Influence of dietary carbohydrate profile on the dairy cow rumen meta-proteome.

Diet starch and fiber contents influence the rumen microbial profile and its fermentation products, yet no information exists about the effects of these dietary carbohydrate fractions on the metabolic activity of these microbes. The objective of this experiment was to evaluate the effects of dietary carbohydrate profile changes on the rumen meta-proteome profile. Eight cannulated Holstein cows were assigned to the study as part of a 4 × 4 Latin square design with a 2 × 2 factorial treatment arrangement including four 28-d periods. Cows received 1 of 4 dietary treatments on a dry matter (DM) basis. Diets included different concentrations of rumen fermentable starch (RFS) and physically effective undigested NDF (peuNDF240) content in the diet: (1) low peuNDF240, low RFS (LNLS); (2) high peuNDF240, low RFS (HNLS); (3) low peuNDF240, high RFS (LNHS); and (4) high peuNDF240, high RFS (HNHS). Rumen fluid samples were collected from each cow on the last 2 d of each period at 3 time points (0600, 1000, and 1400 h). The microbial protein fraction was isolated, isobarically labeled, and analyzed using liquid chromatography combined with tandem mass spectrometry techniques. Product ion spectra were searched using the SEQUEST search on Proteome Discoverer 2.4 (Thermo Scientific) against 71 curated microbe-specific databases. Data were analyzed using PROC MIXED procedure in SAS 9.4 (SAS Institute Inc.). A total of 138 proteins were characterized across 26 of the searched microbial species. In total, 46 proteins were affected by treatments across 17 of the searched microbial species. Of these 46 proteins, 28 were affected by RFS content across 13 microbial species, with 20 proteins having higher abundance with higher dietary RFS and 8 proteins having higher abundance with lower dietary RFS. The majority of these proteins have roles in energetics, carbon metabolism, and protein synthesis. Examples include pyruvate, phosphate dikinase (Ruminococcus albus SY3), 30S ribosomal protein S11 (Clostridium aminophilum), and methyl-coenzyme M reductase subunit α (Methanobrevibacter ruminantium strain 35063), which had higher abundances with higher dietary RFS. Conversely, glutamate dehydrogenase (Butyrivibrio fibrisolvens) and 50S ribosomal protein L5 (Pseudobutyrivibrio ruminis) and L15 (Ruminococcus bromii) had lower abundances with higher dietary RFS content. Among the remaining 18 proteins unaffected by RFS content alone, 5 proteins were affected by peuNDF240 content, and 13 were affected by peuNDF240 × RFS interactions. Our results suggest that the RFS content of the diet may have a greater influence on rumen microbial protein abundances than dietary peuNDF240 content or peuNDF240 × RFS interactions. This research highlights that dietary carbohydrate profile changes can influence rumen microbial protein abundances. Further research is needed to fully characterize the effects of diet on the rumen meta-proteome and manipulate the various roles of rumen microbes. This will aid in designing the strategies to maximize the efficiency of nutrient use in the rumen.

A Novel Mutation of OsPPDKB, Encoding Pyruvate Orthophosphate Dikinase, Affects Metabolism and Structure of Starch in the Rice Endosperm.

Starch, as a main energy storage substance, plays an important role in plant growth and human life. Despite the fact that several enzymes and regulators involved in starch biosynthesis have been identified, the regulating mechanism of starch synthesis is still unclear. In this study, we isolated a rice floury endosperm mutant M14 from a mutant pool induced by 60Co. Both total starch content and amylose content in M14 seeds significantly decreased, and starch thermal and pasting properties changed. Compound starch granules were defected in the floury endosperm of M14 seeds. Map-based cloning and a complementation test showed that the floury endosperm phenotype was determined by a gene of OsPPDKB, which encodes pyruvate orthophosphate dikinase (PPDK, EC 2.7.9.1). Subcellular localization analysis demonstrated that PPDK was localized in chloroplast and cytoplasm, the chOsPPDKB highly expressed in leaf and leaf sheath, and the cyOsPPDKB constitutively expressed with a high expression in developing endosperm. Moreover, the expression of starch synthesis-related genes was also obviously altered in M14 developing endosperm. The above results indicated that PPDK played an important role in starch metabolism and structure in rice endosperm.

Structure and enzyme expression in photosynthetic organs of the atypical C4 grass Arundinella hirta.

In its leaf blade, Arundinella hirta has unusual Kranz cells that lie distant from the veins (distinctive cells; DCs), in addition to the usual Kranz units composed of concentric layers of mesophyll cells (MCs) and bundle sheath cells (BSCs; usual Kranz cells) surrounding the veins. We examined whether chlorophyllous organs other than leaf blades--namely, the leaf sheath, stem, scale leaf, and constituents of the spike--also have this unique anatomy and the C4 pattern of expression of photosynthetic enzymes. All the organs developed DCs to varying degrees, as well as BSCs. The stem, rachilla, and pedicel had C4-type anatomy with frequent occurrence of DCs, as in the leaf blade. The leaf sheath, glume, and scale leaf had a modified C4 anatomy with MCs more than two cells distant from the Kranz cells; DCs were relatively rare. An immunocytochemical study of C3 and C4 enzymes revealed that all the organs exhibited essentially the same C4 pattern of expression as in the leaf blade. In the scale leaf, however, intense expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) occurred in the MCs as well as in the BSCs and DCs. In the leaf sheath, the distant MCs also expressed Rubisco. In Arundinella hirta, it seems that the ratio of MC to Kranz cell volumes, and the distance from the Kranz cells, but not from the veins, affects the cellular expression of photosynthetic enzymes. We suggest that the main role of DCs is to keep a constant quantitative balance between the MCs and Kranz cells, which is a prerequisite for effective C4 pathway operation.

Analysis of leaf proteome after UV-B irradiation in maize lines differing in sensitivity.

UV-B radiation causes diverse morphological and physiological responses in plants, but the underlying mechanisms governing these integrated responses are unknown. In this study, we systematically surveyed responses of maize leaves to UV-B radiation using DIGE 2D gels and identified selected proteins by mass spectrometry and immunodetection analysis. To identify changes in protein accumulation in response to UV-B radiation, a line (b, pl W23) deficient in flavonoid sunscreen compounds and hence similar to commercial corn was used. In addition, its proteome in natural UV-B conditions was compared with that of two maize landraces from high altitudes (Cacahuacintle and Confite Puneño) that have improved UV-B tolerance. Protein patterns in adult maize leaves (Zea mays) were documented after growth for 21 days in sunlight depleted of UV-B radiation or growth in sunlight including an 8-h UV-B supplementation during 1 day in the field. We found that there is a very high correlation between previously documented mRNA accumulation assessed by microarray hybridization and quantitative real time reverse transcription-PCR and protein expression after UV-B irradiation in leaves of W23. Multiple isoforms were confirmed for some proteins; at least one protein, pyruvate, phosphate dikinase, is regulated post-translationally by phosphorylation by UV-B exposure. Proteins differentially regulated by UV-B radiation in W23 with higher levels under similar UV-B conditions in high altitude plants were also identified. These could be genetically fixed traits conferring UV-B tolerance and offer clues to specific adaptations to living in high ambient UV-B conditions.

[Determination of biological substances using bioluminescent reaction based on luciferin-luciferase].

Firefly luciferase catalyzes the oxidation of luciferin in the presence of ATP, magnesium ions and molecular oxygen, with a high quantum yield. Due to its high sensitivity and specificity for ATP, luciferase has been used for bioluminescent detection of ATP in various biological samples. However, it has not been well determined how to apply it in immunoassay. In this article, the use of various enzymes as labels in the design and development of immunoassays, and the combination of PCR/novel bioluminescent pyrophosphate assay, for detecting biomolecules is reviewed.

Primary structure of maize pyruvate, orthophosphate dikinase as deduced from cDNA sequence.

We have isolated two overlapping cDNA clones that encompass the entire structural gene for pyruvate, orthophosphate dikinase from maize. The analysis of the nucleotide sequence has revealed that the cDNA clones include an insert of a total of 3,171 nucleotides without a poly(A) tail and encode a polypeptide that contains 947 amino acid residues and has a molecular weight of 102,673. Comparison of the N-terminal amino acid sequence of purified pyruvate, orthophosphate dikinase protein with that deduced from the nucleotide sequence shows that the mature form of pyruvate, orthophosphate dikinase in the maize chloroplast consists of 876 amino acid residues and has a molecular weight of 95,353. The amino acid composition of the deduced sequence of pyruvate, orthophosphate dikinase is in good agreement with that of the purified enzyme. The region that contains the active and regulatory sites of pyruvate, orthophosphate dikinase can be found in the deduced sequence of amino acids. We have predicted the secondary structure and calculated the hydropathy pattern of this region. The extra 71 residues at the N terminus of the deduced sequence of amino acid residues corresponds to the transit peptide which is indispensable for the transport of the precursor protein into chloroplasts. We have compared the primary structure of the pyruvate, orthophosphate dikinase transit peptide to those of other proteins and found sequences similar to the consensus sequences found in other transit peptides.

Isolation of 3-phosphohistidine from phosphorylated pyruvate, phosphate dikinase.

Pyruvate, phosphate dikinase (EC 2-7-9-1) catalyzes formation of phosphoenolpyruvate, AMP, and inorganic pyrophosphate from pyruvate, ATP, and orthophosphate. A pyrophosphoryl and phosphoryl form of the enzyme is involved in this transfer. The [32P]phosphoryl form of pyruvate, phosphate dikinase was prepared with enzyme isolated from Bacteroides symbiosus. The [32P]phosphoryl enzyme was found to have properties corresponding to a phosphoramidate linkage and this was confirmed by isolation of 3-[32P]phosphohistidine from alkaline hydrolysates of the enzyme. The histidyl residue is considered to be the pyrophosphoryl- and phosphoryl-carrier between the three substrate sites of this enzyme.