Jurassic NLR: Conserved and dynamic evolutionary features of the atypically ancient immune receptor ZAR1.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors generally exhibit hallmarks of rapid evolution, even at the intraspecific level. We used iterative sequence similarity searches coupled with phylogenetic analyses to reconstruct the evolutionary history of HOPZ-ACTIVATED RESISTANCE1 (ZAR1), an atypically conserved NLR that traces its origin to early flowering plant lineages ∼220 to 150 million yrs ago (Jurassic period). We discovered 120 ZAR1 orthologs in 88 species, including the monocot Colocasia esculenta, the magnoliid Cinnamomum micranthum, and most eudicots, notably the Ranunculales species Aquilegia coerulea, which is outside the core eudicots. Ortholog sequence analyses revealed highly conserved features of ZAR1, including regions for pathogen effector recognition and cell death activation. We functionally reconstructed the cell death activity of ZAR1 and its partner receptor-like cytoplasmic kinase (RLCK) from distantly related plant species, experimentally validating the hypothesis that ZAR1 evolved to partner with RLCKs early in its evolution. In addition, ZAR1 acquired novel molecular features. In cassava (Manihot esculenta) and cotton (Gossypium spp.), ZAR1 carries a C-terminal thioredoxin-like domain, and in several taxa, ZAR1 duplicated into 2 paralog families, which underwent distinct evolutionary paths. ZAR1 stands out among angiosperm NLR genes for having experienced relatively limited duplication and expansion throughout its deep evolutionary history. Nonetheless, ZAR1 also gave rise to noncanonical NLRs with integrated domains and degenerated molecular features.

Fully sequencing the cassava full-length cDNA library reveals unannotated transcript structures and alternative splicing events in regions with a high density of single nucleotide variations, insertions-deletions, and heterozygous sequences.

The primary transcript structure provides critical insights into protein diversity, transcriptional modification, and functions. Cassava transcript structures are highly diverse because of alternative splicing (AS) events and high heterozygosity. To precisely determine and characterize transcript structures, fully sequencing cloned transcripts is the most reliable method. However, cassava annotations were mainly determined according to fragmentation-based sequencing analyses (e.g., EST and short-read RNA-seq). In this study, we sequenced the cassava full-length cDNA library, which included rare transcripts. We obtained 8,628 non-redundant fully sequenced transcripts and detected 615 unannotated AS events and 421 unannotated loci. The different protein sequences resulting from the unannotated AS events tended to have diverse functional domains, implying that unannotated AS contributes to the truncation of functional domains. The unannotated loci tended to be derived from orphan genes, implying that the loci may be associated with cassava-specific traits. Unexpectedly, individual cassava transcripts were more likely to have multiple AS events than Arabidopsis transcripts, suggestive of the regulated interactions between cassava splicing-related complexes. We also observed that the unannotated loci and/or AS events were commonly in regions with abundant single nucleotide variations, insertions-deletions, and heterozygous sequences. These findings reflect the utility of completely sequenced FLcDNA clones for overcoming cassava-specific annotation-related problems to elucidate transcript structures. Our work provides researchers with transcript structural details that are useful for annotating highly diverse and unique transcripts and alternative splicing events.

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.

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.

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.

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.

Cassava mosaic disease and its management in Southeast Asia.

KEY MESSAGE: Status of the current outbreak of cassava mosaic disease (CMD) in Southeast Asia was reviewed. Healthy cassava seed production and dissemination systems have been established in Vietnam and Cambodia, along with integrated disease and pest management systems, to combat the outbreak. Cassava (Manihot esculenta Crantz) is one of the most important edible crops in tropical and subtropical regions. Recently, invasive insect pests and diseases have resulted in serious losses to cassava in Southeast Asia. In this review we discuss the current outbreak of cassava mosaic disease (CMD) caused by the Sri Lankan cassava mosaic virus (SLCMV) in Southeast Asia, and summarize similarities between SLCMV and other cassava mosaic begomoviruses. A SATREPS (Science and Technology Research Partnership for Sustainable Development) project "Development and dissemination of sustainable production systems based on invasive pest management of cassava in Vietnam, Cambodia and Thailand", was launched in 2016, which has been funded by The Japan International Cooperation Agency (JICA) and The Japan Science and Technology Agency (JST), Japan. The objectives of SATREPS were to establish healthy seed production and dissemination systems for cassava in south Vietnam and Cambodia, and to develop management systems for plant diseases and insect pests of cassava. To achieve these goals, model systems of healthy seed production in Vietnam and Cambodia have been developed incorporating CMD-resistant planting materials through international networks with The International Center for Tropical Agriculture (CIAT) and The International Institute of Tropical Agriculture (IITA).

Field transcriptome analysis reveals a molecular mechanism for cassava-flowering in a mountainous environment in Southeast Asia.

KEY MESSAGE: The field survey in this article showed in 'KU50', a popular variety and late-branching type of cassava in Southeast Asia, that flowering rarely occurs in normal-field conditions in Southeast Asia but is strongly induced in the dry season in the mountainous region. Flowering time is correlated with the expression patterns of MeFT1 and homologs of Arabidopsis GI, PHYA, and NF-Ys. Cassava (Manihot esculenta Crantz) is a tropical crop that is propagated vegetatively rather than sexually by seed. Flowering rarely occurs in the erect-type variety grown in Southeast Asia, but it is known that cassava produces flowers every year in mountainous regions. Data pertaining to the effect of environmental factors on flowering time and gene expression in cassava, however, is limited. The aim of the present study was to determine the kinds of environmental conditions that regulate flowering time in cassava and the underlying molecular mechanisms. The flowering status of KU50, a popular variety in Southeast Asia and late-branching type of cassava, was monitored in six fields in Vietnam and Cambodia. At non-flowering and flowering field locations in North Vietnam, the two FLOWERING LOCUS T (FT)-like genes, MeFT1 and MeFT2, were characterized by qPCR, and the pattern of expression of flowering-related genes and genes responsive to environmental signals were analyzed by using RNA sequencing data from time-series samples. Results indicate that cassava flowering was induced in the dry season in the mountain region, and that flowering time was correlated with the expression of MeFT1, and homologs of Arabidopsis GI, PHYA, and NF-Ys. Based upon these data, we hypothesize that floral induction in cassava is triggered by some conditions present in the mountain regions during the dry season.

Ethanol-Mediated Novel Survival Strategy against Drought Stress in Plants.

Water scarcity is a serious agricultural problem causing significant losses to crop yield and product quality. The development of technologies to mitigate the damage caused by drought stress is essential for ensuring a sustainable food supply for the increasing global population. We herein report that the exogenous application of ethanol, an inexpensive and environmentally friendly chemical, significantly enhances drought tolerance in Arabidopsis thaliana, rice and wheat. The transcriptomic analyses of ethanol-treated plants revealed the upregulation of genes related to sucrose and starch metabolism, phenylpropanoids and glucosinolate biosynthesis, while metabolomic analysis showed an increased accumulation of sugars, glucosinolates and drought-tolerance-related amino acids. The phenotyping analysis indicated that drought-induced water loss was delayed in the ethanol-treated plants. Furthermore, ethanol treatment induced stomatal closure, resulting in decreased transpiration rate and increased leaf water contents under drought stress conditions. The ethanol treatment did not enhance drought tolerance in the mutant of ABI1, a negative regulator of abscisic acid (ABA) signaling in Arabidopsis, indicating that ABA signaling contributes to ethanol-mediated drought tolerance. The nuclear magnetic resonance analysis using 13C-labeled ethanol indicated that gluconeogenesis is involved in the accumulation of sugars. The ethanol treatment did not enhance the drought tolerance in the aldehyde dehydrogenase (aldh) triple mutant (aldh2b4/aldh2b7/aldh2c4). These results show that ABA signaling and acetic acid biosynthesis are involved in ethanol-mediated drought tolerance and that chemical priming through ethanol application regulates sugar accumulation and gluconeogenesis, leading to enhanced drought tolerance and sustained plant growth. These findings highlight a new survival strategy for increasing crop production under water-limited conditions.

Field-transcriptome analyses reveal developmental transitions during flowering in cassava (Manihot esculenta Crantz).

KEY MESSAGE: We characterized genes that function in the photoperiodic flowering pathway in cassava. Transcriptome analysis of field-grown plants revealed characteristic expression patterns of these genes, demonstrating that field-grown cassava experiences two distinct developmental transitions. Cassava is an important crop for both edible and industrial purposes. Cassava develops storage roots that accumulate starch, providing an important source of staple food in tropical regions. To facilitate cassava breeding, it is important to elucidate how flowering is controlled. Several important genes that control flowering time have been identified in model plants; however, comprehensive characterization of these genes in cassava is still lacking. In this study, we identified genes encoding central flowering time regulators and examined these sequences for the presence or absence of conserved motifs. We found that cassava shares conserved genes for the photoperiodic flowering pathway, including florigen, anti-florigen and its associated transcription factor (GIGANTEA, CONSTANS, FLOWERING LOCUS T, CENTRORADIALIS/TERMINAL FLOWER1 and FD) and florigen downstream genes (SUPRESSOR OF OVEREXPRESSION OF CONSTANS1 and APETALA1/FRUITFUL). We conducted RNA-seq analysis of field-grown cassava plants and characterized the expression of flowering control genes. Finally, from the transcriptome analysis we identified two distinct developmental transitions that occur in field-grown cassava.

Cytosolic GLUTAMINE SYNTHETASE1;1 Modulates Metabolism and Chloroplast Development in Roots.

Nitrogen (N) is an essential macronutrient, and the final form of endogenous inorganic N is ammonium, which is assimilated by Gln synthetase (GS) into Gln. However, how the multiple isoforms of cytosolic GSs contribute to metabolic systems via the regulation of ammonium assimilation remains unclear. In this study, we compared the effects of two rice (Oryza sativa) cytosolic GSs, namely OsGS1;1 and OsGS1;2, on central metabolism in roots using reverse genetics, metabolomic and transcriptomic profiling, and network analyses. We observed (1) abnormal sugar and organic N accumulation and (2) significant up-regulation of genes associated with photosynthesis and chlorophyll biosynthesis in the roots of Osgs1;1 but not Osgs1;2 knockout mutants. Network analysis of the Osgs1;1 mutant suggested that metabolism of Gln was coordinated with the metabolic modules of sugar metabolism, tricarboxylic acid cycle, and carbon fixation. Transcript profiling of Osgs1;1 mutant roots revealed that expression of the rice sigma-factor (OsSIG) genes in the mutants were transiently upregulated. GOLDEN2-LIKE transcription factor-encoding genes, which are involved in chloroplast biogenesis in rice, could not compensate for the lack of OsSIGs in the Osgs1;1 mutant. Microscopic analysis revealed mature chloroplast development in Osgs1;1 roots but not in the roots of Osgs1;2, Osgs1;2-complemented lines, or the wild type. Thus, organic N assimilated by OsGS1;1 affects a broad range of metabolites and transcripts involved in maintaining metabolic homeostasis and plastid development in rice roots, whereas OsGS1;2 has a more specific role, affecting mainly amino acid homeostasis but not carbon metabolism.

Cassava breeding and agronomy in Asia: 50 years of history and future directions.

In Asia, cassava (Manihot esculenta) is cultivated by more than 8 million farmers, driving the rural economy of many countries. The International Center for Tropical Agriculture (CIAT), in partnership with national agricultural research institutes (NARIs), instigated breeding and agronomic research in Asia, 1983. The breeding program has successfully released high-yielding cultivars resulting in an average yield increase from 13.0 t ha-1 in 1996 to 21.3 t ha-1 in 2016, with significant economic benefits. Following the success in increasing yields, cassava breeding has turned its focus to higher-value traits, such as waxy cassava, to reach new market niches. More recently, building resistance to invasive pests and diseases has become a top priority due to the emergent threat of cassava mosaic disease (CMD). The agronomic research involves driving profitability with advanced technologies focusing on better agronomic management practices thereby maintaining sustainable production systems. Remote sensing technologies are being tested for trait discovery and large-scale field evaluation of cassava. In summary, cassava breeding in Asia is driven by a combination of food and market demand with technological innovations to increase the productivity. Further, exploration in the potential of data-driven agriculture is needed to empower researchers and producers for sustainable advancement.

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.

Convergent evolution of polysaccharide debranching defines a common mechanism for starch accumulation in cyanobacteria and plants.

Starch, unlike hydrosoluble glycogen particles, aggregates into insoluble, semicrystalline granules. In photosynthetic eukaryotes, the transition to starch accumulation occurred after plastid endosymbiosis from a preexisting cytosolic host glycogen metabolism network. This involved the recruitment of a debranching enzyme of chlamydial pathogen origin. The latter is thought to be responsible for removing misplaced branches that would otherwise yield a water-soluble polysaccharide. We now report the implication of starch debranching enzyme in the aggregation of semicrystalline granules of single-cell cyanobacteria that accumulate both glycogen and starch-like polymers. We show that an enzyme of analogous nature to the plant debranching enzyme but of a different bacterial origin was recruited for the same purpose in these organisms. Remarkably, both the plant and cyanobacterial enzymes have evolved through convergent evolution, showing novel yet identical substrate specificities from a preexisting enzyme that originally displayed the much narrower substrate preferences required for glycogen catabolism.

Cassava (Manihot esculenta) transcriptome analysis in response to infection by the fungus Colletotrichum gloeosporioides using an oligonucleotide-DNA microarray.

Cassava anthracnose disease (CAD), caused by the fungus Colletotrichum gloeosporioides f. sp. Manihotis, is a serious disease of cassava (Manihot esculenta) worldwide. In this study, we established a cassava oligonucleotide-DNA microarray representing 59,079 probes corresponding to approximately 30,000 genes based on original expressed sequence tags and RNA-seq information from cassava, and applied it to investigate the molecular mechanisms of resistance to fungal infection using two cassava cultivars, Huay Bong 60 (HB60, resistant to CAD) and Hanatee (HN, sensitive to CAD). Based on quantitative real-time reverse transcription PCR and expression profiling by the microarray, we showed that the expressions of various plant defense-related genes, such as pathogenesis-related (PR) genes, cell wall-related genes, detoxification enzyme, genes related to the response to bacterium, mitogen-activated protein kinase (MAPK), genes related to salicylic acid, jasmonic acid and ethylene pathways were higher in HB60 compared with HN. Our results indicated that the induction of PR genes in HB60 by fungal infection and the higher expressions of defense response-related genes in HB60 compared with HN are likely responsible for the fungal resistance in HB60. We also showed that the use of our cassava oligo microarray could improve our understanding of cassava molecular mechanisms related to environmental responses and development, and advance the molecular breeding of useful cassava plants.

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.

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.

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.

Elongated phytoglycogen chain length in transgenic rice endosperm expressing active starch synthase IIa affects the altered solubility and crystallinity of the storage α-glucan.

The relationship between the solubility, crystallinity, and length of the unit chains of plant storage α-glucan was investigated by manipulating the chain length of α-glucans accumulated in a rice mutant. Transgenic lines were produced by introducing a cDNA for starch synthase IIa (SSIIa) from an indica cultivar (SSIIa (I), coding for active SSIIa) into an isoamylase1 (ISA1)-deficient mutant (isa1) that was derived from a japonica cultivar (bearing inactive SSIIa proteins). The water-soluble fraction accounted for >95% of the total α-glucan in the isa1 mutant, whereas it was only 35-70% in the transgenic SSIIa (I)/isa1 lines. Thus, the α-glucans from the SSIIa (I)/isa1 lines were fractionated into soluble and insoluble fractions prior to the following characterizations. X-ray diffraction analysis revealed a weak B-type crystallinity for the α-glucans of the insoluble fraction, while no crystallinity was confirmed for α-glucans in isa1. Concerning the degree of polymerization (DP) ≤30, the chain lengths of these α-glucans differed significantly in the order of SSIIa (I)/isa1 insoluble > SSIIa (I)/isa1 soluble > α-glucans in isa1. The amount of long chains with DP ≥33 was higher in the insoluble fraction α-glucans than in the other two α-glucans. No difference was observed in the chain length distributions of the ß-amylase limit dextrins among these α-glucans. These results suggest that in the SSIIa (I)/isa1 transgenic lines, the unit chains of α-glucans were elongated by SSIIa(I), whereas the expression of SSIIa(I) did not affect the branch positions. Thus, the observed insolubility and crystallinity of the insoluble fraction can be attributed to the elongated length of the outer chains due to SSIIa(I).