Introduction

The plant hormone auxin plays a crucial role in developmental processes throughout the life cycle of a plant, including cell division and elongation, vascular tissue differentiation, root initiation, apical dominance, gravitropic and phototropic responses, fruit ripening, leaf senescence, and abscission of leaves and fruits ¹. How a simple chemical substance can affect such a wide variety of physiological processes has puzzled scientists in this field for many years. Recently, the identification of a gene family encoding proteins called auxin response factors (ARFs) in Arabidopsis ^{2, 3} provides a good entry to study auxin regulation at the molecular level. The ARF proteins can bind to the auxin-responsive cis elements in the promoter regions of members of an auxin-responsive gene family, GH3 ³. The Arabidopsis genome encodes 23 known ARF proteins. Most ARF proteins contain an N-terminal domain for DNA-binding, a central region, and a C-terminal domain for protein-protein interaction ⁴. ARF proteins can either activate or repress downstream genes, primarily depending on the amino acid sequence of their central region ⁵.

Recently, miRNAs have been shown to play important roles in plant development and are widely believed to act as internal signals ^{6, 7, 8, 9}. However, very little is known about the cross talk between miRNA signals and hormones. In Arabidopsis, a number of miRNAs were identified, showing critical roles in developmental regulations ^{7, 10, 11, 12, 13, 14, 15, 16, 17, 18, 17, 18, 19, 20}. Among the reported miRNA functions in Arabidopis, miR160 was found to regulate root cap formation by targeting ARF10 and ARF16, both of which control root cap cell differentiation ²⁰. The findings revealed a possible regulatory mechanism between miRNA and auxin and provide a system to study the interaction between miRNA signals and hormones, particularly the roles of ARFs, in plant development.

Recently, the functions of two of the ARF genes, ARF6 and ARF8, have been shown by analyzing arf6 and arf8 mutant plants. Whereas the arf8-1 mutation only affects hypocotyl elongation in light conditions ²¹, the null mutants arf6-2 and arf8-3 showed slightly enhanced developmental abnormalities. Although both the floral organ number and position are normal in arf6-2 and arf8-3, the fertility in the single mutant plants is often reduced, and the double mutant is sterile ²². Moreover, the sequence of a predicted miRNA, miR167, is complementary to a region of the ARF6 and ARF8 transcripts, within an encoding C-terminal AUXIN/IAA domain ^{23, 24}. However, it is not clear whether miR167 could play a role in downregulating these two ARF genes. Here we report that miR167 can indeed reduce the level of ARF8, but not ARF6 transcripts, in transgenic plants that carried a 35S::MIR167b fusion. We show that the 35S::MIR167b transgenic plants display altered morphology of all floral organs, resembling arf8 single and arf6 arf8 double mutants. These phenotypes of the transgenic plants suggest that unlike ARF8, for which the mRNA level was reduced, ARF6 may be repressed translational. Our results indicate a possible regulatory role of miR167 in plant development. In addition, we find pollen germination in the 35S::MIR167b transgenic plants is defective, indicating that ARF6 and ARF8 may play important roles in pollen germination.

Materials and methods

Construction of a 35S::MIR167b fusion and plant growth

A 185bp fragment for the MIR167b precursor was amplified using polymerase chain reaction (PCR) with genomic DNA from wild-type Col plants with gene-specific primers (5'-CAC AAC TTG TTG CTC AGG-3' and 5'-CAT GTG GAA TGC TTC ATC-3'). This DNA fragment was cloned into a T-vector (TaKaRa, Japan), verified by sequencing, and inserted into the plant transformation vector pMON530 (Monsanto, USA), downstream to the 35S promoter. This construct was introduced into Col plants by Agrobacterium-mediated transformation. The transgenic lines were verified by PCR, using a 35S-specific primer (5'-GCT CCT ACA AAT GCC ATC A-3') and a primer matching the MIR167b precursor sequence (5'-CAT GTG GAA TGC TTC ATC-3'). Plant growth was according to the conditions described previously ³⁹.

RT-PCR

RNA extraction was according to our previous methods ⁴⁰, with inflorescence from 20-day-old seedlings, and reverse transcription was performed with 1 g total RNA, using a kit (Fermentas, Vilnius, Lithuania). PCR was carried out with the following gene-specific primers: 5'-CAA AGG CAA AGG CAA ATC TCC C-3' and 5'-GAC AAC ACC TTC TAG TTG CAT AG-3' for ARF6, and 5'-CAT CAG GAG ATG CTG AAG CTT CC-3' and 5'-CGA GAG AGA TGC GAA CGA ATG GC-3' for ARF8.

MicroRNA filter hybridization

Total RNA was extracted as described previously ⁴¹, with inflorescences of 3-week-old plants. miRNA filter hybridization was according to our previous methods ⁷, with a miR167 antisense probe, 5'-TAG ATC ATG CTG GCA GCT TCA-3'.

Microscopy

Fresh tissues of wild-type and overexpression plants were examined using an SZH10 dissecting microscope, and photographed using a Nikon E995 digital camera (Nikon, Japan). The method for SEM was described previously ³⁹. For pollen observation, stamens were incubated in 1% Lugol for 3 min and were mounted on the glass slide in 75% chloralhydrate, before viewing ⁴².

Pollen germination

Pollen was isolated form the same-stage anthers of wild-type and 35S::MIR172b transgenic plants and was germinated in medium containing 0.36 mg/ml CaCl₂, 0.08 mg/ml H₃BO₃, 20% sucrose, 0.01 mg/ml myo-inositol, 1% gelatin, and 1% noble agar. Glass slides were dipped into warm unsolidified medium, and then cooled to form a thin layer on the slide. Pollen was tapped onto these slides and germinated in a humid chamber for 8-16 h ⁴³.

Results

Generation of 35S::MIR167b transgenic plants

The Arabidopsis genome contains two copies of miR167, MIR167a, and MIR167b (Figure 1A), which are located at different loci ²⁴. Although sequences of the two precursors (pre-miR167 s) are different, these contain the same mature miR167 sequence. To study miR167 functions, we cloned the pre-miR167b sequence, consisting of 21 nucleotide (nt) of mature miR167 flanked by 40 and 36 nt 5' and 3' sequences, respectively. After verification by sequencing, the pre-miR167b fragment was inserted into a plant transformation vector (pMON530), downstream of the constitutive 35S promoter (Figure 1E). This construct was then introduced into Arabidopsis ecotype Columbia (Col) by Agrobacterium-mediated transformation.

Figure 1.

Characterization of the 35S::MIR167b transgenic plants. (A) Pre-miR167 sequences, miR167a (upper panel) and miR167b (lower panel). Mature miR167 sequences are underlined. (B-D) Plant statures of wild-type Col (B), a group I transgenic plant line-14 (C), and a group II transgenic plant line-17 (D). Bars =1 mm in (B) to (C). (E) The structure of 35S::MIR167b fusion. Arrows indicate positions of the primers used in detecting the presence of the 35S::MIR167b transgene. (F) Identification of 35S::MIR167b transgenic plants by PCR. PCR products were from DNAs of nine independent T1 transgenic plants. (G) MicroRNA blot hybridization using a miR167 antisense probe. Among the RNA samples examined, lines 1, 4, 6, 7, 9, 11, and 14 belong to group I transgenic plants, whereas lines 17, 23, and 40 are group II plants. Note that all examined transgenic lines contained over-accumulated miR167, as compared to that in the wild-type plants.

Full figure and legend (51K)

A total of 117 independent transgenic lines were obtained and a subset of these transgenic plants were examined for the presence of the transgene, with primers corresponding to the sequences of the 35S promoter and pre-miR167 (Figure 1E). T-DNA insertions containing 35S::MIR167b were detected in all transgenic plants analyzed (Figure 1F). Among the transgenic plants, 38 lines displayed severe phenotypes with a smaller plant (Figure 1C), in comparison with wild-type plants (Figure 1B). In addition, 36 other lines had less severe phenotypes (Figure 1D), but were clearly shorter than the wild-type plant. We refer to the 38 severe lines and 36 less severe lines as group I and II transgenic plants, respectively. We also obtained 32 lines with phenotypes close to those of the wild-type plant (data not shown), and 11 lines showing different phenotypes possibly unrelated to miRNA167 activities (data not shown). To determine whether the transgenic lines had over-accumulated miR167, we performed RNA blot hybridization using RNA samples from ten independent lines. Our results revealed that the pre-miR167 sequence driven by the 35S promoter can be properly processed into mature miR167, resulting in a much higher miR167 level than that in the wild type (Figure 1G).

35S::MIR167b transgenic plants have the reduced ARF8 transcript level

To determine whether the increased miR167 level could cause degradation of transcripts from ARF6 and/or ARF8, we analyzed mRNA levels of these two genes using RT-PCR. The miR167 sequence is complementary to the ARF6 and ARF8 mRNAs that encode a portion of the conserved C-terminal domain of the proteins, but with four and three mismatches for the ARF6 and the ARF8 pairing regions, respectively, among the 21 nts (Figure 2A). Our RT-PCR results from six independent transgenic lines showed that the levels of ARF8 transcripts were decreased markedly compared to that in the wild type, whereas the ARF6 levels were almost unchanged (Figure 2B). These results strongly support the hypothesis that the ARF8, but not ARF6, mRNAs were degraded in the 35S::MIR167b transgenic plants.

Figure 2.

RT-PCR analyses of ARF6 and ARF8 expression. (A) The miR167 sequence, matching a region of the ARF6 and ARF8 mRNA encoding in the AUX/IAA domain. The letters in lower case indicate mismatches between miR167 and ARF6/ARF8 sequences. (B) RT-PCR of ARF6 and ARF8 using inflorescence of the T1 35S::MIR167b transgenic lines. Note that ARF6 transcript levels were unchanged, whereas the ARF8 levels were reduced markedly.

Full figure and legend (36K)

Phenotypic analyses of the 35S::MIR167b transgenic plants

To understand better the role of miR167 and functions of ARF6 and ARF8 in plant development, we performed phenotypic characterizations of the group I and II 35S::MIR167b transgenic plants. In addition to the shorter plant statures of 35S::MIR167b plants (Figures 1B-1D), group I plants produced completely sterile flowers, although the group II flowers were barely fertile. Compared to the wild-type inflorescence (Figure 3A, left), those of the group I (Figure 3A, middle) and the group II (Figure 3A, right) lines appeared more compacted, with short internodes between flowers and a short pedicel for each flower. Sepals and petals of the 35S::MIR167b flowers always tightly surrounded the gynoecium and barely detached even after the siliques had fully elongated (Figure 3B). Sepals (Figure 3C) and petals (Figure 3D) were usually smaller in size in the 35S::MIR167b plants than those in the wild type. Filaments of the group I stamens were very short (Figure 3E, middle), although filaments of the group II plants (Figure 3E, left) were similar in length to that of the wild type (Figure 3E, right). In addition, the anther color of the group I plants were buff, different from that of the wild-type anthers, and all group I anthers did not release pollens in their maturations (Figure 3E, middle). Compared to the wild type (Figure 3F, left), the infertile gynoecia of the group I plants were very small (Figure 3F, middle). Although the gynoecia of group II plants had a few seeds set, these were also shorter in length (Figure 3F, right) than those in wild-type plants.

Figure 3.

Phenotypes of flower organs of the 35::MIR167b transgenic lines. All floral organs are arranged in the order of Col (left), the group I plant line-14 (center) and the group II plant line-17 (right), except where otherwise indicated. (A) Inflorescence. (B) Flowers. (C) Sepals. (D) Petals. (E) Stamens. (F) Siliques. All group I plants produced siliques with a very small size, and sepals and petals were consistently attached to the siliques even at a stage when the wild-type seeds have matured. Two line-14 siliques are shown, with sepals and petals removed for the right one. (G) Seedlings of 8-day-old wild-type and a T2 group II plant (line-23), showing an elongated hypocotyl in the transgenic plants. Bars =1 mm in (A), (B), (E), (F), and (G), and 0.5 mm in (C) and (D). (H) Measurement of hypocotyl length using 8-day-old seedlings grown under continuous white light at 22 °C. A total of 55 wild-type and 35 T2 line-23 plants were scored. Bars indicate standard error.

Full figure and legend (41K)

It was reported previously that arf8-1 mutation resulted in seedlings with a long hypocotyl in the light ²¹. To test whether the 35S:MIR167b transgene also affected hypocotyl length, we examined hypocotyl length of the T2 seedlings of the group II lines, and found that they had longer hypocotyls than wild-type seedlings (Figure 3G and 3H). This phenotype resembles that in the arf8-1 single mutant ²¹, and other phenotypes described above are very similar to those of the arf6-2 afr8-3 double mutant plants ²². Therefore, it is possible that both ARF6 and ARF8 are repressed in the 35S::MIR167b transgenic plants.

The cellular pattern of floral organs in the 35S::MIR167b plants

The smaller floral organs in the 35S::MIR167b transgenic plants suggest a defect either in cell division or in cell elongation. To understand phenotypic alterations of the 35S::MIR167b transgenic plants at a cellular level, we analyzed floral organs of group I plants using scanning electron microscopy (SEM). In comparison to the wild type (Figure 4A-4C), petal epidermal cells of the group I plants were less elongated (Figure 4E and 4F); the similar situation was observed on the filament, where wild-type epidermal cells (Figure 4G and 4H) were much longer than those on filaments of the group I transgenic plants (Figure 4J and 5K). Compared to the wild-type anther (Figure 4I), the anther of group I plants were always closed (Figure 4L). Interestingly, the replum of the group I plants were deeply dented (Figure 4P and 4Q) as compared to that in the wild-type plants (Figure 4M and 4N). Similar to the short petal and filament cells, the stigma cells of the group I plants were also very short (Figure 4R), in comparison to those in the wild-type plants (Figure 4O). All these results indicate that the ARF6 and ARF8 functions are required for cell elongation in several floral organs.

Figure 4.

SEM of floral organs. (A-F) Petals of 3-week-old wild-type (A-C) and the T1 group I plants (line-6) (D-F). (A) Wild type. (B) A close-up of the boxed region in (A). (C) Further magnification of the epidermal cells in (B). (D) Line-6. (E) A close-up of the boxed region in (D). (F) Further magnification of the epidermal cells in (E). (G) A wild-type stamen. (H) Filamentous epidermal cells of the wild type. (I) A mature wild-type anther with pollen released. (J) A line-6 stamen. (K) Filamentous epidermal cells of line-6. (L) A line-6 anther being indehiscent at the same stage as that in (I). (M-R) Gynoecium phenotypes of wild type and group-I 35::MIR167b transgenic plants. (M) A wild-type gynoecium. (N) A close-up of the boxed replum region in (M). (O) A close-up of the style in (M). (P) A line-6 gynoecium. (Q) A close-up of the boxed replum region in (P). (R) A close-up of the style in (P). Bars = 1 mm in (A) and (D); 50 m in (B) and (E); 10 m in (C) and (F); 0.5 mm in (G), (J), (M), and (P); 20 m in (H), (K), (N), and (Q); 0.2 mm in (I) and (L); and 100 m in (O) and (Q).

Full figure and legend (52K)

Figure 5.

Starch color assay and pollen germination. (A-C) Starch assay of the pollen in the wild type (A), a group I transgenic plant line-14 (B), and a group II transgenic plant line-23 (C). (D-F) Germination of the wild type (D), line-14 (E), and line-17 (F) pollen grains, which were germinated on a medium for 6 h. (F) Measurement of pollen germination rate of the wild type, line-14 and line-17 at the 6^th and 10^th h, respectively, after pollen grains were germinated on the medium.

Full figure and legend (52K)

Pollen germination is defective in the 35S::MIR167b transgenic plants

As described above, group I 35S::MIR167b transgenic plants produced stamens with a short filament (Figure 4J) and an undehisced anther (Figure 4L). These phenotypes provide explanations for the reduced fertility in the 35S::MIR167b transgenic plants ²². In addition, we observed, by potassium iodine staining, that both group I and II anthers produced normal-shaped pollen grains that contained starch (Figure 5B and 5C), similar to those in the wild-type anther (Figure 5A). To determine whether pollen grains of the 35S::MIR167b anther were functionally defective, we first performed reciprocal crosses between wild-type and 35S::MIR167b plants. Our results showed that the 35S::MIR167b gynoecia that were pollinated with the wild-type pollen produced seeds, although the resulted siliques were shorter than normal. On the contrary, when wild-type flowers were used as the female and pollinated with the pollen released from anthers of the 35S::MIR167b plant, no seeds were produced (Table 1). This result indicates that pollen defects likely contributed to the reduced fertility in 35S::MIR167b plants.

Table 1 - Fertility tests by reciprocal crosses between wild-type and 35S::MIR167b transgenic plants.

Full table

To further verify whether pollen grains in the 35S::MIR167b anthers were fertile, we performed pollen germination experiments. A severe transgenic line (line-14) and a less severe transgenic line (line-17), representing group I and II 35S::MIR167b transgenic plants, respectively, were analyzed in this experiment. As compared to the wild-type pollen (Figure 5D and 5G), the germination rate of pollen from line-17 was dramatically reduced (Figure 5F and 5G). More dramatically, germinated pollen grains were not observed among the pollen from line-14 (Figure 5E and 5G). These results indicate that the pollen germination defect is also an important factor leading to the reduced fertility or sterility in the 35S::MIR167b transgenic plants.

Discussion

The Arabidopsis genome contains 23 ARF genes, and different ARF proteins can form homodimers and heterodimers in response to the auxin signal ⁴. ARF proteins are known to act upstream of many physiological effects of auxin. Although a few ARF genes have been characterized functionally ^{21, 22, 25, 26, 27, 28, 29}, the functions for many other ARFs remain unknown. Recent studies indicate that the several ARF genes are negatively regulated via small RNAs at the post-transcriptional level. For example, ARF2, ARF3, and ARF4 are repressed by trans-acting siRNA ^{30, 31, 32, 33}; and ARF10, ARF16, and ARF17 are regulated by miR160 ²⁰. In this study, we show that overexpression of miR167, which matches in sequence to the ARF6 and ARF8 coding region, resulted in phenotypes similar to those of the arf6 arf8 double mutant. These results strongly suggest a likely regulatory relationship between miR167 and ARF6/ARF8. These specific instances highlight a possible general regulation of ARF expression by miRNAs, indicating an importance for small RNAs in auxin signaling responses.

Both the reduced levels of the ARF8 mRNA and the seedling and floral phenotypes of the 35S::MIR167b transgenic plants strongly support the hypothesis that the increased miR167 level resulted in greater extent of miR167-mediated cleavage of the ARF8 transcript. On the other hand, the ARF6 mRNA level was not reduced in the 35S::MIR167b transgenic plants. Nevertheless, the 35S::MIR167b transgenic plants exhibited strong floral phenotypes found only in the arf6 arf8 double mutant, not in the arf8 single mutant. Therefore, it is likely that the ARF6 function is also negatively regulated by the 35S::MIR167b transgene.

It is known that miRNA can target mRNAs for either cleavage or translational repression ³⁴. The repression of protein synthesis by miRNAs is a common mechanism in animals ^{35, 36}, and has also been found in plants ³⁴. For example, the Arabidopsis APETALA2 (AP2) gene encodes a transcription factor that regulates flower development, and a miRNA, miR172, is known to pair to the AP2 transcript to repress the protein translation ^{10, 11}. Although the level of AP2 transcripts remains unchanged in the 35S::MIR172 transgenic lines, the AP2 protein level was dramatically reduced. Therefore, it is possible that miR167-mediated repression of ARF6 function is at the level of translation. The alignments of miR167 with ARF6 and ARF8 contain four and three nt mismatches, respectively. It is known that the degree of miRNA-target sequence matches greatly influences whether the target mRNA is cleaved. In Arabidopsis, for example, the miR165 and miR166 sequences match a PHAVOLUTA (PHV) region by 18 and 17 nt, respectively. Although both miR165 and miR166 can cause PHV transcript cleavage, miR165 supports a higher cleavage activity than miR166 ¹⁴. Therefore, the observation that ARF6 level was not detectably reduced in the 35S::MIR167b transgenic plants may be explained by the fact that fewer base pairs between ARF6 and miRNA167 may result in inefficient cleavage of the ARF6 transcripts.

Our results also revealed that overexpression of the pre-miR167 sequences resulted in the altered floral phenotypes in the 35S::MIR167b transgenic plants, although the leaf phenotypes kept unchanged (data not shown). All floral organs of the transgenic plants were reduced in size and the flowers were sterile, similar to those in the arf6 arf8 double mutant ²². It was reported that auxin is crucial for flower induction ^{37, 38}. Loss of function in PIN1, which encodes an Arabidopsis auxin transporter, results in plant inflorescence without any floral primordium, whereas addition of auxin to the inflorescence tip could rescue this phenotype ³⁸. Our results suggest that auxin may be involved in the regulation of floral organ development. We further performed detailed phenotypic analyses and found that the transgenic plants and the arf6 arf8 double mutant both produced short filaments and indehiscent anthers, which contribute to the fertility defects. In addition, we observed some novel phenotypes in the present study that were not reported in the previous characterizations of arf6 and arf8 single, and afr6 arf8 double mutants. First, cell elongation in the floral organs of transgenic plants, such as petals and filaments, were aberrant. Secondly, pollen germination rate of the transgenic plants was dramatically reduced. The abnormal pollen germination in the 35S::MIR167b transgenic plants is another important factor that contributes to the male sterility, providing strong evidence for another ARF6 and ARF8 function, and a possible auxin action, in regulating reproductive development.

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Acknowledgements

The authors thank X Gao for assistance with the SEM analysis, L Pi for some experimental protocols, and L Yang for discussion of this work. This research was supported by grants from Shanghai Institutes for Biological Sciences for the Plant Reproductive Development to H Ma and from the Shanghai Scientific Committee to H Huang.