Identification of no pollen 1 provides a candidate gene for heterosis utilization in foxtail millet(Setaria italica L.)

Male sterility is a common biological phenomenon in plant kingdom and has been used to generate male-sterile lines, which are important genetic resources for commercial hybrid seed production. Although increasing numbers of male-sterility genes have been identified in rice(Oryza sativa) and Arabidopsis(Arabidopsis thaliana), few male-sterility-related genes have been characterized in foxtail millet(Setaria italica). In this study, we isolated a male-sterile ethyl methanesulfonate-generated mutant in foxtail millet, no pollen 1(sinp1), which displayed abnormal Ubisch bodies, defective pollen exine and complete male sterility. Using bulk segregation analysis, we cloned SiNP1 and confirmed its function with CRISPR/Cas9 genome editing. SiNP1 encoded a putative glucose-methanol-choline oxidoreductase.Subcellular localization showed that the SiNP1 protein was preferentially localized to the endoplasmic reticulum and was predominantly expressed in panicle. Transcriptome analysis revealed that many genes were differentially expressed in the sinp1 mutant, some of which encoded proteins putatively involved in carbohydrate metabolism, fatty acid biosynthesis, and lipid transport and metabolism, which were closely associated with pollen wall development. Metabolome analysis revealed the disturbance of flavonoids metabolism and fatty acid biosynthesis in the mutant. In conclusion, identification of SiNP1 provides a candidate male-sterility gene for heterosis utilization in foxtail millet and gives further insight into the mechanism of pollen reproduction in plants.

OsPRMT6a-mediated arginine methylation of OsJAZ1 regulates jasmonate signaling and spikelet development in rice

Although both protein arginine methylation(PRMT)and jasmonate(JA)signaling are crucial for regulating plant development,the relationship between these processes in the control of spikelet development remains unclear.In this study,we used the CRISPR/Cas9 technology to generate two OsPRMT6a loss-of-function mutants that exhibit various abnormal spikelet structures.Interestingly,we found that OsPRMT6a can methylate arginine residues in JA signal repressors OsJAZ1 and OsJAZ7.We showed that arginine methylation of OsJAZ1 enhances the binding affinity of OsJAZ1 with the JA receptors OsCOI1a and OsCOI1b in the presence of JAs,thereby promoting the ubiquitination of OsJAZ1 by the SCF^(OsCOI1a/OsCOI1b) complex and degradation via the 26S proteasome.This process ultimately releases OsMYC2,a core transcriptional regulator in the JA signaling pathway,to activate or repress JA-responsive genes,thereby maintaining normal plant(spikelet)development.However,in the osprmt6a-1 mutant,reduced arginine methylation of OsJAZ1 impaires the interaction between OsJAZ1 and OsCOI1a/OsCOI1b in the presence of JAs.As a result,OsJAZ1 proteins become more stable,repressing JA responses,thus causing the formation of abnormal spikelet structures.Moreover,we discovered that JA signaling reduces the OsPRMT6a mRNA level in an OsMYC2-dependent manner,thereby establishing a negative feedback loop to balance JA signaling.We further found that OsPRMT6a-mediated arginine methylation of OsJAZ1 likely serves as a switch to tune JA signaling to maintain normal spikelet development under harsh environmental conditions such as high temperatures.Collectively,our study establishes a direct molecular link between arginine methylation and JA signaling in rice.

Establishment of genome-editing system and assembly of a near-complete genome in broomcorn millet

The ancient crop broomcorn millet(Panicum miliaceum L.)is an indispensable orphan crop in semi-arid regions due to its short life cycle and excellent abiotic stress tolerance.These advantages make it an important alternative crop to increase food security and achieve the goal of zero hunger,particularly in light of the uncertainty of global climate change.However,functional genomic and biotechnological research in broomcorn millet has been hampered due to a lack of genetic tools such as transformation and genome-editing techniques.Here,we successfully performed genome editing of broomcorn millet.We identified an elite variety,Hongmi,that produces embryogenic callus and has high shoot regeneration ability in in vitro culture.We established an Agrobacterium tumefaciens-mediated genetic transformation protocol and a clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9-mediated genome-editing system for Hongmi.Using these techniques,we produced herbicide-resistant transgenic plants and edited phytoene desaturase(Pm PDS),which is involved in chlorophyll biosynthesis.To facilitate the rapid adoption of Hongmi as a model line for broomcorn millet research,we assembled a near-complete genome sequence of Hongmi and comprehensively annotated its genome.Together,our results open the door to improving broomcorn millet using biotechnology.

De novo creation of popcorn-like fragrant foxtail millet

Aroma plays a crucial role in the sensory appeal and enjoyment of food,and it significantly impacts the grain quality of cereals,thereby influencing the value and consumption of agricultural products.For example,aromatic rice varieties like Basmati and Jasmine are renowned for their distinct aroma and command almost double the price of regular rice in the market.

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 Ds Red-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.

Engineering Broad-Spectrum Resistance to Bacterial Blight by CRISPR/Cas9-Mediated Precise Homology Directed Repair in Rice

Dear Editor,Rice(Oryza sativa)is one of the most important food crops as well as a model plant for basic research.Rice bacterial blight(BB)caused by Xanthomonas oryzae pv.oryzae(Xoo)is a devastating bacterial disease worldwide(Mew et al.,1993),affecting millions of hectares of rice annually,with an estimated crop loss of as high as 75%.Upon invasion of rice cells,Xoo secretes transcription activator-like effectors(TALEs)to activate the host Sugars Will Eventually be Exported Transporters(SWEET)genes to obtain nutrition for bacterial growth,because the induced SWEET transports more sugars to where the bacteria reside(Boch and Bonas,2010).