Evaluation of potential increase in photosynthetic efficiency of cassava (Manihot esculenta Crantz) plants exposed to elevated carbon dioxide.

Cassava (Manihot esculenta Crantz), an important tropical crop, is affected by extreme climatic events, including rising CO2 levels. We evaluated the short-term effect of elevated CO2 concentration (ECO2 ) (600, 800 and 1000ppm) on the photosynthetic efficiency of 14 cassava genotypes. ECO2 significantly altered gaseous exchange parameters (net photosynthetic rate (P n ), stomatal conductance (g s ), intercellular CO2 (C i ) and transpiration (E )) in cassava leaves. There were significant but varying interactive effects between ECO2 and varieties on these physiological characteristics. ECO2 at 600 and 800ppm increased the P n rate in the range of 13-24% in comparison to 400ppm (ambient CO2 ), followed by acclimation at the highest concentration of 1000ppm. A similar trend was observed in g s and E . Conversely, C i increased significantly and linearly across increasing CO2 concentration. Along with C i , a steady increase in water use efficiency [WUEintrinsic (P n /g s ) and WUEinstantaneous (P n /E )] across various CO2 concentrations corresponded with the central role of restricted stomatal activity, a common response under ECO2 . Furthermore, P n had a significant quadratic relationship with the ECO2 (R 2 =0.489) and a significant and linear relationship with C i (R 2 =0.227). Relative humidity and vapour pressure deficit during the time of measurements remained at 70-85% and ~0.9-1.31kPa, respectively, at 26±2°C leaf temperature. Notably, not a single variety exhibited constant performance for any of the parameters across CO2 concentrations. Our results indicate that the potential photosynthesis can be increased up to 800ppm cassava varieties with high sink capacity can be cultivated under protected cultivation to attain higher productivity.

A high-affinity potassium transporter (MeHKT1) from cassava (Manihot esculenta) negatively regulates the response of transgenic Arabidopsis to salt stress.

BACKGROUND: High-affinity potassium transporters (HKTs) are crucial in facilitating potassium uptake by plants. Many types of HKTs confer salt tolerance to plants through regulating K+ and Na+ homeostasis under salinity stress. However, their specific functions in cassava (Manihot esculenta) remain unclear. RESULTS: Herein, an HKT gene (MeHKT1) was cloned from cassava, and its expression is triggered by exposure to salt stress. The expression of a plasma membrane-bound protein functions as transporter to rescue a low potassium (K+) sensitivity of yeast mutant strain, but the complementation of MeHKT1 is inhibited by NaCl treatment. Under low K+ stress, transgenic Arabidopsis with MeHKT1 exhibits improved growth due to increasing shoot K+ content. In contrast, transgenic Arabidopsis accumulates more Na+ under salt stress than wild-type (WT) plants. Nevertheless, the differences in K+ content between transgenic and WT plants are not significant. Additionally, Arabidopsis expressing MeHKT1 displayed a stronger salt-sensitive phenotype. CONCLUSION: These results suggest that under low K+ condition, MeHKT1 functions as a potassium transporter. In contrast, MeHKT1 mainly transports Na+ into cells under salt stress condition and negatively regulates the response of transgenic Arabidopsis to salt stress. Our results provide a reference for further research on the function of MeHKT1, and provide a basis for further application of MeHKT1 in cassava by molecular biological means.

Exogenous methyl jasmonate induced cassava defense response and enhanced resistance to Tetranychus urticae.

Exogenous application of methyl jasmonate (MeJA) could activate plant defense response against the two-spotted spider mite (TSSM), Tetranychus urticae Koch, in different plants. However, whether MeJA can also serve as an elicitor in cassava (Manihot esculenta Crantz) remains unknown. In this study, induced defense responses were investigated in TSSM-resistant cassava variety C1115 and TSSM-susceptible cassava variety KU50 when applied with MeJA. The performance of TSSM feeding on cassava plants that were pre-treated with various concentrations of MeJA was first evaluated. Subsequently, the activities of antioxidative enzymes (superoxide dismutase and catalase), detoxification enzymes (glutathione S-transferase, cytochrome P450 and carboxylesterase) and digestive enzymes (protease, amylase and invertase) in TSSM were analyzed at days 1, 2, 4 and 8 post-feeding. The results showed that MeJA treatment can induce cassava defense responses to TSSM in terms of reducing egg production and adult longevity as well as slowing development and prolonging the egg stage. Noticeably, C1115 exhibited stronger inhibition of TSSM development and reproduction than KU50. In addition, the activities of all the tested enzymes were induced in both C1115 and KU50, the most in C1115. We conclude that exogenous methyl jasmonate can induce cassava defense responses and enhance resistance to TSSM.

Correlation Between Relative Gene Expression Patterns of Two Flowering locus T (MeFT1 and MeFT2) in Cassava Leaf and Flowering Traits Under Different Flowering Induction Conditions.

<b>Background and Objective:</b> <i>Flowering locus T</i> (<i>FT</i>) genes are involved in the flower induction mechanism in plants as florigen signals. The objective of this study was to study the relationship between the expression of <i>Flowering locus T</i> genes (<i>MeFTs</i>) in cassava and flowering traits under the different flowering induction conditions. <b>Materials and Methods:</b> The experimental design for flowering induction was RCBD for 4 replications. There were 5 treatment factors which were control, red light set from 5 pm to 7 am, 0.5 mM 6-benzyladenine (BA) with 2 mM silver thio-sulfate (STS), paclobutrazol for 6 g/plant and potassium chlorate (KCIO₃) for 250 g/plant. The number of plants with flower bunches and the average number of bunches per plant in two cassava varieties were collected each month from 5-9 months after planting (MAP). The leaf samples were collected from HB80 and R9 varieties at 5-7 MAP for RNA extraction to study <i>MeFT1</i> and <i>MeFT2</i> expression. <b>Results:</b> The results show that <i>MeFT1</i> expression level positively correlated with flowering traits in the same month, while <i>MeFT2</i> expression level positively correlated with flowering traits in the following months. <b>Conclusion:</b> Therefore, expression of <i>MeFT2</i> can be used for the prediction of cassava flowering in the following month which will assist the breeder for the crossing management.

Image-based phenotyping of cassava roots for diversity studies and carotenoids prediction.

Phenotyping to quantify the total carotenoids content (TCC) is sensitive, time-consuming, tedious, and costly. The development of high-throughput phenotyping tools is essential for screening hundreds of cassava genotypes in a short period of time in the biofortification program. This study aimed to (i) use digital images to extract information on the pulp color of cassava roots and estimate correlations with TCC, and (ii) select predictive models for TCC using colorimetric indices. Red, green and blue images were captured in root samples from 228 biofortified genotypes and the difference in color was analyzed using L*, a*, b*, hue and chroma indices from the International Commission on Illumination (CIELAB) color system and lightness. Colorimetric data were used for principal component analysis (PCA), correlation and for developing prediction models for TCC based on regression and machine learning. A high positive correlation between TCC and the variables b* (r = 0.90) and chroma (r = 0.89) was identified, while the other correlations were median and negative, and the L* parameter did not present a significant correlation with TCC. In general, the accuracy of most prediction models (with all variables and only the most important ones) was high (R2 ranging from 0.81 to 0.94). However, the artificial neural network prediction model presented the best predictive ability (R2 = 0.94), associated with the smallest error in the TCC estimates (root-mean-square error of 0.24). The structure of the studied population revealed five groups and high genetic variability based on PCA regarding colorimetric indices and TCC. Our results demonstrated that the use of data obtained from digital image analysis is an economical, fast, and effective alternative for the development of TCC phenotyping tools in cassava roots with high predictive ability.

Character changes and Transcriptomic analysis of a cassava sexual Tetraploid.

BACKGROUND: Cassava (Manihot esculenta Crantz) is an important food crop known for its high starch content. Polyploid breeding is effective in its genetic improvement, and use of 2n gametes in sexual polyploid breeding is one of the potential methods for cassava breeding and improvement. In our study, the cassava sexual tetraploid (ST), which carries numerous valuable traits, was successfully generated by hybridizing 2n female gametes SC5 (♀) and 2n male gametes SC10 (♂). However, the molecular mechanisms remain unclear. To understand these underlying molecular mechanisms behind the phenotypic alterations and heterosis in ST plants, we investigated the differences in gene expression between polyploids and diploids by determining the transcriptomes of the ST plant and its parents during the tuber root enlargement period. We also compared the characters and transcriptomes of the ST plant with its parents. RESULTS: The ST plant was superior in plant height, stem diameter, leaf area, petiole length, plant weight, and root weight than the parent plants, except the leaf number, which was lower. The number of starch granules was higher in the roots of ST plants than those in the parent plants after five months (tuber root enlargement period), which could be due to a higher leaf net photosynthetic rate leading to early filling of starch granules. Based on transcriptome analysis, we identified 2934 and 3171 differentially expressed genes (DEGs) in the ST plant as compared to its female and male parents, respectively. Pathway enrichment analyses revealed that flavonoid biosynthesis and glycolysis/gluconeogenesis were significantly enriched in the ST plants, which might contribute to the colors of petiole (purple-red), root epidermis (dark brown), and tuber starch accumulation, respectively. CONCLUSIONS: After sexual polyploidization, the phenotype of ST has changed significantly in comparison to their diploid parents, mainly manifest as enlarged biomass, yield, early starch filling, deep colored petiole and root epidermis. The tetraploid plants were also mature early due to early starch grain filling. Owing to enriched flavonoid biosynthesis and glycolysis/gluconeogenesis, they are possibly resistant to adversity stresses and provide better yield, respectively.

Identification of cassava alternative splicing-related genes and functional characterization of MeSCL30 involvement in drought stress.

Alternative splicing (AS) is an important post-transcriptional regulation strategy that can increase the proteome diversity and regulate mRNA level in eukaryote. Multi-exon genes can be alternative spliced to generate two or more transcripts, thereby increasing the adaptation to the external stress conditions in planta. However, AS-related proteins were less explored in cassava which is an important staple crop in the tropical area. A total of 365 genes encoding AS-related proteins were identified and renamed in the cassava genome, and the transcriptional and splicing changes of 15 randomly selected genes were systematically investigated in the tissues under diverse abiotic stress conditions. 13 out of 15 genes undergo AS in the tissues and under diverse environmental stress condition. Importantly, the greatest changes of splicing patterns were found in the leaf or in response to temperature stress, indicating that AS-related proteins had their tissue-specific regulation patterns and might be participated in the plant adaptation to temperature stress. We then found that overexpression of MeSCL30 in Arabidopsis enhanced the tolerance to drought stress through maintaining reactive oxygen species (ROS) homeostasis and increasing the expression of drought-responsive genes. Therefore, these findings refined the AS-related protein-coding genes and provided novel insights for manipulation of AS-related genes in order to enhance the resistance to environmental stress in plant.

The metabotyping of an East African cassava diversity panel: A core collection for developing biotic stress tolerance in cassava.

Cassava will have a vital role to play, if food security is to be achieved in Sub-Saharan Africa, especially Central and East Africa. The whitefly Bemisia tabaci poses a major threat to cassava production by small holder farmers in part due to their role as a vector of cassava mosaic begomoviruses (CMBs) and cassava brown streak ipomoviruses (CBSIs). In the present study untargeted metabolomics has been used as a tool to assess natural variation, similarities and attempts to identify trait differentiators among an East African cassava diversity panel that displayed tolerance/resistance to the effects of Bemisia tabaci infestation. The metabolome captured, was represented by 1529 unique chemical features per accession. Principal component analysis (PCA) identified a 23% variation across the panel, with geographical origin/adaption the most influential classification factors. Separation based on resistance and susceptible traits to Bemisia tabaci could also be observed within the data and was corroborated by genotyping data. Thus the metabolomics pipeline represented an effective metabotyping approach. Agglomerative Hierarchical Clustering Analysis (HCA) of both the metabolomics and genotyping data was performed and revealed a high level of similarity between accessions. Specific differentiating features/metabolites were identified, including those potentially conferring vigour to whitefly tolerance on a constitutive manner. The implications of using these cassava varieties as parental breeding material and the future potential of incorporating more exotic donor material is discussed.

Genetic analysis and QTL mapping for multiple biotic stress resistance in cassava.

In Sub-Saharan Africa cassava (Manihot esculenta Crantz) is one of the most important food crops where more than 40% of the population relies on it as their staple carbohydrate source. Biotic constraints such as viral diseases, mainly Cassava Mosaic Disease (CMD) and Cassava Brown Streak Disease (CBSD), and arthropod pests, particularly Cassava Green Mite (CGM), are major constraints to the realization of cassava's full production potential in Africa. To address these problems, we aimed to map the quantitative trait loci (QTL) associated with resistance to CBSD foliar and root necrosis symptoms, foliar CMD and CGM symptoms in a full-sib mapping population derived from the genotypes AR40-6 and Albert. A high-density linkage map was constructed with 2,125 SNP markers using a genotyping-by-sequencing approach. For phenotyping, clonal evaluation trials were conducted with 120 F1 individuals for two consecutive field seasons using an alpha-lattice design at Chambezi and Naliendele, Tanzania. Previously identified QTL for resistance to CBSD foliar symptoms were corroborated, and a new putative QTL for CBSD root necrosis identified (qCBSDRNc14AR) from AR40-6. Two QTL were identified within the region of the previously recognized CMD2 locus from this population in which both parents are thought to possess the CMD2 locus. Interestingly, a minor but consistent QTL, qCGM18AR, for CGM resistance at 3 months after planting stage was also detected and co-localized with a previously identified SSR marker, NS346, linked with CGM resistance. Markers underlying these QTL may be used to increase efficiencies in cassava breeding programs.

Improving root characterisation for genomic prediction in cassava.

Cassava is cultivated due to its drought tolerance and high carbohydrate-containing storage roots. The lack of uniformity and irregular shape of storage roots poses constraints on harvesting and post-harvest processing. Here, we phenotyped the Genetic gain and offspring (C1) populations from the International Institute of Tropical Agriculture (IITA) breeding program using image analysis of storage root photographs taken in the field. In the genome-wide association analysis (GWAS), we detected for most shape and size-related traits, QTL on chromosomes 1 and 12. In a previous study, we found the QTL on chromosome 12 to be associated with cassava mosaic disease (CMD) resistance. Because the root uniformity is important for breeding, we calculated the standard deviation (SD) of individual root measurements per clone. With SD measurements we identified new significant QTL for Perimeter, Feret and Aspect Ratio on chromosomes 6, 9 and 16. Predictive accuracies of root size and shape image-extracted traits were mostly higher than yield trait prediction accuracies. This study aimed to evaluate the feasibility of the image phenotyping protocol and assess GWAS and genomic prediction for size and shape image-extracted traits. The methodology described and the results are promising and open up the opportunity to apply high-throughput methods in cassava.

Genomic and Transcriptomic Analysis Identified Novel Putative Cassava lncRNAs Involved in Cold and Drought Stress.

Long non-coding RNAs (lncRNAs) play important roles in the regulation of complex cellular processes, including transcriptional and post-transcriptional regulation of gene expression relevant for development and stress response, among others. Compared to other important crops, there is limited knowledge of cassava lncRNAs and their roles in abiotic stress adaptation. In this study, we performed a genome-wide study of ncRNAs in cassava, integrating genomics- and transcriptomics-based approaches. In total, 56,840 putative ncRNAs were identified, and approximately half the number were verified using expression data or previously known ncRNAs. Among these were 2229 potential novel lncRNA transcripts with unmatched sequences, 250 of which were differentially expressed in cold or drought conditions, relative to controls. We showed that lncRNAs might be involved in post-transcriptional regulation of stress-induced transcription factors (TFs) such as zinc-finger, WRKY, and nuclear factor Y gene families. These findings deepened our knowledge of cassava lncRNAs and shed light on their stress-responsive roles.

Genetic variation and evolutionary history of a mycorrhizal fungus regulate the currency of exchange in symbiosis with the food security crop cassava.

Most land plants form symbioses with arbuscular mycorrhizal fungi (AMF). Diversity of AMF increases plant community productivity and plant diversity. For decades, it was known that plants trade carbohydrates for phosphate with their fungal symbionts. However, recent studies show that plant-derived lipids probably represent the most essential currency of exchange. Understanding the regulation of plant genes involved in the currency of exchange is crucial to understanding stability of this mutualism. Plants encounter many different AMF genotypes that vary greatly in the benefit they confer to plants. Yet the role that fungal genetic variation plays in the regulation of this currency has not received much attention. We used a high-resolution phylogeny of one AMF species (Rhizophagus irregularis) to show that fungal genetic variation drives the regulation of the plant fatty acid pathway in cassava (Manihot esculenta); a pathway regulating one of the essential currencies of trade in the symbiosis. The regulation of this pathway was explained by clearly defined patterns of fungal genome-wide variation representing the precise fungal evolutionary history. This represents the first demonstrated link between the genetics of AMF and reprogramming of an essential plant pathway regulating the currency of exchange in the symbiosis. The transcription factor RAM1 was also revealed as the dominant gene in the fatty acid plant gene co-expression network. Our study highlights the crucial role of variation in fungal genomes in the trade of resources in this important symbiosis and also opens the door to discovering characteristics of AMF genomes responsible for interactions between AMF and cassava that will lead to optimal cassava growth.

Large-scale analysis of the cassava transcriptome reveals the impact of cold stress on alternative splicing.

Alternative splicing is an essential post-transcriptional regulatory mechanism that can impact mRNA stability and protein diversity of eukaryotic genomes. Although numerous forms of stress-responsive alternative splicing have been identified in model plants, a large-scale study of alternative splicing dynamics under abiotic stress conditions in cassava has not been conducted. Here, we report the parallel employment of isoform-Seq, ssRNA-Seq, and Degradome-Seq to investigate the diversity, abundance, and fate of alternatively spliced isoforms in response to cold and drought stress. We identified 38 164 alternative splicing events, among which 3292 and 1025 events were significantly regulated by cold and drought stress, respectively. Intron retention was the most abundant subtype of alternative splicing. Global analysis of splicing regulators revealed that the number of their alternatively spliced isoforms and the corresponding abundance were specifically modulated by cold stress. We found that 58.5% of cold-regulated alternative splicing events introduced a premature termination codon into the transcripts, and 77.6% of differential alternative splicing events were detected by Degradome-Seq. Our data reveal that cold intensely affects both quantitative and qualitative aspects of gene expression via alternative splicing pathways, and advances our understanding of the high complexity and specificity of gene regulation in response to abiotic stresses. Alternative splicing is responsible for reprogramming of the transcriptome and the sensitivity of cassava plants to cold.

Genome-Wide Identification of Cassava Serine/Arginine-Rich Proteins: Insights into Alternative Splicing of Pre-mRNAs and Response to Abiotic Stress.

Serine/arginine-rich (SR) proteins have an essential role in the splicing of pre-messenger RNA (pre-mRNA) in eukaryote. Pre-mRNA with introns can be alternatively spliced to generate multiple transcripts, thereby increasing adaptation to the external stress conditions in planta. However, pre-mRNA of SR proteins can also be alternatively spliced in different plant tissues and in response to diverse stress treatments, indicating that SR proteins might be involved in regulating plant development and adaptation to environmental changes. We identified and named 18 SR proteins in cassava and systematically studied their splicing and transcriptional changes under tissue-specific and abiotic stress conditions. Fifteen out of 18 SR genes showed alternative splicing in the tissues. 45 transcripts were found from 18 SR genes under normal conditions, whereas 55 transcripts were identified, and 21 transcripts were alternate spliced in some SR genes under salt stress, suggesting that SR proteins might participate in the plant adaptation to salt stress. We then found that overexpression of MeSR34 in Arabidopsis enhanced the tolerance to salt stress through maintaining reactive oxygen species homeostasis and increasing the expression of calcineurin B-like proteins (CBL)-CBL-interacting protein kinases and osmotic stress-related genes. Therefore, our findings highlight the critical role of cassava SR proteins as regulators of RNA splicing and salt tolerance in planta.

The chaperone MeHSP90 recruits MeWRKY20 and MeCatalase1 to regulate drought stress resistance in cassava.

The 90 kDa heat shock protein (HSP90) is widely involved in various developmental processes and stress responses in plants. However, the molecular chaperone HSP90-constructed protein complex and its function in cassava remain elusive. In this study, we report that HSP90 is essential for drought stress resistance in cassava by regulating abscisic acid (ABA) and hydrogen peroxide (H2 O2 ) using two specific protein inhibitors of HSP90 (geldanamycin (GDA) and radicicol (RAD)). Among 10 MeHSP90s, the transcript of MeHSP90.9 is largely induced during drought stress. Further investigation identifies MeWRKY20 and MeCatalase1 as MeHSP90.9-interacting proteins. MeHSP90.9-, MeWRKY20-, or MeCatalase1-silenced plants through virus-induced gene silencing display drought sensitivity in cassava, indicating that they are important to drought stress response. MeHSP90.9 can promote the direct transcriptional activation of MeWRKY20 on the W-box element of MeNCED5 promoter, encoding a key enzyme in ABA biosynthesis. Moreover, MeHSP90.9 positively regulates the activity of MeCatalase1, and MeHSP90.9-silenced cassava leaves accumulate more H2 O2 under drought stress. Taken together, we demonstrate that the MeHSP90.9 chaperone complex is a regulator of drought stress resistance in cassava.

Response of cassava cultivars to African cassava mosaic virus infection across a range of inoculum doses and plant ages.

Cassava production in Africa is constrained by cassava mosaic disease (CMD) that is caused by the Cassava mosaic virus (CMV). The aim of this study was to evaluate the responses of a range of commonly cultivated West African cassava cultivars to varying inoculum doses of African cassava mosaic virus (ACMV). We grafted 10 cultivars of cassava plants with different inoculum doses of CMV (namely two, four, or six CMD-infected buds) when the experimental plants were 8, 10, or 12 weeks old, using non-inoculated plants as controls. Three cultivars showed disease symptoms when grafted with two buds, and four cultivars showed disease symptoms when grafted with four or six buds. Most cultivars became symptomatic six weeks after inoculation, but one ('TMS92/0326') was symptomatic two weeks after inoculation, and two ('Ntollo' and 'Excel') were symptomatic after four weeks. Root weight tended to be lower in the six-bud than in the two-bud dose, and disease severity varied with plant age at inoculation. These results indicate that the level of CMD resistance in cassava cultivars varies with inoculum dose and timing of infection. This will allow appropriate cultivars to be deployed in each production zone of Africa in accordance with the prevalence of CMD.

Involvement of abscisic acid-responsive element-binding factors in cassava (Manihot esculenta) dehydration stress response.

Cassava (Manihot esculenta) is a major staple food, animal feed and energy crop in the tropics and subtropics. It is one of the most drought-tolerant crops, however, the mechanisms of cassava drought tolerance remain unclear. Abscisic acid (ABA)-responsive element (ABRE)-binding factors (ABFs) are transcription factors that regulate expression of target genes involved in plant tolerance to drought, high salinity, and osmotic stress by binding ABRE cis-elements in the promoter regions of these genes. However, there is little information about ABF genes in cassava. A comprehensive analysis of Manihot esculenta ABFs (MeABFs) described the phylogeny, genome location, cis-acting elements, expression profiles, and regulatory relationship between these factors and Manihot esculenta betaine aldehyde dehydrogenase genes (MeBADHs). Here we conducted genome-wide searches and subsequent molecular cloning to identify seven MeABFs that are distributed unevenly across six chromosomes in cassava. These MeABFs can be clustered into three groups according to their phylogenetic relationships to their Arabidopsis (Arabidopsis thaliana) counterparts. Analysis of the 5'-upstream region of MeABFs revealed putative cis-acting elements related to hormone signaling, stress, light, and circadian clock. MeABF expression profiles displayed clear differences among leaf, stem, root, and tuberous root tissues under non-stress and drought, osmotic, or salt stress conditions. Drought stress in cassava leaves and roots, osmotic stress in tuberous roots, and salt stress in stems induced expression of the highest number of MeABFs showing significantly elevated expression. The glycine betaine (GB) content of cassava leaves also was elevated after drought, osmotic, or salt stress treatments. BADH1 is involved in GB synthesis. We show that MeBADH1 promoter sequences contained ABREs and that MeBADH1 expression correlated with MeABF expression profiles in cassava leaves after the three stress treatments. Taken together, these results suggest that in response to various dehydration stresses, MeABFs in cassava may activate transcriptional expression of MeBADH1 by binding the MeBADH1 promoter that in turn promotes GB biosynthesis and accumulation via an increase in MeBADH1 gene expression levels and MeBADH1 enzymatic activity. These responses protect cells against dehydration stresses by preserving an osmotic balance that enhances cassava tolerance to dehydration stresses.

The chloroplast proteome response to drought stress in cassava leaves.

Cassava is an important tropical crop with strong resistance to drought stress. The chloroplast, the site of photosynthesis, is sensitive to stress, and the drought-response proteins in cassava chloroplasts are worthy of investigation. In this study, cassava leaves were collected for ultra-structure observation from plants subjected to different drought stress conditions. Our results showed that drought stress can promote starch accumulation in cassava chloroplasts. To evaluate changes in chloroplast proteins under different drought conditions, two-dimensional electrophoresis was performed using purified chloroplasts, which resulted in the identification of 26 unique chloroplast proteins responsive to drought stress. These drought-responsive proteins are predominantly related to photosynthesis, carbon and nitrogen metabolism, and amino acid metabolism. Among them, most photosynthesis-related proteins are downregulated, with decreases in photosynthetic parameters upon drought stress. Several proteins associated with carbon and nitrogen metabolism, including rubisco and carbonic anhydrase, were upregulated, which might promote drought tolerance in cassava by enhancing the carbohydrate conversion efficiency and protecting the plant from oxidative stress. Our proteomic data not only provide insight into the complement of proteins in cassava chloroplasts but also further our overall understanding of drought-responsive proteins in cassava chloroplasts.

Transcriptomic and proteomic response of Manihot esculenta to Tetranychus urticae infestation at different densities.

Tetranychus urticae (Acari: Tetranychidae) is an extremely serious cassava (Manihot esculenta) pest. Building a genomic resource to investigate the molecular mechanisms of cassava responses to T. urticae is vital for characterizing cassava resistance to mites. Based on the tolerance of cassava varieties to mite infestation (focusing on mite development rate, fecundity and physiology), cassava variety SC8 was selected to analyze transcriptomic and proteomic changes after 5 days of T. urticae feeding. Transcriptomic analysis revealed 698 and 2140 genes with significant expression changes under low and high mite infestation, respectively. More defense-related genes were found in the enrichment pathways at high mite density than at low density. In addition, iTRAQ-labeled proteomic analysis revealed 191 proteins with significant expression changes under low mite infestation. Differentially expressed genes and proteins were mainly found in the following defense-related pathways: flavonoid biosynthesis, phenylpropanoid biosynthesis, and glutathione metabolism under low-density mite feeding and plant hormone signal transduction and plant-pathogen interaction pathways under high-density mite feeding. The plant hormone signal transduction network, involving ethylene, jasmonic acid, and salicylic acid transduction pathways, was explored in relation to the M. esculenta response to T. urticae. Correlation analysis of the transcriptome and proteome generated a Pearson correlation coefficients of R = 0.2953 (P < 0.01), which might have been due to post-transcriptional or post-translational regulation resulting in many genes being inconsistently expressed at both the transcript and protein levels. In summary, the M. esculenta transcriptome and proteome changed in response to T. urticae, providing insight into the general activation of plant defense pathways in response to mite infestation.

Symplasmic phloem unloading and radial post-phloem transport via vascular rays in tuberous roots of Manihot esculenta.

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 improving storage root yield. Here, 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, 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 form the basis for biotechnological approaches aimed at improved phloem loading and enhanced carbohydrate allocation and storage in order to increase tuberous root yield of cassava.