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Genetic basis of nectar guide trichome variation between bumblebee- and self-pollinated monkeyflowers (Mimulus): role of the MIXTA-like gene GUIDELESS

BMC Plant Biology volume 24, Article number: 62 (2024) Cite this article

Abstract

Nectar guide trichomes play crucial ecological roles in bee-pollinated flowers, as they serve as footholds and guides for foraging bees to access the floral rewards. However, the genetic basis of natural variation in nectar guide trichomes among species remains poorly understood. In this study, we performed genetic analysis of nectar guide trichome variation between two closely related monkeyflower (Mimulus) species, the bumblebee-pollinated Mimulus lewisii and self-pollinated M. parishii. We demonstrate that a MIXTA-like R2R3-MYB gene, GUIDELESS, is a major contributor to the nectar guide trichome length variation between the two species. The short-haired M. parishii carries a recessive allele due to non-synonymous substitutions in a highly conserved motif among MIXTA-like MYB proteins. Furthermore, our results suggest that besides GUIDELESS, additional loci encoding repressors of trichome elongation also contribute to the transition from bumblebee-pollination to selfing. Taken together, these results suggest that during a pollination syndrome switch, changes in seemingly complex traits such as nectar guide trichomes could have a relatively simple genetic basis, involving just a few genes of large effects.

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Introduction

Plant-pollinator interactions are ubiquitous and are estimated to involve ~ 170,000 plant and ~ 200,000 animal species in nature [1]. Pollinator-mediated selection is considered a major driver for floral trait diversification and angiosperm speciation [2,3,4,5,6]. Flowers pollinated by a particular type of pollinator often share certain commonalities. For example, many bumblebee-pollinated flowers, especially bilaterally symmetric flowers, contain a ventral petal with variously arranged trichomes and contrasting coloration compared to the other petals, which together serve as nectar guides for the pollinators [7,8,9,10,11,12,13]. Nectar guide trichomes are used as footholds for foraging bees to enter flowers [10, 14] and may trap pollen from insects for subsequent pollen transfer to the stigma when pollinators are scarce, thereby increasing reproductive success [9]. Despite their obvious ecological importance, very little is known about the genetic basis of natural variation in nectar guide trichomes among species.

The basic genetic network controlling trichome development, especially in vegetative tissues, has been extensively studied in several plant model systems. In Arabidopsis, a regulatory complex composed of transcription factors belonging to three families positively regulates trichome initiation, including the R2R3-MYB proteins GLABROUS1 (GL1), AtMYB23 [15], and AtMYB82 [15,16,17], the basic helix-loop-helix (bHLH) proteins GLABRA3 (GL3), ENHANCER OF GLABRA3 (EGL3), TRANSPARENT TESTA 8 (TT8), and AtMYC-1 [18,19,20,21], and the WD40 repeat (WDR) protein TRANSPARENT TESTA GLABRA1 (TTG1) [22]. Transcription factors belonging to the same family are at least partially redundant in function. This MYB-bHLH-WDR (MBW) regulatory complex activates the downstream gene GLABRA2 (GL2), which encodes a homeodomain leucine-zipper (HD-ZIP) protein that determines trichome cell fate in shoots [23, 24]. Additionally, a group of C2H2 zinc finger transcription factors, including GLABROUS INFLORESCENCE STEMS (GIS), GIS2, ZINC FINGER PROTEIN5 (ZFP5), ZFP6, and ZFP8 [25,26,27,28], function as positive regulators of trichome initiation upstream of the MBW regulatory complex. In addition to these activators, a group of single repeat R3-MYBs, including CAPRICE (CPC) [29], TRIPTYCHON (TRY) [30], ENHANCER OF TRY AND CPC1 (ETC1) [31], ETC2 [32], and TRICHOMELESS1 (TCL1) were found to function redundantly as negative regulators of trichome determination by competing with the R2R3-MYBs for bHLH binding [33, 34]. Although most of these genes were identified through mutant analyses, some of them are also known to cause trichome variation among natural accessions of Arabidopsis. For example, a single amino acid substitution in ETC2 is responsible for reduced trichome density on Arabidopsis leaves in natural populations [35]. TCL1 and TRY were also identified as causal genes underlying diversity of trichome patterning in fruits or pedicels in natural Arabidopsis populations [36].

However, recent studies in many crop and medicinal plants, including maize, cotton, tomato, cucumber, melon, and Artemisia annua, suggest that the regulation of trichome development by the MBW complex uncovered in Arabidopsis seems more the exception than the rule. In most other plant species, the core module that positively regulates trichome initiation and elongation contains the MIXTA-like R2R3-MYB belonging to a different subgroup than GL1 [37,38,39,40,41,42,43,44,45,46,47] and class IV HD-ZIP transcription factors [48,49,50,51,52,53]. Other positive regulators of trichome development that genetically or biochemically interact with the MIXTA-like R2R3-MYB or/and HD-ZIP IV proteins include C2H2 zinc-finger proteins (e.g., H in tomato and Tu in cucumber) [54, 55] and MYC1-like bHLH proteins (e.g., SlMYC1 in tomato) [56], whereas negative regulators include B-type cyclin-like and Jasmonate ZIM (JAZ) proteins [51, 56,57,58]. These remarkable progresses in the past decade have set the stage for dissecting the genetic basis underlying nectar guide trichome variation between species with different pollination guilds.

The monkeyflower species Mimulus lewisii and M. parishii are closely related [59, 60] but display different pollination modes. The bumblebee-pollinated M. lewisii bears large showy flowers with long nectar guide trichomes (Fig. 1a, left panel), whereas the self-pollinated M. parishii produces small flowers with very short nectar guide trichomes (Fig. 1a, middle panel). These species are easy to grow in the greenhouse, with short generation time and high fecundity, and are amenable to transgenic manipulation through Agrobacterium-mediated transformation [61, 62]. This study represents the first step towards a detailed dissection of the genetic basis underlying nectar guide trichome variation between the two species. Starting from an F2 population with a wide range of nectar guide trichome lengths (Fig. 1b), we constructed near-isogenic lines (NILs) in the M. lewisii genetic background to reduce the complexity of F2 phenotypes into Mendelian loci. Through bulked segregant analysis, fine-scale genetic mapping, and complementation crosses, we demonstrate that the MIXTA-like gene, GUIDELESS [46], is a major contributor to the nectar guide trichome variation between the two species.

Materials and methods

Plant materials and growth conditions

The M. lewisii inbred line LF10, the guideless mutant, and the M. parishii inbred line Mpar were described previously [46, 61, 62]. All plants were grown in the University of Connecticut EEB research greenhouses under natural light supplemented with sodium vapor lamps, ensuring a 16-hr day length with a light intensity of 110–160 µmol·m− 2·s− 1. Plants were fertilized 2–3 times per week.

Quantification of nectar guide trichome length

Nectar guide tissues at the same site near the throat (marked by a red circle in Fig. 1a) of the corolla were cut into small pieces. The trichomes in these tissues were then imaged under a light microscope, and their lengths were measured using Zeiss ZEN 2.6 lite (blue edition) software.

Fig. 1
figure 1

Phenotypes of nectar guide trichomes. (a) Front view of the whole corolla (upper) and enlarged view of the corolla throat of M. lewisii (Mlew, left), M. parishii (Mpar, middle), and their F1 hybrid (right). Scale bars, 5 mm (top row) and 1 mm (bottom row). The red circle marks the position where nectar guide trichome length were quantified. (b) Front view of F2 hybrids representing the four classes. Scale bars, 4 mm. (c) Light microscopy images of nectar guide trichomes of M. lewisii, M. parishii and F1 hybrids. Scale bars, 200 μm. (d) Quantification of nectar guide trichome length (n = 20 for each genotype). Error bars are 1 SD. Asterisks indicate differences from M. lewisii (** p < 0.01, student’s t test). (e) Frequency distribution of the four classes of F2 individuals

Full size image

NIL construction

To construct NILs with loci controlling nectar guide trichome variation introgressed from M. parishii to M. lewisii, we selected an F2 individual with short trichomes, ZH101 (Fig. 1b), to backcross (BC) to M. lewisii, and then we selfed one BC1 individual that most closely resembled M. lewisii, including the long nectar guide trichomes. The short-haired phenotype reappeared in the selfing population (BC1S1). The same process was repeated for a second time. The short vs. long trichome segregated ~ 1:3 in the BC2S1 population. The short-haired individuals from the BC2S1 population were then subjected to bulked segregant analysis by genome sequencing to identify the causal locus (see the section below). One of the short-haired BC2S1 individuals was backcrossed to M. lewisii again to clean up the residual M. parishii DNA, and one short-haired BC3S1 individual with only the causal locus introgressed from M. parishii was retained as the GP/P NIL (named for the causal gene GUIDELESS; see Results).

Bulked segregant analysis by deep sequencing

To determine which chromosome fragment (s) was introgressed from M. parishii to M. lewisii in the GP/P NIL, we performed bulked segregant analysis, following [63]. Briefly, we pooled DNA samples from 39 short-haired individuals from the ZH101 BC2S1 population, with equal representation from each sample. A small-insert (350-bp) library was prepared for the pooled sample, and 150-bp paired-end reads were generated by Illumina NovaSeq at Novogene (Sacramento, CA), with ~ 80-fold genome coverage. The resulting short reads were mapped to the M. lewisii reference genome (http://mimubase.org/FTP/Genomes/LF10g_v2.0/) with CLC Genomics Workbench 7.0. After SNP calling, the number of homozygous SNPs in 20-kb bins was plotted in a bar graph.

Expression analysis using RT-qPCR

Total RNA was extracted using the Spectrum Plant Total RNA Kit (TRN250, Sigma-Aldrich) and cDNA was synthesized from 500 ng of the DNaseI (Invitrogen) treated RNA using GoScript™ Reverse Transcription Mix (A2791, Promega), then diluted 10-fold before RT-qPCR. To normalize expression levels, the M. lewisii/M. parishii ortholog of Arabidopsis ubiquitin-conjugating enzyme gene (At5g25760), MIUBC, was used as reference gene following [62]. RT-qPCR was performed using iQTMSYBR® Green Supermix (Bio-Rad) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The cDNA samples were amplified for 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Amplification efficiencies for each primer pair were determined using critical threshold values obtained from a dilution series (1:4, 1:8, 1:16, 1:32) of pooled cDNAs. Relative expression of GUIDELESS was calculated using the formula (Etarget)CP (ref)/ (Eref) CP (target). The primers used for RT-(q)PCR are listed in Table S1.

Results

Variation in nectar guide trichome length between M. lewisii and M. parishii

Measurements using the microscope images showed that the nectar guide trichome length of M. lewisii is about 8 times that of M. parishii and about 3 times that of the F1 hybrid (Fig. 1c, d). We visually scored the nectar guide trichome lengths of 264 F2 individuals, and categorized them into four classes (examples shown in Fig. 1b). Class I (14.4%; 38/264) have very short trichomes resembling M. parishii. Class IV (8.3%; 22/264) showed long trichomes similar to M. lewisii. Class II (48.5%; 128/264) and class III (28.8%; 128/264) showed intermediate trichome lengths, with the former closer to M. parishii and the latter closer to M. lewisii (Fig. 1e). The frequency distribution of the F2 phenotypes suggests a non-monogenic basis of nectar guide trichome variation between the two species.

Identification of GUIDELESS as a candidate gene underlying one of the causal loci

To reduce the variation in nectar guide trichome length displayed by the F2 population into mendelian loci, we took a near-isogenic line (NIL) approach. Specifically, we selected the F2 individual ZH101 (Fig. 1b) bearing short nectar guide trichomes, and backcrossed it to M. lewisii. Within the resulting BC2S1 population (two rounds of backcrossing and selfing; see Materials and Methods), approximately 3/4 (111/154) of the individuals had nectar guide trichomes similar to the wild type M. lewisii and ~ 1/4 (43/154) had shorter trichomes. We thus inferred that this short-haired NIL is homozygous for the recessive M. parishii allele at a single causal locus.

To identify the chromosomal location of this locus, we performed a bulked segregant analysis of pooled DNA samples from 39 short-haired individuals in the BC2S1 population. Illumina sequencing of the bulked DNA revealed four relatively small genomic regions homozygous for M. parishii (Fig. 2a), one of which was expected to contain the causal gene. From the BC2S1 population, we selected one short-haired individual that was most similar to M. lewisii and backcrossed it one more time to M. lewisii. Selfing one of the BC3 individuals produced the BC3S1 population. By fine-mapping 96 BC3S1 individuals, we found that a 460-kb DNA segment on chromosome 6 (Chr 6: 1,533,002–1,988,221; between markers MLCP6_200 and MLCP6_250) co-segregated with nectar guide trichome length (Fig. 2b). This segment contains the GUIDELESS gene, which encodes a MIXTA-like R2R3-MYB transcription factor and was previously shown to control nectar guide trichome development in M. lewisii through mutant analysis [46]. Therefore, GUIDELESS was considered the most promising candidate gene. Genotyping the BC3S1 population also allowed us to identify a high-quality NIL that is homozygous for the M. parishii GUIDELESS allele in an otherwise M. lewisii genomic background, named the GP/P NIL (Fig. 2c).

Verification of GUIDELESS as the causal gene

To verify gene causality, we performed a complementation test by crossing the previously characterized loss-of-function guideless mutant (in the M. lewisii LF10 background; Fig. 2c) with the GP/P NIL. If GUIDELESS is the causal gene underlying the short trichome phenotype of the GP/P NIL, we would expect that crossing the null guideless mutant with the GP/P NIL, which is homozygous for the recessive M. parishii allele, should result in F1 progeny (GN_F1) with short trichomes (i.e., the recessive GP allele and the null mutant allele do not complement one another). This was exactly what we observed: the F1 progeny (GN_F1) produced nectar guide trichomes of comparable length to those in the GP/P NIL (Fig. 2c, d). By contrast, the F1 progeny from the cross between the guideless mutant and wild-type M. lewisii (GL_F1) produced nectar guide trichomes comparable to the wild type in length (Fig. 2c, d). These results provide strong evidence that GUIDELESS is indeed the causal gene underlying the GP/P NIL phenotype.

Fig. 2
figure 2

Identification of the causal gene GUIDELESS. (a) Genome scan of the GP/P NIL for regions that are enriched in homozygous SNPs compared with the M. lewisii reference genome. (b) Cross design to generate a fine-scale mapping population (BC3S1), and within this population, the three most informative recombinants reduced the candidate genomic interval to a smaller region (460 Kb) between markers MLCP6_200 and MLCP6_250. Nectar guide trichome length phenotypes (long vs. short) are shown on the right. (c) Front view of the whole corolla and enlarged view of the corolla throat of the GP/P NIL, the guideless mutant, F1 between guideless and the wild-type M. lewisii (GL_F1), and F1 between guideless and the GP/P NIL (GN_F1). Scale bars, 5 mm (top row) and 2 mm (bottom row). (d) Quantification of nectar guide trichome lengths (n = 20 for each genotype). Error bars are 1 SD. Asterisks indicate differences from the wild-type M. lewisii (** p < 0.01, student’s t test). (e) Expression analysis of GUIDELESS by RT-qPCR

Full size image

The observation that nectar guide trichomes in the GP/P NIL are much shorter than those in the wild-type M. lewisii but still substantially longer than those in the null guideless mutant (Fig. 2d) indicates that the GUIDELESS allele in M. parishii is hypomorphic (i.e., partially functional).

Consistent with this inference, expression level of the MpGUIDELESS allele was only slightly lower than that of the MlGUIDELESS allele (Fig. 2e) and the MpGUIDELESS coding DNA does not contain obvious null mutations (e.g., premature stop codons). However, the predicted MpGUIDELESS protein sequence has two amino acid substitutions (“SA” to “TT”) in a highly conserved motif among MIXTA-like R2R3-MYBs (Fig. 3), potentially attenuating protein function.

Fig. 3
figure 3

Multiple alignment of MIXTA-like R2R3-MYB protein sequences. Ml: Mimulus lewisii; Mp: Mimulus parishii; Am: Antirrhinum majus; Ph: Petunia hybrida; At: Arabidopsis thaliana. The signature motif defining the MIXTA-like clade of R2R3-MYBs is marked by a black bar above the alignment. MpGUIDELESS has the two amino acids “SA” in the highly conserved signature motif replaced by “TT”. The two black triangles show the positions of the two introns

Full size image

Other loci underlying nectar guide trichome length variation between M. lewisii and M. parishii

Our measurements showed that the GUIDELESS locus only accounts for ~ 40% of the parental difference in nectar guide trichome length between M. lewisii and M. parishii (Fig. 2d), implying the involvement of other loci underlying trichome length variation. Moreover, the fact that F1 hybrids between M. lewisii and M. parishii have much shorter nectar guide trichomes than M. lewisii (Fig. 1a, c, d), whereas the GUIDELESS NIL is similar to wild-type M. lewisii in a heterozygous state, suggests the existence of at least one other locus negatively regulating nectar guide trichome length, with the M. parishii allele dominant over the M. lewisii allele. In fact, the proportion of F2 individuals with very short trichomes (class I: 14.4%) is remarkably consistent with the expected ratio of a three-loci model. If the M. parishii allele is also dominant at the third locus, individuals with very short trichomes are expected to be homozygous for the M. parishii allele at GUIDELESS, and either homozygous for the M. parishii allele or heterozygous at the other two loci. That is, a ratio of 14.1% (1/4 * 3/4 * 3/4 = 9/64).

Discussion

In this study we analyzed the genetic basis of variation in nectar guide trichome length between two Mimulus species with distinct pollination syndromes. The two species differ by 8-fold in nectar guide trichome length. Our results suggest that the GUIDELESS gene is a major contributor to nectar guide trichome length variation between the bumblebee-pollinated M. lewisii and self-pollinated M. parishii. Compared to other pollinator-associated floral traits such as coloration, nectar guide trichome variation has received little attention. Our study shows that in the M. lewisii-M. parishii system, nectar guide trichome is a typical “complex” trait, as reflected by the continuous variation in the F2 population, but can be dissected into just a few mendelian loci by constructing NILs.

The level of difficulty in generating high quality NILs (e.g., with introgressed chromosome fragments less than 500 kb) and ultimately identifying the causal genes depends on local recombination rates. The GUIDELESS locus has an exceptionally high recombination rate. Genotyping 96 ZH101_BC3S1 individuals was sufficient to narrow down the causal gene to a 460-kb interval (Fig. 2b). The high recombination rate in this region is also consistent with previous bulked segregant analysis of the guideless mutant [46]. Genome sequencing of 100 individuals from an intra-specific F2 population was enough to locate the causal mutation to a 50-kb interval. However, it is not unusual for certain loci to undergo low recombination in inter-specific crosses. For example, in our previous studies of flower color variation between M. lewisii and the closely related M. cardinalis, as well as between M. parishii and M. cardinalis, the generation of high quality NILs of flower color loci required genotyping of 1,000–3,000 individuals from the final mapping population [61,62,63,64]. Generation of high quality NILs at the other inferred nectar guide trichome loci is currently underway. The anticipated NILs will enable us to investigate the genetic interactions among these loci and to ultimately identify the causal genes.

The most recent phylogenomic analysis of M. lewisii, M. parishii, and their close relatives [60] suggests that the self-pollination syndrome of M. parishii is a derived state, including not only short necatar guide trichomes, but also small flower size, inconspicuous coloration, and reduced anther-stigma separation. The inconspicuous coloration of M. parishii flowers was recently shown to be caused by a mutation in the 5’ UTR of an anthocyanin-activating R2R3-MYB gene PELAN [61]. This mutation does not alter protein function or gene transcript level. Instead, it inhibits protein translation, and therefore represents a type of loss-of-function mutation underlying the recessive allele in M. parishii. Although the specific molecular mechanisms are very different, the GUIDELESS allele in M. parishii is somewhat similar to the PELAN case, in that it is also recessive and probably encodes a protein with attenuated function. Similar loss-of-function mutations resulting in decreased trichome formation have also been observed in cucumber, wherein the C2H2 transcription factor Tu is entirely absent, leading to the manifestation of the non-warty fruit trait [55].

By contrast, at the other nectar guide trichome loci, the M. parishii alleles are dominant and, based on the F1 phenotype (Fig. 1a, c, d), most likely encode repressors of trichome elongation. Although transgenic experiments showed that B-type cyclin-like and Jasmonate ZIM (JAZ) proteins can repress trichome initiation in systems other than Arabidopsis [51, 56,57,58], whether these repressors contribute to natural variation in trichome development is unclear. Interestingly, a recent study investigating natural trichome variation in vegetative tissues among snapdragon (Antirrhinum) species identified a glutaredoxin gene as a dominant repressor of trichome development [65]. However, this glutaredoxin gene has acquired its trichome repressing function after the divergence between Antirrhinum and Mimulus [65], and thus is unlikely to cause trichome variation between our focal Mimulus species. Moreover, compared to loss-of-function mutations, the molecular mechanisms generating gain-of-function mutations are much less understood. Further characterization of the dominant repressor loci in M. parishii will not only be significant for the elucidation of negative regulators of trichome development, but also contribute to our understanding of the molecular basis of gain-of-function mutations during phenotypic evolution.

Conclusions

In this study, we performed the first genetic analysis of the variation in nectar guide trichome length between the bumblebee-pollinated M. lewisii and self-pollinated M. parishii. The frequency distribution of F2 phenotypes implies a non-monogenic basis for the variation. Through NIL construction, bulked segregant analysis, fine-scale genetic mapping, and complementation cross, we found that the MIXTA-like R2R3-MYB gene, GUIDELESS, is a major contributor to the nectar guide trichome variation between the two species. Furthermore, we inferred that besides GUIDELESS, at least one other locus encoding repressors of trichome elongation is also responsible for the short trichome length in M. parishii. These results suggest that during a pollination syndrome switch, changes in even seemingly complex traits such as nectar guide trichomes could have a relatively simple genetic basis, and that NIL construction followed by fine-scale genetic mapping is a powerful approach to dissect the genetic basis of these evolutionary transitions.

Data availability

The Illumina sequencing data of 39 short-haired individuals have been deposited to NCBI under the accession number PRJNA1021984. The raw datasets have been deposited to the Dryad Digital Repository: https://datadryad.org/stash/share/7c-E3lKh-zfdObRqsX37XAIWPwilBAitvpmzP3VNP_E (doi:10.5061/dryad.34tmpg4rm) [66]. Supplementary material is available online.

References

  1. Landry C. Mighty mutualisms: the nature of plant-pollinator interactions. Nat Educ Knowl. 2012;3(10):37.

    Google Scholar 

  2. Fenster CB, Armbruster WS, Wilson P, Dudash MR, Thomson JD. Pollination syndromes and floral specialization. Annu Rev Ecol Evol Syst. 2004;35:375–403.

    Article  Google Scholar 

  3. Kay KM, Sargent RD. The role of animal pollination in plant speciation: integrating ecology, geography, and genetics. Annu Rev Ecol Evol Syst. 2009;40:637–56.

    Article  Google Scholar 

  4. Van der Niet T, Peakall R, Johnson SD. Pollinator-driven ecological speciation in plants: new evidence and future perspectives. Ann Bot. 2014;113(2):199–212.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Wei N, Kaczorowski RL, Arceo-Gómez G, O’Neill EM, Hayes RA. Ashman T-L. pollinators contribute to the maintenance of flowering plant diversity. Nature. 2021;597(7878):688–92.

    Article  CAS  PubMed  Google Scholar 

  6. Whittall JB, Hodges SA. Pollinator shifts drive increasingly long nectar spurs in columbine flowers. Nature. 2007;447(7145):706–9.

    Article  CAS  PubMed  Google Scholar 

  7. Faegri K, van der Pijl L. The principles of pollination ecology. Oxford, UK: Pergamon Press; 1979.

    Google Scholar 

  8. Almeida J, Rocheta M, Galego L. Genetic control of flower shape in Antirrhinum majus. Development. 1997;124(7):1387–92.

    Article  CAS  PubMed  Google Scholar 

  9. Arathi H, Kelly JK. Corolla morphology facilitates both autogamy and bumblebee pollination in Mimulus guttatus. Int J Plant Sci. 2004;165(6):1039–45.

    Article  Google Scholar 

  10. Kampny CM. Pollination and flower diversity in Scrophulariaceae. Bot Rev. 1995;61:350–66.

    Article  Google Scholar 

  11. Kolosova N, Sherman D, Karlson D, Dudareva N. Cellular and subcellular localization of S-adenosyl-L-methionine: benzoic acid carboxyl methyltransferase, the enzyme responsible for biosynthesis of the volatile ester methylbenzoate in snapdragon flowers. Plant Physiol. 2001;126(3):956–64.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Owen CR, Bradshaw HD. Induced mutations affecting pollinator choice in Mimulus lewisii (Phrymaceae). Arthropod-Plant Interact. 2011;5:235–44.

    Article  Google Scholar 

  13. Schemsk DW, Bradshaw HD Jr. Pollinator preference and the evolution of floral traits in monkeyflowers (Mimulus). Proc Natl Acad Sci. 1999;96(21):11910–5.

    Article  Google Scholar 

  14. Jachuła J, Konarska A, Denisow B. Micromorphological and histochemical attributes of flowers and floral reward in Linaria vulgaris (Plantaginaceae). Protoplasma. 2018;255:1763–76.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Kirik V, Lee MM, Wester K, Herrmann U, Zheng Z, Oppenheimer D, Schiefelbein J, Hulskamp M. Functional diversification of MYB23 and GL1 genes in trichome morphogenesis and initiation. Development. 2005;132(7):1477–85.

    Article  CAS  PubMed  Google Scholar 

  16. Liang G, He H, Li Y, Ai Q, Yu D. MYB82 functions in regulation of trichome development in Arabidopsis. J Exp Bot. 2014;65(12):3215–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Oppenheimer DG, Herman PL, Sivakumaran S, Esch J, Marks MD. A myb gene required for leaf trichome differentiation in Arabidopsis is expressed in stipules. Cell. 1991;67(3):483–93.

    Article  CAS  PubMed  Google Scholar 

  18. Mae L, Inzé D, Goossens A. Functional specialization of the TRANSPARENT TESTA GLABRA1 network allows differential hormonal control of laminal and marginal trichome initiation in Arabidopsis rosette leaves. Plant Physiol. 2008;148(3):1453–64.

    Article  Google Scholar 

  19. Payne CT, Zhang F, Lloyd AM. GL3 encodes a bHLH protein that regulates trichome development in Arabidopsis through interaction with GL1 and TTG1. Genetics. 2000;156(3):1349–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Zhang F, Gonzalez A, Zhao M, Payne CT, Lloyd A. A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis. Development. 2003;130(20):4859–69.

    Article  CAS  PubMed  Google Scholar 

  21. Zhao H, Wang X, Zhu D, Cui S, Cao LX, Ma Y. A single amino acid substitution in IIIf subfamily of basic helix-loop-helix transcription factor AtMYC1 leads to trichome and root hair patterning defects by abolishing its interaction with partner proteins in Arabidopsis. J Biol Chem. 2012;287(17):14109–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Walker AR, Davison PA, Bolognesi-Winfield AC, James CM, Srinivasan N, Blundell TL, Esch JJ, Marks MD, Gray JC. The TRANSPARENT TESTA GLABRA1 locus, which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis, encodes a WD40 repeat protein. Plant Cell. 1999;11(7):1337–49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Di Cristina M, Sessa G, Dolan L, Linstead P, Baima S, Ruberti I, Morelli G. The Arabidopsis Athb-10 (GLABRA2) is an HD‐Zip protein required for regulation of root hair development. Plant J. 1996;10(3):393–402.

    Article  CAS  PubMed  Google Scholar 

  24. Wang S, Chen J-G. Arabidopsis transient expression analysis reveals that activation of GLABRA2 may require concurrent binding of GLABRA1 and GLABRA3 to the promoter of GLABRA2. Plant Cell Physiol. 2008;49(12):1792–804.

    Article  PubMed  Google Scholar 

  25. Gan Y, Kumimoto R, Liu C, Ratcliffe O, Yu H, Broun P. GLABROUS INFLORESCENCE STEMS modulates the regulation by gibberellins of epidermal differentiation and shoot maturation in Arabidopsis. Plant Cell. 2006;18(6):1383–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Gan Y, Liu C, Yu H, Broun P. Integration of cytokinin and gibberellin signalling by Arabidopsis transcription factors GIS, ZFP8 and GIS2 in the regulation of epidermal cell fate. Development. 2007;134(11):2073–81.

    Article  CAS  PubMed  Google Scholar 

  27. Zhou Z, An L, Sun L, Zhu S, Xi W, Broun P, Yu H, Gan Y. Zinc finger protein5 is required for the control of trichome initiation by acting upstream of zinc finger protein8 in Arabidopsis. Plant Physiol. 2011;157(2):673–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhou Z, Sun L, Zhao Y, An L, Yan A, Meng X, Gan Y. Zinc finger protein 6 (ZFP 6) regulates trichome initiation by integrating gibberellin and cytokinin signaling in Arabidopsis thaliana. New Phytol. 2013;198(3):699–708.

    Article  CAS  PubMed  Google Scholar 

  29. Schellmann S, Schnittger A, Kirik V, Wada T, Okada K, Beermann A, Thumfahrt J, Jürgens G, Hülskamp M. TRIPTYCHON and CAPRICE mediate lateral inhibition during trichome and root hair patterning in Arabidopsis. EMBO J. 2002;21(19):5036–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Schnittger A, Folkers U, Schwab B, Jürgens G, Hülskamp M. Generation of a spacing pattern: the role of TRIPTYCHON in trichome patterning in Arabidopsis. Plant Cell. 1999;11(6):1105–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kirik V, Simon M, Huelskamp M, Schiefelbein J. The ENHANCER OF TRY AND CPC1 gene acts redundantly with TRIPTYCHON and CAPRICE in trichome and root hair cell patterning in Arabidopsis. Dev Biol. 2004;268(2):506–13.

    Article  CAS  PubMed  Google Scholar 

  32. Kirik V, Simon M, Wester K, Schiefelbein J, Hulskamp M. ENHANCER of TRY and CPC 2 (ETC2) reveals redundancy in the region-specific control of trichome development of Arabidopsis. Plant Mol Biol. 2004;55:389–398.

  33. Serna L, Martin C. Trichomes: different regulatory networks lead to convergent structures. Trends Plant Sci. 2006;11(6):274–80.

    Article  CAS  PubMed  Google Scholar 

  34. Wang X, Shen C, Meng P, Tan G, Lv L. Analysis and review of trichomes in plants. BMC Plant Biol. 2021;21(1):1–11.

    Google Scholar 

  35. Hilscher J, Schlötterer C, Hauser M-T. A single amino acid replacement in ETC2 shapes trichome patterning in natural Arabidopsis populations. Curr Biol. 2009;19(20):1747–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Arteaga N, Savic M, Méndez-Vigo B, Fuster-Pons A, Torres-Pérez R, Oliveros JC, Picó FX, Alonso-Blanco C. MYB transcription factors drive evolutionary innovations in Arabidopsis fruit trichome patterning. Plant Cell. 2021;33(3):548–65.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Baumann K, Perez-Rodriguez M, Bradley D, Venail J, Bailey P, Jin H, Koes R, Roberts K, Martin C. Control of cell and petal morphogenesis by R2R3 MYB transcription factors. Development. 2007;134(9):1691–701.

    Article  CAS  PubMed  Google Scholar 

  38. Brockington SF, Alvarez-Fernandez R, Landis JB, Alcorn K, Walker RH, Thomas MM, Hileman LC, Glover BJ. Evolutionary analysis of the MIXTA gene family highlights potential targets for the study of cellular differentiation. Mol Biol Evol. 2013;30(3):526–40.

    Article  CAS  PubMed  Google Scholar 

  39. Chalvin C, Drevensek S, Dron M, Bendahmane A, Boualem A. Genetic control of glandular trichome development. Trends Plant Sci. 2020;25(5):477–87.

    Article  CAS  PubMed  Google Scholar 

  40. Ewas M, Gao Y, Wang S, Liu X, Zhang H, Nishawy EM, Ali F, Shahzad R, Ziaf K, Subthain H. Manipulation of SlMXl for enhanced carotenoids accumulation and drought resistance in tomato. Sci Bull. 2016;61:1413–8.

    Article  CAS  Google Scholar 

  41. Jaffé FW, Tattersall A, Glover BJ. A truncated MYB transcription factor from Antirrhinum majus regulates epidermal cell outgrowth. J Exp Bot. 2007;58(6):1515–24.

    Article  PubMed  Google Scholar 

  42. Machado A, Wu Y, Yang Y, Llewellyn DJ, Dennis ES. The MYB transcription factor GhMYB25 regulates early fibre and trichome development. Plant J. 2009;59(1):52–62.

    Article  CAS  PubMed  Google Scholar 

  43. Perez-Rodriguez M, Jaffe FW, Butelli E, Glover BJ, Martin C. Development of three different cell types is associated with the activity of a specific MYB transcription factor in the ventral petal of Antirrhinum majus flowers. Development. 2005;132(2):359–70.

    Article  CAS  PubMed  Google Scholar 

  44. Tan J, Walford S-A, Dennis ES, Llewellyn D. Trichomes control flower bud shape by linking together young petals. Nat Plants. 2016;2(7):1–5.

    Article  Google Scholar 

  45. Wu H, Tian Y, Wan Q, Fang L, Guan X, Chen J, Hu Y, Ye W, Zhang H, Guo W. Genetics and evolution of MIXTA genes regulating cotton lint fiber development. New Phytol. 2018;217(2):883–95.

    Article  CAS  PubMed  Google Scholar 

  46. Yuan Y-W, Sagawa JM, Di Stilio VS, Bradshaw HD. Bulk segregant analysis of an induced floral mutant identifies a MIXTA-like R2R3 MYB controlling nectar guide formation in Mimulus lewisii. Genetics. 2013;194(2):523–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Shi P, Fu X, Shen Q, Liu M, Pan Q, Tang Y, Jiang W, Lv Z, Yan T, Ma Y. The roles of AaMIXTA 1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua. New Phytol. 2018;217(1):261–76.

    Article  CAS  PubMed  Google Scholar 

  48. Pan Y, Bo K, Cheng Z, Weng Y. The loss-of-function GLABROUS 3 mutation in cucumber is due to LTR-retrotransposon insertion in a class IV HD-ZIP transcription factor gene CsGL3 that is epistatic over CsGL1. BMC Plant Biol. 2015;15(1):1–15.

    Article  CAS  Google Scholar 

  49. Vernoud V, Laigle G, Rozier F, Meeley RB, Perez P, Rogowsky PM. The HD-ZIP IV transcription factor OCL4 is necessary for trichome patterning and anther development in maize. Plant J. 2009;59(6):883–94.

    Article  CAS  PubMed  Google Scholar 

  50. Walford SA, Wu Y, Llewellyn DJ, Dennis ES. Epidermal cell differentiation in cotton mediated by the homeodomain leucine zipper gene, GhHD-1. Plant J. 2012;71(3):464–78.

    Article  CAS  PubMed  Google Scholar 

  51. Wu M, Chang J, Han X, Shen J, Yang L, Hu S, Huang B-B, Xu H, Xu M, Wu S. A HD-ZIP transcription factor specifies fates of multicellular trichomes via dosage-dependent mechanisms in tomato. Dev Cell. 2023;58(4):278–88.

    Article  CAS  PubMed  Google Scholar 

  52. Yang C, Li H, Zhang J, Luo Z, Gong P, Zhang C, Li J, Wang T, Zhang Y, Lu Y, Ye Z. A regulatory gene induces trichome formation and embryo lethality in tomato. Proc Natl Acad Sci. 2011;108(29):11836–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Zhu H, Sun X, Zhang Q, Song P, Hu Q, Zhang X, Li X, Hu J, Pan J, Sun S. GLABROUS (CmGL) encodes a HD-ZIP IV transcription factor playing roles in multicellular trichome initiation in melon. Theor Appl Genet. 2018;131:569–79.

    Article  CAS  PubMed  Google Scholar 

  54. Chang J, Yu T, Yang Q, Li C, Xiong C, Gao S, Xie Q, Zheng F, Li H, Tian Z. Hair, encoding a single C2H2 zinc-finger protein, regulates multicellular trichome formation in tomato. Plant J. 2018;96(1):90–102.

    Article  CAS  PubMed  Google Scholar 

  55. Yang X, Zhang W, He H, Nie J, Bie B, Zhao J, Ren G, Li Y, Zhang D, Pan J. Tuberculate fruit gene Tu encodes a C2H2 zinc finger protein that is required for the warty fruit phenotype in cucumber (Cucumis sativus L). Plant J. 2014;78(6):1034–46.

    Article  CAS  PubMed  Google Scholar 

  56. Hua B, Chang J, Wu M, Xu Z, Zhang F, Yang M, Xu H, Wang LJ, Chen XY, Wu S. Mediation of JA signalling in glandular trichomes by the woolly/SlMYC1 regulatory module improves pest resistance in tomato. Plant Biotechnol J. 2021;19(2):375–93.

    Article  CAS  PubMed  Google Scholar 

  57. Dong B, Wang X, Jiang R, Fang S, Li J, Li Q, Lv ZY, Chen WS. AaCycTL regulates cuticle and Trichome Development in Arabidopsis and Artemisia annua L. Front Plant Sci. 2021;12:808283.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Gao S, Gao Y, Xiong C, Yu G, Chang J, Yang Q, Yang C, Ye Z. The tomato B-type cyclin gene, SlCycB2, plays key roles in reproductive organ development, trichome initiation, terpenoids biosynthesis and Prodenia Litura defense. Plant Sci. 2017;262:103–14.

    Article  CAS  PubMed  Google Scholar 

  59. Beardsley PM, Schoenig SE, Whittall JB, Olmstead RG. Patterns of evolution in western north American mimulus (Phrymaceae). Am J Bot. 2004;91(3):474–89.

    Article  CAS  PubMed  Google Scholar 

  60. Nelson TC, Stathos AM, Vanderpool DD, Finseth FR, Yuan Y-W, Fishman L. Ancient and recent introgression shape the evolutionary history of pollinator adaptation and speciation in a model monkeyflower radiation (Mimulus section Erythranthe). PLoS Genet. 2021;17(2):e1009095.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Liang M, Foster CE, Yuan Y-W. Lost in translation: molecular basis of reduced flower coloration in a self-pollinated monkeyflower (Mimulus) species. Sci Adv. 2022;8(37):eabo1113.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Yuan Y-W, Sagawa JM, Young RC, Christensen BJ, Bradshaw HD Jr. Genetic dissection of a major anthocyanin QTL contributing to pollinator-mediated reproductive isolation between sister species of Mimulus. Genetics. 2013;194(1):255–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Yuan Y-W, Rebocho AB, Sagawa JM, Stanley LE, Bradshaw HD Jr. Competition between anthocyanin and flavonol biosynthesis produces spatial pattern variation of floral pigments between Mimulus species. Proc Natl Acad Sci. 2016;113(9):2448–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Liang M, Chen W, LaFountain AM, Liu Y, Peng F, Xia R, Bradshaw HD, Yuan Y-W. Taxon-specific, phased siRNAs underlie a speciation locus in monkeyflowers. Science. 2023;379(6632):576–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Tan Y, Barnbrook M, Wilson Y, Molnar A, Bukys A, Hudson A. Shared mutations in a novel glutaredoxin repressor of multicellular trichome fate underlie parallel evolution of Antirrhinum species. Curr Biol. 2020;30(8):1357–66.

    Article  CAS  PubMed  Google Scholar 

  66. Chen H, Yuan Y-W. Data from: genetic basis of nectar guide trichome variation between bumblebee- and self-pollinated monkeyflowers (Mimulus): role of the MIXTA-like gene. GUIDELESS Dryad. 2023. https://doi.org/10.5061/dryad.34tmpg4rm.

    Article  Google Scholar 

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Acknowledgements

We thank Matt Opel, Meghan Moriarty, and Chang Liu for plant maintenance in the UConn Botanical Conservatory, and Dr. Pamela K. Diggle for sharing her light microscope.

Funding

This work was supported by the National Science Foundation grants (IOS-1827645, IOS-2319721) to Y-W.Y. H.C. was partially supported by a fellowship from China Scholarship Council.

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  1. Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT, 06269, USA

    Hongfei Chen & Yao-Wu Yuan

  2. Institute for Systems Genomics, University of Connecticut, Storrs, CT, 06269, USA

    Yao-Wu Yuan

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H.C. and Y-W.Y. planned and designed the research; H.C. performed the experiments; H.C. and Y-W.Y. analyzed the data; H.C. and Y-W.Y. wrote the manuscript.

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Correspondence to Hongfei Chen or Yao-Wu Yuan.

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Chen, H., Yuan, YW. Genetic basis of nectar guide trichome variation between bumblebee- and self-pollinated monkeyflowers (Mimulus): role of the MIXTA-like gene GUIDELESS. BMC Plant Biol 24, 62 (2024). https://doi.org/10.1186/s12870-024-04736-y

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BMC Plant Biology

ISSN: 1471-2229

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