Chitinase like proteins (CLPs) are members of the glycosyl hydrolase 18 and 19 families and are grouped into six classes. These proteins are usually known to function in plant defense against pathogens and biotic stresses. CLPs from several plant species, such as chickpea and field bean, have been shown to have anti-fungal activities[22–24]. There is also evidence that CLPs may play a role in plant tolerance to abiotic stresses such as heat, salt, and drought. In Arabidopsis, the hot2 mutant, which has a mutation in a CPL gene, showed tolerance to salt and drought conditions, possibly by preventing over-accumulating Na+ ions.
CLPs also take part in plant organ development, particularly in cellulose metabolism and lignin deposition, thus, they have a significant effect on primary and secondary cell wall development. This effect may also influence the architecture of seed coats, as both lignin and cellulose are involved in seed coat composition. There is evidence to show that lignin deficiency may correlate with seed coat fragility. Another study identified an EMS-derived peanut mutant with a significantly shorter germination time and showed seed-coat cracking, which represented weaker dormancy capacity. The mutant showed lower levels of lignin, anthocyanins, and proanthocyanidins compared to the wild-type. Lignin was also found to affect the permeability of seed coat, hence affecting the longevity of seeds. The transcription profile of the CLP family in our study showed a significant difference between the wild-type and the mutants. The two wheat mutants in our study showed deficiencies in the expression of multiple CLP genes, which may lead to the abnormal deposition of lignin and cellulose (Fig. 6), and eventually affect seed dormancy by changing the permeability of the seed coat (Fig. 7).
CLP genes in plants have been shown to be regulated by transcription factors from families such as WRKY, NAC, and MYB. These transcription factors have similar patterns of regulation according to related studies; they bind directly to the W-box cis-regulatory elements and positively regulate CLP gene activity in the plant defense response against pathogens. Gao et. al isolated a MYB TF, BjMYB1, from Brassica juncea that specifically binds to the W-box-like-4 element in the promoter of the CLP gene BjCHI1, which is responsive to fungal infection. In another study, an NAC transcription factor in rice, OsNAC111, was found to positively regulate several defense-related genes, including two CLP genes. Over-expression of OsNAC111 resulted in enhanced resistance to the rice blast fungus Magnaporthe oryzae. WRKYs were also found to regulate CLP gene expression. Three WRKY TFs were found to bind specifically to the two W-boxes present in the CLP gene CHN48 in tobacco. Subsequent studies showed that CHN48 is positively regulated by these WRKYs in response to elicitor treatment, suggesting a possible involvement of NtWRKYs in the defense response via the regulation of CLP gene transcription. Recent findings have provided more evidence for the relationship between WRKY TFs and CLP gene regulation than for other TFs. Further studies on the regulatory roles of WRKY TFs in wheat may provide an explanation for the significant differences in expression profiles of the CLP family genes between the mutants and the wild-type. In addition, over-expression of a WRKY gene in rice, OsWRKY71, resulted in a significant up-regulation of the expression of multiple CLP genes and the defense response. In this study, the over-expression of OsWRKY71 in rice cells resulted in the up-regulation of 200 genes, 146 of which could also be up-regulated by chitin oligosaccharide elicitor treatment. Sixty genes were down-regulated, 21 of which were also down-regulated by chitin oligosaccharide elicitor treatment. Seven rice CLP genes showed the most significant up-regulation of all the up-regulated genes. Together with the other findings mentioned above, these results indicate that there is a strong relationship between WRKY TFs and CLP genes, and that certain WRKYs may function in the generic regulation of multiple CLP genes. In our study, all the WRKY genes in the target gene group showed reduced transcription in the mutants compared to the wild-type. These results suggest a possible explanation for the transcriptional profile of CLP genes; the deficiency of WRKYs and/or expression of a certain WRKY gene in the mutants has led to the down-regulation of the CLP genes.
In our study, GO analysis also identified a significant enrichment of genes involved in nodulation. This result is consistent with the discovery that CLPs participate in the development of nodules. A related study demonstrated that a CLP gene, Srchi24, is involved in the onset of nodulation in Sesbania rostrata.
ABA and the corresponding synthesis/signaling pathways have been shown to be key regulators of seed dormancy and PHS. However, in our study, we found no significant evidence to show that ABA contributes to the enhanced seed dormancy in the mutants. From the transcriptome data, however, we identified one ABI5-like gene that showed differential expression between the mutants and the wild-type. Further research will be needed to elucidate the possible role of this gene in seed dormancy.
Other transcriptomic studies of seed dormancy in cereals have identified a diversity of dormancy control mechanisms, with variable contributions from the activities of ABA. Consistent with our findings, a previous transcriptomic study of two rice cultivars with different levels of seed dormancy, found no obvious differences in the ABA synthesis and signaling pathways. The seed maturation pathways are significantly correlated with seed dormancy. In another study, however, the strongly dormant rice cultivar N22 and two mutants derived from it, Q4359 and Q4646, which showed weaker dormancy, were used in a microarray study. Genes involved in ABA signaling and GA biosynthesis/signaling showed differential expression between Q4359 and the wild-type N22, indicating that ABA and GA are involved in the seed dormancy process. These findings suggest that the mechanisms of seed dormancy control could be complicated, with ABA signals only partially controlling the regulation.
It should also be noted that although a possible pathway of dormancy regulation involving CLPs has been proposed, we cannot determine whether the overall differential expression of the CLP gene family causes the variation in seed dormancy or merely correlates with it based on our current findings. The group changes in the CLP family and WRKY genes indicate that there could be a mutation in a key upstream regulatory factor in the two wheat mutants, which might be the initiator of the changes in seed dormancy. Follow-up studies will focus on finding the causes of the differential expression of the CLP genes, and possibly the key upstream regulator.