From model to alfalfa:Gene editing to obtain semidwarf and prostrate growth habits

Alfalfa(Medicago sativa L.)is a nutritious forage crop with wide ecological adaptability.The molecular breeding of alfalfa is restricted by its heterozygous tetraploid genome and the difficult genetic manipulation process.Under time and resource constraints,we applied a more convenient approach.We investigated two MtGA3ox genes,MtGA3ox1 and MtGA3ox2,of Medicago truncatula,a diploid legume model species,finding that MtGA3ox1 plays a major role in GA-regulated plant architecture.Mutation of neither gene affected nitrogenase activity.These results suggest that MtGA3ox1 can be used in semidwarf and prostrate alfalfa breeding.Based on the M.truncatula MtGA3ox1 sequence,MsGA3ox1 was cloned from alfalfa,and two knockout targets were designed.An efficient CRISPR/Cas9-based genome editing protocol was used to generate msga3ox1 mutants in alfalfa.We obtained three lines that carried mutations in all four alleles in the T0 generation.Fifteen clonal plants were vegetatively propagated from each transgenic line using shoot cuttings.The plant height and internode length of msga3ox1 null mutants were significantly decreased.The number of total lateral branches,leaf/stem ratio and crude protein content of aerial plant parts of msga3ox1 mutants were significantly increased.Thus,we obtained semi-dwarf and prostrate alfalfa by gene editing.

豆科植物特异SnRK1调控共生固氮的机制

豆科植物和根瘤菌之间的共生固氮(symbiotic nitrogen fixation,SNF)是高度耗能的代谢过程,植物每获取1 g固定的氮(N2)需要消耗5.2~18.8 g的碳源[1].尽管这一行为互利于双方,但共生固氮的有效进行依赖于豆科植物持续的碳源供应[2].豆科新型能量感受器GmNAS1/GmNAP1的发现,为设计高效利用作物周围环境中的碳源、提高共生固氮能力提供了理论支撑[3].然而,豆科植物在缺氮时如何分配能量和碳源实现高耗能共生固氮的分子机制尚不清楚[4].豆科植物是否有独特的能量感受器参与这一过程是科学家们一直探究的问题.

Legume-specific SnRK1 promotes malate supply to bacteroids for symbiotic nitrogen fixation

Nodulation is an energy-expensive behavior driven by legumes by providing carbon sources to bacteroids and obtaining nitrogen sources in return.The energy sensor sucrose nonfermenting 1-related protein kinase 1(SnRK1)is the hub of energy regulation in eukaryotes.However,the molecular mechanism by which SnRK1 coordinates the allocation of energy and substances during symbiotic nitrogen fixation(SNF)remains unknown.In this study,we identified the novel legume-specific SnRK1α4,a member of the SnRK1 family that positively regulates SNF.Phenotypic analysis showed that nodule size and nitrogenase activity increased in SnRK1α4-overexpressing plants and decreased significantly in snrk1α4 mutants.We demonstrated that a key upstream kinase involved in nodulation,Does Not Make Infection 2(DMI2),can phosphorylate SnRK1α4 at Thr175 to cause its activation.Further evidence clarified that SnRK1α4 phosphorylates the malate dehydrogenases MDH1/2 to promote malate production in the cytoplasm,supplying carbon sources to bacteroids.Therefore,our findings reveal an essential role of the DMI2–SnRK1α4–MDH pathway in supplying carbon sources to bacteroids for SNF and provide a new module for constructing cereal crops with SNF.

The Chromosome-Level Genome Sequence of the Autotetraploid Alfalfa and Resequencing of Core Germplasms Provide Genomic Resources for Alfalfa Research

Alfalfa(Medicago sativa)is one of the most important forage crops in the world;however,its molecular genetics and breeding research are hindered due to the lack of a high-quality reference genome.Here,we report a de novo assembled 816-Mb high-quality,chromosome-level haploid genome sequence for‘Zhongmu No.1’alfalfa,a heterozygous autotetraploid.The contig N50 is 3.92 Mb,and 49165 genes are annotated in the genome.The alfalfa genome is estimated to have diverged from M.truncatula approximately 8 million years ago.Genomic population analysis of 162 alfalfa accessions revealed high genetic diversity,weak population structure,and extensive gene flow from wild to cultivated alfalfa.Genome-wide association studies identified many candidate genes associated with important agronomic traits.Furthermore,we showed that MsFTa2,a Flowering Locus T homolog,whose expression is upregulated in salt-resistant germplasms,may be associated with fall dormancy and salt resistance.Taken together,these genomic resources will facilitate alfalfa genetic research and agronomic improvement.

Sensitivity-enhanced surface plasmon resonance sensor utilizing a tungsten disulfide (WS_2) nanosheets overlayer

Tungsten disulfide(WS_2), as a representative layered transition metal dichalcogenide(TMDC) material, possesses important potential for applications in highly sensitive sensors. Here, a sensitivity-enhanced surface plasmon resonance(SPR) sensor with a metal film modified by an overlayer of WS_2 nanosheets is proposed and demonstrated. The SPR sensitivity is related to the thickness of the WS_2 overlayer, which can be tailored by coating a WS_2 ethanol suspension with different concentrations or by the number of times of repeated post-coating.Benefitting from its large surface area, high refractive index, and unique optoelectronic properties, the WS_2 nanosheet overlayer coated on the gold film significantly improves the sensing sensitivity. The highest sensitivity(up to 2459.3 nm∕RIU) in the experiment is achieved by coating the WS_2 suspension once. Compared to the case without a WS_2 overlayer, this result shows a sensitivity enhancement of 26.6%. The influence of the WS_2 nanosheet overlayer on the sensing performance improvement is analyzed and discussed. Moreover, the proposed WS_2 SPR sensor has a linear correlation coefficient of 99.76% in refractive index range of 1.333 to 1.360. Besides sensitivity enhancement, the WS_2 nanosheet overlayer is able to show additional advantages, such as protection of metal film from oxidation, tunability of the resonance wavelength region, biocompatibility, capability of vapor,and gas sensing.

Differentiation trajectories and biofunctions of symbiotic and un-symbiotic fate cells in root nodules of Medicago truncatula

The root nodule is a complex symbiotic nitrogen fixation factory,in which cells are highly heterogeneous.However,the differentiation trajectories and interconnection of nodule cells remain largely unknown.In this study,we set up a modified protocol for nodule protoplast preparation and performed a single-cell RNA sequencing profiling of the indeterminate Medicago truncatula nodule.We designated 13 cell clusters with specific expression patterns in 14-day post inoculation nodules and constructed a spatial and functional cellular map based on experimental data and bioinformatic analyses.Pseudotime analysis further revealed that two groups of apical meristematic cells develop into symbiotic and un-symbiotic fate cells along their particular trajectories.Biofunction analysis of each cell cluster revealed their particularity and interrelation,especially that the un-infected cells in nitrogen fixation zone are also involved in nitrogen assimilation by undertaking the asparagine synthesis.Collectively,our data offer an important resource for investigating the mechanism of nodule organogenesis and symbiotic nitrogen fixation.

The histone methyltransferase SUVR2 promotes DSB repair via chromatin remodeling and liquidliquid phase separation

Maintaining genomic integrity and stability is particularly important for stem cells,which are at the top of the cell lineage origin.Here,we discovered that the plant-specific histone methyltransferase SUVR2 maintains the genome integrity of the root tip stem cells through chromatin remodeling and liquid-liquid phase separation(LLPS)when facing DNA double-strand breaks(DSBs).The histone methyltransferase SUVR2(MtSUVR2)has histone methyltransferase activity and catalyzes the conversion of histone H3 lysine 9 monomethylation(H3K9me1)to H3K9me2/3 in vitro and in Medicago truncatula.Under DNA damage,the proportion of heterochromatin decreased and the level of DSB damage marker y-H2AX increased in suvr2 mutants,indicating that MtSUVR2 promotes the compaction of the chromatin structure through H3K9 methylation modification to protect DNA from damage.Interestingly,MtSUVR2 was induced by DSBs to phase separate and form droplets to localize at the damage sites,and this was confirmed by immunofluorescence and fluorescence recovery after photobleaching experiments.The IDR1 and lowcomplexity domain regions of MtSUVR2 determined its phase separation in the nucleus,whereas the IDR2 region determined the interaction with the homologous recombinase MtRAD51.Furthermore,we found that MtSUVR2 drove the phase separation of MtRAD51 to form"DNA repair bodies,"which could enhance the stability of MtRAD51 proteins to facilitate error-free homologous recombination repair of stem cells.Taken together,our study reveals that chromatin remodeling-associated proteins participate in DNA repair through LLPS.

Recent progress of second harmonic generation based on thin film lithium niobate [Invited]

Recently, nonlinear photonics has attracted considerable interest. Among the nonlinear effects, second harmonic generation(SHG) remains a hot research topic. The recent development of thin film lithium niobate(TFLN) technology has superior performances to the conventional counterparts. Herein, this review article reveals the recent progress of SHG based on TFLN and its integrated photonics. We mainly discuss and compare the different techniques of TFLN-based structures to boost the nonlinear performances assisted by localizing light in nanostructures and structured waveguides.Moreover, our conclusions and perspectives indicate that more efficient methods need to be further explored for higher SHG conversion efficiency on the TFLN platform.

Resonance-assisted light–control–light characteristics of SnS_2 on a microfiber knot resonator with fast response

An all-optical light–control–light functionality with the structure of a microfiber knot resonator (MKR) coated with tin disulfide (SnS_2) nanosheets is experimentally demonstrated. The evanescent light in the MKR [with a resonance Q of ～59,000 and an extinction ratio (ER) of ～26 dB] is exploited to enhance light–matter interaction by coating a two-dimensional material SnS_2 nanosheet onto it. Thanks to the enhanced light–matter interaction and the strong absorption property of SnS_2, the transmitted optical power can be tuned quasi-linearly with an external violet pump light power, where a transmitted optical power variation rate ΔT with respect to the violet light power of ～0.22 dB∕mW is obtained. In addition, the MKR structure possessing multiple resonances enables a direct experimental demonstration of the relationship between resonance properties (such as Q and ER), and the obtained ΔT variation rate with respect to the violet light power. It verifies experimentally that a higher resonance Q and a larger ER can lead to a higher ΔT variation rate. In terms of the operating speed, this device runs as fast as ～3.2 ms. This kind of all-optical light–control–light functional structure may find applications in future all-optical circuitry, handheld fiber sensors, etc.