Project description:Cassava (Manihot esculenta) is one of the most important staple food crops worldwide. Its starchy tuberous roots supply over 800 million people with carbohydrates. Yet, surprisingly little is known about the processes involved in filling of those vital storage organs. A better understanding of cassava carbohydrate allocation and starch storage is key to improve storage root yield. In this work, we studied cassava morphology and phloem sap flow from source to sink using transgenic pAtSUC2::GFP plants, the phloem tracers esculin and 5(6)-carboxyfluorescein diacetate (CFDA), as well as several staining techniques. We show that cassava performs apoplasmic phloem loading in source leaves and symplasmic unloading into phloem parenchyma cells of tuberous roots. We demonstrate that vascular rays play an important role in radial transport from the phloem to xylem parenchyma cells in tuberous roots. Furthermore, enzymatic and proteomic measurements of storage root tissues confirmed high abundance and activity of enzymes involved in the sucrose synthase-mediated pathway and indicated that starch is stored most efficiently in the outer xylem layers of tuberous roots. Our findings represent a first basis for biotechnological approaches aimed at improved phloem loading and enhanced carbohydrate allocation and storage in order to increase tuberous root yield of cassava.
Project description:Tuberous root formation is a complex process consisting of phase changes as well as cell division and elongation for radial growth. We performed an integrated analysis to clarify the relationships among metabolites, phytohormones, and gene transcription during tuberous root formation in cassava (Manihot esculenta Crantz). We also confirmed the effects of the auxin (AUX), cytokinin (CK), abscisic acid (ABA), jasmonic acid (JA), gibberellin (GA), brassinosteroid (BR), salicylic acid (SA), and indole-3-acetic acid conjugated with aspartic acid (IAAsp) on tuberous root development. An integrated analysis of metabolites and gene expression indicated the expression levels of several genes encoding enzymes involved in starch biosynthesis and sucrose metabolism are up-regulated during tuberous root development, which is consistent with the accumulation of starch, sugar phosphates, and nucleotides. An integrated analysis of phytohormones and gene transcripts revealed a relationship among AUX signaling, CK signaling, and BR signaling, with AUX, CK, and BR inducing tuberous root development. In contrast, ABA and JA inhibited tuberous root development. These phenomena might represent the differences between stem tubers (e.g., potato) and root tubers (e.g., cassava). On the basis of these results, a phytohormonal regulatory model for tuberous root development was constructed. This model may be useful for future phytohormonal studies involving cassava.
Project description:microRNAs can play a crucial role in stress response in plants, including biotic stress. Some miRNAs are known to respond to bacterial infection. This work has addressed the role of miRNAs in Manihot esculenta (cassava)-Xanthomonas axonopodis pv. manihotis (Xam) interaction. Illumina sequencing was used for analyzing small RNA libraries from cassava tissue infected and non-infected with Xam. Cassava variety MBRA685 (resistant to Xam-CIO151) Six-week-old plants were inoculated with 36h-old cultures of the aggressive Xanthomonas axonopodis pv. manihotis strain CIO151 in both leaves and stems.
Project description:In this study, β-carotene concentrations in cassava storage roots were enhanced by co-expression of transgenes for deoxyxylulose-5-phosphate synthase (DXS) and bacterial phytoene synthase (crtB), mediated by the patatin type-1 promoter. Storage roots harvested from field-grown plants accumulated carotenoids to ≤50 μg/g DW, a 15- to 20-fold increase relative to roots from non-transgenic plants. Approximately 85-90% of these carotenoids accumulated as all-trans-β-carotene, the most nutritionally efficacious carotenoid. β-carotene-accumulating storage roots displayed delayed onset of post-harvest physiological deterioration, a major constraint limiting utilization of cassava products. Significant metabolite changes were detected in β-carotene enhanced storage roots. Most significantly, an inverse correlation was observed between β-carotene and dry matter contents, with reductions of 50% to 60% of dry matter content in the highest carotenoid accumulating storage roots of different cultivars. Further analysis confirmed concomitant reduction in starch content, and increased levels of total fatty acids, triacylglycerols, soluble sugars, and abscisic acid. Irish potato engineered to co-express DXS and crtB displayed a similar correlation between β-carotene accumulation, reduced dry matter and starch content, and elevated oil and soluble sugars in tubers. Transcriptome analyses revealed reduced expression of starch biosynthetic genes, ADP-glucose pyrophosphorylase genes, in transgenic, carotene-accumulating cassava roots relative to non-transgenic roots. These findings highlight unintended metabolic consequences of provitamin A biofortification of starch-rich organs and point to strategies for redirecting metabolic flux to restore starch production.
Project description:Phytomonas are a large and diverse sub-group of plant-infecting trypanosomatids that are relatively poorly understood. Little is known of their biology or how they have adapted to life inside plants. This study sequenced the genome of the Cassava (Manihot esculenta) infecting species Phytomonas francai to provide additional genome resources and new insight into the biology of this poorly understood group of organisms.
Project description:Cassava (Manihot esculenta) is the food security crop that feeds approximately 800 million people worldwide. Although this crop displays high productivity under drought and poor soil conditions, it is susceptible to disease, postharvest deterioration and the roots contain low nutritional content. Cassava improvement programs are focused on addressing these constraints but are hindered by the crop’s high heterozygosity, difficulty in synchronizing flowering, low seed production and a poor understanding of the physiology of this plant. Among the major food crops, cassava is unique in its ability to develop massive, underground storage roots. Despite the importance of these structures, their basic physiology remains largely unknown, especially the molecular genetic basis of storage root development. Similarly, in cassava, the favored target tissue for transgene integration and genome editing is a friable embryogenic callus (FEC). Little is known concerning gene expression in this tissue, or its relatedness to the somatic organized embryogenic structures (OES) from which it originates. Here, we provide molecular identities for eleven cassava tissue types through RNA sequencing and develop an open access, web-based interface for further interrogation of the data. Through this dataset, we report novel insight into the physiology of cassava and identify promoters able to drive specified tissue expression profiles. The information gained from this study is of value for both conventional and biotechnological improvement programs.
Project description:Mechanisms related to the development of cassava storage roots and starch accumulation remain largely unknown. To evaluate genome-wide expression patterns during cassava tuberization, a 60-mer oligonucleotide microarray representing 20,840 cassava genes was designed to identify differentially expressed transcripts in fibrous root, developing storage root and mature storage root. Using a random variance model and the traditional two-fold change method for statistical analysis, 912 and 3386 differentially expressed genes were identified related to the three different phases. Among 25 significant pathways identified, glycolysis/gluconeogenesis was the most important pathway signature due to its effects on other pathways. Rate-limiting enzymes were identified from each individual pathway, such as pectinesterase, enolase, L-lactate dehydrogenase and aldehyde dehydrogenase in glycolysis/gluconeogenesis, and ADP-glucose pyrophosphorylase, starch branching enzyme and glucan phosphorylase in sucrose and starch metabolism. This study revealed that dynamic changes in at least 16% of the transcriptome, including hundreds of transcription factors, oxidoreductases/transferases/hydrolases, hormone-related genes, and effectors of homeostasis, all of which highlight the complexity of this biological process. The reliability of differentially expressed genes in microarray analysis was further verified by quantitative real-time RT-PCR. The genome-wide transcription analysis facilitates our understanding of the formation of the storage root and deciphers key genes for further cassava improvement. Fibrous roots (FR), developing storage roots (DR) and mature storage roots (MR) were collected for RNA extractions from three independent healthy 4 month-old cassava (cultivar TMS60444) plants in the field .Two RNA samples extracted from stored storage root slices were used as technical repeats (TR) for quality control.
Project description:Mechanisms related to the development of cassava storage roots and starch accumulation remain largely unknown. To evaluate genome-wide expression patterns during cassava tuberization, a 60-mer oligonucleotide microarray representing 20,840 cassava genes was designed to identify differentially expressed transcripts in fibrous root, developing storage root and mature storage root. Using a random variance model and the traditional two-fold change method for statistical analysis, 912 and 3386 differentially expressed genes were identified related to the three different phases. Among 25 significant pathways identified, glycolysis/gluconeogenesis was the most important pathway signature due to its effects on other pathways. Rate-limiting enzymes were identified from each individual pathway, such as pectinesterase, enolase, L-lactate dehydrogenase and aldehyde dehydrogenase in glycolysis/gluconeogenesis, and ADP-glucose pyrophosphorylase, starch branching enzyme and glucan phosphorylase in sucrose and starch metabolism. This study revealed that dynamic changes in at least 16% of the transcriptome, including hundreds of transcription factors, oxidoreductases/transferases/hydrolases, hormone-related genes, and effectors of homeostasis, all of which highlight the complexity of this biological process. The reliability of differentially expressed genes in microarray analysis was further verified by quantitative real-time RT-PCR. The genome-wide transcription analysis facilitates our understanding of the formation of the storage root and deciphers key genes for further cassava improvement.