Gene expression profiles in different Arabidopsis tissue types have been extensively compared to one another in order to identify tissue-specific gene expression, especially as it relates to tissue function [40, 41]. Significantly, the probe signals from a wide range of independent hybridization experiments can be co-normalized and used to identify differentially expressed genes. For example, the Genevestigator Gene Atlas houses co-normalized probe signal values for ~2,000 Arabidopsis hybridization experiments (from many research groups) representing over 60 different Arabidopsis tissues and cell types . This tool is widely used to examine differential gene expression between tissues, as well as between different growth and treatment conditions, all at the same time (tool currently cited 768 times). However, there is currently no report on gene expression profile comparisons between different nectary tissue types or between nectary and non-nectary tissues.
In this study, we systematically interrogated global differences in gene expression between nectaries and non-nectary reference tissues, as well as between nectary types and developmental stages. Functional classification and analysis of genes upregulated in nectaries versus non-nectary tissues (e.g., Additional file 7), along with genes differentially expressed between secretory and non-secretory nectary tissues (e.g., Additional files 9 and 10) may reveal candidate genes involved in nectar production and secretion. Discussed below are several roles these differentially expressed genes and pathways may play in nectary form and function. To the best of our knowledge, this is the first report of a systematic and global interrogation of any nectary transcriptome.
Not surprisingly, a large number of genes involved in sugar metabolism and processing were differentially expressed between nectary and reference tissues (see Figure 6 and Additional file 7), as well as between nectary tissues themselves (see Additional files 9 and 10). This is in agreement with expectations, as simple sugars are the principal solutes in most nectars. In Arabidopsis phloem sap, the primary sugar is sucrose, while hexoses dominate in the nectar. For example, the sucrose/hexose ratio of Arabidopsis (Col-0) nectar is approximately 0.03 . Resultantly, Arabidopsis nectar would be considered hexose-dominant. The compositional differences between Arabidopsis nectar and phloem photosynthate imply that the phloem "pre-nectar" is modified to yield "mature" nectar, and indeed this proposed process has been supported by a number of studies (as reviewed in ). In order to maintain the net flow of carbohydrates from source tissues (e.g. the leaves) to sink tissues like the nectaries, biochemical and physiological processes must be actively maintaining the sink status of nectaries. For example, Bowman  noted starch accumulation in Arabidopsis lateral nectaries (Stage 14); specifically, the guard cells showed the most intense staining. Moreover, according to Baum et al. , starch-containing plastids are visible in Arabidopsis nectary parenchyma cells from the onset of nectary development, which are apparently degraded just prior to anthesis and nectar secretion . It seems likely that both the modification of phloem sap to nectar and the maintenance of nectaries as a sink tissue are interrelated and even involve many of the same genes.
The coordinated control of sugar transport and metabolism in plant cells and tissues is achieved through the action of sugar modifying enzymes and sugar transporters, both of which play roles in establishing and maintaining sugar concentrations across membranes . For example, invertases are a group of enzymes that hydrolyze sucrose into glucose and fructose, which can then be selectively transported across membranes by hexose transporters and/or help create a sucrose gradient. Significantly, nearly all Arabidopsis invertase genes (both intra- and extracellular) appeared to be upregulated in nectaries, while invertase inhibitor genes seemed to be downregulated in actively secreting nectaries (see Additional file 3). In particular, At2g36190, encoding Arabidopsis thaliana CELL WALL INVERTASE 4 (AtCWINV4), was strongly upregulated in nectaries (e.g., Figure 6, Additional file 7). Previously, AtCWINV4 expression was shown to be high in floral tissues ; however, even within floral tissues, expression in nectaries, as observed by microarray, appears pronounced. It is tempting to speculate that this extracellular invertase is at least partly responsible for the hexose-rich nectars observed in Arabidopsis and related members of the Brassicaceae. It may even play a role in maintaining a high intracellular:extracellular sucrose gradient, thus promoting sucrose transport out of nectariferous cells, along with water and other metabolites. Indeed this is likely the case, as cwinv4 T-DNA mutants fail to produce nectar and show marked differences in starch accumulation within flowers (Ruhlmann et al., submitted).
In addition to invertases, we identified a number of genes upregulated in nectaries involved in other aspects of simple sugar metabolism, with some including: sucrose synthase (SUS1, At5g20830; ~5-fold over reference tissues), putative sucrose-phosphate synthase (At5g11110; ~9-fold), putative UDP-glucose 4-epimerase (AT4G23920; ~18-fold), two UDP-glucoronosyl/UDP-glucosyl transferase family proteins (AT5G26310 and AT4G34138; ~14 and 8-fold, respectively) and hexokinase 2 (HXK2, AT2G19860; ~4-fold). Significantly, these genes can be tentatively assigned functions in sucrose synthesis/degradation (based upon TAIR AraCyc database, ), and are likely involved in defining nectar sugar composition. Indeed, the full canonical sucrose biosynthesis pathway was represented by genes upregulated within mature lateral nectaries over individual reference tissues (see Figure 6). Upregulation of both sucrose synthase  and cell wall invertase (Ruhlmann et al., submitted) within Arabidopsis nectaries was experimentally verified previously.
Transcription processes were also highly represented within nectary expressed genes (e.g., see Figure 5 and Additional file 11), with 45 of these genes displaying nectary-enriched expression profiles (see Additional file 7). Members of the YABBY transcription factor gene family–numbering six in Arabidopsis (CRABS CLAW, FILAMENTOUS FLOWER, YABBY3, INNER NO OUTER, YABBY2, and YABBY5)–are determinants of abaxial cell fate in the lateral floral organs . As previously mentioned, CRABS CLAW (At1g69180, CRC) encodes a transcription factor involved in the regulation of carpel and nectary development . CRC is currently the only known gene to be absolutely required for nectary development; here we have identified several other transcription factors preferentially expressed in nectary tissue that could possibly be involved in either restricting CRC expression to the base of the stamens or in some other aspect of nectary development or function. For example, Lee et al.  state that there is a "lack of evidence for any other YABBY gene family member expressing in the nectaries." However, here we evince the preferential expression of YABBY5 (At2g26580) in nectaries, and since this transcription factor belongs to the same family as CRC, it too could potentially be involved in mediating nectary development; it had significantly higher signal probe intensities in nectaries over the reference tissue average (~58-fold), and appeared to have relatively constant expression throughout the nectary tissues examined by microarray and RT PCR (see Figure 4).
In addition to transcription factors specifically upregulated in nectaries, some displayed differences between nectary type or developmental stage. For example, the only gene upregulated 5-fold or more in MLN compared to MMN was At2g16720, a myb family transcription factor; probe signal intensity of this gene was also increased greater than 5-fold in MLN over ILN, and 9-fold over the reference tissue average. Since transcription factors modulate the expression of other genes, the involvement of this single gene in differentiating MLN from other tissues could be substantial. Conversely, At4g28140, a putative AP2 domain-containing transcription factor, was upregulated in MMN compared with MLN (8-fold) and ILN (23-fold), and was also upregulated over all reference tissues examined (~20-fold). A separate myb gene (MYB115; At5g40360) was highly expressed in both MLN and MMN, but not ILN, with an overall probe signal increase in nectaries over reference tissues of ~28-fold. Potentially, these genes are involved in differentiating median from lateral, or immature from mature nectaries.
Related to the identification of upregulated transcription factors described above, promoter motifs are short DNA sequences that transcription factors bind to in order to affect the expression of other genes. This is significant within a biological context, as a single transcription factor can simultaneously govern the expression of many other genes (e.g., ), provided that the promoter regions of the affected genes contain the DNA sequence motif in question. MYB4 and CArGCW8GAT promoter motifs were particularly overrepresented within the promoters of nectary-enriched genes (see Table 4). Significantly, several CArG boxes were previously identified as key regulators of CRC expression within nectaries . The CArG promoter motif (CCWWWWWWGG, where W = A or T) is the canonical target for AGAMOUS and related MADS box proteins, though the CArGCW8GAT motif variant (CWWWWWWWWG) is a known target of AGAMOUS-LIKE MADS BOX PROTEIN 15 (AGL15) specifically. AGL15 is primarily expressed in developing embryos [61, 62], but is apparently expressed at very low levels within nectaries (data not shown). However, several other MADS box-family genes were highly upregulated in nectaries, including AGAMOUS itself, and the functionally redundant SHATTERPROOF genes, AGL1 and AGL5 (see Additional file 7, RT PCR data not shown). These data are consistent with previous findings [27, 28, 30].
The MYB4 binding motif (AMCWAMC) was also highly represented in the promoters of nectary-enriched genes (316 sites within 94 of 96 promoters analyzed). MYB4 is a direct transcriptional repressor of the cinnamate 4-hydroxylase gene (C4H, At2g30490), and can also suppress the expression of chalcone synthase (CHS) when overexpressed . C4H and CHS are involved in the synthesis of hydroxycinnamate esters and flavonoids, respectively, both of which are ultimately known to provide protection from UV-B radiation [63, 64]. Curiously, by microarray MYB4 was highly upregulated within nectaries (see Additional file 7), whereas C4H and CHS were strongly repressed (by a range of 5 to 100-fold) when compared to reference tissues (see Additional file 3), which supports the known functions of MYB4. Nonetheless, it is tempting to speculate that MYB4, or one of the four other myb family proteins upregulated in nectaries (see Additional file 7), may be involved in the regulation or even activation of nectary-specific genes. Indeed, myb family transcription factors were previously implicated in the regulation of the nectary-specific NECTARIN 1 gene in tobacco . While more work needs to be done, the prevalence of MYB4 and CArGCW8GAT promoter motifs within nectary-specific genes suggests that they may provide a basis for regulating nectary-specific gene expression.
Finally, it should be noted that multiple genes involved in aspects of lipid metabolism [e.g., LTP1 (At2g38540) and GPAT5 (At3g11430)], and auxin transport and response [e.g., PIN6 (At1g77110) and CHY1 (At2g30650)], were identified as being highly upregulated in nectaries by both microarray and RT PCR. These findings are significant in that both lipid and auxin processes have been suggested to play roles in nectary development and nectar secretion (e.g., [52, 65, 66]); however, the exact functions these upregulated genes in nectary function is currently unclear.