BackgroundLate embryogenesis abundant (LEA) proteins are involved in protecting higher plants from damage caused by environmental stresses. Foxtail millet (Setaria italica) is an important cereal crop for food and feed in semi-arid areas. However, the molecular mechanisms underlying tolerance to these conditions are not well defined.ResultsHere, we characterized a novel atypical LEA gene named SiLEA14 from foxtail millet. It contains two exons separated by one intron. SiLEA14 was expressed in roots, stems, leaves, inflorescences and seeds at different levels under normal growth conditions. In addition, SiLEA14 was dramatically induced by osmotic stress, NaCl and exogenous abscisic acid. The SiLEA14 protein was localized in the nucleus and the cytoplasm. Overexpression of SiLEA14 improved Escherichia coli growth performance compared with the control under salt stress. To further assess the function of SiLEA14 in plants, transgenic Arabidopsis and foxtail millet plants that overexpressed SiLEA14 were obtained. The transgenic Arabidopsis seedlings showed higher tolerance to salt and osmotic stress than the wild type (WT). Similarly, the transgenic foxtail millet showed improved growth under salt and drought stresses compared with the WT. Taken together, our results indicated that SiLEA14 is a novel atypical LEA protein and plays important roles in resistance to abiotic stresses in plants.ConclusionWe characterized a novel atypical LEA gene SiLEA14 from foxtail millet, which plays important roles in plant abiotic stress resistance. Modification of SiLEA14 expression may improve abiotic stress resistance in agricultural crops.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0290-7) contains supplementary material, which is available to authorized users.

Drought and other types of abiotic stresses negatively affect plant growth and crop yields. The abscisic acid-, stress-, and ripening-induced (ASR) proteins play important roles in the protection of plants against abiotic stress. However, the regulatory pathway of the gene encoding this protein remains to be elucidated. In this study, the foxtail millet (Setaria italica) ASR gene, SiASR4, was cloned and characterized. SiASR4 localized to the cell nucleus, cytoplasm and cytomembrane, and the protein contained 102 amino acids, including an ABA/WDS (abscisic acid/water-deficit stress) domain, with a molecular mass of 11.5 kDa. The abundance of SiASR4 transcripts increased after treatment with ABA, NaCl, and PEG in foxtail millet seedlings. It has been reported that the S. italica ABA-responsive DRE-binding protein (SiARDP) binds to a DNA sequence with a CCGAC core and that there are five dehydration-responsive element (DRE) motifs within the SiASR4 promoter. Our analyses demonstrated that the SiARDP protein could bind to the SiASR4 promoter in vitro and in vivo. The expression of SiASR4 increased in SiARDP-overexpressing plants. SiASR4-transgenic Arabidopsis and SiASR4-overexpressing foxtail millet exhibited enhanced tolerance to drought and salt stress. Furthermore, the transcription of stress-responsive and reactive oxygen species (ROS) scavenger-associated genes was activated in SiASR4 transgenic plants. Together, these findings show that SiASR4 functions in the adaption to drought and salt stress and is regulated by SiARDP via an ABA-dependent pathway.

Lipid transfer proteins (LTPs) are a class of cysteine-rich soluble proteins having small molecular weights. LTPs participate in flower and seed development, cuticular wax deposition, also play important roles in pathogen and abiotic stress responses. A non-specific LTP gene (SiLTP) was isolated from a foxtail millet (Setaria italica) suppression subtractive hybridization library enriched for differentially expressed genes after abiotic stress treatments. A semi-quantitative reverse transcriptase PCR analysis showed that SiLTP was expressed in all foxtail millet tissues. Additionally, the SiLTP promoter drove GUS expression in root tips, stems, leaves, flowers, and siliques of transgenic Arabidopsis. Quantitative real-time PCR indicated that the SiLTP expression was induced by NaCl, polyethylene glycol, and abscisic acid (ABA). SiLTP was localized in the cytoplasm of tobacco leaf epidermal cells and maize protoplasts. The ectopic expression of SiLTP in tobacco resulted in higher levels of salt and drought tolerance than in the wild type (WT). To further assess the function of SiLTP, SiLTP overexpression (OE) and RNA interference (RNAi)-based transgenic foxtail millet were obtained. SiLTP-OE lines performed better under salt and drought stresses compared with WT plants. In contrast, the RNAi lines were much more sensitive to salt and drought compared than WT. Electrophoretic mobility shift assays and yeast one-hybrids indicated that the transcription factor ABA-responsive DRE-binding protein (SiARDP) could bind to the dehydration-responsive element of SiLTP promoter in vitro and in vivo, respectively. Moreover, the SiLTP expression levels were higher in SiARDP-OE plants compared than the WT. These results confirmed that SiLTP plays important roles in improving salt and drought stress tolerance of foxtail millet, and may partly be upregulated by SiARDP. SiLTP may provide an effective genetic resource for molecular breeding in crops to enhance salt and drought tolerance levels.

pF128 drives GUS specifically expressed in transgenic seeds of foxtail millet and Zea mays with higher activity than the constitutive CaMV35S promoter and the maize seed-specific 19Z promoter. Foxtail millet (Setaria italica), a member of the Poaceae family, is an important food and fodder crop in arid regions. Foxtail millet is an excellent C4 crop model owing to its small genome (~490 Mb), self-pollination and availability of a complete genome sequence. F128 was isolated from a cDNA library of foxtail millet immature seeds. Real-time PCR analysis revealed that F128 mRNA was specifically expressed in immature and mature seeds. The highest F128 mRNA level was observed 5 days after pollination and gradually decreased as the seed matured. Sequence analysis suggested that the protein encoded by F128 is likely a protease inhibitor/seed storage protein/lipid-transfer protein. The 1,053 bp 5' flanking sequence of F128 (pF128) was isolated and fused to the GUS reporter gene. The corresponding vector was then transformed into Arabidopsis thaliana, foxtail millet and Zea mays. GUS analysis revealed that pF128 drove GUS expression efficiently and specifically in the seeds of transgenic Arabidopsis, foxtail millet and Zea mays. GUS activity was also detected in Arabidopsis cotyledons. Activity of pF128 was higher than that observed for the constitutive CaMV35S promoter and the maize seed-specific 19 Zein (19Z) promoter. These results indicate that pF128 is a seed-specific promoter. Its application is expected to be of considerable value in plant genetic engineering.

In previous reports, we developed an Agrobacterium-mediated transformation system for foxtail millet. Here, we report optimization of the system through improvement of the regeneration system efficiency and optimization of conditions for gene delivery. Immature inflorescences explants of foxtail millet cv. Jigu 11 varying in length (0.5 to 1.0, 1.1 to 1.5, 1.6 to 2.0 and >2.0 cm) were cultured on modified MS medium for callus induction and regeneration. The highest embryogenic callus-formation efficiency (90.72%) was achieved with 0.5 to 1.0 cm long inflorescences and 25 days old calli induced from 0.5 to 1.0 cm long immature inflorescences gave rise to the highest differentiation frequency (90.93%). In addition, factors affecting T-DNA delivery were examined by transient β-glucuronidase (GUS) expression. Calli induced from younger explants (0.5 to 1.0 cm immature inflorescences) were optimal. Agrobacterium tumefaciens strain LBA4404 performed significantly better than EHA105. Co-cultivation at 22°C with 0.15 g/l dithiothreitol (DTT) in the infection solution and co-cultivation medium led to higher GUS transient expression efficiency than with other treatments. Using this optimized procedure, the lysine-rich protein encoding gene SBgLR from potato was transformed into foxtail millet cv. Jigu 11 with 5.5% transformation efficiency. The procedure described here will be useful for genetic improvement of foxtail millet.