Application of ethanol alleviates heat damage to leaf growth and yield in tomato.

Chemical priming has emerged as a promising area in agricultural research. Our previous studies have demonstrated that pretreatment with a low concentration of ethanol enhances abiotic stress tolerance in Arabidopsis and cassava. Here, we show that ethanol treatment induces heat stress tolerance in tomato (Solanum lycopersicon L.) plants. Seedlings of the tomato cultivar 'Micro-Tom' were pretreated with ethanol solution and then subjected to heat stress. The survival rates of the ethanol-pretreated plants were significantly higher than those of the water-treated control plants. Similarly, the fruit numbers of the ethanol-pretreated plants were greater than those of the water-treated ones. Transcriptome analysis identified sets of genes that were differentially expressed in shoots and roots of seedlings and in mature green fruits of ethanol-pretreated plants compared with those in water-treated plants. Gene ontology analysis using these genes showed that stress-related gene ontology terms were found in the set of ethanol-induced genes. Metabolome analysis revealed that the contents of a wide range of metabolites differed between water- and ethanol-treated samples. They included sugars such as trehalose, sucrose, glucose, and fructose. From our results, we speculate that ethanol-induced heat stress tolerance in tomato is mainly the result of increased expression of stress-related genes encoding late embryogenesis abundant (LEA) proteins, reactive oxygen species (ROS) elimination enzymes, and activated gluconeogenesis. Our results will be useful for establishing ethanol-based chemical priming technology to reduce heat stress damage in crops, especially in Solanaceae.

Ethanol treatment enhances drought stress avoidance in cassava (Manihot esculenta Crantz).

External application of ethanol enhances tolerance to high salinity, drought, and heat stress in various plant species. However, the effects of ethanol application on increased drought tolerance in woody plants, such as the tropical crop "cassava," remain unknown. In the present study, we analyzed the morphological, physiological, and molecular responses of cassava plants subjected to ethanol pretreatment and subsequent drought stress treatment. Ethanol pretreatment induced a slight accumulation of abscisic acid (ABA) and stomatal closure, resulting in a reduced transpiration rate, higher water content in the leaves during drought stress treatment and the starch accumulation in leaves. Transcriptomic analysis revealed that ethanol pretreatment upregulated the expression of ABA signaling-related genes, such as PP2Cs and AITRs, and stress response and protein-folding-related genes, such as heat shock proteins (HSPs). In addition, the upregulation of drought-inducible genes during drought treatment was delayed in ethanol-pretreated plants compared with that in water-pretreated control plants. These results suggest that ethanol pretreatment induces stomatal closure through activation of the ABA signaling pathway, protein folding-related response by activating the HSP/chaperone network and the changes in sugar and starch metabolism, resulting in increased drought avoidance in plants.

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.

Suppressed expression of starch branching enzyme 1 and 2 increases resistant starch and amylose content and modifies amylopectin structure in cassava.

KEY MESSAGE: Suppression of starch branching enzymes 1 and 2 in cassava leads to increased resistant starch content through the production of high-amylose and modification of the amylopectin structure. Cassava (Manihot esculenta Crantz) is a starchy root crop used for human consumption as a staple food and industrial applications. Starch is synthesized by various isoforms of several enzymes. However, the function of starch branching enzymes (SBEs) in starch biosynthesis and mechanisms of starch regulation in cassava have not been understood well. In this study, we aimed to suppress the expression of SBEs in cassava to generate starches with a range of distinct properties, in addition to verifying the functional characteristics of the SBEs. One SBE1, two SBE2, and one SBE3 genes were classified by phylogenetic analysis and amino acid alignment. Quantitative real-time RT-PCR revealed tissue-specific expression of SBE genes in the tuberous roots and leaves of cassava. We introduced RNAi constructs containing fragments of SBE1, SBE2, or both genes into cassava by Agrobacterium-mediated transformation, and assessed enzymatic activity of SBE using tuberous roots and leaves from these transgenic plants. Simultaneous suppression of SBE1 and SBE2 rendered an extreme starch phenotype compared to suppression of SBE2 alone. Degree of polymerization of 6-13 chains in amylopectin was markedly reduced by suppression of both SBE1 and SBE2 in comparison to the SBE2 suppression; however, no change in chain-length profiles was observed in the SBE1 suppression alone. The role of SBE1 and SBE2 may have functional overlap in the storage tissue of cassava. Simultaneous suppression of SBE1 and SBE2 resulted in highly resistant starch with increased apparent amylose content compared to suppression of SBE2 alone. This study provides valuable information for understanding starch biosynthesis and suggests targets for altering starch quality.

Agrobacterium-mediated cassava transformation for the Asian elite variety KU50.

KEY MESSAGE: Cassava genetic transformation has mostly been reported for African cassava varieties, but not for Asian varieties. This is the first report of cassava transformation in Asian elite varieties using friable embryogenic calli. Agrobacterium-mediated cassava transformation via friable embryogenic calli (FEC) has enabled the robust production of transgenic cassava. So far, mostly the model cassava variety 60444 and African varieties have been transformed because of their good production and regeneration from embryogenic tissues. It is important to develop transformation methods for elite Asian cassava varieties to meet the changing needs in one of the world's major cassava production areas. However, a suitable transformation method for the Asian elite variety Kasetsart 50 (KU50) has not been developed. Here, we report a transformation method for KU50, the cultivar with the highest planting area in Thailand and Vietnam. In cassava transformation, the preparation of FEC as the target tissue for transgene integration is a key step. FEC induction from KU50 was improved by using media with reduced nutrients and excess vitamin B1, and somatic embryo and plant regeneration optimized by manipulation of naphthalene acetic acid (NAA), and benzylamino purine (BAP). The transformation efficiency for KU50 was 22%, approximately half that of 60444 at 45%. Transcriptome analysis indicated that the expression of genes related to cell-wall loosening was upregulated in FEC from KU50 compared with 60444, indicating that cell-wall production and assembly were disproportionate in the Asian variety. The transformation system for KU50 reported here will contribute to the molecular breeding of cassava plants for Asian farmers using transgenic and genome-editing technologies.

Intracellular localization of histone deacetylase HDA6 in plants.

Histone modification is an important epigenetic mechanism in eukaryotes. Histone acetyltransferase and deacetylase regulate histone acetylation levels antagonistically, leading to dynamic control of chromatin structure. One of the histone deacetylases, HDA6, is involved in gene silencing in the heterochromatin regions, chromocenter formation, and metabolic adaptation under drought stress. Although HDA6 plays an important role in chromatin control and response to drought stress, its intracellular localization has not been observed in detail. In this paper, we generated transformants expressing HDA6-GFP in the model plant, Arabidopsis thaliana, and the crops, rice, and cassava. We observed the localization of the fusion protein and showed that HDA6-GFP was expressed in the whole root and localized at the nucleus in Arabidopsis, rice, and cassava. Remarkably, HDA6-GFP clearly formed speckles that were actively colocalized with chromocenters in Arabidopsis root meristem. In contrast, such speckles were unlikely to be formed in rice or cassava. Because AtHDA6 directly binds to the acetate synthesis genes, which function in drought tolerance, we performed live imaging analyses to examine the cellular dynamics of pH in roots and the subnuclear dynamics of AtHDA6 responding to acetic acid treatment. The number of HDA6 speckles increased during drought stress, suggesting a role in contributing to drought stress tolerance.

Acetic Acid Treatment Enhances Drought Avoidance in Cassava (Manihot esculenta Crantz).

The external application of acetic acid has recently been reported to enhance survival of drought in plants such as Arabidopsis, rapeseed, maize, rice, and wheat, but the effects of acetic acid application on increased drought tolerance in woody plants such as a tropical crop "cassava" remain elusive. A molecular understanding of acetic acid-induced drought avoidance in cassava will contribute to the development of technology that can be used to enhance drought tolerance, without resorting to transgenic technology or advancements in cassava cultivation. In the present study, morphological, physiological, and molecular responses to drought were analyzed in cassava after treatment with acetic acid. Results indicated that the acetic acid-treated cassava plants had a higher level of drought avoidance than water-treated, control plants. Specifically, higher leaf relative water content, and chlorophyll and carotenoid levels were observed as soils dried out during the drought treatment. Leaf temperatures in acetic acid-treated cassava plants were higher relative to leaves on plants pretreated with water and an increase of ABA content was observed in leaves of acetic acid-treated plants, suggesting that stomatal conductance and the transpiration rate in leaves of acetic acid-treated plants decreased to maintain relative water contents and to avoid drought. Transcriptome analysis revealed that acetic acid treatment increased the expression of ABA signaling-related genes, such as OPEN STOMATA 1 (OST1) and protein phosphatase 2C; as well as the drought response and tolerance-related genes, such as the outer membrane tryptophan-rich sensory protein (TSPO), and the heat shock proteins. Collectively, the external application of acetic acid enhances drought avoidance in cassava through the upregulation of ABA signaling pathway genes and several stress responses- and tolerance-related genes. These data support the idea that adjustments of the acetic acid application to plants is useful to enhance drought tolerance, to minimize the growth inhibition in the agricultural field.

Formation of friable embryogenic callus in cassava is enhanced under conditions of reduced nitrate, potassium and phosphate.

Agrobacterium-mediated transformation is an important research tool for the genetic improvement of cassava. The induction of friable embryogenic callus (FEC) is considered as a key step in cassava transformation. In the present study, the media composition was optimized for enhancing the FEC induction, and the effect of the optimized medium on gene expression was evaluated. In relative comparison to MS medium, results demonstrated that using a medium with reducing nutrition (a 10-fold less concentration of nitrogen, potassium, and phosphate), the increased amount of vitamin B1 (10 mg/L) and the use of picrolam led to reprogram non-FEC to FEC. Gene expression analyses revealed that FEC on modified media increased the expression of genes related to the regulation of polysaccharide biosynthesis and breakdown of cell wall components in comparison to FEC on normal CIM media, whereas the gene expression associated with energy flux was not dramatically altered. It is hypothesized that we reprogram non-FEC to FEC under low nitrogen, potassium and phosphate and high vitamin B1. These findings were more effective in inducing FEC formation than the previous protocol. It might contribute to development of an efficient transformation strategy in cassava.

The Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid Alleviates Salinity Stress in Cassava.

Cassava (Manihot esculenta Crantz) demand has been rising because of its various applications. High salinity stress is a major environmental factor that interferes with normal plant growth and limits crop productivity. As well as genetic engineering to enhance stress tolerance, the use of small molecules is considered as an alternative methodology to modify plants with desired traits. The effectiveness of histone deacetylase (HDAC) inhibitors for increasing tolerance to salinity stress has recently been reported. Here we use the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), to enhance tolerance to high salinity in cassava. Immunoblotting analysis reveals that SAHA treatment induces strong hyper-acetylation of histones H3 and H4 in roots, suggesting that SAHA functions as the HDAC inhibitor in cassava. Consistent with increased tolerance to salt stress under SAHA treatment, reduced Na+ content and increased K+/Na+ ratio were detected in SAHA-treated plants. Transcriptome analysis to discover mechanisms underlying salinity stress tolerance mediated through SAHA treatment reveals that SAHA enhances the expression of 421 genes in roots under normal condition, and 745 genes at 2 h and 268 genes at 24 h under both SAHA and NaCl treatment. The mRNA expression of genes, involved in phytohormone [abscisic acid (ABA), jasmonic acid (JA), ethylene, and gibberellin] biosynthesis pathways, is up-regulated after high salinity treatment in SAHA-pretreated roots. Among them, an allene oxide cyclase (MeAOC4) involved in a crucial step of JA biosynthesis is strongly up-regulated by SAHA treatment under salinity stress conditions, implying that JA pathway might contribute to increasing salinity tolerance by SAHA treatment. Our results suggest that epigenetic manipulation might enhance tolerance to high salinity stress in cassava.

Functional interaction between plastidial starch phosphorylase and starch branching enzymes from rice during the synthesis of branched maltodextrins.

The present study established the way in which plastidial α-glucan phosphorylase (Pho1) synthesizes maltodextrin (MD) which can be the primer for starch biosynthesis in rice endosperm. The synthesis of MD by Pho1 was markedly accelerated by branching enzyme (BE) isozymes, although the greatest effect was exhibited by the presence of branching isozyme I (BEI) rather than by isozyme IIa (BEIIa) or isozyme IIb (BEIIb). The enhancement of the activity of Pho1 by BE was not merely due to the supply of a non-reducing ends. At the same time, Pho1 greatly enhanced the BE activity, possibly by generating a branched carbohydrate substrate which is used by BE with a higher affinity. The addition of isoamylase to the reaction mixture did not prevent the concerted action of Pho1 and BEI. Furthermore, in the product, the branched structure was, at least to some extent, maintained. Based on these results we propose that the interaction between Pho1 and BE is not merely due to chain-elongating and chain-branching reactions, but occurs in a physically and catalytically synergistic manner by each activating the mutual capacity of the other, presumably forming a physical association of Pho1, BEI and branched MDs. This close interaction might play a crucial role in the synthesis of branched MDs and the branched MDs can act as a primer for the biosynthesis of amylopectin molecules.

Functional diversity of isoamylase oligomers: the ISA1 homo-oligomer is essential for amylopectin biosynthesis in rice endosperm.

Rice (Oryza sativa) endosperm has two isoamylase (ISA) oligomers, ISA1 homo-oligomer and ISA1-ISA2 hetero-oligomer. To examine their contribution to starch synthesis, expression of the ISA1 or ISA2 gene was differently regulated in various transgenic plants. Although suppression of ISA2 gene expression caused the endosperm to have only the homo-oligomer, no significant effects were detected on the starch phenotypes. In contrast, ISA2 overexpression led to endosperm having only the hetero-oligomer, and starch synthesis in the endosperm was drastically impaired, both quantitatively and qualitatively, because the starch was devoid of typical starch features, such as thermal and x-ray diffraction properties, and water-soluble highly branched maltodextrins were accumulated. In the ISA2 overexpressed line, about 60% to 70% of the ISA1-ISA2 hetero-oligomer was bound to starch, while the ISA homo- and hetero-oligomers from the wild type were mostly present in the soluble form at the early milking stage of the endosperm. Detailed analysis of the relative amounts of homo- and hetero-oligomers in various lines also led us to the conclusion that the ISA1 homo-oligomer is essential, but not the ISA1-ISA2 oligomer, for starch production in rice endosperm. The relative amounts of ISA1 and ISA2 proteins were shown to determine the ratio of both oligomers and the stoichiometry of both ISAs in the hetero-oligomer. It was noted when compared with the homo-oligomer that all the hetero-oligomers from rice endosperm and leaf and potato (Solanum tuberosum) tuber were much more stable at 40°C. This study provides substantial data on the structural and functional diversity of ISA oligomers between plant tissues and species.

Characterization of the reactions of starch branching enzymes from rice endosperm.

To our knowledge the present paper shows for the first time the kinetic parameters of all the three starch branching enzyme (BE) isozymes, BEI, BEIIa and BEIIb, from rice with both amylopectin and synthetic amylose as glucan substrate. The activities of these BE isozymes with a linear glucan amylose decreased with a decrease in the molar size of amylose, and no activities of BEIIa and BEIIb were found when the degree of polymerization (DP) of amylose was lower than at least 80, whereas BEI had an activity with amylose of a DP higher than approximately 50. Detailed analyses of debranched products from BE reactions revealed the distinct chain length preferences of the individual BE isozymes. BEIIb almost exclusively transferred chains of DP7 and DP6 while BEIIa formed a wide range of short chains of DP6 to around DP15 from outer chains of amylopectin and amylose. On the other hand, BEI formed a variety of short chains and intermediate chains of a DP