Functional Analysis of Rice Long-Chain Acyl-CoA Synthetase 9 (OsLACS9) in the Chloroplast Envelope Membrane.

The long-chain acyl-CoA synthetases (LACSs) are involved in lipid synthesis, fatty acid catabolism, and the transport of fatty acids between subcellular compartments. These enzymes catalyze the critical reaction of fatty acyl chains to fatty acyl-CoAs for the triacylglycerol biosynthesis used as carbon and energy reserves. In Arabidopsis, LACSs are encoded by a family of nine genes, with LACS9 being the only member located in the chloroplast envelope membrane. However, the comprehensive role of LACS9 and its contribution to plant metabolism have not been explored thoroughly. In this study, we report on the identification and characterization of LACS9 mutants in rice plants. Our results indicate that the loss-of-function mutations in OsLACS9 affect the architecture of internodes resulting in dwarf plants with large starch granules in the chloroplast, showing the suppression of starch degradation. Moreover, the plastid localization of α-amylase I-1 (AmyI-1)-a key enzyme involved in starch breakdown in plastids-was suppressed in the lacs9 mutant line. Immunological and confocal laser scanning microscopy analyses showed that OsLACS9-GFP is located in the chloroplast envelope in green tissue. Microscopic analysis showed that OsLACS9s interact with each other in the plastid envelope membrane. Furthermore, OsLACS9 is also one of the proteins transported to plastids without a transit peptide or involvement of the Toc/Tic complex system. To identify the plastid-targeting signal of OsLACS9, the transient expression and localization of a series of N-terminal truncated OsLACS9-green fluorescent protein (GFP) fusion proteins were examined. Truncation analyses identified the N-terminal 30 amino acid residues to be required for OsLACS9 plastid localization. Overall, the data in this study provide an advanced understanding of the function of OsLACS9 and its role in starch degradation and plant growth.

Salt Tolerance Improvement in Rice through Efficient SNP Marker-Assisted Selection Coupled with Speed-Breeding.

Salinity critically limits rice metabolism, growth, and productivity worldwide. Improvement of the salt resistance of locally grown high-yielding cultivars is a slow process. The objective of this study was to develop a new salt-tolerant rice germplasm using speed-breeding. Here, we precisely introgressed the hst1 gene, transferring salinity tolerance from "Kaijin" into high-yielding "Yukinko-mai" (WT) rice through single nucleotide polymorphism (SNP) marker-assisted selection. Using a biotron speed-breeding technique, we developed a BC3F3 population, named "YNU31-2-4", in six generations and 17 months. High-resolution genotyping by whole-genome sequencing revealed that the BC3F2 genome had 93.5% similarity to the WT and fixed only 2.7% of donor parent alleles. Functional annotation of BC3F2 variants along with field assessment data indicated that "YNU31-2-4" plants carrying the hst1 gene had similar agronomic traits to the WT under normal growth condition. "YNU31-2-4" seedlings subjected to salt stress (125 mM NaCl) had a significantly higher survival rate and increased shoot and root biomasses than the WT. At the tissue level, quantitative and electron probe microanalyzer studies indicated that "YNU31-2-4" seedlings avoided Na+ accumulation in shoots under salt stress. The "YNU31-2-4" plants showed an improved phenotype with significantly higher net CO2 assimilation and lower yield decline than WT under salt stress at the reproductive stage. "YNU31-2-4" is a potential candidate for a new rice cultivar that is highly tolerant to salt stress at the seedling and reproductive stages, and which might maintain yields under a changing global climate.

Proteomics Analysis Reveals Non-Controlled Activation of Photosynthesis and Protein Synthesis in a Rice npp1 Mutant under High Temperature and Elevated CO2 Conditions.

Rice nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) catalyzes the hydrolytic breakdown of the pyrophosphate and phosphodiester bonds of a number of nucleotides including ADP-glucose and ATP. Under high temperature and elevated CO2 conditions (HT + ECO2), the npp1 knockout rice mutant displayed rapid growth and high starch content phenotypes, indicating that NPP1 exerts a negative effect on starch accumulation and growth. To gain further insight into the mechanisms involved in the NPP1 downregulation induced starch overaccumulation, in this study we conducted photosynthesis, leaf proteomic, and chloroplast phosphoproteomic analyses of wild-type (WT) and npp1 plants cultured under HT + ECO2. Photosynthesis in npp1 leaves was significantly higher than in WT. Additionally, npp1 leaves accumulated higher levels of sucrose than WT. The proteomic analyses revealed upregulation of proteins related to carbohydrate metabolism and the protein synthesis system in npp1 plants. Further, our data indicate the induction of 14-3-3 proteins in npp1 plants. Our finding demonstrates a higher level of protein phosphorylation in npp1 chloroplasts, which may play an important role in carbohydrate accumulation. Together, these results offer novel targets and provide additional insights into carbohydrate metabolism regulation under ambient and adverse conditions.

N-Glycomic and Microscopic Subcellular Localization Analyses of NPP1, 2 and 6 Strongly Indicate that trans-Golgi Compartments Participate in the Golgi to Plastid Traffic of Nucleotide Pyrophosphatase/Phosphodiesterases in Rice.

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of numerous nucleotides and nucleotide sugars. In many plant species, NPPs are encoded by a small multigene family, which in rice are referred to NPP1-NPP6 Although recent investigations showed that N-glycosylated NPP1 is transported from the endoplasmic reticulum (ER)-Golgi system to the chloroplast through the secretory pathway in rice cells, information on N-glycan composition and subcellular localization of other NPPs is still lacking. Computer-assisted analyses of the amino acid sequences deduced from different Oryza sativa NPP-encoding cDNAs predicted all NPPs to be secretory glycoproteins. Confocal fluorescence microscopy observation of cells expressing NPP2 and NPP6 fused with green fluorescent protein (GFP) revealed that NPP2 and NPP6 are plastidial proteins. Plastid targeting of NPP2-GFP and NPP6-GFP was prevented by brefeldin A and by the expression of ARF1(Q71L), a dominant negative mutant of ADP-ribosylation factor 1 that arrests the ER to Golgi traffic, indicating that NPP2 and NPP6 are transported from the ER-Golgi to the plastidial compartment. Confocal laser scanning microscopy and high-pressure frozen/freeze-substituted electron microscopy analyses of transgenic rice cells ectopically expressing the trans-Golgi marker sialyltransferase fused with GFP showed the occurrence of contact of Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids. Sensitive and high-throughput glycoblotting/mass spectrometric analyses showed that complex-type and paucimannosidic-type glycans with fucose and xylose residues occupy approximately 80% of total glycans of NPP1, NPP2 and NPP6. The overall data strongly indicate that the trans-Golgi compartments participate in the Golgi to plastid trafficking and targeting mechanism of NPPs.

Golgi/plastid-type manganese superoxide dismutase involved in heat-stress tolerance during grain filling of rice.

Superoxide dismutase (SOD) is widely assumed to play a role in the detoxification of reactive oxygen species caused by environmental stresses. We found a characteristic expression of manganese SOD 1 (MSD1) in a heat-stress-tolerant cultivar of rice (Oryza sativa). The deduced amino acid sequence contains a signal sequence and an N-glycosylation site. Confocal imaging analysis of rice and onion cells transiently expressing MSD1-YFP showed MSD1-YFP in the Golgi apparatus and plastids, indicating that MSD1 is a unique Golgi/plastid-type SOD. To evaluate the involvement of MSD1 in heat-stress tolerance, we generated transgenic rice plants with either constitutive high expression or suppression of MSD1. The grain quality of rice with constitutive high expression of MSD1 grown at 33/28 °C, 12/12 h, was significantly better than that of the wild type. In contrast, MSD1-knock-down rice was markedly susceptible to heat stress. Quantitative shotgun proteomic analysis indicated that the overexpression of MSD1 up-regulated reactive oxygen scavenging, chaperone and quality control systems in rice grains under heat stress. We propose that the Golgi/plastid MSD1 plays an important role in adaptation to heat stress.

Optimized Nuclear Pellet Method for Extracting Next-Generation Sequencing Quality Genomic DNA from Fresh Leaf Tissue.

Next-generation sequencing (NGS) is a revolutionary advancement allowing large-scale discovery of functional molecular markers that has many applications, including plant breeding. High-quality genomic DNA (gDNA) is a prerequisite for successful NGS library preparation and sequencing; however, few reliable protocols to obtain such plant gDNA exist. A previously reported nuclear pellet (NP) method enables extraction of high-yielding gDNA from fresh leaf tissue of maize (Zea mays L.), but the quality does not meet the stringent requirements of NGS. In this study, we optimized the NP method for whole-genome sequencing of rice (Oryza sativa L.) through the integration of simple purification steps. The optimized NP method relied on initial nucleus enrichment, cell lysis, extraction, and subsequent gDNA purification buffers. The purification steps used proteinase K, RNase A, phenol/chloroform/isoamyl alcohol (25:24:1), and chloroform/isoamyl alcohol (24:1) treatments for protein digestion and RNA, protein, and phenol removal, respectively. Our data suggest that this optimized NP method allowed extraction of consistently high-yielding and high-quality undegraded gDNA without contamination by protein and RNA. Moreover, the extracted gDNA fulfilled the quality metrics of NGS library preparation for the Illumina HiSeq X Ten platform by the TruSeq DNA PCR-Free Library Prep Kit (Illumina). We provide a reliable step-by-step guide to the extraction of high-quality gDNA from fresh leaf tissues of rice for molecular biologists with limited resources.

Optimized Method of Extracting Rice Chloroplast DNA for High-Quality Plastome Resequencing and de Novo Assembly.

Chloroplasts, which perform photosynthesis, are one of the most important organelles in green plants and algae. Chloroplasts maintain an independent genome that includes important genes encoding their photosynthetic machinery and various housekeeping functions. Owing to its non-recombinant nature, low mutation rates, and uniparental inheritance, the chloroplast genome (plastome) can give insights into plant evolution and ecology and in the development of biotechnological and breeding applications. However, efficient methods to obtain high-quality chloroplast DNA (cpDNA) are currently not available, impeding powerful sequencing and further functional genomics research. To investigate effects on rice chloroplast genome quality, we compared cpDNA extraction by three extraction protocols: liquid nitrogen coupled with sucrose density gradient centrifugation, high-salt buffer, and Percoll gradient centrifugation. The liquid nitrogen-sucrose gradient method gave a high yield of high-quality cpDNA with reliable purity. The cpDNA isolated by this technique was evaluated, resequenced, and assembled de novo to build a robust framework for genomic and genetic studies. Comparison of this high-purity cpDNA with total DNAs revealed the read coverage of the sequenced regions; next-generation sequencing data showed that the high-quality cpDNA eliminated noise derived from contamination by nuclear and mitochondrial DNA, which frequently occurs in total DNA. The assembly process produced highly accurate, long contigs. We summarize the extent to which this improved method of isolating cpDNA from rice can provide practical progress in overcoming challenges related to chloroplast genomes and in further exploring the development of new sequencing technologies.

Ten-year follow-up of familial hypercholesterolemia patients after intensive cholesterol-lowering therapy.

To prevent coronary artery disease, it is necessary for patients with familial hyper-cholesterolemia (FH) to maintain a low cholesterol level. Recently a combination therapy of low-density lipoprotein (LDL) apheresis and statins has been used for FH patients, but their long-term prognosis over 10 years is unknown. In this single center prospective report, 18 FH patients with severe coronary stenosis received LDL apheresis every 2 or 4 weeks and statin therapy for 9.8 +/- 3.0 years. Probucol was given to 17 of the 18 patients. We observed their clinical events as well as coronary stenosis findings and ejection fractions for 10.7 +/- 2.6 years. Total and LDL cholesterol levels before therapy were 345 +/- 46 and 277 +/- 48 mg/dL, respectively. Immediately following LDL-apheresis, these levels decreased to 104 +/- 7.5 and 66 +/- 16 mg/dL, respectively. There were no cardiac deaths and 4 patients were free from any coronary events. There was one noncardiac death. Nonfatal myocardial in-farction occurred in 2 patients and coronary bypass surgery was required in one patient. Twelve patients received additional coronary angioplasty. There was little change in coronary stenosis and ejection fraction following 10 years of the combination therapy. Univariate Cox regression analysis revealed that the calculated mean LDL cholesterol level was the predictive value of treatment efficacy (mean LDL cholesterol < 140 mg/dL, hazard ratio 0.23, P = 0.028). The combination therapy of LDL-apheresis and antilipid drugs delayed the progression of coronary atherosclerosis and prevented a major cardiac event, although complete inhibition was limited to a small group. Additional coronary angioplasty is likely to be required for a favorable clinical outcome in FH patients.