Overexpression of Osta-siR2141 caused abnormal polarity establishment and retarded growth in rice

Abstract

Small RNAs (smRNAs) including miRNAs and siRNAs are critical for gene regulation and plant development. Among the highly diverse siRNAs, trans-acting siRNAs (ta-siRNAs) have been shown to be plant-specific. In Arabidopsis, eight TAS loci belonging to four families (TAS1, TAS2, TAS3, and TAS4) have been identified, and bioinformatics analysis reveals that the sequence of TAS3 is highly conserved in plants. In this study, the function of TAS3 ta-siRNA (tasiR-ARF) has been revealed in rice (Oryza sativa L.) on polarity establishment and stage transition from vegetative to reproductive development by over-expressing Osta-siR2141. Osta-siR2141 replaced miR390 in the miR390 backbone for ectopic expression in rice, and overexpression of Osta-siR2141 caused disturbed vascular bundle development and adaxialization in polarity establishment. Transgenic lines also displayed abnormal shoot apical meristems (SAMs) and retarded growth at the vegetative stage. Molecular analysis revealed that overexpression of Osta-siR2141 resulted in the down-regulation of miR166 and the up-regulation of class III homeodomain-leucine zipper genes (HD-ZIPIIIs) in the vegetative stage but not in the reproductive stage. Moreover, overexpression of Osta-siR2141 in Arabidopsis disturbed polarity establishment and retarded stage transition, suggesting that tasiR-ARF was functionally conserved in rice and Arabidopsis.

Fig. 1.

Analysis of the TAS3 gene and tasiR-ARF in rice. (A) OsTAS3a on chromosome 3 and OsTAS3b on chromosome 5, as marked in red; OsTAS3a-phased tasiR-ARFs were from the 5′D6(+) and 5′D7(+) positions and OsTAS3b-phased from the 5′D7(+) and 5′D8(+) positions. (B) Alignment of tasiR-ARFs in rice and Arabidopsis, shaded nucleotides indicating mismatches. (C) Complementarity of tasiR-ARF and OsARF3s. In OsmiR-ARF(390), Osta-siR2141 from OsTAS3a was used, three red asterisks between ta-siR2141* and ta-siR2141 indicating the introduced mismatches; red asterisks between the OsTAS3a ta-siR2141 and OsARF3-1 site A indicating matching of OstasiR-ARF and OsARF3s.

Fig. 2.

Phenotypes of the transformants. (A) CVS transformants were about 3 cm; there was a ruler on left; (B) Roots of the CVS transformants (left) and transformants from a void vector (right). (C) Thread-like leaves (red arrows) in one CVS transformant. (D) Abnormal phyllotaxy in one CVS transformant. (E) A non-CVS transformant showing twisted leaves and abnormal phyllotaxy. (F) Alternative phyllotaxy in ZH11. (G) Seeds of ZH11. (H) Seeds of the non-CVS transformant. Seeds in (G) and (H) were of 1, 3, 5, and 10 DAPs, respectively (left to right). Bars in (A), (C), and (D) were 0.5 cm, in (B), (E), and (F) were 1 cm, in (G) and (H) were 1 mm.

Fig. 3.

SEM analyses of leaves and sheaths of the CVS transformants. (A) Adaxial surface of a ZH11 leaf, white arrowheads indicating hairs and red arrowheads thorns. (B) Abaxial surface of a ZH11 leaf, red arrows showing water pores. (C) Outer surface of a ZH11 sheath, white arrowheads indicating the vascular bundles. (D) Abaxial surface of the CVS transformant leaf. (E) Outer surface of the CVS transformant sheath, ‘VS’ indicating the vascular bundles. ZH11 was at the five-leaf-stage.

Fig. 4.

Transverse sections of the leaves, sheaths, and roots of the transformants. (A) Half leaf of ZH11; (B) leaf of the CVS transformant, with a white arrowhead indicating the exaggerated epidermial cells, black arrowheads with ‘Bc’ in (A) and (B) indicating the bulliform cells, a black arrowhead indicating bulliform-like cells on the abaxial surface, and red arrowheads in (A) and (B) indicating vascular bundles. (C) ZH11 sheath; (D) sheath of the CVS transformant, with ‘AC’ in (C) and (D) indicating the air cavity. (E) ZH11 root; (F) root of the CVS transformants. (G) ZH11 leaves, left: half leaf blade without midrib, right: midrib with part of leaf blade. (H) Leaf of the non-CVS transformants with red arrows indicating bulliform-like cells on the abaxial surface, a black arrowhead indicating the ectopically formed vascular bundles. (I) Enlarged view of the rectangle in (H). Bars in (A)–(I) were 100 μm. ZH11 in (A), (C), (E) was at the five-leaf-stage, in (G) was at the booting stage.

Fig. 5.

SEM analyses of SAMs of the CVS transformants. (A) SAM of ZH11; (B) SAM of one CVS transformant; M in (A) and (B) indicate shoot apical meristem, P1 in (A) and (B) is the leaf primordia. (C) Enlargement of the P1 region in (A). (D) Enlargement of the P1 region in (B). ZH11 was at the five-leaf-stage.

Fig. 6.

Molecular analyses of the rice and Arabidopsis transformants. (A) Osta-siR2141 Northern blot and RT-PCR analysis of the OsARF3s. (1, 2) leaves of the CVS transformants and ZH11, respectively; (3, 4) roots of the CVS transformants and ZH11, respectively; ZH11 was at the five-leaf-stage and the PCR was processed for 30 cycles for OsARF3s and 25 cycles for actin. (B) OsARF3s were down-regulated in leaves of the CVS and non-CVS transformants. (1, 2) Leaves of two respective CVS transformants, (3, 4) leaves of two respective non-CVS transformants, (5) leaves of the five-leaf-stage ZH11, (6) leaves of booting stage ZH11. Materials for PCR were sampled three times. PCRs were processed for 30 cycles for OsARF3s and 25 cycles for actin. (C) RT-PCR analysis of HD-ZIPIIIs in SAMs of the CVS transformants: (1) five-leaf-stage ZH11, (2) the CVS transformants. (D) RT-PCR analysis of HD-ZIPIIIs in IMs of the non-CVS transformants. (1) IMs of booting stage ZH11, (2) IMs of the non-CVS transformants; IMs were about 0.5 cm and materials for PCR were sampled three times. PCR was processed for 30 cycles for HD-ZIPIIIs and 25 cycles for actin. (E) Northern blot analysis of miR166 in SAMs/IMs of the transformants and anti-sense miR166a was used as probe. (1) SAMs of the CVS transformants, (2) SAMs of the five-leaf-stage ZH11, (3) IMs of the non-CVS transformants, (4) IMs of booting stage ZH11; IMs in (3) and (4) were about 0.5 cm. (F) In situ hybridization of the OsHB3 gene in SAMs of the CVS transformants. (1) ZH11, anti-sense probe; (2) the CVS transformants, anti-sense probe; (3) ZH11, sense probe; (4) the CVS transformants, sense probe. Bars in 1–4 were 100 μm. (G) Molecular analyses of the Arabidopsis transformants. (1) Seedling of Columbia; (2, 3) seedlings of two respective transformants; antisense Osta-siR2141 was used as the probe in the Northern blot; RT-PCR was processed for 36 cycles for ARF3 and ARF4, and for 24 cycles for actin (Ar). (H) Expression of Wx and OsVP1 genes in seeds of the non-CVS transformants. (1, 2, 3, 4) Seeds of ZH11 at about 1–2, 3–4, 5–6, >10 DAP, respectively; (5, 6, 7, 8) seeds of the non-CVS transformants at about 1–2, 3–4, 5–6, >10 DAP, respectively; Materials for PCRs were sampled three times. PCR was processed for 32 cycles for OsVP1, 29 cycles for Wx, 30 cycles for OsARF3s, and 25 cycles for actin.

Fig. 7.

Phenotypes of the Arabidopsis transformants. (A) Leaf of Columbia. (B) Lotus leaf of the transformants. (C) Top and bottom: leaves of Columbia and one transformant arranged in a growth sequence, respectively. (D) Leaf curling and distortion in one transformant. (E) Columbia, showing leaf shape and phyllotaxy. (F) Fused-petiole in one transformant. (G) Opposite phyllotaxy in one transformant. (H) Transformant showing dwarf and cluster. (I) Flower of Columbia. (J) Five-petal flower in one transformant. (K) Silique in Columbia. (L) Infertile silique in one transformant. (M) Disordered silique of one transformant. (N) Gynoecium of Columbia. (O) Gynoecium in one transformant exaggerated. (P) Increased gynoecia in one transformant. (Q, R) SEM analysis of pollen in Columbia and one transformant, showing fertile pollen in (Q) and infertile pollen in (R). Bars in (A), (B), (C), (K), (L), (M), (N), (O), and (P) were 0.5 cm; in (D), (E), (F), (G), and (H) were 1 cm; in (I) and (J) were 1 mm. (This figure is available in colour at JXB online.)