Genomic and Expression Analysis of Cassava (Manihot esculenta Crantz) Chalcone Synthase Genes in Defense against Tetranychus cinnabarinus Infestation.

Cassava is susceptible to mites, especially Tetranychus cinnabarinus. Secondary metabolism products such as flavonoids play an important role as antimicrobial metabolites protecting plants against biotic stressors including fungal, pathogen, bacterial, and pest defense. The chalcone synthase (CHS) is the initial step of the phenylpropanoid pathway for producing flavonoids and is the gatekeeper of the pathway. Until recently, the CHS genes family has not been systematically studied in cassava. Thirty-nine CHS genes were identified from the cassava genome database. Based on phylogenetic and sequence composition analysis, these CHSs were divided into 3 subfamilies. Within the same subfamily, the gene structure and motif compositions of these CHS genes were found to be quite conserved. Duplication events, particularly segmental duplication of the cassava CHS genes, were identified as one of the main driving force of its expansion. Various cis-elements contained in the promoter might regulate the gene expression patterns of MeCHS. Protein-protein interaction (PPI) network analysis showed that MeCHS1 and MeCHS10 protein are more closely related to other family members. The expression of MeCHS genes in young leaves was higher than that in other tissues, and their expression varies even within the same tissue. Coincidentally, these CHS genes of most LAP subclasses were highly expressed in young leaves. The verified MeCHS genes showed consistent with the real-time reverse transcription quantitative PCR (RT-qPCR) and proteomic expression in protected and affected leaves respectively, indicating that these MeCHS genes play crucial roles in the response to T. cinnabarinus. This study is the first to comprehensively expatiate the information on MeCHS family members. These data will further enhance our understanding both the molecular mechanisms and the effects of CHS genes. In addition, the results will help to further clarify the effects on T. cinnabarinus and provide a theoretical basis for the potential functions of the specific CHS gene in resistance to mites and other biotic stress.

Multi-trait selection in multi-environments for performance and stability in cassava genotypes.

Genotype-environment interaction (GEI) presents challenges when aiming to select optimal cassava genotypes, often due to biased genetic estimates. Various strategies have been proposed to address the need for simultaneous improvements in multiple traits, while accounting for performance and yield stability. Among these methods are mean performance and stability (MPS) and the multi-trait mean performance and stability index (MTMPS), both utilizing linear mixed models. This study's objective was to assess genetic variation and GEI effects on fresh root yield (FRY), along with three primary and three secondary traits. A comprehensive evaluation of 22 genotypes was conducted using a randomized complete block design with three replicates across 47 distinct environments (year x location) in Brazil. The broad-sense heritability (H2) averaged 0.37 for primary traits and 0.44 for secondary traits, with plot-based heritability (hmÉ¡2) consistently exceeding 0.90 for all traits. The high extent of GEI variance (σÉ¡xe2) demonstrates the GEI effect on the expression of these traits. The dominant analytic factor (FA3) accounted for over 85% of the total variance, and the communality (ɧ) surpassed 87% for all traits. These values collectively suggest a substantial capacity for genetic variance explanation. In Cluster 1, composed of remarkably productive and stable genotypes for primary traits, genotypes BRS Novo Horizonte and BR11-34-69 emerged as prime candidates for FRY enhancement, while BRS Novo Horizonte and BR12-107-002 were indicated for optimizing dry matter content. Moreover, MTMPS, employing a selection intensity of 30%, identified seven genotypes distinguished by heightened stability. This selection encompassed innovative genotypes chosen based on regression variance index (Sdi2, R2, and RMSE) considerations for multiple traits. In essence, incorporating methodologies that account for stability and productive performance can significantly bolster the credibility of recommendations for novel cassava cultivars.

Water deficit and potassium affect carbon isotope composition in cassava bulk leaf material and extracted carbohydrates.

Cassava (Manihot esculenta Crantz) is an important root crop, which despite its drought tolerance suffers considerable yield losses under water deficit. One strategy to increase crop yields under water deficit is improving the crop's transpiration efficiency, which could be achieved by variety selection and potassium application. We assessed carbon isotope composition in bulk leaf material and extracted carbohydrates (soluble sugar, starch, and cellulose) of selected leaves one month after inducing water deficit to estimate transpiration efficiency and storage root biomass under varying conditions in a greenhouse experiment. A local and improved variety were grown in sand, supplied with nutrient solution with two potassium levels (1.44 vs. 0.04 mM K+) and were subjected to water deficit five months after planting. Potassium application and selection of the improved variety both increased transpiration efficiency of the roots with 58% and 85% respectively. Only in the improved variety were 13C ratios affected by potassium application (up to - 1.8‰ in δ13C of soluble sugar) and water deficit (up to + 0.6‰ in δ13C of starch and soluble sugar). These data revealed a shift in substrate away from transitory starch for cellulose synthesis in young leaves of the improved variety under potassium deficit. Bulk δ13C of leaves that had fully developed prior to water deficit were the best proxies for storage root biomass (r = - 0.62, r = - 0.70) and transpiration efficiency (r = - 0.68, r = - 0.58) for the local and improved variety respectively, making laborious extractions redundant. Results obtained from the youngest fully developed leaf, commonly used as a diagnostic leaf, were complicated by remobilized assimilates in the improved variety, making them less suitable for carbon isotope analysis. This study highlights the potential of carbon isotope composition to assess transpiration efficiency and yield, depending on the chosen sampling strategy as well as to unravel carbon allocation processes.

The Development of Thematic Core Collections in Cassava Based on Yield, Disease Resistance, and Root Quality Traits.

Thematic collections (TCs), which are composed of genotypes with superior agronomic traits and reduced size, offer valuable opportunities for parental selection in plant breeding programs. Three TCs were created to focus on crucial attributes: root yield (CC_Yield), pest and disease resistance (CC_Disease), and root quality traits (CC_Root_quality). The genotypes were ranked using the best linear unbiased predictors (BLUP) method, and a truncated selection was implemented for each collection based on specific traits. The TCs exhibited minimal overlap, with each collection comprising 72 genotypes (CC_Disease), 63 genotypes (CC_Root_quality), and 64 genotypes (CC_Yield), representing 4%, 3.5%, and 3.5% of the total individuals in the entire collection, respectively. The Shannon-Weaver Diversity Index values generally varied but remained below 10% when compared to the entire collection. Most TCs exhibited observed heterozygosity, genetic diversity, and the inbreeding coefficient that closely resembled those of the entire collection, effectively retaining 90.76%, 88.10%, and 88.99% of the alleles present in the entire collection (CC_Disease, CC_Root_quality, and CC_Disease, respectively). A PCA of molecular and agro-morphological data revealed well-distributed and dispersed genotypes, while a discriminant analysis of principal components (DAPC) displayed a high discrimination capacity among the accessions within each collection. The strategies employed in this study hold significant potential for advancing crop improvement efforts.

Development of cassava core collections based on morphological and agronomic traits and SNPS markers.

Cassava (Manihot esculenta Crantz) holds significant importance as one of the world's key starchy crop species. This study aimed to develop core collections by utilizing both phenotypic data (15 quantitative and 33 qualitative descriptors) and genotypic data (20,023 single-nucleotide polymorphisms) obtained from 1,486 cassava accessions. Six core collections were derived through two optimization strategies based on genetic distances: Average accession-to-nearest-entry and Average entry-to-nearest-entry, along with combinations of phenotypic and genotypic data. The quality of the core collections was evaluated by assessing genetic parameters such as genetic diversity Shannon-Weaver Index, inbreeding (Fis), observed (Ho), and expected (Hs) heterozygosity. While the selection of accessions varied among the six core collections, a seventh collection (consolidated collection) was developed, comprising accessions selected by at least two core collections. Most collections exhibited genetic parameters similar to the complete collection, except for those developed by the Average accession-to-nearest-entry algorithm. However, the variations in the maximum and minimum values of Ho, Hs, and Fis parameters closely resembled the complete collection. The consolidated collection and the collection constructed using genotypic data and the Average entry-to-nearest-entry algorithm (GenEN) retained the highest number of alleles (>97%). Although the differences were not statistically significant (above 5%), the consolidated collection demonstrated a distribution profile and mean trait values most similar to the complete collection, with a few exceptions. The Shannon-Weaver Index of qualitative traits exhibited variations exceeding ±10% when compared to the complete collection. Principal component analysis revealed that the consolidated collection selected cassava accessions with a more uniform dispersion in all four quadrants compared to the other core collections. These findings highlight the development of optimized and valuable core collections for efficient breeding programs and genomic association studies.

New protocol for rapid cassava multiplication in field conditions: a perspective on speed breeding.

Despite the economic and social importance, high-yielding cassava cultivars are only released after extensive research, mainly due to the low multiplication rate. This study aimed to assess the impact of using smaller-sized seed cuttings treated with agrochemicals (8MP) compared to the conventional planting size (16 cm) on genetic parameters, agronomic performance, and the ranking of cassava clones based on yield and growth attributes. The evaluation was carried out in clonal evaluation trial (CET), preliminary yield trial (PYT), and uniform yield trials (UYT). Additionally, a new selection scheme for cassava breeding programs was proposed. A total of 169 clones were evaluated, including 154 improved clones at different stages of selection and 15 local varieties used as checks. Field trials were conducted using both sizes of propagative material (8MP and 16 cm) in each phase of the breeding program. The data were analyzed using mixed models, considering the random effects of genotype and genotype-environment interaction (G×E) to determine variances and heritabilities. Bland-Altman concordance and correlation analysis of selection indices were employed to examine the consistency in the ranking of cassava clones using different seed cutting sizes. The distribution of variance components, heritabilities, means, and range of the 8MP and 16 cm trials in different phases of the cassava breeding program exhibited remarkable similarity, thereby enabling a comparative assessment of similar genetic effects. With a selection intensity of 30%, the concordance in clone ranking was 0.41, 0.57, and 0.85 in CET, PYT, and UYT trials, respectively, when comparing the selection based on 8MP and 16 cm trials. It is worth noting that the ranking of the top 15% remained largely unchanged. Based on the findings, proposed changes in the cassava selection scheme involve increasing the number of trials starting from the CET phase, early incorporation of G×E interaction, elimination of the PYT trial, reduction of the breeding cycle from 5 to 3 years, and a decrease in the time required for variety development from 11 to 9 years. These modifications are expected to lead to cost reduction and enhance the effectiveness of cassava breeding programs.

Volatile Organic Compounds from Cassava Plants Confer Resistance to the Whitefly Aleurothrixus aepim (Goeldi, 1886).

Cassava is an essential tuber crop used to produce food, feed, and beverages. Whitefly pests, including Aleurothrixus aepim (Goeldi, 1886) (Hemiptera: Aleyrodidae), significantly affect cassava-based agroecosystems. Plant odours have been described as potential pest management tools, and the cassava clone M Ecuador 72 has been used by breeders as an essential source of resistance. In this study, we analysed and compared the volatile compounds released by this resistant clone and a susceptible genotype, BRS Jari. Constitutive odours were collected from young plants and analysed using gas chromatography-mass spectrometry combined with chemometric tools. The resistant genotype released numerous compounds with previously described biological activity and substantial amounts of the monoterpene (E)-ß-ocimene. Whiteflies showed non-preferential behaviour when exposed to volatiles from the resistant genotype but not the susceptible genotype. Furthermore, pure ocimene caused non-preferential behaviour in whiteflies, indicating a role for this compound in repellence. This report provides an example of the intraspecific variation in odour emissions from cassava plants alongside information on odorants that repel whiteflies; these data can be used to devise whitefly management strategies. A better understanding of the genetic variability in cassava odour constituents and emissions under field conditions may accelerate the development of more resistant cassava varieties.

Adaptations in the transformation of cassava (Manihot esculenta Crantz; Euphorbiaceae) for consumption in the dietary management of diabetes: the case of Palikur, or Parikwene People, from French Guiana.

Introduction: In the French overseas department of French Guiana, in South America, nutrition therapy for the management of diabetes is based on French guidelines. However, this region is demographically diverse and includes several populations of Indigenous Peoples, Parikwene among others, also called Palikur. Due to socio-economical, cultural, and geographical differences, along with distinctions in the local food system, dietary recommendations, which many consider in the context of post-colonial power dynamics, are not well suited to local populations. In the absence of suitable recommendations, it is hypothesized that local populations will adapt their dietary practices considering diabetes as an emerging health problem. Methods: Seventy-five interviews were conducted with community members and Elders, as well as healthcare professionals and administrators providing services to the Parikwene population of Macouria and Saint-Georges de l'Oyapock communes. Data regarding the representation of cassava (Manihot esculenta Crantz) consumption and diabetes were collected via semi-structured interviews and participant observation (i.e., observation and participation in community activities), namely via participating in activities related to the transformation of cassava tubers at swidden and fallow fields. Results and Discussion: Parikwene have adapted the transformation of cassava tubers for their consumption in the management of diabetes.The importance of cassava tubers as a staple and core food to the Parikwene food system was established by identifying it as a cultural keystone species. Narratives illustrated conflicting perceptions regarding the implication of cassava consumption in the development of diabetes. Adaptations to the operational sequence involved in the transformation of cassava tubers led to the production of distinct cassava roasted semolina (i.e., couac), based on organoleptic properties (i.e., sweet, and acidic couac). Preferences for the consumption of acidic couac were grounded in the Parikwene knowledge system, as well as attention to diabetes related symptoms and glucometer readings. Conclusion: These results provide important insights related to knowledge, attitudes, and practices in developing locally and culturally adapted approaches to providing dietary recommendations in the treatment of diabetes.

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.

Fully sequencing the cassava full-length cDNA library reveals unannotated transcript structures and alternative splicing events in regions with a high density of single nucleotide variations, insertions-deletions, and heterozygous sequences.

The primary transcript structure provides critical insights into protein diversity, transcriptional modification, and functions. Cassava transcript structures are highly diverse because of alternative splicing (AS) events and high heterozygosity. To precisely determine and characterize transcript structures, fully sequencing cloned transcripts is the most reliable method. However, cassava annotations were mainly determined according to fragmentation-based sequencing analyses (e.g., EST and short-read RNA-seq). In this study, we sequenced the cassava full-length cDNA library, which included rare transcripts. We obtained 8,628 non-redundant fully sequenced transcripts and detected 615 unannotated AS events and 421 unannotated loci. The different protein sequences resulting from the unannotated AS events tended to have diverse functional domains, implying that unannotated AS contributes to the truncation of functional domains. The unannotated loci tended to be derived from orphan genes, implying that the loci may be associated with cassava-specific traits. Unexpectedly, individual cassava transcripts were more likely to have multiple AS events than Arabidopsis transcripts, suggestive of the regulated interactions between cassava splicing-related complexes. We also observed that the unannotated loci and/or AS events were commonly in regions with abundant single nucleotide variations, insertions-deletions, and heterozygous sequences. These findings reflect the utility of completely sequenced FLcDNA clones for overcoming cassava-specific annotation-related problems to elucidate transcript structures. Our work provides researchers with transcript structural details that are useful for annotating highly diverse and unique transcripts and alternative splicing events.

Cassava pullulanase and its synergistic debranching action with isoamylase 3 in starch catabolism.

Pullulanase (EC 3.2.1.41, PUL), a debranching enzyme belonging to glycoside hydrolase family 13 subfamily 13, catalyses the cleavage of α-1,6 linkages of pullulan and ß-limit dextrin. The present work studied PUL from cassava Manihot esculenta Crantz (MePUL) tubers, an important economic crop. The Mepul gene was successfully cloned and expressed in E. coli and rMePUL was biochemically characterised. MePUL was present as monomer and homodimer, as judged by apparent mass of ~ 84 - 197 kDa by gel permeation chromatography analysis. Optimal pH and temperature were at pH 6.0 and 50 °C, and enzyme activity was enhanced by the addition of Ca2+ ions. Pullulan is the most favourable substrate for rMePUL, followed by ß-limit dextrin. Additionally, maltooligosaccharides were potential allosteric modulators of rMePUL. Interestingly, short-chain maltooligosaccharides (DP 2 - 4) were significantly revealed at a higher level when rMePUL was mixed with cassava isoamylase 3 (rMeISA3), compared to that of each single enzyme reaction. This suggests that MePUL and MeISA3 debranch ß-limit dextrin in a synergistic manner, which represents a major starch catabolising process in dicots. Additionally, subcellular localisation suggested the involvement of MePUL in starch catabolism, which normally takes place in plastids.

Near-infrared spectroscopy for early selection of waxy cassava clones via seed analysis.

Cassava (Manihot esculenta Crantz) starch consists of amylopectin and amylose, with its properties determined by the proportion of these two polymers. Waxy starches contain at least 95% amylopectin. In the food industry, waxy starches are advantageous, with pastes that are more stable towards retrogradation, while high-amylose starches are used as resistant starches. This study aimed to associate near-infrared spectrophotometry (NIRS) spectra with the waxy phenotype in cassava seeds and develop an accurate classification model for indirect selection of plants. A total of 1127 F2 seeds were obtained from controlled crosses performed between 77 F1 genotypes (wild-type, Wx_). Seeds were individually identified, and spectral data were obtained via NIRS using a benchtop NIRFlex N-500 and a portable SCiO device spectrometer. Four classification models were assessed for waxy cassava genotype identification: k-nearest neighbor algorithm (KNN), C5.0 decision tree (CDT), parallel random forest (parRF), and eXtreme Gradient Boosting (XGB). Spectral data were divided between a training set (80%) and a testing set (20%). The accuracy, based on NIRFlex N-500 spectral data, ranged from 0.86 (parRF) to 0.92 (XGB). The Kappa index displayed a similar trend as the accuracy, considering the lowest value for the parRF method (0.39) and the highest value for XGB (0.71). For the SCiO device, the accuracy (0.88-0.89) was similar among the four models evaluated. However, the Kappa index was lower than that of the NIRFlex N-500, and this index ranged from 0 (parRF) to 0.16 (KNN and CDT). Therefore, despite the high accuracy these last models are incapable of correctly classifying waxy and non-waxy clones based on the SCiO device spectra. A confusion matrix was performed to demonstrate the classification model results in the testing set. For both NIRS, the models were efficient in classifying non-waxy clones, with values ranging from 96-100%. However, the NIRS differed in the potential to predict waxy genotype class. For the NIRFlex N-500, the percentage ranged from 30% (parRF) to 70% (XGB). In general, the models tended to classify waxy genotypes as non-waxy, mainly SCiO. Therefore, the use of NIRS can perform early selection of cassava seeds with a waxy phenotype.

Phenotypic diversity and selection in biofortified cassava germplasm for yield and quality root traits.

Increasing carotenoid content and improving other root quality traits has been the focus of cassava biofortification. This study aimed to (i) evaluate the genetic variability for total carotenoid content (TCC), as well as for root yield and root quality attributes; (ii) estimate potentially useful correlations for selection; and (iii) select parents for breeding and estimate the genetic gain. Data from 2011 to 2020 of 265 cassava genotypes with cream and yellow roots were analyzed for dry matter content (DMC), shoot yield, fresh root yield (FRY), dry root yield (DRY), harvest index, average number of roots per plant, starch content, root pulp color, cyanogenic compounds, and TCC. The best linear unbiased predictions showed great phenotypic variation for all traits. Six distinct groups were formed for productive characteristics of root quality, mainly TCC, DMC and FRY. Only TCC showed high broad-sense heritability ( h 2 = 0.72), while the other traits had low to medium magnitude (0.21 ≤ h 2 ≤ 0.60). TCC was strongly correlated with pulp color (r = 0.70), but null significance for DMC. The network analysis identified a clear separation between the agronomic and quality attributes of cassava roots. The selection of the 30 genotypes for recombination in the breeding program has the potential to raise TCC by 27.05% and reduce the cyanogenic compounds content by 23.03%, in addition to increasing FRY and DRY by 22.72% and 22.95%, respectively. This is the first consolidated study on the potential of germplasm for the development biofortified cassava cultivars in Brazil.

Validation of KASP-SNP markers in cassava germplasm for marker-assisted selection of increased carotenoid content and dry matter content.

Provitamin A biofortification and increased dry matter content are important breeding targets in cassava improvement programs worldwide. Biofortified varieties contribute to the alleviation of provitamin A deficiency, a leading cause of preventable blindness common among pre-school children and pregnant women in developing countries particularly Africa. Dry matter content is a major component of dry yield and thus underlies overall variety performance and acceptability by growers, processors, and consumers. Single nucleotide polymorphism (SNP) markers linked to these traits have recently been discovered through several genome-wide association studies but have not been deployed for routine marker-assisted selection (MAS). This is due to the lack of useful information on markers' performances in diverse genetic backgrounds. To overcome this bottleneck, technical and biological validation of the loci associated with increased carotenoid content and dry matter content were carried out using populations independent of the marker discovery population. In the present study, seven previously identified markers for these traits were converted to a robust set of uniplex allele-specific polymerase chain reaction (PCR) assays and validated in two independent pre-breeding and breeding populations. These assays were efficient in discriminating marker genotypic classes and had an average call rate greater than 98%. A high correlation was observed between the predicted and observed carotenoid content as inferred by root yellowness intensity in the breeding (r = 0.92) and pre-breeding (r = 0.95) populations. On the other hand, dry matter content-markers had moderately low predictive accuracy in both populations (r< 0.40) due to the more quantitative nature of the trait. This work confirmed the markers' effectiveness in multiple backgrounds, therefore, further strengthening their value in cassava biofortification to ensure nutritional security as well as dry matter content productivity. Our study provides a framework to guide future marker validation, thus leading to the more routine use of markers in MAS in cassava improvement programs.

Overexpression of leucoanthocyanidin reductase or anthocyanidin reductase elevates tannins content and confers cassava resistance to two-spotted spider mite.

The two-spotted spider mite (TSSM) is a destructive cassava pest. Intensive demonstration of resistance mechanism greatly facilitates the creation of TSSM-resistant cassava germplasm. Gene to metabolite network plays a crucial role in modulating plant resistance, but little is known about the genes and related metabolites which are responsible for cassava resistance to TSSM. Here, a highly resistant (HR) and a highly susceptible (HS) cassava cultivar were used, integrative and comparative transcriptomic and metabolomic analyses between these two cultivars after TSSM infestation revealed that several genes and metabolites were closely related and significantly different in abundance. In particular, the expression of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) genes showed a high positive correlation with most of the metabolites in the tannin biosynthesis pathway. Furthermore, transgenic cassava lines overexpressing either of the genes elevated tannin concentrations and conferred cassava resistance to TSSM. Additionally, different forms of tannins possessed distinct bioactivity on TSSM, of which total condensed tannins (LC50 = 375.68 mg/l) showed maximum lethal effects followed by procyanidin B1 (LC50 = 3537.10 mg/l). This study accurately targets LAR, ANR and specific tannin compounds as critical genes and metabolites in shaping cassava resistance to TSSM, which could be considered as biomarkers for evaluation and creation of pest-resistant cassava germplasm.

CC-type glutaredoxin, MeGRXC3, associates with catalases and negatively regulates drought tolerance in cassava (Manihot esculenta Crantz).

Glutaredoxins (GRXs) are essential for reactive oxygen species (ROS) homeostasis in responses of plants to environment changes. We previously identified several drought-responsive CC-type GRXs in cassava, an important tropical crop. However, how CC-type GRX regulates ROS homeostasis of cassava under drought stress remained largely unknown. Here, we report that a drought-responsive CC-type GRX, namely MeGRXC3, was associated with activity of catalase in the leaves of 100 cultivars (or unique unnamed genotypes) of cassava under drought stress. MeGRXC3 negatively regulated drought tolerance by modulating drought- and abscisic acid-induced stomatal closure in transgenic cassava. It antagonistically regulated hydrogen peroxide (H2 O2 ) accumulation in epidermal cells and guard cells. Moreover, MeGRXC3 interacted with two catalases of cassava, MeCAT1 and MeCAT2, and regulated their activity in vivo. Additionally, MeGRXC3 interacts with a cassava TGA transcription factor, MeTGA2, in the nucleus, and regulates the expression of MeCAT7 through a MeTGA2-MeMYB63 pathway. Overall, we demonstrated the roles of MeGRXC3 in regulating activity of catalase at both transcriptional and post-translational levels, therefore involving in ROS homeostasis and stomatal movement in responses of cassava to drought stress. Our study provides the first insights into how MeGRXC3 may be used in molecular breeding of cassava crops.

NMR-Based Metabolomic Approach for Evaluation of the Harvesting Time and Cooking Characteristics of Different Cassava Genotypes.

Cassava is an important staple food for low-income countries. However, its cooking characteristics are especially affected by genotype. In this study, two groups of genotypes, namely hard to cook (HTC) and easy to cook (ETC), were harvested at different times (9 and 15 months), and evaluated by NMR coupled to chemometrics. Additionally, lignin of these materials was studied by 1H-13C HSQC NMR. The carbohydrates were the most important class of compounds to differentiate the cassava genotypes. The correlation of NMR with cooking time and starch content showed that the higher content of primary metabolites, mostly glucose, can be associated with longer cooking times and reduction of starch, corroborating the metabolic pathways analysis. Furthermore, it was observed that the lignin from cell walls did not differentiate the cooking performance of the genotypes.

The coordination of melatonin and anti-bacterial activity by EIL5 underlies ethylene-induced disease resistance in cassava.

Ethylene and melatonin are widely involved in plant development and environmental stress responses. However, the role of their direct relationship in the immune response and the underlying molecular mechanisms in plants remain elusive. Here, we found that Xanthomonas axonopodis pv. manihotis (Xam) infection increased endogenous ethylene levels, which positively modulated plant disease resistance through activating melatonin accumulation in cassava. In addition, the ethylene-responsive transcription factor ETHYLENE INSENSITIVE LIKE5 (MeEIL5), a positive regulator of disease resistance, was essential for ethylene-induced melatonin accumulation and disease resistance in cassava. Notably, the identification of heat stress transcription factor 20 (MeHsf20) as an interacting protein of MeEIL5 indicated the association between ethylene and melatonin in plant disease resistance. MeEIL5 physically interacted with MeHsf20 to promote the transcriptional activation of the gene encoding N-acetylserotonin O-methyltransferase 2 (MeASMT2), thereby improving melatonin accumulation. Moreover, MeEIL5 promoted the physical interaction of MeHsf20 and pathogen-related gene 3 (MePR3), resulting in improved anti-bacterial activity of MePR3. This study illustrates the dual roles of MeEIL5 in fine-tuning MeHsf20-mediated coordination of melatonin biosynthesis and anti-bacterial activity, highlighting the ethylene-responsive MeEIL5 as the integrator of ethylene and melatonin signals in the immune response in cassava.

LSD3 mediates the oxidative stress response through fine-tuning APX2 activity and the NF-YC15-GSTs module in cassava.

Reactive oxygen species (ROS) overproduction leads to oxidative damage under almost all stress conditions. Lesion-Simulating Disease (LSD), a zinc finger protein, is an important negative regulator of ROS accumulation and cell death in plants. However, the in vivo role of LSD in cassava (Manihot esculenta) and the underlying molecular mechanisms remain elusive. Here, we found that MeLSD3 is essential for the oxidative stress response in cassava. MeLSD3 physically interacted with ascorbate peroxidase 2 (MeAPX2), thereby promoting its enzymatic activity. In addition, MeLSD3 also interacted with the nuclear factor YC15 (MeNF-YC15), which also interacted with nuclear factor YA2/4 (MeNF-YA2/4) and nuclear factor YB18 (MeNF-YB18) to form an MeNF-YC15-MeNF-YA2/4-MeNF-YB18 complex. Notably, MeLSD3 positively modulated the transcriptional activation of the MeNF-YC15-MeNF-YA2/4-MeNF-YB18 complex by interacting with the CCAAT boxes of the promoters of glutathione S-transferases U37/U39 (MeGST-U37/U39), activating their transcription. When one or both of MeLSD3 and the MeNF-YC15-MeNF-YA2/4-MeNF-YB18 complex were co-silenced, cassava showed decreased oxidative stress resistance, while overexpression of MeGST-U37/U39 alleviated the oxidative stress-sensitive phenotype of these silenced plants. This study illustrates the dual roles of MeLSD3 in promoting MeAPX2 activity and MeNF-YC15-MeGST-U37/U39 regulation, which underlie the oxidative stress response in cassava.

CRISPR-Cas9-mediated knockout of CYP79D1 and CYP79D2 in cassava attenuates toxic cyanogen production.

Cassava (Manihot esculenta) is a starchy root crop that supports over a billion people in tropical and subtropical regions of the world. This staple, however, produces the neurotoxin cyanide and requires processing for safe consumption. Excessive consumption of insufficiently processed cassava, in combination with protein-poor diets, can have neurodegenerative impacts. This problem is further exacerbated by drought conditions which increase this toxin in the plant. To reduce cyanide levels in cassava, we used CRISPR-mediated mutagenesis to disrupt the cytochrome P450 genes CYP79D1 and CYP79D2 whose protein products catalyze the first step in cyanogenic glucoside biosynthesis. Knockout of both genes eliminated cyanide in leaves and storage roots of cassava accession 60444; the West African, farmer-preferred cultivar TME 419; and the improved variety TMS 91/02324. Although knockout of CYP79D2 alone resulted in significant reduction of cyanide, mutagenesis of CYP79D1 did not, indicating these paralogs have diverged in their function. The congruence of results across accessions indicates that our approach could readily be extended to other preferred or improved cultivars. This work demonstrates cassava genome editing for enhanced food safety and reduced processing burden, against the backdrop of a changing climate.