Effects of Straw and Biochar Returned to the Soil on Soil Physical Properties and pH Value in Cold Rice Region

[Objectives]In order to explore the feasibility of using straw and biochar returned to the soil to improve soil physical properties and pH value in cold rice regions of China.[Methods]the effects of straw directly returned to the soil and charred straw(biochar)returned to the soil on soil bulk density,porosity,temperature and pH value of cold paddy soil were studied in this paper.[Results]The results showed that compared with conventional production,straw(6 t/ha),a small amount of biochar(2 t/ha)and a large amount of biochar(40 t/ha)returned to the soil reduced paddy soil bulk density at different growth stages by 6.02%-11.86%,2.69%-6.67%and 8.58%-11.32%,respectively,increased total porosity by 7.41%-14.93%,3.19%-8.38%and 9.81%-14.27%,respectively,and increased aeration porosity by 22.28%-192.11%,17.80%-92.11%and 52.44%-157.11%,respectively.Straw and a small amount of biochar returned to the soil had no significant effect on soil temperature and pH value of paddy field,but a large amount of biochar returned to the soil could significantly increase soil temperature by 5.13%-8.79%and pH value by 3.15%-5.96%in the later stage of rice growth.[Conclusions]The straw and biochar returned to the soil could reduce soil bulk density,increase total porosity and aeration porosity,and only a large amount of biochar returned to the soil could significantly increase soil temperature and pH value.

Gene expression profiles in early leaf of rice(Oryza sativa)and foxtail millet(Setaria italica)

Plant anatomy is patterned early during leaf development which suggests studying the spatial–temporal transcriptomes of primordia will help identify critical regulative and functional genes.We successfully isolated the leaf primordia tissues from the C3grass rice and the C4grass foxtail millet by laser capture microdissection(LCM)and studied the gene expression throughout leaf developmental stages.Our data analysis uncovered the conserved expression patterns of certain gene clusters both in rice and foxtail millet during leaf development.We revealed genes and transcription factors involved in vein formation,stomatal development,and suberin accumulation.We identified 79 candidate genes associated with functional regulation of C4anatomy formation.Screening phenotype of the candidate genes revealed that knock-out of a putative polar auxin transport related gene NAL1 resulted significantly reduced veinal space in rice leaf.Our present work provides a foundation for future analyses of genes with novel functions in grasses and their role in leaf development,in particular the role in leaves with a contrasting C3vs.C4biosynthetic pathway.

A simple and efficient CRISPR/Cas9 system permits ultra-multiplex genome editing in plants

The development and maturation of the CRISPR/Cas genome editing system provides a valuable tool for plant functional genomics and genetic improvement.Currently available genome-editing tools have a limited number of targets,restricting their application in genetic research.In this study,we developed a novel CRISPR/Cas9 plant ultra-multiplex genome editing system consisting of two template vectors,eight donor vectors,four destination vectors,and one primer-design software package.By combining the advantages of Golden Gate cloning to assemble multiple repetitive fragments and Gateway recombination to assemble large fragments and by changing the structure of the amplicons used to assemble sg RNA expression cassettes,the plant ultra-multiplex genome editing system can assemble a single binary vector targeting more than 40 genomic loci.A rice knockout vector containing 49 sg RNA expression cassettes was assembled and a high co-editing efficiency was observed.This plant ultra-multiplex genome editing system advances synthetic biology and plant genetic engineering.

Development and drought tolerance assay of marker-free transgenic rice with OsAPX2 using biolistic particle-mediated co-transformation

Abiotic stresses such as drought, salinity, and low temperature cause–losses in rice production worldwide. The emergence of transgenic technology has enabled improvements in the drought resistance of rice plants and helped avert crop damage due to drought stress.Selectable marker genes conferring resistance to antibiotics or herbicides have been widely used to identify genetically modified plants. However, the use of such markers has limited the public acceptance of genetically modified organisms. Marker-free materials (i.e., those containing a single foreign gene) may be more easily accepted by the public and more likely to find common use. In the present study, we created marker-free drought-tolerant transgenic rice plants using particle bombardment. Overall, 842 T_0plants overexpressing the rice ascorbate peroxidase-coding gene OsAPX2 were generated. Eight independentmarker-free lines were identified from T_1 seedlings using the polymerase chain reaction.The molecular characteristics of these lines were examined, including the expression level,copy number, and flanking sequences of OsAPX2, in the T_2 progeny. A simulated drought test using polyethylene glycol and a drought-tolerance test of seedlings confirmed that the marker-free lines carrying OsAPX2 showed significantly improved drought tolerance in seedlings. In the field, the yield of the wild-type plant decreased by 60% under drought conditions compared with normal conditions. However, the transgenic line showed a yield loss of approximately 26%. The results demonstrated that marker-free transgenic lines significantly improved grain yield under drought-stressed conditions.

Effects of Nitrogen Fertilization on Growth and Development and Nitrogen Utilization of Japonica Super Rice

With the super japonica rice Shennong 265 as the tested material, the effects of different nitrogen application amounts and basic tiller fertilizer(BTF)-ear granule fertilizer(EGF) ratios on the yield and nitrogen utilization characteristics of Shennong265 were investigated. The results showed that when the BTF-EGF ratio was 8:2 or 7:3, the yield of Shennong 265 increased with the increased nitrogen application amount; when the BTF-EGF ratio was 6:4, medium nitrogen level was more conducive to improving the yield of Shennong 265; under the condition of same BTF-EGF ratio, with the increased nitrogen application amount, the total nitrogen uptake increased, and the nitrogen physiological efficiency and harvest index decreased. At low and medium nitrogen levels, the higher the proportion of EGF was, the higher the yield, total nitrogen uptake and nitrogen recovery ratio were; at high nitrogen level, the BTF-EGF ratio of 7:3 was more favorable; at the same nitrogen level, the higher the proportion of EGF was, the lower the nitrogen physiological efficiency and harvest index were. Under conditions of nitrogen application level of 255 kg/hm^2 and BTF-EGF ratio of 7:3, the dry matter accumulation in the leaves, stems and spikes of Shennong265 was higher in the late growth period. Higher effective panicle number and grain number per panicle led to higher yield(9581.5 kg/hm^2, 2.4%-20.1% higher than those in the other treatment groups) and higher nitrogen use efficiency.

The Differences of Physiological Characteristics and Yield of Northern Japonica Rice in Different Nitrogen Application Patterns

The research performed analysis on differences of physiological property and yield of Tiejing No. 11 by different nitrogen fertilization patterns. The results indicated that while the nitrogen of basal: tillering: panicle was 6 ∶3∶1, the higher amount of nitrogen improved the chlorophyll content, photosynthetic capacity, effective panicle, grain weight and yield; the medium nitrogen level had advantage under the ratio 4:3:3; nitrogen application at lategrowth stage was suitable for low or medium nitrogen level,which could increase photosynthetic capacity and optimize the yield components and increase yield; under the same ratio for basic tiller and ear fertilizer, the increase of nitrogen could improve total nitrogen uptake and protein content, decrease nitrogen physiological efficiency and milled rice and tasting, but nitrogen recovery and harvest index changes had difference; as application at late growth stage in same nitrogen rate, total nitrogen uptake and protein content increased, nitrogen physiological efficiency and milled rice and amylose content and tasting decreased, nitrogen recovery and harvest index increased under low or medium nitrogen level and decreased under high nitrogen level. Nitrogen application 210 kg/hm^2 and the ratio 4 ∶3 ∶3 of basal:tillering: panicle made the yield increase by 2.8%-11.3%, and this is the optimal mode of nitrogen fertilizer application.

Effects of Straw Utilization Methods on Dry Matter Production and Yield of Japonica Rice in Northern China

To study the effects of straw utilization methods on dry matter production and yield of japonica rice in northern China,taking the super japonica rice Shennong 265 as the test material,using the planting method of seedling transplanting,setting four treatments,namely,the conventional production,directly returning straws to field(6 t/ha),returning straws to field at low amount of biochar(2 t/ha)and returning straws to field at high amount of biochar(40 t/ha),this paper analyzed the changes in production and yield of super japonica rice Shennong265.According to the experimental results,compared with the conventional production,after the straws were directly returned to the field,the dry matter accumulation of japonica rice was insufficient,and the leaf output rate and contribution rate were significantly reduced by 41.19%and 34.69%,respectively;the number of filled grains per panicle,1000-grain weight,and panicles per plant showed negative effect,leading to a decline in the yield;under the condition of returning straws to field at high amount of biochar,the dry matter accumulation showed a decline trend,both the leaf and stem sheath significantly reduced by 21.41%and 17.43%,and the number of filled grains per panicle also declined;under the condition of returning straws to field at low amount of biochar,the dry matter accumulation increased,and the leaf contribution rate increased by 11.68%,the number of filled grains per panicle,1000-grain weight,and panicles per plant showed positive effect,showing the potential of yield increase.In conclusion,returning suitable straw biochar to field(2 t/ha)is favorable for promoting the japonica rice production in northern China.

The genome of Eleocharis vivipara elucidates the genetics of C_(3)–C_(4)photosynthetic plasticity and karyotype evolution in the Cyperaceae

Eleocharis vivipara,an amphibious sedge in the Cyperaceae family,has several remarkable properties,most notably its alternate use of C_(3)photosynthesis underwater and C_(4)photosynthesis on land.However,the absence of genomic data has hindered its utility for evolutionary and genetic research.Here,we present a high-quality genome for E.vivipara,representing the first chromosome-level genome for the Eleocharis genus,with an approximate size of 965.22 Mb mainly distributed across 10 chromosomes.Its Hi–C pattern,chromosome clustering results,and one-to-one genome synteny across two subgroups indicates a tetraploid structure with chromosome count 2n=4x=20.Phylogenetic analysis suggests that E.vivipara diverged from Cyperus esculentus approximately 32.96million years ago(Mya),and underwent a wholegenome duplication(WGD)about 3.5 Mya.Numerous fusion and fission events were identified between the chromosomes of E.vivipara and its close relatives.We demonstrate that E.vivipara has holocentromeres,a chromosomal feature which can maintain the stability of such chromosomal rearrangements.Experimental transplantation and cross-section studies showed its terrestrial culms developed C_(4)Kranz anatomy with increased number of chloroplasts in the bundle sheath(BS)cells.Gene expression and weighted gene co-expression network analysis(WGCNA)showed overall elevated expression of core genes associated with the C_(4)pathway,and significant enrichment of genes related to modified culm anatomy and photosynthesis efficiency.We found evidence of mixed nicotinamide adenine dinucleotide-malic enzyme and phosphoenolpyruvate carboxykinase type C_(4)photosynthesis in E.vivipara,and hypothesize that the evolution of C_(4)photosynthesis predates the WGD event.The mixed type is dominated by subgenome A and supplemented by subgenome B.Collectively,our findings not only shed light on the evolution of E.vivipara and karyotype within the Cyperaceae family,but also provide valuable insights into the transition between C_(3)and C_(4)photosynthesis,offering promising avenues for crop improvement and breeding.

N6-Methyladenine DNA Methylation in Japonica and Indica Rice Genomes and Its Association with Gene Expression,Plant Development, and Stress Responses

N6-Methyladenine (6mA)DNA methylation has recently been implicated as a potential new epigenetic marker in eukaryotes,including the dioot modelArabidopsis thaliana.However,the conservation and divergence of 6mA distribution patterns and functions in plants remain elusive.Here we report high-quality 6mA methylomes at single-nucleotide resolution in rice based on substantially improved genome sequences of two rice cultivars,Nipponbare (Nip;Japonica)and 93-11 (Indica).Analysis of 6mA genomic distribution and its association with transcription suggest that 6mA distribution and function is rather conserved between rice and Arabidopsis.We found that 6mA levels are positively correlated with the expression of key stressrelated genes,which may be responsible for the difference in stress tolerance between Nip and 93-11. Moreover,we showed that mutations in DDM1 cause defects in plant growth and decreased 6mA level. Our results reveal that 6mA is a conserved DNA modification that is positively associated with gene expression and contributes to key agronomic traits in plants.

5-Methylcytosine RNA Methylation in Arabidopsis Thaliana

5-Methylcytosine （m^5C） is a well-characterized DNA modification, and is also predominantly reported in abundant non-coding RNAs in both prokaryotes and eukaryotes. However, the distribution and biological functions of m^5C in plant mRNAs remain largely unknown. Here, we report transcriptome-wide profiling of RNA m^5C in Arabidopsis thaliana by applying m^5C RNA immunoprecipitation followed by a deep- sequencing approach （m^5C-RIP-seq）. LC-MS/MS and dot blot analyses reveal a dynamic pattern of m^5C mRNA modification in various tissues and at different developmental stages, m^5C-RIP-seq analysis identified 6045 m^5C peaks in 4465 expressed genes in young seedlings. We found that m^5C is enriched in coding sequences with two peaks located immediately after start codons and before stop codons, and is associated with mRNAs with low translation activity. We further demonstrated that an RNA （cytosine-5）-methyl- transferase, tRNA-specific methyltransferase 4B （TRM4B）, exhibits m^5C RNA methyltransferase activity. Mutations in TRM4B display defects in root development and decreased m^5C peaks. TRM4B affects the transcript levels of the genes involved in root development, which is positively correlated with their mRNA stability and m^5C levels. Our results suggest that m^5C in mRNA is a new epitranscriptome marker inArabidopsis, and that regulation of this modification is an integral part of gene regulatory networks underlying plant development.

The RNA Editing Factor WSP1 Is Essentia for Chloroplast Development in Rice

Although the multiple organellar RNA editing factors （MORFs） in the plastids of Arabidopsis thaliana have been extensively studied, molecular details underlying how MORFs affect plant development in other species, particularly in rice, remain largely unknown. Here we describe the characterization of wspl, a rice mutant with white-stripe leaves and panicles. Notably, wspl exhibited nearly white immature panicles at the heading stage. Transmission electron microscopy analysis and chlorophyll content measurement re- veale i a chloroplast developmental defect and reduced chlorophyll accumulation in wspl. Positional cloning of WSP1 found a point mutation in OsO4g51280, whose putative product shares high sequence similarity with MORF proteins. Complementation experiments demonstrated that WSP1 was responsible for the variegated phenotypes of wspl. WSP1 is localized to chloroplasts and the point mutation in wspl affected the editing of multiple organellar RNA sites. Owing to the defect in plastid RNA editing, chloroplast ribosome biogenesis and ndhA splicing were also impaired in wspl, which may affect normal chloroplast development in the leaves and panicles at the heading stage. Together, our results demonstrate the importance of rice WSP1 protein in chloroplast development and broaden our knowledge about MORF family members in rice.

Proteomic Study Identifies Proteins Involved in Brassinosteroid Regulation of Rice Growth

Brassinosteroids （BRs） are essential hormones for growth and development of plant. In rice, BRs regulate multiple developmental processes and affect many important traits such as height, leaf angle, fertility and seed filling. We identified brassinosteroid-regulated proteins in rice using proteomic approaches and performed functional analysis of some BR-regulated proteins by overexpression experiments. Using two-dimensional difference gel electrophoresis （2-D DIGE） followed by protein identification by mass spectrometry, we compared proteomic differences in the shoots and roots of the BR-insensitive mutant d61-4 and BR-deficient mutant brd1-3. We identified a large number of proteins differentially expressed in the mutants compared with wild type control. These include a glycine-rich RNA-binding protein （OsGRP1） and a DREPP2 protein, which showed reduced levels in the BR mutants. Overexpression of these two proteins partially suppressed the dwarf phenotype of the Arabidopsis BR-insensitive mutant bri1-5. In contrast to the reduced protein level, the RNA level of OsGRP1 was not significantly affected in the BR mutants or by BR treatment, suggesting BR regulation of OsGRP1 at the posttranslational level. This study identifies many BR-regulated proteins and demonstrates that OsGRP1 functions downstream in the BR signal transduction pathway to promote cell expansion.