Abstract
Floret units in cereals produce grain, directly impacting yield. Here we report mutations in the maize CRABS CLAW (CRC) co-orthologs drooping leaf1 (drl1) and drl2 alter the development of ear and tassel florets. Pistillate florets of drl1 ears appear sterile and display ectopic unfused carpels that fail to enclose an expanded nucellus. Staminate florets of drl1 tassels have extra stamens and retain fertile anthers. Natural variation and transposon alleles of drl2 enhance drl1 floret phenotypes by reducing floral meristem (FM) determinacy. The drl paralogs are co-expressed in lateral floral organ primordia, but not within the FM. Together, the expression patterns and indeterminate mutant FMs suggest that the drl genes regulate FM activity and impose meristem determinacy by a non-cell autonomous signal. Genetic interaction analyses of drl mutants with maize floral mutants indicate that the drl genes are required throughout floret development, illustrating their importance for proper floret patterning in maize.
Introduction
A major goal in plant biology is to understand the factors that regulate meristem activity. Meristems, active, pluripotent stem cell tissues, produce all postembryonic organs of flowering plants1. Meristem determinacy (degree of meristem activity) is a critical factor that shapes vegetative, inflorescence and floral architectures. Vegetative and inflorescence meristems are indeterminate, producing an unspecified number of lateral primordia. Floral meristems (FMs) are generally determinate, initiating a more set number of floral whorls and organs before undergoing terminal differentiation. Commonly, eudicot flowers are composed of four whorls of floral organs (outermost to innermost: sepal, petal, stamen, and carpel). Similarly in the monocots, grass florets (flowers), including those in maize, are arranged in whorls of floral organs, some of which have grass-specific names (outermost to innermost: lemma, palea, stamen and carpel). As each grain is the product of one floret, regulating FM activity is a key agronomic trait.
FMs pattern flowers through the combinatorial activity of four classes of gene functions that dictate organ identity and FM determinacy2,3. In Arabidopsis, carpels are specified by the MADS-box transcription factor AGAMOUS (AG)4. Additionally, AG controls FM determinacy by repressing expression of the stem cell regulator WUSCHEL5,6. In the cereals, where AG orthologs have expanded and undergone subfunctionalization during grass evolution, FM determinacy is regulated redundantly7-9. The maize AG ortholog zea agamous1 (zag1) imposes FM determinacy with a lesser obvious role in regulating floret organ identity as supernumerary carpels develop in pistillate florets of zag1 mutants7. ZAG1 interacts physically with AG-LIKE6 (AGL6) subfamily member BEARDED EAR (BDE)/ZAG3, and zag1; bde double mutants reveal a synergistic interaction in regulating FM determinacy10. The maize indeterminate floral apex1 (ifa1) mutant affects FM determinacy in pistillate and staminate florets, and in the innermost staminate whorl of ifa1 mutants, a nucellus develops at very low penetrance11. ifa1 shows a redundant genetic interaction with zag1 to regulate FM determinacy11. In the bisexual florets of rice, AG orthologs OsMADS3 and OsMADS58 regulate floret organ identity and FM determinacy, respectively8,9.
Arabidopsis YABBY family member CRC is required for proper growth of the gynoecium12. Loss-of-function mutations in CRC consistently result in reduced stylar growth and incomplete medial fusion of carpels. crc mutants occasionally produce three carpels compared with two in wild-type, suggesting CRC is necessary to promote FM determinacy12,13. Expression of CRC is restricted to developing carpels and nectaries13. The rice CRC ortholog DROOPING LEAF (DL) is required for carpel identity, as carpels undergo homeotic transformation to stamens in loss-of-function dl mutants14. Transformed stamens are variable in number, indicating DL also regulates FM determinacy. DL is expressed in carpel primordia of rice florets14. Genetic analysis indicates that DL and the rice AGL6 subfamily member MOSAIC FLORAL ORGAN1 (MFO1)/OsMADS6 redundantly regulate FM determinacy15.
Here we report the maize CRC co-orthologs drl1 and drl2 are required for the development of dimorphic, unisexual ear and tassel florets. drl1 pistillate florets are sterile, display ectopic unfused carpels and have an expanded nucellus. drl1 staminate florets have extra stamens with fertile anthers. Natural variation and transposon alleles of drl2 enhance drl1 floret phenotypes by further reducing FM determinacy. The drl paralogs are co-expressed in lateral organ primordia initiated by the FM, but not within the FM, and interact genetically with other maize floral mutants. Our results demonstrate that the drl genes are required for floret patterning and to impose meristem determinacy via some non-cell autonomous mechanism, and thus, provide critical control of FM activity in the development of grain-bearing structures.
Results
drl1 and drl2 regulate floret development
Maize staminate and pistillate florets are produced on the tassel and ear, respectively16. During tassel and ear development, branching events from multiple meristem types17 ultimately give rise to floret whorls housed in grass-specific spikelets18. The indeterminate inflorescence meristem (IM) of the tassel and ear initiates determinate spikelet pair meristems (SPMs); additionally in the tassel, the IM initiates indeterminate branch meristems (BMs). Each SPM produces a pair of determinate spikelet meristems (SMs), each of which gives rise to two glumes; afterwards, each SM initiates a lower floral meristem (LFM) and then converts identity to an upper floral meristem (UFM). Each determinate LFM and UFM gives rise to a lemma, a palea, two lateral-abaxial lodicules, three stamens and three carpels19. Two lateral-adaxial stamens are spaced widely relative to the medial-abaxial stamen20. Three connately fused carpels form the single pistil; however, only the two lateral-abaxial carpels (indeterminate carpels, Ci) form an elongated silk, whereas growth of the medial-adaxial carpel (determinate carpel, Cd) is limited in growth to envelop the single ovule21,22. The ovule consists of nucellus tissue enclosed mostly by inner and partially by outer integuments, all within a locule formed by the three fused carpels16. After organ initiation, sex determination in the tassel and ear culminates in abortion of the carpel whorl in staminate florets and arrest of stamen primordia in pistillate florets, respectively (Fig. 1a,b)19.
Likely null mutations of drl1 displayed aberrant pistillate and staminate floret morphologies. Macroscopically, drl1 mutant ears appeared sterile, with underdeveloped silks consisting of reduced, unfused carpel walls that failed to enclose an expanded nucellus (Fig. 1c,d). drl1 pistillate phenotypes were reminiscent of the floret phenotypes described for the ifa1 mutant11. We found drl1 and ifa1 to be allelic through genetic complementation (Supplementary Fig. 1) and by sequencing the drl1 locus in ifa1 mutant plants23. Additionally, we observed florets with higher-order branching in drl1-R; zag1-mum1 double mutants, where floret axes displayed iterative secondary and tertiary branch-like lateral growth from the axils of ectopic palea or bracts (Supplementary Fig. 2), corroborating a previously reported synergistic interaction in ifa1; zag1-mum1 double mutant florets11. drl1 and its paralogous genetic enhancer locus, drl2, encode CRC co-orthologs in the YABBY family of transcriptional regulators23.
Genetic combinations between drl1 alleles and the loss of- or low-function drl2-Mo17 natural variant allele (hereafter referred to as drl2-M) or the strong drl2-DsD08 transposon allele23 enhanced all aspects of the drl1 floret phenotype, such that florets from double mutants displayed multiple, expanded nucelli that appeared to originate from sustained FM activity in the upper floret (UF) (Fig. 1c,d and Supplementary Fig. 3). Similarly, an extreme loss of determinacy in florets was also observed in zag1-mum1; drl1-R; drl2-M triple mutants (Supplementary Fig. 2e). The synergistic genetic interactions between drl1 and drl2 mutant and natural variant alleles in florets were dose-sensitive, consistent with dosage effects observed for leaf traits23. Varying the dosage of drl2-M in an F2 population resulted in florets of drl1-R; drl2-M/+ plants being intermediate in severity between drl1-R homozygotes and drl1-R; drl2-M double homozygotes (Fig. 1c). In support of these observations, classification of DRL1 as a “regulator” in a gene regulatory network (GRN) from integrated transcriptome, proteome and phosphoproteome datasets across a developmental atlas for maize24 revealed high-confidence edges with drl1 and drl2 “targets,” indicative of putative auto- and cross-regulation activities, respectively (Supplementary Fig. 4). Collectively, these observations suggest that the drl loci regulate pistillate floret development and FM determinacy in a dose-dependent manner.
In the drl1 mutant tassel, an ectopic stamen appeared periodically in the UF of sessile (3.14 ± 0.06) and pedicellate (3.05 ± 0.04) spikelets (Fig. 1e,g). Histological examination of mature drl1 mutant spikelets revealed the infrequent extra stamens originated internal to the normally placed and numbered lemma and palea in the outer whorl (Fig. 1f). We did not observe a nucellus in drl1 mutant staminate florets. The minor degree of FM indeterminacy in drl1 mutants was enhanced in drl1; drl2 double mutants where stamen number increased in both the UF and lower floret (LF) of sessile (4.49 ± 0.09, P = 1.0 × 10−19; 3.41 ± 0.08, P = 4.6 × 10−6,respectively) and pedicellate (3.92 ± 0.11, P = 3.0 × 10−10; 3.19 ± 0.08, P = 0.015, respectively) spikelets (Fig. 1e-g). Differences were significant between stamen number in the UF and LF within sessile and within pedicellate spikelets (P < 10−6), and for UFs, between sessile and pedicellate spikelets (P < 10−3) (Fig. 1g). These data suggest that the drl genes participate differentially in determinacy pathways of upper and lower staminate FMs, and that ectopic stamens originate from sustained activity of the mutant FM.
drl1; drl2 double mutant florets displayed phenotypes that were not observed in drl1 single mutants. An ectopic primordium with lodicule-like cellular morphology and vascularization was observed occasionally in position of a presumptive, suppressed adaxial-medial lodicule in the UF (Fig. 1f, right panel, arrowhead), indicating a possible role for drl gene products in imposing zygomorphy20,25. We also observed macrohair-like structures along the apical ridge of drl1-R; drl2-M supernumerary anthers (Supplementary Fig. 5). Macrohair production is generally limited to the adaxial epidermis of the adult leaf blade and is frequently used as a morphological marker for leaf polarity26. Though ectopic structures were infrequent, they lacked the multicellular bases of leaf blade macrohairs27 and were consistently associated with supernumerary anthers with altered morphology. Such amorphic anthers had aberrant theca that lacked pollen sacs and were often fused to morphologically normal anthers.
drl1 and drl2 impose FM determinacy
We tracked the developmental basis of drl1 and drl1; drl2 mutant phenotypes in mid- and later-staged pistillate florets with scanning electron microscopy (SEM). Prior to sex determination, the inner whorl of normal UFs consists of a medial-adaxial Cd primordium (determinate carpel) and two lateral-adaxial Ci primordia (indeterminate carpels), all of which are connately fused (Fig. 2a). This gynoecial whorl is flanked by a whorl of three pre-degenerate stamen primordia. The LF lags in development, here with a FM and recently initiated stamen primordia (Fig. 2a). In mid-staged UFs of drl1 mutants, the medial-adaxial Cd failed to initiate (Fig. 2b), resulting in the single protruding nucellus observed in mature drl1 mutant florets (Fig. 1d). drl1 mutant UFs had extra whorls of lateral Ci (Fig. 2c); however, shifts in phyllotaxis between each extra whorl complicated assigning ab- or adaxial orientation relative to the palea axil. The medial-adaxial Cd was similarly suppressed in mid-staged UFs of drl1; drl2 double mutants, yet they displayed multiple whorls of lateral Ci indicating prolonged FM activity (Fig. 2d-f). Additionally, ectopic primordia, interpreted to each be unexpanded nucellus based on position, initiated in the axil of each Ci whorl of drl1; drl2 double mutants (Fig. 2d-f, asterisks), and likely accounts for the multiple, expanded nucelli observed in mature drl1; drl2 double mutant florets (Fig. 1d).
In normal later-staged UFs, lateral-adaxial Ci primordia appear as bi-keeled (paired) and elongate, while the reduced medial-adaxial Cd envelops the ovule (Fig. 2g). Multiple whorls of paired lateral-adaxial Ci primordia were obvious in similarly staged drl1 mutant UFs (Fig. 2h), whereas tri-keeled lateral-adaxial Ci primordia observed in later-staged UFs of drl1; drl2 double mutants indicated the presence of an extra, intra-whorl fused or partially fused Ci primordium (Fig. 2i). Additionally, we often detected involution of the palea along the medial axis in drl1; drl2 double mutant florets (Fig. 2e,i, arrowheads), which may indicate crowding within the inner whorl of the floret. Taken together, these observations document that the drl genes are required for proper elaboration of pistillate florets, including Cd initiation, and to impose FM determinacy.
drl genes are expressed solely in lateral primordia and dynamically throughout inflorescence development
The drl genes are required for carpel development and to impose FM determinacy (Figs 1 and 2). To examine the temporal and spatial patterns of drl transcript accumulation during inflorescence and floret development, we performed RNA in situ hybridization. In median longitudinal sections of the developing ear, drl1 transcripts were detected in the IM periphery, which spatially correspond to cryptic bract anlagen28 (Supplementary Fig. 6a). drl1 transcripts continued to accumulate in outer glume primordia (Fig. 3a), but not in the SM, as marked by accumulation of knotted1 (kn1) transcripts29 (Fig. 3b). The accumulation pattern of drl1 transcripts persisted in later-staged SMs where they were detected in lemma and palea primordia (Fig. 3c,d), expression patterns that were also observed for drl2 (Supplementary Fig. 6b,c). In more advanced pistillate florets, drl1 transcripts accumulated in carpel primordia that had initiated in the UF and LF, but not in the central presumptive ovule primordium of either floret (Fig. 3e,f). In developing staminate florets, drl1 transcripts accumulated in a similar pattern in lateral primordia that were initiated by the FM (Fig. 3g), but not within the FM (Fig. 3h). drl expression dynamics across developing inflorescences were supported using publicly available RNA-seq data (Supplementary Fig. 6d) (www.maizeinflorescence.org). To summarize, the drl genes are expressed in cryptic bracts, in lateral organ primordia initiated by the SM (glumes), and in primordia of outer- (lemma and palea) and inner- (carpels) whorl organs initiated by the FM. drl expression in carpel primordia correlates with the organs that were altered in drl1 and drl1; drl2 mutant florets. The indeterminate FMs observed in drl mutant florets are best explained by misregulation of FM activity, yet drl expression is limited to organs derived from the meristems and is excluded from the meristem. These points strongly suggest that drl regulates meristem activity via a non-cell autonomous mechanism. Consistent with this hypothesis, drl1 and drl1; drl2 mutants also display a dose-dependent reduction in vegetative shoot apical (SAM) meristem size even though the drl genes are expressed in leaf primordia and not in the SAM proper23.
Expression patterns of floret markers in drl1; drl2 mutants indicate sustained FM activity
We performed RNA in situ hybridization with floret marker genes to understand the basis for the indeterminacy and potential organ identity shifts in pistillate florets of drl mutants. We used drl1-R; drl2-M/+ individuals (Fig. 1c) for these analyses. The maize grassy tillers1 (gt1) gene encodes a homeodomain leucine zipper transcription factor that is required to repress carpel growth in staminate florets30. In developing tassel florets, gt1 is expressed in the central gynoecium30. We examined longitudinal sections through later-staged, normal pistillate florets, where gt1 transcripts accumulated in the central gynoecium and in stamen primordia (Fig. 4a).
In contrast, in drl mutant UFs, gt1 transcripts accumulated broadly throughout the presumptive ovule primordium and to a lesser degree throughout the LFM (Fig. 4b,c). Accordingly, GRN subnetwork analysis24 revealed a high-confidence edge score for gt1 as a putative target of DRL1 (Supplementary Fig. 4). The maize APETALA3/DEFICIENS ortholog, silky1 (si1), is required for lodicule and stamen identity, and in staminate and pistillate florets is expressed in developing lodicule and stamen primordia31,32, where si1 transcripts accumulate asymmetrically25. As expected, we found that si1 transcripts accumulated in degenerating stamen primordia in the normal pistillate UF and in the LF, whereas in drl1-R; drl2-M/+ the accumulation pattern was broader and included ectopic primordia in the UF and strong expression throughout the LF (Fig. 4d-f). The zag1 gene regulates FM determinacy with a lesser role in promoting carpel identity; currently, functional analyses have not been reported for its duplicate factor zea mays mads2 (zmm2)7. zag1 and zmm2 expression domains overlap largely throughout the development of pistillate florets, where they mark the FM as well as stamen and carpel primordia32,33.
Expression patterns for zag1 and zmm2 differ in developing staminate florets25. We observed that zag1 transcripts accumulate in stamen and carpel primordia of drl1-R; drl2-M/+ late-staged pistillate UFs and in the LF, whereas in normal florets, accumulation was largely confined to the incipient silk (Fig. 4g-i). Transcripts of zmm2 accumulated in a similar pattern in normal and drl1-R; drl2-M/+ developing pistillate florets (Fig. 4j-l). Interestingly, GRN subnetwork analysis24 revealed high-confidence edge scores for 23 annotated MADS-box genes that included si1, zag1 and zmm2 as putative DRL1 targets; GRN analysis24 also predicted ZAG1 as a putative regulator of drl1 and drl2 genes (Supplementary Fig. 4). The FILAMENTOUS FLOWER homolog, zea yabby15/yabby8 (zyb15/yab8)23,26 is expressed during inflorescence development in cryptic bract primordia28. We observed zyb15 transcript accumulation in glume, lemma, palea and carpel primordia, but not in the FM or in stamen primordia, for both normal and drl1-R; drl2-M/+ developing pistillate UF and LF (Fig. 4m-o). Interestingly, zyb15 transcript accumulation persisted longer in glume primordia compared to drl1 accumulation (Fig. 4m cf. 3e). The kn1 gene is expressed in meristematic cells and is downregulated in cells recruited to form a lateral domain on the flank of the meristem and in lateral organ primordia29. We observed that kn1 transcripts were absent from normal later-staged pistillate UFs that had undergone terminal differentiation to an ovule primodium, whereas in similarly staged drl1 mutant UFs, kn1 transcript accumulation persisted throughout the gynoecial axis, demonstrating the drl1; drl2 FMs are indeterminate (Fig. 4p-r). In summary, the expression patterns of marker genes in developing drl1-R; drl2-M/+ pistillate florets shed light on the genetic basis of indeterminate floret growth in drl1-R; drl2-M double mutants. We observed notable shifts from normal patterns of transcript accumulation for gt1 where transcripts accumulated more broadly throughout the indeterminate pistillate floral axis (Fig. 4b,c), for si1 where the pattern of ectopic accumulation may indicate that in drl1-R; drl2-M mutants, extra stamen primordia initiate in pistillate UFs or lodicule primordia are de-repressed (Fig. 4f), and for kn1, where prolonged transcript accumulation marked the drl1-R; drl2-M/+ indeterminate floral axis (Fig. 4q,r).
drl genes interact with the pistil abortion pathway
Maize is monoecious with unisexual florets borne on the terminal tassel and lateral ear. Tassels resemble ears in the tasselseed1 (ts1) mutant in that feminization of tassel spikelets reduces glume growth, arrests stamen growth, and permits development and growth of a fertile, determinant gynoecial whorl34,35. The ts1 gene encodes a lipoxygenase that may act during the biosynthesis of jasmonic acid36. Laudencia-Chingcuanco and Hake (2002) reported an occasional determinant nucellus in staminate florets of ifa1 mutants. In drl1; drl2 mutants, we observed extra anthers indicative of reduced FM determinacy in staminate florets; however, with multiple expanded nucelli, loss of FM determinacy was more evident in pistillate florets (Fig. 1). To test for an interaction between pathways that regulate FM determinacy and pistil abortion, we examined F2 progeny between drl1-R; drl2-M double mutants and the ts1-Alex mutant. Tassel florets of ts1-Alex; drl1-R; drl2-M triple mutants were feminized like ts1-Alex, but additionally showed many hallmarks of drl1; drl2 double mutant pistillate (ear) florets: reduced, unfused carpels and multiple ectopic nucelli in the UF and LF of the tassel spikelet (Supplementary Fig. 7). Thus, ts1-Alex markedly enhanced the indeterminacy of drl1-R; drl2-M mutant tassel florets (Supplementary Fig. 7b,c). Interestingly, in triple mutants, we consistently observed nucelli in tassel florets were less expanded than nucelli in ear florets, indicating that additional factors may contribute differentially to nucellus growth in ear versus tassel florets. Taken together, the degree of FM indeterminacy differs in drl1; drl2 double mutant staminate and pistillate florets, but that difference diminishes substantially when pistil abortion is suppressed in tassel florets via the ts1-Alex mutation. These observations suggest that the drl genes control stem cell proliferation perhaps by integrating hormonal cues such as from lateral primordia, and that specific integration mechanisms in staminate and pistillate florets may respond to different hormone levels or sensitivities34,35.
drl and knox pathways interact genetically to regulate FM activity
Recessive loss-of-function mutations in kn137, or dominant gain-of-function mutations in the class I kn1-like homeobox (knox) genes Gnarley138 or Liguleless3-O (Lg3-O) (this study, Fig. 5) can displayectopic carpels in pistillate florets, with no reported changes in stamen number in tassel florets. Similarly, pistillate florets of recessive loss-of-function mutations in rough sheath2 (rs2), a negative regulator of knox genes39-41, occasionally display multiple silks, while stamen number remains normal in tassel florets (Supplementary Fig. 8)39. To determine whether the drl genes function in the knox pathway or pathway(s) that regulate KNOX activity, we examined the genetic interactions between drl1; drl2 and Lg3-O or rs2-R mutants.
Stamen number in Lg3-O mutants did not deviate from normal (Fig. 5a cf. 1g). However, we observed significant enhancement of ectopic stamens in UF and LF of both sessile and pedicellate spikelets of Lg3-O; drl1-R; drl2-M triple mutants compared to drl1-R; drl2-M double mutants (Fig. 5a cf. 1g). Similar to what we observed in drl1-R; drl2-M double mutants, for Lg3-O; drl1-R; drl2-M triple mutants the differences in stamen number between UF and LF were highly significant within sessile and pedicellate spikelets (P < 10−2) (Fig. 5a). In pistillate florets of Lg3-O mutants, we found supernumerary carpels that formed partial, extra pistils, which occasionally did not contain an ovule (Fig. 5b), an observation not reported previously42,43.
Dissected pistillate florets from Lg3-O; drl1-R double and Lg3-O; drl1-R; drl2-M triple mutants revealed ectopic, unfused carpels that failed to enclose a central nucellus (Fig. 5c-e).
Interestingly, the multiple nucelli that we observed in triple mutants were reduced in their growth compared to those in drl1-R; drl2-M double mutants (Fig. 5d,e cf. 1d). SEM analysis of developing Lg3-O; drl1-R double mutant pistillate florets revealed carpel elongation from both Ci and Cd gynoecial ridges (Fig. 5f), whereas in Lg3-O; drl1-R; drl2-M triple mutants, multiple whorls of Ci were apparent around the central FM axis (Fig. 5g,h), which led to consumption of the FM in some florets (Fig. 5g). In some Lg3-O; drl1-R; drl2-M triple mutants, the stamen whorl was aberrantly arranged within the palea axil (Fig. 5h). Collectively, these data indicate that misexpression of lg3 enhances aspects of the drl1-R; drl2-M staminate and pistillate floret phenotypes. However, because Lg3-O is characterized by a gain-of-function mutation, an exact functional relationship between the drl genes and lg3 is difficult to interpret. The floret phenotypes in Lg3-O and drl higher-order mutants could indicate that these genes operate in parallel pathways or in the same pathway.
We generated rs2-R; drl1-R; drl2-M triple mutants to ask if the drl genes intersect with, or operate in parallel to, the ASYMMETRIC LEAVES1/PHANTASTICA ortholog rs2, which is required to repress multiple knox gene activities, including lg3, at sites of organ initiation39-41. In pistillate florets, rs2 transcripts accumulate in lateral organs initiated by the SM and FM40 in domains comparable with drl expression (Fig. 3). We found that stamen number in rs2-R did not deviate from normal and observed significant differences in ectopic stamen number between UF and LF within both sessile and pedicellate spikelets of rs2-R; drl1-R; drl2-M triple mutants (P < 10−4; Supplementary Fig. 8a). The differences were greater than those observed in drl1-R; drl2-M double mutants, implying enhancement by rs2-R. Interestingly, the arrangement of inner whorl organs such as lodicules, stamens and ectopic stamen-like structures was irregular in staminate florets of rs2-R; drl1-R; drl2-M triple mutants, and lodicules tended to be amorphic (Supplementary Fig. 8b). Ectopic stamens and stamen-like structures often derived from a central site in the inner whorl and were frequently fused along their filaments. Dissected rs2-R pistillate florets can have multiple silks with fused carpels (Supplementary Fig. 8c)39, whereas rs2-R; drl1-R; drl2-M pistillate florets displayed extreme indeterminacy, with numerous carpelloid-like structures that appeared to derive from multiple origins in the UF (Supplementary Fig. 8d). These carpelloid-like structures were reduced, fleshy, and did not appear to surround a central nucellus, which was conspicuously absent compared to drl1-R; drl2-M pistillate florets (Fig. 1d). Taken together, these results indicate the drl and rs2 genes likely converge on shared targets to regulate staminate and pistillate FM determinacy.
Discussion
Floret architecture in the cereals is a major component of yield. Elegant dissections of Arabidopsis CRC and rice DL gene function have contributed to our understanding of how these YABBY genes regulate floral development across species with perfect, bisexual flowers12,13,14,44. Our results demonstrate that the maize drl genes are required to impose FM determinacy and for proper elaboration of inner whorl organs of staminate and pistillate florets, indicating critical roles for drl genes in regulating stem cell homeostasis and organ growth in dimorphic, unisexual florets (Figs 1 and 2). We hypothesize the drl gene products function non-cell autonomously in or through pathways that signal from lateral primordia, through boundary domains, to regulate developmental programs of the FM. Our findings suggest that non-cell autonomous function of drl interacts differentially with the distinct developmental potentials of staminate UFMs, LFMs and pistillate UFMs (Figs 1-3 and 6). UFs and LFs differentially express key regulators45,46, potentiate differential effects of developmental regulators10, and derive from slightly different developmental trajectories of the SM47. Perhaps akin to the maize ZmFON2-LIKE CLE PROTEIN1 (FCP1)-FASCIATED EAR3 primordia-to-meristem feedback circuit48, a feedback signaling system from SM- and FM-derived lateral primordia involving the drl gene products could provide vital control of stem cell proliferation by integrating hormonal or metabolic cues from incipient and emerging primordia. With some 48 CLE genes currently reported in maize49, it is tempting to speculate that differential interactions between drl and CLE genes and/or gene products could provide non-cell autonomous regulation of FM activity from lateral floral primordia. Indeed, GRN subnetwork analysis24 uncovered high-confidence edge scores for CLE-FCP1 genes as putative DRL1 targets (Supplementary Fig. 4). In broader context, a recent report in Caenorhabditis elegans underscores how non-cell autonomous signaling from somatic to adjacent germline tissue regulates stem cell proliferation in the germline50, implying a common theme in development used to control stem cell proliferation.
Methods
Genetic stocks and plant growth
Maize plants were grown in the field or in the greenhouse. The drl1 and drl2 alleles used in this study were described previously23. drl alleles were backcrossed to A619, B73, Mo17 and W22 inbred lines at least four times. The Lg3-O (B73-5) allele was obtained from Michael Muszynski (Iowa State University). The rs2-R (Mo17-many) and ts1-Alex (Mo17-4) alleles were obtained from Erik Vollbrecht (Iowa State University). The ifa1 (B73-4) allele was obtained from Sarah Hake (UC-Berkeley). The zag1-mum1 (B73-many) allele was obtained from David Jackson (Cold Spring Harbor Laboratory). For quantitative phenotyping, sample sizes per genotype are indicated throughout the manuscript, along with mean ± s.e.m. presented with significance calculated using two-tailed Student’s t tests. All experiments were performed with two or three independent biological replicates.
Genetic interaction analysis
Higher-order mutants were generated using the drl1-R and drl2-M alleles and ifa1, zag1-mum1, ts1-Alex, Lg3-O, or rs2-R alleles. The F1 progeny from these crosses were grown to maturity and self-pollinated or backcrossed. The F2 or BC1 progeny were grown to maturity and screened for the drl1-R and drl2-M alleles by genotype and for the tester mutant alleles by phenotype, except for Lg3-O (Lg3_13-CGTCCATTTCCCATCCCCAA and Lg3_6-CCTTGCGGCACTCGATGTA), rs2-R (rs2_F3-CGCATTATGAGGTGTGGTGG and rs2_R1-CTCCATCTCCAGCTGCTGC) and zag1-mum1 (zag1_F2-GGAATCTGCTAGGCTGAGGC and zag1_R2-GGTCGTTGAAGTCTTTCCGG) alleles, which were genotyped. Genotyping primers for ifa1, drl1-R and drl2-M, as well as DNA isolation and PCR conditions were described previously23.
Histology
Toluidine blue O (TBO) (Sigma) staining was performed on mature spikelets. Briefly, TBO was dissolved in 1% sodium borate (w/v) to make a 1% stock solution (w/v). A 0.5% TBO staining solution was made immediately before use by diluting the stock solution with 1% sodium borate. Microtome sections of 10 µm, adhered to a microscope slide, were deparaffinized in Histo-Clear (National Diagnostics) (2 times, 10 min. each). Slides were passed through a graded ethanol series toward hydration, 1 minute each (100%, 100, 95, 95, 70, 50, distilled water) and stained in 0.5% TBO staining solution for 3 minutes. Slides were then passed through a graded series toward dehydration, 30 seconds each (50%, 70, 95, 95, 100, 100) and Histo-Clear (3 times, 5 min. each). Slides were coverslip mounted with Permount (Fisher).
Scanning Electron Microscopy
Field-grown ears 10 mm in length were fixed with 2% paraformaldehyde and 2% glutaraldehyde in cacodylate buffer (0.1 M) at pH 7.2 for at least 24 hours / 4 ˚ C. After fixation, samples were rinsed 3 times (15 min. each) in cacodylate buffer (0.1 M). Then samples were post-fixed in 1% osmium tetroxide in cacodylate buffer (0.1 M) for 1 hour. After several washes with deionized water, samples were dehydrated through a graded ethanol series (25%, 50, 70, 85, 95, 100) 2 changes each for 15 minutes. Samples were critical point dried using a Denton Vacuum, Inc. Drying Apparatus, Model DCP-1 (Denton Vacuum, Moorestown, NJ). Dried materials were mounted on aluminum stubs with double-sided tape and colloidal silver paint and sputter coated with gold-palladium with a Denton Desk II Sputter Coater (Denton Vacuum, Inc. Moorestown, NJ). Images were captured using a JEOL JSM-5800LV scanning electron microscope at 10 kV (JEOL = Japan Electronic Optics Laboratory, Peabody, MA).
RNA in situ hybridization
Field-grown 10 mm maize ears were fixed overnight at 4 ˚ C in 3.7% FAA. Samples were dehydrated through a graded ethanol series (50%, 70, 85, 95, 100) each 1 hour, with 2 changes in 100% ethanol. Samples were then passed through a graded Histo-Clear (National Diagnostics) series (3:1, 1:1, 1:3 ethanol: Histo-Clear) with 3 changes in 100% Histo-Clear; all changes were 1 hour each. Samples were then embedded in Paraplast®Plus (McCormick Scientific), sectioned, and hybridized as described previously23. Hybridizations were performed using antisense digoxygenin-labeled RNA probes: drl123, drl223, kn129, gt130, si125, zag125, zmm225, and zyb15/yab8 (JS137-CGATCTCTACGCCGCAGC and JS138-GCAGACATACGCAAACATGGG).
Accession numbers
Maize: drl1, GRMZM2G088309; drl2, GRMZM2G102218; gt1, GRMZM2G005624; kn1, GRMZM2G017087; lg3, GRMZM2G087741; rs2, GRMZM2G403620; si1, GRMZM2G139073; ts1, GRMZM2G104843; zyb15/yab8, GRMZM2G529859; zag1, GRMZM2G052890; zmm2, GRMZM2G152862.
Author contributions
J.S. designed and performed the experiments, and analyzed the data with guidance from E.V.
J.S. wrote the article with edits from E.V.
Acknowledgements
We thank Harry Horner and Tracey Stewart at the Iowa State University Bessey Microscopy Facility for assistance with scanning electron microscopy and Pete Lelonek for plant care. Many thanks to Clint Whipple for generously sharing gt1, si1, zag1 and zmm2 probes for RNA in situ hybridization, and to Beth Thompson for discussions on ifa1. We thank Justin Walley for helpful discussions on incorporating GRN analysis. We are grateful for the many former undergraduate students, especially Sarah Briggs, Emery Peyton and Charlie Beeler, for their help in our summer genetics nurseries. We also thank Jim Cahill for the Lg3-O genotyping assay. Many thanks to Erin Irish and Erica Unger-Wallace for insightful discussions and comments on the manuscript. This work was supported by the National Science Foundation (IOS-1238202).