MicroRNA 157-targeted SPL genes regulate floral organ size and ovule production in cotton
© The Author(s). 2017
Received: 13 September 2016
Accepted: 23 December 2016
Published: 10 January 2017
microRNAs (miRNAs) have been involved in regulation of diverse spectrum of plant development processes in many species. In cotton, few miRNAs have been well characterised in floral organ development. Floral organ, which should be finely tuned, is a crucial factor affecting the yield of cotton. Therefore, it is well worth revealing the function of miRNAs in regulation of floral organ development. Here, we report the role of miRNA156/157 in regulation of floral organ size in cotton.
Over-expression of the GhmiRNA157 precursor in cotton (Gossypium hirsutum) resulted in smaller floral organs, fewer ovules and decreased seed production due to suppression of cell proliferation and cell elongation. Five SQUAMOSA promoter-binding protein-like (SPL) genes were identified as targets of GhmiRNA157 using a RNA ligase-mediated rapid amplification of cDNA end approach, and the expression level of miR157-targeted GhSPLs decreased in the miR157 over-expression lines, indicating the presence of the miR157/SPL axis in cotton. Two MADS-box genes, orthologs of AtAGL6 and SITDR8, which are associated with floral organ development and reproductive production, were repressed in the miR157 over-expression lines. In addition, auxin-inducible genes were also down-regulated, and auxin signal visualized by a DR5::GUS reporter was attenuated in the miR157 over-expression lines.
Our results indicate that the miR157/SPL axis controls floral organ growth and ovule production by regulating MADS-box genes and auxin signal transduction. The work further elucidates the mechanism of floral organ development and provides helpful molecular basis for improvement of cotton yield.
KeywordsFloral organ size Gossypium hirsutum MADS-box transcription factor miR157 SPL gene Ovule production
Seed number is an important factor in crop yield. Because seeds are derived from fertilized ovules, development of the floral organs, especially the gynoecium, which bears ovules, directly affects the final seed number. Although environmental factors can modulate floral organ development, intrinsic mechanisms control the final size of the floral organs .
After the meristematic primordium is established, organ growth can be divided into two phases: cell division followed by cell expansion [2–5]. Many genes, such as the transcription factors GRFs/GIFs and JAGGED, cytochrome P450 (KLU), ubiquitin receptor DA1 and the E3 ubiquitin ligases DA2 and BB, regulate cell proliferation rate or control the timing of proliferation arrest to maintain normal cell numbers and final organ size [6–10]. Other genes, such as the transcription factor bHLH and mediator complex subunit 8, regulate the cell expansion rate to control the cell area, which affects organ size and shape [11, 12].
Moreover, hormones, such as jasmonic acid, gibberellin, brassinosteroids, and cytokinin, have been reported to be involved in the regulatory network of organ size [3–5, 13, 14]. Auxin plays a very important role in floral organ size and development. Auxin signals directly induce expression of the gene AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE (ARGOS), which further upregulates the downstream gene transcription factor AINTEGUMENTA (ANT) . Over-expression of ARGOS or ANT can extend the period of cell division, resulting in larger leaves and floral organs with more seeds per silique in Arabidopsis. Conversely, mutation of ARGOS or ANT decreased the final organ size and seed number [15–17]. In addition, SMALL AUXIN UP RNA (SAUR) was induced by auxin to promote cell elongation and final organ size . Several auxin response factors, which mediate the transcriptional response to auxin, regulate floral organ size and development. ARF8 was reported to suppress petal cell proliferation, cell elongation and final petal size through interaction with the bHLH transcription factor . ARF2 could repress ANT transcription to limit cell proliferation and organ size . In addition to controlling floral organ size, auxin also regulates normal development of floral organs. In mutants defective in auxin biosynthesis and transport, the gynoecium is a thin and round stalk with diminished or no valve tissues of the ovary, indicating that auxin is necessary for the early establishment of carpel primordium . MP (ARF5) which, mediates auxin signalling, could control ovule primordial formation by regulating ANT, CUC1 and CUC2 expression .
MADS-box genes are major players in floral organ differentiation and development. Floral homeotic proteins from MIKC-type MADS-box genes form a combinatorial quaternary complex to control differentiation of the distinct floral organs, which was used to explain the principle of the ABC (D)E model [23, 24]. In addition to floral organ identity, MIKC-type MADS-box genes also control floral organ size and shape by regulating cell division and expansion . Through chromatin immunoprecipitation (ChIP), many growth regulatory genes were shown to be targets of MADS-box transcription factors [26–28]. For example, E class MADS-box gene (SEP3) could directly bind GRF genes and JAGGED gene, which regulate cell division. In addition, MADS-box transcription factors interact with other transcription factors, such as ARF2 and SPL8 .
Three MADS-box transcription factor genes, APETALA1 (AP1), FRUITFULL (FUL), and SUPPRESSOR OF CONSTANS OVEREXPRESSION 1 (SOC1), were directly induced by miR156/157-targeted SPLs [30–32]. By promoting AP1, FUL and SOC1 expression, miR156/157-targeted SPLs could accelerate phase transition. Different miR156/157-targeted OsSPLs have been reported to regulate tiller and panicle architecture and grain size in rice [33–36]. MiR156s with highly similar miR157s are collectively referred to as miR156/157 family. Many studies have reported that miR156/157 family could regulate root development, increase tolerance to heat or salt stress, and promote trichome distribution and shoot regenerative capacity [37–40]. Here, we identified another role of miR156/157 family in the regulation of floral organ growth and ovule production through over-expression of a miR157 precursor in cotton. At least five miR156/157-targeted SPLs and two MADS-box transcription factors, which are orthologs of AtAGL6 and SITDR8, were down-regulated in the over-expression lines. Meanwhile, auxin signalling was attenuated in the miR157 over-expression lines. We hypothesized that the miR157/SPL axis may regulate MADS-box transcription factors and affect auxin signal transduction, finally regulating floral organ growth and ovule production.
Over-expression of GhmiR157 in cotton reduced flower size and seed production and altered plant architecture
MiR156/157 family is one of the most conserved miRNA families in the plant kingdom and has many functions in plant development [41, 42]. Through small RNA sequencing, the GhmiR156/157 family was profiled in cotton [43–46].
Over-expression of GhmiR157 in cotton suppressed floral organ development
Size of floral organs in Null and miR157 over-expressing lines
Petal area (mm2)
965.29 ± 67.83a
328.77 ± 87.07b
258.49 ± 59.48b
Bract area (mm2)
553.71 ± 72.36a
339.60 ± 110.91b
342.29 ± 83.92b
Stigma length (mm)
19.26 ± 0.99a
10.43 ± 1.26b
10.85 ± 1.23b
66.56 ± 6.68a
17.60 ± 3.22b
17.64 ± 2.87b
Sepal length (mm)
27.80 ± 1.57a
20.87 ± 2.42b
19.85 ± 1.68b
Sepal width (mm)
16.87 ± 2.75a
12.69 ± 1.30b
11.77 ± 3.11b
Ectopic expression of a GhmiR157 precursor in Arabidopsis also arrested flower development
Over-expression of GhmiR157 in cotton attenuated female fertility
RNA-sequencing analysis of developing floral buds
In the up-regulated genes of Control, some are transcription factors, such as SPL and MADS-box genes. Interestingly, all the differentially expressed SPLs have been predicted as targets of GhmiR157, but the abundance of non-miR157-targeted SPLs was not different between Control and OV12 (Additional files 6 and 7). There are 59 members of the SPL family, which could be categorized into eight subgroups based on orthologous genes in Arabidopsis, and five of eight subgroups could be predicted as targets of GhmiR157 (Additional files 8 and 7). Five SPLs, which were selected from five subgroups, were further verified as GhmiR157 targets using RLM-RACE (Fig. 5n). Real-time PCR was performed to validate the abundance of these five SPLs. The results showed that compared to Control, three SPLs (Gh_A10G2217, Gh_A11G0344 and Gh_A13G0749) were significantly down-regulated in OV12 and OV38, and the expression level of other SPLs (Gh_A01G2095 and Gh_A01G1281) modestly decreased in OV12 and OV38 (Fig. 5d-h). These data demonstrated that GhmiR157-targeted SPLs were generally down-regulated in over-expression lines.
MADS-box transcription factor genes, such as AtSOC1, AtAP1 and AtFUL, were shown to be directly up-regulated by SPLs in Arabidopsis [30–32]. Our data showed that three MADS-box transcription factors, Gh_A11G0755, Gh_D08G1430 and Gh_A11G0343, which were orthologs of AtSOC1, AtAGL6 and SITDR8, respectively, were significantly down-regulated in OV12 and OV38 compared to Control (Fig. 5i-k). The levels of Gh_D13G0878 and Gh_A07G0605, which are orthologs of AtAP1 and AtFUL, were slightly lower in OV12 and OV38 than Control (Fig. 5l and m). Similarly, AtAP1, AtFUL, AtSOC1, and AtAGL6 were also down-regulated in 35S::GhmiR157 Arabidopsis transgenic lines compared with the wild type (Additional file 9). All the data indicated that MADS-box genes, as candidate downstream genes of GhmiR157-targeted SPLs, were down-regulated in over-expression lines.
Expression pattern of GhmiR157 and miR157-targeted GhSPLs during floral bud development
The expression patterns of eight differentially expressed miR157-targeted GhSPLs (Fig. 5 and Additional file 6) were analysed during floral organ development. And statistically significant differences of expression level among floral organ development stages was analysed, based on analysis of variance. Since GhmiR157 could trigger degradation of its targeted mRNAs, the expression pattern of most miR157-targeted GhSPLs showed a decreasing trend during floral organ development. Gh_A01G0447, Gh_A01G2095, Gh_A11G0344, and Gh_A13G0749 were obviously down-regulated during the floral bud developmental stages (Fig. 6d, e, h and l). The abundance of Gh_A10G2217 and Gh_A11G2811 also slightly decreased (Fig. 6c and f). However, expression patterns of Gh_A04G1331 and Gh_A01G1281 did not show an obvious trend during floral bud development (Fig. 6g and m). According to negative correlation of expression pattern between GhmiR157 and most miR157-targeted GhSPLs, it is reasonable to speculate that GhmiR157 could trigger degradation of its targeted mRNAs to suppress the expression of miR157-targeted GhSPLs during floral organ development.
Auxin signalling was attenuated in the over-expression lines
Recently, a lot of miRNAs have been identified through small RNA sequencing in cotton [43–46]. However, few of their function have been verified in cotton. In this study, we found that over-expression of GhmiR157 in cotton could arrest cell proliferation and cell expansion, which repressed floral organ development and reduced the final organ size (Figs. 1 and 2, Table 1 and Additional file 4). Since floral bud growth was arrested at very early emergence, ovule primordium establishment may be repressed in the over-expression line, which resulted in reduced ovule production. All these results demonstrated that GhmiR157 may play an important role in floral organ development, although the mechanism needs to be further elucidated. Ectopic expression of the GhmiR157 precursor in Arabidopsis also reduced the petal area, gynoecium length and ovule number, which was similar to the phenotype in cotton. Since short gynoecium and few ovules were also found in 35S:AtMIR156b transgenic plants in Arabidopsis , it appears that the regulatory function in floral organ size and growth is conserved between species for miR156/157. Over-expressing the AtMIR156b precursor in tomato did not reduce floral organ size but resulted in severe fruit development defects, indicating that the miR156/157 family plays a major role in reproductive organ development among different species .
SPL transcription factors have been reported as miR156/157 targets in many species , and degradome sequencing also demonstrated that GhSPLs are targets of miR156/157 in cotton [43, 56]. Additionally, in all the differentially expressed genes between the over-expression line and Control, only SPL transcription factors were predicted as miR157 targets, and five GhSPLs were further verified as miR157 targets using RLM-RACE (Fig. 5n and Additional file 7). The expression patterns of GhmiR157 and several targeted GhSPLs were generally negatively correlated during the floral organ developmental stages (Fig. 6). Therefore, it is reasonable to presume that GhSPLs as miR157 targets play important roles in floral organ development.
In Arabidopsis, AtSPLs could directly promote MADS-box transcription factors, such as AtSOC1, AtAP1, and AtFUL, to control phase transition [30–32]. In our study, the AtSOC1 ortholog and two other MADS-box transcription factors, the AtAGL6 ortholog and SITDR8 ortholog, were significantly down-regulated in the over-expression lines compared to Control (Fig. 5i-k and Additional file 6). The AGL6 clade of MADS-box genes is very similar to the closely related E class MADS-box genes and could serve as a scaffold to interact with other A, B, C and D class MADS-box transcription factors to form combinatorial quaternary complexes [57–59]. Dominant loss of function of the AGL6 clade of MADS-box genes by fusing a conserved suppressing motif to the proteins resulted in much smaller floral organs and partial sterility in Arabidopsis [58, 60]. SITDR8 is another clade of MADS-box genes, which was not found in Arabidopsis . Dominant loss of function of SITDR8 leads to alteration of ovary shape and seedless fruits . MADS-box transcription factors play a crucial role in floral organ differentiation and development, and over-expressing miR157 in cotton resulted in smaller floral organs, fewer ovules and seeds, and attenuated female fertility, which partially reproduced the phenotype of mutants of these two genes in Arabidopsis and tomato. Therefore, it is likely that the miR157/SPL axis regulated orthologs of AtAGL6 and SITDR8 in cotton to control normal floral organ growth. However, direct regulation of these two clades of MADS-box genes in cotton by miR157-targeted GhSPLs should be verified in the future.
In addition to MADS-box transcription factors, several auxin-inducible genes were down-regulated in over-expression lines compared with Control (Fig. 7a–e). Moreover, auxin signalling, which could be monitored by the DR5::GUS reporter, was also lower in the over-expression lines than Control (Fig. 7g and h). However, free IAA content was not lower (Fig. 7f). Therefore, decreased auxin signalling in the over-expression lines was not due to IAA content but defects in signal transduction. The mechanism of how the miR157/SPL axis functions in auxin signal transduction is not clear. Interestingly, it was reported that the E class MADS-box transcription factor SEPALLATA3 could bind several ARF recognition motifs, for example, the AtGH3.3 promoter region . Another study reported that MADS-box transcription factors could interact with ARF2 . Given that AGL6 is functionally similar to SEPALLATA3, and the ortholog of AtAGL6 is down-regulated in over-expression lines, it is reasonable to hypothesize that MADS-box transcription factors, which may be regulated by miR157-targeted SPLs, possibly serve as ARF-like or ARF partner-like molecules to transduce auxin signalling to regulate normal organ differentiation and growth.
Floral organs are crucial factors affecting the harvest of many crops and are complex reproductive organs that are regulated by many transcription factors and hormones . Here, we found that the miR157/SPL axis could affect floral growth and size formation through regulating MADS-box genes and auxin signal transduction. The work further illuminates molecular basis of floral organ development, which is helpful for improvement of cotton yield. Future studies of the crosstalk between the miR157/SPL axis and other factors in the regulation of floral organ development should be performed.
Plant materials and RNA isolation
Gossypium hirsutum cv. YZ1 was used as the wild type and transgenic receptor. All transgenic and non-transgenic cotton plants were grown in the experiment field and greenhouse at Huazhong Agricultural University in Wuhan using standard farming management practices in accordance with relevant national approvals for biotechnology research. The floral buds with lengths ≤ 2 mm and flower buds at minus one day post-anthesis (−1 DPA) were harvested, immediately immersed in liquid nitrogen and stored at −80 °C. Total RNA was extracted using a thiocyanate method .
Arabidopsis thaliana ecotype Columbia was used as the wild type and transgenic receptor. Plants were grown in a greenhouse at 20 °C-22 °C under long-day conditions (16 h light/8 h dark). Inflorescences were harvested, immersed in liquid nitrogen and stored at −80 °C. Total RNA was extracted using TRIzol® reagent according to the protocol (Thermo Fisher Scientific).
Plasmid construction and genetic transformation
A 372 bp genomic sequence containing a miR157 precursor from Gossypium hirsutum (Additional file 1) was cloned and ligated into the pGWB402 vector to overexpress the miR157 precursor . A DR5 promoter fragment was ligated into the pGWB433 vector to construct the DR5::GUS vector . The oligonucleotides for generating the plasmids described above are listed in Additional file 10.
Agrobacterium tumefaciens (GV3101) carrying the vector was used to transform hypocotyls of Gossypium hirsutum cv. YZ1. The infected hypocotyls were treated as described previously . Agrobacterium tumefaciens (GV3101)-mediated transformation of Arabidopsis thaliana ecotype Columbia plants was performed by the floral dip method .
Southern blotting, northern blotting and qRT-PCR analysis
Southern blotting was performed as follows: genomic DNA isolation, enzyme digestion, electrophoresis and hybridization. The detailed methods were described previously . The PCR-generated NPTII fragment was used as the probe. The relevant primers are listed in Additional file 10.
Northern blotting of miRNA was performed according to a previous report . First, 20 μg total RNA was electrophoresed in a 15% denaturing polyacrylamide gel containing 8 M of urea and transferred to an Immobilon-Ny + membrane (Merck Millipore). Then, the probes were labelled with γ32P-ATP using T4 polynucleotide kinase (New England BioLabs). After hybridization and membrane wash, the blot was exposed to a phosphor Imager screen, and the signal was detected in Cyclone Plus Phosphor Imager (PerkinElmer).
To quantify mRNA expression, 3 μg total RNA was reverse-transcribed to cDNA using SuperScript II reverse transcriptase (Invitrogen). qRT-PCR was performed using a 7500 real-time system (Applied Biosystems) with Sso-Fast EvaGreen Supermix With Low ROX (Bio-Rad). The relative expression levels (R.E.L.) were calculated using the 2-ΔCT method. HISTONE3 (AF024716) and AtACT7 (AT5G09810.1) were used as endogenous reference genes in cotton and Arabidopsis respectively.
Morphological and cellular analysis
For kinematic analysis of cotton petal development, petals were manually dissected from the first-node floral bud on the second branches back to the first-node floral bud on the ninth branches when the first-node flower on the first branches opened.
For measurements of petal, sepal and bract area, the organs were flattened and scanned to produce a digital image. ImageJ software (https://imagej.nih.gov/ij/download.html) was used to calculate the organ areas. The cotton ovule, seed and anther number and stigma length were calculated manually. For measurement of ovule number in Arabidopsis, the gynoecium was washed in 70% ethanol twice and cleared in chloral hydrate:dH2O:glycerine (8:3:1). Ovules were observed using differential interference contrast microscopy (ZEISS).
Petal cell size was measured on the adaxial side at the top of petal. Average cell size was calculated from the number of cells per unit area of images from microscopy (ZEISS). Petal cell number was calculated according to cell size and petal area.
Bioinformatic analysis of sequencing data
Floral buds (length ≤ 2 mm) were sampled from Control and miRNA157-over-expressing OV12 plants growing in a greenhouse. RNA libraries were generated and sequenced via Illumina HiSeqTM 2000 at the Beijing Genomics Institute (BGI) in Shenzhen. After sequencing, the raw reads were filtered into clean reads and then mapped to the reference genome of Gossypium hirsutum  using Bowtie . Gene expression levels were quantified using the software package RNASeq by Expectation Maximization . Differentially expressed genes between the Control and OV12 lines with two biological repeats were screened using the NOISeq package .
miR157 target prediction was performed on a website tool using the default criterion .
RNA ligase-mediated rapid amplification of cDNA end (RLM-RACE)
RLM-RACE was performed with a GeneRacer kit (Invitrogen) to map the cleavage sites of target transcripts. Total RNA (5 μg) from floral buds (length ≤ 2 mm) was ligated to RNA adapters without calf intestine alkaline phosphatase. The cDNAs were transcribed using the GeneRacer Oligo dT primer. The PCR was performed with 5ˈ adaptor primers and 3ˈ gene-specific primers according to the manufacturer’s instructions. RACE products were cloned, and approximately 10 inserts were sequenced and analysed.
Histochemical analysis and quantification of GUS activity
Floral buds (length ≤ 2 mm) and ovules (−1 DPA) were incubated in the GUS staining buffer at 37 °C for 4 h and then washed in 75% ethanol one or more times. Stained samples were photographed using a stereomicroscope (Leica Microsystems). The staining buffer contained 0.9 g L−1 5-bromo-4-chloro-3-indolylglucuronide, 50 mM sodium phosphate buffer (pH 7.0), 20% (v/v) methanol and 100 mg L−1 chloromycetin.
For quantification of GUS activity, total protein from the samples was extracted using GUS extraction buffer containing 50 mM potassium phosphate buffer at pH 7.0, 10 mM ethylenediaminetetraacetic acid, 0.1% sodium laurylsarcosine, 0.1% Triton X-100 and 10 mM β-mercaptoethanol. The homogenate was centrifuged, and the supernatant was collected to measure GUS activity as described previously .
Quantification of endogenous IAA
Floral buds (100 mg fresh weight) at −1 DPA were homogenized in 1 mL of 80% (vol/vol) methanol containing 10 ng/mL 2H5-IAA (OIChemlm Ltd, CAS: 76937-78-5) as the internal standard and then shaken at 4 °C overnight. The supernatant was evaporated and redissolved in 10% (vol/vol) methanol and subsequently filtered through a 0.22 μm nylon membrane. The quantification of endogenous IAA was performed according to a previous report .
- AP1 :
- FUL :
- SAUR :
SMALL AUXIN UP RNA
- SOC1 :
SUPPRESSOR OF CONSTANS OVEREXPRESSION 1
- SPL :
SQUAMOSA Promoter- Binding Protein- Like
We are grateful to Xu Jian (Department of Biological Science, National University of Singapore) for kindly providing the DR5 promoter and Nakagawa Tsuyoshi (Shimane University) for kindly providing the pGWB402 vector. We also thank Liu Hongbo and Li Dongqin (National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University) for their assistance with the determination of IAA content.
This work was financially supported by the Program of Introducing Talents of Discipline to Universities in China (grant no. B14032) and the Fundamental Research Funds for the Central Universities (no. 2013YB06).
Availability of data and materials
RNA sequence data from floral buds (length ≤ 2 mm) sampled from Control and miRNA157-over-expressing OV12 plants growing in a greenhouse has been submitted to The NCBI Sequence Read Archive (BioProject: PRJNA341749).
NL, LT and XZ designed the research; NL performed experiments and analyzed data; LW, HH and JX supply materials; NL wrote the manuscript and XZ revised the maunscript. All the Authors critically read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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