Dataset Information

Conserved residues in the wheat (Triticum aestivum) NAM-A1 NAC domain are required for protein binding and when mutated lead to delayed peduncle and flag leaf senescence.

ABSTRACT: BACKGROUND:NAC transcription factors contain five highly conserved subdomains which are required for protein dimerisation and DNA binding. Few residues within these subdomains have been identified as essential for protein function, and fewer still have been shown to be of biological relevance in planta. Here we use a positive regulator of senescence in wheat, NAM-A1, to test the impact of missense mutations at specific, highly conserved residues of the NAC domain on protein function. RESULTS:We identified missense mutations in five highly conserved residues of the NAC domain of NAM-A1 in a tetraploid TILLING population. TILLING lines containing these mutations, alongside synonymous and non-conserved mutation controls, were grown under glasshouse conditions and scored for senescence. Four of the five mutations showed a significant and consistent delay in peduncle senescence but had no consistent effects on flag leaf senescence. All four mutant alleles with the delayed senescence phenotype also lost the ability to interact with the homoeolog NAM-B1 in a yeast two-hybrid assay. Two of these residues were previously shown to be involved in NAC domain function in Arabidopsis, suggesting conservation of residue function between species. Three of these four alleles led to an attenuated cell death response compared to wild-type NAM-A1 when transiently over-expressed in Nicotiana benthamiana. One of these mutations was further tested under field conditions, in which there was a significant and consistent delay in both peduncle and leaf senescence. CONCLUSIONS:We combined field and glasshouse studies of a series of mutant alleles with biochemical analyses to identify four residues of the NAC domain which are required for NAM-A1 function and protein interaction. We show that mutations in these residues lead to a gradient of phenotypes, raising the possibility of developing allelic series of mutations for traits of agronomic importance. We also show that mutations in NAM-A1 more severely impact peduncle senescence, compared to the more commonly studied flag leaf senescence, highlighting this as an area deserving of further study. The results from this integrated approach provide strong evidence that conserved residues within the functional domains of NAC transcription factors have biological significance in planta.

SUBMITTER: Harrington SA

PROVIDER: S-EPMC6749658 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Conserved residues in the wheat (Triticum aestivum) NAM-A1 NAC domain are required for protein binding and when mutated lead to delayed peduncle and flag leaf senescence.

Harrington Sophie A SA Overend Lauren E LE Cobo Nicolas N Borrill Philippa P Uauy Cristobal C

BMC plant biology 20190918 1

<h4>Background</h4>NAC transcription factors contain five highly conserved subdomains which are required for protein dimerisation and DNA binding. Few residues within these subdomains have been identified as essential for protein function, and fewer still have been shown to be of biological relevance in planta. Here we use a positive regulator of senescence in wheat, NAM-A1, to test the impact of missense mutations at specific, highly conserved residues of the NAC domain on protein function.<h4> ...[more]

PMID: 31533618

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Project description:BACKGROUND: MicroRNAs (miRNAs) regulate various biological processes in plants. Considerable data are available on miRNAs involved in the development of rice, maize and barley. In contrast, little is known about miRNAs and their functions in the development of wheat. In this study, five small RNA (sRNA) libraries from wheat seedlings, flag leaves, and developing seeds were developed and sequenced to identify miRNAs and understand their functions in wheat development. RESULTS: Twenty-four known miRNAs belonging to 15 miRNA families were identified from 18 MIRNA loci in wheat in the present study, including 15 miRNAs (9 MIRNA loci) first identified in wheat, 13 miRNA families (16 MIRNA loci) being highly conserved and 2 (2 MIRNA loci) moderately conserved. In addition, fifty-five novel miRNAs were also identified. The potential target genes for 15 known miRNAs and 37 novel miRNAs were predicted using strict criteria, and these target genes are involved in a wide range of biological functions. Four of the 15 known miRNA families and 22 of the 55 novel miRNAs were preferentially expressed in the developing seeds with logarithm (log2) of the fold change of 1.0 ~ 7.6, and half of them were seed-specific, suggesting that they participate in regulating wheat seed development and metabolism. From 5 days post-anthesis to 20 days post-anthesis, miR164 and miR160 increased in abundance in the developing seeds, whereas miR169 decreased, suggesting their coordinating functions in the different developmental stages of wheat seed. Moreover, 8 known miRNA families and 28 novel miRNAs exhibited tissue-biased expression in wheat flag leaves, with the logarithm of the fold changes of 0.1 ~ 5.2. The putative targets of these tissue-preferential miRNAs were involved in various metabolism and biological processes, suggesting complexity of the regulatory networks in different tissues. Our data also suggested that wheat flag leaves have more complicated regulatory networks of miRNAs than developing seeds. CONCLUSIONS: Our work identified and characterised wheat miRNAs, their targets and expression patterns. This study is the first to elucidate the regulatory networks of miRNAs involved in wheat flag leaves and developing seeds, and provided a foundation for future studies on specific functions of these miRNAs.

Project description:Background: MicroRNAs regulate various biological processes in plants. Considerable data are available on miRNAs involved in the development of rice, maize and barley. In contrast, little is known about miRNAs and their functions in the development of wheat. In this study, five small RNA (sRNA) libraries from wheat seedlings, flag leaves, and developing seeds were developed and sequenced to identify miRNAs and understand their functions in wheat development. Results: Twenty-four known miRNAs belonging to 15 miRNA families were identified from 18 MIRNA loci in wheat in the present study, including 15 (9 MIRNA loci) first identified in wheat, 13 miRNA families (16 MIRNA loci) being highly conserved and 2 (2 MIRNAs loci) moderately conserved. In addition, fifty-five novel miRNAs were also identified. The potential target genes for 15 known miRNAs and 37 novel miRNAs were predicted using strict criteria, and these target genes are involved in a wide range of biological functions. Four of the 15 known miRNA families and 22 of the 55 novel miRNAs were preferentially expressed in the developing seeds with logarithm of the fold change of 1.0～7.6, and half of them were seed-specific, suggesting that they participate in regulating wheat seed development and metabolism. From 5 days post-anthesis to 20 days post-anthesis, miR164 and miR160 increased in abundance in developing seeds, whereas miR169 decreased, suggesting their coordinating functions in the different developmental stages of wheat seed. Moreover, eight known miRNA families and 28 novel miRNAs exhibited tissue-biased expression in wheat flag leaves, with the logarithm of the fold changes of 0.5～5.2. The putative targets of these tissue-preferential miRNAs were involved in various metabolism and biological processes, suggesting complexity of the regulatory networks in different tissues. Our data also suggested that wheat flag leaves have more complicated regulatory networks of miRNAs than developing seeds. Conclusions: Our work identified and characterised wheat miRNAs, their targets and expression patterns. This study is the first to elucidate the regulatory networks of miRNAs involved in wheat flag leaves and developing seeds, and provided a foundation for future studies on specific functions of these miRNAs.

Dataset Information

Conserved residues in the wheat (Triticum aestivum) NAM-A1 NAC domain are required for protein binding and when mutated lead to delayed peduncle and flag leaf senescence.

Publications

Conserved residues in the wheat (Triticum aestivum) NAM-A1 NAC domain are required for protein binding and when mutated lead to delayed peduncle and flag leaf senescence.

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