Tissue specific expression of UMAMIT amino acid transporters in wheat
Posted on: Sunday, April 3rd, 2022 | ID: #715

Identification of TaUMAMIT genes

Wheat UMAMIT homologs were searched using Interpro and Pfam programs21,22. Interpro search of all 44 Arabidopsis UMAMIT genes classified them as WAT1-related family (IPR30183), and most carried EamA domain (IPR00620). Searching wheat proteins (Ensembl v47) for IPR30183 and IPR00620 yielded 194 and 192 translated sequences respectively. Among those, 182 sequences belonged to WAT1-related family (IPR30183) and contained EamA domain (IPR00620). Pfam search of Arabidopsis UMAMIT genes identified two repeats of PF00892 motifs in full-length proteins (EamA domain). Pfam search of wheat proteins with PF00892 yielded an identical list of 194 proteins with the Interpro search using IPR00620. Blastp search using all AtUMAMIT genes, at the e-value cutoff of 1e−5, yielded 186 entries, all of which were included in the list found in the Interpro search. In total, 204 genes have been identified. The domain structure of full-length UMAMITs is shown in Fig. S1.

In addition, low confidence genes in the Refseq v1.1 cDNA annotation have also been mined. A tblastn search using all AtUMAMIT protein sequences at e-value cutoff of 1e−5, yielded an additional 28 genes, of which 12 were partial genes. Corresponding intervals for all low confidence proteins were identified in IWGSCv1.0 genome, and gene models were examined using the reads mapped to the corresponding intervals in CRAM files for 308 publicly available RNAseq datasets (Table S1). Of the 28 low confidence genes, 10 were significantly expressed, allowing the gene models to be examined. The longest isoform for each was chosen and searched for conserved domains with Interpro. Nine out of 10 included domain IPR30184 (WAT1-related). These proteins were added to the list of high confidence proteins, resulting in the list of 213 TaUMAMIT genes. As expected for hexaploidy wheat, the majority of genes were found in all three subgenomes; among 213 proteins, 153 were found as triads, whereas 60 were not found in at least one of the three genomes (Table S2). In total, 98 distinct groups of homeologs were identified, among which 51 were found in triads (Table S3).

Most of the UMAMIT family proteins analyzed so far are broad specificity amino acid transporters, except for Arabidopsis WAT1, which is a vacuolar auxin transporter17,23. To identify the clades to which the TaUMAMIT proteins belong, a phylogenetic tree including the sequences from six representative species (sequences found in Table S4) was constructed (Fig. 1). Previously published phylogenetic analyses of AtUMAMITs identified seven clades13,24, and a more recent study identified three additional clades while merging the previously identified clades I, II, and VI into a single clade (D)25. Genes belonging to clades I to IV, VII, IX, and X were found in both Arabidopsis and wheat. Clades I and III includes previously characterized amino acid transporters13,14,15,16. Two other clades include previously documented genes; clade V includes Arabidopsis WAT117,23, and clade IX includes RTP1/UMMAIT36, which has been identified as a negative regulator of SA-mediated pathogen response26. Clade VI, which contains many wheat and rice UMAMIT members, did not contain any Arabidopsis members. Clade IX, on the other hand, did not contain any UMAMIT from wheat, confirming the previous report showing that this clade is dicot-specific. In summary, all but one clade of UMAMITs found in Arabidopsis were conserved in wheat. TaUMAMIT genes were distributed on all 21 chromosomes and the unassigned scaffold. Some of the TaUMAMITs were found in multiple tandem duplications, such as the lower arm of chromosome 6, where 6 or 7 tandem repeats of TaUMAMITs belonging to clade III are found (Fig. 2). In some cases, subgenome-specific duplication was also observed. Overall, the results suggest that UMAMITs have been mostly conserved since the formation of hexaploidy in wheat.

Figure 1figure 1

Phylogneitc relationship between UMAMITs of wheat and other species. The wheat gene IDs are abbreviated as; TraesCS1A02G019700 = Ta1A019700, indicated by blue dots. UMAMITs from other species are indicated as follows: Arabidopsis thaliana, no abbreviations (UMAMIT); Physco, Physcomitrella patens; Selagi, Selaginella moellendorffii; Pinus, Pinus pinaster; Picea, Picea abies; Os, Oryza sativa. Arabidopsis UMAMITs are indicated with triangles. The clade IDs have been assigned according to the numbers based previous publications13,24. The corresponding clade in Zhao et al. is indicated in parenthesis25.

Figure 2figure 2

Chromosomal locations of TaUMAMIT genes. The gene IDs are color coded by the clades as indicated in the inset. Low confidence genes identified in this study are indicated with _M. Un represents the unassigned scaffold.

According to the positions in the phylogenetic trees, we have assigned names to the gene models (Tables S2 and S3).

Tissue expression of UMAMITs

In order to understand the tissue specificity of TaUMAMITs, an RNAseq dataset prepared from grain, leaf, root, spike and stem tissues at three different developmental stages each4 was aligned to the IWGSCv1.0 genome. Reads mapped to the UMAMIT genes annotated in the published transcriptome (v. 47), as well as to the manually annotated UMAMITs identified in this study were counted. In total, the expression of 91 out of 98 homeologous groups was detected in at least one tissue. The largest cluster of genes (cluster 1 in Fig. 3) was mainly expressed in the roots. Some genes such as TaUMAMIT22 and 23 were specific to an early stage of development (Z10, first leaf through coleoptile), whereas the majority of genes in this group showed higher expression at later stages (Z13, three leaves stage; Z39, flag leaf stage) of development. The homeologous groups expressed the most were TaUMAMIT17 and 39 (average TPM through three stages at 968.4 and 676.9, respectively), which were expressed highly in all three developmental stages. Homeologous groups belonging to cluster 2 are largely specific to stem tissues, most of which were expressed higher in the later stage (Z65, during anthesis) compared to the earlier stage (Z32, second node detectable). Likewise, many genes that are mainly expressed in leaves (cluster 3) are induced during the grain filling (Z71), as observed for TaUMAMIT50 and TaUMAMIT42. Together, these TaUMAMITs induced during reproductive growth in the vegetative tissues could be involved in nitrogen remobilization. Clusters 4 and 5 genes are expressed mainly in the reproductive tissues (spikes and grains, respectively). Multiple homeologous groups belonging to clade III (Figs. 1, 3, TaUMAMIT11, 18, 20 and 21) are highly specific to the spikes at Z65 (mid anthesis, early grain development). Clade III genes in Arabidopsis have been previously shown to be involved in grain filling5,6, therefore the function of clade III UMAMITs might be conserved between Arabidopsis and wheat. Likewise, TaUMAMIT1, 4, 5, 6, 8 and 9, belonging to clade II that includes previously characterized AtUMAMITs involved in grain filling7, were found to be expressed at water ripe, medium milk and soft dough stages of grain filling (Z71 Z75,and Z85 respectively).

Figure 3figure 3

Expression of UMAMIT genes in different organs. The heatmap is scaled by row (each gene). For each gene, expression values were averaged for all representing wheat subgenomes. Growth stages are expressed in Zadoks growth scale (i.e. Z71 represents Zadoks stage 71). UMAMIT genes without expression in any of the cell type has been excluded from the figure.

Grain development

Amino acids are the main carrier of N delivered to the developing seeds via the phloem8. In wheat, amino acids are released from the nucellar projection on the maternal side to be picked up by the filial tissues such as the embryo, transfer cells and the aleurone layer surrounding the endosperm9. To identify the UMAMITs involved in the amino acid transfer process described above, a publicly available RNAseq data set derived from whole endosperm at 10 days post-anthesis (DPA), starchy endosperm at 20 and 30 DPA, aleurone layer at 20 and 30 DPA, and transfer cells at 20DPA10 were queried as described above. The cell type that expressed the largest number of UMAMIT genes was transfer cells (Fig. 4, cluster 3), with 18 genes being largely specific to this cell type. Cluster 4 genes were also expressed in transfer cells, with expression detected in 10DPA whole endosperm. TaUMAMIT92 and 17 were the most highly expressed triads in the transfer cells (average TPM 264.5 and 96.1, respectively). Another cell type with prominent UMAMIT expression was aleurone layer cells, with 14 genes largely specific to this cell type (Fig. 4 cluster 5). TaUMAMIT51 and 43 were the most abundant triads in aleurone cells (72.9 and 45.9 TPM, respectively in 20 DPA). Starchy endosperm cells also expressed several UMAMITs, most of which were also developmentally regulated (Fig. 4 clusters 2 and 5).

Figure 4figure 4

Expression of UMAMIT genes in different cell types of developing grains. The heatmap is scaled by row (each gene). For each gene, the expression values were averaged for all representing wheat subgenomes. WE Whole endosperm, WE Starchy endosperm, AL Aleurone layer, TC Transfer cells. UMAMIT genes without expression in any of the cell type has been excluded from the figure.

Previous studies found transfer cells and aleurone layer cells to be also expressing multiple amino acid importers, signifying the potential role for these cell types in amino acid transport11. The expression of concentrative amino acid importers and UMAMITs within the same tissue type begs a question; since UMAMITs characterized so far function as bidirectional transporters5,6,7,12, the co-expression of UMAMIT transporters and concentrative amino acid importers could potentially lead to a futile cycle in which amino acid entering the cell through an amino acid importer is lost through UMAMIT transporters. However, the currently available data set does not allow for cellular resolution. Likely, the tissues examined consist of more than one cell type with distinct functions, as revealed by recent single-cell RNAseq studies showing that the cells in the vascular bundle expressing mainly sugar and amino acid importers are distinct from those expressing the exporters13,14. In addition, polar localization of transporters within the same cell type might allow for the directional transport, as previously found for both plant15,16,18 and animal19,20 systems. Some of the UMAMITs might also be involved in amino acid transport across the intracellular membranes, analogous to AtUMAMIT247. Additional studies of UMAMIT localization within this tissue type will help to understand the path of amino acid transport and the roles of UMAMIT.

Senescence

During grain filling, as much as 90% of nitrogen is remobilized from the leaves to the developing grains21, and remobilized N accounts for ~ 70% of total grain N22. During reproductive growth, RuBisCO and other chloroplastic proteins in the source leaves are degraded to amino acids, which are transported to the grains23. Therefore, high amino acid export activity is expected at the source leaves during grain filling. To identify the TaUMAMITs potentially involved in amino acid export from senescing leaves, RNAseq data set including post-anthesis leaves was queried17. Among 55 homeologous groups expressed, 16 groups (Fig. 5, cluster 2) were induced at the 23 and 26 days after anthesis (DAA), at which stage chlorophyll contents decrease significantly17. The second cluster (Fig. 5, cluster 1) on the other hand, showed higher expression in the earlier stages and was downregulated during senescence.

Figure 5figure 5

TaUMAMIT expression in senescing leaves. The heatmap is scaled by row (each gene). For each gene, the expression values were averaged for all representing wheat subgenomes. DAA Days after anthesis.

These results, therefore, seem to show that the UMAMIT genes involved in the amino acid export from actively photosynthesizing leaves are likely different from those mediating the export at the later stage of senescence. Those TaUMAMITs upregulated in the late senescence stage might be a part of the gene network mediated by a NAC transcription factors such as NAM-B1, which promotes senescence and increases N transfer to the grains27,28, or other pathways induced during cell death29. Interestingly, an ectopic expression of Arabidopsis UMAMIT has been shown to induce SA-mediated stress response and cell death30. Whether TaUMAMITs are expressed in response to senescence, or involved in promoting cell death in the senescing tissues remains to be seen.

Earlier studies examining the leaf N content after anthesis showed a gradual decline in N throughout the filling stage and not a sharp decline at the late senescing stage31,31,33, and phloem amino acid content remains high (~ 900 mM detected in aphid stylet exudation) throughout early and mid grain filling stages despite some changes in composition34. Therefore, it is likely that amino acid export out of the flag leaf is also a continuous process, and is mediated not only by those expressed in the late senescence stage but also by those expressed in the earlier stages. Understanding the main players in leaf N export will require additional biochemical analysis to identify the active amino acid exporters, as well as detailed temporal analysis of leaf N loss and the expression profile of TaUMAMITs.

Expression and amino acid transport activity of selected TaUMAMITs expressed in grains

To validate the expression of some of the TaUMAMITs in grains, four strongly expressed TaUMAMIT genes were selected (Fig. 6a). The expression pattern of TaUMAMIT17 (clade III), 30 (clade IV), 51 (clade VII), and 92 (clade VI) was examined using qRT-PCR, using a primer set that detects all three homeologs. As expected, all of them were significantly expressed in grains at both 5 and 14 days after flowering (DAF), representing early- and mid-grain filling stages, respectively (Fig. 6b). TaUMAMIT17 accumulation was similar between five and 14 DAF. For TaUMAMIT30, 51, and 92, the expression levels differed between the two time points; TaUMAMIT30 and 51 expression was higher at 5DAF whereas the level of TaUMAMIT92 was higher at 14DAF (Fig. 6b). In addition, TaUMAMIT17, 30 and 51 were expressed in leaf, stem and root tissues. The expression level of TaUMAMIT17 and 51 in the stem was higher at 14 DAF compared to 5 DAF (Fig. 6b).

Figure 6figure 6

Expression of selected TaUMAMITs in grain tissues. (a) Expression of TaUMAMIT17, 30, 51, 92 in various grain tissues. The TPM values for each subgenome are indicated. AL Aleurone layer, CE Central endosperm, SE Starchy endosperm, TC Transfer cells, WE Whole endosperm, DPA Days after anthesis. (b) Expression of selected TaUMAMITs in developing grains, leaf, stem and root. The mRNA levels detected with RT-qPCR are expressed as the relative amount to the reference gene, TaGAPDH. Statistical difference according to Student’s t-test is indicated by asterisk (p < 0.05). All experiments were conducted at n = 3. DAF Days after flowering.

To test their functions as amino acid exporters, one homeolog each of TaUMAMIT17, 30, 51, 92 were selected (Table S5) and expressed in the yeast strain 22Δ10α which lacks all known endogenous amino acid importers12. The cells were grown in a minimal medium containing ammonium as the sole nitrogen source, and the excretion of L-amino acids to the medium was measured using a colorimetric assay. The results indicated that TaUMAMIT17 has a strong activity for amino acid export, resulting in > 100 fold concentration of amino acids in the growth media compared to the control expressing the empty vector, and higher than cells expressing previously characterized AtUMAMIT14 and 18 genes (Fig. 7). TaUMAMIT17 belongs to clade III, to which many Arabidopsis UMAMITs with known amino acid export activity belong5,6,12. Expression of TaUMAMIT30 (clade IV) and 51 (clade VII) did not result in a statistically significant increase in amino acid excretion compared to the WT, despite them belonging to the clades that include Arabidopsis members that show some activity as amino acid exporters25.The amino acid secretion from yeast expressing clade I, V, and VII transporters was lower than those expressing clade III genes from Arabidopsis in the yeast expression system. TaUMAMIT92 belongs to clade VI, which largely consists of monocot members. TaUMAMIT92 did not show a significant amino acid transport activity in our assay. Therefore, whether transporters in these clades indeed function as amino acid exporters remain to be seen.

Figure 7figure 7

TaUMAMIT17 exports amino acid secretion in yeast. Amino acid secretion from 22Δ10α cells expressing TaUMAMIT17D, AtUMAMIT14 and AtUMAMIT18, compared the vector control (pDR196). Statistical difference according to Student’s t-test is shown with asterisks (n ≥ 5).

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