Autotrophic growth of Escherichia coli is achieved by a small number of genetic changes.

Synthetic autotrophy is a promising avenue to sustainable bioproduction from CO2. Here, we use iterative laboratory evolution to generate several distinct autotrophic strains. Utilising this genetic diversity, we identify that just three mutations are sufficient for Escherichia coli to grow autotrophically, when introduced alongside non-native energy (formate dehydrogenase) and carbon-fixing (RuBisCO, phosphoribulokinase, carbonic anhydrase) modules. The mutated genes are involved in glycolysis (pgi), central-carbon regulation (crp), and RNA transcription (rpoB). The pgi mutation reduces the enzyme's activity, thereby stabilising the carbon-fixing cycle by capping a major branching flux. For the other two mutations, we observe down-regulation of several metabolic pathways and increased expression of native genes associated with the carbon-fixing module (rpiB) and the energy module (fdoGH), as well as an increased ratio of NADH/NAD+ - the cycle's electron-donor. This study demonstrates the malleability of metabolism and its capacity to switch trophic modes using only a small number of genetic changes and could facilitate transforming other heterotrophic organisms into autotrophs.

Widespread dissolved inorganic carbon-modifying toolkits in genomes of autotrophic Bacteria and Archaea and how they are likely to bridge supply from the environment to demand by autotrophic pathways.

Using dissolved inorganic carbon (DIC) as a major carbon source, as autotrophs do, is complicated by the bedeviling nature of this substance. Autotrophs using the Calvin-Benson-Bassham cycle (CBB) are known to make use of a toolkit comprised of DIC transporters and carbonic anhydrase enzymes (CA) to facilitate DIC fixation. This minireview provides a brief overview of the current understanding of how toolkit function facilitates DIC fixation in Cyanobacteria and some Proteobacteria using the CBB and continues with a survey of the DIC toolkit gene presence in organisms using different versions of the CBB and other autotrophic pathways (reductive citric acid cycle, Wood-Ljungdahl pathway, hydroxypropionate bicycle, hydroxypropionate-hydroxybutyrate cycle, and dicarboxylate-hydroxybutyrate cycle). The potential function of toolkit gene products in these organisms is discussed in terms of CO2 and HCO3- supply from the environment and demand by the autotrophic pathway. The presence of DIC toolkit genes in autotrophic organisms beyond those using the CBB suggests the relevance of DIC metabolism to these organisms and provides a basis for better engineering of these organisms for industrial and agricultural purposes.

Host-symbiont stress response to lack-of-sulfide in the giant ciliate mutualism.

The mutualism between the thioautotrophic bacterial ectosymbiont Candidatus Thiobius zoothamnicola and the giant ciliate Zoothamnium niveum thrives in a variety of shallow-water marine environments with highly fluctuating sulfide emissions. To persist over time, both partners must reproduce and ensure the transmission of symbionts before the sulfide stops, which enables carbon fixation of the symbiont and nourishment of the host. We experimentally investigated the response of this mutualism to depletion of sulfide. We found that colonies released some initially present but also newly produced macrozooids until death, but in fewer numbers than when exposed to sulfide. The symbionts on the colonies proliferated less without sulfide, and became larger and more rod-shaped than symbionts from freshly collected colonies that were exposed to sulfide and oxygen. The symbiotic monolayer was severely disturbed by growth of other microbes and loss of symbionts. We conclude that the response of both partners to the termination of sulfide emission was remarkably quick. The development and the release of swarmers continued until host died and thus this behavior contributed to the continuation of the association.

Phylogenetically and functionally diverse microorganisms reside under the Ross Ice Shelf.

Throughout coastal Antarctica, ice shelves separate oceanic waters from sunlight by hundreds of meters of ice. Historical studies have detected activity of nitrifying microorganisms in oceanic cavities below permanent ice shelves. However, little is known about the microbial composition and pathways that mediate these activities. In this study, we profiled the microbial communities beneath the Ross Ice Shelf using a multi-omics approach. Overall, beneath-shelf microorganisms are of comparable abundance and diversity, though distinct composition, relative to those in the open meso- and bathypelagic ocean. Production of new organic carbon is likely driven by aerobic lithoautotrophic archaea and bacteria that can use ammonium, nitrite, and sulfur compounds as electron donors. Also enriched were aerobic organoheterotrophic bacteria capable of degrading complex organic carbon substrates, likely derived from in situ fixed carbon and potentially refractory organic matter laterally advected by the below-shelf waters. Altogether, these findings uncover a taxonomically distinct microbial community potentially adapted to a highly oligotrophic marine environment and suggest that ocean cavity waters are primarily chemosynthetically-driven systems.

Intelligent self-control of carbon metabolic flux in SecY-engineered Escherichia coli for xylitol biosynthesis from xylose-glucose mixtures.

Xylitol is a salutary sugar substitute that has been widely used in the food, pharmaceutical, and chemical industries. Co-fermentation of xylose and glucose by metabolically engineered cell factories is a promising alternative to chemical hydrogenation of xylose for commercial production of xylitol. Here, we engineered a mutant of SecY protein-translocation channel (SecY [ΔP]) in xylitol-producing Escherichia coli JM109 (DE3) as a passageway for xylose uptake. It was found that SecY (ΔP) channel could rapidly transport xylose without being interfered by XylB-catalyzed synthesis of xylitol-phosphate, which is impossible for native XylFGH and XylE transporters. More importantly, with the coaction of SecY (ΔP) channel and carbon catabolite repression (CCR), the flux of xylose to the pentose phosphate (PP) pathway and the xylitol synthesis pathway in E. coli could be automatically controlled in response to glucose, thereby ensuring that the mutant cells were able to fully utilize sugars with high xylitol yields. The E. coli cell factory developed in this study has been proven to be applicable to a broad range of xylose-glucose mixtures, which is conducive to simplifying the mixed-sugar fermentation process for efficient and economical production of xylitol.

Multisubstrate DNA stable isotope probing reveals guild structure of bacteria that mediate soil carbon cycling.

Soil microorganisms determine the fate of soil organic matter (SOM), and their activities compose a major component of the global carbon (C) cycle. We employed a multisubstrate, DNA-stable isotope probing experiment to track bacterial assimilation of C derived from distinct sources that varied in bioavailability. This approach allowed us to measure microbial contributions to SOM processing by measuring the C assimilation dynamics of diverse microorganisms as they interacted within soil. We identified and tracked 1,286 bacterial taxa that assimilated 13C in an agricultural soil over a period of 48 d. Overall 13C-assimilation dynamics of bacterial taxa, defined by the source and timing of the 13C they assimilated, exhibited low phylogenetic conservation. We identified bacterial guilds composed of taxa that had similar 13C assimilation dynamics. We show that C-source bioavailability explained significant variation in both C mineralization dynamics and guild structure, and that the growth dynamics of bacterial guilds differed significantly in response to C addition. We also demonstrate that the guild structure explains significant variation in the biogeographical distribution of bacteria at continental and global scales. These results suggest that an understanding of in situ growth dynamics is essential for understanding microbial contributions to soil C cycling. We interpret these findings in the context of bacterial life history strategies and their relationship to terrestrial C cycling.

Holocene life and microbiome profiling in ancient tropical Lake Chalco, Mexico.

Metagenomic and traditional paleolimnological approaches are suitable to infer past biological and environmental changes, however, they are often applied independently, especially in tropical regions. We combined both approaches to investigate Holocene Prokaryote and Eukaryote diversity and microbial metabolic pathways in ancient Lake Chalco, Mexico. Here, we report on diversity among a large number of lineages (36,722 OTUs) and functional diversity (27,636,243 non-clustered predicted proteins, and 6,144 annotated protein-family genes). The most abundant domain is Bacteria (81%), followed by Archaea (15%) and Eukarya (3%). We also determined the diversity of protein families and their relationship to metabolic pathways. The early Holocene (> 11,000 cal years BP) lake was characterized by cool, freshwater conditions, which later became warmer and hyposaline (11,000-6,000 cal years BP). We found high abundances of cyanobacteria, and fungi groups associated with mature forests in these sediments. Bacteria and Archaea include mainly anaerobes and extremophiles that are involved in the sulfur, nitrogen, and carbon cycles. We found evidence for early human impacts, including landscape modifications and lake eutrophication, which began ~ 6,000 cal years BP. Subsaline, temperate conditions were inferred for the past 5,000 years. Finally, we found nitrogen-fixing bacteria and protein-family genes that are linked to contaminated environments, as well as several fungal pathogens of crops in near-surface sediments.

Stress-Mediated Reprogramming of Prostate Cancer One-Carbon Cycle Drives Disease Progression.

One-carbon (1C) metabolism has a key role in metabolic programming with both mitochondrial (m1C) and cytoplasmic (c1C) components. Here we show that activating transcription factor 4 (ATF4) exclusively activates gene expression involved in m1C, but not the c1C cycle in prostate cancer cells. This includes activation of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) expression, the central player in the m1C cycle. Consistent with the key role of m1C cycle in prostate cancer, MTHFD2 knockdown inhibited prostate cancer cell growth, prostatosphere formation, and growth of patient-derived xenograft organoids. In addition, therapeutic silencing of MTHFD2 by systemically administered nanoliposomal siRNA profoundly inhibited tumor growth in preclinical prostate cancer mouse models. Consistently, MTHFD2 expression is significantly increased in human prostate cancer, and a gene expression signature based on the m1C cycle has significant prognostic value. Furthermore, MTHFD2 expression is coordinately regulated by ATF4 and the oncoprotein c-MYC, which has been implicated in prostate cancer. These data suggest that the m1C cycle is essential for prostate cancer progression and may serve as a novel biomarker and therapeutic target. SIGNIFICANCE: These findings demonstrate that the mitochondrial, but not cytoplasmic, one-carbon cycle has a key role in prostate cancer cell growth and survival and may serve as a biomarker and/or therapeutic target.

Rubisco proton production can drive the elevation of CO2 within condensates and carboxysomes.

Membraneless organelles containing the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are a common feature of organisms utilizing CO2 concentrating mechanisms to enhance photosynthetic carbon acquisition. In cyanobacteria and proteobacteria, the Rubisco condensate is encapsulated in a proteinaceous shell, collectively termed a carboxysome, while some algae and hornworts have evolved Rubisco condensates known as pyrenoids. In both cases, CO2 fixation is enhanced compared with the free enzyme. Previous mathematical models have attributed the improved function of carboxysomes to the generation of elevated CO2 within the organelle via a colocalized carbonic anhydrase (CA) and inwardly diffusing HCO3-, which have accumulated in the cytoplasm via dedicated transporters. Here, we present a concept in which we consider the net of two protons produced in every Rubisco carboxylase reaction. We evaluate this in a reaction-diffusion compartment model to investigate functional advantages these protons may provide Rubisco condensates and carboxysomes, prior to the evolution of HCO3- accumulation. Our model highlights that diffusional resistance to reaction species within a condensate allows Rubisco-derived protons to drive the conversion of HCO3- to CO2 via colocalized CA, enhancing both condensate [CO2] and Rubisco rate. Protonation of Rubisco substrate (RuBP) and product (phosphoglycerate) plays an important role in modulating internal pH and CO2 generation. Application of the model to putative evolutionary ancestors, prior to contemporary cellular HCO3- accumulation, revealed photosynthetic enhancements along a logical sequence of advancements, via Rubisco condensation, to fully formed carboxysomes. Our model suggests that evolution of Rubisco condensation could be favored under low CO2 and low light environments.

Brockarchaeota, a novel archaeal phylum with unique and versatile carbon cycling pathways.

Geothermal environments, such as hot springs and hydrothermal vents, are hotspots for carbon cycling and contain many poorly described microbial taxa. Here, we reconstructed 15 archaeal metagenome-assembled genomes (MAGs) from terrestrial hot spring sediments in China and deep-sea hydrothermal vent sediments in Guaymas Basin, Gulf of California. Phylogenetic analyses of these MAGs indicate that they form a distinct group within the TACK superphylum, and thus we propose their classification as a new phylum, 'Brockarchaeota', named after Thomas Brock for his seminal research in hot springs. Based on the MAG sequence information, we infer that some Brockarchaeota are uniquely capable of mediating non-methanogenic anaerobic methylotrophy, via the tetrahydrofolate methyl branch of the Wood-Ljungdahl pathway and reductive glycine pathway. The hydrothermal vent genotypes appear to be obligate fermenters of plant-derived polysaccharides that rely mostly on substrate-level phosphorylation, as they seem to lack most respiratory complexes. In contrast, hot spring lineages have alternate pathways to increase their ATP yield, including anaerobic methylotrophy of methanol and trimethylamine, and potentially use geothermally derived mercury, arsenic, or hydrogen. Their broad distribution and their apparent anaerobic metabolic versatility indicate that Brockarchaeota may occupy previously overlooked roles in anaerobic carbon cycling.

Untargeted Metabolomics Unveil Changes in Autotrophic and Mixotrophic Galdieria sulphuraria Exposed to High-Light Intensity.

The thermoacidophilic red alga Galdieria sulphuraria has been optimizing a photosynthetic system for low-light conditions over billions of years, thriving in hot and acidic endolithic habitats. The growth of G. sulphuraria in the laboratory is very much dependent on light and substrate supply. Here, higher cell densities in G. sulphuraria under high-light conditions were obtained, although reductions in photosynthetic pigments were observed, which indicated this alga might be able to relieve the effects caused by photoinhibition. We further describe an extensive untargeted metabolomics study to reveal metabolic changes in autotrophic and mixotrophic G. sulphuraria grown under high and low light intensities. The up-modulation of bilayer lipids, that help generate better-ordered lipid domains (e.g., ergosterol) and keep optimal membrane thickness and fluidity, were observed under high-light exposure. Moreover, high-light conditions induced changes in amino acids, amines, and amide metabolism. Compared with the autotrophic algae, higher accumulations of osmoprotectant sugars and sugar alcohols were recorded in the mixotrophic G. sulphuraria. This response can be interpreted as a measure to cope with stress due to the high concentration of organic carbon sources. Our results indicate how G. sulphuraria can modulate its metabolome to maintain energetic balance and minimize harmful effects under changing environments.

Repurposing the Endogenous Type I-E CRISPR/Cas System for Gene Repression in Gluconobacter oxydans WSH-003.

Gluconobacter oxydans is well-known for its incomplete oxidizing capacity and has been widely applied in industrial production. However, genetic tools in G. oxydans are still scarce compared with model microorganisms, limiting its metabolic engineering. This study aimed to develop a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system based on the typical type I-E endogenous CRISPR/CRISPR-associated proteins (Cas) system in G. oxydans WSH-003. The nuclease Cas3 in this system was inactivated naturally and hence did not need to be knocked out. Subsequently, the CRISPRi effect was verified by repressing the expression of fluorescent proteins, revealing effective multiplex gene repression. Finally, the endogenous CRISPRi system was used to study the role of the central carbon metabolism pathway, including the pentose phosphate pathway (PPP) and Entner-Doudoroff pathway (EDP), in G. oxydans WSH-003. This was done to demonstrate a metabolic engineering application. The PPP was found to be important for cell growth and the substrate conversion rate. The development of the CRISPRi system enriched the gene regulation tools in G. oxydans and promoted the metabolic engineering modification of G. oxydans to improve its performance. In addition, it might have implications for metabolic engineering modification of other genetically recalcitrant strains.

Tumor Reliance on Cytosolic versus Mitochondrial One-Carbon Flux Depends on Folate Availability.

Folate metabolism supplies one-carbon (1C) units for biosynthesis and methylation and has long been a target for cancer chemotherapy. Mitochondrial serine catabolism is considered the sole contributor of folate-mediated 1C units in proliferating cancer cells. Here, we show that under physiological folate levels in the cell environment, cytosolic serine-hydroxymethyltransferase (SHMT1) is the predominant source of 1C units in a variety of cancers, while mitochondrial 1C flux is overly repressed. Tumor-specific reliance on cytosolic 1C flux is associated with poor capacity to retain intracellular folates, which is determined by the expression of SLC19A1, which encodes the reduced folate carrier (RFC). We show that silencing SHMT1 in cells with low RFC expression impairs pyrimidine biosynthesis and tumor growth in vivo. Overall, our findings reveal major diversity in cancer cell utilization of the cytosolic versus mitochondrial folate cycle across tumors and SLC19A1 expression as a marker for increased reliance on SHMT1.

Roles of FERONIA-like receptor genes in regulating grain size and quality in rice.

Grain yield and quality are critical factors that determine the value of grain crops. In this study, we analyzed the functions of 12 FERONIA-like receptor (FLR) family members in rice and investigated their effects on grain size and quality. We found that FLR1, FLR2 and FLR8 negatively regulated grain size, and FLR15 positively regulated grain size. flr1 mutants had a higher cell number and an accelerated rate of grain filling compared to wild-type plants, which led to grains with greater widths. A mechanism underlying the regulation of grain size by FLR1 is that FLR1 is associated with OsRac1 Rho-like GTPase, a positive regulator of grain size. Regarding grain quality, the flr1 mutant had a higher percentage of chalkiness compared with wild-type plants, and seeds carrying mutations in flr3 and flr14 had endosperms with white floury cores. To elucidate the possible mechanism underlying this phenomenon, we found that FLR1 was constitutively expressed during endosperm development. RNA-seq analysis identified 2,367 genes that were differentially expressed in the flr1 mutant, including genes involved in starch and sucrose metabolism and carbon fixation. In this study, we identified the roles played by several FLR genes in regulating grain size and quality in rice and provided insights into the molecular mechanism governing the FLR1-mediated regulation of grain size.

Screening of native microalgae species for carbon fixation at the vicinity of Malaysian coal-fired power plant.

Global warming has become a serious issue nowadays as the trend of CO2 emission is increasing by years. In Malaysia, the electricity and energy sector contributed a significant amount to the nation's CO2 emission due to fossil fuel use. Many research works have been carried out to mitigate this issue, including carbon capture and utilization (CCUS) technology and biological carbon fixation by microalgae. This study makes a preliminary effort to screen native microalgae species in the Malaysian coal-fired power plant's surrounding towards carbon fixation ability. Three dominant species, including Nannochloropsis sp., Tetraselmis sp., and Isochrysis sp. were identified and tested in the laboratory under ambient and pure CO2 condition to assess their growth and CO2 fixation ability. The results indicate Isochrysis sp. as the superior carbon fixer against other species. In continuation, the optimization study using Response Surface Methodology (RSM) was carried out to optimize the operating conditions of Isochrysis sp. using a customized lab-scale photobioreactor under simulated flue gas exposure. This species was further acclimatized and tested under actual flue gas generated by the power plant. Isochrysis sp. had shown its capability as a carbon fixer with CO2 fixation rate of 0.35 gCO2/L day under actual coal-fired flue gas exposure after cycles of acclimatization phase. This work is the first to demonstrate indigenous microalgae species' ability as a carbon fixer under Malaysian coal-fired flue gas exposure. Thus, the findings shall be useful in exploring the microalgae potential as a biological agent for carbon emission mitigation from power plants more sustainably.

New viral biogeochemical roles revealed through metagenomic analysis of Lake Baikal.

BACKGROUND: Lake Baikal is the largest body of liquid freshwater on Earth. Previous studies have described the microbial composition of this habitat, but the viral communities from this ecosystem have not been characterized in detail. RESULTS: Here, we describe the viral diversity of this habitat across depth and seasonal gradients. We discovered 19,475 bona fide viral sequences, which are derived from viruses predicted to infect abundant and ecologically important taxa that reside in Lake Baikal, such as Nitrospirota, Methylophilaceae, and Crenarchaeota. Diversity analysis revealed significant changes in viral community composition between epipelagic and bathypelagic zones. Analysis of the gene content of individual viral populations allowed us to describe one of the first bacteriophages that infect Nitrospirota, and their extensive repertoire of auxiliary metabolic genes that might enhance carbon fixation through the reductive TCA cycle. We also described bacteriophages of methylotrophic bacteria with the potential to enhance methanol oxidation and the S-adenosyl-L-methionine cycle. CONCLUSIONS: These findings unraveled new ways by which viruses influence the carbon cycle in freshwater ecosystems, namely, by using auxiliary metabolic genes that act upon metabolisms of dark carbon fixation and methylotrophy. Therefore, our results shed light on the processes through which viruses can impact biogeochemical cycles of major ecological relevance. Video Abstract.

Rhizodegradation of PAHs differentially altered by C3 and C4 plants.

Pyrosequencing of 16S ribosomal RNA (rRNA) was employed to characterize bacterial communities colonizing the rhizosphere of plants with C3 and C4 photosynthetic pathways grown in soil contaminated with polycyclic aromatic hydrocarbons (PAHs) after 60 and 120 days. The results of this study exhibited a clear difference in bacterial diversity between the rhizosphere and non-rhizosphere samples and between the rhizospheres of the C3 and C4 plants after 120 days. In both C3 and C4 rhizospheres, an incremental change in PAHs degrading bacterial genera was observed in the 120th day samples compared to the 60th day ones. Among the PAHs degrading bacterial genera, Pseudomonas showed good resistance to PAHs in the 120th day rhizosphere of both C3 and C4 plants. Conversely, the genus Sphingomonas showed sensitivity to PAHs in the 120th day rhizosphere soils of C3 plants only. Also, a significant increase in the PAHs degrading genera was observed at 120th day in the C4 rhizosphere in comparison to the C3 rhizosphere, which was reflected in a reduced PAHs concentration measured in the soil remediated with C4 plants rather than C3 plants. These results suggest that the rhizoremediation of PAHs was primarily governed by the plant photosystems, which led to differences in root secretions that caused the variation in bacterial diversity seen in the rhizospheres. This study is the first report to demonstrate the greater effectiveness of C4 plants in enhancing the PAHs degrading bacterial community than C3 plants.

Assessment of transcriptomic constraint-based methods for central carbon flux inference.

MOTIVATION: Determining intracellular metabolic flux through isotope labeling techniques such as 13C metabolic flux analysis (13C-MFA) incurs significant cost and effort. Previous studies have shown transcriptomic data coupled with constraint-based metabolic modeling can determine intracellular fluxes that correlate highly with 13C-MFA measured fluxes and can achieve higher accuracy than constraint-based metabolic modeling alone. These studies, however, used validation data limited to E. coli and S. cerevisiae grown on glucose, with significantly similar flux distribution for central metabolism. It is unclear whether those results apply to more diverse metabolisms, and therefore further, extensive validation is needed. RESULTS: In this paper, we formed a dataset of transcriptomic data coupled with corresponding 13C-MFA flux data for 21 experimental conditions in different unicellular organisms grown on varying carbon substrates and conditions. Three computational flux-balance analysis (FBA) methods were comparatively assessed. The results show when uptake rates of carbon sources and key metabolites are known, transcriptomic data provides no significant advantage over constraint-based metabolic modeling (average correlation coefficients, transcriptomic E-Flux2 0.725 and SPOT 0.650 vs non-transcriptomic pFBA 0.768). When uptake rates are unknown, however, predictions obtained utilizing transcriptomic data are generally good and significantly better than those obtained using constraint-based metabolic modeling alone (E-Flux2 0.385 and SPOT 0.583 vs pFBA 0.237). Thus, transcriptomic data coupled with constraint-based metabolic modeling is a promising method to obtain intracellular flux estimates in microorganisms, particularly in cases where uptake rates of key metabolites cannot be easily determined, such as for growth in complex media or in vivo conditions.

Working with Randy: The Diacylglycerol Acyltransferase Story.

Vegetable oils are one of the main agricultural commodities. Demand has been increasing steadily over the last five decades and, with finite land available, it is vital that we increase productivity. My laboratory has focused on the regulation of plant lipid metabolism and, as part of this work, we identified diacylglycerol acyltransferase (DGAT) as important at regulating carbon flux during oil accumulation. This led to collaborations with Randy Weselake's research group when we quantified the importance of DGAT in oilseed rape by using flux control analysis. Later, with David Taylor, we showed that over-expression of DGAT boosted oil accumulation in field-grown crops by around 8%. These studies led to a multitude of experiments with different oil crops, such as oil palm and soybean, as well as many rewarding collaborations with Randy.

Molecular Mechanism of Vegetative Growth Advantage in Allotriploid Populus.

Allotriploid poplar has a prominent vegetative growth advantage that impacts dramatically on lumber yield. The growth regulation is complex which involves abundant genes, metabolic and signaling pathways, while the information about the functional control process is very little. We used high-throughput sequencing and physiological index measurement to obtain a global overview of differences between allotriploid and diploid Populus. The genes related to plant growth advantage show a higher expression compared to diploid, and most of them are revolved around hormones, photosynthesis and product accumulation. Thus, allotriploid Populus showed more efficient photosynthesis, carbon fixation, sucrose and starch synthesis, and metabolism as well as augmented biosynthesis of auxin, cytokinin, and gibberellin. These data enable the connection of metabolic processes, signaling pathways, and specific gene activity, which will underpin the development of network models to elucidate the process of triploid Populus advantage growth.