Pyruvate (PA), as a key metabolic node, plays a decisive role in maintaining postharvest quality. However, the effect of exogenous pyruvate on postharvest physiological deterioration (PPD) in cassava has not yet been studied. In this study, exogenous PA significantly delayed PPD in fresh-cut cassava in a concentration-dependent manner, an effect not attributable to pH changes. Treatment with 5% PA completely inhibited browning for 48 h and was therefore used for mechanistic studies. PA reduced the levels of H2O2, O-2(center dot-) , and malondialdehyde (MDA). Although superoxide dismutase (SOD) activity was suppressed, PA significantly enhanced catalase (CAT) and ascorbate peroxidase (APX) activities and elevated the AsA/DHA and GSH/GSSG ratios, indicating a remodeling of cellular redox homeostasis. Metabolomics identified 1451 differentially accumulated metabolites, and transcriptomics revealed 16926 differentially expressed genes. KEGG enrichment analysis showed that PAspecific metabolites and genes were mainly enriched in pyruvate metabolism, the TCA cycle, glutathione metabolism, and cutin, suberin, and wax biosynthesis. Correlation network analysis screened 11 metabolites and 30 carbohydrate and energy metabolism-related genes, from which five core genes (LDHB, SIR1, PK, FBA2, and G6PD) were identified. Eight antioxidant enzyme genes (CATA1, GR, GPX2, GPX6, PER48, PER5, PERX, and PIOX) were also characterized. In hormone signaling, PA regulated 29 key genes involved in the jasmonic acid, ethylene, abscisic acid, and salicylic acid pathways, and stabilized JA-Ile accumulation. In conclusion, exogenous pyruvate delays cassava PPD by coordinately regulating carbohydrate and energy metabolism, the antioxidant system, and multiple hormone signaling networks. The identified core genes provide potential molecular targets for precise regulation of postharvest deterioration in cassava.

Cassava (Manihot esculenta Crantz), a tropical tuberous crop, faces severe postharvest physiological deterioration (PPD) that critically impacts its storage duration and economic value. The glyoxalase (GLY) pathway is an antioxidant defense mechanism in plants. This study investigated the function of MeGLYI-13 in regulating cassava PPD, via constitutive overexpression of MeGLYI-13, utilizing integrated physiological and transcriptomic analyses. The results indicated that overexpression of MeGLYI-13 in cassava delayed PPD occurrence, increased GLYI, APX, GR, and POD enzyme activities, as well as GSH and AsA levels, while reducing H2O2, MDA, and MG accumulation. Transcriptomic analysis revealed that MeGLYI-13 overexpression upregulated genes associated with antioxidant systems, the energy metabolism pathways. The MeGLYI-13 protein could interact with the MeFBP protein in the Calvin cycle. Two transcription factors, MADS and MYB, in the DEGs were predicted to be the common transcription factors of MeGLYI-13 and MeFBP. The above results indicate that MeGLYI-13 enhances antioxidant capacity and interacts with MeFBP to promote the Calvin cycle, reducing the accumulation of MG precursors, thereby reducing damage to cells and delaying the occurrence of cassava PPD. This process may be regulated by MADS and MYB transcription factors.

Enhancing resistant starch (RS) content in cassava is vital for developing nutritionally improved, functional food crops. In this study, targeted mutagenesis of the MeSSI gene via CRISPR/Cas9 was conducted to investigate its role in starch biosynthesis and RS accumulation. MeSSI knockout lines exhibited a 6.74-fold increase in RS content and a 16.42% elevation in amylose levels compared to the wild-type, without compromising total starch content or root yield. Starch structural analysis revealed an increased number of smaller granules per amyloplast and a shift in amylopectin chain-length distribution, characterized by reduced short chains (DP 6-12) and enrichment of intermediate and long chains, resulting in a lower branching degree. These modifications were associated with enhanced thermal stability and altered pasting behavior. Transcriptomic profiling indicated compensatory upregulation of AGPase subunits, and glycolytic genes, suggesting a reprogramming of carbon metabolism to sustain starch accumulation. This work identifies MeSSI as a key determinant of amylopectin fine structure and RS formation, providing a precise genome-editing strategy to improve the nutritional profile of cassava.

Cassava (Manihot esculenta Crantz) is a staple crop in tropical and subtropical regions, valued for its high starch content in roots. Effective genetic transformation and genome editing of cassava require efficient screening methods for transgenic and edited plants. In this study, a visual selection marker system using an R2R3-MYB transcription factor anthocyanin 1 gene (HbAN1, LOC110667474) from a rubber tree (Hevea brasiliensis Müll. Arg.) has been developed to facilitate the identification of transgenic cassava plants. Transgenic cassava lines expressing HbAN1 accumulated anthocyanins in their leaves, allowing for easy visual identification without the need for destructive assays or specialized equipment. Importantly, the accumulation of anthocyanins did not affect the regeneration or transformation efficiency of cassava. Additionally, the AR-CRISPR/Cas9-gRNA system with the HbAN1 gene as a marker produced MeCDD4 gene-edited cassava mutants with purple leaves, demonstrating successful editing. This anthocyanin-based visual reporter (AR) system will provide an effective tool for genetic transformation and genome editing in cassava.

Amyloplasts are the sites of starch synthesis and accumulation. Little is known about amyloplast division and its effects on the size, structure, and physicochemical properties of starch granules. In this study, we created mutants of plastid division-related gene MeMinD by CRISPR/Cas9 technology, leading to the disruption of normal division of amyloplasts in cassava storage roots. The memind mutants exhibited significantly enlarged amyloplasts with an increased number of starch granules, and broader range of granule sizes. The loss of MeMinD function led to transcriptional reprogramming of gene expressions related to starch-synthesizing enzymes, affecting the fine structure of starch. Starch in memind mutant storage roots showed a significantly decreased proportion of shorter amylopectin chains and an increased proportion of medium and long chains, which ultimately led to a significant increase in apparent amylose content (AAC) in memind mutants compared to that in WT. The changes in starch granule size and structure resulted in a significant increase in onset temperature (To), peak temperature (Tp), and conclusion temperature (Tc) of the gelatinization process, extending the time to reach peak temperature. These data suggest that regulating amyloplast division affects starch accumulation in cassava, presenting an effective strategy for developing novel cassava starch.

Mutation of the MATRILINEAL gene in sugarcane induces in vivo maternal haploids and suggests prospects for sugarcane breeding.

Coix lacryma-jobi L. is one of the most economically and medicinally important corns. This study constructed a high-density genetic linkage map of C. lacryma-jobi based on a cross between the parents 'Qianyi No. 2' × 'Wenyi No. 2' and their F2 progeny through high-throughput sequencing and the construction of a specific-locus amplified fragment (SLAF) library. After pre-processing, 325.49 GB of raw data containing 1628 M reads were obtained. A total of 22,944 high-quality SLAFs were identified, among which 3952 SLAFs and 3646 polymorphic markers met the requirements for the construction of a genetic linkage map. The integrated map contained 3605 high-quality SLAFs, which were grouped into ten genetic linkage groups. The total length of the map was 1620.39 cM, with an average distance of 0.45 cM and an average of 360.5 markers per linkage group. This report presents the first high-density genetic map of C. lacryma-jobi. This map was constructed using an F2 population and SLAF-seq approach, which allows the development of a large number of polymorphic markers in a short period. These results provide a platform for precise gene/quantitative trait locus (QTL) mapping, map-based gene separation, and molecular breeding in C. lacryma-jobi. They also help identify a target gene for tracking, splitting quantitative traits, and estimating the phenotypic effects of each QTL for QTL mapping. They are of great significance for improving the efficiency of discovering and utilizing excellent gene resources of C. lacryma-jobi.

MeFtsZ2-1 is a key gene for plant plastid division, but the mechanism by which MeFtsZ2-1 affects pigment accumulation in cassava ( Manihot esculenta Crantz) through plastids remains unclear. We found that MeFtsZ2-1 overexpression in cassava (OE) exhibited darker colors of leaves, with increased levels of anthocyanins and carotenoids. Further observation via Transmission Electron Microscopy (TEM) revealed no apparent defects in chloroplast structure but an increase in the number of plastoglobule in OE leaves. RNA-seq results showed 1582 differentially expressed genes (DEGs) in leaves of OE. KEGG pathway analysis indicated that these DEGs were enriched in pathways related to flavonoid, anthocyanin, and carotenoid biosynthesis. This study reveals the role of MeFtsZ2-1 in cassava pigment accumulation from a physiological and transcriptomic perspective, providing a theoretical basis for improving cassava quality.

Cassava starch is a widely used raw material for industrial production and food source for people. However, cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam) results in severe yield losses and is the most destructive bacterial disease in all worldwide cassava-growing regions. Xam11 is a highly pathogenic subspecies from China that infects the Chinese local cassava South China No. 8 (SC8) cultivar with marked symptoms. This study showed that the transcription activator-like effector TALE20(Xam11) of Xam11 strain regulates the expression of disease-susceptibility gene MeSWEET10a by binding to the EBETALE20 region of the MeSWEET10a promoter in cassava cultivar SC8. CRISPR/Cas9-generated mutations of the EBETALE20 region resulted in a significant reduction in MeSWEET10a expression after infection by Xam11, correlating with reduced disease symptoms, smaller lesion sizes and decreased bacterial proliferation compared with the wild type. Importantly, the edited plants maintained normal growth, development and yield characteristics under greenhouse conditions. The results lay a research foundation for breeding resistant cassava cultivar SC8 to bacterial blight.

Soluble starch synthases (SSs) play important roles in the synthesis of cassava starch. However, the expression characteristics of the cassava SSs genes have not been elucidated. In this study, the MeSSIII-1 gene and its promoter, from SC8 cassava cultivars, were respectively isolated by PCR amplification. MeSSIII-1 protein was localized to the chloroplasts. qRT-PCR analysis revealed that the MeSSIII-1 gene was expressed in almost all tissues tested, and the expression in mature leaves was 18.9 times more than that in tuber roots. MeSSIII-1 expression was induced by methyljasmonate (MeJA), abscisic acid (ABA), and ethylene (ET) hormones in cassava. MeSSIII-1 expression patterns were further confirmed in proMeSSIII-1 transgenic cassava. The promoter deletion analysis showed that the -264 bp to -1 bp MeSSIII-1 promoter has basal activity. The range from -1228 bp to -987 bp and -488 bp to -264 bp significantly enhance promoter activity. The regions from -987 bp to -747 bp and -747 bp to -488 bp have repressive activity. These findings will provide an important reference for research on the potential function and transcriptional regulation mechanisms of the MeSSIII-1 gene and for further in-depth exploration of the regulatory network of its internal functional elements.

Formic acid is reported to act as a food preservative and feed additive, but its effects on controlling postharvest physiological deterioration (PPD) development in cassava are unclear. In this study, we assessed the effectiveness of different concentrations of formic acid in attenuating PPD occurrence in fresh-cut cassava. The results showed that the concentration of 0.1% (v/v) formic acid could significantly delay the occurrence of PPD, and that the higher the concentration of formic acid supplied, the later the occurrence of PPD symptoms. The physiological and biochemical analysis of 0.5%-formic-acid-treated cassava slices revealed that formic acid decreased the degradation of starch, inhibited the accumulation of hydrogen peroxide (H2O2), malondialdehyde (MDA), and water-soluble pectin in cassava slices with PPD development, and increased the activities of the antioxidant enzymes ascorbate peroxidase (APX) and glutathione reductase (GR). A microscopic observation showed that the formic acid treatment inhibited the enlargement of the intercellular space during the cassava PPD process, which suggests that the formation of an intercellular layer of the cell wall was inhibited by formic acid. This study thus revealed the mechanism used by formic acid to extend the cassava shelf life; however, a detailed evaluation of the possible side effects on, for example, the cyanide content will be needed to categorically ensure the safety of this method.

The sugar transporter (STP) gene family is a key regulator of plant development, which is crucial for the efficient transport and utilization of sugars during plant growth and development. In this study, we identified the MeSTP7 gene, which is highly expressed in cassava fibrous roots, early storage roots, and under hormonal treatment, including IAA, MeJA, ABA, and GA3, and abiotic stressors, such as mannitol and NaCl. A strong response was observed with exoqenous IAA. Transfecting MeSTP7 into Arabidopsis promoted early seedling growth, particularly in lateral root development. The content of endogenous hormones (IAA and MeJA) as well as soluble sugars (sucrose, fructose, and glucose) was elevated in transgenic Arabidopsis. Hormone treatments with IAA, MeJA, GA3, and ABA on transgenic Arabidopsis revealed that transgenic Arabidopsis responded positively to added 20 μM IAA. They also exhibited co-induced regulation of lateral root formation by GA3, MeJA, and ABA. qRT-PCR analysis showed that overexpression of MeSTP7 upregulated the expression of IAA14, ARF7, and ARF19 in Arabidopsis. Under IAA treatment, the expression of these genes was similarly upregulated but downregulated under MeJA treatment. These results suggest that MeSTP7 may promote Arabidopsis seedling development by increasing the content of sucrose, glucose, and fructose in roots, which in turn influences IAA-based hormonal signaling.

Foods with high amylose and resistant starch (RS) contents have great potential to enhance human health. In this study, cassava soluble starch synthase MeSSIII-1 gene mutants were generated using CRISPR/Cas9 system. The results showed that the storage roots of messiii-1 mutants had higher contents of amylose, RS, and total starch than those in CK. The rates of small and large-sized starch granules were increased. Additionally, amylopectin starch in messiii-1 mutants had a higher proportion of medium- and long- chains, and a lower proportion of short-chains than those in CK. The onset, peak, and conclusion temperatures of starch gelatinization in messiii-1 mutants were significantly lower than those in CK, and the peak viscosity, trough viscosity and final viscosity all increased. MeSSIII-1 mutation could increase the contents of sucrose, glucose, and fructose in cassava storage roots. We hypothesize that these soluble sugars serve a dual role: they provide the necessary carbon source for starch synthesis and act as sugar signals to trigger the transcriptional reprogramming of genes involved in starch biosynthesis. This process results in a collective enhancement of amylose, RS, and total starch contents, accompanied by changes in starch granule morphology, fine structure, and physicochemical properties.

The AT-hook motif nuclear-localized (AHL) family is pivotal for the abiotic stress response in plants. However, the function of the cassava AHL genes has not been elucidated. Promoters, as important regulatory elements of gene expression, play a crucial role in stress resistance. In this study, the promoter of the cassava MeAHL31 gene was cloned. The MeAHL31 protein was localized to the cytoplasm and the nucleus. qRT-PCR analysis revealed that the MeAHL31 gene was expressed in almost all tissues tested, and the expression in tuber roots was 321.3 times higher than that in petioles. Promoter analysis showed that the MeAHL31 promoter contains drought, methyl jasmonate (MeJA), abscisic acid (ABA), and gibberellin (GA) cis-acting elements. Expression analysis indicated that the MeAHL31 gene is dramatically affected by treatments with salt, drought, MeJA, ABA, and GA3. Histochemical staining in the proMeAHL31-GUS transgenic Arabidopsis corroborated that the GUS staining was found in most tissues and organs, excluding seeds. Beta-glucuronidase (GUS) activity assays showed that the activities in the proMeAHL31-GUS transgenic Arabidopsis were enhanced by different concentrations of NaCl, mannitol (for simulating drought), and MeJA treatments. The integrated findings suggest that the MeAHL31 promoter responds to the abiotic stresses of salt and drought, and its activity is regulated by the MeJA hormone signal.

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.

Light quality is highly important for growth control of in vitro plant cultures. Here, we investigated the effect of blue light (BL), red light (RL) and combined red and blue light (RBL) on in vitro cassava growth. Our results indicate that RL facilitated radial elongation of cassava and increased stomatal conductance as well as glucose, sucrose, fructose and starch content in leaves and cellulose content in the stem. It also enhanced SOD and POD activities but decreased the stomatal density and chlorophyll and carotenoid content in leaves. In addition, RL leads to shorter palisade cells, denser chloroplasts and more starch granules. These phenotypic changes were inverted following BL treatment. The expression levels of photosynthesis-related genes MeLHCA1, MeLHCA3, MePSB27-2, MePSBY, MePETE1 and MePNSL2 in leaves were at their lowest following RL treatment, while the expression levels of MePSB27-2, MePSBY, MePETE1 and MePNSL2 were at their highest after BL treatment. The phenotypic changes after RBL treatment were between the values observed for the RL and BL treatments alone. Moreover, the responses of SC8 and SC9 cassava varieties to light quality were largely conserved. As such, we believe that the results of this study lay the foundation for controlling the in vitro growth of cassava seedlings by light quality.

Although zinc and copper are the two essential nutrients necessary for plant growth, their excessive accumulation in soil not only causes environmental pollution but also seriously threatens human health and inhibits plant growth. The breeding of plants with novel zinc or copper toxicity tolerance capacities represents one strategy to address this problem. Glyoxalase I (GLYI) family genes have previously been suggested to be involved in the resistance to a wide range of abiotic stresses, including those invoked by heavy metals. Here, a MeGLYI-13 gene cloned from a cassava SC8 cultivar was characterized with regard to its potential ability in resistance to zinc or copper stresses. Sequence alignment indicated that MeGLYI-13 exhibits sequence differences between genotypes. Transient expression analysis revealed the nuclear localization of MeGLYI-13. A nuclear localization signal (NLS) was found in its C-terminal region. There are 12 Zn2+ binding sites and 14 Cu2+ binding sites predicted by the MIB tool, of which six binding sites were shared by Zn2+ and Cu2+. The overexpression of MeGLYI-13 enhanced both the zinc and copper toxicity tolerances of transformed yeast cells and Arabidopsis seedlings. Taken together, our study shows the ability of the MeGLYI-13 gene to resist zinc and copper toxicity, which provides genetic resources for the future breeding of plants resistant to zinc and copper and potentially other heavy metals.

Cassava is one of the most versatile tuberous-root crops on Earth. However, the postharvest storage properties of cassava tuberous root mean that it is perishable through a process known as postharvest physiological deterioration (PPD), which seriously affects its starch quality. Therefore, a comprehensive understanding of the transcriptional regulatory activity of cassava against the PPD response is necessary in order to extract key molecular mechanisms related to PPD tolerance. In this study, we found that RYG1 tuberous roots showed delayed PPD compared to those of SC8. In addition, RYG1 roots maintained a more stable cell wall structure after storage than those of SC8. The transcriptome changes in tuberous roots were analyzed for both RYG1 and SC8 after 21 days of storage (SR and SS) compared to fresh (FR and FS) by the RNA-Seq method. The total number of differentially expressed genes (DEGs) in the various comparisons of these four samples ranged from 68 to 3847. Of these, a total of 2008 co-DEGs in SR vs. SS were shared by either SR vs. FR or SS vs. FS. GO and KEGG enrichment analysis revealed that upregulated co-DEGs in SR vs. SS were mainly enriched in photosynthesis, protein processing, hormone and cutin, suberine and wax biosynthesis. By contrast, the downregulated co-DEGs were mainly related to cell wall organization, starch and sucrose metabolism, galactose metabolism, phenylpropanoid biosynthesis, diterpenoid biosynthesis, cysteine and methionine metabolism and flavonoid biosynthesis. The protein–protein interaction (PPI) networks of the co-DEGs showed a complex interaction of genes in different pathways, and 16 hub genes were characterized to have a degree in excess of 15, among which eight genes were associated with photosynthesis. These results provide new information for the study of cassava resistance to PPD and lay a foundation for the further molecular breeding of storage-tolerant cassava varieties.

BACKGROUND:Plant virus vectors designed for virus-mediated protein overexpression (VOX), virus-induced gene silencing (VIGS), and genome editing (VIGE) provide rapid and cost-effective tools for functional genomics studies, biotechnology applications and genome modification in plants. We previously reported that a cassava common mosaic virus (CsCMV, genus Potexvirus)-based VIGS vector was used for rapid gene function analysis in cassava. However, there are no VOX and VIGE vectors available in cassava.RESULTS:In this study, we developed an efficient VOX vector (CsCMV2-NC) for cassava by modifying the CsCMV-based VIGS vector. Specifically, the length of the duplicated putative subgenomic promoter (SGP1) of the CsCMV CP gene was increased to improve heterologous protein expression in cassava plants. The modified CsCMV2-NC-based VOX vector was engineered to express genes encoding green fluorescent protein (GFP), bacterial phytoene synthase (crtB), and Xanthomonas axonopodis pv. manihotis (Xam) type III effector XopAO1 for viral infection tracking, carotenoid biofortification and Xam virulence effector identification in cassava. In addition, we used CsCMV2-NC to deliver single guide RNAs (gMePDS1/2) targeting two loci of the cassava phytoene desaturase gene (MePDS) in Cas9-overexpressing transgenic cassava lines. The CsCMV-gMePDS1/2 efficiently induced deletion mutations of the targeted MePDS with the albino phenotypes in systemically infected cassava leaves.CONCLUSIONS:Our results provide a useful tool for rapid and efficient heterologous protein expression and guide RNA delivery in cassava. This expands the potential applications of CsCMV-based vector in gene function studies, biotechnology research, and precision breeding for cassava.