Altered Expression of OsAAP3 Influences Rice Lesion Mimic and Leaf Senescence by Regulating Arginine Transport and Nitric Oxide Pathway

Persistent lesion mimic can cause leaf senescence, affecting grain yield in crops. However, knowledge about the regulation of lesion mimic and leaf senescence in crop plants is still limited. Here, we report that the amino acid transporter OsAAP3, a negative regulator of tiller bud elongation and rice grain yield, is involved in lesion mimic and leaf senescence. Altered expression of OsAAP3 can initiate the nitric oxide signaling pathway through excessive accumulation of arginine in rice leaves, influencing ROS accumulation, antioxidant enzymes activities, proline concentration, and malondialdehyde concentration. This finally triggers cell death which ultimately leads to lesion mimic and leaf senescence by regulating the degradation of chloroplast and the expression abundance of components in the photosynthetic pathway. Overall, the results not only provide initial insights into the regulatory role of amino acid transport genes in rice growth and development, but also help to understand the factors regulating the leaf senescence.     

BRITTLE CULM17, a Novel Allele of TAC4, Affects the Mechanical Properties of Rice Plants

Lodging resistance of rice (Oryza sativa L.) has always been a hot issue in agricultural production. A brittle stem mutant, osbc17, was identified by screening an EMS (Ethylmethane sulfonate) mutant library established in our laboratory. The stem segments and leaves of the mutant were obviously brittle and fragile, with low mechanical strength. Examination of paraffin sections of flag leaf and internode samples indicated that the number of cell layers in mechanical tissue of the mutant was decreased compared with the wild type, Pingtangheinuo, and scanning electron microscopy revealed that the mechanical tissue cell walls of the mutant were thinner. Lignin contents of the internodes of mature-stage rice were significantly lower in the mutant than in the wild type. By the MutMap method, we found candidate gene OsBC17, which was located on rice chromosome 2 and had a 2433 bp long coding sequence encoding a protein sequence of 810 amino acid residues with unknown function. According to LC-MS/MS analysis of intermediate products of the lignin synthesis pathway, the accumulation of caffeyl alcohol in the osbc17 mutant was significantly higher than in Pingtangheinuo. Caffeyl alcohol can be polymerized to the catechyl lignin monomer by laccase ChLAC8; however, ChLAC8 and OsBC17 are not homologous proteins, which suggests that the osbc17 gene is involved in this process by regulating laccase expression.     

LPS1, Encoding Iron-Sulfur Subunit SDH2-1 of Succinate Dehydrogenase,Affects Leaf Senescence and Grain Yield in Rice

The iron-sulfur subunit (SDH2) of succinate dehydrogenase plays a key role in electron transport in plant mitochondria. However, it is yet unknown whether SDH2 genes are involved in leaf senescence and yield formation. In this study, we isolated a late premature senescence mutant, lps1, in rice (Oryza sativa). The mutant leaves exhibited brown spots at late tillering stage and wilted at the late grain-filling stage and mature stage. In its premature senescence leaves, photosynthetic pigment contents and net photosynthetic rate were reduced; chloroplasts and mitochondria were degraded. Meanwhile, lps1 displayed small panicles, low seed-setting rate and dramatically reduced grain yield. Gene cloning and complementation analysis suggested that the causal gene for the mutant phenotype was OsSDH2-1 (LOC_Os08g02640), in which single nucleotide mutation resulted in an amino acid substitution in the encoded protein. OsSDH2-1 gene was expressed in all organs tested, with higher expression in leaves, root tips, ovary and anthers. OsSDH2-1 protein was targeted to mitochondria. Furthermore, reactive oxygen species (ROS), mainly H₂O₂, was excessively accumulated in leaves and young panicles of lps1, which could cause premature leaf senescence and affect panicle development and pollen function. Taken together, OsSDH2-1 plays a crucial role in leaf senescence and yield formation in rice.     

Leaf Mutant 7 Encoding Heat Shock Protein OsHSP40 Regulates Leaf Size in Rice

Leaf size is an important agronomic trait directly affecting yield in rice, and thus understanding the genes determining leaf size is important in breeding. In this study, one Leaf Mutant 7 (lm7) with small leaf size was isolated using ethyl methane sulphonate (EMS) mutagenesis from the japonica Zhenggeng 1925. MutMap by whole genome resequencing of phenotypic bulks revealed that LM7 is likely located in the 133 kb region on chromosome 7 using F₂ population from a cross between lm7 and wild-type (WT) Zhenggeng 1925. The candidate gene encoding heat shock protein OsHSP40 for LM7 was functionally validated. Disruption of this gene in Oshsp40 mutants significantly reduced the leaf size compared with that of WT in rice. Microscopic examination showed that OsHSP40 modulated leaf size via regulating the veins formation and cell size/cell number. Nucleotide diversity analysis indicated that a single nucleotide polymorphism (SNP) variation of C to T in the coding region of OsHSP40 may cause small leaves among rice accessions. Therefore, the natural variation of OsHSP40 contributing to leaf size might be useful for rice breeding.     

Programmed Editing of Rice (Oryza sativa L.) OsSPL16 Gene Using CRISPR/Cas9 Improves Grain Yield by Modulating the Expression of Pyruvate Enzymes and Cell Cycle Proteins

Rice (Oryza sativa L.) is one of the major crops in the world and significant increase in grain yield is constant demand for breeders to meet the needs of a rapidly growing population. The size of grains is one of major components determining rice yield and a vital trait for domestication and breeding. To increase the grain size in rice, OsSPL16/qGW8 was mutagenized through CRISPR/Cas9, and proteomic analysis was performed to reveal variations triggered by mutations. More specifically, mutants were generated with two separate guide RNAs targeting recognition sites on opposite strands and genomic insertions and deletions were characterized. Mutations followed Mendelian inheritance and homozygous and heterozygous mutants lacking any T-DNA and off-target effects were screened. The mutant lines showed a significant increase in grain yield without any change in other agronomic traits in T₀, T₁, and T₂ generations. Proteomic screening found a total of 44 differentially expressed proteins (DEPs), out of which 33 and 11 were up and downregulated, respectively. Most of the DEPs related to pyruvate kinase, pyruvate dehydrogenase, and cell division and proliferation were upregulated in the mutant plants. Pathway analysis revealed that DEPs were enriched in the biosynthesis of secondary metabolites, pyruvate metabolism, glycolysis/gluconeogenesis, carbon metabolism, ubiquinone and other terpenoid-quinone biosynthesis, and citrate cycle. Gene Ontology (GO) analysis presented that most of the DEPs were involved in the pyruvate metabolic process and pyruvate dehydrogenase complex. Proteins related to pyruvate dehydrogenase E1 component subunit alpha-1 displayed higher interaction in the protein-protein interaction (PPI) network. Thus, the overall results revealed that CRISPR/Cas9-guided OsSPL16 mutations have the potential to boost the grain yield of rice. Additionally, global proteome analysis has broad applications for discovering molecular components and dynamic regulation underlying the targeted gene mutations.     

Enhanced microwave absorption and electromagnetic shielding property of (1-x)K0.5Na0.5NbO3 ~ xAl2O3 nano-ceramics

(1-x) K_0.5Na_0.5NbO₃ ~ xAl₂O₃ (x = 0, 0.2, 0.4, 0.6) ceramics were prepared via a traditional solid-state reaction method. The phase structure, micro-morphology, dielectric properties and electromagnetic properties of ceramic samples were studied and analyzed. Results indicate that all the samples are similar to K_0.5Na_0.5NbO₃ (KNN) in perovskite structure. With the increase of Al₂O₃ content, the X-ray diffraction peaks move to a large angle region, suggesting the substitution of niobium ions by aluminium ions and the distortion of the KNN lattice with a new phase arising. With the increase of Al₂O₃ content the grain size reduces and the dielectric constant decrease, yielding to the decrease of the electromagnetic shielding performance of ceramic. When the x is 0.4, the minimum value of reflectivity of sample is −28 dB at the frequency of 11.6 GHz. It can be concluded that both the grain size and Al₂O₃ content can obviously affect the electromagnetic properties of ceramics, which can be easily turned through a multi-layer SiO₂ heterojunction structure.     

OsBRKq1, Related Grain Size Mapping,and Identification of Grain Shape Based on QTL Mapping in Rice

The world population is growing rapidly, and food shortage remains a critical issue. Quantitative trait locus (QTL) mapping is a statistical analytical method that uses both phenotypic and genotypic data. The purpose of QTL mapping is to determine the exact gene location for various complex traits. Increasing grain weight is a way to increase yield in rice. Genes related to grain size were mapped using the Samgang/Nagdong double haploid (SNDH) populations. Grain sizes were diversely distributed in SNDH 113 populations, and OsBRKq1 was detected on chromosome 1 in an analysis of QTL mapping that used 1000 grain weight, grain length, and grain width. OsBRKq1 exhibited high sequence similarity with the brassinosteroid leucine-rich repeat-receptor kinases of Arabidopsis thaliana and Zea mays. It was also predicted to have a similar function because of its high homology. OsBRKq1 interacts with various grain-size control genes. Among the SNDH populations, the analysis of the relative expression level during the panicle formation stage of OsBRKq1 in panicles of SNDH117, which has the largest grain size, and SNDH6, which has the smallest grain size, the relative expression level was significantly increased in SNDH117 panicles. SNDH populations have been advancing generations for 10 years; various genetic traits have been fixed and are currently being used as bridging parents. Therefore, the stable expression level of OsBRKq1 was confirmed via QTL mapping. In the future, OsBRKq1 can be effectively used to increase the yield of rice and solve food problems by increasing the size of seeds.     

Identifying QTLs for Grain Size in a Colossal Grain Rice (Oryza sativa L.) Line,and Analysis of Additive Effects of QTLs

Grain size is an important component of quality and harvest traits in the field of rice breeding. Although numerous quantitative trait loci (QTLs) of grain size in rice have been reported, the molecular mechanisms of these QTLs remain poorly understood, and further research on QTL observation and candidate gene identification is warranted. In our research, we developed a suite of F₂ intercross populations from a cross of 9311 and CG. These primary populations were used to map QTLs conferring grain size, evaluated across three environments, and then subjected to bulked-segregant analysis-seq (BSA-seq). In total, 4, 11, 12 and 14 QTLs for grain length (GL), grain width (GW), 1000-grain weight (TGW), and length/width ratio (LWR), respectively, were detected on the basis of a single-environment analysis. In particular, over 200 splicing-related sites were identified by whole-genome sequencing, including one splicing-site mutation with G>A at the beginning of intron 4 on Os03g0841800 (qGL3.3), producing a smaller open reading frame, without the third and fourth exons. A previous study revealed that the loss-of-function allele caused by this splicing site can negatively regulate rice grain length. Furthermore, qTGW2.1 and qGW2.3 were new QTLs for grain width. We used the near-isogenic lines (NILs) of these GW QTLs to study their genetic effects on individuals and pyramiding, and found that they have additive effects on GW. In summary, these discoveries provide a valuable genetic resource, which will facilitate further study of the genetic polymorphism of new rice varieties in rice breeding.     

Influence of pulse plating parameters on the synthesis and preferred orientation of nanocrystalline zinc from zinc sulfate electrolytes

The influence of pulse electrodeposition parameters (current on-time T_on, current off-time T_off, and pulse current density J_p) was investigated on the surface morphology and grain size of zinc electrodeposited from a sulfate bath containing polyacrylamide and thiourea additives. The grain size and surface morphology of zinc deposits were studied by field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM), and the preferred orientation of the deposits was studied by X-ray diffraction. At constant current off-time and pulse current density, the grain size decreased asymptotically with increasing current on-time. In contrast, increase in the current off-time at constant current on-time and pulse current density resulted in grain growth. A progressive decrease of the grain size was observed with increasing pulse current density at constant current on-time and off-time. Nanocrystalline zinc with an average grain size of 38 nm was obtained at a pulse current density of 1200 mA/cm². The crystallographic orientations developed were correlated with the change in the cathodic overpotential, the angle between the preferred oriented plane and the lowest energy of formation plane (0 0 0 2), and the pulse electrodeposition parameters.     

Submergence Gene Sub1A Transfer into Drought-Tolerant japonica Rice DT3 Using Marker-Assisted Selection

Flash flooding is a major environmental stressor affecting rice production worldwide. DT3 is a drought-tolerant, recurrent parent with a good yield, edible quality, and agronomic traits akin to those of an elite Taiwanese variety, Taiken9 (TK9). Progenies carrying Sub1A can enhance submergence stress tolerance and can be selected using the marker-assisted backcross (MAB) breeding method. For foreground selection, Sub1A and SubAB1 were utilized as markers on the BC₂F₁, BC₃F₁, and BC₃F₂ generations to select the submergence-tolerant gene, Sub1A. Background selection was performed in the Sub1A-BC₃F₂ genotypes, and the percentages of recurrent parent recovery within individuals ranged from 84.7–99.55%. BC₃F₃ genotypes (N = 100) were evaluated for agronomic traits, yield, and eating quality. Four of the eleven BC₃F₄ lines showed good yield, yield component, grain, and eating quality. Four BC₃F₄ lines, SU39, SU40, SU89, and SU92, exhibited desirable agronomic traits, including grain quality and palatability, consistent with those of DT3. These genotypes displayed a high survival rate between 92 and 96%, much better compared with DT3 with 64%, and demonstrated better drought tolerance compared to IR64 and IR96321-345-240. This study provides an efficient and precise MAB strategy for developing climate-resilient rice varieties with good grain quality for flood-prone regions.     

A Novel Allele Encoding 7-Hydroxymethyl Chlorophyll a Reductase Confers Bacterial Blight Resistance in Rice

Rice spotted leaf mutants are helpful to investigate programmed cell death (PCD) and defense response pathways in plants. Using a map-based cloning strategy, we characterized novel rice spotted leaf mutation spl^HM143 that encodes a 7-hydroxymethyl chlorophyll a reductase (OsHCAR). The wild-type (WT) allele could rescue the mutant phenotype, as evidenced by complementation analysis. OsHCAR was constitutively expressed at all rice tissues tested and its expression products localized to chloroplasts. The mutant exhibited PCD and leaf senescence with increased H₂O₂ (hydrogen peroxide) accumulation, increased of ROS (reactive oxygen species) scavenging enzymes activities and TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling) -positive nuclei, upregulation of PCD related genes, decreased chlorophyll (Chl) contents, downregulation of photosynthesis-related genes, and upregulation of senescence-associated genes. Besides, the mutant exhibited enhanced bacterial blight resistance with significant upregulation of defense response genes. Knockout lines of OsHCAR exhibited spotted leaf phenotype, cell death, leaf senescence, and showed increased resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) coupled with upregulation of five pathogenesis-related marker genes. The overexpression of OsHCAR resulted in increased susceptibility to Xoo with decreased expression of pathogenesis-related marker genes. Altogether, our findings revealed that OsHCAR is involved in regulating cell death and defense response against bacterial blight pathogen in rice.     

Improved Transformation and Regeneration of Indica Rice: Disruption of SUB1A as a Test Case via CRISPR-Cas9

Gene editing by use of clustered regularly interspaced short palindromic repeats (CRISPR) has become a powerful tool for crop improvement. However, a common bottleneck in the application of this approach to grain crops, including rice (Oryza sativa), is efficient vector delivery and calli regeneration, which can be hampered by genotype-dependent requirements for plant regeneration. Here, methods for Agrobacterium-mediated and biolistic transformation and regeneration of indica rice were optimized using CRISPR-Cas9 gene-editing of the submergence tolerance regulator SUBMERGENCE 1A-1 gene of the cultivar Ciherang-Sub1. Callus induction and plantlet regeneration methods were optimized for embryogenic calli derived from immature embryos and mature seed-derived calli. Optimized regeneration (95%) and maximal editing efficiency (100%) were obtained from the immature embryo-derived calli. Phenotyping of T₁ seeds derived from the edited T₀ plants under submergence stress demonstrated inferior phenotype compared to their controls, which phenotypically validates the disruption of SUB1A-1 function. The methods pave the way for rapid CRISPR-Cas9 gene editing of recalcitrant indica rice cultivars.     

The relationship between microstructure and quality factor for (Al,Mg,Ta)O2 microwave dielectrics

The relationship between microstructures and quality factor (Q) of (1−x)(Al_1/2Ta_1/2)O₂–x(Mg_1/3Ta_2/3)O₂ ceramics was investigated. The extrinsic loss of microwave dielectrics depended on cations ordering, grain size, and porosity. (Al_1/2Ta_1/2)O₂ has a disordered structure and (Mg_1/3Ta_2/3)O₂ has an ordered trirutile structure. As (Mg_1/3Ta_2/3)O₂ content increased, (1−x)(Al_1/2Ta_1/2)O₂–x(Mg_1/3Ta_2/3)O₂ ceramics revealed an ordered phase and were of single phase for x>0.6. The increase of the ordered phase and grain size enhanced the Q. When ordering was completed at (Mg_1/3Ta_2/3)O₂ concentration over 60 mol%, the grain size was a major factor in the increase the Q value. In contrast the porosity degraded the Q value. Therefore, the Q value depended on order/disorder, the porosity, and the grain size in that order.     

Functional Characterization of MdTAC1a Gene Related to Branch Angle in Apple (Malus x domestica Borkh.)

The Tiller Angle Control 1 (TAC1) gene belongs to the IGT family, which mainly controls plant branch angle, thereby affecting plant form. Two members of MdTAC1 are identified in apple; the regulation of apple branch angle by MdTAC1 is still unclear. In this study, a subcellular localization analysis detected MdTAC1a in the nucleus and cell membrane, but MdTAC1b was detected in the cell membrane. Transgenic tobacco by overexpression of MdTAC1a or MdTAC1b showed enlarged leaf angles, the upregulation of several genes, such as GA 2-oxidase (GA2ox), and a sensitive response to light and gravity. According to a qRT-PCR analysis, MdTAC1a and MdTAC1b were strongly expressed in shoot tips and vegetative buds of weeping cultivars but were weakly expressed in columnar cultivars. In the MdTAC1a promoter, there were losses of 2 bp in spur cultivars and 6 bp in weeping cultivar compared with standard and columnar cultivars. An InDel marker specific to the MdTAC1a promoter was developed to distinguish apple cultivars and F₁ progeny. We identified a protein, MdSRC2, that interacts with MdTAC1a, whose encoding gene which was highly expressed in trees with large branch angles. Our results indicate that differences in the MdTAC1a promoter are major contributors to branch-angle variation in apple, and the MdTAC1a interacts with MdSRC2 to affect this trait.