Ribosome collisions induce mRNA cleavage and ribosome rescue in bacteria.

Ribosome rescue pathways recycle stalled ribosomes and target problematic mRNAs and aborted proteins for degradation1,2. In bacteria, it remains unclear how rescue pathways distinguish ribosomes stalled in the middle of a transcript from actively translating ribosomes3-6. Here, using a genetic screen in Escherichia coli, we discovered a new rescue factor that has endonuclease activity. SmrB cleaves mRNAs upstream of stalled ribosomes, allowing the ribosome rescue factor tmRNA (which acts on truncated mRNAs3) to rescue upstream ribosomes. SmrB is recruited to ribosomes and is activated by collisions. Cryo-electron microscopy structures of collided disomes from E. coli and Bacillus subtilis show distinct and conserved arrangements of individual ribosomes and the composite SmrB-binding site. These findings reveal the underlying mechanisms by which ribosome collisions trigger ribosome rescue in bacteria.

Dynamic relationships among pathways producing hydrocarbons and fatty acids of maize silk cuticular waxes.

The hydrophobic cuticle is the first line of defense between aerial portions of plants and the external environment. On maize (Zea mays L.) silks, the cuticular cutin matrix is infused with cuticular waxes, consisting of a homologous series of very long-chain fatty acids (VLCFAs), aldehydes, and hydrocarbons. Together with VLC fatty-acyl-CoAs (VLCFA-CoAs), these metabolites serve as precursors, intermediates, and end-products of the cuticular wax biosynthetic pathway. To deconvolute the potentially confounding impacts of the change in silk microenvironment and silk development on this pathway, we profiled cuticular waxes on the silks of the inbreds B73 and Mo17, and their reciprocal hybrids. Multivariate interrogation of these metabolite abundance data demonstrates that VLCFA-CoAs and total free VLCFAs are positively correlated with the cuticular wax metabolome, and this metabolome is primarily affected by changes in the silk microenvironment and plant genotype. Moreover, the genotype effect on the pathway explains the increased accumulation of cuticular hydrocarbons with a concomitant reduction in cuticular VLCFA accumulation on B73 silks, suggesting that the conversion of VLCFA-CoAs to hydrocarbons is more effective in B73 than Mo17. Statistical modeling of the ratios between cuticular hydrocarbons and cuticular VLCFAs reveals a significant role of precursor chain length in determining this ratio. This study establishes the complexity of the product-precursor relationships within the silk cuticular wax-producing network by dissecting both the impact of genotype and the allocation of VLCFA-CoA precursors to different biological processes and demonstrates that longer chain VLCFA-CoAs are preferentially utilized for hydrocarbon biosynthesis.

Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices.

Mass spectrometry-based metabolomics approaches can enable detection and quantification of many thousands of metabolite features simultaneously. However, compound identification and reliable quantification are greatly complicated owing to the chemical complexity and dynamic range of the metabolome. Simultaneous quantification of many metabolites within complex mixtures can additionally be complicated by ion suppression, fragmentation and the presence of isomers. Here we present guidelines covering sample preparation, replication and randomization, quantification, recovery and recombination, ion suppression and peak misidentification, as a means to enable high-quality reporting of liquid chromatography- and gas chromatography-mass spectrometry-based metabolomics-derived data.

Systematic molecular analyses for 115 karyotypically normal men with isolated non-obstructive azoospermia.

STUDY QUESTION: Do copy-number variations (CNVs) in the azoospermia factor (AZF) regions and monogenic mutations play a major role in the development of isolated (non-syndromic) non-obstructive azoospermia (NOA) in Japanese men with a normal 46, XY karyotype? SUMMARY ANSWER: Deleterious CNVs in the AZF regions and damaging sequence variants in eight genes likely constitute at least 8% and approximately 8% of the genetic causes, respectively, while variants in other genes play only a minor role. WHAT IS KNOWN ALREADY: Sex chromosomal abnormalities, AZF-linked microdeletions, and monogenic mutations have been implicated in isolated NOA. More than 160 genes have been reported as causative/susceptibility/candidate genes for NOA. STUDY DESIGN, SIZE, DURATION: Systematic molecular analyses were conducted for 115 patients with isolated NOA and a normal 46, XY karyotype, who visited our hospital between 2017 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied 115 unrelated Japanese patients. AZF-linked CNVs were examined using sequence-tagged PCR and multiplex ligation-dependent probe amplification, and nucleotide variants were screened using whole exome sequencing (WES). An optimized sequence kernel association test (SKAT-O), a gene-based association study using WES data, was performed to identify novel disease-associated genes in the genome. The results were compared to those of previous studies and our in-house control data. MAIN RESULTS AND THE ROLE OF CHANCE: Thirteen types of AZF-linked CNVs, including the hitherto unreported gr/gr triplication and partial AZFb deletion, were identified in 63 (54.8%) cases. When the gr/gr deletion, a common polymorphism in Japan, was excluded from data analyses, the total frequency of CNVs was 23/75 (30.7%). This frequency is higher than that of the reference data in Japan and China (11.1% and 14.7%, respectively). Known NOA-causative AZF-linked CNVs were found in nine (7.8%) cases. Rare damaging variants in known causative genes (DMRT1, PLK4, SYCP2, TEX11, and USP26) and hemizygous/multiple-heterozygous damaging variants in known spermatogenesis-associated genes (TAF7L, DNAH2, and DNAH17) were identified in nine cases (7.8% in total). Some patients carried rare damaging variants in multiple genes. SKAT-O detected no genes whose rare damaging variants were significantly accumulated in the patient group. LIMITATIONS, REASONS FOR CAUTION: The number of participants was relatively small, and the clinical information of each patient was fragmentary. Moreover, the pathogenicity of identified variants was assessed only by in silico analyses. WIDER IMPLICATIONS OF THE FINDINGS: This study showed that various AZF-linked CNVs are present in more than half of Japanese NOA patients. These results broadened the structural variations of AZF-linked CNVs, which should be considered for the molecular diagnosis of spermatogenic failure. Furthermore, the results of this study highlight the etiological heterogeneity and possible oligogenicity of isolated NOA. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Grants from the Japan Society for the Promotion of Science (21K19283 and 21H0246), the Japan Agency for Medical Research and Development (22ek0109464h0003), the National Center for Child Health and Development, the Canon Foundation, the Japan Endocrine Society, and the Takeda Science Foundation. The results of this study were based on samples and patient data obtained from the International Center for Reproductive Medicine, Dokkyo Medical University Saitama Medical Center, Koshigaya, Japan. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Genetic regulation and manipulation of nicotine biosynthesis in tobacco: strategies to eliminate addictive alkaloids.

Tobacco (Nicotiana tabacum L.) is a widely cultivated crop of the genus Nicotiana. Due to the highly addictive nature of tobacco products, tobacco smoking remains the leading cause of preventable death and disease. There is therefore a critical need to develop tobacco varieties with reduced or non-addictive nicotine levels. Nicotine and related pyridine alkaloids biosynthesized in the roots of tobacco plants are transported to the leaves, where they are stored in vacuoles as a defense against predators. Jasmonate, a defense-related plant hormone, plays a crucial signaling role in activating transcriptional regulators that coordinate the expression of downstream metabolic and transport genes involved in nicotine production. In recent years, substantial progress has been made in molecular and genomics research, revealing many metabolic and regulatory genes involved in nicotine biosynthesis. These advances have enabled us to develop tobacco plants with low or ultra-low nicotine levels through various methodologies, such as mutational breeding, genetic engineering, and genome editing. We review the recent progress on genetic manipulation of nicotine production in tobacco, which serves as an excellent example of plant metabolic engineering with profound social implications.

Seed-coat protective neolignans are produced by the dirigent protein AtDP1 and the laccase AtLAC5 in Arabidopsis.

Lignans/neolignans are generally synthesized from coniferyl alcohol (CA) in the cinnamate/monolignol pathway by oxidation to generate the corresponding radicals with subsequent stereoselective dimerization aided by dirigent proteins (DIRs). Genes encoding oxidases and DIRs for neolignan biosynthesis have not been identified previously. In Arabidopsis thaliana, the DIR AtDP1/AtDIR12 plays an essential role in the 8-O-4' coupling in neolignan biosynthesis by unequivocal structural determination of the compound missing in the atdp1 mutant as a sinapoylcholine (SC)-conjugated neolignan, erythro-3-{4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-hydroxymethylethoxy]-3,5-dimethoxyphenyl}acryloylcholine. Phylogenetic analyses showed that AtDP1/AtDIR12 belongs to the DIR-a subfamily composed of DIRs for 8-8' coupling of monolignol radicals. AtDP1/AtDIR12 is specifically expressed in outer integument 1 cells in developing seeds. As a putative oxidase for neolignan biosynthesis, we focused on AtLAC5, a laccase gene coexpressed with AtDP1/AtDIR12. In lac5 mutants, the abundance of feruloylcholine (FC)-conjugated neolignans decreased to a level comparable to those in the atdp1 mutant. In addition, SC/FC-conjugated neolignans were missing in the seeds of mutants defective in SCT/SCPL19, an enzyme that synthesizes SC. These results strongly suggest that AtDP1/AtDIR12 and AtLAC5 are involved in neolignan biosynthesis via SC/FC. A tetrazolium penetration assay showed that seed coat permeability increased in atdp1 mutants, suggesting a protective role of neolignans in A. thaliana seeds.

Retrograde sulfur flow from glucosinolates to cysteine in Arabidopsis thaliana.

Specialized (secondary) metabolic pathways in plants have long been considered one-way routes of leading primary metabolite precursors to bioactive end products. Conversely, endogenous degradation of such "end" products in plant tissues has been observed following environmental stimuli, including nutrition stress. Therefore, it is of general interest whether specialized metabolites can be reintegrated into primary metabolism to recover the invested resources, especially in the case of nitrogen- or sulfur-rich compounds. Here, we demonstrate that endogenous glucosinolates (GLs), a class of sulfur-rich plant metabolites, are exploited as a sulfur source by the reallocation of sulfur atoms to primary metabolites such as cysteine in Arabidopsis thaliana Tracer experiments using 34S- or deuterium-labeled GLs depicted the catabolic processing of GL breakdown products in which sulfur is mobilized from the thioglucoside group in GL molecules, potentially accompanied by the release of the sulfate group. Moreover, we reveal that beta-glucosidases BGLU28 and BGLU30 are the major myrosinases that initiate sulfur reallocation by hydrolyzing particular GL species, conferring sulfur deficiency tolerance in A. thaliana, especially during early development. The results delineate the physiological function of GL as a sulfur reservoir, in addition to their well-known functions as defense chemicals. Overall, our findings demonstrate the bidirectional interaction between primary and specialized metabolism, which enhances our understanding of the underlying metabolic mechanisms via which plants adapt to their environments.

Defective cytokinin signaling reprograms lipid and flavonoid gene-to-metabolite networks to mitigate high salinity in Arabidopsis.

Cytokinin (CK) in plants regulates both developmental processes and adaptation to environmental stresses. Arabidopsis histidine phosphotransfer ahp2,3,5 and type-B Arabidopsis response regulator arr1,10,12 triple mutants are almost completely defective in CK signaling, and the ahp2,3,5 mutant was reported to be salt tolerant. Here, we demonstrate that the arr1,10,12 mutant is also more tolerant to salt stress than wild-type (WT) plants. A comprehensive metabolite profiling coupled with transcriptome analysis of the ahp2,3,5 and arr1,10,12 mutants was conducted to elucidate the salt tolerance mechanisms mediated by CK signaling. Numerous primary (e.g., sugars, amino acids, and lipids) and secondary (e.g., flavonoids and sterols) metabolites accumulated in these mutants under nonsaline and saline conditions, suggesting that both prestress and poststress accumulations of stress-related metabolites contribute to improved salt tolerance in CK-signaling mutants. Specifically, the levels of sugars (e.g., trehalose and galactinol), amino acids (e.g., branched-chain amino acids and γ-aminobutyric acid), anthocyanins, sterols, and unsaturated triacylglycerols were higher in the mutant plants than in WT plants. Notably, the reprograming of flavonoid and lipid pools was highly coordinated and concomitant with the changes in transcriptional levels, indicating that these metabolic pathways are transcriptionally regulated by CK signaling. The discovery of the regulatory role of CK signaling on membrane lipid reprogramming provides a greater understanding of CK-mediated salt tolerance in plants. This knowledge will contribute to the development of salt-tolerant crops with the ability to withstand salinity as a key driver to ensure global food security in the era of climate crisis.

Cut-off value for anti-Müllerian hormone in the diagnostic criteria for polycystic ovary syndrome in the Japanese population.

AIM: To establish cut-off values for anti-Müllerian hormone (AMH) and antral follicle count (AFC) in the diagnostic criteria for polycystic ovary syndrome (PCOS) applicable to the Japan Society of Obstetrics and Gynecology (JSOG) 2024 criteria and the Rotterdam/International Evidence-Based Guideline for the assessment and management of PCOS (IEBG) 2023 criteria based on a nationwide survey, respectively, taking into account age, assays, and structure of the diagnostic criteria. METHODS: Data were collected for 986 PCOS cases and 965 control cases using a national survey in Japan and used to establish cut-off values for AMH and AFC. RESULTS: Serum AMH levels were significantly higher in the PCOS group compared to the control group. Serum AMH showed a significant negative correlation with age and significant positive correlation with AFC in both groups. In multiple regression analysis, serum AMH level was independently affected by AFC and total testosterone. AMH cut-off values suitable for the JSOG 2024 criteria and the Rotterdam/IEBG 2023 criteria were separately established for the 20-29 and 30-39 years of age groups, respectively, and for Access, Lumipulse and Elecsys/ECLusys, respectively. AFC cut-off values suitable for the JSOG 2024 criteria and Rotterdam/IEBG 2023 criteria were also established separately. AFC exhibited statistically greater variability than AMH. CONCLUSION: The serum AMH level is the biochemical representation of ovarian findings in PCOS and considered objective and highly reliable. Therefore, it could serve as a surrogate for AFC as a marker of polycystic ovarian morphology in diagnostic criteria.

Ratoon rice research: Review and prospect for the tropics.

Context: With increasing labor shortage and production costs, water scarcity and climate change, there is increased interest in ratooning as a green, resource-efficient technology to boost sustainable rice production, especially in China. Since the performance of ratoon rice (regenerating a second crop from the stubble left in the fields after the main harvest) and the impact of agronomic practices on its yield have shown mixed results across the world, a better understanding is needed to determine under which conditions ratoon rice performs well. Objective: The objectives are (i) to quantify variation in rice yield of main and ratoon crops, (ii) to assess genetic variation in and impact of agronomic practices on rice yield, focusing on the yield of ratoon crop and total yield (main and ratoon crops), and (iii) review of economic and environmental benefits of ratoon rice in comparison with single and double rice cropping. Methods: In researching ratoon rice, we compiled a database from 68 studies published from 2000 to 2023. Descriptive data analysis was performed. Results: Studies from non-tropical regions account for about 70%. Large variation exists in the yield of ratoon crop across the studies, with lower yield from the tropics than non-tropics. The ratio of yield of ratoon crop to that of main crop also varied widely from 0.13 to 0.67 with 0.36 and 0.5 in tropics and non-tropics, respectively. The yield of ratoon crop was positively related to the yield of main crop, crop duration and nitrogen fertilizer application rate, which were generally higher in non-tropics. Hybrid varieties out-yielded inbred varieties in both main and ratoon crops in non-tropical regions. Direct seeding and AWD had a positive impact on the yield of ratoon crop. The impact of stubble cutting height was mixed. While agronomic nitrogen use efficiency (AEN) during entire ratoon rice cropping was similar to that reported for single rice cropping in previous studies, AEN for ratoon crop in tropical regions tended to be lower than those from previous studies on single rice cropping. Ratoon rice cropping reduced labor input and production cost and increased net economic return compared with double rice cropping. Conclusions: We propose a research agenda, with the focus on improvement of genetic and agronomic practices to explore the potential of ratoon rice cropping, especially in the tropics. Implications: This study provides insight into the progress in ratoon rice research over the past two decades globally, and specifically in the tropics.

Reinfusion of peritoneal fluid elevates the level of plasma D-dimer in patients with early-onset ovarian hyperstimulation syndrome.

Purpose: This study aimed to elucidate the factors that affect the dynamics of blood D-dimer in ovarian hyperstimulation syndrome (OHSS). Methods: We retrospectively reviewed medical records from two hospitals and extracted data obtained during assisted reproductive technology and OHSS treatment. Blood D-dimer levels during hospitalization were plotted against body weight. Other factors possibly related to blood D-dimer levels were also analyzed. Results: The analysis included 10 patients with OHSS admitted between January 2013 and June 2023. In all patients, blood D-dimer levels increased significantly when they convalesced from OHSS and lost weight. None of the patients showed clinical signs of thrombosis, which was confirmed using imaging tests in 8 of 10 patients. Two patients underwent cell-free and concentrated ascites reinfusion therapy (CART), and their blood D-dimer levels increased dramatically after the procedure. Conclusion: Weight change and CART are associated with blood D-dimer dynamics in OHSS. Our results show that elevated blood D-dimer levels in patients with OHSS do not always represent the presence of thrombosis. Reinfusion of pooled D-dimer in ascites may explain the D-dimer surge during the recovery phase or after CART in these patients. Our study provides new perspectives on the clinical implications of D-dimer during OHSS.

Natural and induced variations in transcriptional regulator genes result in low-nicotine phenotypes in tobacco.

In tobacco, the homologous ETHYLENE RESPONSE FACTOR (ERF) transcription factors ERF199 and ERF189 coordinate the transcription of multiple metabolic genes involved in nicotine biosynthesis. Natural alleles at the NIC1 and NIC2 loci greatly affect alkaloid accumulation and overlap with ERF199 and ERF189 in the tobacco genome, respectively. In this study, we identified several low-nicotine tobacco varieties lacking ERF199 or ERF189 from a tobacco germplasm collection. We characterized the sequence of these new nic1 and nic2 alleles, as well as the previously defined alleles nic1-1 and nic2-1. Moreover, we examined the influence of different nic alleles on alkaloid contents and expression levels of genes related to nicotine biosynthesis. We also demonstrated that the deletion of a distal genomic region attenuates ERF199 expression, resulting in a moderately negative effect on the alkaloid phenotype. Our study provides new insights into the regulation of nicotine biosynthesis and novel genetic resources to breed low-nicotine tobacco.

Excretion of triacylglycerol as a matrix lipid facilitating apoplastic accumulation of a lipophilic metabolite shikonin.

Plants produce a large variety of lipophilic metabolites, many of which are secreted by cells and accumulated in apoplasts. These compounds often play a role to protect plants from environmental stresses. However, little is known about how these lipophilic compounds are secreted into apoplastic spaces. In this study, we used shikonin-producing cultured cells of Lithospermum erythrorhizon as an experimental model system to analyze the secretion of lipophilic metabolites, taking advantage of its high production rate and the clear inducibility in culture. Shikonin derivatives are lipophilic red naphthoquinone compounds that accumulate exclusively in apoplastic spaces of these cells and also in the root epidermis of intact plants. Microscopic analysis showed that shikonin is accumulated in the form of numerous particles on the cell wall. Lipidomic analysis showed that L. erythrorhizon cultured cells secrete an appreciable portion of triacylglycerol (24-38% of total triacylglycerol), composed predominantly of saturated fatty acids. Moreover, in vitro reconstitution assay showed that triacylglycerol encapsulates shikonin derivatives with phospholipids to form lipid droplet-like structures. These findings suggest a novel role for triacylglycerol as a matrix lipid, a molecular component involved in the secretion of specialized lipophilic metabolites.

Fruit setting rewires central metabolism via gibberellin cascades.

Fruit set is the process whereby ovaries develop into fruits after pollination and fertilization. The process is induced by the phytohormone gibberellin (GA) in tomatoes, as determined by the constitutive GA response mutant procera However, the role of GA on the metabolic behavior in fruit-setting ovaries remains largely unknown. This study explored the biochemical mechanisms of fruit set using a network analysis of integrated transcriptome, proteome, metabolome, and enzyme activity data. Our results revealed that fruit set involves the activation of central carbon metabolism, with increased hexoses, hexose phosphates, and downstream metabolites, including intermediates and derivatives of glycolysis, the tricarboxylic acid cycle, and associated organic and amino acids. The network analysis also identified the transcriptional hub gene SlHB15A, that coordinated metabolic activation. Furthermore, a kinetic model of sucrose metabolism predicted that the sucrose cycle had high activity levels in unpollinated ovaries, whereas it was shut down when sugars rapidly accumulated in vacuoles in fruit-setting ovaries, in a time-dependent manner via tonoplastic sugar carriers. Moreover, fruit set at least partly required the activity of fructokinase, which may pull fructose out of the vacuole, and this could feed the downstream pathways. Collectively, our results indicate that GA cascades enhance sink capacities, by up-regulating central metabolic enzyme capacities at both transcriptional and posttranscriptional levels. This leads to increased sucrose uptake and carbon fluxes for the production of the constituents of biomass and energy that are essential for rapid ovary growth during the initiation of fruit set.

Estimating nutrient concentrations and uptake in rice grain in sub-Saharan Africa using linear mixed-effects regression.

Context or problem: Quantification of nutrient concentrations in rice grain is essential for evaluating nutrient uptake, use efficiency, and balance to develop fertilizer recommendation guidelines. Accurate estimation of nutrient concentrations without relying on plant laboratory analysis is needed in sub-Saharan Africa (SSA), where farmers do not generally have access to laboratories. Objective or research question: The objectives are to 1) examine if the concentrations of macro- (N, P, K, Ca, Mg, S) and micronutrients (Fe, Mn, B, Cu) in rice grain can be estimated using agro-ecological zones (AEZ), production systems, soil properties, and mineral fertilizer application (N, P, and K) rates as predictor variables, and 2) to identify if nutrient uptakes estimated by best-fitted models with above variables provide improved prediction of actual nutrient uptakes (predicted nutrient concentrations x grain yield) compared to average-based uptakes (average nutrient concentrations in SSA x grain yield). Methods: Cross-sectional data from 998 farmers' fields across 20 countries across 4 AEZs (arid/semi-arid, humid, sub-humid, and highlands) in SSA and 3 different production systems: irrigated lowland, rainfed lowland, and rainfed upland were used to test hypotheses of nutrient concentration being estimable with a set of predictor variables among above-cited factors using linear mixed-effects regression models. Results: All 10 nutrients were reasonably predicted [Nakagawa's R2 ranging from 0.27 (Ca) to 0.79 (B), and modeling efficiency ranging from 0.178 (Ca) to 0.584 (B)]. However, only the estimation of K and B concentrations was satisfactory with a modeling efficiency superior to 0.5. The country variable contributed more to the variation of concentrations of these nutrients than AEZ and production systems in our best predictive models. There were greater positive relationships (up to 0.18 of difference in correlation coefficient R) between actual nutrient uptakes and model estimation-based uptakes than those between actual nutrient uptakes and average-based uptakes. Nevertheless, only the estimation of B uptake had significant improvement among all nutrients investigated. Conclusions: Our findings suggest that with the exception of B associated with high model EF and an improved uptake over the average-based uptake, estimates of the macronutrient and micronutrient uptakes in rice grain can be obtained simply by using average concentrations of each nutrient at the regional scale for SSA. Implications: Further investigation of other factors such as the timing of fertilizer applications, rice variety, occurrence of drought periods, and atmospheric CO2 concentration is warranted for improved prediction accuracy of nutrient concentrations.

Exome-based genome-wide screening of rare variants associated with the risk of polycystic ovary syndrome.

Purpose: Genetic factors associated with the risk of polycystic ovary syndrome (PCOS) remain largely unknown. Here, we conducted an optimal sequence kernel association test (SKAT-O), an exome-based rare variant association study, to clarify whether rare variants in specific genes contribute to the development of PCOS. Methods: SKAT-O was performed using exome data of 44 Japanese patients with PCOS and 301 control women. We analyzed frequencies of rare probably damaging variants in the genome. Results: Rare variants of GSTO2 were more commonly identified in the patient group than in the control group (6/44 vs. 1/301; Bonferroni-corrected p-value, 0.028), while the frequencies of variants in other genes were comparable between the two groups. The identified GSTO2 variants were predicted to affect the function, structure, stability, hydrophobicity, and/or the formation of intrinsically disordered regions of the protein. GSTO2 encodes a glutathione transferase that mediates the oxidative stress response and arsenic metabolism. Previously, common variants in GSTO2 and its paralog GSTO1 were associated with the risk of PCOS. Conclusions: The results indicate that there are no genes whose rare variants account for a large fraction of the etiology of PCOS, although rare damaging variants in GSTO2 may constitute a risk factor in some cases.

Characterization of C-26 aminotransferase, indispensable for steroidal glycoalkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in members of the Solanaceae, such as Solanum tuberosum (potato) and Solanum lycopersicum (tomato). The major potato SGAs are α-solanine and α-chaconine, which are biosynthesized from cholesterol. Previously, we have characterized two cytochrome P450 monooxygenases and a 2-oxoglutarate-dependent dioxygenase that function in hydroxylation at the C-22, C-26 and C-16α positions, but the aminotransferase responsible for the introduction of a nitrogen moiety into the steroidal skeleton remains uncharacterized. Here, we show that PGA4 encoding a putative γ-aminobutyrate aminotransferase is involved in SGA biosynthesis in potatoes. The PGA4 transcript was expressed at high levels in tuber sprouts, in which SGAs are abundant. Silencing the PGA4 gene decreased potato SGA levels and instead caused the accumulation of furostanol saponins. Analysis of the tomato PGA4 ortholog, GAME12, essentially provided the same results. Recombinant PGA4 protein exhibited catalysis of transamination at the C-26 position of 22-hydroxy-26-oxocholesterol using γ-aminobutyric acid as an amino donor. Solanum stipuloideum (PI 498120), a tuber-bearing wild potato species lacking SGA, was found to have a defective PGA4 gene expressing the truncated transcripts, and transformation of PI 498120 with functional PGA4 resulted in the complementation of SGA production. These findings indicate that PGA4 is a key enzyme for transamination in SGA biosynthesis. The disruption of PGA4 function by genome editing will be a viable approach for accumulating valuable steroidal saponins in SGA-free potatoes.

Genetic divergence in transcriptional regulators of defense metabolism: insight into plant domestication and improvement.

KEY MESSAGE: A number of mutational changes in transcriptional regulators of defense metabolism have occurred during plant domestication and improvement. Plant domestication and improvement entail genetic changes that underlie divergence in development and metabolism, providing a tremendous model of biological evolution. Plant metabolism produces numerous specialized alkaloids, terpenoids, phenolics, and cyanogenic glucosides with indispensable roles in defense against herbivory and microbial infection. Many compounds toxic or deterrent to predators have been eliminated through domestication and breeding. Series of genes involved in defense metabolism are coordinately regulated by transcription factors that specifically recognize cis-regulatory elements in promoter regions of downstream target genes. Recent developments in DNA sequencing technologies and genomic approaches have facilitated studies of the metabolic and genetic changes in chemical defense that have occurred via human-mediated selection, many of which result from mutations in transcriptional regulators of defense metabolism. In this article, we review such examples in almond (Prunus dulcis), cucumber (Cucumis sativus), pepper (Capsicum spp.), potato (Solanum tuberosum), quinoa (Chenopodium quinoa), sorghum (Sorghum bicolor), and related species and discuss insights into the evolution and regulation of metabolic pathways for specialized defense compounds.

Integrative omics approaches revealed a crosstalk among phytohormones during tuberous root development in cassava.

KEY MESSAGE: Integrative omics approaches revealed a crosstalk among phytohormones during tuberous root development in cassava. Tuberous root formation is a complex process consisting of phase changes as well as cell division and elongation for radial growth. We performed an integrated analysis to clarify the relationships among metabolites, phytohormones, and gene transcription during tuberous root formation in cassava (Manihot esculenta Crantz). We also confirmed the effects of the auxin (AUX), cytokinin (CK), abscisic acid (ABA), jasmonic acid (JA), gibberellin (GA), brassinosteroid (BR), salicylic acid, and indole-3-acetic acid conjugated with aspartic acid on tuberous root development. An integrated analysis of metabolites and gene expression indicated the expression levels of several genes encoding enzymes involved in starch biosynthesis and sucrose metabolism are up-regulated during tuberous root development, which is consistent with the accumulation of starch, sugar phosphates, and nucleotides. An integrated analysis of phytohormones and gene transcripts revealed a relationship among AUX signaling, CK signaling, and BR signaling, with AUX, CK, and BR inducing tuberous root development. In contrast, ABA and JA inhibited tuberous root development. These phenomena might represent the differences between stem tubers (e.g., potato) and root tubers (e.g., cassava). On the basis of these results, a phytohormonal regulatory model for tuberous root development was constructed. This model may be useful for future phytohormonal studies involving cassava.

Tandem Gene Duplication of Dioxygenases Drives the Structural Diversity of Steroidal Glycoalkaloids in the Tomato Clade.

Cultivated tomato (Solanum lycopersicum) contains α-tomatine, a steroidal glycoalkaloid (SGA), which functions as a defense compound to protect against pathogens and herbivores; interestingly, wild species in the tomato clade biosynthesize a variety of SGAs. In cultivated tomato, the metabolic detoxification of α-tomatine during tomato fruit ripening is an important trait that aided in its domestication, and two distinct 2-oxoglutarate-dependent dioxygenases (DOXs), a C-23 hydroxylase of α-tomatine (Sl23DOX) and a C-27 hydroxylase of lycoperoside C (Sl27DOX), are key to this process. There are tandemly duplicated DOX genes on tomato chromosome 1, with high levels of similarity to Sl23DOX. While these DOX genes are rarely expressed in cultivated tomato tissues, the recombinant enzymes of Solyc01g006580 and Solyc01g006610 metabolized α-tomatine to habrochaitoside A and (20R)-20-hydroxytomatine and were therefore named as habrochaitoside A synthase (HAS) and α-tomatine 20-hydroxylase (20DOX), respectively. Furthermore, 20DOX and HAS exist in the genome of wild tomato S. habrochaites accession LA1777, which accumulates habrochaitoside A in its fruits, and their expression patterns were in agreement with the SGA profiles in LA1777. These results indicate that the functional divergence of α-tomatine-metabolizing DOX enzymes results from gene duplication and the neofunctionalization of catalytic activity and gene expression, and this contributes to the structural diversity of SGAs in the tomato clade.