Genome-wide analysis of the metallothionein gene family in cassava reveals its role in response to physiological stress through the regulation of reactive oxygen species.

BACKGROUND: Cassava (Manihot esculenta Crantz) is widely planted in tropical and several subtropical regions in which drought, high temperatures, and other abiotic stresses occur. Metallothionein (MT) is a group of conjugated proteins with small molecular weight and rich in cysteine. These proteins play a substantial role in response to physiological stress through the regulation of reactive oxygen species (ROS). However, the biological functions of MT genes in cassava are unknown. RESULTS: A total of 10 MeMT genes were identified in the cassava genome. The MeMTs were divided into 3 groups (Types 2-4) based on the contents and distribution of Cys residues. The MeMTs exhibited tissue-specific expression and located on 7 chromosomes. The MeMT promoters contain some hormones regulatory and stresses responsiveness elements. MeMTs were upregulated under hydrogen peroxide (H2O2) treatment and in respond to post-harvest physiological deterioration (PPD). The results were consistent with defense-responsive cis-acting elements in the MeMT promoters. Further, four of MeMTs were selected and silenced by using the virus-induced gene silencing (VIGS) method to evaluate their functional characterization. The results of gene-silenced cassava suggest that MeMTs are involved in oxidative stress resistance, as ROS scavengers. CONCLUSION: We identified the 10 MeMT genes, and explore their evolutionary relationship, conserved motif, and tissue-specific expression. The expression profiles of MeMTs under three kinds of abiotic stresses (wounding, low-temperature, and H2O2) and during PPD were analyzed. The tissue-specific expression and the response to abiotic stresses revealed the role of MT in plant growth and development. Furthermore, silenced expression of MeMTs in cassava leaves decreased its tolerance to ROS, consistent with its predicted role as ROS scavengers. In summary, our results suggest an important role of MeMTs in response to physiological stress as well as species adaptation via the regulation of ROS homeostasis.

Magnesium chelatase subunit D is not only required for chlorophyll biosynthesis and photosynthesis, but also affecting starch accumulation in Manihot esculenta Crantz.

BACKGROUND: Magnesium chelatase plays an important role in photosynthesis, but only a few subunits have been functionally characterized in cassava. RESULTS: Herein, MeChlD was successfully cloned and characterized. MeChlD encodes a magnesium chelatase subunit D, which has ATPase and vWA conservative domains. MeChlD was highly expressed in the leaves. Subcellular localization suggested that MeChlD:GFP was a chloroplast-localized protein. Furthermore, the yeast two-hybrid system and BiFC analysis indicated that MeChlD interacts with MeChlM and MePrxQ, respectively. VIGS-induce silencing of MeChlD resulted in significantly decreased chlorophyll content and reduction the expression of photosynthesis-related nuclear genes. Furthermore, the storage root numbers, fresh weight and the total starch content in cassava storage roots of VIGS-MeChlD plants was significantly reduced. CONCLUSION: Taken together, MeChlD located at the chloroplast is not only required for chlorophyll biosynthesis and photosynthesis, but also affecting the starch accumulation in cassava. This study expands our understanding of the biological functions of ChlD proteins.

Integrated Metabolomic and Transcriptomic Analyses Reveals Sugar Transport and Starch Accumulation in Two Specific Germplasms of Manihot esculenta Crantz.

As a starchy and edible tropical plant, cassava (Manihot esculenta Crantz) has been widely used as an industrial raw material and a dietary source. However, the metabolomic and genetic differences in specific germplasms of cassava storage root were unclear. In this study, two specific germplasms, M. esculenta Crantz cv. sugar cassava GPMS0991L and M. esculenta Crantz cv. pink cassava BRA117315, were used as research materials. Results showed that sugar cassava GPMS0991L was rich in glucose and fructose, whereas pink cassava BRA117315 was rich in starch and sucrose. Metabolomic and transcriptomic analysis indicated that sucrose and starch metabolism had significantly changing metabolites enrichment and the highest degree of differential expression genes, respectively. Sugar transport in storage roots may contribute to the activities of sugar, which will eventually be exported to transporters (SWEETs), such as (MeSWEET1a, MeSWEET2b, MeSWEET4, MeSWEET5, MeSWEET10b, and MeSWEET17c), which transport hexose to plant cells. The expression level of genes involved in starch biosynthesis and metabolism were altered, which may result in starch accumulation. These results provide a theoretical basis for sugar transport and starch accumulation and may be useful in improving the quality of tuberous crops and increasing yield.

Cassava breeding and agronomy in Asia: 50 years of history and future directions.

In Asia, cassava (Manihot esculenta) is cultivated by more than 8 million farmers, driving the rural economy of many countries. The International Center for Tropical Agriculture (CIAT), in partnership with national agricultural research institutes (NARIs), instigated breeding and agronomic research in Asia, 1983. The breeding program has successfully released high-yielding cultivars resulting in an average yield increase from 13.0 t ha-1 in 1996 to 21.3 t ha-1 in 2016, with significant economic benefits. Following the success in increasing yields, cassava breeding has turned its focus to higher-value traits, such as waxy cassava, to reach new market niches. More recently, building resistance to invasive pests and diseases has become a top priority due to the emergent threat of cassava mosaic disease (CMD). The agronomic research involves driving profitability with advanced technologies focusing on better agronomic management practices thereby maintaining sustainable production systems. Remote sensing technologies are being tested for trait discovery and large-scale field evaluation of cassava. In summary, cassava breeding in Asia is driven by a combination of food and market demand with technological innovations to increase the productivity. Further, exploration in the potential of data-driven agriculture is needed to empower researchers and producers for sustainable advancement.

Comparative physiology and transcriptome analysis allows for identification of lncRNAs imparting tolerance to drought stress in autotetraploid cassava.

BACKGROUND: Polyploidization, pervasive among higher plant species, enhances adaptation to water deficit, but the physiological and molecular advantages need to be investigated widely. Long non-coding RNAs (lncRNAs) are involved in drought tolerance in various crops. RESULTS: Herein, we demonstrate that tetraploidy potentiates tolerance to drought stress in cassava (Manihot esculenta Crantz). Autotetraploidy reduces transpiration by lesser extent increasing of stomatal density, smaller stomatal aperture size, or greater stomatal closure, and reducing accumulation of H2O2 under drought stress. Transcriptome analysis of autotetraploid samples revealed down-regulation of genes involved in photosynthesis under drought stress, and less down-regulation of subtilisin-like proteases involved in increasing stomatal density. UDP-glucosyltransferases were increased more or reduced less in dehydrated leaves of autotetraploids compared with controls. Strand-specific RNA-seq data (validated by quantitative real time PCR) identified 2372 lncRNAs, and 86 autotetraploid-specific lncRNAs were differentially expressed in stressed leaves. The co-expressed network analysis indicated that LNC_001148 and LNC_000160 in autotetraploid dehydrated leaves regulated six genes encoding subtilisin-like protease above mentioned, thereby result in increasing the stomatal density to a lesser extent in autotetraploid cassava. Trans-regulatory network analysis suggested that autotetraploid-specific differentially expressed lncRNAs were associated with galactose metabolism, pentose phosphate pathway and brassinosteroid biosynthesis, etc. CONCLUSION: Tetraploidy potentiates tolerance to drought stress in cassava, and LNC_001148 and LNC_000160 mediate drought tolerance by regulating stomatal density in autotetraploid cassava.

Induction of apomixis by dimethyl sulfoxide (DMSO) and genetic identification of apomictic plants in cassava.

Apomixis, or asexual seed formation, is of great value for plant breeding and seed production, and is desirable in modern agriculture, but natural apomixis occurs in cassava at very low frequency. In present study, apomixis was induced by the treatments of female flower buds with 1%, 1.5% and 2% (v/v) dimethyl sulfoxide (DMSO) and the results showed that 1.5% DMSO treatment was most effective for the induction of apomictic seed formation in cassava cultivar SC5 with the highest percentages of fruit set and true apomictic seeds. The germinated seedlings resembled their parents and displayed no morphological characteristics of cassava polyploid. Flow cytometry and chromosome counting showed that these plants were uniform diploids. Analysis of 34 DMSO-induced cassava progenies by the expressed sequence tag-simple sequence repeat (EST-SSR) and sequence-related amplified polymorphism (SRAP) markers showed that three true apomictic seeds were obtained from the group of SC5 treated with 1.5% DMSO.

Genome-Wide Identification, Expression, and Functional Analysis of the Sugar Transporter Gene Family in Cassava (Manihot esculenta).

The sugar transporter (STP) gene family encodes monosaccharide transporters that contain 12 transmembrane domains and belong to the major facilitator superfamily. STP genes play critical roles in monosaccharide distribution and participate in diverse plant metabolic processes. To investigate the potential roles of STPs in cassava (Manihot esculenta) tuber root growth, genome-wide identification and expression and functional analyses of the STP gene family were performed in this study. A total of 20 MeSTP genes (MeSTP1-20) containing the Sugar_tr conserved motifs were identified from the cassava genome, which could be further classified into four distinct groups in the phylogenetic tree. The expression profiles of the MeSTP genes explored using RNA-seq data showed that most of the MeSTP genes exhibited tissue-specific expression, and 15 out of 20 MeSTP genes were mainly expressed in the early storage root of cassava. qRT-PCR analysis further confirmed that most of the MeSTPs displayed higher expression in roots after 30 and 40 days of growth, suggesting that these genes may be involved in the early growth of tuber roots. Although all the MeSTP proteins exhibited plasma membrane localization, variations in monosaccharide transport activity were found through a complementation analysis in a yeast (Saccharomyces cerevisiae) mutant, defective in monosaccharide uptake. Among them, MeSTP2, MeSTP15, and MeSTP19 were able to efficiently complement the uptake of five monosaccharides in the yeast mutant, while MeSTP3 and MeSTP16 only grew on medium containing galactose, suggesting that these two MeSTP proteins are transporters specific for galactose. This study provides significant insights into the potential functions of MeSTPs in early tuber root growth, which possibly involves the regulation of monosaccharide distribution.

Cassava postharvest physiological deterioration: a complex phenomenon involving calcium signaling, reactive oxygen species and programmed cell death.

Postharvest physiological deterioration (PPD) of cassava (Manihot esculenta) storage roots is a complex physiological and biochemical process which involve many regulatory networks linked with specific proteins modulation and signaling transduction pathways. However, it is poorly understood regarding biological regulation, and the interactions among protein groups and signals to determine PPD syndrome in cassava storage roots. This review sheds some light on the possible molecular mechanisms involved in reactive oxygen species (ROS), calcium signaling transduction, and programmed cell death (PCD) in cassava PPD syndrome. A model for predicting crosstalk among calcium signaling, ROS and PCD is suggested to fine-tune PPD syndrome. This would clues to cassava molecular breeding to alleviate the PPD effects on the shelf-life.

Natural variation in expression of genes associated with carotenoid biosynthesis and accumulation in cassava (Manihot esculenta Crantz) storage root.

BACKGROUND: Cassava (Manihot esculenta Crantz) storage root provides a staple food source for millions of people worldwide. Increasing the carotenoid content in storage root of cassava could provide improved nutritional and health benefits. Because carotenoid accumulation has been associated with storage root color, this study characterized carotenoid profiles, and abundance of key transcripts associated with carotenoid biosynthesis, from 23 landraces of cassava storage root ranging in color from white-to-yellow-to-pink. This study provides important information to plant breeding programs aimed at improving cassava storage root nutritional quality. RESULTS: Among the 23 landraces, five carotenoid types were detected in storage root with white color, while carotenoid types ranged from 1 to 21 in storage root with pink and yellow color. The majority of storage root in these landraces ranged in color from pale-to-intense yellow. In this color group, total ß-carotene, containing all-E-, 9-Z-, and 13-Z-ß-carotene isomers, was the major carotenoid type detected, varying from 26.13 to 76.72 %. Although no α-carotene was observed, variable amounts of a α-ring derived xanthophyll, lutein, was detected; with greater accumulation of α-ring xanthophylls than of ß-ring xanthophyll. Lycopene was detected in a landrace (Cas51) with pink color storage root, but it was not detected in storage root with yellow color. Based on microarray and qRT-PCR analyses, abundance of transcripts coding for enzymes involved in carotenoid biosynthesis were consistent with carotenoid composition determined by contrasting HPLC-Diode Array profiles from storage root of landraces IAC12, Cas64, and Cas51. Abundance of transcripts encoding for proteins regulating plastid division were also consistent with the observed differences in total ß-carotene accumulation. CONCLUSIONS: Among the 23 cassava landraces with varying storage root color and diverse carotenoid types and profiles, landrace Cas51 (pink color storage root) had low LYCb transcript abundance, whereas landrace Cas64 (intense yellow storage root) had decreased HYb transcript abundance. These results may explain the increased amounts of lycopene and total ß-carotene observed in landraces Cas51 and Cas64, respectively. Overall, total carotenoid content in cassava storage root of color class representatives were associated with spatial patterns of secondary growth, color, and abundance of transcripts linked to plastid division. Finally, a partial carotenoid biosynthesis pathway is proposed.

The Comparatively Proteomic Analysis in Response to Cold Stress in Cassava Plantlets.

Cassava (Manihot esculenta Crantz) is a tropical root crop and sensitive to low temperature. However, it is poorly to know how cassava can modify its metabolism and growth to adapt to cold stress. An investigation aimed at a better understanding of cold-tolerant mechanism of cassava plantlets was carried out with the approaches of physiology and proteomics in the present study. The principal component analysis of seven physiological characteristics showed that electrolyte leakage (EL), chlorophyll content, and malondialdehyde (MDA) may be the most important physiological indexes for determining cold-resistant abilities of cassava. The genome-wide proteomic analysis showed that 20 differential proteins had the same patterns in the apical expanded leaves of cassava SC8 and Col1046. They were mainly related to photosynthesis, carbon metabolism and energy metabolism, defense, protein synthesis, amino acid metabolism, signal transduction, structure, detoxifying and antioxidant, chaperones, and DNA-binding proteins, in which 40 % were related with photosynthesis. The remarkable variation in photosynthetic activity and expression level of peroxiredoxin is closely linked with expression levels of proteomic profiles. Moreover, analysis of differentially expressed proteins under cold stress is an important step toward further elucidation of mechanisms of cold stress resistance.

Expression Pattern and Functional Analysis of MebHLH149 Gene in Response to Cassava Bacterial Blight.

The significant reduction in cassava (Manihot esculenta Crantz) yields attributed to cassava bacterial blight (CBB) constitutes an urgent matter demanding prompt attention. The current study centered on the MebHLH149 transcription factor, which is acknowledged to be reactive to CBB and exhibits augmented expression levels, as indicated by laboratory transcriptome data. Our exploration, encompassing Xanthomonas phaseoli pv. manihotis strain CHN01 (Xpm CHN01) and hormone stress, disclosed that the MebHLH149 gene interacts with the pathogen at the early stage of infection. Furthermore, the MebHLH149 gene has been discovered to be responsive to the plant hormones abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA), intimating a potential role in the signaling pathways mediated by these hormones. An analysis of the protein's subcellular localization suggested that MebHLH149 is predominantly located within the nucleus. Through virus-induced gene silencing (VIGS) in cassava, we discovered that MebHLH149-silenced plants manifested higher disease susceptibility, less ROS accumulation, and significantly larger leaf spot areas compared to control plants. The proteins MePRE5 and MePRE6, which are predicted to interact with MebHLH149, demonstrated complementary downregulation and upregulation patterns in response to silencing and overexpression of the MebHLH149 gene. This implies a potential interaction between MebHLH149 and these proteins. Both MePRE5 and MePRE6 genes are involved in the initial immune response to CBB. Notably, MebHLH149 was identified as a protein that physically interacts with MePRE5 and MePRE6. Based on these findings, it is hypothesized that the MebHLH149 gene likely functions as a positive regulator in the defense mechanisms of cassava against CBB.

The Potential of Co-Fermentation of Whole-Plant Cassava with Piper sarmentosum: A Comprehensive Study of Fermentation Quality, Antioxidant Activity, Bacterial Community Structure, and Microbial Ecological Networks in Novel Foods.

The objective of this study was to explore the preservation of food products through the co-fermentation of whole-plant cassava and Piper sarmentosum (PS) without additives. We assessed fermentation quality, antioxidant activity, bacterial community structure, function profile, and microbial ecological network features. Our results demonstrate that co-fermentation of whole-plant cassava with 10% PS significantly improves food quality. The co-fermented samples exhibited enhanced lactic acid concentrations and increased antioxidant activity, with reduced pH values and concentrations of acetic acid, butyric acid, and ammonia-N(NH3-N) compared to whole-plant cassava fermented alone. In addition, PS addition also optimized microbial community structure by elevating the total abundance of lactic acid bacteria and influenced bacterial predicted functions. Furthermore, our analysis of co-occurrence networks reveals that co-fermentation impacts microbial network features, including module numbers and bacterial relative abundances, leading to altered complexity and stability of the networks. Moreover, out study also highlights the impact of ferment undesirable bacteria like Pseudomonas aeruginosa and unclassified_Muribaculaceae playing crucial roles in microbial network complexity and stability. These findings provide valuable insights into the anaerobic fermentation process and offers strategies for regulating food fermentation quality.

A major QTL identification and candidate gene analysis of watermelon fruit cracking using QTL-seq and RNA-seq.

Fruit cracking decreases the total production and the commercial value of watermelon. The molecular mechanisms of fruit cracking are unknown. In this study, 164 recombinant inbred lines (RILs) of watermelon, derived from the crossing of the WQ1 (cracking-sensitive) and WQ2 (cracking-tolerant) lines, were sequenced using specific length amplified fragment sequencing (SLAF-seq). A high-density genetic linkage map was constructed with 3,335 markers spanning 1,322.74 cM, at an average 0.40 cM across whole-genome flanking markers. The cracking tolerance capacity (CTC), depth of fruit cracking (DFC), rind thickness (RT), and rind hardness (RH) were measured for quantitative trait locus (QTL) analysis. Of the four traits analyzed, one major QTL with high phenotypic variation (41.04%-61.37%) was detected at 76.613-76.919 cM on chromosome 2, which contained 104 annotated genes. Differential gene expression analysis with RNA sequencing (RNA-seq) data between the two parents identified 4,508 differentially expressed genes (DEGs). Comparison of the genes between the QTL region and the DEGs obtained eight coexisting genes. Quantitative real-time PCR (qRT-PCR) analysis revealed that these genes were significant differentially expressed between the two parents. These results provide new insights into the identification of QTLs or genes and marker-assisted breeding in watermelon.

Cassava Foliage Effects on Antioxidant Capacity, Growth, Immunity, and Ruminal Microbial Metabolism in Hainan Black Goats.

Cassava (Manihot esculenta Crantz) foliage is a byproduct of cassava production characterized by high biomass and nutrient content. In this study, we investigated the effects of cassava foliage on antioxidant capacity, growth performance, and immunity status in goats, as well as rumen fermentation and microbial metabolism. Twenty-five Hainan black goats were randomly divided into five groups (n = 5 per group) and accepted five treatments: 0% (T1), 25% (T2), 50% (T3), 75% (T4), and 100% (T5) of the cassava foliage silage replaced king grass, respectively. The feeding experiment lasted for 70 d (including 10 d adaptation period and 60 d treatment period). Feeding a diet containing 50% cassava foliage resulted in beneficial effects for goat growth and health, as reflected by the higher average daily feed intake (ADFI), average daily gain (ADG) and better feed conversion rate (FCR), as well as by the reduced serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), and triglycerides (TG). Meanwhile, cassava foliage improved antioxidant activity by increasing the level of glutathion peroxidase (GSH-Px), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC) and lowering malondialdehyde (MDA). Moreover, feeding cassava foliage was also beneficial to immunity status by enhancing complement 3 (C3), complement 4 (C4), immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM). Furthermore, the addition of dietary cassava foliage also altered rumen fermentation, rumen bacterial community composition, and metabolism. The abundance of Butyrivibrio_2 and Prevotella_1 was elevated, as were the concentrations of beneficial metabolites such as butyric acid; there was a concomitant decline in metabolites that hindered nutrient metabolism and harmed host health. In summary, goats fed a diet containing 50% cassava foliage silage demonstrated a greater abundance of Butyrivibrio_2, which enhanced the production of butyric acid; these changes led to greater antioxidant capacity, growth performance, and immunity in the goats.

Spraying chitosan on cassava roots reduces postharvest deterioration by promoting wound healing and inducing disease resistance.

Postharvest damage makes cassava roots vulnerable to pathogen infections and decay, which significantly hinders the development of the cassava industry. The objective of this study was to assess the antibacterial properties of chitosan in vitro, as well as its effect on wound healing and resistance in cassava roots. The findings demonstrated that the bacteriostatic effect of chitosan became increasingly prominent as the concentration of chitosan enhanced. Chitosan at a concentration of 0.5 mg/mL was revealed to significantly inhibit the germination of P. palmivora spores and damage to their structure. Moreover, chitosan activated the transcription of crucial genes and enzyme activities associated with the phenylpropane metabolism pathway in cassava roots, thus promoting rapid lignin accumulation and resulting in the early formation of a fracture layer. Chitosan was also found to enhance cassava root resistance by promoting the expression of pathogenesis-related proteins and the accumulation of flavonoids and total phenols. After 48 h of inoculation, cassava roots treated with chitosan exhibited a 51.4 % and 53.4 % decrease in lesion area for SC9 and SC6 varieties, respectively. The findings of this study offer a novel approach for managing postharvest deterioration of cassava roots.

Genome-wide identification and characterization of 14-3-3 gene family related to negative regulation of starch accumulation in storage root of Manihot esculenta.

The 14-3-3 protein family is a highly conservative member of the acid protein family and plays an important role in regulating a series of important biological activities and various signal transduction pathways. The role of 14-3-3 proteins in regulating starch accumulation still remains largely unknown. To investigate the properties of 14-3-3 proteins, the structures and functions involved in starch accumulation in storage roots were analyzed, and consequently, 16 Me14-3-3 genes were identified. Phylogenetic analysis revealed that Me14-3-3 family proteins are split into two groups (ε and non-ε). All Me14-3-3 proteins contain nine antiparallel α-helices. Me14-3-3s-GFP fusion protein was targeted exclusively to the nuclei and cytoplasm. In the early stage of starch accumulation in the storage root, Me14-3-3 genes were highly expressed in high-starch cultivars, while in the late stage of starch accumulation, Me14-3-3 genes were highly expressed in low-starch cultivars. Me14-3-3 I, II, V, and XVI had relatively high expression levels in the storage roots. The transgenic evidence from Me14-3-3II overexpression in Arabidopsis thaliana and the virus-induced gene silencing (VIGS) in cassava leaves and storage roots suggest that Me14-3-3II is involved in the negative regulation of starch accumulation. This study provides a new insight to understand the molecular mechanisms of starch accumulation linked with Me14-3-3 genes during cassava storage root development.

Integrative analysis of metabolome and transcriptome reveals the mechanism of color formation in cassava (Manihot esculenta Crantz) leaves.

Cassava (Manihot esculenta Crantz) leaves are often used as vegetables in Africa. Anthocyanins possess antioxidant, anti-inflammatory, anti-cancer, and other biological activities. They are poor in green leaves but rich in the purple leaves of cassava. The mechanism of anthocyanin's accumulation in cassava is poorly understood. In this study, two cassava varieties, SC9 with green leaves and Ziyehuangxin with purple leaves (PL), were selected to perform an integrative analysis using metabolomics and transcriptomics. The metabolomic analysis indicated that the most significantly differential metabolites (SDMs) belong to anthocyanins and are highly accumulated in PL. The transcriptomic analysis revealed that differentially expressed genes (DEGs) are enriched in secondary metabolites biosynthesis. The analysis of the combination of metabolomics and transcriptomics showed that metabolite changes are associated with the gene expressions in the anthocyanin biosynthesis pathway. In addition, some transcription factors (TFs) may be involved in anthocyanin biosynthesis. To further investigate the correlation between anthocyanin accumulation and color formation in cassava leaves, the virus-induced gene silencing (VIGS) system was used. VIGS-MeANR silenced plant showed the altered phenotypes of cassava leaves, partially from green to purple color, resulting in a significant increase of the total anthocyanin content and reduction in the expression of MeANR. These results provide a theoretical basis for breeding cassava varieties with anthocyanin-rich leaves.

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.

The transcription factor MebHLH18 in cassava functions in decreasing low temperature-induced leaf abscission to promote low-temperature tolerance.

The reactive oxygen species (ROS) signal regulates stress-induced leaf abscission in cassava. The relationship between the function of the cassava transcription factor bHLH gene and low temperature-induced leaf abscission is still unclear. Here, we report that MebHLH18, a transcription factor, involved in regulating low temperature-induced leaf abscission in cassava. The expression of the MebHLH18 gene was significantly related to low temperature-induced leaf abscission and POD level. Under low temperatures, the levels of ROS scavengers in different cassava genotypes were significantly different in the low temperature-induced leaf abscission process. Cassava gene transformation showed that MebHLH18 overexpression significantly decreased the low temperature-induced leaf abscission rate. Simultaneously, interference expression increased the rate of leaf abscission under the same conditions. ROS analysis showed a connection between the decrease in the low temperature-induced leaf abscission rate caused by MebHLH18 expression and the increase in antioxidant activity. A Genome-wide association studies analysis showed a relationship between the natural variation of the promoter region of MebHLH18 and low temperature-induced leaf abscission. Furthermore, studies showed that the change in MebHLH18 expression was caused by a single nucleotide polymorphism variation in the promoter region upstream of the gene. The high expression of MebHLH18 led to a significant increase in POD activity. The increased POD activity decreased the accumulation of ROS at low temperatures and the rate of leaf abscission. It indicates that the natural variation in the promoter region of MebHLH18 increases antioxidant levels under low temperatures and slows down low temperature-induced leaf abscission.

Systematic Analysis of bHLH Transcription Factors in Cassava Uncovers Their Roles in Postharvest Physiological Deterioration and Cyanogenic Glycosides Biosynthesis.

The basic helix-loop-helix (bHLH) proteins are a large superfamily of transcription factors, and play a central role in a wide range of metabolic, physiological, and developmental processes in higher organisms. However, systematic investigation of bHLH gene family in cassava (Manihot esculenta Crantz) has not been reported. In the present study, we performed a genome-wide survey and identified 148 MebHLHs genes were unevenly harbored in 18 chromosomes. Through phylogenetic analyses along with Arabidopsis counterparts, these MebHLHs genes were divided into 19 groups, and each gene contains a similar structure and conserved motifs. Moreover, many cis-acting regulatory elements related to various defense and stress responses showed in MebHLH genes. Interestingly, transcriptome data analyses unveiled 117 MebHLH genes during postharvest physiological deterioration (PPD) process of cassava tuberous roots, while 65 MebHLH genes showed significantly change. Meanwhile, the relative quantitative analysis of 15 MebHLH genes demonstrated that they were sensitive to PPD, suggesting they may involve in PPD process regulation. Cyanogenic glucosides (CGs) biosynthesis during PPD process was increased, silencing of MebHLH72 and MebHLH114 showed that linamarin content was significantly decreased in the leaves. To summarize, the genome-wide identification and expression profiling of MebHLH candidates pave a new avenue for uderstanding their function in PPD and CGs biosynthesis, which will accelerate the improvement of PPD tolerance and decrease CGs content in cassava tuberous roots.