Autophagy is a cellular degradation process that is highly evolutionarily-conserved in yeast, plants, and animals. In plants, autophagy plays important roles in regulating intracellular degradation and recycling of amino acids in response to nutrient starvation, senescence, and other environmental stresses. Foxtail millet (Setaria italica) has strong resistance to stresses and has been proposed as an ideal material for use in the study of the physiological mechanisms of abiotic stress tolerance in plants. Although the genome sequence of foxtail millet (Setaria italica) is available, the characteristics and functions of abiotic stress-related genes remain largely unknown for this species. A total of 37 putative ATG (autophagy-associated genes) genes in the foxtail millet genome were identified. Gene duplication analysis revealed that both segmental and tandem duplication events have played significant roles in the expansion of the ATG gene family in foxtail millet. Comparative synteny mapping between the genomes of foxtail millet and rice suggested that the ATG genes in both species have common ancestors, as their ATG genes were primarily located in similar syntenic regions. Gene expression analysis revealed the induced expression of 31 SiATG genes by one or more phytohormone treatments, 26 SiATG genes by drought, salt and cold, 24 SiATG genes by darkness and 25 SiATG genes by nitrogen starvation. Results of qRT-PCR showing that among 37 SiATG genes, the expression level of SiATG8a was the highest after nitrogen starvation treatment 24 h, suggesting its potential role in tolerance to nutrient starvation. Moreover, the heterologous expression of SiATG8a in rice improved nitrogen starvation tolerance. Compared to wild type rice, the transgenic rice performed better and had higher aboveground total nitrogen content when the plants were grown under nitrogen starvation conditions. Our results deepen understanding about the characteristics and functions of ATG genes in foxtail millet and also identify promising new genetic resources that should be of use in future efforts to develop varieties of foxtail millet and other crop species that have resistance to nitrogen deficiency stress.

Pentatricopeptide repeat (PPR) proteins are encoded by a large gene family of approximately 450 members in Arabidopsis and 477 in rice, which characterized by tandem repetitions of a degenerate 35 amino acid characteristic sequence motifs. A large majority of the PPR genes in the higher plants are localized in organelles. Their functions remain as yet largely unknown. The majority of characterized PPR proteins have been found to function in modulating the expression plastid and mitochondrial genes in plants. Here, a genome-wide identification and comparison of the PPR genes from 5 organisms was performed, including the moss Physcomitrella patens, the lycophyte Selaginella moellendorffii, the eudicot Arabidopsis, and the monocots rice and foxtail millet. It appears that the expansion of this gene family prior to the divergence of the euphyllophytes and the lycophytes in land plants. The duplication and divergence rates of the foxtail millet PPR genes (SiPPRs) showed that the expansion period of this gene family around 400 Mya, and indicated that genome segmental duplication was very likely the primary mechanism underlying the expansion of the PPR gene family in vascular plants. An analysis of a complete set of SiPPR genes/proteins that included classification, chromosomal location, orthologous relationships, duplication analysis, and auxiliary motifs is presented. Expression analysis of the SiPPR genes under stress conditions revealed that the expression of 24 SiPPR genes was responsive to abiotic stress. Subcellular localization analysis of 11 PPR proteins indicated that 5 proteins were localized to chloroplasts, that 4 were localized to mitochondria, and that 2 were localized to the cytoplasm. Our results contribute to a more comprehensive understanding the roles of PPR proteins and will be useful in the prioritization of particular PPR proteins for subsequent functional validation studies in foxtail millet.

Alfalfa (Medicago sativa L.) is a perennial forage legume that is widely distributed throughout the world, and cold stress is an important environmental factor limiting the growth and production of alfalfa in cold regions. However, little is known of the molecular mechanisms regarding cold tolerance in alfalfa. Here, we conducted physiological metabolism assays and pan-transcriptome sequencing on eight cultivars of alfalfa under cold stress conditions. The results of the RNA-seq analysis showed that the genes are “oxidoreductase activity” and “transcription regulator activity”, suggesting that genes with such functions are more likely to play important roles in the response to cold stress by alfalfa. In addition, to identify specific gene modules and hub genes in response to alfalfa cold stress, we applied weighted gene co-expression network (WGCNA) analyses to the RNA-seq data. Our results indicate that the modules of genes that focus on the ATPase complex, ribosome biogenesis, are more likely to be involved in the alfalfa response to cold stress. It is important to note that we identified two fibronectin (FIB) genes as hub genes in alfalfa in response to cold stress and that they negatively regulate alfalfa response to chilling stress, and it is possible that dormant alfalfa is more effective at down-regulating FIB expression and therefore more resistant to cold stress.