Expression profiling of Nuclear Factor-Y (NF-Y) transcription factors during dehydration and salt stress in finger millet reveals potential candidate genes for multiple stress tolerance.

MAIN CONCLUSION: Expression profiling of NF-Y transcription factors during dehydration and salt stress in finger millet genotypes contrastingly differing in tolerance levels identifies candidate genes for further characterization and functional studies. The Nuclear Factor-Y (NF-Y) transcription factors are known for imparting abiotic stress tolerance in different plant species. However, there is no information on the role of this transcription factor family in naturally drought-tolerant crop finger millet (Eleusine coracana L.). Therefore, interpretation of expression profiles against drought and salinity stress may provide valuable insights into specific and/or overlapping expression patterns of Eleusine coracana Nuclear Factor-Y (EcNF-Y) genes. Given this, we identified 59 NF-Y (18 NF-YA, 23 NF-YB, and 18 NF-YC) encoding genes and designated them EcNF-Y genes. Expression profiling of these genes was performed in two finger millet genotypes, PES400 (dehydration and salt stress tolerant) and VR708 (dehydration and salt stress sensitive), subjected to PEG-induced dehydration and salt (NaCl) stresses at different time intervals (0, 6, and 12 h). The qRT-PCR expression analysis reveals that the six EcNF-Y genes namely EcNF-YA1, EcNF-YA5, EcNF-YA16, EcNF-YB6, EcNF-YB10, and EcNF-YC2 might be associated with tolerance to both dehydration and salinity stress in early stress condition (6 h), suggesting the involvement of these genes in multiple stress responses in tolerant genotype. In contrast, the transcript abundance of finger millet EcNF-YA5 genes was also observed in the sensitive genotype VR708 under late stress conditions (12 h) of both dehydration and salinity stress. Therefore, the EcNF-YA5 gene might be important for adaptation to salinity and dehydration stress in sensitive finger millet genotypes. Therefore, this gene could be considered as a susceptibility determinant, which can be edited to impart tolerance. The phylogenetic analyses revealed that finger millet NF-Y genes share strong evolutionary and functional relationship to NF-Ys governing response to abiotic stresses in rice, sorghum, maize, and wheat. This is the first report of expression profiling of EcNF-Ys genes identified from the finger millet genome and reveals potential candidate for enhancing dehydration and salt tolerance.

Integration of transcriptomic and proteomic analyses for finger millet [Eleusine coracana (L.) Gaertn.] in response to drought stress.

Drought is one of the most significant abiotic stresses that affects the growth and productivity of crops worldwide. Finger millet [Eleusine coracana (L.) Gaertn.] is a C4 crop with high nutritional value and drought tolerance. However, the drought stress tolerance genetic mechanism of finger millet is largely unknown. In this study, transcriptomic (RNA-seq) and proteomic (iTRAQ) technologies were combined to investigate the finger millet samples treated with drought at different stages to determine drought response mechanism. A total of 80,602 differentially expressed genes (DEGs) and 3,009 differentially expressed proteins (DEPs) were identified in the transcriptomic and proteomic levels, respectively. An integrated analysis, which combined transcriptome and proteome data, revealed the presence of 1,305 DEPs were matched with the corresponding DEGs (named associated DEGs-DEPs) when comparing the control to samples which were treated with 19 days of drought (N1-N2 comparison group), 1,093 DEGs-DEPs between control and samples which underwent rehydration treatment for 36 hours (N1-N3 comparison group) and 607 DEGs-DEPs between samples which were treated with drought for 19 days and samples which underwent rehydration treatment for 36 hours (N2-N3 comparison group). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified 80 DEGs-DEPs in the N1-N2 comparison group, 49 DEGs-DEPs in the N1-N3 comparison group, and 59 DEGs-DEPs in the N2-N3 comparison group, which were associated with drought stress. The DEGs-DEPs which were drought tolerance-related were enriched in hydrolase activity, glycosyl bond formation, oxidoreductase activity, carbohydrate binding and biosynthesis of unsaturated fatty acids. Co-expression network analysis revealed two candidate DEGs-DEPs which were found to be centrally involved in drought stress response. These results suggested that the coordination of the DEGs-DEPs was essential to the enhanced drought tolerance response in the finger millet.

Genome and Transcriptome sequence of Finger millet (Eleusine coracana (L.) Gaertn.) provides insights into drought tolerance and nutraceutical properties.

BACKGROUND: Finger millet (Eleusine coracana (L.) Gaertn.) is an important staple food crop widely grown in Africa and South Asia. Among the millets, finger millet has high amount of calcium, methionine, tryptophan, fiber, and sulphur containing amino acids. In addition, it has C4 photosynthetic carbon assimilation mechanism, which helps to utilize water and nitrogen efficiently under hot and arid conditions without severely affecting yield. Therefore, development and utilization of genomic resources for genetic improvement of this crop is immensely useful. RESULTS: Experimental results from whole genome sequencing and assembling process of ML-365 finger millet cultivar yielded 1196 Mb covering approximately 82% of total estimated genome size. Genome analysis showed the presence of 85,243 genes and one half of the genome is repetitive in nature. The finger millet genome was found to have higher colinearity with foxtail millet and rice as compared to other Poaceae species. Mining of simple sequence repeats (SSRs) yielded abundance of SSRs within the finger millet genome. Functional annotation and mining of transcription factors revealed finger millet genome harbors large number of drought tolerance related genes. Transcriptome analysis of low moisture stress and non-stress samples revealed the identification of several drought-induced candidate genes, which could be used in drought tolerance breeding. CONCLUSIONS: This genome sequencing effort will strengthen plant breeders for allele discovery, genetic mapping, and identification of candidate genes for agronomically important traits. Availability of genomic resources of finger millet will enhance the novel breeding possibilities to address potential challenges of finger millet improvement.

Optimization of the malting process for nutritional improvement of finger millet and amaranth flours in the infant weaning food industry.

Malting is a beneficial approach to improve the nutritional value of cereals used in infant preparations. Malted finger millet and amaranth might be considered as potentially appropriate gluten-free alternatives for common wheat-based weaning products, especially in case of those suffering from celiac disease. In this study, the effects of germination temperature and duration on the main nutrients of malted finger millet and amaranth, are evaluated and optimized. Grains were germinated for 24, 36 and 48 h at 22, 26 and 30 °C. In the case of finger millet, germinating for 48 h at 30 °C resulted into 17% increase in protein availability, 10% increase in total energy and 60% reduction in resistant starch (RS). For amaranth, germinating for 48 h at 26 °C was preferable, resulting in 8% increase in protein availability, 11% increase in total energy, 70% reduction in RS and a 10% increase in the linoleic acid.

Chitosan nanoparticle induced defense responses in fingermillet plants against blast disease caused by Pyricularia grisea (Cke.) Sacc.

The in vitro antifungal properties of chitosan nanoparticle and its role in protection of fingermillet plants from blast disease were evaluated. Chitosan nanoparticle inhibited the radial growth of Pyricularia grisea indicating the antifungal property. Application of chitosan nanoparticle delayed blast symptom expression on fingermillet leaves for 25days while it was on 15day in control plants. Chitosan naoparticle was able to induce the reactive oxygen species and the level of peroxidase actvitiy in leaves of fingermillet, which might be the reason for delayed symptom. The treated plants showed reduced disease incidence when compared to untreated control plants. These results suggested the role of chitosan nanoparticle in protecting fingermillet plants from P. grisea infection.

Growth in Turface® clay permits root hair phenotyping along the entire crown root in cereal crops and demonstrates that root hair growth can extend well beyond the root hair zone.

In cereal crops, root hairs are reported to function within the root hair zone to carry out important roles in nutrient and water absorption. Nevertheless, these single cells remain understudied due to the practical challenges of phenotyping these delicate structures in large cereal crops growing on soil or other growth systems. Here we present an alternative growth system for examining the root hairs of cereal crops: the use of coarse Turface® clay alongside fertigation. This system allowed for root hairs to be easily visualized along the entire lengths of crown roots in three different cereal crops (maize, wheat, and finger millet). Surprisingly, we observed that the root hairs in these crops continued to grow beyond the canonical root hair zone, with the most root hair growth occurring on older crown root segments. We suggest that the Turface® fertigation system may permit a better understanding of the changing dynamics of root hairs as they age in large plants, and may facilitate new avenues for crop improvement below ground. However, the relevance of this system to field conditions must be further evaluated in other crops.

Salt tolerance and activity of antioxidative enzymes of transgenic finger millet overexpressing a vacuolar H(+)-pyrophosphatase gene (SbVPPase) from Sorghum bicolor.

A vacuolar proton pyrophosphatase cDNA clone was isolated from Sorghum bicolor (SbVPPase) using end-to-end gene-specific primer amplification. It showed 80-90% homology at the nucleotide and 85-95% homology at the amino acid level with other VPPases. The gene was introduced into expression vector pCAMBIA1301 under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter and transformed into Agrobacterium tumifaciens strain LBA4404 to infect embryogenic calli of finger millet (Eleusine coracana). Successful transfer of SbVPPase was confirmed by a GUS histochemical assay and PCR analysis. Both, controls and transgenic plants were subjected to 100 and 200mM NaCl and certain biochemical and physiological parameters were studied. Relative water content (RWC), plant height, leaf expansion, finger length and width and grain weight were severely reduced (50-70%), and the flowering period was delayed by 20% in control plants compared to transgenic plants under salinity stress. With increasing salt stress, the proline and chlorophyll contents as well as the enzyme activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR) increased by 25-100% in transgenics, while malondialdehyde (MDA) showed a 2-4-fold decrease. The increased activities of antioxidant enzymes and the reduction in the MDA content suggest efficient scavenging of reactive oxygen species (ROS) in transgenics and, as a consequence, probably alleviation of salt stress. Also, the leaf tissues of the transgenics accumulated 1.5-2.5-fold higher Na(+) and 0.4-0.8-fold higher K(+) levels. Together, these results clearly demonstrate that overexpression of SbVPPase in transgenic finger millet enhances the plant's performance under salt stress.

Identification of genes involved in carbon metabolism from Eleusine coracana (L.) for understanding their light-mediated entrainment and regulation.

KEY MESSAGE: The study would be helpful in understanding the synchronization of genes of a pathway and its effect on carbon metabolism which can be further utilized for better agronomic performance. Finger millet (Eleusine coracana) is a C4 crop with high nitrogen use efficiency (NUE) said to be organic by default. Being carbon and nitrogen mutually exclusive, in the present study, it was investigated how light regulates the expression of genes of carbon metabolism and photosynthesis in two finger millet genotypes (GE 3885 and GE 1437) with differing grain protein content (13.8 and 6.2%). Different genes associated with carbon metabolism were isolated (Cab, RBCS, PEPC, PPDK, PEPC-k, ME, SPS, PK, 14-3-3 and SnRK1) and the co-expression of Dof1 and these genes was investigated under different light-dark conditions. The deduced protein sequences of isolated genes showed relationship of marked variations with their homolog which might corresponds to difference in photosynthetic efficiency between finger millet and other plants. In 24 h day-night conditions, the identified genes exhibited diurnal rhythm in both genotypes with different time of peak expression. In dark, the expression of identified genes in both genotypes oscillated with varied amplitude indicating their control by an endogenous clock. However, Cab, RBCS and PPDK showed no oscillations suggesting that genes are light inducible. Exceptionally, ME transcript showed differential response within genotypes. Upon illumination, genes were induced within the measured period indicating that light is a signal involved in the entrainment of these genes. Exception was ME and SnRK1 in GE 1437. We conclude that expression of Dof1 in higher grain protein genotype was more consistent with the expression of carbon metabolism genes under study suggesting that Dof1 differentially regulates the expression of these light inducible genes and simultaneously controls the grain protein content in finger millet genotypes.

A somaclonal line SE7 of finger millet (Eleusine coracana) exhibits modified cytokinin homeostasis and increased grain yield.

The SE7 somaclonal line of finger millet (Eleusine coracana) achieved increased grain yield in field trials that apparently resulted from a higher number of inflorescences and seeds per plant, compared with the wild type. Levels of endogenous cytokinins, especially those of highly physiologically active iso-pentenyl adenine, were increased during early inflorescence development in SE7 plants. Transcript levels of cytokinin-degrading enzymes but not of a cytokinin-synthesizing enzyme were also decreased in young leaves, seedlings, and initiating inflorescences of SE7. These data suggest that attenuated degradation of cytokinins in SE7 inflorescences leads to higher cytokinin levels that stimulate meristem activity and result in production of more inflorescences. Gene expression was compared between SE7 and wild-type young inflorescences using the barley 12K cDNA array. The largest fraction of up-regulated genes in SE7 was related to transcription, translation, and cell proliferation, cell wall assembly/biosynthesis, and to growth regulation of young and meristematic tissues including floral formation. Other up-regulated genes were associated with protein and lipid degradation and mitochondrial energy production. Down-regulated genes were related to pathogen defence and stress response, primary metabolism, glycolysis, and the C:N balance. The results indicate a prolonged proliferation phase in SE7 young inflorescences characterized by up-regulated protein synthesis, cytokinesis, floral formation, and energy production. In contrast, wild-type inflorescences are similar to a more differentiated status characterized by regulated protein degradation, cell elongation, and defence/stress responses. It is concluded that attenuated degradation of cytokinins in SE7 inflorescences leads to higher cytokinin levels, which stimulate meristem activity, inflorescence formation, and seed set.

The avoidance and aggregative movements of mesophyll chloroplasts in C(4) monocots in response to blue light and abscisic acid.

In C(4) plants, mesophyll (M) chloroplasts are randomly distributed along the cell walls, whereas bundle sheath chloroplasts are located in either a centripetal or centrifugal position. It was reported previously that only M chloroplasts aggregatively redistribute to the bundle sheath side in response to extremely strong light or environmental stresses. The aggregative movement of M chloroplasts is also induced in a light-dependent fashion upon incubation with abscisic acid (ABA). The involvement of reactive oxygen species (ROS) and red/blue light in the aggregative movement of M chloroplasts are examined here in two distinct subtypes of C(4) plants, finger millet and maize. Exogenously applied hydrogen peroxide or ROS scavengers could not change the response patterns of M chloroplast movement to light and ABA. Blue light irradiation essentially induced the rearrangement of M chloroplasts along the sides of anticlinal walls, parallel to the direction of the incident light, which is analogous to the avoidance movement of C(3) chloroplasts. In the presence of ABA, most of the M chloroplasts showed the aggregative movement in response to blue light but not red light. Together these results suggest that ROS are not involved in signal transduction for the aggregative movement, and ABA can shift the blue light-induced avoidance movement of C(4)-M chloroplasts to the aggregative movement.

Efficient callus formation and plant regeneration of goosegrass [Eleusine indica (L.) Gaertn.].

Efficient methods in totipotent callus formation, cell suspension culture establishment and whole-plant regeneration have been developed for the goosegrass [ Eleusine indica (L.) Gaertn.] and its dinitroaniline-resistant biotypes. The optimum medium for inducing morphogenic calli consisted of N6 basal salts and B5 vitamins supplemented with 1-2 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l(-1) glycine, 100 mg l(-1) asparagine, 100 mg l(-1) casein hydrolysate, 30 g l(-1) sucrose and 0.6% agar, pH 5.7. The presence of organogenic and embryogenic structures in these calli was histologically documented. Cell suspension cultures derived from young calli were established in a liquid medium with the same composition. Morphogenic structures of direct shoots and somatic embryos were grown into rooted plantlets on medium containing MS basal salts, B5 vitamins, 1 mg l(-1) kinetin (Kn) and 0.1 mg l(-1) indole-3-acetic acid (IAA), 3% sucrose, 0.6% agar, pH 5.7. Calli derived from the R-biotype of E. indica possessed a high resistance to trifluralin (dinitroaniline herbicide) and cross-resistance to a structurally non-related herbicide, amiprophosmethyl (phosphorothioamidate herbicide), as did the original resistant plants. Embryogenic cell suspension culture was a better source of E. indica protoplasts than callus or mesophyll tissue. The enzyme solution containing 1.5% cellulase Onozuka R-10, 0.5% driselase, 1% pectolyase Y-23, 0.5% hemicellulase and N(6) mineral salts with an additional 0.2 M KCl and 0.1 M CaCl(2) (pH 5.4-5.5) was used for protoplast isolation. The purified protoplasts were cultivated in KM8p liquid medium supplemented with 2 mg l(-1) 2,4-D and 0.2 mg l(-1) Kn.