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MiR172-APETALA2-like genes integrate vernalization and plant age to control flowering time in wheat

['Juan M. Debernardi', 'Department Of Plant Sciences', 'University Of California', 'Davis', 'California', 'United States Of America', 'Howard Hughes Medical Institute', 'Chevy Chase', 'Maryland', 'Daniel P. Woods']

Date: 2022-06

Plants possess regulatory mechanisms that allow them to flower under conditions that maximize reproductive success. Selection of natural variants affecting those mechanisms has been critical in agriculture to modulate the flowering response of crops to specific environments and to increase yield. In the temperate cereals, wheat and barley, the photoperiod and vernalization pathways explain most of the natural variation in flowering time. However, other pathways also participate in fine-tuning the flowering response. In this work, we integrate the conserved microRNA miR172 and its targets APETALA2-like (AP2L) genes into the temperate grass flowering network involving VERNALIZATION 1 (VRN1), VRN2 and FLOWERING LOCUS T 1 (FT1 = VRN3) genes. Using mutants, transgenics and different growing conditions, we show that miR172 promotes flowering in wheat, while its target genes AP2L1 (TaTOE1) and AP2L5 (Q) act as flowering repressors. Moreover, we reveal that the miR172-AP2L pathway regulates FT1 expression in the leaves, and that this regulation is independent of VRN2 and VRN1. In addition, we show that the miR172-AP2L module and flowering are both controlled by plant age through miR156 in spring cultivars. However, in winter cultivars, flowering and the regulation of AP2L1 expression are decoupled from miR156 downregulation with age, and induction of VRN1 by vernalization is required to repress AP2L1 in the leaves and promote flowering. Interestingly, the levels of miR172 and both AP2L genes modulate the flowering response to different vernalization treatments in winter cultivars. In summary, our results show that conserved and grass specific gene networks interact to modulate the flowering response, and that natural or induced mutations in AP2L genes are useful tools for fine-tuning wheat flowering time in a changing environment.

Reproductive success is essential for species survival, and in cultivated crops to maximize yield. Plants can sense and integrate different internal and environmental signals to ensure that flowering occurs under optimal conditions. In the temperate cereals, wheat and barley, specific mechanisms have evolved that guarantee flowering is promoted by the longer days of spring only after the plants have been exposed to the cold days of winter, a process called vernalization. In this work, we characterized the interactions between the vernalization requirement and a conserved pathway that integrates plant age into flowering regulation. This pathway involves the sequential action of two microRNAs, miR156 and miR172. In spring wheat cultivars, miR156 expression decreases with plant age, while miR172 expression increases. This results in the downregulation of its targets, the APETALA2-like (AP2L) flowering repressors, and the induction of flowering. In winter wheat cultivars, however, the induction of miR172 and the downregulation of AP2L1 is decoupled from miR156, and induction of the VERNALIZATION1 gene by vernalization is required to repress AP2L1 and promote flowering. Our results show that natural or induced mutations in the AP2L genes are useful tools for fine-tuning wheat flowering time in a changing environment.

Funding: J.D. acknowledges support from the Howard Hughes Medical Institute (HHMI Researcher Funding, https://www.hhmi.org/ ) and by competitive Grants 2022-67013-36209 and 2022-68013-36439 (WheatCAP) from the United States Department of Agriculture, National Institute of Food and Agriculture ( https://nifa.usda.gov/ ). J.M.D. was supported by a fellowship (LT000590/2014-L) of the Human Frontier Science Program ( hfsp.org ). D.P.W is a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation ( http://www.lsrf.org/ ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Debernardi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Our previous studies focused on the role of miR172 and AP2L genes in wheat spike development [ 66 , 84 ]. In this work, we focus on the role of the miR172-AP2L module in the regulation of the flowering transition through its interaction with the temperate grasses specific flowering pathway involving the VRN1-VRN2-FT1 genetic feedback loop. Using a combination of mutants, transgenics, and different growth conditions, we show that miR172 promotes the flowering transition in wheat, while its targets AP2L genes work as repressors of this transition. In addition, we show that miR172 and AP2L genes regulate the expression of VRN1-VRN2-FT1 genes in leaves and modulate the flowering response in spring and winter wheats under different environmental conditions. Finally, we describe mutations in these genes that could be useful tools to fine-tune wheat flowering time in a changing environment.

The expression of miR172 in vegetative tissues is controlled by the environment and the developmental status of the plant [ 58 , 59 , 62 , 64 , 68 , 76 – 78 ]. As plants age, the expression levels of miR172 increase. This is part of a conserved network that involves another conserved miRNA named miR156, which controls the juvenile to adult vegetative phase transition and flowering competence in several species [ 76 , 78 ]. In this so-called plant age pathway, miR156 is expressed at high levels in juvenile stages and represses the expression of a group of SQUAMOSA-PROMOTER-BINDING-like (SPL) genes [ 79 ]. As plants grow and in response to the increase in sugar levels [ 80 , 81 ], miR156 expression goes down resulting in an increased expression of SPLs, which in turn activate the expression of miR172 in the leaves. The increased levels of miR172 result in repression of AP2L genes expression, which promotes adult character traits in leaves and also flowering competence [ 78 , 82 , 83 ].

The repression of AP2L genes by miR172 is also important to control the timing of the flowering transition. Ectopic expression of MIR172 genes phenocopies the rapid flowering of mutants in multiple AP2L genes in Arabidopsis, rice and wheat [ 56 , 58 , 64 – 66 ]. On the other hand, plants with reduced activity of miR172, including Arabidopsis CRISPR mutants in multiple miR172 loci [ 70 , 71 ], plants expressing artificial target mimics against miR172 (henceforth MIM172) or miR172-resistant versions of AP2L genes [ 56 , 64 – 66 , 75 ], all display a delayed flowering phenotype.

The expression of AP2L genes is regulated at the post-transcriptional level by miR172, which is an ancient and conserved miRNA in plants [ 54 ]. Across flowering plant diversification, the expression of miR172 increases during and after the transition to flowering [ 58 , 59 , 62 , 64 , 68 – 71 ]. In reproductive tissues, miR172 controls AP2L expression to regulate inflorescence and flower development [ 72 , 73 ]. In wheat inflorescences, this regulation is important to control spikelet density, floret number, and the free-threshing character of the spike in domesticated wheat [ 66 , 74 ].

In Arabidopsis, AP2L transcription factors repress FT transcription in the leaves as well as other flowering activator genes in the SAM of juvenile plants [ 55 – 57 ]. In addition, AP2L proteins interact with CO-like proteins and inhibit CO activity [ 57 ]. As a result, Arabidopsis plants with mutations in multiple AP2L members are rapid flowering under both LD and SD conditions [ 56 , 58 , 59 ]. Interestingly, in the perennial species Arabis alpina (A. alpina), a close relative of Arabidopsis, the AP2L gene PERPETUAL FLOWERING2 (PEP2) is a flowering repressor that controls the vernalization response and contributes to the perennial life cycle [ 60 , 61 ]. The function of AP2L genes as flowering repressors is also conserved in monocot species. In maize (Zea mays), overexpression of the AP2L genes Glossy15 and ZmTARGET OF EAT1 (ZmTOE1 or Related to APETALA2.7, ZmRAP2.7) delays flowering [ 62 , 63 ]. In rice (Oryza sativa), the AP2L genes INDETERMINATE SPIKELET 1 (OsIDS1), SUPERNUMERARY BRACT (SNB) and OsTOE1 also act as flowering repressors [ 64 , 65 ]. In wheat, loss-of-function mutations in the domestication gene Q (also named AP2L5 = wheat ortholog of IDS1) accelerate flowering [ 66 , 67 ].

In addition to the major photoperiodic and vernalization genes, additional genes and pathways integrate multiple signals that impact flowering, such as the circadian clock, the nutritional and developmental status of the plant and a variety of biotic and abiotic stressors [ 7 , 45 – 51 ]. For example, members of the APETALA2-like (AP2L) family (euAP2 lineage) of transcription factors have been shown to act as flowering repressors [ 52 , 53 ]. These transcription factors, defined by the presence of two AP2-like domains in tandem, are highly conserved in plants. They play important roles in the regulation of the flowering transition by integrating information related to the age of the plant and environmental signals [ 54 ].

The expression of VRN1 in the leaves promotes the repression of VRN2 [ 41 ]. Thus, at the onset of spring as day length is extended, VRN2 expression levels are low due to the induction of VRN1 by the previous winter’s cold temperatures allowing PPD1 to induce the expression of FT1 in the leaves. In addition, a positive feedback loop between VRN1 and FT1 results in elevated levels of VRN1 in the presence of elevated FT1 levels and vice versa [ 42 – 44 ]. Therefore, in winter plants the repression of FT1 by VRN2 in the fall guarantees that promotion of flowering by long days is blocked until plants have been exposed to winter temperatures, and the positive feedback loop in the spring secures the commitment to flowering (reviewed in [ 43 ]).

In wheat, FT1 expression is also regulated by the vernalization pathway. A current molecular model of vernalization in temperate grasses consists of three large-effect loci acting in a regulatory loop including VRN1, VRN2 and FT1 (also known as VRN3 in wheat, [ 9 ]). VRN2, which is a LD flowering repressor, antagonizes the role of PPD1 as a LD flowering promoter, preventing the induction of FT1 in the leaves during the fall [ 29 ]. VRN2 encodes a protein containing a zinc finger motif and a CCT domain and is orthologous to rice GHD7 [ 30 , 31 ] but has no known orthologs in eudicots [ 29 , 31 ]. Wheat and barley plants harboring loss-of-function VRN2 alleles display a spring growth habit [ 29 , 32 – 35 ]. VRN1 encodes a MADS box transcription factor of the SQUAMOSA clade [ 36 , 37 ], and its expression in leaves and apices is induced by vernalization [ 36 ]. Winter wheat varieties with a functional but recessive vrn1 allele require several weeks of vernalization to induce VRN1 and acquire competence to flower [ 36 , 38 , 39 ]. In contrast, spring wheat varieties carrying dominant Vrn1 alleles, which are expressed in the absence of cold temperature, bypass the vernalization requirement [ 38 , 40 , 41 ].

Mutations in the PPD-H1 coding region in barley result in non-functional or hypomorphic alleles with reduced ability to induce flowering under LD [ 19 ]. By contrast, deletions in the promoter regions of PPD-A1 (Ppd-A1a allele) and PPD-D1 (Ppd-D1a allele) homeologs in wheat result in increased PPD1 expression and accelerated heading under short day (SD) relative to the ancestral Ppd1b allele [ 25 , 26 ]. As a result, plants carrying the Ppd1a alleles show reduced photoperiod response and are designated as photoperiod insensitive (PI), whereas plants carrying the Ppd1b allele are designated as photoperiod sensitive (PS) [ 25 , 26 ]. In addition to PPD1, temperate grasses have a parallel photoperiod pathway that involves CONSTANS (CO)-like genes, CO1 and CO2 [ 22 ]. However, while in other species CO-like genes play a dominant role in photoperiod perception [ 6 , 12 ], in wheat and barley CO-like genes have a limited effect that is more relevant in the absence of PPD1 or when PPD1 has reduced function [ 22 , 27 , 28 ].

The major determinant of the photoperiodic regulation of FT1 in wheat and barley is PHOTOPERIOD1 (PPD1 or PRR37) [ 19 ]. PPD1 encodes a protein with a pseudo-receiver domain and a CONSTANS, CONSTANS-like, TIMING OF CAB EXPRESSION 1 (CCT) domain that promotes FT1 expression under LD conditions [ 19 ]. PPD1 is the result of a grass specific duplication event (PRR37/PPD1 and PRR73) that is independent of the PRR3-PRR7 duplication in Arabidopsis thaliana (Arabidopsis) [ 20 ]. The two Arabidopsis genes are part of the circadian clock, but in the temperate grasses PPD1 has a more specialized role in the photoperiod pathway [ 19 , 21 ]. PPD1 expression in the leaves is regulated by the circadian clock and phytochrome-mediated light signaling pathways, ensuring that PPD1 can only promote FT1 expression under LD conditions [ 3 , 19 , 21 – 24 ].

In the temperate grasses, vernalization and photoperiod pathways converge in the transcriptional activation of FLOWERING LOCUS T (FT)-like genes in the leaves [ 1 , 9 ] and allelic variation at the FT1 locus in both barley and wheat is responsible for differences in heading time [ 9 – 11 ]. The central role of FT-like genes in initiating flowering appears to be conserved across flowering plants [ 2 , 12 ], and the timing of flowering depends largely on changes in FT expression in leaves. FT encodes a mobile protein that travels from the leaves to the shoot apical meristem (SAM) [ 13 , 14 ] where it interacts with the bZIP transcription factor FD to activate floral genes and transform the vegetative meristem into a floral meristem [ 15 – 18 ].

Comparisons of flowering regulatory networks in different plant species revealed several conserved genes and gene families, but also clade specific genes [ 6 – 8 ]. Different plant clades have evolved flowering network architectures that include novel components, extensive variation in gene expression patterns and/or interactions among conserved genes. Still, a common feature of these networks is that they converge on a small number of floral integrator genes that initiate the early stages of flowering [ 6 ].

The precise control of flowering is central to plant reproductive success, and in cultivated cereals to maximize grain yield. Plants have evolved mechanisms that integrate various endogenous and environmental signals such as changes in day-length and temperature that enable them to flower under conditions that optimize seed production. Flowering takes place during a particular time of the year in response to perception of seasonal cues, such as changes in the length of the day or the night by a process called photoperiodism [ 1 ]. Temperate grasses, which include the agronomically important crops wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), flower in the spring or early summer in response to shorter nights (longer photoperiods) and are referred to as long-day (LD) plants (e.g. [ 2 , 3 ]). Many plants adapted to temperate climates also have a winter annual or biennial life cycle strategy [ 4 ]. These plants become established in the fall, overwinter, and flower rapidly in the spring. Essential to this adaptive strategy is that flowering does not occur prior to winter, during which flowering would not lead to successful reproduction. Thus, these plants have evolved regulatory mechanisms to prevent fall flowering and sense the passing of winter to establish competence to flower [ 5 ]. The process by which flowering is promoted by a long exposure to cold temperatures is known as vernalization [ 5 ].

Results

miR172 promotes the flowering transition in spring wheat To study the role of miR172 in the control of the flowering transition in wheat, we first analyzed transgenic lines with altered levels of miR172 that were previously generated in the tetraploid wheat variety Kronos (Triticum turgidum subsp. durum) [66] (Fig 1A). Kronos has a spring growth habit determined by the Vrn-A1c allele (deletion in intron 1), and a reduced photoperiod response conferred by the Ppd-A1a allele [41]. We selected and compared independent T 2 transgenic lines overexpressing either miR172 (UBI pro :miR172) or a target mimic against miR172 (MIM172). Quantification of miR172 levels by qRT-PCR in a fully expanded fifth leaf confirmed a 4-fold increase in miR172 expression levels in UBI pro :miR172 plants compared to wild type, and a 20-fold reduction in MIM172 transgenic plants (Fig 1B and Data A in S1 Data). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. miR172 promotes flowering in wheat by repressing AP2L genes. (A) Six-week-old wild type Kronos plants (Wt) compared to UBI pro :miR172 and MIM172 transgenic plants in the same genetic background grown under LD. Scale bar = 10 cm. (B) Box plots showing miR172 expression levels in the 5th leaf of UBI pro :miR172, wild type Kronos (Wt) and MIM172 plants grown under LD. Mature miR172 levels were determined by qRT-PCR using the small nucleolar RNA 101 (SnoR) as internal reference. Data correspond to four independent biological replicates. (C-D) Box plots showing days to heading (C; n ≥ 7) and number of leaves produced by the main tiller (D; n ≥ 7) in the same genotypes and conditions as in B. (E) Schematic diagrams showing the gene structures of AP2L5 and AP2L1. Exons are indicated as dark grey boxes, AP2 domains are indicated in light grey, and the miR172-target sites in red. The positions of the TILLING mutations K3946 (AP2L5) and K863 (AP2L1) in the A homeologs are indicate above the gene structures, and the natural variant with 2 nucleotides deletion (AP2L5) and CAD161 splicing mutant (AP2L1) in the B homeologs below. (F) Box plots showing days to heading under LD conditions for wild type (Wt), plants harboring mutations in only one AP2L1 homeolog (aaBB and AAbb), in both AP2L1 homeologs (ap2l1-null = aabb), ap2l5-null mutants, and apl2l1 ap2l5 combined null mutants (n ≥ 8). (G) Box plots showing days to heading under LD conditions for an F 2 population segregating for the K4254 mutation in the miR172-target site of AP2L-A1 gene (n ≥ 10).; Wt = homozygous wild type plants, mut = homozygous K4254 mutants, het = heterozygous plants. The interaction between the wild type miR172-target site and miR172 is shown in the right. The TILLING line K4254 has a G>A mutation in the miR172 target site that reduces the free energy of interaction with miR172. (H-I) Box plots showing the expression levels of AP2L1 in the 5th leaves (H; n = 4) and days to heading (I; n ≥ 5) for wild type Kronos (Wt) and three independent UBI pro :AP2L-B1 transgenic lines grown under LD. (J-M) Wild type Kronos (Wt), UBI pro :AP2L1 (transgenic line #5), MIM172 and MIM172 UBI pro :AP2L-B1#5 plants grown under LD. (J) Eight-week-old plants. Scale bar = 10 cm. (K-M) Box plots showing days to heading (K; n ≥ 6), number of leaves produced by the main tiller (L; n ≥ 5) and expression levels of AP2L1 in the 5th leaves (M). Different letters above the box plots indicate significant differences based on Tukey tests (P < 0.05), except for panels (D), (F) and (L) where non-parametric Kruskal-Wallis tests were used (Data A in S1 Data). https://doi.org/10.1371/journal.pgen.1010157.g001 Under LD conditions (16 h light / 8 h dark), plants overexpressing miR172 headed 4 days earlier (Fig 1C) and produced 0.6 fewer leaves than the wild type (Fig 1D), whereas MIM172 plants headed 8 days later and produced 1.4 more leaves than the wild type (Fig 1C and 1D). Under SD conditions (8 h light / 16h dark) Kronos plants headed in approximately 80 days (S1A and S1B Fig). UBI pro :miR172 plants flowered earlier than the wild type, and MIM172 plants flowered later than the wild type, similarly to the LD conditions. However, the differences in heading time between UBI pro :miR172 and MIM172 plants were larger under SD (40 days and 5 leaves, S1A–S1C Fig) than under LD (12 days and two leaves, Fig 1C and 1D). These results show that in spring wheat, miR172 accelerates the transition to flowering both under LD and SD conditions.

Mutations in AP2L genes lead to an acceleration of flowering We next explored the role of AP2L transcription factors, which are known targets of miR172 in the regulation of flowering time [54]. In wheat, we previously identified four AP2L genes with miR172 target sequences (named AP2L1, AP2L2, AP2L5, and AP2L7; [66,84]; S1 Table). Loss-of-function AP2L5 mutants (Fig 1E) showed an early flowering phenotype [66,84] (Fig 1F), whereas the heading time of a null mutant for AP2L2 was not different to the wild type control [84]. We identified another AP2L gene (named AP2L1), which is orthologous with TOE1 (TARGET OF EARLY ACTIVATION TAGGED (EAT) 1), a known flowering repressor in Arabidopsis and maize [58,63,84]. Therefore, in this work we further characterized the function of AP2L1. We identified TILLING lines with loss-of-function mutations in both the A (K863) and B (CAD0161) homeologs and crossed them to generate an ap2l1-null mutant in Kronos (Fig 1E, see Material and Methods). Under LD conditions, the ap2l1-null plants headed 5 days earlier than both wild type control and lines harboring mutations in only one AP2L1 homeolog (Fig 1F). The acceleration of flowering was similar to an ap2l5-null mutant grown in the same chamber. We also generated and tested ap2l5 ap2l1 combined null mutant plants in the same growing conditions. The combined null mutant headed significantly earlier than each of the single gene null mutants and 10 days earlier than the wild type (Fig 1F). These results suggest overlapping and additive roles for these two miR172-targets in the control of flowering time in spring wheat. The ap2l1-null mutant did not show any of the spikelet or floret phenotypes previously described for the ap2l5-null mutant [66,84] (S2 Fig).

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