Barley accumulates proanthocyanidins (PA), a type of flavonoid compound, in the testa layer. The accumulated PA have a detrimental effect on the quality of beer and pearled barley. Therefore, controlling PA accumulation in the testa layer is an important breeding objective in barley. PA accumulation is regulated by a ternary complex consisting of MYB, basic helix-loop-helix (bHLH), and WD40 transcription factors (MBW complex) in various plant species. The MBW complex regulates the transcription of structural genes encoding enzymes in the flavonoid biosynthesis pathway. Although HvMYB10 and HvWD40-1 have been identified as regulators of PA accumulation in the barley testa layer, the bHLH transcription factor regulating it remains unclear. In this study, we isolated a novel PA-defective mutant, TK113, using sodium azide mutagenesis. Expression analysis revealed that the expression levels of multiple structural genes decreased in the seed coat of TK113. Single nucleotide polymorphism (SNP)-index analysis with a candidate gene approach identified a mutation causing amino acid substitution in HvTT8, which encodes the bHLH transcription factor, of TK113 associated with the PA-defective phenotype. The mutation was located in a highly conserved domain among TT8 orthologs, indicating that it is the most likely causal mutation for the PA-defective phenotype of TK113.

Chrysanthemum (Chrysanthemum morifolium Ramat.) is a short-day plant, and flowering is stimulated when the photoperiod is shorter than a variety-specific threshold (critical day length). In Japan, summer-to-autumn-flowering cultivars (SA-cvs.) flower from July to September. Little research has been conducted to understand why SA-cvs. bloom earlier than autumn-flowering cultivars (A-cvs.). We conducted a comparative study of the relationship between the photoperiodic response of flowering and the gene expression of florigen FLOWERING LOCUS T-like 3 (FTL3) and antiflorigen anti-florigenic FT/TFL1 (AFT). SA-cvs. had a longer critical day length than A-cvs. However, in both groups, a decrease in AFT and increase in FTL3 were consistently observed below the critical day length when flowering was promoted. The opposite responses (less flowering, low FTL3, and high AFT) were observed for longer than the critical day lengths. This indicated that flowering in SA-cvs. was controlled by the regulation of AFT/FTL3 expression, similar to that in A-cvs. Next, we studied the mechanism that causes a variation in critical day lengths. In SA-cvs., the photosensitive phase, which occurs at night, occurs earlier than that in A-cvs. This indicates a variation in the endogenous time-keeping mechanism. This was supported by the fact that the circadian rhythmicity of leaf movement was weaker in SA-cvs. than that in A-cvs. Thus, variation in the endogenous time-keeping mechanism may cause a longer critical day length and earlier flowering time in SA-cvs.

DNA markers serve as essential tools in breeding selection and genetic analysis. However, developing DNA markers can be time-consuming and labor-intensive due to the need to identify polymorphisms between cultivars/lines and to design suitable primers. To address these challenges, we have developed DNAMarkMaker, a tool designed to automate the process of primer design for Amplification Refractory Mutation System (ARMS) and Cleaved Amplified Polymorphic Sequences (CAPS) markers, utilizing resequencing data. One key feature of DNAMarkMaker is its user-friendly graphical user interface (GUI), ensuring its accessibility and ease of use, even for researchers not well-versed in bioinformatics. We confirmed DNAMarkMaker's applicability by developing DNA markers for rice, potato, and turnip-each representing distinct genome structures: homozygous diploid, heterozygous autotetraploid, and heterozygous diploid, respectively. DNAMarkMaker will contribute to the rapid and efficient development of DNA markers, accelerating breeding and genetic analysis in various crops.

The progenies of polyploid crops inherit multiple sets of homoeologous chromosomes through various combinations, which impedes the identification of the quantitative trait loci (QTL) governing agronomic traits and the implementation of DNA marker-assisted breeding. Previously, we developed a whole-genome sequencing-based polyploid QTL-seq method that utilizes comprehensively extracted simplex polymorphisms for QTL mapping. Here, we verified the detection of duplex QTLs by modifying the analytical settings to explore the QTLs governing tuber flesh color and starch phosphorus content using tetraploid potato (Solanum tuberosum L.). The F1 progenies were obtained from a cross between 'Touya' (TY) and 'Benimaru' (BM). A single TY-derived QTL responsible for yellow flesh color was identified around a β-carotene hydroxylase gene on chromosome 3 using simplex polymorphisms, and a BM-derived QTL associated with decreased starch phosphorus content near a starch synthase II gene on chromosome 2 was detected using duplex polymorphisms. Furthermore, linked DNA markers were developed at the QTL sites. For the latter QTL, plexity-distinguishable markers were developed using quantitative PCR, fragment analysis, and amplicon sequencing. These revealed the allele dosage-dependent effect of the reduced starch phosphorus content. Thus, the polyploid QTL-seq pipeline can explore versatile QTLs beyond simplex, facilitating DNA marker-assisted breeding in various polyploid crops.

Sweetpotato (Ipomoea batatas) includes diverse cultivars with flesh textures ranging from dry to moist. Moist-fleshed cultivars often contain starch with a lower gelatinization temperature (GT). To elucidate the genetic determinants of flesh texture and starch GT, we conducted a QTL analysis using F1 progenies obtained from a cross between dry-fleshed and moist-fleshed cultivars, 'Benikomachi' (BK) and 'Amahazuki' (AH), by using an updated polyploid QTL-seq pipeline. Flesh texture was assessed based on the wet area ratio (WAR) observed on the cut surface of steamed tubers, as progenies with dry and moist flesh exhibited low and high WAR values, respectively, demonstrating a strong correlation. Three QTLs were found to regulate the WAR. Notably, two AH-derived alleles at 4.30 Mb on Itr_chr05 and 21.01 Mb on Itr_chr07, along with a BK-derived allele at 2.89 Mb on Itr_chr15, were associated with increased WAR. Starch GT, which displayed no correlation with either flesh texture or WAR, was distinctly influenced by two QTLs: a GTincreasing BK-derived allele at 1.74 Mb on Itr_chr05 and a GT-decreasing AH-derived allele at 30.16 Mb on Itr_chr12. Consequently, we developed DNA markers linked to WAR, offering a promising avenue for the targeted breeding of sweetpotato with the desired flesh textures.

We have identified a unique genetic locus for seed shattering in Italian ryegrass that has an exceedingly large effect and shows partial dominance for reduced seed shattering. Genetic improvement of seed retention in forage grasses can contribute to improving their commercial seed production. The objective of this study was to identify the genetic loci responsible for seed shattering in Italian ryegrass (Lolium multiflorum Lam.) using F2 and F3 progeny from a cross between a reduced shattering genotype and a self-fertile shattering genotype. High negative correlations (− 0.622 in F2 and − 0.737 in F3) were found between two methods of measuring shattering: (1) the percentage of seed shattering obtained by manually stripping the spike and (2) the non-basal floret breaking tensile strength (BTS). On the other hand, basal floret BTS showed a non-significant (F2) or low (− 0.226 in F3) correlation with the percentage of seed shattering by stripping. We identified a quantitative trait locus (QTL) near the start of linkage group 2, designated as qSH2.1, which was associated with both seed shattering measured by stripping and non-basal floret BTS with exceptionally high LOD values (11.0–34.0); in addition, we detected five minor QTLs. qSH2.1 explained about 2/3 of the total variation in the percentage of seed shattering by stripping at the late dough stage in the F2 population. The reduced shattering trait was partially dominant, in contrast to the genetic mode in many previous reports on other crops. Candidate orthologs for the previously reported seed shattering genes were not found near the qSH2.1 locus in the ryegrass genome, suggesting that this QTL may be due to a yet-undiscovered gene.

Potato (Solanum tuberosum L.) and sweetpotato (Ipomoea batatas L.), which are nutritionally and commercially important tuberous crops, possess a perplexing heredity because of their autopolyploid genomes. To reduce cross-breeding efforts for selecting superior cultivars from progenies with innumerable combinations of traits, DNA markers tightly linked to agronomical traits are required. To develop DNA markers, we developed a method for quantitative trait loci (QTL) mapping using whole-genome next-generation sequencing (NGS) in autopolyploid crops. To apply the NGS-based bulked segregant method, QTL-seq was modified. (1) Single parent-specific simplex (unique for one homologous chromosome) single-nucleotide polymorphisms (SNPs), which present a simple segregation ratio in the progenies, were exploited by filtering SNPs by SNP index (allele frequency). (2) Clusters of SNPs, which were inherited unevenly between bulked progenies with opposite phenotypes, especially those with an SNP index of 0 for the bulk that did not display the phenotypes of interest, were explored. These modifications allowed for separate tracking of alleles located on each of the multiple homologous chromosomes. By applying this method, clusters of SNPs linked to the potato cyst nematode resistance H1 gene and storage root anthocyanin (AN) content were identified in tetraploid potato and hexaploid sweetpotato, respectively, and completely linked DNA markers were developed at the site of the presented SNPs. Thus, polyploid QTL-seq is a versatile method that is free from specialized manipulation for sequencing and construction of elaborate linkage maps and facilitates rapid development of tightly linked DNA markers in autopolyploid crops, such as potato and sweetpotato.

A newly identified chemical, 4-{3-[(3,5-dichloro-2-hydroxybenzylidene)amino]propyl}-4,5-dihydro-1H-pyrazol-5-one (BAPP) was characterized as a plant immunity activator. BAPP enhanced disease resistance in rice against rice blast disease and expression of a defense-related gene without growth inhibition. Moreover, BAPP was able to enhance disease resistance in dicotyledonous tomato and Arabidopsis plants against bacterial pathogen without growth inhibition, suggesting that BAPP could be a candidate as an effective plant activator. Analysis using Arabidopsis sid2-1 and npr1-2 mutants suggested that BAPP induced systemic acquired resistance (SAR) by stimulating between salicylic acid biosynthesis and NPR1, the SA receptor protein, in the SAR signaling pathway.

High temperature (HT) during the grain developing stage causes deleterious effects on rice quality resulting in mature grains with a chalky appearance. Phospholipase D (PLD) plays an important role in plants, including responses to environmental stresses. OsPLDα1, α3 and β2-knockdown (KD) plants showed decreased production of chalky grains at HT. HT ripening increased H2O2 accumulated in the developing grains. However, the increase was canceled by the knockdown of OsPLDβ2. Expression levels of OsCATA which is one of three rice catalase genes, in developing grains of OsPLDβ2-KD plants at 10 DAF were increased compared with that in vector-controls in HT growth conditions. Overexpression of OsCATA markedly suppressed the production of chalky grains in HT growth conditions. These results suggested that OsPLDβ2 functions as a negative regulator of the induction of OsCATA and is involved in the production of chalky grains in HT growth conditions.

Analysis Reveals a Novel Regulatory Network of Genes Involved in Heat-Induced Grain Chalk in Rice Endosperm Tsutomu Ishimaru*, Sabiha Parween, Yuhi Saito, Takanari Shigemitsu, Hiromoto Yamakawa, Mikio Nakazono, Takehiro Masumura, Naoko K. Nishizawa, Motohiko Kondo and Nese Sreenivasulu* NARO Institute of Crop Science, NARO, 2-1-18 Kannondai, Tsukuba, Ibaraki, 305-8518 Japan Hokuriku Research Station, Central Region Agricultural Research Center, National Agriculture and Food Research Organization (CARC/NARO), 1-2-1 Inada, Joetsu, Niigata, 941-0193 Japan International Rice Research Institute (IRRI), DAPO Box 7777, Metro Manila, The Philippines Graduate School of Life and Environmental Science, Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto, 606-8522 Japan Graduate School of Bioagricultural Sciences, Nagoya University, Furo, Chikusa, Nagoya, 464-8601 Japan Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-38 Suematsu, Nonoichi, Ishikawa, 921-8836 Japan Present address: Agriculture, Forestry and Fisheries Research Council, Kasumigaseki, Chiyoda, Tokyo, 100-8950 Japan *Corresponding authors: Tsutomu Ishimaru, E-mail, cropman@affrc.go.jp; Fax, +81-25-524-8578; Nese Sreenivasulu, E-mail, n.sreenivasulu@irri.org; Fax, +63-2-580-5699. (Received May 31, 2018; Accepted November 27, 2018)

Abstract α-Amylases have been of interest in diverse fields for many years because of their importance in basic biology, agriculture, and industry. Starch hydrolysis in plants has been studied extensively in germinating cereal seeds. It is generally accepted that α-amylases are secretory enzymes with a pivotal role in the breakdown of starch reserves in the endosperm. Intriguingly, however, recent investigations reveal that some α-amylases degrade starch in the plastids of living cells. The recent solving of the crystal structure of rice AmyI-1 isoform shows that the binding pocket of starch binding site 1 situated outside of the active site cleft interacts with the substances other than oligosaccharides. These findings provided novel insights into structural and cell biological aspects of α-amylase functions in intracellular transport, organelle targeting, and organ-specific actions. Under global warming, abnormal high temperatures during rice grain filling increase grain chalkiness, resulting in yield loss. Intensive “omics” analyses of developing caryopses and mature grains grown under heat stress showed the downregulation of starch synthesis enzymes and the upregulation of α-amylases. Transgenic studies using ectopic overexpression and suppression of α-amylase revealed that α-amylase is a key factor in grain chalkiness. Here we discuss unique new functions of α-amylase in rice cells.

Heat stress occurrence during seed filling leads to the formation of a chalky portion in the limited zone of the starchy endosperm of rice grains. In this study, isolation of aleurone, dorsal, central and lateral tissues of developing endosperm by laser-microdissection (LM) coupled with gene expression analysis of a 44 K microarray was performed to identify key regulatory genes involved in the formation of milky-white (MW) and white-back (WB) grains during heat stress. Gene regulatory network analysis classified the genes changed under heat stress into five modules. The most distinct expression pattern was observed in modules where most of the small heat shock proteins and cellular organization genes were changed under heat stress in dorsal aleurone cells and dorsal starchy endosperm zones. The histological observation supported the significant increase in cell number and size of dorsal aleurone cells in WB grains. With regard to the central starchy endosperm zone, preferential down-regulation of high molecular weight heat shock proteins (HMW HSPs), including a prominent member encoding endoplasmic reticulum (ER) chaperones, by heat stress was observed, while changes in expression of starch biosynthesis genes were minimal. Characterization of transgenic plants suppressing endosperm lumenal binding protein gene (BiP1), an ER chaperone preferentially down-regulated at the MW zone under heat stress, showed evidence of forming the chalky grains without disturbing the expression of starch biosynthesis genes. The present LM-based comprehensive expression analysis provides novel inferences that HMW HSPs play an important role in controlling redox, nitrogen and amino acid metabolism in endosperm leading to the formation of MW and WB chalky grains under heat stress.

Susumu Hiragai' 4, Hireyuki Ito i, Katsutomo Sasaki i' 4, Hiromoto Yamakawa2' 4, Ichiro Mitsuhara3' 4, Hiroaki Toshima i, Hirokazu Matsui i, Mamoru Honma i and Yuko 0hashi3i 4i S i Department ofApptiedBioscience, Graduate Schoot ofAgriculture, Hbkkaido University, Stmporo, 060-8589 Jopan 2 institute ofBiotogical Sciences, Uhiversity of 7lsukuba, Tlsukuba, Ibaraki, 3C15-85Z2 Japan i DE:partment ofMbiecular Geneties, Ntitional Ihstitute ofAgrobiological Resources, 7lsukuba, Ibaraki, 305-86{12 Jlu)an ` Core Research for Evolutional Science and TZichnology rCREST), Chiyoda-ku, fokyo, IOJ-O062 Japan

A bacterial endophyte Azospirillum sp. B510 induces systemic disease resistance in the host without accompanying defense-related gene expression. To elucidate molecular mechanism of this induced systemic resistance (ISR), involvement of ethylene (ET) was examined using OsEIN2-knockdown mutant rice. Rice blast inoculation assay and gene expression analysis indicated that ET signaling is required for endophyte-mediated ISR in rice.Abbreviations: ACC: 1-aminocyclopropane-1-carboxylic acid; EIN2: ethylene-insensitive protein 2; ET: ethylene; ISR: induced systemic resistance; JA: jasmonic acid; RNAi: RNA interference; SA: salicylic acid; SAR: systemic acquired resistance

Summary Physicochemical properties of storage starch largely determine rice grain quality and food characteristics. Therefore, modification of starch property is effective to fine‐tune cooked rice textures. To obtain new resources with modified starch property as breeding materials, we screened a mutant population of a japonica cultivar Nipponbare and found two independent mutant lines, altered gelatinization (age)1 and age2, with moderate changes in starch gelatinization property. A combination of conventional genetic analyses and the latest mapping method, MutMapPlus, revealed that both of these lines harbour novel independent mutant alleles of starch branching enzyme IIb (BEIIb) gene. In age1, amino acid substitution of Met‐723 to Lys completely abolished BEIIb enzyme activity without significant reduction in its protein level. A transposon insertion in an intron of BEIIb gene reduced BEIIb protein level and activity in age2. Production of a series of the mutant lines by combining age alleles and indica‐type starch synthase IIa allele established stepwise alteration of the physicochemical properties of starch including apparent amylose content, thermal property, digestibility by α‐amylase and branched structures of amylopectin. Consistent with the alteration of starch properties, the results of a sensory evaluation test demonstrated that warm cooked rice of the mutants showed a variety of textures without marked reduction in overall palatability. These results suggest that a series of the mutant lines are capable of manipulation of cooked rice textures.

Because environmental stress can reduce crop growth and yield, the identification of genes that enhance agronomic traits is increasingly important. Previous screening of full-length cDNA overexpressing (FOX) rice lines revealed that OsTIFY11b, one of 20 TIFY proteins in rice, affects plant size, grain weight, and grain size. Therefore, we analyzed the effect of OsTIFY11b and nine other TIFY genes on the growth and yield of corresponding TIFY-FOX lines. Regardless of temperature, grain weight and culm length were enhanced in lines overexpressing TIFY11 subfamily genes, except OsTIFY11e. The TIFY-FOX plants exhibited increased floret number and reduced days to flowering, as well as reduced spikelet fertility, and OsTIFY10b, in particular, enhanced grain yield by minimizing decreases in fertility. We suggest that the enhanced growth of TIFY-transgenic rice is related to regulation of the jasmonate signaling pathway, as in Arabidopsis. Moreover, we discuss the potential application of TIFY overexpression for improving crop yield.

α-Amylase is a starch-hydrolyzing enzyme (EC 3.2.1.1) indispensable for germination of cereal seeds, but it is also expressed during the ripening stage. Previous studies demonstrated that the enzyme is activated in developing rice seeds under extremely hot weather and triggers a loss of grain quality by hindering the accumulation of storage starch in the endosperm. Since inactive or, preferably, heat-labile α-amylases are preferable for breeding premium rice, we developed a method for rapid screening of inactive and temperature-sensitive mutants of the enzyme by combining the random mutagenesis by error-prone PCR and an on-filter activity test of the recombinant enzyme expressed by Escherichia coli. This technique was applied to a major α-amylase in the developing seed, Amy3D, and the activity of the isolated mutant enzymes was verified with both the bacteria-expressed recombinant proteins and the extract from the endosperm overexpressing each of them. Then, we identified several substitutions leading to loss of the activity of amino acid residues (Leu28, Asp112, Cys149, Trp201, Asp204, Gly295, Leu300 and Cys342), as well as a variety of heat-sensitive substitutions of Asp83, Asp187 and Glu252. Furthermore, variations of the heat-labile enzymes were created by combining these heat-sensitive mutations. The effects of the respective mutations and their relationship to the structure of the enzyme molecule are discussed.

Global warming impairs grain filling in rice and reduces starch accumulation in the endosperm, leading to chalky-appearing grains, which damages their market value. We found previously that high temperature-induced expression of starch-lytic α-amylases during ripening is crucial for grain chalkiness. Because the rice genome carries at least eight functional α-amylase genes, identification of the α-amylase(s) that contribute most strongly to the production of chalky grains could accelerate efficient breeding. To identify α-amylase genes responsible for the production of chalky grains, we characterized the histological expression pattern of eight α-amylase genes and the influences of their overexpression on grain appearance and carbohydrate components through a series of experiments with transgenic rice plants. The promoter activity of most α-amylase genes was elevated to various extents at high temperature. Among them, the expression of Amy1A and Amy3C was induced in the internal, especially basal to dorsal, region of developing endosperm, whereas that of Amy3D was confined near the ventral aleurone. These regions coincided with the site of occurrence of chalkiness, which was in clear contrast to conventionally known expression patterns of the enzyme in the scutellum and aleurone during seed germination. Furthermore, overexpression of α-amylase genes, except for Amy3E, in developing endosperm produced various degrees of chalky grains without heat exposure, whereas that of Amy3E yielded normal translucent grains, as was the case in the vector control, even though Amy3E-overexpressing grains contained enhanced α-amylase activities. The weight of the chalky grains was decreased due to reduced amounts of starch, and microscopic observation of the chalky part of these grains revealed that their endosperm consisted of loosely packed round starch granules that had numerous pits on their surface, confirming the hydrolysis of the starch reserve by α-amylases. Moreover, the chalky grains contained increased amounts of soluble sugars including maltooligosaccharides at the expense of starch. The integrated analyses proposed that expression of Amy1A, Amy3C, and Amy3D at the specific regions of the developing endosperm could generate the chalkiness. This finding provides the fundamental knowledge to narrow down the targets for the development of high temperature-tolerant premium rice.