Phylogenetic Analysis Of The NAP Transcription Factor In Plants And Its Regulation To Leaf Senescence

The NAP (NAC-Like, Activated by AP3/PI) subfamily is an important plant-specific transcription factor and it belongs to the NAC family. The NAP subfamily can control plant growth and development, respond to various plant stresses and regulate leaf senescence. Nowadays, it has become a hot topic that NAP subfamily plays important roles in the crop yield and quality through regulating leaf senescence. Most of researches about NAP subfamily concentrate on model plants such as Arabidopsis and rice, but there were limited reports on others e.g. cotton. Cotton is an important economic crop in the world, but comprehensive studies about NAP members in cotton are lacking. Thus, our study analyzed molecular evolution and species-specific expansion of NAP subfamily in maize and other plants. Then, we focus on a novel NAP member (GhNAP) in cotton. Numerous researches revealed GhNAP’s role in leaf senescence and its key regulation mechanisms in order to illustrate the evolution, structure and leaf senescence on GhNAP. The results and conclusions are summarized as follows.1. In the current study,124NAC members were identified in Zea mays and were phylogenetically clustered into13distinct subfamilies. The whole genome duplication (WGD), especially an additional WGD event, may lead to expanding ZmNAC members. Different subfamily has different expansion rate, and NAC subfamily preference was found during the expansion in maize. Moreover, the duplication events might occur after the divergence of the lineages of Z mays and S. italica, and segmental duplication seemed to be the dominant pattern for the gene duplication in maize. The expansion of ZmNAC members may be also related to the gain and loss of introns. Furthermore, the restriction of functional divergence was discovered after most of the gene duplication events. Besides, NAP subfamily had relatively limited members, and there were not any duplication events in NAP subfamily. The gene structure and intro phase was relatively conserved in NAP member, and the functional divergence of NAP members was limited in maize and other plants. All of results indicated that the structure and function of NAP subfamily were relatively conserved in maize.2. Through genome-wide identification of NAP subfamily from39plant species,197NAP proteins were identified from31vascular plants, but no NAP members were found in8non-vascular plants. All NAP proteins were phylogenetically classified into two groups (NAP I and NAP II), and the origin time of the NAP I group might be relatively later than that of the NAP II group. Furthermore, species-specific gene duplications, caused by segmental duplication events, resulted in the expansion of the NAP subfamily after species-divergence. Different group has different expansion rate, and NAP group preference was found during the expansion in plants. Moreover, the expansion of NAP proteins may be related to the gain and loss of introns. Besides, functional divergence was limited after the gene duplication. Abscisic acid (ABA) might play an important role in leaf senescence which is regulated by NAP subfamily.3. With in silicon cloning and traditional PCR cloning methods, we isolated a GhNAP transcription factor (the homolog of Arabidopsis AtNAP) from Gossypium hirsutum. GhNAP has the typical structure of NAP subfamily. GhNAP has a highly conserved N-terminal domain (NAC domain), which can be further divided into five distinct subdomains (A-E). Meanwhile, a conserved novel subdomain (subdomain NAP I) existed in its divergent TAR region. Besides, GhNAP clustered with AtNAP, and both belonged to the NAP I group of NAP subfamily. Then a complementation test indicated that GhNAP could restore the Arabidopsis atnap null mutant phenotype to the normal wild-type phenotype. These results collectively indicated that GhNAP is a homolog of AtNAP in cotton.4. The multiple copies often existed in Gossypium hirsutum genome. In our study, GhNAP has four copies (GhNAP_a, GhNAP_b, GhNAP_c and GhNAP_d), and each has three exons and two introns. GhNAP_a and GhNAP_b might come from A genome of the Old World diploids, while GhNAP_c and GhNAP_d might originate from D genome of New World diploids. The expansion of NAP member in Gossypium hirsutum may occur after interspecific hybrids, not in the ancestor. Besides, the GhNAP copy in A subgenome converted easily to the GhNAP copy in the D subgenome. Moreover, NAP functional divergence was limited in the Gossypium hirsutum, Gossypium arboretum and Gossypium raimondii, but the function might be a little divergent among different GhNAP copies.5. In Arabidopsis, overexpression of GhNAP could cause precocious senescence, while GhNAPi line could delay leaf senescence. In cotton, the interference of GhNAP expression showed an obvious delay in leaf senescence. The similar phenomena also existed in the dark-induced leaf senescence. Thus, GhNAP can be regarded as an ideal marker to depict leaf senescence in cotton, and GhNAP played crucial roles in leaf senescence in cotton under natural and dark environment. Besides, GhNAP has influence on yield and quality in cotton through regulating leaf senescence.6. GhNAP showed transcriptional activation activity in yeast, and the transcriptional activation domain existed in the relatively divergent TAR region of GhNAP. In addition, GhNAP was a nuclear protein. These results indicated that GhNAP may function as a transcription factor in cotton. Besides, we found that GhNAP may regulate leaf senescence through ABA-mediated pathways. Moreover, the YIH assay showed that GhNAP did not bind directly to the promoters of GhSAG113, indicating that GhSAG113was not the target gene of GhNAP. Thus, the regulation model between GhNAP and ABA in cotton differs from the ABA-AtNAP-SAG113regulatory chain in Arabidopsis during leaf senescence.Overall, the comparative analysis of NAP members in maize and other plants indicated that the structure and function of NAP subfamily are relatively conserved in plants. Then, a NAP I member (GhNAP) was isolated from Gossypium hirsutum, and it has four copies. Our results showed that GhNAP in cotton was closely associated with leaf senescence via ABA-mediated pathways. Down-regulation of GhNAP in cotton could delay leaf senescence, and effected on the cotton yield and fiber quality. The finding presented here opened a new avenue for the researchers to further investigate the structure and function of NAP subfamily in plants, and provided a candidate gene in the plant breeding.