Integrative omics approaches revealed a crosstalk among phytohormones during tuberous root development in cassava.

KEY MESSAGE: Integrative omics approaches revealed a crosstalk among phytohormones during tuberous root development in cassava. 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, and indole-3-acetic acid conjugated with aspartic acid 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.

Enhancing of anthracnose disease resistance indicates a potential role of antimicrobial peptide genes in cassava.

Cassava (Manihot esculenta Crantz.) is an important economic crop in tropical countries. Demands for using cassava in food, feed and biofuel industries have been increasing worldwide. Cassava anthracnose disease, caused by Colletotrichum gloeosporioides f.sp. manihotis (CAD), is considered a major problem in cassava production. To minimize the effects of such disease, this study investigated the response of cassava to attack by CAD and how the plants defend themselves against this threat. Genome-wide identification of antimicrobial peptide genes (AMPs) and their expression in response to fungal infection was performed in the resistant cassava cultivar (Huay Bong 60; HB60) in comparison with the highly susceptible cultivar (Hanatee; HN). A total of 114 gene members of AMP were identified in the cassava genome database. Fifty-six gene members were selected for phylogenetic tree construction and analysis of putative cis-acting elements in their promoter regions. Differential expression profiles of six candidate genes were observed in response to CAD infection of both cassava cultivars. Upregulation of snakins, MeSN1 and MeSN2 was found in HB60, whereas MeHEL, Me-AMP-D2 and MeLTP2 were highly induced in HN. The MeLTP1 gene was not expressed in either cultivar. HB60 showed a reduced severity rating in comparison to HN after CAD infection. The biomembrane permeability test of fungal CAD was strongly affected after treatment with protein extract derived from CAD-infected HB60. Altogether, these findings suggest that snakins have a potential function in the CAD defense response in cassava. These results could be useful for cassava improvement programs to fight fungal pathogen.

Genome-wide analysis of aquaporin gene family and their responses to water-deficit stress conditions in cassava.

Cassava (Manihot esculenta Crantz) is an important economic crop in tropical countries. Although cassava is considered a drought-tolerant crop that can grow in arid areas, the impact of drought can significantly reduce the growth and yield of cassava storage roots. The discovery of aquaporin molecules (AQPs) in plants has resulted in a paradigm shift in the understanding of plant-water relationships, whereas the relationship between aquaporin and drought resistance in cassava still remains elusive. To investigate the potential role of aquaporin in cassava under water-deficit conditions, 45 putative MeAQPs were identified in the cassava genome. Six members of MeAQPs, containing high numbers of water stress-responsive motifs in their promoter regions, were selected for a gene expression study. Two cassava cultivars, which showed different degrees of responses to water-deficit stress, were used to test in in vitro and potted plant systems. The differential expression of all candidate MeAQPs were found in only leaves from the potted plant system were consistent with the relative water content and with the stomatal closure profile of the two cultivars. MePIP2-1 and MePIP2-10 were up-regulated and this change in their expression might regulate a special signal for water efflux out of guard cells, thus inducing stomatal closure under water-deficit conditions. In addition, the expression profiles of genes in the ABA-dependent pathway revealed an essential correlation with stomatal closure. The potential functions of MeAQPs and candidate ABA-dependent pathway genes in response to water deficit in the more tolerant cassava cultivar were discussed.

Gene Co-Expression Analysis Inferring the Crosstalk of Ethylene and Gibberellin in Modulating the Transcriptional Acclimation of Cassava Root Growth in Different Seasons.

Cassava is a crop of hope for the 21st century. Great advantages of cassava over other crops are not only the capacity of carbohydrates, but it is also an easily grown crop with fast development. As a plant which is highly tolerant to a poor environment, cassava has been believed to own an effective acclimation process, an intelligent mechanism behind its survival and sustainability in a wide range of climates. Herein, we aimed to investigate the transcriptional regulation underlying the adaptive development of a cassava root to different seasonal cultivation climates. Gene co-expression analysis suggests that AP2-EREBP transcription factor (ERF1) orthologue (D142) played a pivotal role in regulating the cellular response to exposing to wet and dry seasons. The ERF shows crosstalk with gibberellin, via ent-Kaurene synthase (D106), in the transcriptional regulatory network that was proposed to modulate the downstream regulatory system through a distinct signaling mechanism. While sulfur assimilation is likely to be a signaling regulation for dry crop growth response, calmodulin-binding protein is responsible for regulation in the wet crop. With our initiative study, we hope that our findings will pave the way towards sustainability of cassava production under various kinds of stress considering the future global climate change.

Genome-wide analysis reveals phytohormone action during cassava storage root initiation.

Development of storage roots is a process associated with a phase change from cell division and elongation to radial growth and accumulation of massive amounts of reserve substances such as starch. Knowledge of the regulation of cassava storage root formation has accumulated over time; however, gene regulation during the initiation and early stage of storage root development is still poorly understood. In this study, transcription profiling of fibrous, intermediate and storage roots at eight weeks old were investigated using a 60-mer-oligo microarray. Transcription and gene expression were found to be the key regulating processes during the transition stage from fibrous to intermediate roots, while homeostasis and signal transduction influenced regulation from intermediate roots to storage roots. Clustering analysis of significant genes and transcription factors (TF) indicated that a number of phytohormone-related TF were differentially expressed; therefore, phytohormone-related genes were assembled into a network of correlative nodes. We propose a model showing the relationship between KNOX1 and phytohormones during storage root initiation. Exogeneous treatment of phytohormones N (6) -benzylaminopurine and 1-Naphthaleneacetic acid were used to induce the storage root initiation stage and to investigate expression patterns of the genes involved in storage root initiation. The results support the hypothesis that phytohormones are acting in concert to regulate the onset of cassava storage root development. Moreover, MeAGL20 is a factor that might play an important role at the onset of storage root initiation when the root tip becomes swollen.

Biochemical characterization of a new glycosylated protease from Euphorbia cf. lactea latex.

A dimeric protease designated as EuP-82 was purified from Euphorbia cf. lactea latex. Since its proteolytic activity was inhibited by a serine protease specific inhibitor (PMSF), EuP-82 was classified as a serine protease. N-glycan deglycosylation tests revealed that EuP-82 was a glycosylated protein. MALDI-TOF MS showed that EuP-82 was a homodimer, which was its active form. The optimal conditions for fibrinogenolytic activity were at pH 11 and 35 °C. EuP-82 enzyme had broad range of pH stability from 4 to 12. Moreover, the enzyme was still active in the presence of reducing agent (ß-mercaptoethanol). EuP-82 was a proline-rich enzyme (about 20.69 mol%). Increased proline production can be found in higher plants in response to both biotic and abiotic stresses, high proline in the molecule of EuP-82 might stabilize its activity, structure and folding. Based on the N-terminal amino acid sequences and peptide mass fingerprint (PMF) of EuP-82, the enzyme was identified as a new serine protease. The digested products from EuP-82 cleavage of human fibrinogen were analyzed by SDS-PAGE and PMF. The results confirmed that EuP-82 could digest all subunits of human fibrinogen. EuP-82 cleaved fibrinogen with a Michaelis constant (Km) of 3.30 ± 0.26 µM; a maximal velocity (Vmax) of 400.9 ± 0.85 ng min(-1); and a catalytic efficiency (Vmax/Km) of 121.5 ± 9.25 ng µM(-1) min(-1). EuP-82 has potential for use in medicinal treatment, for example thrombosis, since the enzyme had fibrinogenolytic activity and high stability.

Gene flow and genetic structure of Bactrocera carambolae (Diptera, Tephritidae) among geographical differences and sister species, B. dorsalis, inferred from microsatellite DNA data.

The Carambola fruit fly, Bactrocera carambolae, is an invasive pest in Southeast Asia. It has been introduced into areas in South America such as Suriname and Brazil. Bactrocera carambolae belongs to the Bactrocera dorsalis species complex, and seems to be separated from Bactrocera dorsalis based on morphological and multilocus phylogenetic studies. Even though the Carambola fruit fly is an important quarantine species and has an impact on international trade, knowledge of the molecular ecology of Bactrocera carambolae, concerning species status and pest management aspects, is lacking. Seven populations sampled from the known geographical areas of Bactrocera carambolae including Southeast Asia (i.e., Indonesia, Malaysia, Thailand) and South America (i.e., Suriname), were genotyped using eight microsatellite DNA markers. Genetic variation, genetic structure, and genetic network among populations illustrated that the Suriname samples were genetically differentiated from Southeast Asian populations. The genetic network revealed that samples from West Sumatra (Pekanbaru, PK) and Java (Jakarta, JK) were presumably the source populations of Bactrocera carambolae in Suriname, which was congruent with human migration records between the two continents. Additionally, three populations of Bactrocera dorsalis were included to better understand the species boundary. The genetic structure between the two species was significantly separated and approximately 11% of total individuals were detected as admixed (0.100 ≤ Q ≤ 0.900). The genetic network showed connections between Bactrocera carambolae and Bactrocera dorsalis groups throughout Depok (DP), JK, and Nakhon Sri Thammarat (NT) populations. These data supported the hypothesis that the reproductive isolation between the two species may be leaky. Although the morphology and monophyly of nuclear and mitochondrial DNA sequences in previous studies showed discrete entities, the hypothesis of semipermeable boundaries may not be rejected. Alleles at microsatellite loci could be introgressed rather than other nuclear and mitochondrial DNA. Bactrocera carambolae may be an incipient rather than a distinct species of Bactrocera dorsalis. Regarding the pest management aspect, the genetic sexing Salaya5 strain (SY5) was included for comparison with wild populations. The SY5 strain was genetically assigned to the Bactrocera carambolae cluster. Likewise, the genetic network showed that the strain shared greatest genetic similarity to JK, suggesting that SY5 did not divert away from its original genetic makeup. Under laboratory conditions, at least 12 generations apart, selection did not strongly affect genetic compatibility between the strain and wild populations. This knowledge further confirms the potential utilization of the Salaya5 strain in regional programs of area-wide integrated pest management using SIT.

A protein from Aloe vera that inhibits the cleavage of human fibrin(ogen) by plasmin.

A protease inhibitor protein with the molecular mass of 11,804.931 Da (analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) was isolated from Aloe vera leaf gel and designated as AVPI-12. The isoelectric point of the protein is about 7.43. The first ten amino acid sequence from the N-terminal was found to be R-D-W-A-E-P-N-D-G-Y, which did not match other protease inhibitors in database searches and other publications, indicating AVPI-12 is a novel protease inhibitor. The band protein of AVPI-12 migrated further on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) than reducing SDS-PAGE. This result indicated that the molecule of AVPI-12 did not contain interchain disulfide bonds, but appeared to have intrachain disulfide bonds instead. AVPI-12 strongly resisted digestion by the serine proteases human plasmin and bovine trypsin. The protein could protect the γ-subunit of human fibrinogen from plasmin and trypsin digestion, similar to the natural plasma serine protease inhibitor α2-macroglobulin. The protein also could protect the γ-subunit of fibrinogen from the cysteine protease papain. AVPI-12 also exhibited dose-dependent inhibition of the fibrinogenolytic activity of plasmin, similar to α2-macroglobulin. The fibrinolytic inhibitory activity of AVPI-12 and the small-angle X-ray scattering showed that the protein could protect human fibrin clot from complete degradation by plasmin. The inhibition of the fibrinogenolytic and fibrinolytic activities of plasmin by AVPI-12 suggests that the inhibitor has potential for use in antifibrinolytic treatment.

Computational identification of microRNAs and their targets in cassava (Manihot esculenta Crantz.).

MicroRNAs (miRNAs) are a newly discovered class of noncoding endogenous small RNAs involved in plant growth and development as well as response to environmental stresses. miRNAs have been extensively studied in various plant species, however, only few information are available in cassava, which serves as one of the staple food crops, a biofuel crop, animal feed and industrial raw materials. In this study, the 169 potential cassava miRNAs belonging to 34 miRNA families were identified by computational approach. Interestingly, mes-miR319b was represented as the first putative mirtron demonstrated in cassava. A total of 15 miRNA clusters involving 7 miRNA families, and 12 pairs of sense and antisense strand cassava miRNAs belonging to six different miRNA families were discovered. Prediction of potential miRNA target genes revealed their functions involved in various important plant biological processes. The cis-regulatory elements relevant to drought stress and plant hormone response were identified in the promoter regions of those miRNA genes. The results provided a foundation for further investigation of the functional role of known transcription factors in the regulation of cassava miRNAs. The better understandings of the complexity of miRNA-mediated genes network in cassava would unravel cassava complex biology in storage root development and in coping with environmental stresses, thus providing more insights for future exploitation in cassava improvement.

Arabidopsis-derived shrimp viral-binding protein, PmRab7 can protect white spot syndrome virus infection in shrimp.

White spot syndrome virus is currently the leading cause of production losses in the shrimp industry. Penaeus monodon Rab7 protein has been recognized as a viral-binding protein with an efficient protective effect against white spot syndrome infection. Plant-derived recombinant PmRab7 might serve as an alternative source for in-feed vaccination, considering the remarkable abilities of plant expression systems. PmRab7 was introduced into the Arabidopsis thaliana T87 genome. Arabidopsis-derived recombinant PmRab7 showed high binding activity against white spot syndrome virus and a viral envelope, VP28. The growth profile of Arabidopsis suspension culture expressing PmRab7 (ECR21# 35) resembled that of its counterpart. PmRab7 expression in ECR21# 35 reached its maximum level at 5 mg g(-1) dry weight in 12 days, which was higher than those previously reported in Escherichia coli and in Pichia. Co-injection of white spot syndrome virus and Arabidopsis crude extract containing PmRab7 in Litopenaeus vannamei showed an 87% increase in shrimp survival rate at 5 day after injection. In this study, we propose an alternative PmRab7 source with higher production yield, and cheaper culture media costs, that might serve the industry's need for an in-feed supplement against white spot syndrome infection.