Genome evolution and initial breeding of the Triticeae grass Leymus chinensis dominating the Eurasian Steppe.

Leymus chinensis, a dominant perennial grass in the Eurasian Steppe, is well known for its remarkable adaptability and forage quality. Hardly any breeding has been done on the grass, limiting its potential in ecological restoration and forage productivity. To enable genetic improvement of the untapped, important species, we obtained a 7.85-Gb high-quality genome of L. chinensis with a particularly long contig N50 (318.49 Mb). Its allotetraploid genome is estimated to originate 5.29 million years ago (MYA) from a cross between the Ns-subgenome relating to Psathyrostachys and the unknown Xm-subgenome. Multiple bursts of transposons during 0.433-1.842 MYA after genome allopolyploidization, which involved predominantly the Tekay and Angela of LTR retrotransposons, contributed to its genome expansion and complexity. With the genome resource available, we successfully developed a genetic transformation system as well as the gene-editing pipeline in L. chinensis. We knocked out the monocot-specific miR528 using CRISPR/Cas9, resulting in the improvement of yield-related traits with increases in the tiller number and growth rate. Our research provides valuable genomic resources for Triticeae evolutionary studies and presents a conceptual framework illustrating the utilization of genomic information and genome editing to accelerate the improvement of wild L. chinensis with features such as polyploidization and self-incompatibility.

Acyl carrier protein OsMTACP2 confers rice cold tolerance at the booting stage.

Low temperatures occurring at the booting stage in rice (Oryza sativa L.) often result in yield loss by impeding male reproductive development. However, the underlying mechanisms by which rice responds to cold at this stage remain largely unknown. Here, we identified MITOCHONDRIAL ACYL CARRIER PROTEIN 2 (OsMTACP2), the encoded protein of which mediates lipid metabolism involved in the cold response at the booting stage. Loss of OsMTACP2 function compromised cold tolerance, hindering anther cuticle and pollen wall development, resulting in abnormal anther morphology, lower pollen fertility, and seed setting. OsMTACP2 was highly expressed in tapetal cells and microspores during anther development, with the encoded protein localizing to both mitochondria and the cytoplasm. Comparative transcriptomic analysis revealed differential expression of genes related to lipid metabolism between the wild type and the Osmtacp2-1 mutant in response to cold. Through a lipidomic analysis, we demonstrated that wax esters, which are the primary lipid components of the anther cuticle and pollen walls, function as cold-responsive lipids. Their levels increased dramatically in the wild type but not in Osmtacp2-1 when exposed to cold. Additionally, mutants of two cold-induced genes of wax ester biosynthesis, ECERIFERUM1 and WAX CRYSTAL-SPARSE LEAF2, showed decreased cold tolerance. These results suggest that OsMTACP2-mediated wax ester biosynthesis is essential for cold tolerance in rice at the booting stage.

Blocking miR528 function promotes tillering and regrowth in switchgrass.

MiRNAs have been reported to be the key regulators involving a wide range of biological processes in diverse plant species, but their functions in switchgrass, an important biofuel and forage crop, are largely unknown. Here, we reported the novel function of miR528, which has expanded to four copies in switchgrass, in controlling biomass trait of tillering number and regrowth rate after mowing. Blocking miR528 activity by expressing short tandem target mimic (STTM) increased tiller number and regrowth rate after mowing. The quadruple pvmir528 mutant lines derived from genome editing also showed such improved traits. Degradome and RNA-seq analysis, combined with in situ hybridization assay revealed that up-regulation of two miR528 targets coding for Cu/Zn-SOD enzymes, might be responsible for the improved traits of tillering and regrowth in pvmir528 mutant. Additionally, natural variations in the miR528-SOD interaction exist in C3 and C4 monocot species, implying the distinct regulatory strength of the miR528-SOD module during monocot evolution. Overall, our data illuminated a novel role of miR528 in controlling biomass traits and provided a new target for genetic manipulation-mediated crop improvement.

Control of OsARF3a by OsKANADI1 contributes to lemma development in rice.

In rice (Oryza sativa), the lemma and palea protect the internal organs of the floret，provide nutrients for seed development, and determine grain size. We previously revealed that a trans-acting small interfering RNA targeting AUXIN RESPONSE FACTORS (tasiR-ARF) regulates lemma polarity establishment via post-transcriptional repression of AUXIN RESPONSE FACTORS (ARFs) in rice. TasiR-ARF formation requires RNA-DEPENDENT RNA POLYMERASE 6 (RDR6). However, the underlying molecular mechanism of the tasiR-ARF-ARF regulon in lemma development remains unclear. Here, by genetic screening for suppressors of the thermosensitive mutant osrdr6-1, we identified three suppressors, huifu 1 (hf1), hf9, and hf17. Mapping-by-sequencing revealed that HF1 encodes a MYB transcription factor belonging to the KANADI1 family. The hf1 mutation partially rescued the osrdr6-1 lemma defect but not the defect in tasiR-ARF levels. DNA affinity purification sequencing analysis identified 17 725 OsKANADI1-associated sites, most of which contain the SPBP-box binding motif (RGAATAWW) and are located in the promoter, protein-coding, intron, and intergenic regions. Moreover, we found that OsKANADI1 could directly bind to the intron of OsARF3a in vitro and in vivo and promote OsARF3a expression at the transcriptional level. In addition, hf9 and hf17 are intragenic suppressors containing mutations in OsRDR6 that partially rescue tasiR-ARF levels by restoring OsRDR6 protein levels. Collectively, our results demonstrate that OsKANADI1 and tasiR-ARFs synergistically maintain the proper expression of OsARF3a and thus contribute to rice lemma development.

Efficient CRISPR/Cas9-mediated genome editing in sheepgrass (Leymus chinensis).

The lack of genome editing platforms has hampered efforts to study and improve forage crops that can be grown on lands not suited to other crops. Here, we established efficient Agrobacterium-mediated clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) genome editing in a perennial, stress-tolerant forage grass, sheepgrass (Leymus chinensis). By screening for active single-guide RNAs (sgRNAs), accessions that regenerate well, suitable Agrobacterium strains, and optimal culture media, and co-expressing the morphogenic factor TaWOX5, we achieved 11% transformation and 5.83% editing efficiency in sheepgrass. Knocking out Teosinte Branched1 (TB1) significantly increased tiller number and biomass. This study opens avenues for studying gene function and breeding in sheepgrass.

Pseudouridylation of chloroplast ribosomal RNA contributes to low temperature acclimation in rice.

Ribosomal RNAs (rRNAs) undergo many modifications during transcription and maturation; homeostasis of rRNA modifications is essential for chloroplast biogenesis in plants. The chloroplast acts as a hub to sense environmental signals, such as cold temperature. However, how RNA modifications contribute to low temperature responses remains unknown. Here we reveal that pseudouridine (Ψ) modification of rice chloroplast rRNAs mediated by the pseudouridine synthase (OsPUS1) contributes to cold tolerance at seedling stage. Loss-function of OsPUS1 leads to abnormal chloroplast development and albino seedling phenotype at low temperature. We find that OsPUS1 is accumulated upon cold and binds to chloroplast precursor rRNAs (pre-rRNAs) to catalyse the pseudouridylation on rRNA. These modifications on chloroplast rRNAs could be required for their processing, as the reduction of mature chloroplast rRNAs and accumulation of pre-rRNAs are observed in ospus1-1 at low temperature. Therefore, the ribosome activity and translation in chloroplasts is disturbed in ospus1-1. Furthermore, transcriptome and translatome analysis reveals that OsPUS1 balances growth and stress-responsive state, preventing excess reactive oxygen species accumulation. Taken together, our findings unveil a crucial function of Ψ in chloroplast ribosome biogenesis and cold tolerance in rice, with potential applications in crop improvement.

Genetic Dissection of Germinability under Low Temperature by Building a Resequencing Linkage Map in japonica Rice.

Among all cereals, rice is highly sensitive to cold stress, especially at the germination stage, which adversely impacts its germination ability, seed vigor, crop stand establishment, and, ultimately, grain yield. The dissection of novel quantitative trait loci (QTLs) or genes conferring a low-temperature germination (LTG) ability can significantly accelerate cold-tolerant rice breeding to ensure the wide application of rice cultivation through the direct seeding method. In this study, we identified 11 QTLs for LTG using 144 recombinant inbred lines (RILs) derived from a cross between a cold-tolerant variety, Lijiangxintuanheigu (LTH), and a cold-sensitive variety, Shennong265 (SN265). By resequencing two parents and RIL lines, a high-density bin map, including 2,828 bin markers, was constructed using 123,859 single-nucleotide polymorphisms (SNPs) between two parents. The total genetic distance corresponding to all 12 chromosome linkage maps was 2,840.12 cm. Adjacent markers were marked by an average genetic distance of 1.01 cm, corresponding to a 128.80 kb physical distance. Eight and three QTL alleles had positive effects inherited from LTH and SN265, respectively. Moreover, a pleiotropic QTL was identified for a higher number of erected panicles and a higher grain number on Chr-9 near the previously cloned DEP1 gene. Among the LTG QTLs, qLTG3 and qLTG7b were also located at relatively small genetic intervals that define two known LTG genes, qLTG3-1 and OsSAP16. Sequencing comparisons between the two parents demonstrated that LTH possesses qLTG3-1 and OsSAP16 genes, and SN-265 owns the DEP1 gene. These comparison results strengthen the accuracy and mapping resolution power of the bin map and population. Later, fine mapping was done for qLTG6 at 45.80 kb through four key homozygous recombinant lines derived from a population with 1569 segregating plants. Finally, LOC_Os06g01320 was identified as the most possible candidate gene for qLTG6, which contains a missense mutation and a 32-bp deletion/insertion at the promoter between the two parents. LTH was observed to have lower expression levels in comparison with SN265 and was commonly detected at low temperatures. In conclusion, these results strengthen our understanding of the impacts of cold temperature stress on seed vigor and germination abilities and help improve the mechanisms of rice breeding programs to breed cold-tolerant varieties.

Ribosomal RNA Biogenesis and Its Response to Chilling Stress in Oryza sativa.

Ribosome biogenesis is crucial for plant growth and environmental acclimation. Processing of ribosomal RNAs (rRNAs) is an essential step in ribosome biogenesis and begins with transcription of the rDNA. The resulting precursor-rRNA (pre-rRNA) transcript undergoes systematic processing, where multiple endonucleolytic and exonucleolytic cleavages remove the external and internal transcribed spacers (ETS and ITS). The processing sites and pathways for pre-rRNA processing have been deciphered in Saccharomyces cerevisiae and, to some extent, in Xenopus laevis, mammalian cells, and Arabidopsis (Arabidopsis thaliana). However, the processing sites and pathways remain largely unknown in crops, particularly in monocots such as rice (Oryza sativa), one of the most important food resources in the world. Here, we identified the rRNA precursors produced during rRNA biogenesis and the critical endonucleolytic cleavage sites in the transcribed spacer regions of pre-rRNAs in rice. We further found that two pre-rRNA processing pathways, distinguished by the order of 5' ETS removal and ITS1 cleavage, coexist in vivo. Moreover, exposing rice to chilling stress resulted in the inhibition of rRNA biogenesis mainly at the pre-rRNA processing level, suggesting that these energy-intensive processes may be reduced to increase acclimation and survival at lower temperatures. Overall, our study identified the pre-rRNA processing pathway in rice and showed that ribosome biogenesis is quickly inhibited by low temperatures, which may shed light on the link between ribosome biogenesis and environmental acclimation in crop plants.

Integrated analysis of phenome, genome, and transcriptome of hybrid rice uncovered multiple heterosis-related loci for yield increase.

Hybrid rice is the dominant form of rice planted in China, and its use has extended worldwide since the 1970s. It offers great yield advantages and has contributed greatly to the world's food security. However, the molecular mechanisms underlying heterosis have remained a mystery. In this study we integrated genetics and omics analyses to determine the candidate genes for yield heterosis in a model two-line rice hybrid system, Liang-you-pei 9 (LYP9) and its parents. Phenomics study revealed that the better parent heterosis (BPH) of yield in hybrid is not ascribed to BPH of all the yield components but is specific to the BPH of spikelet number per panicle (SPP) and paternal parent heterosis (PPH) of effective panicle number (EPN). Genetic analyses then identified multiple quantitative trait loci (QTLs) for these two components. Moreover, a number of differentially expressed genes and alleles in the hybrid were mapped by transcriptome profiling to the QTL regions as possible candidate genes. In parallel, a major QTL for yield heterosis, rice heterosis 8 (RH8), was found to be the DTH8/Ghd8/LHD1 gene. Based on the shared allelic heterozygosity of RH8 in many hybrid rice cultivars, a common mechanism for yield heterosis in the present commercial hybrid rice is proposed.

Dicer-like 3 produces transposable element-associated 24-nt siRNAs that control agricultural traits in rice.

Transposable elements (TEs) and repetitive sequences make up over 35% of the rice (Oryza sativa) genome. The host regulates the activity of different TEs by different epigenetic mechanisms, including DNA methylation, histone H3K9 methylation, and histone H3K4 demethylation. TEs can also affect the expression of host genes. For example, miniature inverted repeat TEs (MITEs), dispersed high copy-number DNA TEs, can influence the expression of nearby genes. In plants, 24-nt small interfering RNAs (siRNAs) are mainly derived from repeats and TEs. However, the extent to which TEs, particularly MITEs associated with 24-nt siRNAs, affect gene expression remains elusive. Here, we show that the rice Dicer-like 3 homolog OsDCL3a is primarily responsible for 24-nt siRNA processing. Impairing OsDCL3a expression by RNA interference caused phenotypes affecting important agricultural traits; these phenotypes include dwarfism, larger flag leaf angle, and fewer secondary branches. We used small RNA deep sequencing to identify 535,054 24-nt siRNA clusters. Of these clusters, ∼82% were OsDCL3a-dependent and showed significant enrichment of MITEs. Reduction of OsDCL3a function reduced the 24-nt siRNAs predominantly from MITEs and elevated expression of nearby genes. OsDCL3a directly targets genes involved in gibberellin and brassinosteroid homeostasis; OsDCL3a deficiency may affect these genes, thus causing the phenotypes of dwarfism and enlarged flag leaf angle. Our work identifies OsDCL3a-dependent 24-nt siRNAs derived from MITEs as broadly functioning regulators for fine-tuning gene expression, which may reflect a conserved epigenetic mechanism in higher plants with genomes rich in dispersed repeats or TEs.

An epiallele of rice AK1 affects photosynthetic capacity.

Epigenetic gene variants, termed epialleles, can broaden genetic and phenotypic diversity in eukaryotes. Here, we identify a natural epiallele of OsAK1, which encodes a rice adenylate kinase. The Epi-ak1 plants show albino in young leaf and panicle with abnormal chloroplast structures. We found that no nucleotide sequence variation but hypermethylation at promoter region caused silencing of OsAK1 (Os08g01770) in Epi-ak1 plants. OsAK1 localizes to chloroplast and many genes associated with photosynthesis processes were downregulated in Epi-ak1. Thus, the work identified a novel rice epiallele caused by DNA methylation changes, shedding light on significant roles of DNA methylation on rice development.

Epigenetic Mutation of RAV6 Affects Leaf Angle and Seed Size in Rice.

Heritable epigenetic variants of genes, termed epialleles, can broaden genetic and phenotypic diversity in eukaryotes. Epialleles may also provide a new source of beneficial traits for crop breeding, but very few epialleles related to agricultural traits have been identified in crops. Here, we identified Epi-rav6, a gain-of-function epiallele of rice (Oryza sativa) RELATED TO ABSCISIC ACID INSENSITIVE3 (ABI3)/VIVIPAROUS1 (VP1) 6 (RAV6), which encodes a B3 DNA-binding domain-containing protein. The Epi-rav6 plants show larger lamina inclination and smaller grain size; these agronomically important phenotypes are inherited in a semidominant manner. We did not find nucleotide sequence variation of RAV6. Instead, we found hypomethylation in the promoter region of RAV6, which caused ectopic expression of RAV6 in Epi-rav6 plants. Bisulfite analysis revealed that cytosine methylation of four CG and two CNG loci within a continuous 96-bp region plays essential roles in regulating RAV6 expression; this region contains a conserved miniature inverted repeat transposable element transposon insertion in cultivated rice genomes. Overexpression of RAV6 in the wild type phenocopied the Epi-rav6 phenotype. The brassinosteroid (BR) receptor BR INSENSITIVE1 and BR biosynthetic genes EBISU DWARF, DWARF11, and BR-DEFICIENT DWARF1 were ectopically expressed in Epi-rav6 plants. Also, treatment with a BR biosynthesis inhibitor restored the leaf angle defects of Epi-rav6 plants. This indicates that RAV6 affects rice leaf angle by modulating BR homeostasis and demonstrates an essential regulatory role of epigenetic modification on a key gene controlling important agricultural traits. Thus, our work identifies a unique rice epiallele, which may represent a common phenomenon in complex crop genomes.

NOT2 proteins promote polymerase II-dependent transcription and interact with multiple MicroRNA biogenesis factors in Arabidopsis.

MicroRNAs (miRNAs) play key regulatory roles in numerous developmental and physiological processes in animals and plants. The elaborate mechanism of miRNA biogenesis involves transcription and multiple processing steps. Here, we report the identification of a pair of evolutionarily conserved NOT2_3_5 domain-containing-proteins, NOT2a and NOT2b (previously known as At-Negative on TATA less2 [NOT2] and VIRE2-INTERACTING PROTEIN2, respectively), as components involved in Arabidopsis thaliana miRNA biogenesis. NOT2 was identified by its interaction with the Piwi/Ago/Zwille domain of DICER-LIKE1 (DCL1), an interaction that is conserved between rice (Oryza sativa) and Arabidopsis thaliana. Inactivation of both NOT2 genes in Arabidopsis caused severe defects in male gametophytes, and weak lines show pleiotropic defects reminiscent of miRNA pathway mutants. Impairment of NOT2s decreases the accumulation of primary miRNAs and mature miRNAs and affects DCL1 but not HYPONASTIC LEAVES1 (HYL1) localization in vivo. In addition, NOT2b protein interacts with polymerase II and other miRNA processing factors, including two cap binding proteins, CBP80/ABH1, CBP20, and SERRATE (SE). Finally, we found that the mRNA levels of some protein coding genes were also affected. Therefore, these results suggest that NOT2 proteins act as general factors to promote the transcription of protein coding as well as miRNA genes and facilitate efficient DCL1 recruitment in miRNA biogenesis.

Control of transposon activity by a histone H3K4 demethylase in rice.

Transposable elements (TEs) are ubiquitously present in plant genomes and often account for significant fractions of the nuclear DNA. For example, roughly 40% of the rice genome consists of TEs, many of which are retrotransposons, including 14% LTR- and ∼1% non-LTR retrotransposons. Despite their wide distribution and abundance, very few TEs have been found to be transpositional, indicating that TE activities may be tightly controlled by the host genome to minimize the potentially mutagenic effects associated with active transposition. Consistent with this notion, a growing body of evidence suggests that epigenetic silencing pathways such as DNA methylation, RNA interference, and H3K9me2 function collectively to repress TE activity at the transcriptional and posttranscriptional levels. It is not yet clear, however, whether the removal of histone modifications associated with active transcription is also involved in TE silencing. Here, we show that the rice protein JMJ703 is an active H3K4-specific demethylase required for TEs silencing. Impaired JMJ703 activity led to elevated levels of H3K4me3, the misregulation of numerous endogenous genes, and the transpositional reactivation of two families of non-LTR retrotransposons. Interestingly, loss of JMJ703 did not affect TEs (such as Tos17) previously found to be silenced by other epigenetic pathways. These results indicate that the removal of active histone modifications is involved in TE silencing and that different subsets of TEs may be regulated by distinct epigenetic pathways.

Water conservation pattern of Fangcheng River Basin in Beibu Gulf and its response to precipitation.

Assessing the spatiotemporal patterns of watershed water conservation under the influence of the South Asian monsoon climate and its response to precipitation is essential for revealing the evolving patterns of water conservation under different temporal scales. Following the principles of water balance and using the Soil and Water Assessment Tool (SWAT) model, we investigated the spatiotemporal patterns of water conservation and its response to precipitation in the Fangcheng River Basin of Beibu Gulf. The results showed that water conservation in Fangcheng River Basin calculated by SWAT model were 1637.4 mm·a-1, accounting for 50.7% of the mean annual precipitation. The variation of water conservation in different sub-basins was obviously different. Sub-basins with high forest coverage and steep slopes exhibited higher water conservation, while sub-basins with other land use types (such as cropland and grassland), gentle slopes, and intense human activities showed lower water conservation. At the monthly scale, both water conservation and its variation showed similar response characteristics to precipitation in the basin. The response of water conservation variation to sub-precipitation events could be classified into two types. For the short-term rainfall events (duration≤2 days), water conservation variation showed a linear relationship. For the medium to long-term rainfall events (2 days

Telomere-to-telomere genome of the allotetraploid legume Sesbania cannabina reveals transposon-driven subgenome divergence and mechanisms of alkaline stress tolerance.

Alkaline soils pose an increasing problem for agriculture worldwide, but using stress-tolerant plants as green manure can improve marginal land. Here, we show that the legume Sesbania cannabina is very tolerant to alkaline conditions and, when used as a green manure, substantially improves alkaline soil. To understand genome evolution and the mechanisms of stress tolerance in this allotetraploid legume, we generated the first telomere-to-telomere genome assembly of S. cannabina spanning ∼2,087 Mb. The assembly included all centromeric regions, which contain centromeric satellite repeats, and complete chromosome ends with telomeric characteristics. Further genome analysis distinguished A and B subgenomes, which diverged approximately 7.9 million years ago. Comparative genomic analysis revealed that the chromosome homoeologs underwent large-scale inversion events (>10 Mb) and a significant, transposon-driven size expansion of the chromosome 5A homoeolog. We further identified four specific alkali-induced phosphate transporter genes in S. cannabina; these may function in alkali tolerance by relieving the deficiency in available phosphorus in alkaline soil. Our work highlights the significance of S. cannabina as a green tool to improve marginal lands and sheds light on subgenome evolution and adaptation to alkaline soils.

An inducible CRISPR activation tool for accelerating plant regeneration.

The inducible CRISPR activation (CRISPR-a) system offers unparalleled precision and versatility for regulating endogenous genes, making it highly sought after in plant research. In this study, we developed a chemically inducible CRISPR-a tool for plants called ER-Tag by combining the LexA-VP16-ER inducible system with the SunTag CRISPR-a system. We systematically compared different induction strategies and achieved high efficiency in target gene activation. We demonstrated that guide RNAs can be multiplexed and pooled for large-scale screening of effective morphogenic genes and gene pairs involved in plant regeneration. Further experiments showed that induced activation of these morphogenic genes can accelerate regeneration and improve regeneration efficiency in both eudicot and monocot plants, including alfalfa, woodland strawberry, and sheepgrass. Our study expands the CRISPR toolset in plants and provides a powerful new strategy for studying gene function when constitutive expression is not feasible or ideal.

Dynamic root microbiome sustains soybean productivity under unbalanced fertilization.

Root-associated microbiomes contribute to plant growth and health, and are dynamically affected by plant development and changes in the soil environment. However, how different fertilizer regimes affect quantitative changes in microbial assembly to effect plant growth remains obscure. Here, we explore the temporal dynamics of the root-associated bacteria of soybean using quantitative microbiome profiling (QMP) to examine its response to unbalanced fertilizer treatments (i.e., lacking either N, P or K) and its role in sustaining plant growth after four decades of unbalanced fertilization. We show that the root-associated bacteria exhibit strong succession during plant development, and bacterial loads largely increase at later stages, particularly for Bacteroidetes. Unbalanced fertilization has a significant effect on the assembly of the soybean rhizosphere bacteria, and in the absence of N fertilizer the bacterial community diverges from that of fertilized plants, while lacking P fertilizer impedes the total load and turnover of rhizosphere bacteria. Importantly, a SynCom derived from the low-nitrogen-enriched cluster is capable of stimulating plant growth, corresponding with the stabilized soybean productivity in the absence of N fertilizer. These findings provide new insights in the quantitative dynamics of the root-associated microbiome and highlight a key ecological cluster with prospects for sustainable agricultural management.

Rice RNA-dependent RNA polymerase 6 acts in small RNA biogenesis and spikelet development.

Higher plants have evolved multiple RNA-dependent RNA polymerases (RDRs), which work with Dicer-like (DCL) proteins to produce different classes of small RNAs with specialized molecular functions. Here we report that OsRDR6, the rice (Oryza sativa L.) homolog of Arabidopsis RDR6, acts in the biogenesis of various types and sizes of small RNAs. We isolated a rice osrdr6-1 mutant, which was temperature sensitive and showed spikelet defects. This mutant displays reduced accumulation of tasiR-ARFs, the conserved trans-acting siRNAs (tasiRNAs) derived from the TAS3 locus, and ectopic expression of tasiR-ARF target genes, the Auxin Response Factors (including ARF2 and ARF3/ETTIN). The loss of tasiR-mediated repression of ARFs in osrdr6-1 can explain its morphological defects, as expression of two non-targeted ARF3 gene constructs (ARF3muts) in a wild-type background mimics the osrdr6 and osdcl4-1 mutant phenotypes. Small RNA high-throughput sequencing also reveals that besides tasiRNAs, 21-nucleotide (nt) phased small RNAs are also largely dependent on OsRDR6. Unexpectedly, we found that osrdr6-1 has a strong impact on the accumulation of 24-nt phased small RNAs, but not on unphased ones. Our work uncovers the key roles of OsRDR6 in small RNA biogenesis and directly illustrates the crucial functions of tasiR-ARFs in rice development.

Roles of DCL4 and DCL3b in rice phased small RNA biogenesis.

Higher plants have evolved multiple proteins in the RNase III family to produce and regulate different classes of small RNAs with specialized molecular functions. In rice (Oryza sativa), numerous genomic clusters are targeted by one of two microRNAs (miRNAs), miR2118 and miR2275, to produce secondary small interfering RNAs (siRNAs) of either 21 or 24 nucleotides in a phased manner. The biogenesis requirements or the functions of the phased small RNAs are completely unknown. Here we examine the rice Dicer-Like (DCL) family, including OsDCL1, -3a, -3b and -4. By deep sequencing of small RNAs from different tissues of the wild type and osdcl4-1, we revealed that the processing of 21-nucleotide siRNAs, including trans-acting siRNAs (tasiRNA) and over 1000 phased small RNA loci, was largely dependent on OsDCL4. Surprisingly, the processing of 24-nucleotide phased small RNA requires the DCL3 homolog OsDCL3b rather than OsDCL3a, suggesting functional divergence within DCL3 family. RNA ligase-mediated 5' rapid amplification of cDNA ends and parallel analysis of RNA ends (PARE)/degradome analysis confirmed that most of the 21- and 24-nucleotide phased small RNA clusters were initiated from the target sites of miR2118 and miR2275, respectively. Furthermore, the accumulation of the two triggering miRNAs requires OsDCL1 activity. Finally, we show that phased small RNAs are preferentially produced in the male reproductive organs and are likely to be conserved in monocots. Our results revealed significant roles of OsDCL4, OsDCL3b and OsDCL1 in the 21- and 24-nucleotide phased small RNA biogenesis pathway in rice.