FLOURY ENDOSPERM19 encoding a class I glutamine amidotransferase affects grain quality in rice

As a staple food for more than half of the world’s population, the importance of rice is self-evident. Compared with ordinary rice, rice cultivars with superior eating quality and appearance quality are more popular with consumers due to its unique taste and ornamental value, even if their price is much higher. Appearance quality and CEQ (cooking and eating quality) are two very important aspects in the evaluation of rice quality. Here, we performed a genome-wide association study on chalkiness rate in a diverse panel of 533 cultivated rice accessions. We identified a batch of potential chalky genes and prioritize one (LOC_Os03g48060) for functional analyses. Two floury outer endosperm mutants (flo19-1 and flo19-2) were generated through editing LOC_Os03g48060 (named as FLO19 in this study), which encodes a class I glutamine amidotransferase. The different performance of the two mutants in various storage substances directly led to completely different changes in CEQ. The mutation of FLO19 gene caused the damage of carbon and nitrogen metabolism in rice, which affected the normal growth and development of rice, including decreased plant height and yield loss by decreased grain filling rate. Through haplotype analysis, we identified a haplotype of FLO19 that can improve both CEQ and appearance quality of rice, Hap2, which provides a selection target for rice quality improvement, especially for high-yield indica rice varieties.

Neutron crystallographic study of heterotrimeric glutamine amidotransferase CAB

Heterotrimeric glutamine amidotransferase CAB (GatCAB) possesses an ammonia-self-sufficient mechanism in which ammonia is produced and used in the inner complex by GatA and GatB, respectively. The X-ray structure of GatCAB revealed that the two identified active sites of GatA and GatB are markedly distant, but are connected in the complex by a channel of 30 Å in length. In order to clarify whether ammonia is transferred through this channel in GatCAB by visualizing ammonia, neutron diffraction studies are indispensable. Here, GatCAB crystals were grown to approximate dimensions of 2.8 × 0.8 × 0.8 mm (a volume of 1.8 mm3) with the aid of a polymer using microseeding and macroseeding processes. Monochromatic neutron diffraction data were collected using the neutron single-crystal diffractometer BIODIFF at the Heinz Maier-Leibnitz Zentrum, Germany. The GatCAB crystals belonged to space group P212121, with unit-cell parameters a = 74.6, b = 94.5, c = 182.5 Å and with one GatCAB complex (molecular mass 119 kDa) in the asymmetric unit. This study represented a challenge in current neutron diffraction technology.

Choline Catabolism in Burkholderia thailandensis Is Regulated by Multiple Glutamine Amidotransferase 1-Containing AraC Family Transcriptional Regulators

ABSTRACTBurkholderia thailandensisis a soil-dwelling bacterium that shares many metabolic pathways with the ecologically similar, but evolutionarily distant,Pseudomonas aeruginosa. Among the diverse nutrients it can utilize is choline, metabolizable to the osmoprotectant glycine betaine and subsequently catabolized as a source of carbon and nitrogen, similar toP. aeruginosa. Orthologs of genes in the choline catabolic pathway in these two bacteria showed distinct differences in gene arrangement as well as an additional orthologous transcriptional regulator inB. thailandensis. In this study, we showed that multiple glutamine amidotransferase 1 (GATase 1)-containing AraC family transcription regulators (GATRs) are involved in regulation of theB. thailandensischoline catabolic pathway (gbdR1,gbdR2, andsouR). Using genetic analyses and sequencing the transcriptome in the presence and absence of choline, we identified the likely regulons ofgbdR1(BTH_II1869) andgbdR2(BTH_II0968). We also identified a functional ortholog forP. aeruginosasouR, a GATR that regulates the metabolism of sarcosine to glycine. GbdR1 is absolutely required for expression of the choline catabolic locus, similar toP. aeruginosaGbdR, while GbdR2 is important to increase expression of the catabolic locus. Additionally, theB. thailandensisSouR ortholog (BTH_II0994) is required for catabolism of choline and its metabolites as carbon sources, whereas inP. aeruginosa, SouR function can by bypassed by GbdR. The strategy employed byB. thailandensisrepresents a distinct regulatory solution to control choline catabolism and thus provides both an evolutionary counterpoint and an experimental system to analyze the acquisition and regulation of this pathway during environmental growth and infection.IMPORTANCEMany proteobacteria that occupy similar environmental niches have horizontally acquired orthologous genes for metabolism of compounds useful in their shared environment. The arrangement and differential regulation of these components can help us understand both the evolution of these systems and the potential roles these pathways have in the biology of each bacterium. Here, we describe the transcriptome response ofBurkholderia thailandensisto the eukaryote-enriched molecule choline, identify the regulatory pathway governing choline catabolism, and compare the pathway to that previously described forPseudomonas aeruginosa. These data support a multitiered regulatory network inB. thailandensis, with conserved orthologs in the select agentsBurkholderia pseudomalleiandBurkholderia mallei, as well as the opportunistic lung pathogens in theBurkholderia cepaciaclade.

Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein fromStaphylococcus aureuspeptidoglycan

Amidation of peptidoglycan is an essential feature inStaphylococcus aureusthat is necessary for resistance to β-lactams and lysozyme. GatD, a 27 kDa type I glutamine amidotransferase-like protein, together with MurT ligase, catalyses the amidation reaction of the glutamic acid residues of the peptidoglycan ofS. aureus. The native and the selenomethionine-derivative proteins were crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol, sodium acetate and calcium acetate. The crystals obtained diffracted beyond 1.85 and 2.25 Å, respectively, and belonged to space groupP212121. X-ray diffraction data sets were collected at Diamond Light Source (on beamlines I02 and I04) and were used to obtain initial phases.