Genome-wide survey of the F-box/Kelch (FBK) members and molecular identification of a novel FBK gene TaAFR in wheat.

F-box proteins play critical roles in plant responses to biotic/abiotic stresses. In the present study, a total of 68 wheat F-box/Kelch (TaFBK) genes, unevenly distributed across 21 chromosomes and encoding 74 proteins, were identified in EnsemblPlants. Protein sequences were compared with those of Arabidopsis and three cereal species by phylogenetic and domain analyses, where the wheat sequences were resolved into 6 clades. In silico analysis of a digital PCR dataset revealed that TaFBKs were expressed at multiple developmental stages and tissues, and in response to drought and/or heat stresses. The TaFBK19 gene, a homolog of the Attenuated Far-Red Response (AFR) genes in other plant species, and hence named TaAFR, was selected for further analysis. Reverse-transcription quantitative real-time PCR (RT-qPCR) was carried out to determine tissue-specific, hormone and stress (abiotic/biotic) responsive expression patterns. Of interest, TaAFR was expressed most abundantly in the leaves, and its expression in response to leaf rust variants suggests a potential role in compatible vs incompatible rust responses. The protein was predicted to localize in cytosol, but it was shown experimentally to localize in both the cytosol and the nucleus of tobacco. A series of protein interaction studies, starting with a yeast-2-hybrid (Y2H) library screen (wheat leaf infected with incompatible leaf rust pathogens), led to the identification of three TaAFR interacting proteins. Skp1/ASK1-like protein (Skp1) was found to interact with the F-box domain of TaAFR, while ADP-ribosylation factor 2-like isoform X1 (ARL2) and phenylalanine ammonia-lyase (PAL) were shown to interact with its Kelch domain. The data presented herein provides a solid foundation from which the function and metabolic network of TaAFR and other wheat FBKs can be further explored.

Identification and expression analysis of some wheat F-box subfamilies during plant development and infection by Puccinia triticina.

As one of the largest protein families in plants, F-box proteins are involved in many important cellular processes. Until now, a limited number of investigations have been conducted on wheat F-box genes due to its variable structure and large and polyploid genome. Classification, identification, structural analysis, evolutionary relationship, and chromosomal distribution of some wheat F-box genes are described in the present study. A total number of 1013 potential F-box proteins which are encoded by 409 genes was identified in wheat, and classified into 12 subfamilies based on their C-terminal domain structures. Furthermore, proteins with identical or similar C-terminal domain were clustered together. Location of 409 F-box genes was identified on all 21 wheat chromosomes but showed an uneven distribution. Segmental duplication was the main reason for the increase in the number of wheat F-box genes. Gene expression analysis based on digital PCR showed that most of the F-box genes were highly expressed in the later development stages of wheat, including the formation of spike, grain, flag leaf, and participated in drought stress (DS), heat stress (HS), and their combination (HD). Of the nine F-box genes we investigated using quantitative PCR (qPCR) following fungal pathogen infection, five were involved in wheat resistance to the infection by leaf rust pathogen and one in the susceptible response. These results provide important information on wheat F-box proteins for further functional studies, especially the proteins that played roles in response to heat and drought stresses and leaf rust pathogen infection.