We previously identified a large number of genes in Arabidopsis with nectary-enriched expression profiles (Kram et al., 2009), and have subsequently used a large-scale reverse genetics approach to identify factors controlling nectar production in the Brassicaceae. This report describes initial efforts to characterize the involvement of PIN6 and MYB57 in Arabidopsis nectary function.
The canonical function of the PIN family of proteins is polar auxin transport, which controls differential growth and cellular response via establishment of auxin gradients (Feraru and Friml, 2008; Kleine-Vehn and Friml, 2008; Krecek et al., 2009; Petrasek and Friml, 2009; Robert and Friml, 2009; Vanneste and Friml, 2009; Friml, 2010; Grunewald and Friml, 2010; Wabnik et al., 2011a). PIN6 has been characterized as an irregular PIN protein that does not follow the typical intron–exon motif of most characterized PINs. Specifically, PIN6 has a shorter hydrophilic loop in the middle of its protein structure; however, like PIN1 and PIN4, it has ten transmembrane domains (Paponov et al. 2005) and demonstrates in vitro auxin transporter activity (Petrasek et al., 2006). Nonetheless, a biological function has not yet been ascribed to PIN6.
The role of PIN6 in the nectary auxin response
Microarray and reporter assays demonstrated that PIN6 is highly up-regulated in nectaries (Figures 1 and 5), with other PIN genes being expressed at very low levels (Table 1 and Table S1). This specificity of expression suggested a very particular role for PIN6 and auxin in nectary function, which was supported by several findings in this study as discussed below.
Three independent pin6 T–DNA mutant alleles with differing PIN6 expression levels were examined here. Interestingly, the level of PIN6 expression positively correlated with nectar production (Figure 2). To obtain an explanation for this phenotype, we first examined floral and nectary morphology. An attractive initial hypothesis was that PIN6 activity may affect nectary size, which in turn would affect nectar production. Indeed, pin6–2 often did have smaller nectaries and petals that failed to fully expand; however, no noticeable differences in nectary size or morphology were observed for pin6–1 (knock-up mutant) or pin6–3 (knock-down), even though they had contrasting nectar secretion phenotypes.
These results suggest that PIN6 and auxin may play a direct role in the regulation of nectar production rather than singularly affecting nectary development, a conclusion supported by several findings in this study. For example, a very strong DR5:GFP signal was observed in post-anthesis nectaries of the Col–0 background, which co-localized with PIN6–GFP at nectary tips (Figure 5). This observation is consistent with previous studies suggesting that the floral nectaries of multiple plant species produce indoleacetic acid (IAA) immediately prior to anthesis (Endress, 1994; Aloni et al., 2006). Aloni et al. (2006) used DR5:GUS expression analyses as a proxy to follow the initiation and progression of free auxin production in floral organs throughout development, including nectaries. The authors suggested that free IAA has two primary functions in flower development: (i) promotion of growth within the organs that produce auxin, and (ii) repression of development in adjacent organs that do not produce auxin (Aloni et al., 2006). One conclusion of their study was that auxin production shifts from anthers to nectaries at anthesis. It was suggested that IAA derived from anthers in pre-anthesis Arabidopsis flowers prevents nectar secretion until anthesis, whereupon nectaries become the primary sites of auxin synthesis in flowers (Aloni et al., 2006). However, it is important to note that DR5-based reporters are only a proxy for the auxin response, not auxin synthesis itself. Thus, nectaries may sequester auxin from surrounding tissues instead of directly synthesizing auxin, thereby controlling auxin homeostasis and response. Indeed, an analysis of the RNA-seq data in Table S2 indicates that genes involved in IAA biosynthesis (Mano and Nemoto, 2012) are expressed at very low levels (Table S4, most of these genes are near or in the lowest quartile of all genes for total RNA-seq counts). This analysis suggests that nectaries may not produce large amounts of free IAA; however, more studies are required to determine whether or not nectaries are active sites of auxin synthesis.
Another piece of evidence for a role for PIN6 and auxin in nectary function was that the auxin-dependent DR5:GFP signal in both the pin6–1 (knock-up) and pin6–2 (knock-out) alleles was significantly decreased in mature lateral nectaries (even when the nectary morphology was normal in pin6–2), whereas median nectaries appeared unaffected (Figure 5). This result was unexpected because: (i) pin6–1 and pin6–2 have opposite expression levels and nectar secretion phenotypes relative to wild-type Col–0, and (ii) PIN6 is highly expressed in both median and lateral nectaries, so it is expected that both nectary types would be affected equally. PIN6 is most closely related to PIN5, which was previously localized to the ER membrane (Mravec et al., 2009), thus differing from the plasma membrane localization of other described PINs. Mravec et al. (2009) also showed via transient expression assays that PIN6 and PIN8 appear to be located in the ER of tobacco BY–2 cells. The observed localization patterns of PIN6pro:PIN6–GFP (Figure 5e,f) are consistent with the previously suggested ER localization; unfortunately, attempts to observe co-localization of PIN6–GFP with ER-specific dyes (e.g. ER-Tracker Red) and even diamidino-2-phenylindole (DAPI; nuclear stain) were unsuccessful because the thick cuticle covering the nectaries prevented staining in sub-epidermal cells.
The presence of PIN6 on the ER membrane suggests that it may play a role in intracellular auxin homeostasis, as demonstrated for PIN5 (Mravec et al., 2009) and later expanded upon from a mechanistic perspective by Wabnik et al. (2011b). Thus, the function of PIN6 may be to sequester auxin from the cytosol into the lumen of the ER, thereby modulating cytosolic auxin concentrations and its cellular availability for auxin-dependent processes such as the SCFTIR1/AFB signaling pathway (Gray et al., 2001; Dharmasiri et al., 2005; Kepinski and Leyser, 2005). This idea is supported by the finding that, in the pin6–1 mutant, which has nearly a twofold increase in PIN6 transcript (Figure 2), the DR5:GFP signal is significantly reduced in lateral nectaries (Figure 5), and did not respond to exogenously applied auxin (Figure 6). Thus, the increased PIN6 expression in pin6–1 may result in decreased cytosolically available auxin and a concomitant reduction in SCFTIR1/AFB signaling. This notion is further supported by the finding that the auxin co-receptor mutant tir1–1 (Ruegger et al., 1998) phenocopied pin6–1 with regard to nectar secretion, secreting significantly higher amounts of nectar than Col–0 (Figure 2).
Additionally, one may expect auxin-responsive genes to be down-regulated in pin6–2 nectaries (due to decreased DR5:GFP signal in pin6–2 nectaries, Figure 5); indeed, seven of 271 genes with more than twofold higher counts in Col–0 versus pin6–2 nectaries were annotated as being auxin-responsive via GO annotation at http://arabidopsis.org/tools/bulk/go/index.jsp (data from Table S2; only genes with counts in the top half of all genes expressed in nectaries were analyzed due to low counts in remaining genes). These genes included At4g30080 (auxin response factor 16), At4g36740 (homeobox protein 40), At3g11820 (syntaxin of plants 121), At4g23570 (phosphatase-related), At4g37390 (auxin-responsive GH3 family protein), At5g35735 (auxin-responsive family protein) and At2g33830 (dormancy/auxin-associated family protein). However, enrichment of auxin-responsive genes was not statistically significant, which suggests that at least part of the auxin response in nectaries may be independent of transcriptional processes.
As mentioned above, whereas exogenously applied auxin (via peduncles placed in 100 μm NAA in 10% sucrose solutions) resulted in a large increase in nectar production in Col–0, it caused a significant reduction in pin6–1, no change in pin6–2, and a small but statistically insignificant increase in pin6–3 (Figure 6). The results we observed with wild-type flowers were consistent with a previous study in which exogenous auxin and gibberellic acid (GA3) (applied by spraying, not in cultured flowers) caused significant increases in nectar volume, nectar sugar concentration, dry nectar sugar mass, insect visitation abundance and seed yield in two species closely related to Arabidopsis (Brassica campestris and Brassica oleracea) (Mishra and Sharma, 1988). Other studies on excised flowers of snapdragon (Antirrhinum majus) supported a conflicting role for auxin in inhibiting nectar secretion (Shuel, 1959, 1964, 1978); however, under some conditions, exogenous IAA resulted in an increase in nectar production, suggesting a dual role for auxin in nectar production (Shuel, 1964). Whether these conflicting observations result from species differences (Brassicaceae versus snapdragon) or experimental design (the snapdragon studies used 500 μm IAA, whereas this study used 100 μm NAA) are unclear; however, we observed a positive correlation between NAA concentration and nectar production in our sucrose feeding experiments up to 100 μm NAA in 10% sucrose solutions, with a sharp decline at higher NAA concentrations (Peter M. Klinkenberg and Clay J. Carter, unpublished data). Significantly, through 14C-labeled IAA and sucrose experiments, Shuel (1978) concluded that exogenously applied auxin affects the secretory process itself within nectaries, rather than the movement of sugars to nectaries.
In contrast to NAA application, the auxin transport inhibitor NPA caused a significant decrease in nectar production in Col–0, but not in any of the pin6 mutants (Figure 6). The accumulation of PIN6 and auxin at nectary tips was also intriguing, and suggests that PIN6 may also play a role in defining nectary polarity. The reason for PIN6 expression in the distal nectary is uncertain; however, other nectary-enriched genes, such as CELL WALL INVERTASE 4 and a sesquiterpine synthase (At5g44630), appear to be expressed throughout the entire nectary (Tholl et al., 2005; Ruhlmann et al., 2010), suggesting that sub-domains exist within the Arabidopsis nectary. Cumulatively, these results indicate that fine-tuned control of auxin concentration in the sub-epidermal nectary parenchyma is essential for proper nectary function.
As to why median nectaries still have a strong DR5:GFP signal in pin6 mutant backgrounds, it should be noted that lateral nectaries secrete >99% of nectar in Arabidopsis, and there are also clear differences in gene expression and development between median and lateral nectaries (Davis et al., 1998; Kram and Carter, 2009; Kram et al., 2009). For example, a cupin-family gene, At1g74820, was previously found to be highly up-regulated in median versus lateral nectaries (Kram et al., 2009). RNA-seq analysis in this study identified At1g74820 as being expressed at a level that was 24-fold higher in the mature lateral nectaries of pin6–2 compared with Col–0 (Table S2). The cupin family of genes includes the auxin receptor AUXIN BINDING PROTEIN 1 (Shi and Yang, 2011) and other auxin binding proteins (Ohmiya, 2002). Thus, it is possible that At1g74820 may play a role in regulating the auxin response in nectaries.
The role of PIN6 and MYB57 in short stamen development
We also found that the pin6–2 and myb57–2 mutants partially phenocopied one another in having significantly reduced nectar production, nectary size and short stamen presence; however, differences between these two lines included that pin6–2 had petals that failed to fully expand (Figure 3), whereas myb57–2 had petaloid short stamens (when short stamens were present). Since PIN6 is expressed at normal levels in both myb57–2 whole flowers and mature lateral nectaries, it is unlikely that MYB57 directly regulates PIN6 expression (Figure S1 and Table S2). However, the anther portion of petaloid stamens in myb57–2 stained strongly in the DR5:GUS background in mature flowers, and PIN6 was also found to be mis-expressed in myb57–2 petaloid stamens instead of only being expressed in the nectaries of wild-type Col-0 flowers. As PIN6 is also expressed in immature stamens of wild-type plants (Figure 1), which coincides with auxin production or response in stamens at this stage (Aloni et al., 2006), these results are not necessarily surprising. It is currently unknown whether PIN6 activity is required for the petaloid short stamen phenotype observed in myb57–2, or whether PIN6 mis-expression in myb57–2 short stamens is a consequence of the petaloid phenotype. Nonetheless, the observed role of MYB57 in the proper development of short stamens is consistent with previous results, as MYB57 involvement in stamen elongation, through redundant action with MYB21 and MYB24, has been demonstrated previously (Cheng et al., 2009). Interestingly, MYB21 and MYB24 are also highly expressed in nectaries and are required for proper floral maturation (Reeves et al., 2012). Indeed, myb21 null mutants do not secrete nectar (Peter M. Klinkenberg and Clay J. Carter, unpublished data).
Cumulatively, these observations suggest the existence of an indirect link between MYB57 and PIN6, as well as a general role for PIN6 in the auxin response in mature flowers. The fact that small under-developed nectaries occur when short stamens are absent is consistent with previous results. Most floral jasmonic acid appears to be produced in stamen filaments (Ishiguro et al., 2001), and is not only required for floral maturation as a whole, including nectaries (Cheng et al., 2009; Reeves et al., 2012), but may also induce floral nectar secretion (Radhika et al., 2010). Indeed, when short stamens are present in pin6–2 and myb57–2, nectary morphology occasionally appears normal (e.g. Figure 5b). The link between stamen presence and nectary development and maturation is unclear; however, one previously proposed model is that jasmonic acid is transported downwards from filaments to the rest of the flower for maturation and expansion of other organs, such as petals (Ishiguro et al., 2001). Regardless, PIN6 activity is clearly required for proper nectary function, even when nectary morpology is normal, as demonstrated by the reduced nectar secretion phenotype of pin6–3 (Figure 2).