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Lessons Learned From Plant Research in Low Earth Orbit

This presentation is part of the pre-ASGSR workshop that will be held in Houston, Texas on November 9, 2022. The overall objective of the workshop is to discuss conducting biology experiments beyond low Earth orbit (LEO) and present the challenges and the paths forward. In part, the workshop will highlight lessons learned in plant space biology, some of the plant spaceflight stressors that are anticipated to cause biological changes beyond LEO and discuss what is currently known about these changes, and how results can be leveraged for future missions to the Moon and Mars. Platforms that can be utilized to conduct plant science experiments beyond LEO will also be discussed.

Plant Space Biology↗
DOE OSTI2023

Comparative Genomics and Transcriptomics Analyses Reveal Divergent Plant Biomass-Degrading Strategies in Fungi

Plant biomass is one of the most abundant renewable carbon sources, which holds great potential for replacing current fossil-based production of fuels and chemicals. In nature, fungi can efficiently degrade plant polysaccharides by secreting a broad range of carbohydrate-active enzymes (CAZymes), such as cellulases, hemicellulases, and pectinases. Due to the crucial role of plant biomass-degrading (PBD) CAZymes in fungal growth and related biotechnology applications, investigation of their genomic diversity and transcriptional dynamics has attracted increasing attention. In this project, we systematically compared the genome content of PBD CAZymes in six taxonomically distant species, Aspergillus niger, Aspergillus nidulans, Penicillium subrubescens, Trichoderma reesei, Phanerochaete chrysosporium, and Dichomitus squalens, as well as their transcriptome profiles during growth on nine monosaccharides. Considerable genomic variation and remarkable transcriptomic diversity of CAZymes were identified, implying the preferred carbon source of these fungi and their different methods of transcription regulation. In addition, the specific carbon utilization ability inferred from genomics and transcriptomics was compared with fungal growth profiles on corresponding sugars, to improve our understanding of the conversion process. This study enhances our understanding of genomic and transcriptomic diversity of fungal plant polysaccharide-degrading enzymes and provides new insights into designing enzyme mixtures and metabolic engineering of fungi for related industrial applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Metagenomes and metagenome-assembled genomes from a nutrient removal plant at Los Angeles County Sanitation Districts (LACSD) that transitioned from high to low dissolved oxygen

Operating biological nutrient removal (BNR) wastewater treatment plants with low dissolved oxygen (DO) conditions can reduce energy costs. We report on five metagenomes and 492 metagenome-assembled genomes (MAGs) obtained from samples collected at the Pomona water reclamation plant before and after a DO reduction from 3.5 to 0.7 mg/L.

dissolved oxygen↗
DOE OSTI2024

Genomes of multicellular algal sisters to land plants illuminate signaling network evolution

Zygnematophyceae are the algal sisters of land plants. Here we sequenced four genomes of filamentous Zygnematophyceae, including chromosome-scale assemblies for three strains of Zygnema circumcarinatum . We inferred traits in the ancestor of Zygnematophyceae and land plants that might have ushered in the conquest of land by plants: expanded genes for signaling cascades, environmental response, and multicellular growth. Zygnematophyceae and land plants share all the major enzymes for cell wall synthesis and remodifications, and gene gains shaped this toolkit. Co-expression network analyses uncover gene cohorts that unite environmental signaling with multicellular developmental programs. Our data shed light on a molecular chassis that balances environmental response and growth modulation across more than 600 million years of streptophyte evolution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Draft genome of multiple resistance donor plant Sinapis alba: An insight into SSRs, annotations and phylogenetics

Sinapis alba is a wild member of the Brassicaceae family reported to possess genetic resistance against major biotic and abiotic stresses of oilseed brassicas. However, the resistance nature of S. alba was not exploited generously due to the unavailability of usable genome sequences in public databases. Therefore, the present study was conducted to assemble the first draft genome from raw whole genome shotgun sequences with annotation and develop simple sequence repeat markers for molecular genetics and marker-assisted breeding. Results The raw genome sequences had 96x coverage on the Illumina platform with 170 Gbp data. The developed assembly by SOAPdenovo2 has ~459 Mbp genome size covered in 403,423 contigs with an average size of 1138.04 bp. The assembly was BLASTX with Arabidopsis thaliana which showed 32.9% positive hits between both plants. The top hit species distribution analysis showed the highest similarity with A. thaliana. A total of 809,597 GO level annotations were recorded after BLASTX results, and 34,012 sequences were annotated with different enzyme codes grouped under seven classes. The gene prediction tool AUGUSTUS identified 113,107 probable genes with an average size of 684 bp. The biochemical pathway annotation assigned 16,119 potential genes to 152 KEGG maps and 1751 enzyme codes. The development of potential SSRs from the de-novo assembly yielded 70731 unique primer pairs. Out of 159 randomly selected SSR markers for validation, 149 successfully amplified in S. alba. However, 10 SSR markers did not amplify during the validation experiment. Conclusion The annotated genome assembly with a large number of SSRs was developed in the present study. To the best of our knowledge, this is the first report of S. alba genome assembly development, annotation, and SSRs mining to date. The data presented here will be a very important resource for future crop improvement programs, especially for resistant breeding.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Cellulose Nanocrystals are a Renewable and Biocompatible Nanocarrier of Agrochemicals Directly to Plant Cells

More sustainable agricultural practices are critical to meeting humanity's food needs while minimizing adverse environmental impacts. Engineered nanomaterials used as nanocarriers promise to reduce the volume of agrochemicals required for crop production but concerns about nanocontamination of agricultural products necessitate the discovery of naturally occurring alternatives. Here, in this study, plant‐derived cellulose nanocrystals (CNC) as a “green” alternative to man‐made nanomaterials for the economical delivery of agrochemicals to plants are suggested. Using confocal microscopy and fluorescently labeled CNC, it is demonstrated that CNC can successfully penetrate the plant cell wall and enter cells without causing any negative effects on the overall plant phenotype, genome, and metabolome. The efficiency of CNC to deliver chemicals is demonstrated through in vitro tests involving CNC covalently conjugated with 2,4‐dichlorophenoxyacetic acid (2,4‐D). Plant cell culture experiments confirmed that CNC can be used as a 2,4‐D nanocarrier and reduces the volume of plant growth regulators needed to cultivate plant cells. This work creates a new platform for cyclical agricultural practices where CNC extracted from agricultural waste can be used for the direct delivery of agrochemicals to crops, thereby reducing the environmental burden created by both agricultural waste and excess herbicides.

Cellulose nanocrystals↗
NASA NTRS2005

Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays

Using a maskless photolithography method, we produced DNA oligonucleotide microarrays with probe sequences tiled throughout the genome of the plant Arabidopsis thaliana. RNA expression was determined for the complete nuclear, mitochondrial, and chloroplast genomes by tiling 5 million 36-mer probes. These probes were hybridized to labeled mRNA isolated from liquid grown T87 cells, an undifferentiated Arabidopsis cell culture line. Transcripts were detected from at least 60% of the nearly 26,330 annotated genes, which included 151 predicted genes that were not identified previously by a similar genome-wide hybridization study on four different cell lines. In comparison with previously published results with 25-mer tiling arrays produced by chromium masking-based photolithography technique, 36-mer oligonucleotide probes were found to be more useful in identifying intron-exon boundaries. Using two-dimensional HPLC tandem mass spectrometry, a small-scale proteomic analysis was performed with the same cells. A large amount of strongly hybridizing RNA was found in regions "antisense" to known genes. Similarity of antisense activities between the 25-mer and 36-mer data sets suggests that it is a reproducible and inherent property of the experiments. Transcription activities were also detected for many of the intergenic regions and the small RNAs, including tRNA, small nuclear RNA, small nucleolar RNA, and microRNA. Expression of tRNAs correlates with genome-wide amino acid usage.

Arabidopsis/genetics↗
DOE OSTI2024

Anaerobic fungi contain abundant, diverse, and transcriptionally active Long Terminal Repeat retrotransposons

Long Terminal Repeat (LTR) retrotransposons are a class of repetitive elements that are widespread in the genomes of plants and many fungi. LTR retrotransposons have been associated with rapidly evolving gene clusters in plants and virulence factor transfer in fungal-plant parasite-host interactions. We report here the abundance and transcriptional activity of LTR retrotransposons across several species of the early-branching Neocallimastigomycota, otherwise known as the anaerobic gut fungi (AGF). The ubiquity of LTR retrotransposons in these genomes suggests key evolutionary roles in these rumen-dwelling biomass degraders, whose genomes also contain many enzymes that are horizontally transferred from other rumen-dwelling prokaryotes. Up to 10% of anaerobic fungal genomes consist of LTR retrotransposons, and the mapping of sequences from LTR retrotransposons to transcriptomes shows that the majority of clusters are transcribed, with some exhibiting expression greater than 104 reads per kilobase million mapped reads (rpkm). Many LTR retrotransposons are strongly differentially expressed upon heat stress during fungal cultivation, with several exhibiting a nearly three-log10 fold increase in expression, whereas growth substrate variation modulated transcription to a lesser extent. We show that some LTR retrotransposons contain carbohydrate-active enzymes (CAZymes), and the expansion of CAZymes within genomes and among anaerobic fungal species may be linked to retrotransposon activity. We further discuss how these widespread sequences may be a source of promoters and other parts towards the bioengineering of anaerobic fungi.

Genetics & Heredity↗
DOE OSTI2025

Populus PtrbHLH011 Is a Transcriptional Co‐Regulator Involved in the Activation of Cell Wall Biosynthesis by Iron Deprivation

The lack of a mechanistic understanding of the environmental plasticity of secondary cell wall (SCW) biosynthesis restricts large-scale biomass and bioenergy production on marginal lands. Using Populus (poplar), a key bioenergy crop, we discovered that iron deprivation, a prevalent abiotic stress on marginal lands, stimulates SCW biosynthesis in stems. We identified the transcription factor PtrbHLH011 as a critical regulator underlying this response. Through integrated analyses involving phenotypic characterisation of PtrbHLH011 knockout and overexpression plants, functional genomics and molecular investigations, we established that PtrbHLH011 functions as a central regulator of SCW biosynthesis, iron homeostasis and flavonoid biosynthesis by directly repressing essential genes in these pathways. Iron deprivation downregulates PtrbHLH011 expression, subsequently activating these biosynthetic pathways. Notably, cytosine base editing-based knockout of PtrbHLH011 significantly enhanced plant growth, yielding up to a 110% increase in stem diameter and a 300% increase in leaf iron content. These findings present a novel regulatory mechanism linking environmental iron availability to SCW biosynthesis and illustrate a practical strategy to improve biomass yield on iron-deficient marginal lands. Furthermore, our mechanistic insights into PtrbHLH011 target recognition and regulation provide a valuable foundation for precise manipulation of gene regulatory networks, facilitating the development of high-performance bioenergy crops adapted to marginal environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Virus-induced gene editing of stomatal regulators in Nicotiana benthamiana enables rapid functional genomics

Virus-induced gene editing (VIGE) holds promise as a rapid and scalable approach for functional genomics in plants. Here, we apply a tobacco rattle virus (TRV)-based single-guide RNA (sgRNA) delivery system to target key regulators of stomatal development in Nicotiana benthamiana using transgenic Cas9-expressing lines. sgRNAs fused to a mobile RNA element and co-delivered with TRV enabled both somatic and heritable genome editing across orthologs of STOMAGEN, EPF2, YODA, and SPEECHLESS. Somatic editing frequencies reached up to 95%, and heritable tetra-allelic mutations were recovered in multiple target genes. Mutants exhibited significant, gene-specific changes in stomatal density, with corresponding effects on leaf temperature indicative of altered evaporative cooling. Additionally, sgRNAs fused to an AmCyan reporter enabled visualization of virus-infected tissues, allowing stomatal phenotyping in edited M0 sectors. This TRV-based platform facilitates functional assessment of genes influencing stomatal patterning and offers a powerful tool for dissecting gene function in a developmentally and physiologically relevant context.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Annotated genome sequence of a fast-growing diploid clone of red alder ( Alnus rubra Bong.)

Abstract Red alder (Alnus rubra Bong.) is an ecologically significant and important fast-growing commercial tree species native to western coastal and riparian regions of North America, having highly desirable wood, pigment, and medicinal properties. We have sequenced the genome of a rapidly growing clone. The assembly is nearly complete, containing the full complement of expected genes. This supports our objectives of identifying and studying genes and pathways involved in nitrogen-fixing symbiosis and those related to secondary metabolites that underlie red alder's many interesting defense, pigmentation, and wood quality traits. We established that this clone is most likely diploid and identified a set of SNPs that will have utility in future breeding and selection endeavors, as well as in ongoing population studies. We have added a well-characterized genome to others from the order Fagales. In particular, it improves significantly upon the only other published alder genome sequence, that of Alnus glutinosa. Our work initiated a detailed comparative analysis of members of the order Fagales and established some similarities with previous reports in this clade, suggesting a biased retention of certain gene functions in the vestiges of an ancient genome duplication when compared with more recent tandem duplications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Ancient Origin of Acetyltransferases Catalyzing O -acetylation of Plant Cell Wall Polysaccharides

Abstract Members of the domain of unknown function 231/trichome birefringence–like (TBL) family have been shown to be O-acetyltransferases catalyzing the acetylation of plant cell wall polysaccharides, including pectins, mannan, xyloglucan and xylan. However, little is known about the origin and evolution of plant cell wall polysaccharide acetyltransferases. Here, we investigated the biochemical functions of TBL homologs from Klebsormidium nitens, a representative of an early divergent class of charophyte green algae that are considered to be the closest living relatives of land plants, and Marchantia polymorpha, a liverwort that is an extant representative of an ancient lineage of land plants. The genomes of K. nitens and Marchantia polymorpha harbor two and six TBL homologs, respectively. Biochemical characterization of their recombinant proteins expressed in human embryonic kidney 293 cells demonstrated that the two K. nitens TBLs exhibited acetyltransferase activities acetylating the pectin homogalacturonan (HG) and hence were named KnPOAT1 and KnPOAT2. Among the six M. polymorpha TBLs, five (MpPOAT1 to 5) possessed acetyltransferase activities toward pectins and the remaining one (MpMOAT1) catalyzed 2-O- and 3-O-acetylation of mannan. While MpPOAT1,2 specifically acetylated HG, MpPOAT3,4,5 could acetylate both HG and rhamnogalacturonan-I. Consistent with the acetyltransferase activities of these TBLs, pectins isolated from K. nitens and both pectins and mannan from M. polymorpha were shown to be acetylated. These findings indicate that the TBL genes were recruited as cell wall polysaccharide O-acetyltransferases as early as in charophyte green algae with activities toward pectins and they underwent expansion and functional diversification to acetylate various cell wall polysaccharides during evolution of land plants.

Cell Biology↗
DOE OSTI2024

SIVB's 2024 In Vitro Biology Meeting Proceedings

SIVB's 2024 World Congress on In Vitro Biology took place in Saint Louis, Missouri, from June 8th to 12th, 2024. The conference featured renowned speakers from academic and non-academic institutions who will present recent advancements in critical areas like plant transformation, genome editing, synthetic biology, advanced breeding technologies, cellular agriculture, future food sources, chromosome engineering, epigenetics, artificial intelligence, and machine learning. The Society for In Vitro Biology (SIVB) has always considered the education and professional development of young researchers as an integral component of its mission. The 2024 World Congress program, along with SIVB’s student initiatives, was customized to foster scientific growth and professional development among students and young scientists empowering them in their professional journeys. The recording of the DOE supported "Single Cell RNA Sequencing" workshop was made publicly available at https://youtu.be/A0UnuYwefwg for easy retrieval and reference of all information shared during the live event, thereby increasing accessibility and knowledge transfer. Their are 14 articles in the proceedings and the full list of files is located at https://link.springer.com/journal/11626/volumes-and-issues/60-1/supplement.

10 SYNTHETIC FUELS↗
DOE OSTI2025

Genomic factors limiting the diversity of Saccharomycotina plant pathogens

The Saccharomycotina fungi have evolved to inhabit a vast diversity of habitats over their 400-million-year evolution. There are, however, only a few known fungal pathogens of plants in this subphylum, primarily belonging to the genera Eremothecium and Geotrichum. We compared the genomes of 12 plant-pathogenic Saccharomycotina strains to 360 plant-associated strains to identify features unique to the phytopathogens. Characterization of the oxylipin synthesis genes, a compound believed to be involved in Eremothecium pathogenicity, did not reveal any differences in gene presence within or between the plant-pathogenic and plant-associated strains. A reverse-ecological approach, however, revealed that plant pathogens lack several metabolic enzymes known to assist other phytopathogens in overcoming plant defenses. This includes L-rhamnose metabolism, formamidase and nitrilase genes. This result suggests that the Saccharomycotina plant pathogens are limited to infecting ripening fruits as they are without the necessary enzymes to degrade common phytohormones and secondary metabolites produced by plants.

Saccharomycotina, fungi, phytopathogen, reverse ec↗
DOE OSTI2021

The tepary bean genome provides insight into evolution and domestication under heat stress

Tepary bean (Phaseolus acutifolis A. Gray), native to the Sonoran Desert, is highly adapted to heat and drought. It is a sister species of common bean (Phaseolus vulgaris L.), the most important legume protein source for direct human consumption, and whose production is threatened by climate change. Here, we report on the tepary genome including exploration of possible mechanisms for resilience to moderate heat stress and a reduced disease resistance gene repertoire, consistent with adaptation to arid and hot environments. Extensive collinearity and shared gene content among these Phaseolus species will facilitate engineering climate adaptation in common bean, a key food security crop, and accelerate tepary bean improvement.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

Genomic insights into the origin, domestication and diversification of Brassica juncea

Despite early domestication around 3000 BC, the evolutionary history of the ancient allotetraploid species Brassica juncea (L.) Czern & Coss remains uncertain. Here, we report a chromosome-scale de novo assembly of a yellow-seeded B. juncea genome by integrating long-read and short-read sequencing, optical mapping and Hi-C technologies. Nuclear and organelle phylogenies of 480 accessions worldwide supported that B. juncea is most likely a single origin in West Asia, 8,000–14,000 years ago, via natural interspecific hybridization. Subsequently, new crop types evolved through spontaneous gene mutations and introgressions along three independent routes of eastward expansion. Selective sweeps, genome-wide trait associations and tissue-specific RNA-sequencing analysis shed light on the domestication history of flowering time and seed weight, and on human selection for morphological diversification in this versatile species. Our data provide a comprehensive insight into the origin and domestication and a foundation for genomics-based breeding of B. juncea.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2002

Plant adaptation to low atmospheric pressures: potential molecular responses

There is an increasing realization that it may be impossible to attain Earth normal atmospheric pressures in orbital, lunar, or Martian greenhouses, simply because the construction materials do not exist to meet the extraordinary constraints imposed by balancing high engineering requirements against high lift costs. This equation essentially dictates that NASA have in place the capability to grow plants at reduced atmospheric pressure. Yet current understanding of plant growth at low pressures is limited to just a few experiments and relatively rudimentary assessments of plant vigor and growth. The tools now exist, however, to make rapid progress toward understanding the fundamental nature of plant responses and adaptations to low pressures, and to develop strategies for mitigating detrimental effects by engineering the growth conditions or by engineering the plants themselves. The genomes of rice and the model plant Arabidopsis thaliana have recently been sequenced in their entirety, and public sector and commercial DNA chips are becoming available such that thousands of genes can be assayed at once. A fundamental understanding of plant responses and adaptation to low pressures can now be approached and translated into procedures and engineering considerations to enhance plant growth at low atmospheric pressures. In anticipation of such studies, we present here the background arguments supporting these contentions, as well as informed speculation about the kinds of molecular physiological responses that might be expected of plants in low-pressure environments.

NASA Discipline Plant Biology↗