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. 2006 Jul;61(4-5):781-93.
doi: 10.1007/s11103-006-0049-0.

Floral patterning defects induced by Arabidopsis APETALA2 and microRNA172 expression in Nicotiana benthamiana

Sizolwenkosi Mlotshwa  1 , Zhiyong Yang, Yunju Kim, Xuemei Chen
Affiliations

Floral patterning defects induced by Arabidopsis APETALA2 and microRNA172 expression in Nicotiana benthamiana

Sizolwenkosi Mlotshwa et al. Plant Mol Biol. 2006 Jul.

Abstract

Floral patterning and morphogenesis are controlled by many transcription factors including floral homeotic proteins, by which floral organ identity is determined. Recent studies have uncovered widespread regulation of transcription factors by microRNAs (miRNAs), approximately 21-nucleotide non-coding RNAs that regulate protein-coding RNAs through transcript cleavage and/or translational inhibition. The regulation of the floral homeotic gene APETALA2 (AP2) by miR172 is crucial for normal Arabidopsis flower development and is likely to be conserved across plant species. Here we probe the activity of the AP2/miR172 regulatory circuit in a heterologous Solanaceae species, Nicotiana benthamiana. We generated transgenic N. benthamiana lines expressing Arabidopsis wild type AP2 (35S::AP2), miR172-resistant AP2 mutant (35S::AP2m3) and MIR172a-1 (35S::MIR172) under the control of the cauliflower mosaic virus 35S promoter. 35S::AP2m3 plants accumulated high levels of AP2 mRNA and protein and exhibited floral patterning defects that included proliferation of numerous petals, stamens and carpels indicating loss of floral determinacy. On the other hand, nearly all 35S::AP2 plants accumulated barely detectable levels of AP2 mRNA or protein and were essentially non-phenotypic. Overall, the data indicated that expression of the wild type Arabidopsis AP2 transgene was repressed at the mRNA level by an endogenous N. benthamiana miR172 homologue that could be detected using Arabidopsis miR172 probe. Interestingly, 35S::MIR172 plants had sepal-to-petal transformations and/or more sepals and petals, suggesting interference with N. benthamiana normal floral homeotic gene function in perianth organs. Our studies uncover the potential utility of the Arabidopsis AP2/miR172 system as a tool for manipulation of floral architecture and flowering time in non-model plants.

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Figures

Fig. 1
Fig. 1
Analysis of AP2 transgene expression in 35S∷AP2, 35S∷AP2m3, 35S∷MIR172 & Vector transgenic lines. (A) RNA gel blot analysis of AP2 mRNA levels. Total RNA was isolated from floral buds of transgenic lines (indicated above each blot) 15 days post flowering and separated into high and low molecular weight fractions. 10 μg of HMW RNA was used in Northern blotting with P32-labelled full length Arabidopsis AP2 cDNA as the probe. Ethidium bromide stained rRNA is shown as loading control for each blot. (B) Protein gel blot analysis of AP2 protein levels in transgenic lines analyzed in Fig. 1A. Total protein was isolated from part of the ground floral bud tissue used to isolate RNA that was used in Fig. 1A and probed with Arabidopsis AP2 polyclonal antibodies. Wild-type Arabidopsis (Landsberg erecta–Ler) was included as a positive control showing two closely migrating AP2 forms that are absent in the Arabidopsis ap2-2 mutant. The anti-AP2 antibody recognized an unknown protein from N. benthamiana that migrated together with the slower migrating form of Arabidopsis AP2 (upper arrow), since the band was present in vector alone lines. In most 35S∷AP2m3 lines, both bands increased in intensity indicating that both Arabidopsis AP2 protein forms accumulated to higher levels as compared to vector lines. In the second and third panels, the two bands in Ler were slightly offset in mobility from the rest of the bands due to the “smiling” of the gels. Ponceau S-stained RUBISCO is shown as a loading control for each blot
Fig. 2
Fig. 2
RNA gel blot analysis of miR172 levels in 35S∷AP2, 35S∷AP2m3, 35S∷MIR172 & Vector transgenic lines analyzed in Fig. 1. (A) Levels of miR172 15 days post flowering. The predominant slow migrating RNA species, stained with ethidium bromide prior to blotting, is shown as loading control. (B) Levels of miR172 in early compared to very late flowers of 35S∷MIR172 lines. The relative levels of miR172 are indicated for each sample, with levels in vector control samples designated as 1.0
Fig. 3
Fig. 3
Phenotypes of 35S∷AP2 & 35S∷AP2m3 lines. Whole flower side view (A) and top view (A″), and stigma (A″) of vector line. Whole flower side view (B) and top view (B′), and stigma (B″) of 35S∷AP2 flowers that have a mild phenotype associated with moderate accumulation of AP2 mRNA and protein [e.g. 35S∷AP2 lines 1, 4, & 7 (Fig. 1)]. Whole flower side view (C & D) and top view (C′ & D′), and stigma (C″ & D″) of 35S∷AP2m3 flowers that have a strong phenotype associated with high accumulation of AP2 mRNA and protein (observed for nearly all 35S∷AP2m3 lines analyzed in Fig. 1). Sepals were removed to reveal reduction in petal size of phenotypic 35S∷AP2 (G) and 35S∷AP2m3 (F) lines as compared with the wild type phenotype of vector lines (E). Petals were further removed to reveal enlarged carpels of strongly phenotypic 35S∷AP2m3 lines (I) as compared to midly affected carpels of 35S∷AP2 (J) and non-affected carpels of vector lines (H). All flowers were photographed 15 days post flowering. Each unit on the ruler is equivalent to 1 mm for all images (Fig. 1 through Fig. 5)
Fig. 4
Fig. 4
Loss of floral determinacy in severely phenotypic 35S∷AP2m3 lines. Vector: Top view (A) and section (B) of maturing ovary, and top view (C) of very late flower. 35S∷AP2: Top view (D) and section (E) of maturing ovary, and top view (F) of very late flower. D–F are from mildly phenotypic 35S∷AP2 lines described in Fig. 3. 35S∷AP2m3: A subset of phenotypic 35S∷AP2m3 lines with highest levels of AP2 protein (lines 1, 4, 6, 14, & 16 in Fig. 1B) were severely phenotypic, producing flowers with numerous stamens and carpelloid structures (G & H) and numerous petals (I, very late flower). Very late flowers (C, F & I) were collected from plants for photography 2 months post flowering. The rest were collected 15 days post flowering. (J) Semi-quantitative RT-PCR analyses of levels of NbAG (left panel) and NbWUS (right panel) in vector, 35S∷AP2 & 35S∷AP2m3 lines. For both panels, lanes 1–8 correspond to samples from PCR cycles 12, 15, 18, 21, 24, 27, 30 and 40, respectively. Lane C (control): reverse transcriptase-lacking cDNA synthesis reaction used as PCR template. Lane M (marker): 100 bp DNA ladder
Fig. 5
Fig. 5
Phenotypes of 35S∷MIR172 lines. (A) Vector line as compared with 35S∷MIR172 flowers with increased number of sepals and petals typical of a subset of flowers of moderately over-expressing 35S∷MIR172 lines 2, 3, 4 & 9 shown in Fig. 2. (B) Vector line as compared with phenotypic 35S∷MIR172 flower with sepal-to-petal transformations and ripples at the base of the first whorl perianth (inset) typical of highly over-expressing 35S∷MIR172 lines 6, 7 & 11 shown in Fig. 2. (C) Vector line as compared with 35S∷MIR172 flowers that show a range of partial to complete sepal-to-petal transformations associated with highly over-expressing 35S∷MIR172 lines 6, 7 & 11. In addition to sepalto-petal transformations, these lines also have an increased number of sepals and petals for a subset of flowers (not shown)
Fig. 6
Fig. 6
Expression analysis of NbAP2-like1 and B function genes NbGLO and NbDEF. (A) Semi-quantitative RT-PCR analysis of levels of NbDEF (left panel) and NbGLO (right panel) in young sepals of vector and 35S∷MIR172 line # 7. Lanes 1–8, C & M are as described in Fig. 4J. (B) Alignment of the NbAP2-like1 (NbAP2L1) target site with Arabidopsis AP2 (AtAP2) and AP2 cDNAs of Solanaceae species P. hybrida (PhAP2) and L. esculentum (LeAP2). The 21-nucleotide core target site is indicated in bold, with non-conserved nucleotides within the 21-nucleotide core target site boxed. The sequence of Arabidopsis miR172 (AtmiR172a-1) is shown below the alignment, with 3 mismatched nucleotides shown in bold and the mismatch that is unique only to the AtmiR172/NbAP2L1 pair underlined. (C) RNA gel blot analysis of NbAP2-like1 in vector & 35S∷MIR172 lines. Total RNA was isolated from newly formed sepals of vector & 35S∷MIR172 T1 lines (indicated above the blot). 10 μg of HMW RNA was resolved on denaturing gels, blotted onto membranes (Amersham) and probed with 32P-labelled NbAP2L1 antisense RNA probe. a, full length NbAP2L1 mRNA (~2.0 kb); b, putative 5′ cleavage fragment (~1.2 kb); c, putative 3′ cleavage fragment (~0.6 kb). Ethidium bromide stained rRNA is shown as loading control. The relative levels of the ~2 kb full-length NbAP2L1 mRNA are indicated for each sample (levels in vector control samples are designated as 1.0)
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