The longest isoforms of wheat UMAMIT genes (213 total) and Arabidopsis UMAMIT genes (44 total) were used for the analysis. Additionally, UMAMIT genes in four representative species (Physcomitrella patens, Selaginella moellendorffii, Arabidopsis thaliana, Oryza sativa) previously published were included31. The sequences were aligned using MUSCLE35, with gap open penalty of − 2.9, gap extend 0 and hydrophobicity multiplier 1.2, UPGMA clustering option. The aligned sequence was used to build a phylogenetic tree with RAxML version 8.236, using rapid bootstrap analysis (1000 iterations) and GAMMA model of rate heterogeneity using empirical base frequencies and the LG substitution model.
Wheat RNAseq projects publicly available on NCBI Sequence Read Archive (SRA) database ()36 were surveyed and datasets with the following SRA study IDs were selected for further processing: ERP004714 (spatiotemporal development), ERP004505 (grain development), and SRP166963 (leaf senescence). For each SRA study, the read counts for high confidence UMAMI genes were obtained from iRAP pipeline (v1.0.1)37—mapped data available at European Nucleotide Archive (ENA)38 (), obtained using RNASeq-er REST API39. The pipeline uses FASTX (v0.0.13) for filtering and adapter trimming, FASTQC (v0.11.7) for quality check, HISAT2 (v2.1.0)40 for alignment and featureCounts (v1.6.2)41 for quantification. The mapping quality for every dataset was confirmed to be above 75%. For determining counts of low confidence UMAMIT genes, the HISAT240 mapped CRAM alignment and corresponding CRAI index files from the ENA database were used to count the reads within the corresponding intervals identified in this studyHyderabad Stocks. The wheat genome assembly and GTF version 41 were downloaded from the Ensembl Plants website (ftp://ftp.ensemblgenomes.org/pub/plants/release-41). SAMTools (v1.9)42 was used to extract the reads within gene boundaries for the low confidence UMAMIT genesGuoabong Wealth Management. FeatureCounts41 from Subread package (version 1.6.2) was then used with the GTF file corresponding to the low confidence UMAMIT genes to count the mapped reads. The read counts for the high- and low-confidence genes were processed in featureCounts, then normalized using GeTMM43, where gene length was obtained by summing lengths of unique exons for each gene from the GTF files. Mean was taken across replicates and the resulting expression was summed for each gene in the triad. For triads missing one or more genes in the triad, only the mean expression of available genes was added. This was followed by log2 transformation in GeTMM with an offset of 1 to reduce the dynamic range for creating the heatmap. The R-package pheatmap (v1.0.12)44 was used to generate the heatmaps by scaling the mean sample expression by row to show peak tissue expression. The row clustering was performed using the ward.D method and Euclidean distance11Surat Investment. The genes with zero variance across all samples were removed to resolve errors resulting from clustering in pheatmap.Chennai Stock
TaUMAMIT sequences were synthesized (Twist Bioscience, USA) and cloned into the pTwist_ENTR vector. TaUMAMIT17 and 92 were codon-optimized to avoid GC-rich sequences. The sequences of synthesized fragments are found in Table S5. All TaUMAMITs were introduced into the yeast expression vector pDR196-f1GW45 via gateway cloning (Invitrogen, USA).
TaUMAMITs amino acid export activity was determined via yeast cell secretion in liquid media according to the procedure previously described7,12. Briefly, 22Δ10α (genotype MATα gap1-1 put4-1 uga4-1 can1::HisG lyp1-alp1::HisG hip1::HisG dip5::HisG gnp1Δ agp1Δ ura3-1) expressing individual UMAMIT proteins were grown in a minimum medium46 overnight at 28C. On the next day, the cultures were diluted to OD600 = 0.1 in 5 mL of minimal medium, then incubated at 28C for 5.5 h. The cultures were spun down and the supernatant was flitered using a 10 kDa exclusion membrane. Amino acid concentration of the supernatants was determined using L-Amino Acid Quantitation Kit (Sigma-Aldrich, USA) following the manufacturer’s instruction.Hyderabad Wealth Management
Wheat (Triticum aestivum cv. TAM11447) plants were grown in 2018 at Texas A&M Research and Extension Center in Bushland, TX (35° 06′ N, 102° 27′ W). Standard agronomic practices were performed according to Texas A&M AgriLife extension recommendations. Spikes at five and 14 days after flowering were harvested and stored in liquid N2. The grains were mechanically isolated from the glumes while soaking in liquid N2, and stored at − 80 °C until use. Flag leaves and stems were harvested from plants grown at 28 °C with a 12 h light cycle in a greenhouse, at five and 14 days after flowering. Root tissues were collected from 10 day-old seedlings germinated on filter paper moistened with distilled water.
Chennai Investment
