Proteomic analysis of leaves and roots during drought stress and recovery in Setaria italica L.

Drought is a major environmental factor that limits agricultural crop productivity and threatens food security. Foxtail millet is a model crop with excellent abiotic stress tolerance and is consequently an important subject for obtaining a better understanding of the molecular mechanisms underlying plant responses to drought and recovery. Here the physiological and proteomic responses of foxtail millet (cultivar Yugu1) leaves and roots to drought treatments and recovery were evaluated. Drought-treated foxtail millet exhibited increased relative electrolyte leakage and decreased relative water content and chlorophyll content compared to control and rewatering plants. A global analysis of protein profiles was evaluated for drought-treated and recovery treatment leaves and roots. We also identified differentially abundant proteins in drought and recovery groups, enabling comparisons between leaf and root tissue responses to the conditions. The principal component analysis suggested a clear distinction between leaf and root proteomes for the drought-treated and recovery treatment plants. Gene Ontology enrichment and co-expression analyses indicated that the biological responses of leaves differed from those in roots after drought and drought recovery. These results provide new insights and data resources to investigate the molecular basis of tissue-specific functional responses of foxtail millet during drought and recovery, thereby significantly informing crop breeding.

A straight-forward seed production technology system for foxtail millet (Setaria italica).

For autogamous crops, a precondition for using heterosis is to produce sufficient pure male-sterile female parents that can be used to produce hybrid seeds. To date, cytoplasmic male sterility (CMS) and environment-sensitive genic male sterility (EGMS) have been used commercially to exploit heterosis for autogamous species. However, neither CMS nor EGMS has been established for foxtail millet (Setaria italica). Here, we report on the establishment and application of a seed production technology (SPT) system for this crop. First, we established a DsRed-based SPT system, but found that it was unsuitable because it required the use of a fluorescent device for seed sorting. Instead, we constructed an SPT system with de novo betalain biosynthesis as the selection marker. This allowed us to distinguish transgenic seeds with the naked eye, thereby facilitating the identification of SPT maintainer line seeds. In this system, a seed sorter was not required to obtain sufficient seeds. The key point of the strategy is that the seed pool of the SPT maintainer line is propagated by artificial identification and harvesting of male-fertile individuals in the field, and the male-sterile line seed pool for hybrid production is produced and propagated by free pollination of male-sterile plants with the SPT maintainer line. In a field experiment, we obtained 423.96 kg male-sterile line seeds per acre, which is sufficient to plant 700.18 acres of farmland for hybrid seed production or male-sterile line reproduction. Our study therefore describes a powerful tool for hybrid seed production in foxtail millet, and demonstrates how the SPT system can be used for a small-grained crop with high reproduction efficiency.

Molecular evolution, diversification, and expression assessment of MADS gene family in Setaria italica, Setaria viridis, and Panicum virgatum.

KEY MESSAGE: This paper sheds light on the evolution and expression patterns of MADS genes in Setaria and Panicum virgatum. SiMADS51 and SiMADS64 maybe involved in the ABA-dependent pathway of drought response. The MADS gene family is a key regulatory factor family that controls growth, reproduction, and response to abiotic stress in plants. However, the molecular evolution of this family is rarely reported. Here, a total of 265 MADS genes were identified in Setaria italica (foxtail millet), Setaria viridis (green millet), and Panicum virgatum (switchgrass) and analyzed by bioinformatics, including physicochemical characteristics, subcellular localization, chromosomal position and duplicate, motif distribution, genetic structure, genetic evolvement, and expression patterns. Phylogenetic analysis was used to categorize these genes into M and MIKC types. The distribution of motifs and gene structure were similar for the corresponding types. According to a collinearity study, the MADS genes have been mostly conserved during evolution. The principal cause of their expansion is segmental duplication. However, the MADS gene family tends to shrink in foxtail millet, green millet, and switchgrass. The MADS genes were subjected to purifying selection, but several positive selection sites were also identified in three species. And most of the promoters of MADS genes contain cis-elements related to stress and hormonal response. RNA-seq and quantitative Real-time PCR (qRT-PCR) analysis also were examined. SiMADS genes expression levels are considerably changed in reaction to various treatments, following qRT-PCR analysis. This sheds fresh light on the evolution and expansion of the MADS family in foxtail millet, green millet, and switchgrass, and lays the foundation for further research on their functions.

Domestication-associated PHYTOCHROME C is a flowering time repressor and a key factor determining Setaria as a short-day plant.

Phytochromes play vital roles in the regulation of flowering time, but little is known in Panicoideae species, especially the C4 model Setaria. Here, genomic variations of PHYTOCHROME C (PHYC) between wild and cultivated Setaria gene pools were analysed and three SiphyC mutants were identified. The function of SiPHYC was verified by CRISPR-Cas9 approach and transcriptome sequencing. Furthermore, efficiency of indoor cultivation of SiphyC mutants were systematically evaluated. An extreme purified selection of PHYC was detected in wild to cultivated domestication process of Setaria. SiphyC mutants and knockout transgenic plants showed an early heading date and a loss of response to short-day photoperiod. Furthermore, variable expression of SiFTa, SiMADS14 and SiMADS15 might be responsible for promoting flowering of SiphyC mutants. Moreover, SiphyC mutant was four times that of the indoor plot ratio of wild-type and produced over 200 seeds within 45 d per individual. Our results suggest that domestication-associated SiPHYC repressed flowering and determined Setaria as a short-day plant, and SiphyC mutants possess the potential for creating efficient indoor cultivation system suitable for research on Setaria as a model, and either for maize or sorghum as well.

A mini foxtail millet with an Arabidopsis-like life cycle as a C4 model system.

Foxtail millet (Setaria italica) is an important crop species and an emerging model plant for C4 grasses. However, functional genomics research on foxtail millet is challenging because of its long generation time, relatively large stature and recalcitrance to genetic transformation. Here we report the development of xiaomi, a rapid-cycling mini foxtail millet mutant as a C4 model system. Five to six generations of xiaomi can be grown in a year in growth chambers due to its short life cycle and small plant size, similar to Arabidopsis. A point mutation in the Phytochrome C (PHYC) gene was found to be causal for these characteristics. PHYC encodes a light receptor essential for photoperiodic flowering. A reference-grade xiaomi genome comprising 429.94 Mb of sequence was assembled and a gene-expression atlas from 11 different tissues was developed. These resources, together with an established highly efficient transformation system and a multi-omics database, make xiaomi an ideal model system for functional studies of C4 plants.

DROOPY LEAF1 controls leaf architecture by orchestrating early brassinosteroid signaling.

Leaf architecture directly determines canopy structure, and thus, grain yield in crops. Leaf droopiness is an agronomic trait primarily affecting the cereal leaf architecture but the genetic basis and underlying molecular mechanism of this trait remain unclear. Here, we report that DROOPY LEAF1 (DPY1), an LRR receptor-like kinase, plays a crucial role in determining leaf droopiness by controlling the brassinosteroid (BR) signaling output in Setaria, an emerging model for Panicoideae grasses. Loss-of-function mutation in DPY1 led to malformation of vascular sclerenchyma and low lignin content in leaves, and thus, an extremely droopy leaf phenotype, consistent with its preferential expression in leaf vascular tissues. DPY1 interacts with and competes for SiBAK1 and as a result, causes a sequential reduction in SiBRI1-SiBAK1 interaction, SiBRI1 phosphorylation, and downstream BR signaling. Conversely, DPY1 accumulation and affinity of the DPY1-SiBAK1 interaction are enhanced under BR treatment, thus preventing SiBRI1 from overactivation. As such, those findings reveal a negative feedback mechanism that represses leaf droopiness by preventing an overresponse of early BR signaling to excess BRs. Notably, plants overexpressing DPY1 have more upright leaves, thicker stems, and bigger panicles, suggesting potential utilization for yield improvement. The maize ortholog of DPY1 rescues the droopy leaves in dpy1, suggesting its conserved function in Panicoideae. Together, our study provides insights into how BR signaling is scrutinized by DPY1 to ensure the upward leaf architecture.

Identification and function analyses of senescence-associated WRKYs in wheat.

Leaf senescence is a positive, highly regulated, complex process, and transcription factors play important roles in the regulation of this process. We identified and characterized 116 WRKYs from the wheat genome database. Thirteen TaWRKYs were confirmed as senescence-associated genes. We focused on TaWRKY7, which is up-regulated in the natural leaf senescence process. TaWRKY7 is expressed in different tissues of wheat and is localized in the nucleus. It shows transcriptional activation activity in yeast cells. The ectopic over-expression of TaWRKY7 in Arabidopsis (Arabidopsis thaliana) significantly promoted early leaf senescence under darkness treatment and prevented leaf moisture losses. TaWRKY7 played important roles in the senescence process and was involved in abiotic stress responses. Our transcriptomic and genetic studies on WRKYs suggest that WRKY transcription factors are a type of vital regulator in leaf senescence in wheat (Triticum aestivum L.).

Signal transduction in leaf senescence.

Leaf senescence is a complex developmental phase that involves both degenerative and nutrient recycling processes. It is characterized by loss of chlorophyll and the degradation of proteins, nucleic acids, lipids, and nutrient remobilization. The onset and progression of leaf senescence are controlled by an array of environmental cues (such as drought, darkness, extreme temperatures, and pathogen attack) and endogenous factors (including age, ethylene, jasmonic acid, salicylic acid, abscisic acid, and cytokinin). This review discusses the major breakthroughs in signal transduction during the onset of leaf senescence, in dark- and drought-mediated leaf senescence, and in various hormones regulating leaf senescence achieved in the past several years. Various signals show different mechanisms of controlling leaf senescence, and cross-talks between different signaling pathways make it more complex. Key senescence regulatory networks still need to be elucidated, including cross-talks and the interaction mechanisms of various environmental signals and internal factors.

Physiological and molecular changes of detached wheat leaves in responding to various treatments.

Leaf senescence is induced or accelerated when leaves are detached. However, the senescence process and expression pattern of senescence-associated genes (SAGs) when leaves are detached are not clearly understood. To detect senescence-associated physiological changes and SAG expression, wheat (Triticum aestivum L.) leaves were detached and treated with light, darkness, low temperature (4 °C), jasmonic acid (JA), abscisic acid (ABA), and salicylic acid (SA). The leaf phenotypes, chlorophyll content, delayed fluorescence (DF), and expression levels of two SAGs, namely, TaSAG3 and TaSAG5, were analyzed. Under these different treatments, the detached leaves turned yellow with different patterns and varying chlorophyll content. DF significantly decreased after the dark, ABA, JA and SA treatments. TaSAG3 and TaSAG5, which are expressed in natural senescent leaves, showed different expression patterns under various treatments. However, both TaSAG3 and TaSAG5 were upregulated after leaf detachment. Our results revealed senescence-associated physiological changes and molecular differences in leaves, which induced leaf senescence during different stress treatments.