Table_3.XLSX

<p>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.</p

Table_4.XLSX

<p>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.</p

Table_5.XLSX

<p>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.</p

Table_1.XLSX

<p>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.</p

Additional file 1: Table S1. of Proteomic and Glycomic Characterization of Rice Chalky Grains Produced Under Moderate and High-temperature Conditions in Field System

Proteomic comparison of chalky and perfect grains harvested in 2009 and 2010. s.d., standard deviation; # AAs, the number of amino acids; MW, the calculated molecular weight; pI, the calculated isoelectric point; ΣCoverage, the percentage of sequence coverage. (XLSX 62343 kb

Genetic and isotope ratio mass spectrometric evidence for the occurrence of starch degradation and cycling in illuminated Arabidopsis leaves

<div><p>Although there is a great wealth of data supporting the occurrence of simultaneous synthesis and breakdown of storage carbohydrate in many organisms, previous ¹³CO₂ pulse-chase based studies indicated that starch degradation does not operate in illuminated Arabidopsis leaves. Here we show that leaves of <i>gwd</i>, <i>sex4</i>, <i>bam4</i>, <i>bam1/bam3</i> and <i>amy3/isa3/lda</i> starch breakdown mutants accumulate higher levels of starch than wild type (WT) leaves when cultured under continuous light (CL) conditions. We also show that leaves of CL grown <i>dpe1</i> plants impaired in the plastidic disproportionating enzyme accumulate higher levels of maltotriose than WT leaves, the overall data providing evidence for the occurrence of extensive starch degradation in illuminated leaves. Moreover, we show that leaves of CL grown <i>mex1/pglct</i> plants impaired in the chloroplastic maltose and glucose transporters display a severe dwarf phenotype and accumulate high levels of maltose, strongly indicating that the MEX1 and pGlcT transporters are involved in the export of starch breakdown products to the cytosol to support growth during illumination. To investigate whether starch breakdown products can be recycled back to starch during illumination through a mechanism involving ADP-glucose pyrophosphorylase (AGP) we conducted kinetic analyses of the stable isotope carbon composition (δ¹³C) in starch of leaves of ¹³CO₂ pulsed-chased WT and AGP lacking <i>aps1</i> plants. Notably, the rate of increase of δ¹³C in starch of <i>aps1</i> leaves during the pulse was exceedingly higher than that of WT leaves. Furthermore, δ¹³C decline in starch of <i>aps1</i> leaves during the chase was much faster than that of WT leaves, which provides strong evidence for the occurrence of AGP-mediated cycling of starch breakdown products in illuminated Arabidopsis leaves.</p></div

Leaves of different starch breakdown mutants display a high starch content phenotype when cultured under continuous light conditions.

<p>(A) Iodine staining and (B) starch content in leaves of WT and the indicated starch breakdown mutants cultured under CL conditions. Leaves were harvested at the 18 days after sowing (DAS) growth stage. In “B” values represent the means ± SE determined from three independent experiments using 6 plants in each experiment. Asterisks indicate significant differences with respect to WT plants according to Student´s t-tests (<i>p</i><0.05).</p

Amyloglucosidase releases carbon compounds other than starch glucose molecules from Arabidopsis leaf ethanol precipitates.

<p>The graphic shows the total carbon (TOC) and the starch carbon content in amyloglucosidase digests of WT (Col-<i>0</i>) and <i>aps1</i> leaves. Values represent the means ± SE determined from three independent experiments using 6 plants in each experiment.</p

Maltose content in leaves of WT (Col-<i>0</i>), <i>aps1</i>, <i>pgm</i>, <i>aps1/pgm</i>, <i>mex1/aps1</i> and <i>mex1/pgm</i> plants.

<p>Leaves of the indicated plants were harvested at the 18 DAS growth stage. Values obtained using HPAEC-PAD and GC-MS are represented with white and grey columns, respectively. Values represent the means ± SE determined from three independent experiments using 6 plants in each experiment. Asterisks indicate significant differences according to Student´s t-tests (*<i>P</i><0.05, <i>aps1</i>, <i>pgm</i> and <i>aps1/pgm</i> vs. Col-<i>0</i>; **<i>P</i><0.05, <i>mex1/aps1</i> vs. <i>aps1;</i> ***<i>P</i><0.05, <i>mex1/pgm</i> vs. <i>pgm</i>). Values correspond to plants cultured under continuous light (CL) conditions. Essentially the same results were obtained using plants cultured under long day (LD) conditions (not shown).</p

δ¹³C kinetics in starch is slower than that of sucrose in WT plants cultured in ¹³CO₂-enriched environment.

<p>The graphic represents the values of δ¹³C in starch and the indicated soluble sugars of leaves of 26 DAS WT (Col-<i>0</i>) plants exposed to ¹³C enriched CO₂ for 5 hours and then chased for 15 additional hours. Plants were cultured in growth cabinets under long day (LD) conditions. The grey area indicates the ¹³CO₂ pulse period. Values represent the means ± SE determined from three independent experiments using 6 plants in each experiment.</p