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Editorial: Proceedings of ICPSBBB 2018 - 2nd International Conference on Plant Synthetic Biology, Bioengineering and Biotechnology

Plant synthetic biology combines engineering principles with plant biology toward the design, construction, and testing of plants with new traits (Liu and Stewart, 2015). Synthetic biology applies engineering principles, using defined genetic parts with the aim of achieving predictable outcomes. The field aims at providing fuels, pharmaceuticals, bioactive metabolites and increased crop yield. Synthetic biology is increasingly forming a framework in genetic transformation of plants allowing us to improve traits and crop performance. The conference on Plant Synthetic Biology, Bioengineering, and Biotechnology covers broad aspects of plant synthetic biology, ranging from tools to the design of predictable functions and precision genome editing, to re-designed photosynthesis, plant bioengineering techniques, and rational design of novel plant traits. This Frontiers in Plant Science virtual issue on the “Research Topic Proceedings of ICPSBBB 2018 - 2nd International Conference on Plant Synthetic Biology, Bioengineering and Biotechnology” consists of 8 publications, including four reviews and four original research articles, which fall into the following general topics: quantitative and genetic parts, evolution, expression of transgenes and epigenetic changes, engineering of stress tolerance and bioproduction.

59 BASIC BIOLOGICAL SCIENCES↗

Behavioral and biological interactions with small groups in confined microsocieties

Requirements for high levels of human performance in the unfamiliar and stressful environments associated with space missions necessitate the development of research-based technological procedures for maximizing the probability of effective functioning at all levels of personnel participation. Where the successful accomplishment of such missions requires the coordinated contributions of several individuals collectively identified with the achievement of a common objective, the conditions for characterizing a team, crew, or functional group are operationally defined. For the most part, studies of group performances under operational conditions which emphasize relatively long exposure to extended mission environments have been limited by the constraints imposed on experimental manipulations to identify critical effectiveness factors. On the other hand, laboratory studies involving relatively brief exposures to contrived task situations have been considered of questionable generality to operational settings requiring realistic group objectives.

Brady, J. V.↗

Stoichiometry of the large conductance bacterial mechanosensitive channel of E. coli. A biochemical study

MscL, a 15 kDa transmembrane protein, is the only component involved in the formation of a 3 nS channel in the inner membrane of Escherichia coli that opens in response to mechanical or osmotic stress. While previous data had suggested that the functional MscL complex might be a hexamer, a recent crystallographic study of the MscL homologue from M. tuberculosis reveals a pentameric structure. The present work further examines the stoichiometry of the E. coli MscL using a variety of biochemical approaches. Detergent-purified 6His-MscL in solution and MscL in the membrane could be chemically crosslinked with the products displaying ladderlike patterns on SDS gels. Three crosslinking agents (EDC, DMS, and DMA) used at saturating concentrations invariably generated pentamers as the largest product. DSS produced additional bands corresponding to larger complexes although the pentamer band appeared to be the predominant product at high levels of crosslinker. It is not clear whether these extra bands reflect a difference in the crosslinking chemistry of DSS or whether its spacer arm is the longest of those used, or a combination of both facts. For the detergent-solubilized 6His-MscL both sedimentation equilibrium and gel chromatography showed the presence of multiple species. Thus the longer spacer arm could permit both intra- and intercomplex linkages. Nonetheless, the patterns obtained with all agents are consistent with and strongly suggest a pentameric organization for the MscL channel. Expression of MscL as genetically engineered double or triple subunit tandems yields low numbers of functional channels as compared to expressed monomers. The double-tandem assemblies must have an even number of subunits and crosslinking in the membrane confirmed hexamerization. Gel chromatography clearly demonstrated that the channels formed from the double tandems were larger than those formed from WT MscL, consistent with the native channel being pentameric. The observation that both double and triple tandems form channels of normal conductance implies that the pentameric assembly is to some degree independent of the number of subunit repeats in the polypeptide precursor. The channel is thus a pentameric core with the 'extra' subunits left out of the functional complex. From sedimentation equilibrium and size-exclusion chromatography, we also conclude that MscL complexes are not in a dynamic equilibrium with monomers, but are pre-assembled; and thus, their gating properties must result from changes in the conformation of the entire complex induced by the mechanical stress.

NASA Discipline Cell Biology↗

Longitudinal genome-wide association study reveals early QTL that predict biomass accumulation under cold stress in sorghum

Sorghum bicolor is a promising cellulosic feedstock crop for bioenergy due to its high biomass yields. However, early growth phases of sorghum are sensitive to cold stress, limiting its planting in temperate environments. Cold adaptability is crucial for cultivating bioenergy and grain sorghum at higher latitudes and elevations, or for extending the growing season. Identifying genes and alleles that enhance biomass accumulation under early cold stress can lead to improved sorghum varieties through breeding or genetic engineering. We conducted image-based phenotyping on 369 accessions from the sorghum Bioenergy Association Panel (BAP) in a controlled environment with early cold treatment. The BAP includes diverse accessions with dense genotyping and varied racial, geographical, and phenotypic backgrounds. Daily, non-destructive imaging allowed temporal analysis of growth-related traits and water use efficiency (WUE). A genome-wide association study (GWAS) was performed to identify genomic intervals and genes associated with cold stress response. The GWAS identified transient quantitative trait loci (QTL) strongly associated with growth-related traits, enabling an exploration of the genetic basis of cold stress response at different developmental stages. This analysis of daily growth traits, rather than endpoint traits, revealed early transient QTL predictive of final phenotypes. The study identified both known and novel candidate genes associated with growth-related traits and temporal responses to cold stress. The identified QTL and candidate genes contribute to understanding the genetic mechanisms underlying sorghum's response to cold stress. These findings can inform breeding and genetic engineering strategies to develop sorghum varieties with improved biomass yields and resilience to cold, facilitating earlier planting, extended growing seasons, and cultivation at higher latitudes and elevations.

59 BASIC BIOLOGICAL SCIENCES↗

The major trimeric antenna complexes serve as a site for qH-energy dissipation in plants

Plants and algae are faced with a conundrum: harvesting sufficient light to drive their metabolic needs while dissipating light in excess to prevent photodamage, a process known as nonphotochemical quenching. A slowly relaxing form of energy dissipation, termed qH, is critical for plants’ survival under abiotic stress; however, qH location in the photosynthetic membrane is unresolved. Here, we tested whether we could isolate subcomplexes from plants in which qH was induced that would remain in an energy-dissipative state. Interestingly, we found that chlorophyll (Chl) fluorescence lifetimes were decreased by qH in isolated major trimeric antenna complexes, indicating that they serve as a site for qH-energy dissipation and providing a natively quenched complex with physiological relevance to natural conditions. Next, we monitored the changes in thylakoid pigment, protein, and lipid contents of antenna with active or inactive qH but did not detect any evident differences. Finally, we investigated whether specific subunits of the major antenna complexes were required for qH but found that qH was insensitive to trimer composition. Because we previously observed that qH can occur in the absence of specific xanthophylls, and no evident changes in pigments, proteins, or lipids were detected, we tentatively propose that the energy-dissipative state reported here may stem from Chl–Chl excitonic interaction.

59 BASIC BIOLOGICAL SCIENCES↗

Oxygen stress mitigation for microalgal biomass productivity improvement in outdoor raceway ponds

Dissolved oxygen (DO) concentrations at supersaturated levels are commonly observed in outdoor microalgae cultures due to photosynthesis during periods of rapid growth. Paradoxically, the photosynthetically generated oxygen is inhibitory and lowers biomass productivity, therefore, increasing the cost of microalgal biofuels. In this study, photosynthetic rates of Monoraphidium minutum 26B-AM and Scenedesmus obliquus UTEX393, two of the top DISCOVR strains, were compared at three DO levels, 0, 8, and 30 mg L -1 . Increasing DO from 0 to 30 mg L -1 caused a 36.6% and 26.1% reduction in photosynthetic rate for M. minutum 26B-AM and S. obliquus UTEX393, respectively. To improve the outdoor pond biomass productivity of each strain, three oxygen stress mitigation methods were tested. The first method raised the dissolved bicarbonate concentration by an order of magnitude to favor bicarbonate transport into the cell, increasing the CO 2 :O 2 ratio near RuBisCO, thus reducing photorespiration. Although this method increased the observed photosynthesis rate of M. minutum 26B-AM by 26% and S. obliquus UTEX393 by 13% in laboratory testing, it failed to improve biomass productivity under outdoor pond conditions for both strains. The second method added sodium sulfite as an oxygen scavenger to chemically lower the DO concentration in the culture. Chemical reduction of dissolved molecular oxygen did not significantly improve the biomass productivity of M. minutum 26B-AM; however, a 10% improvement in biomass productivity for S. obliquus UTEX393 was observed. The final method sparged cultures with air to strip oxygen out of the liquid medium. This did not improve the biomass productivity of M. minutum 26B-AM, likely due to unfavorable weather conditions during cold season cultivation; however, an improvement of 36% in biomass productivity was achieved for S. obliquus UTEX393 during the warm season cultivation. Of the three tested methods, air-sparging was the most effective and practical for improving outdoor biomass production.

59 BASIC BIOLOGICAL SCIENCES↗

Plant ecological genomics at the limits of life in the Atacama Desert

The Atacama Desert in Chile—hyperarid and with high–ultraviolet irradiance levels—is one of the harshest environments on Earth. Yet, dozens of species grow there, including Atacama-endemic plants. Herein, we establish the Talabre–Lejía transect (TLT) in the Atacama as an unparalleled natural laboratory to study plant adaptation to extreme environmental conditions. We characterized climate, soil, plant, and soil–microbe diversity at 22 sites (every 100 m of altitude) along the TLT over a 10-y period. We quantified drought, nutrient deficiencies, large diurnal temperature oscillations, and pH gradients that define three distinct vegetational belts along the altitudinal cline. Further, we deep-sequenced transcriptomes of 32 dominant plant species spanning the major plant clades, and assessed soil microbes by metabarcoding sequencing. The top-expressed genes in the 32 Atacama species are enriched in stress responses, metabolism, and energy production. Moreover, their root-associated soils are enriched in growth-promoting bacteria, including nitrogen fixers. To identify genes associated with plant adaptation to harsh environments, we compared 32 Atacama species with the 32 closest sequenced species, comprising 70 taxa and 1,686,950 proteins. To perform phylogenomic reconstruction, we concatenated 15,972 ortholog groups into a supermatrix of 8,599,764 amino acids. Using two codon-based methods, we identified 265 candidate positively selected genes (PSGs) in the Atacama plants, 64% of which are located in Pfam domains, supporting their functional relevance. For 59/184 PSGs with an Arabidopsis ortholog, we uncovered functional evidence linking them to plant resilience. As some Atacama plants are closely related to staple crops, these candidate PSGs are a “genetic goldmine” to engineer crop resilience to face climate change.

54 ENVIRONMENTAL SCIENCES↗

Phosphate Availability Modulates Root Exudate Composition and Rhizosphere Microbial Community in a Teosinte and a Modern Maize Cultivar

Domestication and breeding have affected interactions between plants and their microbiomes in ways that are only beginning to be understood but may have important implications for recruitment of rhizosphere microorganisms, particularly under stress conditions. We investigated the responses of a modern maize (Zea mays subsp. mays) cultivar and its wild relative, teosinte (Z. mays subsp. parviglumis), to different phosphate availabilities. We appraised responses of the plant-microbial holobiont to phosphate stresses by profiling root exudate metabolomes, and microbial communities in the root endosphere and rhizosphere. We also performed plate assays to quantify phosphate-solubilizing microorganisms from the rhizosphere. Although root exudate metabolite profiles were distinct between the teosinte and modern maize under high phosphate, both plants shifted exudate compositions in response to phosphate stress toward a common metabolite profile. Root and rhizosphere microbial communities also responded significantly to both plant type and the phosphate availability. A subset of bacterial and fungal taxa were differentially abundant under the different phosphate conditions, with each of the three conditions favoring different taxa. Both teosinte and maize rhizospheres harbored phosphate-solubilizing microorganisms under all growth conditions. These results suggest that the root exudation response to phosphate stress was conserved through the domestication of maize from teosinte, shifting exudation levels of specific metabolites. Although microbial communities also shifted, plate-based assays did not detect selective recruitment of phosphate solubilizers in response to phosphate availability.

59 BASIC BIOLOGICAL SCIENCES↗

Plant endomembranes and cytoskeleton: moving targets in immunity

Pathogens attack plant cells to divert resources toward pathogen proliferation. To resist pathogens, plant cells rely on multilayered signaling pathways that hinge upon the secretory pathway for the synthesis and trafficking of pathogen sensors and defense molecules. In recent years, significant strides have been made in the understanding of the functional relationship between pathogen response and membrane traffic. Furthermore, we discuss how the plant cytoskeleton and endomembranes are targeted by pathogen effectors and highlight an emerging role of membrane contact sites in biotic stress responses.

59 BASIC BIOLOGICAL SCIENCES↗

Different threats, same response

Plants can experience a range of challenges, from osmotic stress to pathogen attack, requiring different types of responses. Here, despite this variety, two recent studies of plant transcriptomes reveal a surprising commonality in the genes induced by stress.

59 BASIC BIOLOGICAL SCIENCES↗

Structure of 3'-PO 4 /5'-OH RNA ligase RtcB in complex with a 5'-OH oligonucleotide

RtcB enzymes comprise a widely distributed family of manganese- and GTP-dependent RNA repair enzymes that join 2',3'-cyclic phosphate ends to 5'-OH ends via RtcB-(histidinyl-N)–GMP, RNA 3'-phosphate, and RNA 3 'pp 5 'G intermediates. RtcB can ligate either 5'-OH RNA or 5'-OH DNA strands in vitro. The nucleic acid contacts of RtcB are uncharted. In this work, we report a 2.7 Å crystal structure of Pyrococcus horikoshii RtcB in complex with a 6-mer 5'-OH DNA oligonucleotide HO A 1 pT 2 pG 3 pT 4 pC 5 pC 6 , which reveals enzymic contacts of Asn202 to the terminal 5'-OH nucleophile; Arg238 to the A1pT2 and T2pG3 phosphates; Arg190 and Gln194 to the T 2 pG 3 phosphate; and an Arg190 π-cation interaction with the G 3 nucleobase. The structural insights affirm functional studies of E. coli RtcB that implicated the conserved counterpart of Arg238 in engagement of the 5'-OH strand for ligation. The essential active site Cys98 that coordinates two manganese ions is oxidized to cysteine sulfonic acid in our structure, raising the prospect that RtcB activity might be sensitive to modulation during oxidative stress.

59 BASIC BIOLOGICAL SCIENCES↗

The effect of increasing temperature on crop photosynthesis: from enzymes to ecosystems

Abstract As global land surface temperature continues to rise and heatwave events increase in frequency, duration, and/or intensity, our key food and fuel cropping systems will likely face increased heat-related stress. A large volume of literature exists on exploring measured and modelled impacts of rising temperature on crop photosynthesis, from enzymatic responses within the leaf up to larger ecosystem-scale responses that reflect seasonal and interannual crop responses to heat. This review discusses (i) how crop photosynthesis changes with temperature at the enzymatic scale within the leaf; (ii) how stomata and plant transport systems are affected by temperature; (iii) what features make a plant susceptible or tolerant to elevated temperature and heat stress; and (iv) how these temperature and heat effects compound at the ecosystem scale to affect crop yields. Throughout the review, we identify current advancements and future research trajectories that are needed to make our cropping systems more resilient to rising temperature and heat stress, which are both projected to occur due to current global fossil fuel emissions.

59 BASIC BIOLOGICAL SCIENCES↗

Transcriptional and translational landscape of Candida auris in response to caspofungin

Candida auris has emerged as a serious worldwide threat by causing opportunistic infections that are frequently resistant to one or more conventional antifungal medications resulting in high mortality rates. Against this backdrop, health warnings around the world have focused efforts on understanding C. auris fungal biology and effective prevention and treatment approaches to combat this fungus. To date, there is little information about the differentially expressed genes when this fungus is treated with conventional antifungals, and caspofungin is a standard echinocandin deployed in the therapy against C. auris. In this work, we treated two distinct strains of C. auris for 24h with caspofungin, and the cellular responses were evaluated at the morphological, translational and transcriptional levels. We first observed that the echinocandin caused morphological alterations, aggregation of yeast cells, and modifications in the cell wall composition of C. auris. Transcriptomic analysis revealed an upregulation of genes related to the synthesis of the cell wall, ribosome, and cell cycle after exposure to caspofungin. Supporting these findings, the integrated proteomic analysis showed that caspofungin-treated cells were enriched in ribosome-related proteins and cell wall, especially mannoproteins. Altogether, these results provide further insights into the biology of C. auris and expands our understanding regarding the antifungal activity of caspofungin and reveal cellular targets, as the mannose metabolism, that can be further explored for the development of novel antifungals.

59 BASIC BIOLOGICAL SCIENCES↗

Mediated Electrochemical Probing: A Systems-Level Tool for Redox Biology

Biology uses well-known redox mechanisms for energy harvesting (e.g., respiration), biosynthesis, and immune defense (e.g., oxidative burst), and now we know biology uses redox for systems-level communication. Currently, we have limited abilities to “eavesdrop” on this redox modality, which can be contrasted with our abilities to observe and actuate biology through its more familiar ionic electrical modality. In this Perspective, we argue that the coupling of electrochemistry with diffusible mediators (electron shuttles) provides a unique opportunity to access the redox communication modality through its electrical features. Here, we highlight previous studies showing that mediated electrochemical probing (MEP) can “communicate” with biology to acquire information and even to actuate specific biological responses (i.e., targeted gene expression). We suggest that MEP may reveal an extent of redox-based communication that has remained underappreciated in nature and that MEP could provide new technological approaches for redox biology, bioelectronics, clinical care, and environmental sciences.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Redundant roles of four ZIP family members in zinc homeostasis and seed development in Arabidopsis thaliana

Zinc (Zn) is essential for normal plant growth and development. The Zn-regulated transporter, iron-regulated transporter (IRT)-like protein (ZIP) family members are involved in Zn transport and cellular Zn homeostasis throughout the domains of life. In this study, we have characterized four ZIP transporters from Arabidopsis thaliana (IRT3, ZIP4, ZIP6, and ZIP9) to better understand their functional roles. The four ZIP proteins can restore the growth defect of a yeast Zn uptake mutant and are upregulated under Zn deficiency. Single and double mutants show no phenotypes under Zn-sufficient or Zn-limited growth conditions. In contrast, triple and quadruple mutants show impaired growth irrespective of external Zn supply due to reduced Zn translocation from root to shoot. All four ZIP genes are highly expressed during seed development, and siliques from all single and higher-order mutants exhibited an increased number of abnormal seeds and decreased Zn levels in mature seeds relative to wild type. The seed phenotypes could be reversed by supplementing the soil with Zn. Finally, our data demonstrate that IRT3, ZIP4, ZIP6, and ZIP9 function redundantly in maintaining Zn homeostasis and seed development in A. thaliana.

59 BASIC BIOLOGICAL SCIENCES↗

The survivor strain: isolation and characterization of Phormidium yuhuli AB48, a filamentous phototactic cyanobacterium with biotechnological potential

Despite their recognized potential, current applications of cyanobacteria as microbial cell factories remain in early stages of development. This is partly due to the fact that engineered strains are often difficult to grow at scale. This technical challenge contrasts with the dense and highly productive cyanobacteria populations thriving in many natural environments. It has been proposed that the selection of strains pre-adapted for growth in industrial photobioreactors could enable more productive cultivation outcomes. Here, we described the initial morphological, physiological, and genomic characterization of Phormidium yuhuli AB48 isolated from an industrial photobioreactor environment. P. yuhuli AB48 is a filamentous phototactic cyanobacterium with a growth rate comparable to Synechocystis sp . PCC 6803. The isolate forms dense biofilms under high salinity and alkaline conditions and manifests a similar nutrient profile to Arthrospira platensis ( Spirulina ). We sequenced, assembled, and analyzed the P. yuhuli AB48 genome, the first closed circular isolate reference genome for a member of the Phormidium genus. We then used cultivation experiments in combination with proteomics and metabolomics to investigate growth characteristics and phenotypes related to industrial scale cultivation, including nitrogen and carbon utilization, salinity, and pH acclimation, as well as antibiotic resistance. These analyses provide insight into the biological mechanisms behind the desirable growth properties manifested by P. yuhuli AB48 and position it as a promising microbial cell factory for industrial-scale bioproduction[221, 1631].

59 BASIC BIOLOGICAL SCIENCES↗