, tolerance to cations toxicity, and plant–microbe interactions. Therefore, methodologies for ...the rapid and precise quantification of OAs are necessary to be incorporated in the analysis of roots and their exudates. The spatial location of root exudates is also important to understand the molecular mechanisms directing OA production and release into the rhizosphere. Here, we report the development of two complementary methodologies for OA determination, which were employed to evaluate the effect of inorganic ortho‐phosphate (Pi) deficiency and aluminum toxicity on OA excretion by Arabidopsis roots. OA exudation by roots is considered a core response to different types of abiotic stress and for the interaction of roots with soil microbes, and for decades has been a target trait to produce plant varieties with increased capacities of Pi uptake and Al tolerance. Using targeted ultra‐performance liquid chromatography coupled with high‐resolution tandem mass spectrometry (UPLC‐HRMS/MS), we achieved the quantification of six OAs in root exudates at sub‐micromolar detection limits with an analysis time of less than 5 min per sample. We also employed targeted (MS/MS) matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to detect the spatial location of citric and malic acid with high specificity in roots and exudates. Using these methods, we studied OA exudation in response to Al toxicity and Pi deficiency in Arabidopsis seedlings overexpressing genes involved in OA excretion. Finally, we show the transferability of the MALDI‐MSI method by analyzing OA excretion in Marchantia polymorpha gemmalings subjected to Pi deficiency.
Significance Statement
We developed high‐resolution tandem mass spectrometry methods for high‐throughput quantification of six small organic acids in root exudates and for the in situ localization of malate and citrate in plant tissues and its exudates with high specificity. Employing these methods, we were able to detect changes in the exudation rate and localization patterns of organic acids in response to P deficiency in roots of Arabidopsis plants.
Summary
The Mediator (MED) complex plays a key role in the recruitment and assembly of the transcription machinery for the control of gene expression. Here, we report on the role of MEDIATOR18 ...(MED18) subunit in root development, auxin signaling and meristem cell viability in Arabidopsis thaliana seedlings. Loss‐of‐function mutations in MED18 reduce primary root growth, but increase lateral root formation and root hair development. This phenotype correlates with alterations in cell division and elongation likely caused by an increased auxin response and transport at the root tip, as evidenced by DR5:GFP, pPIN1::PIN1‐GFP, pPIN2::PIN2‐GFP and pPIN3::PIN3‐GFP auxin‐related gene expression. Noteworthy, med18 seedlings manifest cell death in the root meristem, which exacerbates with age and/or exposition to DNA‐damaging agents, and display high expression of the cell regeneration factor ERF115. Cell death in the root tip was reduced in med18 seedlings grown in darkness, but remained when only the shoot was exposed to light, suggesting that MED18 acts to protect root meristem cells from local cell death, and/or in response to root‐acting signal(s) emitted by the shoot in response to light stimuli. These data point to MED18 as an important component for auxin‐regulated root development, cell death and cell regeneration in root meristems.
Significance statement
Plant growth and adaptation rely on the activity of meristems. Here, we show that the MEDIATOR subunit 18 (MED18) is critical for viability of root proliferative cells controlling the balance between cell death and differentiation, and demonstrate that light perception in the shoot influences the stability and DNA repair mechanisms in root meristems.
The presence of Bacillus in very diverse environments reflects the versatile metabolic capabilities of a widely distributed genus. Traditional phylogenetic analysis based on limited gene sampling is ...not adequate for resolving the genus evolutionary relationships. By distinguishing between core and pan-genome, we determined the evolutionary and functional relationships of known Bacillus.
Our analysis is based upon twenty complete and draft Bacillus genomes, including a newly sequenced Bacillus isolate from an aquatic environment that we report for the first time here. Using a core genome, we were able to determine the phylogeny of known Bacilli, including aquatic strains whose position in the phylogenetic tree could not be unambiguously determined in the past. Using the pan-genome from the sequenced Bacillus, we identified functional differences, such as carbohydrate utilization and genes involved in signal transduction, which distinguished the taxonomic groups. We also assessed the genetic architecture of the defining traits of Bacillus, such as sporulation and competence, and showed that less than one third of the B. subtilis genes are conserved across other Bacilli. Most variation was shown to occur in genes that are needed to respond to environmental cues, suggesting that Bacilli have genetically specialized to allow for the occupation of diverse habitats and niches.
The aquatic Bacilli are defined here for the first time as a group through the phylogenetic analysis of 814 genes that comprise the core genome. Our data distinguished between genomic components, especially core vs. pan-genome to provide insight into phylogeny and function that would otherwise be difficult to achieve. A phylogeny may mask the diversity of functions, which we tried to uncover in our approach. The diversity of sporulation and competence genes across the Bacilli was unexpected based on previous studies of the B. subtilis model alone. The challenge of uncovering the novelties and variations among genes of the non-subtilis groups still remains. This task will be best accomplished by directing efforts toward understanding phylogenetic groups with similar ecological niches.
Summary
High‐temperature bioconversion of lignocellulose into fermentable sugars has drawn attention for efficient production of renewable chemicals and biofuels, because competing microbial ...activities are inhibited at elevated temperatures and thermostable cell wall degrading enzymes are superior to mesophilic enzymes. Here, we report on the development of a platform to produce four different thermostable cell wall degrading enzymes in the chloroplast of Chlamydomonas reinhardtii. The enzyme blend was composed of the cellobiohydrolase CBM3GH5 from C. saccharolyticus, the β‐glucosidase celB from P. furiosus, the endoglucanase B and the endoxylanase XynA from T. neapolitana. In addition, transplastomic microalgae were engineered for the expression of phosphite dehydrogenase D from Pseudomonas stutzeri, allowing for growth in non‐axenic media by selective phosphite nutrition. The cellulolytic blend composed of the glycoside hydrolase (GH) domain GH12/GH5/GH1 allowed the conversion of alkaline‐treated lignocellulose into glucose with efficiencies ranging from 14% to 17% upon 48h of reaction and an enzyme loading of 0.05% (w/w). Hydrolysates from treated cellulosic materials with extracts of transgenic microalgae boosted both the biogas production by methanogenic bacteria and the mixotrophic growth of the oleaginous microalga Chlorella vulgaris. Notably, microalgal treatment suppressed the detrimental effect of inhibitory by‐products released from the alkaline treatment of biomass, thus allowing for efficient assimilation of lignocellulose‐derived sugars by C. vulgaris under mixotrophic growth.
Summary
Microalgal cultivation that takes advantage of solar energy is one of the most cost‐effective systems for the biotechnological production of biofuels, and a range of high value products, ...including pharmaceuticals, fertilizers and feed. However, one of the main constraints for the cultivation of microalgae is the potential contamination with biological pollutants, such as bacteria, fungi, zooplankton or other undesirable microalgae. In closed bioreactors, the control of contamination requires the sterilization of the media, containers and all materials, which increases the cost of production, whereas open pond systems severely limits the number of species that can be cultivated under extreme environmental conditions to prevent contaminations. Here, we report the metabolic engineering of Chlamydomonas reinhardtii to use phosphite as its sole phosphorus source by expressing the ptxD gene from Pseudomonas stutzeri WM88, which encodes a phosphite oxidoreductase able to oxidize phosphite into phosphate using NAD as a cofactor. Engineered C. reinhardtii lines are capable of becoming the dominant species in a mixed culture when fertilized with phosphite as a sole phosphorus source. Our results represent a new platform for the production of microalgae, potentially useful for both closed photobioreactors and open pond systems without the need for using sterile conditions nor antibiotics or herbicides to prevent contamination with biological pollutants.
Plants adapt to soil injury and biotic stress via cell regeneration. In Arabidopsis, root tip damage by genotoxic agents, antibiotics, UV light and cutting induces a program that recovers the missing ...tissues through activation of stem cells and involves ethylene response factor 115 (ERF115), which triggers cell replenishment. Here, we show that mutation of the gene encoding an MED18 subunit of the transcriptional MEDIATOR complex and chromate Cr(VI), an environmental pollutant, synergistically trigger a developmental program that enables the splitting of the meristem in vivo to produce twin roots. Expression of the quiescent centre gene marker WOX5, auxin‐inducible DR5:GFP reporter and the ERF115 factor traced the changes in cell identity during the conversion of single primary root meristems into twin roots and were induced in an MED18 and chromate‐dependent manner during the root twinning events, which also required auxin redistribution and signalling mediated by IAA14/SOLITARY ROOT (SLR1). Splitting of the root meristem allowed dichotomous root branching in Arabidopsis, a poorly understood process in which stem cells may act to enable whole organ regeneration.
This manuscript describes the twinning of the primary root meristem, also termed root fasciation, in Arabidopsis med18 mutants treated with chromate. Root regeneration takes place through cell re‐specification programs, which correlates with quiescent centre, auxin re‐distribution and cell renewal.
Predictions on the lifetime of phosphate (Pi) reserves usually take into account only the need for crop production. A recent report predicts the global need of chemical Pi fertilizer for sustaining ...productivity in cropland and grassland. Here we discuss the implications of these predictions for the lifetime of Pi reserves.
Summary
Phosphate (Pi)‐deficient soils are a major limitant factor for crop production in many regions of the world. Despite that plants have innovated several developmental and biochemical ...strategies to deal with this stress, there are still massive extensions of land which combine several abiotic stresses, including phosphate starvation, that limit their use for plant growth and food production. In several plant species, a genetic programme underlies the biochemical and developmental responses of the organism to cope with low phosphate (Pi) availability. Both protein‐ and miRNA‐coding genes involved in the adaptative response are transcriptionally activated upon Pi starvation. Several of the responsive genes have been identified as transcriptional targets of PHR1, a transcription factor that binds a conserved cis‐element called PHR1‐binding site (P1BS). Our group has previously described and characterized a minimal genetic arrangement that includes two P1BS elements, as a phosphate‐responsive enhancer (EZ2). Here, we report the engineering and successful use of a phosphate‐dependent bidirectional promoter, which has been designed and constructed based on the palindromic sequences of the two P1BS elements present in EZ2. This bidirectional promoter has a potential use in both plant in vitro approaches and in the generation of improved crops adapted to Pi starvation and other abiotic stresses.
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