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.

Identification of a Dominant Chlorosis Phenotype Through a Forward Screen of the Triticum turgidum cv. Kronos TILLING Population

“…F 2 populations of the selected TILLING lines (backcrossed to cv. Kronos) were sown at Church Farm in March 2016 and grown as described previously (Harrington et al, 2019b). Briefly, individual F 2 seeds were hand-sown in 6 × 6 1 m 2 grids, leaving approximately 17 cm between each plant (Supplementary Figure 1B).…”

“…Plants were scored for senescence across the different field trials as detailed previously (Harrington et al, 2019b). Briefly, when scoring individual plants, all phenotyping was carried out on the main tiller, tagged upon heading.…”

Identification of Transcription Factors Regulating Senescence in Wheat through Gene Regulatory Network Modelling

“…Furthermore, the NAM-A2 homeologs NAM-B2 (TraesCS2B02G228900) and NAM-D2 (TraesCS2D02G214100) were also among the top 36 candidate genes. NAM-A2 is a paralog of NAM-A1, which regulates senescence and nutrient remobilization (Uauy et al, 2006;Avni et al, 2014;Harrington et al, 2019). The homeolog of NAM-A2, NAM-B2, was previously found to cause a slight delay in senescence (Pearce et al, 2014), but NAM-A2 has not been previously characterized and so was a strong candidate TF that might regulate senescence.…”

“…For NAM-A2, we could not identify any mutations that would cause truncations, so instead we selected three missense mutations that were in highly conserved domains and were thus expected to play important roles in protein function (Fig. 5A), as has recently been shown for NAM-A1 (Harrington et al, 2019). These were located in the A, C and D NAC subdomain and were predicted to be highly deleterious according to SIFT (Sorting Intolerant From Tolerant; Ng and Henikoff, 2003) and Position-Specific Scoring Matrix scores for the NAM family (pfam02365; Marchler-Bauer et al, 2017).…”

“…We found significant delays in flag leaf and peduncle senescence in NAM-A2/NAM-B2 double mutants, indicating that the genes predicted by the network play roles in senescence. The peduncle senescence phenotype indicates that this approach can identify genes that regulate senescence across different tissues, not only in the flag leaf, and may reflect that monocarpic senescence in wheat is a developmental process regulated across the whole plant (Harrington et al, 2019). Ongoing work is currently characterizing the additional candidate genes through the development of wheat double mutants for phenotypic characterization.…”