Genome-wide identification and evolutionary and expression 1 analysis of the wheat amino acid transporter, an important 2 gene family involved in abiotic stresses

16 17 Background: Amino acid transporters (AATs), which transport amino acids across 18 cellular membranes, play important roles in alleviating plant damage under stresses as 19 well as in plant growth and development. Although this family has been 20 systematically studied in many plant species, little is known about the AAT genes in 21 bread wheat ( Triticum aestivum L.) due to its complex genome sequence. 22 23 Results: In this study, a total of 296 AAT genes were identified from the latest wheat 24 genome sequence (IWGSC v1.1) and classified into twelve distinct subfamilies based 25 upon their sequence composition and phylogenetic relationship. Wheat AAT family 26 members showed significant heterogeneity in chromosome distribution, with 27 relatively high density in specific chromosomal regions. Comparison the number 28 variation of gene copies and transmembrane regions of AAT genes in different 29 sub-genome showed that the functional adaptation of the wheat AAT family during 30 wheat polyploidization was driven mainly by sequence mutations rather than copy 31 number variation. In addition, it was confirmed that changes in gene structure and 32 protein conserved domains played important roles in the functional differentiation of 33 the AAT family. Finally, the expression profiles of these TaAAT genes under heat, 34 drought and salt stress and in the development stage of wheat showed that the 35 expression of TaAATs exhibited abundant and distinct expression patterns under 36 different abiotic stresses or in different tissues, and several important candidate AAT 37 genes that may affect abiotic stress response and grain quality were also identified. 38 39 Conclusions : In this study, a total of 297 AAT proteins were systematically identified 40 and characterized. Our study highlighted the important roles of gene duplication 41 events in the expansion and functional differentiation of the wheat AAT family. The 42 expression profiles of TaAATs revealed their importance for the grain development of 43 wheat and their response to biotic and abiotic stresses. Our study also provided a 44 theoretical basis for the further functional identification and utilization of the AAT 45 gene family in wheat or other crops.


23
Results: In this study, a total of 296 AAT genes were identified from the latest wheat genes that may affect abiotic stress response and grain quality were also identified.
Background 50 Amino acids are not only indispensable for the formation of proteins but also the main 51 carrier for nitrogen exchange in plants [1,2]. The obtainment of organic nitrogen 52 depends to a large extent on the transport of amino acids, especially in processes of 53 seed germination and seedling growth, mainly by amino acid transporters (AATs) [3,   genes' temporal and spatial expression characteristics and their responses to abiotic 131 stresses, this study will provide a basis for further functional analysis of the wheat 132 AAT genes, as well as a better understanding of the molecular mechanisms underlying 133 the wheat AAT genes' regulation of wheat growth and stress responses.

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Identification of AAT gene family in wheat 138 Initially, a total of 307 putative wheat AAT transcripts were identified in the wheat 139 genome by local BLASTP and HMMER searches. Twenty of the transcripts 140 corresponded to 10 genes, and we selected the longest transcripts as candidates. One  Table S1. The length of the putative wheat AAT proteins ranged from 318 to 1002, with 150 isoelectric points (pI) ranging from 5.00 to 8.96 and molecular weights (Mw) ranging 151 from 33.5 to 52.5 kD. The homologous AAT genes from different wheat sub-genomes 152 showed no significant difference in protein characteristics.

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To better understand the transmembrane structure of the AAT family, TMHMM 154 Server 2.0 was used to predict the putative transmembrane (TM) regions. The number 155 of TM regions in TaAAT proteins ranged from 6 to 15, and AAT genes belonging to the 156 same subfamily showed a similar distribution (Fig. 1, Additional file 2: Table S1).

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Among them, CAT subfamily members contained the most TM regions, ranging from 158 13 to 15, while the AAP and LHT subfamily members contained the fewest TM regions, 159 ranging from 7 to 11 and 7 to 10, respectively (Fig. 1). In addition, it was found that 19 160 groups of homologous genes derived from the A, B and D sub-genomes were mutated 161 in TM number, accounting for approximately 20% (19 of 93) of the total complete 162 homologous gene groups identified in this study, which confirmed that during the 163 process of wheat genome doubling, the homologous genes from different sub-genomes 164 had great TM number variation, to achieve functional synergy and suitability.

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Chromosomal distribution and duplication analysis of AAT genes in wheat 167 To study the relationship between AAT gene family expansion and gene duplication in    (Table 2). In all species, the proportion of tandemly duplicated genes in 206 the AAP subfamily was very high, ranging from 25% to 52.63%, which confirmed the

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The alignment of the TaANT members is shown in Fig. 6 as an example. All 286 ANT family member protein sequences derived from the wheat A sub-genome were 287 used for multiple sequence alignments. The overall identity of the protein sequences 288 of these genes was 61.97%. There were six conserved motifs in TaANTs, including 289 motifs 1, 7, 9, 2, 13 and 5 (Fig. 6). There was a very high correlation between the 290 conserved motifs and the transmembrane regions, in which motifs 1, 7, 2, 5, 13 291 corresponded to TM1, TM2, TM7, TM9 and TM8, respectively, and motif 9 292 corresponded to TM3. All of the conserved sequences were located around the 293 transmembrane domains, suggesting that the stabilization of the transmembrane 294 domains played an integral role in the normal functioning of ANT family proteins.

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The unequal distribution of TM numbers in different subfamilies also confirmed that 296 TM affected the functional specificity of different subfamilies.

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Analysis of cis-regulatory elements and three-dimensional modeling 299 The cis-regulatory elements predicted in promoter regions of all AAT genes were 300 mainly classified into three categories: tissue specificity, stress response, and hormone 301 response. A large number of stress response cis-regulatory regulatory elements were 302 found in all gene promoter regions, indicating that the AAT genes responded strongly 303 to stress, and multiple GA, ABA and other hormone response elements were found, 304 indicating that these genes might be involved in multiple hormone signal pathways. In 305 addition, multiple transcription factor binding sites, such as MYB, were observed in 306 the promoter regions, confirming that these genes might be regulated by a variety of 307 transcription factors.

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All predicted AAT proteins contained multiple α-helices and coil structures (Fig.   309   7). The multiple α-helix structures ensured the efficient and stable transmembrane 310 transport of AAT proteins. Most AAT proteins had similar three-dimensional 311 structures, and the closer the phylogenetic relationship was between genes, the closer 312 the three-dimensional structures of the proteins were, such as LHT and ProT (Fig. 7). which suggested that most occurred before the formation of hexaploid wheat (Fig. 2).

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In general, the expansion of AAT gene family in wheat mainly relies on WGD and 408 tandem duplication events.  (Table 1, Additional file 2: Table S1). All of these  and TaAAP18 were down-regulated, confirming that the response of the first two genes 527 to salt stress can be maintained for a longer period of time, while the latter two genes 528 may play a role mainly in the early stage. In general, TaAATs showed different response 529 modes to abiotic stresses, including enthusiasm for response, generality of response, 530 and differences in response duration, which may greatly improve the adaptability of 531 wheat to abiotic stresses.

533
We identified 296 AAT gene family members in the wheat genome. Similar to other 534 identified species, all wheat AAT genes were classified into two families -the AAAP 535 and APC families. The AAAP family was subdivided into 8 subfamilies, while the APC 536 family was divided into 4 subfamilies. We demonstrate that the expansion of the wheat 537 AAT gene family is primarily due to WGD and tandem duplication events, while 538 tandem repeat events have greatly determined the functional differentiation of AAT 539 family members. We systematically outlined the chromosomal distribution, gene 540 structure, phylogeny and conserved motifs of AAT family members in wheat and 541 annotated all its AAT genes. We further evaluated the expression patterns of wheat AAT 542 gene family members in different tissues and their responses to three conventional 543 abiotic stresses, heat, drought and salt stress. We also identified several important 544 candidate genes that might affect grain quality and root amino acid transport. Our work 545 will provide a comprehensive framework for the study of the wheat AAT family and 546 will also contribute to the functional analysis and utilization of wheat AAT genes.

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The X-axis indicates 14 tissues at three stages, and the Y-axis represents the TPM 907 value. The full names for X-axis tissue abbreviations are shown in Fig. 8.