Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Sep;139(1):296-305.
doi: 10.1104/pp.105.063420. Epub 2005 Aug 26.

Loss of function of OsDCL1 affects microRNA accumulation and causes developmental defects in rice

Bin Liu  1 , Pingchuan Li, Xin Li, Chunyan Liu, Shouyun Cao, Chengcai Chu, Xiaofeng Cao
Affiliations

Loss of function of OsDCL1 affects microRNA accumulation and causes developmental defects in rice

Bin Liu et al. Plant Physiol. 2005 Sep.

Abstract

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are two types of noncoding RNAs involved in developmental regulation, genome maintenance, and defense in eukaryotes. The activity of Dicer or Dicer-like (DCL) proteins is required for the maturation of miRNAs and siRNAs. In this study, we cloned and sequenced 66 candidate rice (Oryza sativa) miRNAs out of 1,650 small RNA sequences (19 to approximately 25 nt), and they could be further grouped into 21 families, 12 of which are newly identified and three of which, OsmiR528, OsmiR529, and OsmiR530, have been confirmed by northern blot. To study the function of rice DCL proteins (OsDCLs) in the biogenesis of miRNAs and siRNAs, we searched genome databases and identified four OsDCLs. An RNA interference approach was applied to knock down two OsDCLs, OsDCL1 and OsDCL4, respectively. Strong loss of function of OsDCL1IR transformants that expressed inverted repeats of OsDCL1 resulted in developmental arrest at the seedling stage, and weak loss of function of OsDCL1IR transformants caused pleiotropic developmental defects. Moreover, all miRNAs tested were greatly reduced in OsDCL1IR but not OsDCL4IR transformants, indicating that OsDCL1 plays a critical role in miRNA processing in rice. In contrast, the production of siRNA from transgenic inverted repeats and endogenous CentO regions were not affected in either OsDCL1IR or OsDCL4IR transformants, suggesting that the production of miRNAs and siRNAs is via distinct OsDCLs.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Representatives of newly identified miRNAs from rice. A, Predicted fold-back structure of OsmiR528, OsmiR529a, OsmiR529b, and OsmiR530 precursors from rice. B, Expression patterns of novel rice miRNAs. The samples from different tissues of wild-type rice, including root, leaf, panicle, and callus, are indicated above. The tRNA bands were visualized by ethidium bromide staining of gels and served as internal loading controls.
Figure 2.
Figure 2.
Phylogenetic relationships of DCL proteins in higher plants and animals. Full-length protein sequences were used for phylogenetic analyses. Abbreviations and accession numbers were as follows: OsDCL1 to OsDCL4 are four putative DCL proteins from rice. The sequences were derived from http://www.chromdb.org/ and partially confirmed by RT-PCR. DCL1 to DCL4 are four Arabidopsis DCL proteins. DCL1 (NM_099986), DCL2 (NM_111200), DCL3 (NM_114260), and DCL4 (NM_122039) correspond to predicted protein sequences from Arabidopsis At1g01040, At3003300, At3g43920, and At5g20320 genes, respectively. Dicer-1 (NM_079729), Drosophila melanogaster DCR-1; Dicer-2 (NM_079054), D. melanogaster DCR-2; hDicer (NM_177438), human Dicer-1; mDicer (NM_148948), mouse Dicer1.
Figure 3.
Figure 3.
A, Schematic representation of conserved motifs among four DCL proteins in rice. Regions used in the RNAi knockdown are labeled. B, Diagram of OsDCL RNAi constructs. Fragments containing respective OsDCL genes in sense and antisense orientations separated by an unrelated intron were cloned under the rice Actin1 promoter.
Figure 4.
Figure 4.
Specificity of RNAi in OsDCL1IR and OsDCL4IR transformants. RT-PCR analyses were performed for the OsDCL1, OsDCL2, and OsDCL4 loci in a control plant and two OsDCL1IR (A) and OsDCL4IR (B) transformants, respectively. Equal amount of cDNAs was determined by RT-PCR with 28 and 30 cycles at the Actin locus; the same amount of cDNAs was used to amplify 36 and 38 cycles at OsDCL1, OsDCL2, and OsDCL4 loci, respectively.
Figure 5.
Figure 5.
Morphology of OsDCL1IR transformants showing pleiotropic phenotypes. A, Wild type and strong loss of function of OsDCL1IR transformants. The OsDCL1IR transformant showed severe dwarfism, rolled and curly leaves, and tortuous shoots. B, The strong loss of function of OsDCL1IR transformant showed developmental arrest at the young seedling stage. C, The weak loss of function of OsDCL1IR transformants displayed pleiotropic phenotypes at the seedling stage. D and E, Roots from OsDCL1IR transformants (D) and control (E) plants. The weak OsDCL1IR transformants had fewer adventitious roots. F and G, Cross sections of roots from OsDCL1IR transformants (F) and control plants (G). Ectopically developed chloroplasts were found in OsDCL1IR transformants roots; however, the cell number and overall cellular organization of weak loss of function of OsDCL1IR transformant roots did not change.
Figure 6.
Figure 6.
The OsDCL1 gene is essential for miRNAs accumulation. Small-sized RNAs from different tissues were separated in polyacrylamide gels, and blots were probed with antisense oligenucleotides complementary to mRNA sequences. The samples from leaf tissue of OsDCL1IR transformants and different tissues of wild type, including root, leaf, panicle, and callus are indicated. The tRNA bands were visualized by ethidium bromide staining of gels and served as internal loading controls. Each probe is listed.
Figure 7.
Figure 7.
siRNA and miRNA accumulation in Osdcl1IR and Osdcl4IR transformants. To detect siRNA, each OsDCL inverted repeats region and endogenous CentO regions was labeled by T7 RNA polymerase, and miRNA was detected as previously described. Hybridization was performed sequentially to the same blot. The tRNA bands were visualized by ethidium bromide staining of gels and serve as internal loading controls. Leaf and flower RNA samples were isolated from control plants, OsDCL1IR, and OsDCL4IR transformants as indicated.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adai A, Johnson C, Mlotshwa S, Archer-Evans S, Manocha V, Vance V, Sundaresan V (2005) Computational prediction of miRNAs in Arabidopsis thaliana. Genome Res 15: 78–91 - PMC - PubMed
    1. Ambros V, Bartel B, Bartel DP, Burge CB, Carrington JC, Chen X, Dreyfuss G, Eddy SR, Griffiths-Jones S, Marshall M, et al (2003) A uniform system for microRNA annotation. RNA 9: 277–279 - PMC - PubMed
    1. Bartel B, Bartel DP (2003) MicroRNAs: at the root of plant development? Plant Physiol 132: 709–717 - PMC - PubMed
    1. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116: 281–297 - PubMed
    1. Baulcombe D (2004) RNA silencing in plants. Nature 431: 356–363 - PubMed

Publication types

MeSH terms

LinkOut - more resources