IC4R012-miRNA-2009-20017947

Project Title

• Over-expression of miR172 causes loss of spikelet determinacy and floral organ abnormalities in rice (Oryza sativa)

The Background of This Project

• Regulation of gene expression by microRNAs (miRNAs) plays a crucial role in many developmental and physiological processes in plants. miRNAs act to repress expression of their target genes via mRNA cleavage or translational repression. Dozens of miRNA families have been identified in rice, 21 of which are conserved between rice and Arabidopsis. miR172 is a conserved miRNA family which has been shown to regulate expression of APETALA2 (AP2)-like transcription factors in Arabidopsis and maize. The rice genome encodes five AP2-like genes predicted to be targets of miR172. To determine whether these rice AP2-like genes are regulated by miR172 and investigate the function of the target genes, the researchers studied the effect of over-expressing two members of the miR172 family on rice plant development.

Plant Culture & Treatment

• All experiments were performed using rice (Oryza sativa spp. japonica) cultivar Nipponbare. Rice tissue samples were collected from plants grown in a controlled glasshouse at 25 ± 3°C with 16 hours of light, except the twoleaf-stage shoots and roots that were collected from young seedlings grown in Petri dishes at 28°C. For miR172 overexpression transgenic lines, mature leaves (for northern blot) and panicle samples (for qRT-PCR) were collected from T 0 plants. The two-leaf-stage shoot sample included shoot apices and all leaves. The 10-leaf-stage shoot apex sample included the basal ~0.5 cm part of young leaves that are ~1 cm in length. Two-, four- and ten-leaf-stage samples were used to represent juvenile, intermediate and adult vegetative stage, respectively. Panicles with a length of less than 0.5 cm and 0.5-4 cm represent differentiation stage of spikelets and florets, respectively. Booting panicle was representative of developed panicle.

Research Findings

• The mature miR172a-d sequences differ only in their 5' and 3' bases and therefore hybridization with a miR172a probe is likely to detect expression of all mature miR172 sequences. In wild-type plants, miR172 expression varied considerably between organs and developmental stages.Mature miR172 accumulation increased significantly in leaves but not in roots as plants grew, reaching a maximum in the flag leaf (Figure 1A).Similar expression patterns of miR172 have also been observed in vegetative tissues of Arabidopsis and maize [13,17], suggesting that miR172 has a conserved role during vegetative development. In reproductive tissues, miR172 was consistently expressed although its abundance reduced gradually during panicle development (Figure 1B). Expression of miR172 was below the detection limit in 10 DAF (daysafter-fertilization) grains (Figure 1B).

'Figure 1 lation of miR172 in wild-type RNA gel blot analysis of accumulation of miR172 in wild-type plants. A, Accumulation of miR172 in vegetative tissues. 2L-S and 2L-R: shoot and root of two-leaf stage seedlings. 4L: the 4th leaf. 10L: the 10th leaf. 10L-SA: shoot apex of 10-leaf stage seedlings. 10L-R: 10-leaf stage root. FL: flag leaf.B, Accumulation of miR172 in reproductive tissues and grains. ≤ 0.5P, 0.5-1P, 1-2P and 2-4P: developing panicles with a length of ≤ 0.5 cm, 0.5-1 cm, 1-2 cm and 2-4 cm, respectively. BP: booting panicle. Em, En and Pe: embryo,endosperm and pericarp of 10 DAF grains, respectively.'

• Expression of SNB(Os07g13170) was highest in developing panicles (<4 cm in length), in which differentiation of the spikelet and floral organs is progressing; expression of SNB was also high in roots from 10-leaf plants (Figure 2A). Os03g60430 was highly expressed in developing panicles and also in young seedlings (2L-S) (Figure 2B). In contrast expression of Os05g03040 was highest in young seedlings and roots(Figure 2C). All these three genes had a very low expression level in embryo, endosperm and pericarp of 10 DAF grains (Figure 2A, B, C). Expression of SNB and Os03g60430 showed an inverse correlation with the abundance of miR172 in two-leaf shoots, leaf four and leaf ten,but generally the expression of miR172 was not inversely correlated with the expression of its targets in the tissues analyzed (Figure 2A, B, C).

'qRT-PCR analyses of miR172 target genes in wild-type plants Figure 2 qRT-PCR analyses of miR172 target genes in wildtype plants. A primer pair spanning the miR172 target site was used to quantify expression of the uncleaved target mRNAs. For each gene, relative fold expression difference is shown by using the expression level detected in flag leaf as the reference. Error bars represent standard deviation of the expression ratio. 2L-S and 2L-R: shoot and root of two-leaf stage seedlings. 4L: the 4th leaf. 10L: the 10th leaf. 10L-SA:shoot apex of 10-leaf stage seedlings. 10L-R: 10-leaf stage root. FL: flag leaf. ≤ 0.5P, 0.5-1P, 1-2P and 2-4P: developing panicles with a length of ≤ 0.5 cm, 0.5-1 cm, 1-2 cm and 2-4cm, respectively. BP: booting panicle. Em: embryo. En: endosperm.'

• Cleavage of Os04g55560 was detected in a mixed sample of shoot and grain as well as in booting panicles; cleavage of Os06g43220 was only detected in the mixed sample with a low frequency (most likely contributed by young seedlings as accumulation of miR172 was below the detection limit in 10 DAF grains); and cleavage of SNB was only detected in booting panicles. No cleavage was detected for Os03g60430 or Os05g03040 in any of the samples analyzed (Figure 3).

'Analysis of miR172-mediated Figure 3 cleavage of target genes Analysis of miR172-mediated cleavage of target genes. 5' RACE was used to map the miR172-mediated cleavage sites in the predicted targets. The expected cleavage site is indicated by an arrow. Nucleotides that differ among miR172 family members or their targets are shown in bold italic. The cleavage frequencies (number of clones with the expected cleavage site/total number of clones sequenced)detected in the indicated tissues are shown to the right of the sequence alignment. BP: booting panicle. nd: no RACE product detected.'

Labs working on this Project

• CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia

Corresponding Author

• Qian-Hao Zhu:qianhao.zhu@csiro.au & Narayana M Upadhyaya:narayana.upadhyaya@csiro.au & Frank Gubler: frank.gubler@csiro.au & Chris A Helliwell:chris.helliwell@csiro.au