Low Temperature Enhances Plant Immunity via Salicylic Acid Pathway Genes That Are Repressed by Ethylene.

Temperature has a large impact on plant immune responses. Earlier studies identified intracellular immune receptor nucleotide-binding leucine-rich repeat (NLR) genes and salicylic acid (SA) as targets of high-temperature inhibition of plant immunity. Here, we report that moderately low temperature enhances immunity to the bacterial pathogen Pseudomonas syringae in Arabidopsis (Arabidopsis thaliana). This enhancement is dependent on SA signaling and is accompanied by up-regulation of multiple SA biosynthesis and signaling genes at lower temperature. SA signaling is repressed by jasmonic acid and ethylene at both normal and low temperatures. The inhibition of SA biosynthesis by ethylene, while mainly through ISOCHORISMATE SYNTHASE1/SALICYLIC ACID-INDUCTION DEFICIENT2 (ICS1/SID2) at normal temperature, is through ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5)/SID1, ICS2, and ICS1/SID2 at lower temperature. The repression by ethylene is mediated by a direct regulation of the ethylene response transcription factor ETHYLENE INSENSITIVE3 (EIN3) on multiple SA biosynthesis and signaling genes. Thus, low temperature enhances the SA pathway to promote immunity and at the same time uses ethylene to repress multiple SA regulators to achieve fine-tuned immune responses.

Identification and Expression Analyses of the Special 14-3-3 Gene Family in Papaya and its Involvement in Fruit Development, Ripening, and Abiotic Stress Responses.

Plant 14-3-3 proteins play key roles in regulating growth, development, and stress responses. However, little is known about this gene family in papaya (Carica papaya L.). We characterized eight 14-3-3 genes from the papaya genome and designed them as CpGRF1-8. Based on phylogenetic, conserved motif, and gene structure analyses, papaya CpGRFs were divided into ε and non-ε groups. Expression analysis showed differential and class-specific transcription patterns in different organs. Quantitative real-time polymerase chain reaction analysis showed that most CpGRFs had large changes in expression during fruit development and ripening. This indicated that the CpGRFs were involved in regulating fruit development and ripening. Significant expression changes occurred after cold, salt, and drought treatments in papaya seedlings, indicating that CpGRFs were also involved in signaling responses to abiotic stress. These results provide a transcription profile of 14-3-3 genes in organs, during fruit development and ripening and in response to stress. Some highly expressed, fruit-specific, and stress-responsive candidate CpGRFs will be identified for further genetic improvement of papayas.

Genome-wide discovery and functional prediction of salt-responsive lncRNAs in duckweed.

BACKGROUND: Salt significantly depresses the growth and development of the greater duckweed, Spirodela polyrhiza, a model species of floating aquatic plants. Physiological responses of this plant to salt stress have been characterized, however, the roles of long noncoding RNAs (lncRNAs) remain unknown. RESULTS: In this work, totally 2815 novel lncRNAs were discovered in S. polyrhiza by strand-specific RNA sequencing, of which 185 (6.6%) were expressed differentially under salinity condition. Co-expression analysis indicated that the trans-acting lncRNAs regulated their co-expressed genes functioning in amino acid metabolism, cell- and cell wall-related metabolism, hormone metabolism, photosynthesis, RNA transcription, secondary metabolism, and transport. In total, 42 lncRNA-mRNA pairs that might participate in cis-acting regulation were found, and these adjacent genes were involved in cell wall, cell cycle, carbon metabolism, ROS regulation, hormone metabolism, and transcription factor. In addition, the lncRNAs probably functioning as miRNA targets were also investigated. Specifically, TCONS_00033722, TCONS_00044328, and TCONS_00059333 were targeted by a few well-studied salt-responsive miRNAs, supporting the involvement of miRNA and lncRNA interactions in the regulation of salt stress responses. Finally, a representative network of lncRNA-miRNA-mRNA was proposed and discussed to participate in duckweed salt stress via auxin signaling. CONCLUSIONS: This study is the first report on salt-responsive lncRNAs in duckweed, and the findings will provide a solid foundation for in-depth functional characterization of duckweed lncRNAs in response to salt stress.

Highly dynamic, coordinated, and stage-specific profiles are revealed by a multi-omics integrative analysis during tuberous root development in cassava.

Cassava (Manihot esculenta) is an important starchy root crop that provides food for millions of people worldwide, but little is known about the regulation of the development of its tuberous root at the multi-omics level. In this study, the transcriptome, proteome, and metabolome were examined in parallel at seven time-points during the development of the tuberous root from the early to late stages of its growth. Overall, highly dynamic and stage-specific changes in the expression of genes/proteins were observed during development. Cell wall and auxin genes, which were regulated exclusively at the transcriptomic level, mainly functioned during the early stages. Starch biosynthesis, which was controlled at both the transcriptomic and proteomic levels, was mainly activated in the early stages and was greatly restricted during the late stages. Two main branches of lignin biosynthesis, coniferyl alcohol and sinapyl alcohol, also functioned during the early stages of development at both the transcriptomic and proteomic levels. Metabolomic analysis further supported the stage-specific roles of particular genes/proteins. Metabolites related to lignin and flavonoid biosynthesis showed high abundance during the early stages, those related to lipids exhibited high abundance at both the early and middle stages, while those related to amino acids were highly accumulated during the late stages. Our findings provide a comprehensive resource for broadening our understanding of tuberous root development and will facilitate future genetic improvement of cassava.

Genomic analyses of heat stress transcription factors (HSFs) in simulated drought stress response and storage root deterioration after harvest in cassava.

Heat shock factors (HSFs) play crucial roles in various plant stress responses. However, the current knowledge about HSFs in cassava, an important crop, is still insufficient. In this research, we identified 32 cassava HSF genes (MeHSFs) and clustered them into three groups (A, B, C) based on phylogenetic analysis and structural characteristics. Conserved motif analyses showed that MeHSFs display domains characteristic to HSF transcription factors. Gene structure analyses suggested that 29 MeHSFs contained only two exons. All identified 32 cassava MeHSFs were distributed on 13 chromosomes. Their expression profiles revealed that the different MeHSFs were expressed differentially in different tissues, most high expression genes belonged to group A. The similar MeHSFs were up-regulated after treatment with both PEG and abscisic acid (ABA), which implied that these MeHSFs may participate in resistance to simulated drought stress associated with the ABA signaling pathway. In addition, several MeHSFs were induced during postharvest physiological deterioration (PPD) in cassava. Our results provided basic but important knowledge for future gene function analysis of MeHSFs toward efforts in improving tolerance to abiotic stress and PPD in cassava.

De novo assembly, transcriptome characterization, and simple sequence repeat marker development in duckweed Lemna gibba.

Lemna gibba is a species of duckweed showing great potential in bioenergy production and wastewater treatment. However, the relevant transcriptomic and genomic resources are very limited for this species, which dramatically hinders its genetic diversity and genome mapping researches. In this work, ~ 233.5 million clean reads were generated from L. gibba by Illumina paired-end sequencing, and subsequently they were de novo assembled into 131,870 unigenes, of which 61,622 were annotated and 43,319 were expressed with Fragments Per Kilobase of transcript per Million fragments mapped (FPKM) > 5. In total, 19,297 simple sequence repeats (SSRs) were identified from 15,261 SSR-containing unigenes. Dinucleotide (78.4%) were the most abundant SSRs, followed by tri- (14.9%), tetra- (4.1%), and penta-nucleotides (1.5%). The top three motifs were AG/CT (69.9%), AC/GT (6.5%), and ATC/ATG (4.9%). Further analysis revealed that the presence of SSR motif was independent of the expression level for a given gene. Based on the sequence of these SSR-containing unigenes, a total of 10,292 SSR markers were developed, of which only 2671 were further retained after removing those derived from unannotated or extra-low expressed (e.g., FPKM ≤ 5) unigenes. Finally, a subset of 70 SSR markers was randomly selected and examined in nine diverse L. gibba genotypes for the PCR amplification and polymorphism, as well as in other duckweed species for the inter-specifically amplifiability. This work is the first report on the transcriptome-based large-scale SSR markers development and analysis in L. gibba. The transcriptome generated and the SSR markers developed in this work will provide a valuable resource for genetic diversity assessment in L. gibba and also for species relationship investigation in Lemnaceae family.

Strand-specific RNA-seq based identification and functional prediction of drought-responsive lncRNAs in cassava.

BACKGROUND: Long noncoding RNAs (lncRNAs) have emerged as playing crucial roles in abiotic stress responsive regulation, however, the mechanism of lncRNAs underlying drought-tolerance remains largely unknown in cassava, an important tropical and sub-tropical root crop of remarkable drought tolerance. RESULTS: In this study, a total of 833 high-confidence lncRNAs, including 652 intergenic and 181 anti-sense lncRNAs, were identified in cassava leaves and root using strand-specific RNA-seq technology, of which 124 were drought-responsive. Trans-regulatory co-expression network revealed that lncRNAs exhibited tissue-specific expression patterns and they preferred to function differently in distinct tissues: e.g., cell-related metabolism, cell wall, and RNA regulation of transcription in folded leaf (FL); degradation of major carbohydrate (CHO) metabolism, calvin cycle and light reaction, light signaling, and tetrapyrrole synthesis in full expanded leaf (FEL); synthesis of major CHO metabolism, nitrogen-metabolism, photosynthesis, and redox in bottom leaf (BL); and hormone metabolism, secondary metabolism, calcium signaling, and abiotic stress in root (RT). In addition, 27 lncRNA-mRNA pairs referred to cis-acting regulation were identified, and these lncRNAs regulated the expression of their neighboring genes mainly through hormone metabolism, RNA regulation of transcription, and signaling of receptor kinase. Besides, 11 lncRNAs were identified acting as putative target mimics of known miRNAs in cassava. Finally, five drought-responsive lncRNAs and 13 co-expressed genes involved in trans-acting, cis-acting, or target mimic regulation were selected and confirmed by qRT-PCR. CONCLUSIONS: These findings provide a comprehensive view of cassava lncRNAs in response to drought stress, which will enable in-depth functional analysis in the future.

Transcriptomic Analysis of Leaf Sheath Maturation in Maize.

The morphological development of the leaf greatly influences plant architecture and crop yields. The maize leaf is composed of a leaf blade, ligule and sheath. Although extensive transcriptional profiling of the tissues along the longitudinal axis of the developing maize leaf blade has been conducted, little is known about the transcriptional dynamics in sheath tissues, which play important roles in supporting the leaf blade. Using a comprehensive transcriptome dataset, we demonstrated that the leaf sheath transcriptome dynamically changes during maturation, with the construction of basic cellular structures at the earliest stages of sheath maturation with a transition to cell wall biosynthesis and modifications. The transcriptome again changes with photosynthesis and lignin biosynthesis at the last stage of sheath tissue maturation. The different tissues of the maize leaf are highly specialized in their biological functions and we identified 15 genes expressed at significantly higher levels in the leaf sheath compared with their expression in the leaf blade, including the BOP2 homologs GRMZM2G026556 and GRMZM2G022606, DOGT1 (GRMZM2G403740) and transcription factors from the B3 domain, C2H2 zinc finger and homeobox gene families, implicating these genes in sheath maturation and organ specialization.

The Class III Peroxidase (POD) Gene Family in Cassava: Identification, Phylogeny, Duplication, and Expression.

The class III peroxidase (POD) enzymes participate in plant development, hormone signaling, and stress responses. However, little is known about the POD family in cassava. Here, we identified 91 cassava POD genes (MePODs) and classified them into six subgroups using phylogenetic analysis. Conserved motif analysis demonstrated that all MePOD proteins have typical peroxidase domains, and gene structure analysis showed that MePOD genes have between one and nine exons. Duplication pattern analysis suggests that tandem duplication has played a role in MePOD gene expansion. Comprehensive transcriptomic analysis revealed that MePOD genes in cassava are involved in the drought response and postharvest physiological deterioration. Several MePODs underwent transcriptional changes after various stresses and related signaling treatments were applied. In sum, we characterized the POD family in cassava and uncovered the transcriptional control of POD genes in response to various stresses and postharvest physiological deterioration conditions. These results can be used to identify potential target genes for improving the stress tolerance of cassava crops.

The Late Embryogenesis Abundant Protein Family in Cassava (Manihot esculenta Crantz): Genome-Wide Characterization and Expression during Abiotic Stress.

Late embryogenesis abundant (LEA) proteins, as a highly diverse group of polypeptides, play an important role in plant adaptation to abiotic stress; however, LEAs from cassava have not been studied in cassava. In this study, 26 LEA members were genome-wide identified from cassava, which were clustered into seven subfamily according to evolutionary relationship, protein motif, and gene structure analyses. Chromosomal location and duplication event analyses suggested that 26 MeLEAs distributed in 10 chromosomes and 11 MeLEA paralogues were subjected to purifying selection. Transcriptomic analysis showed the expression profiles of MeLEAs in different tissues of stem, leaves, and storage roots of three accessions. Comparative transcriptomic analysis revealed that the function of MeLEAs in response to drought may be differentiated in different accessions. Compared with the wild subspecies W14, more MeLEA genes were activated in cultivated varieties Arg7 and SC124 after drought treatment. Several MeLEA genes showed induction under various stresses and related signaling treatments. Taken together, this study demonstrates the transcriptional control of MeLEAs in tissue development and the responses to abiotic stress in cassava and identifies candidate genes for improving crop resistance to abiotic stress.

Maize network analysis revealed gene modules involved in development, nutrients utilization, metabolism, and stress response.

BACKGROUND: The advent of big data in biology offers opportunities while poses challenges to derive biological insights. For maize, a large amount of publicly available transcriptome datasets have been generated but a comprehensive analysis is lacking. RESULTS: We constructed a maize gene co-expression network based on the graphical Gaussian model, using massive RNA-seq data. The network, containing 20,269 genes, assembles into 964 gene modules that function in a variety of plant processes, such as cell organization, the development of inflorescences, ligules and kernels, the uptake and utilization of nutrients (e.g. nitrogen and phosphate), the metabolism of benzoxazionids, oxylipins, flavonoids, and wax, and the response to stresses. Among them, the inflorescences development module is enriched with domestication genes (like ra1, ba1, gt1, tb1, tga1) that control plant architecture and kernel structure, while multiple other modules relate to diverse agronomic traits. Contained within these modules are transcription factors acting as known or potential expression regulators for the genes within the same modules, suggesting them as candidate regulators for related biological processes. A comparison with an established Arabidopsis network revealed conserved gene association patterns for specific modules involved in cell organization, nutrients uptake & utilization, and metabolism. The analysis also identified significant divergences between the two species for modules that orchestrate developmental pathways. CONCLUSIONS: This network sheds light on how gene modules are organized between different species in the context of evolutionary divergence and highlights modules whose structure and gene content can provide important resources for maize gene functional studies with application potential.

The core regulatory network of the abscisic acid pathway in banana: genome-wide identification and expression analyses during development, ripening, and abiotic stress.

BACKGROUND: Abscisic acid (ABA) signaling plays a crucial role in developmental and environmental adaptation processes of plants. However, the PYL-PP2C-SnRK2 families that function as the core components of ABA signaling are not well understood in banana. RESULTS: In the present study, 24 PYL, 87 PP2C, and 11 SnRK2 genes were identified from banana, which was further supported by evolutionary relationships, conserved motif and gene structure analyses. The comprehensive transcriptomic analyses showed that banana PYL-PP2C-SnRK2 genes are involved in tissue development, fruit development and ripening, and response to abiotic stress in two cultivated varieties. Moreover, comparative expression analyses of PYL-PP2C-SnRK2 genes between BaXi Jiao (BX) and Fen Jiao (FJ) revealed that PYL-PP2C-SnRK2-mediated ABA signaling might positively regulate banana fruit ripening and tolerance to cold, salt, and osmotic stresses. Finally, interaction networks and co-expression assays demonstrated that the core components of ABA signaling were more active in FJ than in BX in response to abiotic stress, further supporting the crucial role of the genes in tolerance to abiotic stress in banana. CONCLUSIONS: This study provides new insights into the complicated transcriptional control of PYL-PP2C-SnRK2 genes, improves the understanding of PYL-PP2C-SnRK2-mediated ABA signaling in the regulation of fruit development, ripening, and response to abiotic stress, and identifies some candidate genes for genetic improvement of banana.

The Discrepant and Similar Responses of Genome-Wide Transcriptional Profiles between Drought and Cold Stresses in Cassava.

Background: Cassava, an important tropical crop, has remarkable drought tolerance, but is very sensitive to cold. The growth, development, and root productivity of cassava are all adversely affected under cold and drought. Methods: To profile the transcriptional response to cold and drought stresses, cassava seedlings were respectively subjected to 0, 6, 24, and 48 h of cold stress and 0, 4, 6, and 10 days of drought stress. Their folded leaves, fully extended leaves, and roots were respectively investigated using RNA-seq. Results: Many genes specifically and commonly responsive to cold and drought were revealed: genes related to basic cellular metabolism, tetrapyrrole synthesis, and brassinosteroid metabolism exclusively responded to cold; genes related to abiotic stress and ethylene metabolism exclusively responded to drought; and genes related to cell wall, photosynthesis, and carbohydrate metabolism, DNA synthesis/chromatic structure, abscisic acid and salicylic acid metabolism, and calcium signaling commonly responded to both cold and drought. Discussion: Combined with cold- and/or drought-responsive transcription factors, the regulatory networks responding to cold and drought in cassava were constructed. All these findings will improve our understanding of the specific and common responses to cold and drought in cassava, and shed light on genetic improvement of cold and drought tolerance in cassava.

Physiological Investigation and Transcriptome Analysis of Polyethylene Glycol (PEG)-Induced Dehydration Stress in Cassava.

Cassava is an important tropical and sub-tropical root crop that is adapted to drought environment. However, severe drought stress significantly influences biomass accumulation and starchy root production. The mechanism underlying drought-tolerance remains obscure in cassava. In this study, changes of physiological characters and gene transcriptome profiles were investigated under dehydration stress simulated by polyethylene glycol (PEG) treatments. Five traits, including peroxidase (POD) activity, proline content, malondialdehyde (MDA), soluble sugar and soluble protein, were all dramatically induced in response to PEG treatment. RNA-seq analysis revealed a gradient decrease of differentially expressed (DE) gene number in tissues from bottom to top of a plant, suggesting that cassava root has a quicker response and more induced/depressed DE genes than leaves in response to drought. Overall, dynamic changes of gene expression profiles in cassava root and leaves were uncovered: genes related to glycolysis, abscisic acid and ethylene biosynthesis, lipid metabolism, protein degradation, and second metabolism of flavonoids were significantly induced, while genes associated with cell cycle/organization, cell wall synthesis and degradation, DNA synthesis and chromatin structure, protein synthesis, light reaction of photosynthesis, gibberelin pathways and abiotic stress were greatly depressed. Finally, novel pathways in ABA-dependent and ABA-independent regulatory networks underlying PEG-induced dehydration response in cassava were detected, and the RNA-Seq results of a subset of fifteen genes were confirmed by real-time PCR. The findings will improve our understanding of the mechanism related to dehydration stress-tolerance in cassava and will provide useful candidate genes for breeding of cassava varieties better adapted to drought environment.

Genome-Wide Identification and Expression Analyses of Aquaporin Gene Family during Development and Abiotic Stress in Banana.

Aquaporins (AQPs) function to selectively control the flow of water and other small molecules through biological membranes, playing crucial roles in various biological processes. However, little information is available on the AQP gene family in bananas. In this study, we identified 47 banana AQP genes based on the banana genome sequence. Evolutionary analysis of AQPs from banana, Arabidopsis, poplar, and rice indicated that banana AQPs (MaAQPs) were clustered into four subfamilies. Conserved motif analysis showed that all banana AQPs contained the typical AQP-like or major intrinsic protein (MIP) domain. Gene structure analysis suggested the majority of MaAQPs had two to four introns with a highly specific number and length for each subfamily. Expression analysis of MaAQP genes during fruit development and postharvest ripening showed that some MaAQP genes exhibited high expression levels during these stages, indicating the involvement of MaAQP genes in banana fruit development and ripening. Additionally, some MaAQP genes showed strong induction after stress treatment and therefore, may represent potential candidates for improving banana resistance to abiotic stress. Taken together, this study identified some excellent tissue-specific, fruit development- and ripening-dependent, and abiotic stress-responsive candidate MaAQP genes, which could lay a solid foundation for genetic improvement of banana cultivars.

Extensive post-transcriptional regulation revealed by integrative transcriptome and proteome analyses in salicylic acid-induced flowering in duckweed (Lemna gibba).

Duckweed is an aquatic model plant with tremendous potential in industrial and agricultural applications. Duckweed rarely flowers which significantly hinders the resource collection and heterosis utilization. Salicylic acid (SA) can significantly induce duckweed to flower; however, the underlying regulatory mechanisms remain largely unknown. In this work, transcriptome and proteome were conducted in parallel to examine the expression change of genes and proteins in Lemna gibba under SA treatment. A high-quality reference transcriptome was generated using Iso-Seq strategy, yielding 42,281 full-length transcripts. A total of 422, 423, and 417 differentially expressed genes (DEGs), as well as 213, 51, and 92 differentially expressed proteins (DEPs), were identified at flower induction, flower initiation, and flowering stages by ssRNA-seq and iTRAQ methods. Most DEGs and DEPs were only regulated at either the transcriptomic or proteomic level. Additionally, DEPs exhibited low expression correlations with the corresponding mRNAs, suggesting that post-transcriptional regulation plays a pivotal role in SA-induced flowering in L. gibba. Specifically, the genes related to photosynthesis, stress, and hormone metabolism were mainly regulated at the mRNA level, those associated with mitochondrial electron transport / ATP synthesis, nucleotide synthesis, and secondary metabolism were regulated at the protein level, while those related to redox metabolism were regulated at the mRNA and/or protein levels. The post-transcriptional regulation of genes relevant to hormone synthesis, transcription factors, and flowering was also extensively analyzed and discussed. This is the first study of integrative transcriptomic and proteomic analyses in duckweed, providing novel insights of post-transcriptional regulation in SA-induced flowering of L. gibba.

The regulation mechanism of ethephon-mediated delaying of postharvest physiological deterioration in cassava storage roots based on quantitative acetylproteomes analysis.

Ethylene plays diverse roles in post-harvest processes of horticultural crops. However, its impact and regulation mechanism on the postharvest physiological deterioration (PPD) of cassava storage roots is unknown. In this study, a notable delay in PPD of cassava storage roots was observed when ethephon was utilized as an ethylene source. Physiological analyses and quantitative acetylproteomes were employed to investigate the regulation mechanism regulating cassava PPD under ethephon treatment. Ethephon was found to enhance the reactive oxygen species (ROS) scavenging system, resulting in a significant decrease in H2O2 and malondialdehyde (MDA) content. The comprehensive acetylome analysis identified 12,095 acetylation sites on 4403 proteins. Subsequent analysis demonstrated that ethephon can regulate the acetylation levels of antioxidant enzymes and members of the energy metabolism pathways. In summary, ethephon could enhance the antioxidant properties and regulate energy metabolism pathways, leading to the delayed PPD of cassava.

MaDREB1F confers cold and drought stress resistance through common regulation of hormone synthesis and protectant metabolite contents in banana.

Adverse environmental factors severely affect crop productivity. Improving crop resistance to multiple stressors is an important breeding goal. Although CBFs/DREB1s extensively participate in plant resistance to abiotic stress, the common mechanism underlying CBFs/DREB1s that mediate resistance to multiple stressors remains unclear. Here, we show the common mechanism for MaDREB1F conferring cold and drought stress resistance in banana. MaDREB1F encodes a dehydration-responsive element binding protein (DREB) transcription factor with nuclear localization and transcriptional activity. MaDREB1F expression is significantly induced after cold, osmotic, and salt treatments. MaDREB1F overexpression increases banana resistance to cold and drought stress by common modulation of the protectant metabolite levels of soluble sugar and proline, activating the antioxidant system, and promoting jasmonate and ethylene syntheses. Transcriptomic analysis shows that MaDREB1F activates or alleviates the repression of jasmonate and ethylene biosynthetic genes under cold and drought conditions. Moreover, MaDREB1F directly activates the promoter activities of MaAOC4 and MaACO20 for jasmonate and ethylene syntheses, respectively, under cold and drought conditions. MaDREB1F also targets the MaERF11 promoter to activate MaACO20 expression for ethylene synthesis under drought stress. Together, our findings offer new insight into the common mechanism underlying CBF/DREB1-mediated cold and drought stress resistance, which has substantial implications for engineering cold- and drought-tolerant crops.

Metabolic GWAS-based dissection of genetic basis underlying nutrient quality variation and domestication of cassava storage root.

BACKGROUND: Metabolites play critical roles in regulating nutritional qualities of plants, thereby influencing their consumption and human health. However, the genetic basis underlying the metabolite-based nutrient quality and domestication of root and tuber crops remain largely unknown. RESULTS: We report a comprehensive study combining metabolic and phenotypic genome-wide association studies to dissect the genetic basis of metabolites in the storage root (SR) of cassava. We quantify 2,980 metabolic features in 299 cultivated cassava accessions. We detect 18,218 significant marker-metabolite associations via metabolic genome-wide association mapping and identify 12 candidate genes responsible for the levels of metabolites that are of potential nutritional importance. Me3GT, MeMYB4, and UGT85K4/UGT85K5, which are involved in flavone, anthocyanin, and cyanogenic glucoside metabolism, respectively, are functionally validated through in vitro enzyme assays and in vivo gene silencing analyses. We identify a cluster of cyanogenic glucoside biosynthesis genes, among which CYP79D1, CYP71E7b, and UGT85K5 are highly co-expressed and their allelic combination contributes to low linamarin content. We find MeMYB4 is responsible for variations in cyanidin 3-O-glucoside and delphinidin 3-O-rutinoside contents, thus controlling SR endothelium color. We find human selection affects quercetin 3-O-glucoside content and SR weight per plant. The candidate gene MeFLS1 is subject to selection during cassava domestication, leading to decreased quercetin 3-O-glucoside content and thus increased SR weight per plant. CONCLUSIONS: These findings reveal the genetic basis of cassava SR metabolome variation, establish a linkage between metabolites and agronomic traits, and offer useful resources for genetically improving the nutrition of cassava and other root crops.

Integrated Metabolomic and Transcriptomic Analyses Reveal Novel Insights of Anthocyanin Biosynthesis on Color Formation in Cassava Tuberous Roots.

Yellow roots are of higher nutritional quality and better appearance than white roots in cassava, a crucial tropical and subtropical root crop. In this work, two varieties with yellow and white cassava roots were selected to explore the mechanisms of color formation by using comparative metabolome and transcriptome analyses during seven developmental stages. Compared with the white-rooted cassava, anthocyanins, catechin derivatives, coumarin derivatives, and phenolic acids accumulated at higher levels in yellow-rooted cassava. Anthocyanins were particularly enriched and displayed different accumulation patterns during tuberous root development. This was confirmed by metabolic comparisons between five yellow-rooted and five white-rooted cassava accessions. The integrative metabolomic and transcriptomic analysis further revealed a coordinate regulation of 16 metabolites and 11 co-expression genes participating in anthocyanin biosynthesis, suggesting a vital role of anthocyanin biosynthesis in yellow pigmentation in cassava tuberous roots. In addition, two transcriptional factors, i.e., MeMYB5 and MeMYB42, were also identified to co-express with these anthocyanin biosynthesis genes. These findings expand our knowledge on the role of anthocyanin biosynthesis in cassava root color formation, and offer useful information for the genetic breeding of yellow-rooted cassava in the future.