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At least 1,585 records · Page 88
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Eukaryotic RNA-guided endonucleases evolved from a unique clade of bacterial enzymes

Abstract RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and a TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12s. In eukaryotes, TnpB homologs occur as two distinct types, Fanzor1s and Fanzor2s. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, which revealed that both Fanzor1s and Fanzor2s stem from a single lineage of IS607 TnpBs with unusual active site arrangement. The widespread nature of Fanzors implies that the properties of this particular lineage of IS607 TnpBs were particularly suited to adaptation in eukaryotes. Biochemical analysis of an IS607 TnpB and Fanzor1s revealed common strategies employed by TnpBs and Fanzors to co-evolve with their cognate transposases. Collectively, our results provide a new model of sequential evolution from IS607 TnpBs to Fanzor2s, and Fanzor2s to Fanzor1s that details how genes of prokaryotic origin evolve to give rise to new protein families in eukaryotes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A bromodomain-independent mechanism of gene regulation by the BET inhibitor JQ1: direct activation of nuclear receptor PXR

Abstract Bromodomain and extraterminal (BET) proteins are extensively studied in multiple pathologies, including cancer. BET proteins modulate transcription of various genes, including those synonymous with cancer, such as MYC. Thus, BET inhibitors are a major area of drug development efforts. (+)-JQ1 (JQ1) is the prototype inhibitor and is a common tool to probe BET functions. While showing therapeutic promise, JQ1 is not clinically usable, partly due to metabolic instability. Here, we show that JQ1 and the BET-inactive (−)-JQ1 are agonists of pregnane X receptor (PXR), a nuclear receptor that transcriptionally regulates genes encoding drug-metabolizing enzymes such as CYP3A4, which was previously shown to oxidize JQ1. A PXR-JQ1 co-crystal structure identified JQ1′s tert-butyl moiety as a PXR anchor and explains binding by (−)-JQ1. Analogs differing at the tert-butyl lost PXR binding, validating our structural findings. Evaluation in liver cell models revealed both PXR-dependent and PXR-independent modulation of CYP3A4 expression by BET inhibitors. We have characterized a non-BET JQ1 target, a mechanism of physiological JQ1 instability, a biological function of (−)-JQ1, and BET-dependent transcriptional regulation of drug metabolism genes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut

Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements. In our study, we investigated the tRNA:aaRS pair that installs the 22nd amino acid, pyrrolysine (tRNAPyl:PylRS). Pyrrolysyl-tRNA synthetases (PylRSs) are naturally encoded in some archaeal and bacterial genomes to acylate tRNAPyl with pyrrolysine. Their large amino acid binding pocket and poor recognition of the tRNA anticodon have been instrumental in incorporating > 200 noncanonical amino acids. PylRS enzymes can be divided into three classes based on their genomic structure. Two classes contain both an N-terminal and C-terminal domain, however the third class (ΔpylSn) lacks the N-terminal domain. In this study we explored the tRNA identity elements for a ΔpylSn tRNAPyl from Candidatus Methanomethylophilus alvus which drives the orthogonality seen with its cognate PylRS (MaPylRS). From aminoacylation and translation assays we identified five key elements in ΔpylSn tRNAPyl necessary for MaPylRS activity. The absence of a base (position 8) and a G–U wobble pair (G28:U42) were found to affect the high-resolution structure of the tRNA, while molecular dynamic simulations led us to acknowledge the rigidity imparted from the G–C base pairs (G3:C70 and G5:C68).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

TroR is the primary regulator of the iron homeostasis transcription network in the halophilic archaeon Haloferax volcanii

Abstract Maintaining the intracellular iron concentration within the homeostatic range is vital to meet cellular metabolic needs and reduce oxidative stress. Previous research revealed that the haloarchaeon Halobacterium salinarum encodes four diphtheria toxin repressor (DtxR) family transcription factors (TFs) that together regulate the iron response through an interconnected transcriptional regulatory network (TRN). However, the conservation of the TRN and the metal specificity of DtxR TFs remained poorly understood. Here we identified and characterized the TRN of Haloferax volcanii for comparison. Genetic analysis demonstrated that Hfx. volcanii relies on three DtxR transcriptional regulators (Idr, SirR, and TroR), with TroR as the primary regulator of iron homeostasis. Bioinformatics and molecular approaches revealed that TroR binds a conserved cis-regulatory motif located ∼100 nt upstream of the start codon of iron-related target genes. Transcriptomics analysis demonstrated that, under conditions of iron sufficiency, TroR repressed iron uptake and induced iron storage mechanisms. TroR repressed the expression of one other DtxR TF, Idr. This reduced DtxR TRN complexity relative to that of Hbt. salinarum appeared correlated with natural variations in iron availability. Based on these data, we hypothesize that variable environmental conditions such as iron availability appear to select for increasing TRN complexity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Homologous recombination shapes the architecture and evolution of bacterial genomes

Homologous recombination is a key evolutionary force that varies considerably across bacterial species. However, how the landscape of homologous recombination varies across genes and within individual genomes has only been studied in a few species. Here, we used Approximate Bayesian Computation to estimate the recombination rate along the genomes of 145 bacterial species. Our results show that homologous recombination varies greatly along bacterial genomes and shapes many aspects of genome architecture and evolution. The genomic landscape of recombination presents several key signatures: rates are highest near the origin of replication in most species, patterns of recombination generally appear symmetrical in both replichores (i.e. replicational halves of circular chromosomes) and most species have genomic hotspots of recombination. Furthermore, many closely related species share conserved landscapes of recombination across orthologs indicating that recombination landscapes are conserved over significant evolutionary distances. We show evidence that recombination drives the evolution of GC-content through increasing the effectiveness of selection and not through biased gene conversion, thereby contributing to an ongoing debate. Finally, we demonstrate that the rate of recombination varies across gene function and that many hotspots of recombination are associated with adaptive and mobile regions often encoding genes involved in pathogenicity.

Torrance, Ellis L [University of North Carolina, G↗
DOE OSTI2024

Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB

Mitomycin C (MMC) repair factor A (mrfA) and factor B (mrfB), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium Bacillus subtilis. Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 Å, in what appears to be an inactive state. In this conformation, a predicted α-helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg 2+ ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ-family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg 2+ -dependent 3'–5' DNA exonuclease. We show that Leu113 aids in coordinating the 3' end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC-induced DNA adducts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Comparative mitogenomics of kingdom Fungi – evolutionary insights and metagenomic applications

Mitochondria are essential components of eukaryotic cells, responsible for ATP production through oxidative phosphorylation. Despite their biological importance, unique challenges have hindered the adoption of automated mitochondrial genome (mitogenome) annotation methods, obstructing mitochondrial comparative genomics in a broad evolutionary context. Using Fungi as a study system and a Joint Genome Institute (JGI) annotated high-quality reference set, we observed broad patterns of mitochondrial evolution across the kingdom. We found that the median fungal mitogenome size is 58 kb and identified exceptionally large examples over 1 Mb in Pezizomycetes. All 14 expected oxidative phosphorylation protein-coding genes, plus rps3, were generally conserved. We found evidence of major evolutionary transitions within the Ascomycota, including the transfer of mitochondrially encoded atp8 and atp9 to the nuclear genomes across the Pezizomycotina and shifts in mitogenome tRNA patterns across the kingdom. We found substantial concordance between mitochondrial and nuclear evolution, enabling us to document 3131 total fungal mitogenomes from JGI-derived metagenomic datasets. We also identified 6467 total undeclared mitogenomes embedded in Genbank fungal nuclear assemblies. We provide interactive tools for mitogenome analysis through the JGI MycoCosm platform. Collectively, this work generated nearly 10 000 new fungal mitogenome annotations, providing a foundation and resources for future exploration of comparative fungal mitogenomics.

Ahrendt, Steven R. [USDOE Joint Genome Institute (↗
DOE OSTI2025

Antitoxin control of optimal transcriptional repression in the atypical HigB–HigA toxin–antitoxin system from Proteus vulgaris

Abstract Bacterial toxin–antitoxin (TA) pairs transcriptionally autoregulate their expression via a repression/derepression mechanism in response to changing environmental conditions. The structural diversity of TA systems influences the mechanisms of transcriptional regulation. Here, we define the molecular mechanism for the plasmid-encoded HigB–HigA TA pair originally identified in a post-operative infection with antibiotic-resistant Proteus vulgaris. We determine DNA binding and promoter activity by the HigB–HigA complex supported by structural biology and molecular dynamics simulations of an elusive DNA operator–TA repressor complex. To define the optimal oligomeric TA repressor–DNA operator complex required for derepression, we engineered a dedicated trimeric HigB–HigA2 complex that represses transcription more than 26-fold as compared to the tetrameric HigB2–HigA2. These results expand the known diversity of how the HigB–HigA TA family is autoregulated.

Biochemistry & Molecular Biology↗
DOE OSTI2022

Tonoplast Sucrose Trafficking Modulates Starch Utilization and Water Deficit Behavior in Poplar Leaves

Leaf osmotic adjustment by the active accrual of compatible organic solutes (e.g. sucrose) contributes to drought tolerance throughout the plant kingdom. In Populus tremula x alba, PtaSUT4 encodes a tonoplast sucrose–proton symporter, whose downregulation by chronic mild drought or transgenic manipulation is known to increase leaf sucrose and turgor. While this may constitute a single drought tolerance mechanism, we now report that other adjustments which can occur during a worsening water deficit are damped when PtaSUT4 is constitutively downregulated. Specifically, we report that starch use and leaf relative water content (RWC) dynamics were compromised when plants with constitutively downregulated PtaSUT4 were subjected to a water deficit. Leaf RWC decreased more in wild-type and vector control lines than in transgenic PtaSUT4-RNAi (RNA-interference) or CRISPR (clustered regularly interspersed short palindromic repeats) knockout (KO) lines. The control line RWC decrease was accompanied by increased PtaSUT4 transcript levels and a mobilization of sucrose from the mesophyll-enriched leaf lamina into the midvein. The findings suggest that changes in SUT4 expression can increase turgor or decrease RWC as different tolerance mechanisms to reduced water availability. Evidence is presented that PtaSUT4-mediated sucrose partitioning between the vacuole and the cytosol is important not only for overall sucrose abundance and turgor, but also for reactive oxygen species (ROS) and antioxidant dynamics. Interestingly, the reduced capacity for accelerated starch breakdown under worsening water-deficit conditions was correlated with reduced ROS in the RNAi and KO lines. A role for PtaSUT4 in the orchestration of ROS, antioxidant, starch utilization and RWC dynamics during water stress and its importance in trees especially, with their high hydraulic resistances, is considered.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Genome-wide association of the metabolic shifts underpinning dark-induced senescence in Arabidopsis

Dark-induced senescence provokes profound metabolic shifts to recycle nutrients and to guarantee plant survival. To date, research on these processes has largely focused on characterizing mutants deficient in individual pathways. Here, we adopted a time-resolved genome-wide association-based approach to characterize dark-induced senescence by evaluating the photochemical efficiency and content of primary and lipid metabolites at the beginning, or after 3 or 6 days in darkness. We discovered six patterns of metabolic shifts and identified 215 associations with 81 candidate genes being involved in this process. Among these associations, we validated the roles of four genes associated with glycine, galactinol, threonine, and ornithine levels. We also demonstrated the function of threonine and galactinol catabolism during dark-induced senescence. Intriguingly, we determined that the association between tyrosine contents and TYROSINE AMINOTRANSFERASE 1 influences enzyme activity of the encoded protein and transcriptional activity of the gene under normal and dark conditions, respectively. Moreover, the single-nucleotide polymorphisms affecting the expression of THREONINE ALDOLASE 1 and the amino acid transporter gene AVT 1 B, respectively, only underlie the variation in threonine and glycine levels in the dark. Taken together, these results allow us to present a very detailed model of the metabolic aspects of dark-induced senescence, as well as the process itself.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Chlamydomonas mutants lacking chloroplast TRIOSE PHOSPHATE TRANSPORTER3 are metabolically compromised and light sensitive

Abstract Modulation of photoassimilate export from the chloroplast is essential for controlling the distribution of fixed carbon in the cell and maintaining optimum photosynthetic rates. In this study, we identified chloroplast TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR 2 (CreTPT2) and CreTPT3 in the green alga Chlamydomonas (Chlamydomonas reinhardtii), which exhibit similar substrate specificities but whose encoding genes are differentially expressed over the diurnal cycle. We focused mostly on CreTPT3 because of its high level of expression and the severe phenotype exhibited by tpt3 relative to tpt2 mutants. Null mutants for CreTPT3 had a pleiotropic phenotype that affected growth, photosynthetic activities, metabolite profiles, carbon partitioning, and organelle-specific accumulation of H2O2. These analyses demonstrated that CreTPT3 is a dominant conduit on the chloroplast envelope for the transport of photoassimilates. In addition, CreTPT3 can serve as a safety valve that moves excess reductant out of the chloroplast and appears to be essential for preventing cells from experiencing oxidative stress and accumulating reactive oxygen species, even under low/moderate light intensities. Finally, our studies indicate subfunctionalization of the TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR (CreTPT) transporters and suggest that there are differences in managing the export of photoassimilates from the chloroplasts of Chlamydomonas and vascular plants.

Huang, (黄伟超), Weichao (ORCID:0000000189403110)↗
DOE OSTI2021

Biotin attachment domain-containing proteins mediate hydroxy fatty acid-dependent inhibition of acetyl CoA carboxylase

Hundreds of naturally occurring specialized fatty acids (FAs) have potential as desirable chemical feedstocks if they could be produced at large scale by crop plants; however, transgenic expression of their biosynthetic genes has generally been accompanied by dramatic reductions in oil yield. For example, expression of castor (Ricinus communis) FA hydroxylase (FAH) in the Arabidopsis thaliana FA elongation mutant fae1 resulted in a 50% reduction of FA synthesis rate that was attributed to inhibition of acetyl-CoA carboxylase (ACCase) by an undefined mechanism. Here, we tested the hypothesis that the ricinoleic acid-dependent decrease in ACCase activity is mediated by biotin attachment domain-containing (BADC) proteins. BADCs are inactive homologs of biotin carboxy carrier protein that lack a biotin cofactor and can inhibit ACCase. Arabidopsis contains three BADC genes. To reduce expression levels of BADC1 and BADC3 in fae1/FAH plants, a homozygous badc1,3/fae1/FAH line was created. The rate of FA synthesis in badc1,3/fae1/FAH seeds doubled relative to fae1/FAH, restoring it to fae1 levels, increasing both native FA and HFA accumulation. Total FA per seed, seed oil content, and seed yield per plant all increased in badc1,3/fae1/FAH, to 5.8 µg, 37%, and 162 mg, respectively, relative to 4.9 µg, 33%, and 126 mg, respectively, for fae1/FAH. Transcript levels of FA synthesis-related genes, including those encoding ACCase subunits, did not significantly differ between badc1,3/fae1/FAH and fae1/FAH. These results demonstrate that BADC1 and BADC3 mediate ricinoleic acid-dependent inhibition of FA synthesis. We propose that BADC-mediated FAS inhibition as a general mechanism that limits FA accumulation in specialized FA-accumulating seeds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The SvFUL2 transcription factor is required for inflorescence determinacy and timely flowering in Setaria viridis

Inflorescence architecture in cereal crops directly impacts yield potential through regulation of seed number and harvesting ability. Extensive architectural diversity found in inflorescences of grass species is due to spatial and temporal activity and determinacy of meristems, which control the number and arrangement of branches and flowers, and underlie plasticity. Timing of the floral transition is also intimately associated with inflorescence development and architecture, yet little is known about the intersecting pathways and how they are rewired during development. Here, we show that a single mutation in a gene encoding an AP1/FUL-like MADS-box transcription factor significantly delays flowering time and disrupts multiple levels of meristem determinacy in panicles of the C 4 model panicoid grass, Setaria viridis. Previous reports of AP1/FUL-like genes in cereals have revealed extensive functional redundancy, and in panicoid grasses, no associated inflorescence phenotypes have been described. In S. viridis, perturbation of SvFul2, both through chemical mutagenesis and gene editing, converted a normally determinate inflorescence habit to an indeterminate one, and also repressed determinacy in axillary branch and floral meristems. Our analysis of gene networks connected to disruption of SvFul2 identified regulatory hubs at the intersection of floral transition and inflorescence determinacy, providing insights into the optimization of cereal crop architecture.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Cellulose synthesis complexes are homo-oligomeric and hetero-oligomeric in Physcomitrium patens

The common ancestor of seed plants and mosses contained homo-oligomeric cellulose synthesis complexes (CSCs) composed of identical subunits encoded by a single CELLULOSE SYNTHASE (CESA) gene. Seed plants use different CESA isoforms for primary and secondary cell wall deposition. Both primary and secondary CESAs form hetero-oligomeric CSCs that assemble and function in planta only when all the required isoforms are present. The moss Physcomitrium (Physcomitrella) patens has seven CESA genes that can be grouped into two functionally and phylogenetically distinct classes. Previously, we showed that PpCESA3 and/or PpCESA8 (class A) together with PpCESA6 and/or PpCESA7 (class B) form obligate hetero-oligomeric complexes required for normal secondary cell wall deposition. Here, we show that gametophore morphogenesis requires a member of class A, PpCESA5, and is sustained in the absence of other PpCESA isoforms. Further, PpCESA5 also differs from the other class A PpCESAs as it is able to self-interact and does not co-immunoprecipitate with other PpCESA isoforms. These results are consistent with the hypothesis that homo-oligomeric CSCs containing only PpCESA5 subunits synthesize cellulose required for gametophore morphogenesis. Analysis of mutant phenotypes also revealed that, like secondary cell wall deposition, normal protonemal tip growth requires class B isoforms (PpCESA4 or PpCESA10), along with a class A partner (PpCESA3, PpCESA5, or PpCESA8). Thus, P. patens contains both homo-oligomeric and hetero-oligomeric CSCs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Mass spectrometry-based technologies for probing the 3D world of plant proteins

Abstract Over the past two decades, mass spectrometric (MS)-based proteomics technologies have facilitated the study of signaling pathways throughout biology. Nowhere is this needed more than in plants, where an evolutionary history of genome duplications has resulted in large gene families involved in posttranslational modifications and regulatory pathways. For example, at least 5% of the Arabidopsis thaliana genome (ca. 1,200 genes) encodes protein kinases and protein phosphatases that regulate nearly all aspects of plant growth and development. MS-based technologies that quantify covalent changes in the side-chain of amino acids are critically important, but they only address one piece of the puzzle. A more crucially important mechanistic question is how noncovalent interactions—which are more difficult to study—dynamically regulate the proteome’s 3D structure. The advent of improvements in protein 3D technologies such as cryo-electron microscopy, nuclear magnetic resonance, and X-ray crystallography has allowed considerable progress to be made at this level, but these methods are typically limited to analyzing proteins, which can be expressed and purified in milligram quantities. Newly emerging MS-based technologies have recently been developed for studying the 3D structure of proteins. Importantly, these methods do not require protein samples to be purified and require smaller amounts of sample, opening the wider proteome for structural analysis in complex mixtures, crude lysates, and even in intact cells. These MS-based methods include covalent labeling, crosslinking, thermal proteome profiling, and limited proteolysis, all of which can be leveraged by established MS workflows, as well as newly emerging methods capable of analyzing intact macromolecules and the complexes they form. In this review, we discuss these recent innovations in MS-based “structural” proteomics to provide readers with an understanding of the opportunities they offer and the remaining challenges for understanding the molecular underpinnings of plant structure and function.

Plant Sciences↗
DOE OSTI2022

H-lignin can be deposited independently of CINNAMYL ALCOHOL DEHYDROGENASE C and D in Arabidopsis

Abstract Lignin contributes substantially to the recalcitrance of biomass toward saccharification. To circumvent this problem, researchers have genetically altered lignin, although, in a number of cases, these efforts have resulted in an undesirable yield penalty. Recent findings have shown that by knocking out two subunits (MED5A and MED5B) of the transcriptional regulatory complex Mediator, the stunted growth phenotype of mutants in p-coumaroyl shikimate 3′-hydroxylase, reduced epidermal fluorescence 8-1 (ref8-1), can be alleviated. Furthermore, these plants synthesize a lignin polymer almost entirely derived from p-coumaryl alcohol. Plants deficient in cinnamyl alcohol dehydrogenase (CAD) are notable in that they primarily incorporate coniferaldehyde and sinapaldehyde into their lignin. We tested the hypothesis that by stacking mutations in the genes encoding for the CAD paralogs C and D on an Arabidopsis (Arabidopsis thaliana) med5a/5b ref8-1 genetic background, the biosynthesis of p-coumaryl alcohol would be blocked, making p-coumaraldehyde available for polymerization into a novel kind of lignin. The med5a/5b ref8-1 cadc cadd plants are viable, but lignin analysis demonstrated that they continue to synthesize p-hydroxyphenyl lignin despite being mutated for the CADs typically considered to be required for monolignol biosynthesis. In addition, enzyme activity tests showed that even in the absence of CADC and CADD, there is high CAD activity in stems. We tested the potential involvement of other CADs in p-coumaraldehyde biosynthesis in the quintuple mutant by mutating them using the CRISPR/Cas9 system. Lignin analysis demonstrated that the resulting hextuple mutant plants continue to deposit p-coumaryl alcohol-derived lignin, demonstrating a route for the synthesis of p-hydroxyphenyl lignin in Arabidopsis independent of four CAD isoforms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Tetratricopeptide repeat protein SlREC2 positively regulates cold tolerance in tomato

Abstract Cold stress is a key environmental constraint that dramatically affects the growth, productivity, and quality of tomato (Solanum lycopersicum); however, the underlying molecular mechanisms of cold tolerance remain poorly understood. In this study, we identified REDUCED CHLOROPLAST COVERAGE 2 (SlREC2) encoding a tetratricopeptide repeat protein that positively regulates tomato cold tolerance. Disruption of SlREC2 largely reduced abscisic acid (ABA) levels, photoprotection, and the expression of C-REPEAT BINDING FACTOR (CBF)-pathway genes in tomato plants under cold stress. ABA deficiency in the notabilis (not) mutant, which carries a mutation in 9-CIS-EPOXYCAROTENOID DIOXYGENASE 1 (SlNCED1), strongly inhibited the cold tolerance of SlREC2-silenced plants and empty vector control plants and resulted in a similar phenotype. In addition, foliar application of ABA rescued the cold tolerance of SlREC2-silenced plants, which confirms that SlNCED1-mediated ABA accumulation is required for SlREC2-regulated cold tolerance. Strikingly, SlREC2 physically interacted with β-RING CAROTENE HYDROXYLASE 1b (SlBCH1b), a key regulatory enzyme in the xanthophyll cycle. Disruption of SlBCH1b severely impaired photoprotection, ABA accumulation, and CBF-pathway gene expression in tomato plants under cold stress. Taken together, this study reveals that SlREC2 interacts with SlBCH1b to enhance cold tolerance in tomato via integration of SlNCED1-mediated ABA accumulation, photoprotection, and the CBF-pathway, thus providing further genetic knowledge for breeding cold-resistant tomato varieties.

Zhang, Ying (ORCID:0000000169841164)↗
DOE OSTI2023

Altered cell wall hydroxycinnamate composition impacts leaf- and canopy-level CO2 uptake and water use in rice

Abstract Cell wall properties play a major role in determining photosynthetic carbon uptake and water use through their impact on mesophyll conductance (CO2 diffusion from substomatal cavities into photosynthetic mesophyll cells) and leaf hydraulic conductance (water movement from xylem, through leaf tissue, to stomata). Consequently, modification of cell wall (CW) properties might help improve photosynthesis and crop water use efficiency (WUE). We tested this using 2 independent transgenic rice (Oryza sativa) lines overexpressing the rice OsAT10 gene (encoding a “BAHD” CoA acyltransferase), which alters CW hydroxycinnamic acid content (more para-coumaric acid and less ferulic acid). Plants were grown under high and low water levels, and traits related to leaf anatomy, CW composition, gas exchange, hydraulics, plant biomass, and canopy-level water use were measured. Alteration of hydroxycinnamic acid content led to statistically significant decreases in mesophyll CW thickness (−14%) and increased mesophyll conductance (+120%) and photosynthesis (+22%). However, concomitant increases in stomatal conductance negated the increased photosynthesis, resulting in no change in intrinsic WUE (ratio of photosynthesis to stomatal conductance). Leaf hydraulic conductance was also unchanged; however, transgenic plants showed small but statistically significant increases in aboveground biomass (AGB) (+12.5%) and canopy-level WUE (+8.8%; ratio of AGB to water used) and performed better under low water levels than wild-type plants. Our results demonstrate that changes in CW composition, specifically hydroxycinnamic acid content, can increase mesophyll conductance and photosynthesis in C3 cereal crops such as rice. However, attempts to improve photosynthetic WUE will need to enhance mesophyll conductance and photosynthesis while maintaining or decreasing stomatal conductance.

59 BASIC BIOLOGICAL SCIENCES↗