Polymorphic homoeolog of key gene of RdDM pathway, ARGONAUTE4_9 class is associated with pre-harvest sprouting in wheat (Triticum aestivum L.)

Abstract

Resistance to pre-harvest sprouting (PHS) is an important objective for the genetic improvement of many cereal crops, including wheat. Resistance, or susceptibility, to PHS is mainly influenced by seed dormancy, a complex trait. Reduced seed dormancy is the most important aspect of seed germination on a spike prior to harvesting, but it is influenced by various environmental factors including light, temperature and abiotic stresses. The basic genetic framework of seed dormancy depends on the antagonistic action of abscisic acid (ABA) and gibberellic acid (GA) to promote dormancy and germination. Recent studies have revealed a role for epigenetic changes, predominantly histone modifications, in controlling seed dormancy. To investigate the role of DNA methylation in seed dormancy, we explored the role of ARGONAUTE4_9 class genes in seed development and dormancy in wheat. Our results indicate that the two wheat AGO4_9 class genes i.e. AGO802 and AGO804 map to chromosomes 3L and 1L are preferentially expressed in the embryos of developing seeds [corrected]. Differential expressions of AGO802-B in the embryos of PHS resistant and susceptible varieties also relates with DNA polymorphism in various wheat varieties due to an insertion of a SINE-like element into this gene. DNA methylation patterns of the embryonic tissue from six PHS resistant and susceptible varieties demonstrate a correlation with this polymorphism. These results suggest a possible role for AGO802-B in seed dormancy and PHS resistance through the modulation of DNA methylation.

Figure 1. Comparing wheat homoeologs.

DNA sequence comparison of wheat homoeologs of AGO802 and AGO804. The three wheat AGO802 homoeologs are almost 100% identical for the sequence and AGO804 has sequence homology of about 98% among its three homoeologs.

Figure 2. Phylogenetic comparison of ARGONAUTE4_9.

Comparison of ARGONAUTE4_9 class of genes using a Phylogenetic tree from wheat, barley, rice, maize, sorghum and Arabidopsis.

Figure 3. Mapping of AGO802 and AGO804 in wheat.

Absence of amplification in lanes Dt3AS, Dt3BS and Dt3DS in AGO802, suggest its map location on chromosome arm 3S. While in AGO804, absence of amplification in Dt1AS, Dt1BS and Dt1DS lanes, confirms its map location on chromosome 1S.

Figure 4. Expression analysis of AGO802 and AGO804.

Expression of AGO802 and AGO804 homoeologs in embryos during seed development in Chinese Spring cultivar. QRT-expression analysis was performed at six different stages consisting of ovaries at 5 DAP stages and embryos at 10, 15, 20, 25 and 30 DAP, shown on X-axis of graph as 1–6 respectively.

Figure 5. Analysis of a retro-transposon insertion in the AGO802B transcripts.

Polymorphism as observed on agarose gel in different wheat varieties with varying reaction to pre-harvest sprouting, due to insertion of a retro-transposon insertion in the AGO802B transcripts.

Figure 6. Methylation differences between wheat varieties.

DNA extracted from the embryos of mature seeds was digested with Msp I restriction enzyme and probed with 5S rDNA. PHS resistant varieties RL4137, Snowbird, AC Domain, AC Karma show reduced methylation as compared to a PHS susceptible variety AC Andrew. DNA methylation in CDC Teal, a variety with medium PHS reaction was also less, compared to AC Andrew but more than the PHS resistant varieties.

Figure 7. The AGO4 proteins in different wheat varieties.

Total proteins from the embryos of mature seed were extracted and resolved on SDS-PAGE. Argonaute 4 antibodies detected AGO4 proteins in PHS resistant and susceptible varieties. Differential protein concentration was observed in resistant RL4137, Snowbird, AC Domain, AC Karma and susceptible varieties (CDC Teal and AC Andrew). Immunoblot and protein loading lanes correspond to coomassie gel.