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) exhibits remarkable capacity for heavy metal accumulation, yet the molecular mechanisms governing lead (Pb²⁺) sequestration in tuberous roots remain poorly understood. This study investigates the role of pectin methylesterase inhibitor 1 (MePMEI1) in modulating pectin methylesterification status and Pb²⁺ accumulation in cassava. Through generation of MePMEI1 overexpressing lines and CRISPR/Cas9-mediated mepmei1 knockout mutants, we demonstrate that MePMEI1 overexpression significantly reduces pectin methylesterification degree, resulting in substantial accumulation of low-methylesterified pectin within cell walls. This modified pectin composition dramatically increases Pb²⁺ binding capacity in root tissues, particularly within the meristematic zone, while simultaneously enhancing plant tolerance to Pb stress. Mechanistically, MePMEI1-mediated pectin remodeling thickens cell walls, improves structural integrity, and activates antioxidant defense systems, evidenced by elevated catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) activities with concomitant reduction in malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) accumulation under Pb²⁺ exposure. Notably, mepmei1 mutants exhibited compromised Pb²⁺ sequestration and reduced stress tolerance compared to wild-type plants. Our findings establish MePMEI1 as a central regulator in the pectin-dependent Pb²⁺ immobilization pathway, providing crucial molecular insights for developing cassava cultivars with minimized Pb²⁺ accumulation in edible tuberous roots while maintaining phytoremediation potential for contaminated soils.

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.

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.

Postharvest physiological deterioration (PPD) reduces the availability and economic value of fresh produces, resulting in the waste of agricultural products and becoming a worldwide problem. Therefore, many studies have been carried out at the anatomical structural, physiological and biochemical levels and molecular levels of PPD of fresh produces to seek ways to manage the postharvest quality of fresh produce. The cell wall is the outermost structure of a plant cell and as such represents the first barrier to prevent external microorganisms and other injuries. Many studies on postharvest quality of crop storage organs relate to changes in plant cell wall-related components. Indeed, these studies evidence the non-negligible role of the plant cell wall in postharvest storage ability. However, the relationship between cell wall metabolism and postharvest deterioration of fresh produces has not been well summarized. In this review, we summarize the structural changes of cell walls in different types of PPD, metabolic changes, and the possible molecular mechanism regulating cell wall metabolism in PPD of fresh produce. This review provides a basis for further research on delaying the occurrence of PPD of fresh produce.

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.

Cold stress is a limiting stress factor that limits plant distribution and development; however, polyploid plants have specific characteristics such as higher resistance to abiotic stress, especially cold stress, that allow them to overcome this challenge. The cultivated cultivar Ziziphus jujuba Mill. 'Yueguang' (YG) and its autotetraploid counterpart ‘Hongguang’ (HG) exhibit differential cold tolerance. However, the underlying molecular mechanism and methods to enhance their cold tolerance remain unknown. Anatomical structure and physiological analysis indicated YG had a higher wood bark ratio, and xylem ratio under cold treatment compared to HG. However, the half-lethal temperature (LT50), cortex ratio, and malondialdehyde (MDA) content were significantly decreased in YG than HG, which indicated YG was cold tolerant than HG. Transcriptome analysis showed that 2084, 1725, 2888, and 2934 differentially expressed genes (DEGs) were identified in HC vs YC, H20 vs Y20, Y20 vs YC, and H20 vs HC treatment, respectively. Meanwhile, KEGG enrichment analysis of DEGs showed that several metabolic pathways, primarily plant hormone signal transduction and the MAPK signaling pathway, were involved in the differential regulation of cold tolerance between YG and HG. Furthermore, exogenous abscisic acid (ABA) and brassinolide (BR) treatments could improve their cold tolerance through increased SOD and POD activities, decreased relative electrical conductivity, and MDA content. All of these findings suggested that plant hormone signal transduction, particularly ABA and BR, might have an important role in the regulation of differential cold tolerance between YG and HG, laying the foundation for further improving cold tolerance in jujube and examining the molecular mechanisms underlying differences in cold tolerance among different ploidy cultivars.

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.

3′,5′-Cyclic adenosine monophosphate(cAMP)is an important metabolite that is specifically enriched in jujube.However,the effect of cAMP on jujube cellular responses has not been comprehensively studied.Here,we established jujube cell suspension cultures and investigated the calcium influx in response to cAMP treatment through protoplast isolation and fluorescence intensity.Firstly,cAMP treatment could promote jujube growth and increase the content of endogenous cAMP.Using transcriptome analysis with transgenic Arabidopsis plants overexpressing adenylate cyclase(ZjAC)as a positive control,we identified 60 calcium-related differential expressed genes(DEGs)that contributed to the calcium signaling and inter-or intra-cellular responses.Pharmacological treatments such as cAMP and the calcium ionophore A23187 could induce ZjAC expression,the accumulation of cAMP and calcium influx in jujube cells,while ethylene glycol tetraacetic acid(EGTA)or bithionol treatment inhibited these changes.Moreover,the calcium channels and transporters in calcium influx,such as the ZjCNGC2 channel and the mitogen activated protein(MAP)kinase pathway,could be activated by cAMP treatment.In summary,our findings demonstrated that cAMP biosynthesis is dependent on calcium influx and the amplifying effect between calcium and cAMP may be involved in intracellular signal induction,which might contribute to the growth and development of jujube.

愈伤组织是进行植物离体培养与生物技术研究的常用材料.近年来,枣愈伤组织培养及其应用研究取得了重要进展.对枣茎段、叶片、花药和胚等不同组织的愈伤诱导培养体系及其在植株离体再生、多倍体诱导、遗传转化、悬浮细胞系建立、原生质体培养等领域的应用研究进展进行了综述,对今后的相关研究工作进行了展望,以期为枣愈伤组织培养的技术优化和科学利用提供借鉴和参考.

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.

Sour jujube has strong adaptability to the environment,including drought and barrenness resistance,with high economic value.Callus is a common material for ex-vivo cultivation,but few reports on the establishment of sour jujube tissue cultivating system were shown.In the current study,twenty sour jujubes were selected to explore the effect of genotypes,explants and hormone ratios on the induction of callus,bud and root.The results showed that the optimal callus inducing medium in sour jujube fast propagation system was MS+3.0 mg/L TDZ+0.5 mg/L NAA.The optimal bud inducing medium of sour jujube callus was MS+1.5 mg/L 6-BA+0.5 mg/L IB A,MS+2.0 mg/L 6-BA+0.2 mg/L IBA.The optimal root inducing medium of sour jujube was 1/2 MS+1.0 mg/L IBA and 1/2 MS+1.0 mg/L IBA+0.4 mg/L NAA.The rapid tissue cultivating system suitable for most sour jujubes was established,which could provide important theoretical guidance for large-scale tissue cultivation of sour jujube in the future.

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.

LBD基因家族是指含有侧生器官边界(lateral organ boundaries,LOB)结构域的一种植物特异性转录因子家族,在植物的生长发育和代谢调控等方面起着至关重要的作用.木薯MeLOB36基因是LBD基因家族成员,前期研究发现该基因在木薯根尖、体细胞胚、脆性胚性愈伤组织表达量较高,推测其参与了木薯植株的形态建成.为了鉴定MeLOB36基因的功能,本研究利用在线软件CRISPR-Pv2.0在保守区域设计靶标MeLOB36 基因的 sgRNA,构建 MeLOB36 基因的编辑载体 pCAMBIA1301-Cas9-MeLOB36-sgRNA.将重组载体转化LBA4404根癌农杆菌后侵染木薯脆性胚性愈伤组织,通过PCR扩增MeLOB36基因编辑靶点及潜在脱靶位点区段序列,进行Sanger测序.结果表明,MeLOB36基因被成功编辑,且未脱靶.本研究有助于进一步获得MeLOB36基因的突变体,从而鉴定MeLOB36转录因子对木薯植株形态建成的影响.