Interactions of OsMADS1 with Floral Homeotic Genes in Rice Flower Development

During reproductive development, rice plants develop unique flower organs which determine the final grain yield. OsMADS1, one of SEPALLATA-like MADS-box genes, has been unraveled to play critical roles in rice floral organ identity specification and floral meristem determinacy. However, the molecular mechanisms underlying interactions of OsMADS1 with other floral homeotic genes in regulating flower development remains largely elusive. In this work, we studied the genetic interactions of OsMADS1 with B-, C-, and D-class genes along with physical interactions among their proteins. We show that the physical and genetic interactions between OsMADS1 and OsMADS3 are essential for floral meristem activity maintenance and organ identity specification; while OsMADS1 physically and genetically interacts with OsMADS58 in regu- lating floral meristem determinacy and suppressing spikelet meristem reversion. We provided important genetic evidence to support the neofunctionalization of two rice C-class genes （OsMADS3 and OsMADS58） during flower development. Gene expression profiling and quantitative RT-PCR analyses further revealed that OsMADS1 affects the expression of many genes involved in floral identity and hormone signaling, and chromatin immunoprecipitation （ChlP）-PCR assay further demonstrated that OsMADS17 is a direct target gene of OsMADS1. Taken together, these results reveal that OsMADS1 has diversified regulatory functions in specifying rice floral organ and meristem identity, probably through its genetic and physical interactions with different floral homeotic regulators.

Investigation of CRISPR/Cas9-induced SD1 rice mutants highlights the importance of molecular characterization in plant molecular breeding

Although Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)/CRISPR-associated 9(Cas9)system has been widely used for basic research in model plants,its application for applied breeding in crops has faced strong regulatory obstacles,due mainly to a poor understanding of the authentic output of this system,particularly in higher generations.In this study,different from any previous studies,we investigated in detail the molecular characteristics and production performance of CRISPR/Cas9-generated SD1(semi-dwarf 1)mutants from T2 to T4 generations,of which the selection of T1 and T2 was done only by visual phenotyping for semidwarf plants.Our data revealed not only on-and off-target mutations with small or lager indels but also exogenous elements in T2 plants.All indel mutants passed stably to T3 or T4 without additional modifications independent on the presence of Cas9,while some lines displayed unexpected hereditary patterns of Cas9 or some exogenous elements.In addition,effects of various SD1 alleles on rice height and yield differed depending on genetic backgrounds.Taken together,our data showed that the CRISPR/Cas9 system is effective in producing homozygous mutants for functional analysis,but it may be not as precise as expected in rice,and that early and accurate molecular characterization and screening must be carried out for generations before transitioning of the CRISPR/Cas9 system from laboratory to field.

A versatile biosensing platform coupling CRISPR-Cas12a and aptamers for detection of diverse analytes

Rapid and sensitive detection of various analytes is in high demand.Apart from its application in genome editing,CRISPR-Cas also shows promises in nucleic acid detection applications.To further exploit the potential of CRISPR-Cas for detection of diverse analytes,we present a versatile biosensing platform that couples the excellent affinity of aptamers for broad-range analytes with the collateral single-strand DNA cleavage activity of CRISPR-Cas12 a.We demonstrated that the biosensors developed by this platform can be used to detect protein and small molecule in human serum with a complicated background,i.e.,the tumor marker alpha fetoprotein and cocaine with the detection limits of 0.07 fmol/L and 0.34 lmol/L,respectively,highlighting the advantages of simplicity,sensitivity,short detection time,and low cost compared with the state-of-the-art biosensing approaches.Altogether,this biosensing platform with plug-and-play design show great potential in the detection of diverse analytes.

OsMADS32 interacts with PI-like proteins and regulates rice flower development

OsMADS32 is a monocot specific MIKCc type MADS‐box gene that plays an important role in regulating rice floral meristem and organs identity, a crucial process for reproductive success and rice yield. However, its underlying mechanism of action remains to be clarified. Here, we characterized a hypomorphic mutant allele of OsMADS32/CFO1, cfo1‐3 and identified its function in controlling rice flower development by bioinformatics and protein‐protein interaction analysis. The cfo1‐3 mutant produces defective flowers, including loss of lodicule identity, formation of ectopic lodicule or hull‐like organs and decreased stamen number, mimicking phenotypes related to the mutation of B class genes. Molecular characterization indicated that mis‐splicing of OsMADS32 transcripts in the cfo1‐3 mutant resulted in an extra eight amino acids in the K‐domain of OsMADS32 protein. By yeast two hybrid and bimolecular fluorescence comple-mentation assays, we revealed that the insertion of eight amino acids or deletion of the internal region in the K1 subdomain of OsMADS32 affects the interaction between OsMADS32 with PISTILLATA （PI）‐like proteins OsMADS2 and OsMADS4. This work provides new insight into the mecha-nism by which OsMADS32 regulates rice lodicule and stamen identity, by interaction with two PI‐like proteins via its K domain.

Integrating PCR-free amplification and synergistic sensing for ultrasensitive and rapid CRISPR/Cas12a-based SARS-CoV-2 antigen detection

Antigen detection provides particularly valuable information for medical diagnoses;however,the current detection methods are less sensitive and accurate than nucleic acid analysis.The combination of CRISPR/Cas12a and aptamers provides a new detection paradigm,but sensitive sensing and stable amplification in antigen detection remain challenging.Here,we present a PCR-free multiple trigger dsDNA tandem-based signal amplification strategy and a de novo designed dual aptamer synergistic sensing strategy.Integration of these two strategies endowed the CRISPR/Cas12a and aptamer-based method with ultra-sensitive,fast,and stable antigen detection.In a demonstration of this method,the limit of detection was at the single virus level(0.17 fM,approximately two copies/μL)in SARS-CoV-2 antigen nucleocapsid protein analysis of saliva or serum samples.The entire procedure required only 20 min.Given our system’s simplicity and modular setup,we believe that it could be adapted reasonably easily for general applications in CRISPR/Cas12a-aptamer-based detection.

RNA-seq profiling the transcriptome of secondary seed dormancy in canola(Brassica napus L.)

Secondary seed dormancy(SSD)is responsible for volunteer plants in canola fields,which causes a series of problems in canola production and serves as an important trait for the environmental safety assessment of transgenic canola.A canola cultivar with strong SSD was used to establish insight into seed transcriptomes in its secondarily dormant seeds and control seeds without dormancy by RNA-seq analysis,aiming to determine the molecular ecological characterizations of SSD.A dataset(more than 4 Gb)of valid sequences was obtained from each sample,which was combined to carry out the de novo assembling.The assembled sequences consisted of 314,261fragments with length[100 bp,including 29,740 long transcripts of length C 500 bp.Functional annotation indicated that 1,641 long transcripts could be categorized into 24 cluster of orthologous groups of proteins(COGs)and 16,515 transcripts were linked to 2,648 gene ontology(GO)terms.There were 452 long transcripts with significantly different expression identified by a threshold of[2-fold expression change(P\0.001)between samples,among which 343 transcripts were unambiguously homologous to Arabidopsis genes.The plant hormones abscisic acid and gibberellins were known as the pivotal regulators of seed dormancy and germination.Although genes responsible for either biosynthesis or signaling of each hormone could be widely verified from the SSD transcriptome,theirs expression evidences failed to correlate with the induction of SSD.Based on the enriched terms of gene ontology and KEGG orthology,as well as the expression models of candidate genes of SSD,we proposed that fatty acid metabolism might implicate in SSD in canola.The information reported here may play a significant role in further understanding of environmental safety assessment of SSD in transgenic canola.