Macromolecular α-glucans with α-1,3/α-1,4 branching structures produced using dual glycosyltransferases: Elucidation of physicochemical and slowly digestible properties.

Amylosucrase from Neisseria polysaccharea (NpAS) produces the linear amylose-like α-glucans by the elongation property from sucrose, and 4,3-α-glucanotransferase from Lactobacillus fermentum NCC 2970 (4,3-αGT) newly synthesizes the α-1,3 linkages after cleaving the α-1,4 linkages by the glycosyltransferring property. This study focused on the synthesis of high molecular α-1,3/α-1,4-linked glucans by combining NpAS and 4,3-αGT and analyzed their structural and digestive properties. The enzymatically synthesized α-glucans have a molecular weight of >1.6 × 107 g mol-1, and the α-4,3 branching ratios on the structures increased as the amount of 4,3-αGT increased. The synthesized α-glucans were hydrolyzed to linear maltooligosaccharides and α-4,3 branched α-limit dextrins (α-LDx) by human pancreatic α-amylase, and the amounts of produced α-LDx were increased depending on the ratio of synthesized α-1,3 linkages. In addition, approximately 80 % of the synthesized products were partially hydrolyzed by mammalian α-glucosidases, and the glucose generation rates decelerated as the amounts of α-1,3 linkages increased. In conclusion, new types of α-glucans with α-1,4 and α-1,3 linkages were successfully synthesized by a dual enzyme reaction. These can be utilized as slowly digestible and prebiotic ingredients in the gastrointestinal tract due to their novel linkage patterns and high molecular weights.

The thickening and modification of the galactan-enriched layer during primary phloem fibre development in Cannabis sativa.

Primary phloem fibres (PPFs) have higher fibre quality and are economically more important for the textile sector than secondary phloem fibres. Both the chemical composition and mechanical structure of the secondary cell wall mainly influence the quality of bast fibres. We investigated the thickening of the galactan-enriched (Gn) layer and its modification process into a gelatinous (G)-layer, which is the largest portion of the secondary cell wall, during the development of the PPF in Cannabis sativa. Stem segments of hemp collected at 17, 29, 52 and 62 days after sowing were comparatively examined using light microscopy, scanning electron microscopy and transmission electron microscopy. The initial cells of PPF started the proliferation and differentiation at 17 days, but the secondary cell wall thickening had already commenced before the 29 days. Both the G- and Gn-layer were rapidly added onto the S-layer of PPFs; thus, the secondary cell wall thickness increased approximately 2-fold at 52 days (from the 29-day mark), and 8-fold at 62 days. The cortical microtubule arrays appeared adjacent to the plasma membrane of PPF cells related to the cellulose synthesis. Additionally, cross-sectioned microfibrils were observed on Gn-layer as the cluster of tiny spots. At 62 days, the specific stratification structure consisting of several lamellae occurred on the G-layer of the secondary cell wall. The secondary cell wall thickened remarkably at 52 days through 62 days so that the mature secondary cell wall consisted of three distinctive layers, the S-, G- and Gn-layer. Cortical microtubule arrays frequently appeared adjacent to the plasma membrane together with cellulose microfibrils on secondary cell wall. The G-layer of PPF at 62 days exhibited the characteristic stratification structure, which demonstrates the modification of the Gn-layer into the G-layer.

Genetic Diversity and Population Structure of Nutria (Myocastor coypus) in South Korea.

The nutria (Myocastor coypus) is an invasive alien species that have had major adverse effects on biodiversity and the agricultural economy in wetland habitats. Since 2014, the Ministry of Environment in South Korea has been carrying out the Nutria Eradication Project, and we investigated nutria distribution and genetic diversity of nutria populations in South Korea. We estimated that 99.2% of nutria habitats are in the mid-lower Nakdong River regions. To further analyze the genetic diversity in eight major nutria populations, we performed a genetic analysis using microsatellite markers. Genetic diversity levels of the eight nutria populations in South Korea were relatively lower than those in other countries. The probability of migration direction among nutria populations was predicted from genetic distance analysis. Genetic structure analysis showed little difference among the nutria populations in South Korea. These results suggest that nutrias in South Korea originated from a single population. Our results provide important data for establishing management strategies for the successful eradication of nutria populations in South Korea, as well as in other countries with alien invasive species.

AtPR5K2, a PR5-Like Receptor Kinase, Modulates Plant Responses to Drought Stress by Phosphorylating Protein Phosphatase 2Cs.

Cell surface receptors perceive signals from the environment and transfer them to the interior of the cell. The Arabidopsis thaliana PR5 receptor-like kinase (AtPR5K) subfamily consists of three members with extracellular domains that share sequence similarity with the PR5 proteins. In this study, we characterized the role of AtPR5K2 in plant drought-stress signaling. AtPR5K2 is predominantly expressed in leaves and localized to the plasma membrane. The atpr5k2-1 mutant showed tolerance to dehydration stress, while AtPR5K2-overexpressing plants was hypersensitive to drought. Bimolecular fluorescence complementation assays showed that AtPR5K2 physically interacted with the type 2C protein phosphatases ABA-insensitive 1 (ABI1) and ABI2 and the SNF1-related protein kinase 2 (SnRK2.6) proteins, all of which are involved in the initiation of abscisic acid (ABA) signaling; however, these interactions were inhibited by treatments of exogenous ABA. Moreover, AtPR5K2 was found to phosphorylate ABI1 and ABI2, but not SnRK2.6. Taken together, these results suggest that AtPR5K2 participates in ABA-dependent drought-stress signaling through the phosphorylation of ABI1 and ABI2.

Functional Mechanisms Underlying the Antimicrobial Activity of the Oryza sativa Trx-like Protein.

Plants are constantly subjected to a variety of environmental stresses and have evolved regulatory responses to overcome unfavorable conditions that might reduce or adversely change a plant's growth or development. Among these, the regulated production of reactive oxygen species (ROS) as a signaling molecule occurs during plant development and pathogen defense. This study demonstrates the possible antifungal activity of Oryza sativa Tetratricopeptide Domain-containing thioredoxin (OsTDX) protein against various fungal pathogens. The transcription of OsTDX was induced by various environmental stresses known to elicit the generation of ROS in plant cells. OsTDX protein showed potent antifungal activity, with minimum inhibitory concentrations (MICs) against yeast and filamentous fungi ranging between 1.56 and 6.25 and 50 and 100 µg/mL, respectively. The uptake of SYTOX-Green into fungal cells and efflux of calcein from artificial fungus-like liposomes suggest that its killing mechanism involves membrane permeabilization and damage. In addition, irregular blebs and holes apparent on the surfaces of OsTDX-treated fungal cells indicate the membranolytic action of this protein. Our results suggest that the OsTDX protein represents a potentially useful lead for the development of pathogen-resistant plants.

Hydrophilic Linear Peptide with Histidine and Lysine Residues as a Key Factor Affecting Antifungal Activity.

Increases in the numbers of immunocompromised patients and the emergence of drug-resistance fungal pathogens have led to the need for new, safe, efficacious antifungal agents. In this study, we designed a histidine-lysine-lysine (HKK) motif and synthesized six HKK peptides with repetitions of the motif. These peptides showed length-dependent antifungal activity against drug-susceptible and drug-resistant fungal pathogens via membranolytic or non-membranolytic action. None of the peptides were cytotoxic to rat erythrocytes or NIH3T3 mouse embryonic fibroblasts. Short-length peptides were directly translocated in fungal cytosol and reacted with mitochondria, resulting in apoptosis. Membrane-permeabilizing activity occurred in the presence of long peptides, and peptides were able to transfer to the cytosol and induce reactive oxygen species. Our results suggest that peptides composed only of cationic amino acids may be good candidates as antifungal agents.

Pathogen associated molecular pattern (PAMP)-triggered immunity is compromised under C-limited growth.

In the interaction between plants and pathogens, carbon (C) resources provide energy and C skeletons to maintain, among many functions, the plant immune system. However, variations in C availability on pathogen associated molecular pattern (PAMP) triggered immunity (PTI) have not been systematically examined. Here, three types of starch mutants with enhanced susceptibility to Pseudomonas syringae pv. tomato DC3000 hrcC were examined for PTI. In a dark period-dependent manner, the mutants showed compromised induction of a PTI marker, and callose accumulation in response to the bacterial PAMP flagellin, flg22. In combination with weakened PTI responses in wild type by inhibition of the TCA cycle, the experiments determined the necessity of C-derived energy in establishing PTI. Global gene expression analyses identified flg22 responsive genes displaying C supply-dependent patterns. Nutrient recycling-related genes were regulated similarly by C-limitation and flg22, indicating re-arrangements of expression programs to redirect resources that establish or strengthen PTI. Ethylene and NAC transcription factors appear to play roles in these processes. Under C-limitation, PTI appears compromised based on suppression of genes required for continued biosynthetic capacity and defenses through flg22. Our results provide a foundation for the intuitive perception of the interplay between plant nutrition status and pathogen defense.

TsHKT1;2, a HKT1 homolog from the extremophile Arabidopsis relative Thellungiella salsuginea, shows K(+) specificity in the presence of NaCl.

Cellular Na(+)/K(+) ratio is a crucial parameter determining plant salinity stress resistance. We tested the function of plasma membrane Na(+)/K(+) cotransporters in the High-affinity K(+) Transporter (HKT) family from the halophytic Arabidopsis (Arabidopsis thaliana) relative Thellungiella salsuginea. T. salsuginea contains at least two HKT genes. TsHKT1;1 is expressed at very low levels, while the abundant TsHKT1;2 is transcriptionally strongly up-regulated by salt stress. TsHKT-based RNA interference in T. salsuginea resulted in Na(+) sensitivity and K(+) deficiency. The athkt1 mutant lines overexpressing TsHKT1;2 proved less sensitive to Na(+) and showed less K(+) deficiency than lines overexpressing AtHKT1. TsHKT1;2 ectopically expressed in yeast mutants lacking Na(+) or K(+) transporters revealed strong K(+) transporter activity and selectivity for K(+) over Na(+). Altering two amino acid residues in TsHKT1;2 to mimic the AtHKT1 sequence resulted in enhanced sodium uptake and loss of the TsHKT1;2 intrinsic K(+) transporter activity. We consider the maintenance of K(+) uptake through TsHKT1;2 under salt stress an important component supporting the halophytic lifestyle of T. salsuginea.

Identification and molecular properties of SUMO-binding proteins in Arabidopsis.

Reversible conjugation of the small ubiquitin modifier (SUMO) peptide to proteins (SUMOylation) plays important roles in cellular processes in animals and yeasts. However, little is known about plant SUMO targets. To identify SUMO substrates in Arabidopsis and to probe for biological functions of SUMO proteins, we constructed 6xHis-3xFLAG fused AtSUMO1 (HFAtSUMO1) controlled by the CaMV35S promoter for transformation into Arabidopsis Col-0. After heat treatment, an increased sumoylation pattern was detected in the transgenic plants. SUMO1-modified proteins were selected after two-dimensional gel electrophoresis (2-DE) image analysis and identified using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We identified 27 proteins involved in a variety of processes such as nucleic acid metabolism, signaling, metabolism, and including proteins of unknown functions. Binding and sumoylation patterns were confirmed independently. Surprisingly, MCM3 (At5G46280), a DNA replication licensing factor, only interacted with and became sumoylated by AtSUMO1, but not by SUMO1ΔGG or AtSUMO3. The results suggest specific interactions between sumoylation targets and particular sumoylation enzymes.

NKS1, Na(+)- and K(+)-sensitive 1, regulates ion homeostasis in an SOS-independent pathway in Arabidopsis.

An Arabidopsis thaliana mutant, nks1-1, exhibiting enhanced sensitivity to NaCl was identified in a screen of a T-DNA insertion population in the genetic background of Col-0 gl1sos3-1. Analysis of the genome sequence in the region flanking the T-DNA left border indicated two closely linked mutations in the gene encoded at locus At4g30996. A second allele, nks1-2, was obtained from the Arabidopsis Biological Resource Center. NKS1 mRNA was detected in all parts of wild-type plants but was not detected in plants of either mutant, indicating inactivation by the mutations. Both mutations in NKS1 were associated with increased sensitivity to NaCl and KCl, but not to LiCl or mannitol. NaCl sensitivity was associated with nks1 mutations in Arabidopsis lines expressing either wild type or null alleles of SOS1, SOS2 or SOS3. The NaCl-sensitive phenotype of the nks1-2 mutant was complemented by expression of a full-length NKS1 allele from the CaMV35S promoter. When grown in medium containing NaCl, nks1 mutants accumulated more Na(+) than wild type and K(+)/Na(+) homeostasis was perturbed. It is proposed NKS1, a plant-specific gene encoding a 19kDa endomembrane-localized protein of unknown function, is part of an ion homeostasis regulation pathway that is independent of the SOS pathway.

Specific domain structures control abscisic acid-, salicylic acid-, and stress-mediated SIZ1 phenotypes.

SIZ1 (for yeast SAP and MIZ1) encodes the sole ortholog of mammalian PIAS (for protein inhibitor of activated STAT) and yeast SIZ SUMO (for small ubiquitin-related modifier) E3 ligases in Arabidopsis (Arabidopsis thaliana). Four conserved motifs in SIZ1 include SAP (for scaffold attachment factor A/B/acinus/PIAS domain), PINIT (for proline-isoleucine-asparagine-isoleucine-threonine), SP-RING (for SIZ/PIAS-RING), and SXS (for serine-X-serine, where X is any amino acid) motifs. SIZ1 contains, in addition, a PHD (for plant homeodomain) typical of plant PIAS proteins. We determined phenotypes of siz1-2 knockout mutants transformed with SIZ1 alleles carrying point mutations in the predicted domains. Domain SP-RING is required for SUMO conjugation activity and nuclear localization of SIZ1. Salicylic acid (SA) accumulation and SA-dependent phenotypes of siz1-2, such as diminished plant size, heightened innate immunity, and abscisic acid inhibition of cotyledon greening, as well as SA-independent basal thermotolerance were not complemented by the altered SP-RING allele of SIZ1. The SXS domain also controlled SA accumulation and was involved in greening and expansion of cotyledons of seedlings germinated in the presence of abscisic acid. Mutations of the PHD zinc finger domain and the PINIT motif affected in vivo SUMOylation. Expression of the PHD and/or PINIT domain mutant alleles of SIZ1 in siz1-2 promoted hypocotyl elongation in response to sugar and light. The various domains of SIZ1 make unique contributions to the plant's ability to cope with its environment.

Functional analysis of the stress-inducible soybean calmodulin isoform-4 (GmCaM-4) promoter in transgenic tobacco plants.

The transcription of soybean (Glycine max) calmodulin isoform-4 (GmCaM-4) is dramatically induced within 0.5 h of exposure to pathogen or NaCl. Core cis-acting elements that regulate the expression of the GmCaM-4 gene in response to pathogen and salt stress were previously identified, between -1,207 and -1,128 bp, and between -858 and -728 bp, in the GmCaM-4 promoter. Here, we characterized the properties of the DNA-binding complexes that form at the two core cis-acting elements of the GmCaM-4 promoter in pathogen-treated nuclear extracts. We generated GUS reporter constructs harboring various deletions of approximately 1.3-kb GmCaM-4 promoter, and analyzed GUS expression in tobacco plants transformed with these constructs. The GUS expression analysis suggested that the two previously identified core regions are involved in inducing GmCaM-4 expression in the heterologous system. Finally, a transient expression assay of Arabidopsis protoplasts showed that the GmCaM-4 promoter produced greater levels of GUS activity than did the CaMV35S promoter after pathogen or NaCl treatments, suggesting that the GmCaM-4 promoter may be useful in the production of conditional gene expression systems.