Nectar Chemistry Part One: Pollinator Macronutrients

Lipids, Carbohydrates & Amino Acids (Plus Non-Protein Amino Acids & Plant Immunity Proteins)

Nectars are carbohydrates predominantly composed of three sugars: sucrose, glucose and fructose. The non-carbohydrate component is minor and differs by species in terms of amino acid, alkaloid, phenols, lipids & secondary metabolite composition, amongst others. Trace elements (vitamins, minerals, alkaloids and other -oids/-enols) are present as well as water, which can be deemed a “nutrient without (direct) nutritional function”.

I have written on how bees make honey (spoiler: it’s from nectar) and how Apis mellifera digests pollen. This article is on nectar’s macronutrient chemistry: carbohydrates (many), amino acids (few) and lipids (fewer still, unless you’re in Patagonia or a yellow loosestrife). There are also non-protein amino acids like GABA and taurine (if a bee was to visit a flower that produced nectar containing taurine & caffeine then he/she is drinking redbull), and nectar proteins involved in plant defence against microbial intruders.

One more thing: Did you know that the common tobacco plant Nicotiana tabacum is a model organism in molecular biology, plant pathology, and genetic engineering? No? Have a puff in its honour. Yes? Do it anyway.

Nectar is a Sugary Secretion Whose Production Depends on Biotic & Abiotic Factors

“Nectar” is the name given to the largely-sugary liquid produced by plants. The “largely sugary” aspect skips over the many trace elements that make each plant’s nectar unique: variabilities in sugar ratios, amino acids, organic acids, minerals, (very small amounts of) lipids, proteins, enzymes, biogenic amines, and the secondary metabolites- alkaloids, phenolics and so on. Nectar is produced daily, sometimes replenished multiple times a day after its removal by pollinators. Nectar’s composition depends on:

• A flower’s age, which influences the quality and quantity of nectar.
  • The number of visitors, as well as their microbial secretions have a direct influence on quality.
    • I wonder if nectar thieves cause a flower to behave differently relative to a “cooperative” visitor?
• The day’s weather; during extremes a plant may not produce nectar at all.
  • Beekeepers are told that plants don’t produce nectar when “it is too hot or too wet (or too cold?) because it’s unlikely that a pollinator will pick it up”. This is true in the case of the forager caste, as they will shift their focus towards maintenance tasks to keep the hive alive.

You can sum up the nectar variations as being based on biotic and abiotic factors: a tomato grown from seed in my backyard will have a different nectar (and pollinator) profile than one grown from the same seed in a pot on a rooftop in downtown LA (even when adjusted for cost of living). These intra-specific variations are reduced in greenhouses, as demonstrated by Canto and colleagues, who showed that columbines (Ranunculaceae family) showed consistently high sucrose, low fructose & (much, much) lower glucose levels from nectar in greenhouse-grown plants relative to their wild counterparts (Canto et al., 2007).

Their field-grown counterparts had large variations in nectary sugars with sucrose dominance¹, followed by fructose and then glucose. I’ve included the authors’ ternary diagram below to help you visualise it.

Nectar Quality is Influenced by Multiple Compounds, Not Just Sugar Content

When beekeepers hear of plants with “high quality nectar”, how are they defining the “quality”? From the beekeeper’s perspective, it means one with a high sugar content (and subsequently “high energy content”), or a high liquid volume which will keep the foragers busy and the middle aged bees buzzy as they make the most of this “nectar flow”.

What of the bee’s perspective? If a “high quality nectar” was the one with the highest sugars, then they would be happy to gorge themselves on sugar syrup feeds ad infinitum, yet they do not, as colonies tend to ignore supplemental feeds during a nectar flow. This implies the availability of  higher-quality forage, even if it is “less “energy-rich” than the syrup (the syrup has both “quality” [dissolved sugars] and “quantity” [“volume of liquid”]). It is the non-sugar components that the bees (or pollinators) value, since sugar, while important, is not the only benefit to pollinators. See summary below from Nicolson, 2022:

Why is Nectar Mostly Sucrose, Some Fructose & A Little Bit Glucose?

A sucrose molecule is made up of one fructose and one glucose molecule. As with the columbines example above, plant nectars are mostly sucrose, followed by fructose and then glucose. Why? Because when a plant photosynthesises it produces glucose (remember that highschool equation?). Fructose forms by enzymatic reshuffling of the glucose molecule, and the two are joined together via a condensation reaction to form sucrose, a more-stable (‘less reactive”) sugar that is transported out of the leaves (the “sources”) into the roots, fruits, or flowers (the “sinks”). At the sinks, it is enzymatically broken down (“hydrolysed”) back to its component sugars glucose and fructose, for use by the plant (e.g. respiration) or stored as starch.

Sugar is transported in the phloem, which provides it to the nectar tissue (there are exceptions). At nectaries, it can be temporary stored as starch for on-demand secretion or broken down by plant cell wall enzymes (invertases) into glucose and fructose in a controlled manner to maintain a concentration gradient (this keeps the sucrose flowing out of the phloem and into the nectaries). One would think that the breakdown of sucrose would lead to nectar with equal parts fructose-glucose, and again this is not the case, as fructose concentrations are low, and glucose even lower, suggesting that the plants reabsorb these sugars rather than keeping them in nectaries, with a bias to glucose as this is simplest carbohydrate unit of energy.

Other sugars exist, however they are minor – sorbitol, ribose, mannitol, arabinose, lactose (in Delphinium peregrinum; violet larkspur), mannose and so on (e.g. Petanidou, 2005 in the Mediterranean biome). Exceptions exist in the Proteaceae family in Australia and South Africa (proteas, banksias, grevilleas…), whose nectar sugar concentrations contained up to 39% xylose, a five-carbon pentose sugar (the ones we’ve covered above are hexose – six-carbon sugars; Nicolson & Van Wyk, 1998).

Amino Acids in Nectar are Negligible. Perhaps They Influence Pollinator Behaviour?

In beekeeping literature, pollen is referenced as the amino acid source for bees and nectar is purely carbohydrates, and this is correct if you’re solely discussing beekeeping literature. You’re here for more biology, so you’ll be (assumedly) unsurprised to know that nectar contains trace amounts of amino acids, with concentrations ranging from 1,000-100,000 times lower than the sugars² (see three-plant comparison by Lohaus & Schwerdtfeger, 2014).

Such small concentrations lead to large variations in the amino acid composition across plant species – of the 20 amino acids, one plant may have more non-essential aminos (for both plant and pollinator), and another the opposite. What is considered an essential amino acid to a bee is not the case for a bat, a bird, a butterfly, a beetle, a blue-tongued lizard, a beagle, a bandicoot or your cousin Robert (“Bobby” to friends and family).

A pollinator does not consume nectar for its amino acid content, so why are there variations in their levels? Why are there amino acids at all? Consider that flowers visited by moths & butterflies (Lepidoptera) tend to have more nectar aminos than those pollinated by other animals. Could this be because Lepidoptera lacks other protein (“nitrogen”) sources, as this order does not consume pollen in any of their life stages? (Mevi-Schutz et al., 2005). A bee or beetle can consume pollen for protein, whereas a bird or bat (or cousin Bobby) can consume animals in addition to plants for their protein source.

Certain Amino Acids are More Attractive Than Others

There are studies showing pollinators preferring a sugar feeder with lower sucrose concentrations if it had a certain amino acid. Before elaborating, you should realise that what’s possible in a laboratory is muddled in real life, hence the literature’s open-ended language: “may”, “could”, “possibly” etc…. Nectar is a mixture, and where laboratory results show an amino to be attractive, in the mixture it may end becoming a repellent, and vice versa.

With this caveat, an umami taste receptor (AmGr10) was described in honey bees being more responsive to glutamate, aspartate, asparagine, arginine, lysine, and glutamine, with “significantly high-expression level[s] in the fat body”, suggesting a physiological “monitor” role on entire bee body’s amino acid content (the fat bodies synthesise haemolymph proteins & metabolise macronutrients; Lim et al., 2019).

AmGr10 is also expressed in the mouthparts, which suggests it contributes towards determining the quality of a pollen’s amino acid content, and authors suggest that “AmGr10 located on the mouthparts is responsible for the sensitivity of honey bees to a broad spectrum of amino acids through cooperative interaction with purine-ribonucleotides [AMP, GMP, IMP etc…]” (Lim et al., 2019). They also acknowledge the lab-vs-field results confounder, in that the receptors are tasting the fatty acids that coat pollen, rather than a “pure” amino acid derived from pollen as in lab settings.

Another honey bee field experiment found that foragers preferred pollen from rapeseed (Brassica napus) over broad beans (Vicia faba), as the former contained more of the “most” essential amino acids relative to latter (Cook et al., 2003). This preference was evident in the bees who had prior foraging experience on rapeseed, as their subsequent foraging trips showed higher preference for it over the humble bean. Foragers who had prior broad exposure to broad bean or rosebay willowherb (a control) showed no differences in the number of landings made on either of the two test plants.

Rapeseed had higher level of leucine, valine & isoleucine (BCAAs!) relative to the beans, and despite the beans having more “mid-tier” amino acids, the bees showed a strong preference for those “most essential” or “high-tier” aminos, thus favouring rapeseed (which also had one non-protein amino: GABA; Cook et al., 2003). In laboratory settings, honey bees had the strongest preferences for sugar solutions containing phenylalanine, an amino acid frequently found at high concentrations in floral nectar (at least in the Mediterranean; Petandiou et al., 2006 & Hendriksma, Oxman & Shafir, 2016).

Non-Protein Amino Acids Contribute to Plant-Pollinators Interactions

There are twenty amino acids involved in protein synthesis and a few thousand non-protein amino acids (“NPAA”) involved in multiple processes in both plants and pollinators. From a plant perspective, NPAAs provide protection from potential microbial pathogens (elaborated below) and “entice” pollinators to become frequent visitors by bestowing them with “health benefits” (reviewed in Nepi, 2013). GABA is the most abundant nectar NPAA and is also one of the more-abundant compounds that benefits plants and animals. In plants, GABA is involved in development, stress defence and organism-organism interactions, whereas in pollinators it regulates nerve and muscle activity, and promotes feeding behaviours in insects (“phagostimulation”).

As you now know, GABA is not found alone, instead as part of a mixture alongside other pollen compounds. It could be that the interactions within the mixture are what shape nectar’s attractiveness. To quote Nepi’s 2013 article: “Despite a lack of information about the metabolism of non-protein amino acids and their fate following ingestion by feeding insects, a complex, ecophysiological picture is beginning to emerge. Proline [an amino acid] powers the take-off of the insect from the flower, sugars propel its prolonged flight and NPAAs (such as taurine, GABA and β-alanine) may promote the highly efficient functioning of flight muscles. Meanwhile, insects are forced to move from flower to flower by the combined phagostimulatory activity of proline, phenylalanine and GABA that maintain a high feeding rate. Excessive excitation due to hunger can be reduced by the intake of inhibitory neurotransmitters (as GABA) that keep the insect calm.”

Influence of Pollinator Visitations on Nectar Concentrations: Microbial Transfer

A sugary medium like nectar is susceptible to microbial infiltration, which can be transferred by the visiting pollinators (I am told animals do not wash before they eat). Colonisation by yeasts and bacteria alters the nectar chemistry: the consumption of floral sugars reduces its nutritional content, and the subsequent release of microbial metabolites adds new compounds to the nectar (themselves depending on the microbe species): butyric acid, acetone, butanol, acetic acid or good ol’ ethanol³.

The control of microbes in nectaries may be done by the plant’s secondary metabolites (e.g. nicotine, caffeine, or citric acid), yet growth inhibition is weak at physiological concentrations (Fridman et al., 2011), and in some cases the opposite occurred whereby microbes consumed these secondary metabolites to further alter nectar’s chemistry (granted these were in laboratory settings; Vannette & Fukami, 2016).

Could the high sucrose concentration of nectars be exerting a preservative effect on the compound, as in honey? The research is unclear. With honey, the high-sugar low-water content makes it hygroscopic, pulling the moisture out of an enterprising bacterial or yeast cell attempting to consume it, leading to death by dehydration. This protects honey from spoilage and when properly stored, makes for clickbaity headlines like “Ancient Egyptian Honey Still Edible: Bees Are Magic”.

Nectar as a Contributor to Plant Immune Defences

Several proteins have been isolated from nectar with a “possible” role in defence, with the tobacco plants being most well studied (Nicotiana spp.; go figure). In one study, the proteins related to the nectar redox cycle (which generate hydrogen peroxide as a form of defence), accounted for 8.3% of the total proteins identified in Nicotiana, whereas the defence/anti-stress proteins accounted for 34% (Silva et al., 2020). Chitinases, endoglucanases, lipid transfer proteins and glucosidases are some of the defence proteins identified in Nicotiana nectars:

• Chitinases and endoglucanases catalyse the hydrolysis of glycosidic bonds in complex sugars. Involved in defence against several phytopathogens through synergistic effects in the microbial cell wall.
• Two classes of lipid transfer proteins were identified as being related to insectivorous resistance (as in, microbes transferred from insects, or “stopping nectar thieves” type of resistance?). A fungal presence triggers the activation of these proteins, so they can be thought of as “antifungal” defence.
  • A lipid transfer protein was able to prevent microbial growth in Brassica rapa nectar (Schmitt et al., 2018)
• Osmotin-like proteins act by inhibition of a microbe’s defensive cell wall, cytotoxicity, cell wall binding and increasing membrane permeabilization (“leakage” [yes it’s spelt with a z]) of the microbes.
• Trypsin proteinase inhibitors protect plants by inhibiting the digestive proteases [protein-breaking enzymes] of herbivorous insects, harming the pollinators in the process. They are highly expressed in the anthers of the American black nightshade Solanum americanum, possiblyas a way of limiting the amount of pollen collected by pollinators or robbers, and leaving some for the next visitor (Hartl et al.,  2011).

Nectar Lipids: Minor Roles to Play

If you thought nectar having amino acids is outlandish, then you’ll be more (or less) surprised to know it also has lipids, which I’ll to conclude part one. Hawkmoths (in this case, Manduca sexta) use the free fatty acids found in nectar as metabolic fuel for flight (Levin, McCue & Davidowitz, 2017). Solitary bees from the genus Macropis collect floral oils from yellow loosestrife flowers (Lysimachia sp.) in addition to pollen and in place of nectar, since these plants do not produce nectar (I am struggling to find a reference for this claim).

Lipids are secreted by epithelial cells, with the major constituents being β-acetoxy fatty acids and diglycerides (From the textbook by Nicolson & Thornburg, 2007). They are energy-dense and would be prized by pollinators, however they are metabolically expensive for the plant to produce. Some notable, high-lipid nectars come from Jacarandas (Jacaranda mimosifolia) and the cactus Trichocereus andalgalensis. Both species are common to the Patagonian region, and a survey of plants from this area showed ~30-50% of species accumulate lipids in their nectar, probably owing to the extreme environmental conditions of the region, which suggests that a high-energy food source is required to encourage pollinator visits.

This ends part one. I thought I could cover nectar in a single post, I was wrong – part two discusses the secondary metabolites, the water content, inorganic ions and other topics that will undoubtedly pop up. Stay tuned.

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Footnotes

1. Why the sucrose dominance? The authors discuss two articles by Baker (1975) & Freeman et al., (1984), which found that sucrose-dominant nectars were associated with flowers pollinated by hummingbirds or long-tongued insects (Freeman’s study samples were from southwestern U.S.): to paraphrase: In general, the sugar composition of nectars varies widely. If a group of plants visited by similar pollinators were to produce a sugar composition with less variability than a random sample, you can suspect a convergence [as in, convergent evolution, whereby the unrelated species become similar over time owing to selection similar pressures {here: the same group of pollinators}] (Freeman et al., 1984). For the record, columbine species are primarily pollinated by insects and birds, and I have elaborated on sucrose dominance below. ↩︎
2. There are also non-protein amino acids, like taurine, GABA and β-alanine which occur at high concentrations in certain nectars, however given their non-protein-forming-roles, I’m going to keep it simple and classify them as “secondary metabolites” (seething biochemists are welcome). ↩︎
3. A rather-hilarious example is described in the Malaysian Pentailed treeshrews (Ptilocercus lowii), who overconsume nectar from the bertam palm (Eugeissona tristis). Now these little mammals pollinate the palms, who entice the pollinators by producing alcoholic nectar as a result of sugar fermentation by yeasts. Seven other small mammal species were attracted to this alcoholic nectar at all stages of the day (dawn, day & dusk), with the visitation frequencies doubling during the night… party at my palm place? The end result is a strong pollinator-plant relationship: pollen is transferred between palm plants in exchange for intoxicated little critters. Further reading: Weins et al., 2008 ↩︎