Systems analyses of key metabolic modules of floral and extrafloral nectaries of cotton

Nectar is a primary reward mediating plant-animal mutualisms to improve plant fitness and reproductive success. In Gossypium hirsutum (cotton), four distinct trichomatic nectaries develop, one floral and three extrafloral. The secreted floral and extrafloral nectars serve different purposes, with the floral nectar attracting bees to promote pollination and the extrafloral nectar attracting predatory insects as a means of indirect resistance from herbivores. Cotton therefore provides an ideal system to contrast mechanisms of nectar production and nectar composition between floral and extrafloral nectaries. Here, we report the transcriptome, ultrastructure, and metabolite spatial distribution using mass spectrometric imaging of the four cotton nectary types throughout development. Additionally, the secreted nectar metabolomes were defined and were jointly composed of 197 analytes, 60 of which were identified. Integration of theses datasets support the coordination of merocrine-based and eccrine-based models of nectar synthesis. The nectary ultrastructure supports the merocrine-based model due to the abundance of rough endoplasmic reticulum positioned parallel to the cell walls and profusion of vesicles fusing to the plasma membranes. The eccrine-based model which consist of a progression from starch synthesis to starch degradation and to sucrose biosynthesis was supported by gene expression data. This demonstrates conservation of the eccrine-based model for the first time in both trichomatic and extrafloral nectaries. Lastly, nectary gene expression data provided evidence to support de novo synthesis of amino acids detected in the secreted nectars. One sentence summary The eccrine-based model of nectar synthesis and secretion is conserved in both trichomatic and extrafloral nectaries determined by a system-based comparison of cotton (Gossypium hirsutum) nectaries.

nectaries contain virtually no vacuoles (Fig. 4C, E), while at the secretory stage the distal two-208 thirds of the papillae cells become highly vacuolated, especially the head cells (Fig. 4D, H). 209 In contrast to the bracteal and circumbracteal nectaries, the pre-secretory stalk and head cells 210 of foliar and floral nectaries contain large, circular vacuoles in section (Fig. 4A, G), and by the 211 secretory stage these vacuoles become smaller, and more numerous within the cells of the 212 distal two-thirds of the papillae (Fig. 4B, H).   components, which are amino acids, sugar alcohols, lipids, diols, organic acids, esters, and 244 aromatics.

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The major constituents of the four nectars are similar, being hexose-dominant, with an 246 equal molar ratio of fructose:glucose (Table 1). However, the four different nectar types can 247 be distinguished based on the minor nectar metabolites, particularly between the floral and the 248 extrafloral nectars (Supplemental Fig. 1 and 2). Variation between the three nectar categories 249 is clearly illustrated by sucrose abundance, which differs significantly between floral, 250 reproductive extrafloral, and vegetative extrafloral nectars (Table 1). These compositional values. This dataset was refined by applying two selection filters in order to reduce the number 267 of ion-analytes and begin the process of idetifying the chemical nature of each ion. One of 268 these filters evaluated the "reliability" of ion-detection from nectar tissue associated pixels. 269 Namely, ions that were detectable in 5 out of 10 near-adjoining pixels, which were positioned 270 over papillae gland or nectariferous parenchyma nectar tissues were were considered reliable 271 and were retained. The second filter compared the ion-strengths of each ion from tissue 272 associated pixels to the signal strength obtained from non-tissue pixels, retaining only those 273 ions that showed 2-times greater signal strength from tissue-pixels compared to background 274 signal obtained from pixels devoid of tissue. Implementing these criteria reduced the dataset  Approximately 60% of these tentatively identified analytes are phenolic type metabolites, and 281 they are localized near the vasculature within the subnectariferous parenchyma and the 282 epidermis ( Fig. 9). This distribution matches the distribution of subcellular bodies that stain 283 heavily with osmium tetroxide and are visualized by TEM (Fig. 7), confirming their identity 284 as polyphenolic compounds.    Figure 11A). These genes however, did not demonstrate any temporal change

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Collectively therefore, these data suggest that cotton nectaries actively assimilate inorganic 555 nitrogen into the amide moiety of glutamine, which functions as the amino group donor for 556 synthesis of additional amino acids such as alanine, glycine, and branched chain amino acids.   The upregulation of SWEET9 and CWINV4 at the secretory stages of nectary 597 development is also supportive of the eccrine model, and the relative expression levels of these 598 two genes appears to be predictive of whether the nectar product will be hexose-rich. As with   In this study, we analyzed four types of nectaries, the floral, bracteal, and 663 circumbracteal nectaries collected from flowers, and foliar nectaries collected from leaves.

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The developmental trajectory of each nectary type was defined relative to nectar secretion, and to 320 °C, followed by a 5 min hold at 320 °C.

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The second analysis method focused on the less abundant constituents of the nectar, 689 which were extracted from a 5-µL aliquot of nectar sample that was spiked with 0.5 µg 690 nonadecanoic acid and 1 µg ribitol, as internal standards. Hot methanol (2.5 mL) was added to 691 the nectar, and the mixture was incubated at 60 °C for 10 min. Following sonication for 10 min 692 at 4 °C, chloroform (2.5 mL) and water (1.5 mL) were sequentially added, and the mixture was  fractions were set to a helium gas flow rate of 1 mL min -1 , 2 µL injection, with a temperature 704 gradient of 80 °C to 320 °C increasing at a rate of 5 °C min -1 , followed by a 9 min hold at 320 705 °C. The polar fractions were analyzed using a "heart-cut" method which diverted gas flow to 706 an FID detector during elution times for fructose, glucose, and sucrose. Deconvolution and 707 integration of resulting spectra were performed with AMDIS (Automated Mass Spectral 708 Deconvolution and Identification System) software (Stein, 1999). Analyte peaks were 709 identified by comparing mass spectra and retention indices to the NIST14 Mass Spectral  The reads were mapped to the UTX-JGI Gossypium hirsutum genome (v1.1) and 827 predicted transcripts using NCBI's BLASTN (Camacho et al., 2009). The UTX-JGI annotation 828 was used to map read counts to Arabidopsis genes (Araport 11). Read counts were upper-829 quartile normalized, and pairwise differential expression tests were performed using a negative     Table 1 | Predominant sugars, and amino acids in G. hirsutum nectars. Different superscript 875 letters indicate statistically significant differences in abundance (q-value < 0.05).