Molecular analysis of the NAC gene family in rice

Genes that encode products containing a NAC domain, such as NO APICAL MERISTEM (NAM) in petunia, CUP-SHAPED COTYLEDON2 (CUC2) and NAP in Arabidopsis thaliana, have crucial functions in plant development. We describe here molecular aspects of the OsNAC genes that encode proteins with NAC domains in rice (Oryza sativa L.). Sequence analysis revealed that the NAC genes in plants can be divided into several subfamilies, such as the NAM, ATAF, and OsNAC3 subfamilies. In rice, OsNAC1 and OsNAC2 are classified in the NAM subfamily, which includes NAM and CUC2, while OsNAC5 and OsNAC6 fall into the ATAF subfamily. In addition to the members of these subfamilies, the rice genome contains the NAC genes OsNAC3, OsNAC4 (both in the OsNAC3 subfamily), OsNAC7, and OsNAC8. These results and Southern analysis indicate that the OsNAC genes constitute a large gene family in the rice genome. Each OsNAC gene is expressed in a specific pattern in different organs, suggesting that this family has diverse and important roles in rice development.     

GRAB proteins,novel members of the NAC domain family,isolated by their interaction with a geminivirus protein

Geminiviruses encode a few proteins and depend on cellular factors to complete their replicative cycle. As a way to understand geminivirus-host interactions, we have searched for cellular proteins which interact with viral proteins. By using the yeast two-hybrid technology and the wheat dwarf geminivirus (WDV) RepA protein as a bait, we have isolated a family of proteins which we termed GRAB (for Geminivirus Rep A-binding). We report here the molecular characterization of two members, GRAB1 and GRAB2. We have found that the 37 C-terminal amino acids of RepA are required for interaction with GRAB proteins. This region contains residues conserved in an equivalent region of the RepA proteins encoded by other viruses of the WDV subgroup. The N-terminal domain of GRAB proteins is necessary and sufficient to interact with WDV RepA. GRAB proteins contain an unique acidic C-terminal domain while their N-terminal domain, of ca. 170 amino acids, are highly conserved in all of them. Interestingly, this conserved N-terminal domain of GRAB proteins exhibits a significant amino acid homology to the NAC domain present in proteins involved in plant development and senescence. GRAB1 and GRAB2 mRNAs are present in cultured cells and roots but are barely detectable in leaves. GRAB expression inhibits WDV DNA replication in cultured wheat cells. Our studies highlight the importance that the pathway(s) mediated by GRAB proteins, as well as by other NAC domain-containing proteins, might have on geminivirus DNA replication in connection to plant growth, development and senescence pathways.     

The NAC domain mediates functional specificity of CUP-SHAPED COTYLEDON proteins

In higher plants, although several genes involved in shoot apical meristem (SAM) formation and organ separation have been isolated, the molecular mechanisms by which they function are largely unknown. CUP-SHAPED COTYLEDON (CUC) 1 and CUC2 are examples of two such genes that encode the NAC domain proteins. This study investigated the molecular basis for their activities. Nuclear localization assays indicated that green fluorescent protein (GFP)-CUC proteins accumulate in the nucleus. Yeast one-hybrid and transient expression assays demonstrated that the C-terminal domain (CTD) of the CUC has transactivation activity. Domain-swapping experiments revealed that the functional specificity of the CUC for promoting adventitious shoot formation resides in the highly conserved NAC domain, not in the CTD in which motifs specific to the CUC subfamily are located. Taken together, these observations suggest that CUC proteins transactivate the target genes involved in SAM formation and organ separation through a specific interaction between the NAC domain and the promoter region of the target genes.     

互花米草NAC转录因子家族的鉴定与表达分析

互花米草(Spartina alterniflora)作为一种海岸带盐生植物,高度耐盐胁迫,但因为缺少参考基因组,其耐盐的分子机制却尚未见报道。NAC家族蛋白是植物特有的转录因子,调控植物的生长发育和胁迫应答。为了鉴定互花米草NAC蛋白(SaNAC)并探究它们与互花米草生长发育及胁迫响应之间的关系,本研究以互花米草三代全长转录组数据为参考,通过与水稻(Oryza sativa)、拟南芥(Arabidopsis thaliana)和玉米(Zea mays)NAC蛋白序列进行比对,并结合保守功能域进一步筛选,最终找到62个SaNAC蛋白。从蛋白序列比对、进化、motif预测、同源性比较、亚细胞定位、组织表达以及非生物胁迫下的基因差异表达等方面分别对互花米草NAC家族成员进行分析,结果发现SaNAC蛋白均含有保守的NAM结构域,且在进化上与水稻NAC家族具有一定的相似性;SaNAC家族中的两个蛋白SaNAC9和SaNAC49在细胞核表达;另外,本研究还发现互花米草SaNAC基因表达具有高度组织和胁迫应答差异性。这些结果表明互花米草NAC转录因子家族不仅具有保守的功能域,而且在调控互花米草的生长发育和非生物胁迫响应过程中具有重要的作用。