Over-expression of microRNA171 affects phase transitions and floral meristem determinancy in barley
© Curaba et al.; licensee BioMed Central Ltd. 2013
Received: 12 November 2012
Accepted: 2 January 2013
Published: 7 January 2013
The transitions from juvenile to adult and adult to reproductive phases of growth are important stages in the life cycle of plants. The regulators of these transitions include miRNAs, in particular miR156 and miR172 which are part of a regulatory module conserved across the angiosperms. In Arabidopsis miR171 represses differentiation of axillary meristems by repressing expression of SCARECROW-LIKE(SCL) transcription factors, however the role of miR171 has not been examined in other plants.
To investigate the roles of mir171 and its target genes in a monocot, the Hvu pri-miR171a was over-expressed in barley (Hordeum vulgare L. cv. Golden promise) leading to reduced expression of at least one HvSCL gene. The resulting transgenic plants displayed a pleiotropic phenotype which included branching defects, an increased number of short vegetative phytomers and late flowering. These phenotypes appear to be the consequence of changes in the organisation of the shoot meristem. In addition, the data show that miR171 over-expression alters the vegetative to reproductive phase transition by activating the miR156 pathway and repressing the expression of the TRD (THIRD OUTER GLUME) and HvPLA1 (Plastochron1) genes.
Our data suggest that some of the roles of miR171 and its target genes that have been determined in Arabidopsis are conserved in barley and that they have additional functions in barley including activation of the miR156 pathway.
KeywordsBarley miR171 Scarecrow-like Phase change Meristems Flowering time
During their life cycle, flowering plants undergo three developmental phases, the juvenile and adult vegetative stages and a reproductive phase. During the last decade it has become clear that microRNAs (miRNAs) are important regulators of transitions between these phases. miRNAs are small regulatory RNA molecules which trigger the post-transcriptional repression of target genes through a base-pairing mechanism . There are some 20 miRNA families that are highly conserved in flowering plants. Many of these conserved miRNA families control crucial developmental processes through the down-regulation of conserved transcription factor-encoding genes.
The miRNAs with the best-defined roles in regulating phase changes in the shoot meristem are miR156 and miR172 which form a regulatory module that is widely conserved in plants. In Arabidopsis, rice and maize, miR156 regulates shoot branching, leaf initiation and juvenile-to-adult phase transition through the down-regulation of several SPL genes [2–9]. In grass plants, over-expression of miR156 promotes vegetative branching producing an increased number of tillers but inhibits inflorescence branching resulting in a reduced number of spikelets [2–5, 9]. Detailed analysis of miR156 function in the maize inflorescence has shown that it has a role in establishing axillary meristem boundaries by spatially restricting expression of the SPL gene Tasselsheath4 (TSH4) to the bract. This leads to the allocation of more cells to the outgrowth of spikelet meristems at the expense of leaf initiation [3, 10]. Recent studies have demonstrated cross talk between the miR156 and miR172 pathways, which appear to have conserved roles in coordinating the timing of vegetative phase changes and competency to flower in all angiosperms . During vegetative shoot development in Arabidopsis and maize, miR156 expression gradually decreases and is inversely correlated with that of miR172 [2, 12, 13]. Over-expression of miR156 leads to a decrease in miR172 abundance in young shoots [2, 13]. In Arabidopsis miR156 down-regulates AtSPL9/10 which in turn directly activates the transcription of MIR172b. The AtSPL3/4/5 genes are regulated both transcriptionally and post-transcriptionally by miR172  and miR156, , respectively. In maize, miR172 is thought to repress the maintenance of juvenile traits and promote the onset of reproductive development by down-regulating Glossy15 (GL15). miR172 expression reaches a maximum in reproductive shoots and promotes floral initiation and flower development by repressing expression of AP2-Like (AP2L) genes [12, 13, 17–23]. In maize and barley, loss of miR172 function promotes the formation of ectopic undifferentiated branches in the place of spikelet meristems (SMs) which in barley eventually lead to the formation of a nascent spike [12, 24]. In barley miR172 has also been shown to be responsible for the cleistogamy phenotype by repressing an AP2-like gene, Cleistogamy1 (Cly1), in the lodicules, preventing the outgrowth and opening of the floret .
miR171 is a well conserved miRNA family known to regulate members of the SCARECROW-LIKE (SCL) transcription factor family. The SCLs belong to a subclass of the highly conserved GRAS family composed of homologs of GAI, RGA and SCR . Among other functions, GRAS proteins are known to be involved in GA responses controlling flowering and regulating apical meristem development [26, 27]. In Arabidopsis, there are three MIR171 genes (a, b and c) which are predicted to regulate three SCL6 genes (SCLII, III, IV, also known as HAIRY MERISTEM (HAM) and LOST MERISTEMS (LOM), [28–30]. The expression domains of the miR171 family members and the SCL6-II/III mRNAs overlap, suggesting a redundant function for both miRNA and target mRNAs [30–32]. miR171a is most highly expressed in the inflorescence where it regulates SCL6-III and SCL6-IV expression through mRNA cleavage [29, 33]. Arabidopsis plants over-expressing miR171c (OE171c) and the triple scl6 mutant show similar pleiotropic phenotypes, including altered shoot branching, plant height, chlorophyll accumulation, primary root elongation, flower structure, leaf shape and patterning, indicating that miR171 acts mainly by down-regulating SCL6 genes to control a wide range of developmental processes during shoot development . The reduced branching phenotype observed in OE171c plants can be rescued by the expression of a miR171c-resistant version of SCL6-II, III, or IV, suggesting that the three targets have partially redundant function. SCL6-IV, which is expressed at the boundary between the axillary meristem and the SAM, seems to have a different function to the two other SCL6 genes, being linked to branching but not SAM maintenance [30, 32]. A more detailed analysis of SCL6-II/III showed that these genes are expressed in the peripheral zone and vascular tissues of the SAM and promote the maintenance of the pool of meristematic cells and the differentiation of the axillary meristem [28, 30].
Although studies in Arabidopsis have revealed important roles for miR171 and its SCL targets, their roles in monocot plants are unknown. This study investigated the role of the miR171-SCL module in controlling plant architecture in barley (Hordeum vulgare L. cv Golden promise). Over-expression of miR171 affects expression of meristem identity genes suggesting a conservation of the role identified in Arabidopsis. In addition a delay in the transition to reproductive growth involving miR156 was observed which suggests that there are monocot specific functions for miR171 and its target genes.
Results and discussion
Conserved molecular functions of miR171 in barley
In barley two mature miR171 sequences (hvu-miR171a/b) have been identified  which differ by one central nucleotide. The only miRNA precursor identified is that for hvu-miR171a (Additional file 1). There are nine rice, fourteen maize and four Brachypodium miR171 family members in miRBase, indicating that barley is likely to have additional miR171 genes.
Mis-expression of miR171 in barley leads to pleiotropic phenotypes
These observations suggest that barley miR171 can regulate axillary meristem development and the timing of the juvenile to adult phase transtition. In Arabidopsis, miR171 acts mainly through the down-regulation of the SCL6-II/III/IV genes; over-expression of miR171 or loss of SCL6 function represses axillary meristem differentiation resulting in reduced shoot branching [30, 32], and eventually a complete developmental arrest under SD conditions . This phenotype correlates with the observations here and suggests that miR171 regulates shoot branching through a conserved mechanism between monocots and dicots. However, in contrast to the OE171 phenotype in barley, OE171 in Arabidopsis also promotes stem elongation , represses leaf initiation  and only slightly delays flowering , suggesting that the roles of miR171 and its targets evolved differently for these processes in barley and Arabidopsis.
Mis-regulation of gene expression in OE171 barley plants
We determined the tissue specificity of HvSCL expression by RT-qPCR (Figure 1B). HvSCL is expressed in the shoot, basal node, green spike and ovary. The strong expression of HvSCL in green spike and ovary overlaps with the highest miR171 abundance, at least at the tissue level, suggesting that miR171 acts to dampen HvSCL expression rather than restrict it . These data correlate with previous observations in Arabidopsis where both miR171 and SCL target mRNAs are predominantly detected in the same tissues .
RNA in situ hybridization of Arabidopsis shoot meristem tissues shows that SCL6-II/III are mostly expressed in the peripheral zone and the vascular strands of developing axillary buds where they promote cell differentiation and help maintain the polar organization of the SAM . The maintenance of the SAM requires the expression of WUSCHEL (WUS) and the knotted-like gene SHOOT MERISTEMLESS (STM) to prevent the differentiation of the meristematic cells located in the central zone. In the double mutant scl6-II scl6-III grown under short day conditions, the spatial expression patterns of WUS and STM are altered . A WUS-related homeobox gene, HvWUS (U21_34817), and a class I Knotted-like gene, HvKN1 (TC279468) were identified in barley EST databases. Quantification of their relative expression in the inflorescences of OE171 plants shows a reduction for both genes compared to the WT (Figure 5A). This effect may reflect a disorganization of the SAM and loss of maintenance of meristem cells, which correlates with miR171 function in Arabidopsis and could explain the reduction of shoot branching observed in barley OE171.
The OE171 phenotype resembles that of OE156 in maize [2, 12]. In maize, miR156 controls plant architecture by regulating at least 2 SPL genes, Teosinte Glume Architecture1 (TGA1) and Tasselsheath4 (TSH4)[10, 12]. The dominant Corngrass1 (Cg1) mutant, in which zma-miR156b/c is over-expressed, shows an extended vegetative program characterized by an increased number of leaves and the development of large bract leaves in the inflorescences . Over-expression of zma-miR156b/c also causes the reduction of zma-miR172 abundance which is known to control juvenile-to-adult phase transition through the down-regulation of Glossy15 (GL15) and spikelet meristem determinancy through the repression of IDS1 and SID1[12, 19]. In barley, the phenotype associated with loss of miR172 function is similar to the OE171 plants, blocking spikelet meristem determinacy and exhibiting ectopic branch structures from the base and the end of the spike  (Figure 4A). These phenotypic similarities suggest that miR171 is linked to the miR156-miR172 pathway in barley.
The effect of OE171 on miR156 and miR172 expression was examined using inflorescence tissue from T1 OE171 and WT plants grown under LD conditions (Figure 5B). miR156 abundance was higher in OE171 lines but no change in miR172 expression was detected. Using the same tissues, we quantified the relative abundance of two barley mRNAs, coding for an SPL gene predicted to be targeted by miR156, HvSPL (U21_18637) and a potential target of miR172, HvAP2L (U21_18652) (Figure 5A). As expected, HvSPL was down-regulated in OE171, reinforcing the hypothesis that miR171 acts at least partially through the up-regulation of miR156. Surprisingly, HvAP2L was up-regulated in OE171, suggesting that miR171 regulates HvAP2L expression independently from miR172. This last observation correlates with previous work in Arabidopsis reporting that TOE3 (an AP2-like gene containing a miR172 binding site) expression gradually increases along with miR171 abundance in the developing shoot . Additionally, AP2 was recently shown to directly activate MIR156e expression in Arabidopsis , suggesting that the up-regulation of HvAP2L in OE171 could be responsible for the increasing abundance of miR156.
The increased accumulation of miR156 in OE171 did not cause a reduction of miR172 abundance in contrast with previous observations in Arabidopsis and maize [2, 13]. However, observations by Jung et al.  showed that OE156 driven by an inducible promoter in Arabidopsis did not noticeably affect miR172 abundance. The authors proposed that miR156 principally affects miR172 during the early vegetative stage by repressing MIR172b expression and that the increasing miR172 abundance during the late vegetative stage is driven by other MIR172 genes independent of miR156. This correlates with the observation that miR172 abundance continues to increase during later vegetative stages while the miR156 level stays at a constant minimum level [13, 14]. In agreement with this hypothesis, a decrease in miR172 expression was detected in the young leaves of OE171 T0 plants which over-accumulated miR156 (Figure 2B). Maize OE156 plants did not show the same flowering defect as for loss of miR172 function [2, 12], suggesting that the function of miR172 in the inflorescence is independent of miR156.
The phenotype of the barley OE171 plants was also reminiscent of mutants in maize, rice and barley genes that affect the rate of leaf initiation. We did not observe any clear changes in the leaf initiation rate in OE171 (Additional file 6) but similarities in other phenotypes led us to examine these genes in OE171 plants. In Arabidopsis the leaf initiation rate is likely to be controlled by two independent pathways, one involving miR156-SPLs and the other involving two orthologs of the rice PLASTOCHRON1 (PLA1) gene which encode cytochrome P450 enzymes [39, 40]. In rice, loss of PLA1 function causes an extension of the vegetative program characterized by an increased number of short vegetative phytomers and the production of vegetative shoots (with the emergence of new leaves) instead of primary rachis branches , a phenotype that closely resembles OE171 in barley. More recently it has been shown that PLA1 expression is under control of the transcription factor NECK LEAF 1 (NL1), which when mutated causes a similar phenotype to that of the pla1 mutant . NL1 is orthologous to Third Outer Glume (TRD) in barley and Tasselsheath-1 (TSH-1) in maize. In both trd and tsh-1 mutants the repression of bract growth is suppressed, producing leaves that sheath the inflorescence and a reduction in the number of inflorescence branches . Therefore the regulation of TRD and PLA1 by miR171 in barley was investigated. TRD was previously cloned using degenerate PCR (GU722206,  and one barley EST homolog to PLA1 was found, HvPLA1 (U21_15786). The mRNA abundance for both genes was significantly reduced in the inflorescences of OE171 compared to the WT (Figure 5A). These results suggest that miR171 coordinates vegetative phase changes in barley through the regulation of two potentially distinct pathways. It is proposed that miR171 acts up-stream of miR156 and TRD which in turn may regulate HvPLA1 expression as was observed for their orthologs in rice .
Overall, the data presented here suggest roles for miR171 and its targets in regulating shoot development in barley. Over-expression of miR171 results in an extended vegetative phase characterised by an increased number of leaves and the initiation of indeterminate axillary meristems instead of spikelet meristems. Additionally, OE171 plants have a reduced number of tillers emerging from the axillary meristems of the crown (under SD conditions) and a delay in the differentiation of spikelet meristems into floral organs. A model is proposed in which miR171 is an upstream regulator that coordinates the timing of shoot development in barley through three independent pathways. First, the results, together with current knowledge from Arabidopsis, suggest that miR171 affects meristem maintenance and axillary meristem differentiation in barley through the down-regulation of HvSCL[28, 30], and consequently affects the expression of meristem specific genes such as the homologs of WUS and KN1 analysed in this study. Secondly, miR171 could repress vegetative phase transitions in barley through the upregulation of miR156, a known regulator of the transition from juvenile to adult phases across the angiosperms [2, 6–9, 13, 45]. Interestingly, OE171 and OE156 in Arabidopsis show opposite effects on leaf initiation [30, 32, 40], suggesting that the possible connection between the miR171 and miR156 pathways may be monocotyledon specific. Thirdly, miR171 promotes vegetative traits in barley through a secondary pathway, independent from miR156 , that involves TRD and HvPLA1[42, 43]. These apparent additional roles for miR171 and its targets in barley shoot development may represent an important evolutionary difference between monocot and dicot plants.
The sequence corresponding to pri-miR171a was amplified using the primers MIR171-1F/2R (Additional file 7) and cloned into the pENTR/D topo vector (Invitrogen). The construct was then recombined (according to the manufacturer’s instructions) into the Agrobacterium binary vector pWBVec8  modified to contain a Gateway cassette down-stream of the Ubiquitin promoter. The construct was then introduced into the Agrobacterium strain AGL1 which was used to transform Golden Promise barley plants.
Plant transformation and growing conditions
The transformation was performed as described . In brief, barley plants were grown at 12°C / 16h light and immature grains were harvested at about DPA20 (when the embryo turned from translucent to white and the scutellum is about 2mm in diameter). After sterilization of the grains, the embryos were isolated and the axis was removed. Dissected scutella (approximately 150) were put in contact with a fresh culture of AGL1-MIR171a for 3 days on antibiotic-free media. Transformed calli were selected on hygromycin containing media. The resulting T0 plants were transferred into soil and grown in naturally lit phytotron glasshouses. T1 plants were selected for the presence of the transgene and grown with air temperature set at 17°C/9°C day/night cycle under long day (LD) and short day (SD) conditions with 16 and 10 hours of day light, respectively.
RT-PCR reactions were performed as described . In brief, total RNA was extracted using TRIzol reagent (Invitrogen) and 4μg was used for first-strand cDNA synthesis using Oligo(dT) primers and the Super-Script III RT kit (Invitrogen). PCRs were performed according to the manufacturer on an ABI 7900 HT Fast Real-Time PCR System (Applied Biosystems). 1μl of 1:10 diluted template cDNA was used in a 10μl reaction. The amplification program was: 15″ at 95°C, (15″ at 95°C, 30″ at 60°C, 30″ at 72°C) for 35 cycles, followed by a thermal denaturing step. Relative transcript levels were calculated with the ΔΔCt method (Applied Biosystems) using the ACTIN gene as a reference. Each value presented in the histograms represents an average from three independent replicates plus/minus Standard Deviation. Forward and reverse primer sequences are presented in Additional file 7.
RNA gel blot analysis
40 μg of total RNA, extracted using TRIzol reagent (Invitrogen) and separated on a denaturing 15% polyacryamide gel containing 7M urea at 120V for 2hr. RNA loading was based on spectroscopic measurement and confirmed by ethidium bromide staining of the gel prior to transfer. RNA was electrophoretically transferred to Zeta-probe GT membrane (BioRad) at 40V for 90 min and fixed by UV crosslinking. The membrane was first incubated in hybridization buffer [Na2PO4-pH7.2 125 mM, NaCl 250mM, SDS 7%, formamide 50%] for 4h at 42°C and then incubated in the presence of 32P-end-labeled oligonucleotide probes at 42°C overnight. The membrane was washed in [2× SSC, 0.2% SDS] at 42°C and the radioactivity was detected by phosphorimager. Oligonucleotide probe sequences are presented in Additional file 7.
RNA ligase-mediated 5′ rapid amplification of cDNA ends (RLM 5′-RACE) was performed using the GeneRacer kit (Invitrogen). The manufacturer’s protocol was followed for 5′ end analysis with exception of the 5′ de-capping step. In brief, total RNA was isolated from 2 week-old shoot tissues and ligated to a 5′ end RNA adaptor before being reverse transcribed using an oligo(dT) primer. The PCR reactions were performed using gene specific reverse primers (Additional file 7). The amplicons were gel purified, cloned into the pGEM-T vector (Promega) and sequenced.
We would like to thank Sue Allen, Anna Wielopolska and Kerrie Ramm for their excellent technical assistance and Drs Tony Millar and Scott Boden for their critical reading of the manuscript.
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