TaMIR397-6A and -6B Homoeologs Encode Active miR397 Contributing to the Regulation of Grain Size in Hexaploid Wheat.

Wheat is one of the most important food crops globally, and understanding the regulation of grain size is crucial for wheat breeding to achieve a higher grain yield. MicroRNAs (miRNAs) play vital roles in plant growth and development. However, the miRNA-mediated mechanism underlying grain size regulation remains largely elusive in wheat. Here, we report the characterization and functional validation of a miRNA, TamiR397a, associated with grain size regulation in wheat. The function of three TaMIR397 homoeologs was determined through histochemical ß-glucuronidase-dependent assay. MiRNA expression was detected using quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the function of TamiR397a was validated through its transgenic overexpression and repression in wheat. It was found that TaMIR397-6A and TaMIR397-6B encode active TamiR397a. The expression profiling indicated that TamiR397a was differentially expressed in various tissues and gradually up-regulated during grain filling. The inhibition of TamiR397a perturbed grain development, leading to a decrease in grain size and weight. Conversely, the overexpression of TamiR397a resulted in increased grain size and weight by accelerating the grain filling process. Transcriptome analysis revealed that TamiR397a regulates a set of genes involved in hormone response, desiccation tolerance, regulation of cellular senescence, seed dormancy, and seed maturation biological processes, which are important for grain development. Among the down-regulated genes in the grains of the TamiR397a-overexpressing transgenic plants, 11 putative targets of the miRNA were identified. Taken together, our results demonstrate that TamiR397a is a positive regulator of grain size and weight, offering potential targets for breeding wheat with an increased grain yield.

Characterization of thioredoxin gene TaTrxh9 associated with heading-time regulation in wheat.

Thioredoxins (Trxs) are thiol-disulfide oxidoreductase proteins that play important roles in a spectrum of processes linking redox regulation and signaling in plants. However, little is known about Trxs and their biological functions in wheat, one of the most important food crops worldwide. This study reports the identification and functional characterization of an h-type Trx gene, TaTrxh9, in wheat. Three homoeologs of TaTrxh9 were identified and the sequences in the coding region were highly consistent among the homoeologs. Protein characterization showed that a conserved Trx_family domain, as well as a typical active site with a dithiol signature (WCGPC), was included in TaTrxh9. Structural modeling demonstrated that TaTrxh9 could fold into a canonical thioredoxin structure consisting of five-stranded antiparallel beta sheets sandwiched between four alpha helices. The insulin disulfide reduction assay demonstrated that TaTrxh9 was catalytically active in vitro. TaTrxh9 overexpression in the Arabidopsis mutant trxh9 complemented the abnormal growth phenotypes of the mutant, suggesting is functionality in vivo. The transcription level of TaTrxh9 was higher in leaf tissues and it was differentially expressed during the development of wheat plants. Interestingly, barley stripe mosaic virus-mediated suppression of TaTrxh9 shortened the seedling-heading period of wheat. Furthermore, CRISPR-Cas9 mediated gene knockout confirmed that the TaTrxh9 mutation resulted in early heading of wheat. To our knowledge, this study is the first to report that Trxh is associated with heading-time regulation, which lays a foundation for further exploring the biological function of TaTrxh9 and provides new ideas for molecular breeding focusing on early heading in wheat.

Direct calibration of neutron detectors for laser-driven nuclear reaction experiments with a gated neutron source.

Nowadays, the sustained technological progress in high-intensity lasers is opening up the possibility of super-intense laser pulses to trigger or substantially influence nuclear reactions. However, it is a big challenge to quantitatively measure the reaction products because of the interference of electromagnetic pulses induced by high-intensity lasers. Fast scintillation detectors are widely chosen for fast neutron detection. The calibration of neutron detectors is crucial to measuring the yield of neutron products. Since one large signal superimposed by a number of neutron signals appears during a short period, it is difficult to directly and precisely calibrate the detectors' response for a single neutron. In the present work, we developed a direct calibration method with a gated fission neutron source 252Cf to solve this problem. This work demonstrates that the gated fission neutron source approach, with a unique "Pulse Shape Discrimination & Time of Flight window" function, has the highest background-γ-rejection and improves the confidence level of the final results for both liquid and plastic scintillator. Compared with the result of Compton edge method and neutron beam method, the gated fission neutron source method achieves much cleaner neutron signals and avoids interference caused by the modeling accuracy of the neutron detectors. This approach can be widely used in laser-driven nuclear physics experiments with higher accuracy for neutron detection.

Cloning and characterization of TaMBD6 homeologues encoding methyl-CpG-binding domain proteins in wheat.

DNA methylation is a major epigenetic marker in plants that plays a crucial role in transcriptional and developmental regulation. The DNA methylation 'code' is thought to be 'read' by a set of proteins containing methyl-CpG-binding domain (MBD). However, little is known about MBD genes in common wheat (Triticum aestivum L.). Here, we report the isolation and characterization of TaMBD6 and its homeologues (TaMBD6_A, TaMBD6_B, and TaMBD6_D) in hexaploid wheat. The cDNA was quite different among the three homeologues and InDel mutations were detected in 5'-UTR and coding region. Two types of TRs (tandem repeats) -- TR1 (57 bp) and TR2 (39 bp) -- occurred in the coding region. TaMBD6_B harbored five copies of TR1 and two copies of TR2. In contrast, TaMBD6_A lacked 30 bp between the 2nd and 3rd copy of TR1, while TaMBD6_D was missing two copies of TR1 but had three copies of TR2. TaMBD6_A, TaMBD6_B, and TaMBD6_D encoded 435, 446, and 420 amino acids, respectively. Structural analysis of TaMBD6 protein indicated that each of the three homeologues had an identical MBD domain at the N-terminal, as well as a typical nuclear localization signal. Although genomics analysis showed that two introns were included, the length of the first intron varied from 3100 bp to 3471 bp and their sequences were very different. Expression analysis demonstrated that the transcription level of TaMBD6 began to increase gradually in developing grains at 15 days after pollination while decreasing significantly in endosperm and embryo tissues during germination. Expression of TaMBD6 appeared to be positively correlated with starch metabolism in the endosperm but was negatively correlated with embryo formation and sprouting. We were also interested to learn that TaMBD6 homeologues were differentially expressed in developing wheat plants and that their expression patterns were variously affected by vernalization treatment. Further investigation revealed that TaMBD6 was induced by prolonged chilling, indicating that the protein is potentially involved in regulating the developmental transition from vegetative to reproductive stages. Although the homeologues generally showed similar differential expression patterns, TaMBD6_D and TaMBD6_B contribute more to the processes of grain development and germination while TaMBD6_A is predominant in mature plants.