Is polyphenol induction simply a result of altered carbon and nitrogen accumulation?

Abstract

Carbon translocation in plants is shaped by phyllotaxis and regulated by source/sink interactions that respond to the demands of growth and defense. We have studied this extensively in poplar saplings, and recently showed that unlike carbon import, nitrogen is not translocated to sink leaves in response to application of jasmonic acid. Here we report that this is also true for young trees in the field. We discuss the importance of transport processes in establishing local C:N ratios, and suggest that the JA-induced flow of C but not N to sink tissues, and their corresponding increases in C-based defenses, may simply reflect a plant adaptation to handle excess reduced carbon and energy.

Keywords: :

• ¹³C
• ¹⁵N
• carbon and nitrogen balance
• invertase
• jasmonate (JA)
• polyphenols
• Populus
• sink strength

This article refers to:

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Local increases in sink strength are essential components of plant responses to grazing, mechanical wounding, infection, hormones and artificial elicitors in a wide range of plant species.¹ These responses involve rapid increases in the local activities of cell wall invertase enzymes (CWI; EC 3.2.1.26), which boost phloem unloading and carbohydrate (CHO) transport to the elicited tissues.^2-8 We have found that both insect grazing and exogenous jasmonate (JA) trigger increased sink strength, including a 3-fold increase in the import of ¹³C-labeled CHOs from orthostichously linked source leaves to sink leaves in poplar saplings.^1,3,4

We recently found that unlike carbon import, nitrogen translocation was not increased by JA treatment in poplar saplings.¹ We also examined the impact of sink strength on nitrogen translocation in larger, more complex poplar trees, using whole branches, longer transport distances and multiple sampling intervals. We measured ¹⁵N flow rates in 4 y-old hybrid poplar trees (OP-367) cultivated at the Dickinson College Agricultural Research Center in Boiling Springs, PA in June and July of 2010. On each tree, two opposing lateral branches were selected and randomly designated as primary (1°) and secondary (2°) branches. The branches were similar, originating at similar truck heights and of similar lengths. For three days, the 1° lateral branches received 5mM JA on the 20 most apical leaves and the 2° branches received nothing. On the morning of day two, we added ¹⁵N as a soil drench and harvested leaves 12h, 24h, 48h and 72h later to determine the flow of ¹⁵N into 1°and 2° branches. At each interval, two leaves from each 1þand 2þ branch were harvested – one sink leaf and one source leaf – and combined for isotope analysis. Here we found that JA treatment, which generally increases local sink strength, led to significantly lower rates of ¹⁵N import at 24h, 48h and 72h compared with rates observed for opposing (untreated) branches on the same trees (Fig. 1). Altogether, we have found that while the supply of CHOs to elicited tissues is related to local sink strength, nitrogen movement is neither related to sink strength nor is it constrained by plant architecture.

Figure 1. Import of ¹⁵N to lateral branches of four-year old hybrid poplar trees over a four day period. JA-treated lateral branches did not import additional ¹⁵N as a component of the defense responses. In fact, ¹⁵N import was lower in these branches (closed circles) compared with control branches located on the same trees (open circles). Points represent the mean import of 4–5 branches with +/1 SE error bars. At each time point, import was compared via two-tailed t-tests. * indicates p < 0.10.

These observations make sense in light of the fact that poplar’s main response to elicitation involves carbon-based metabolites such as polyphenols.⁹ Increased N transport in response to localized stress has been observed mainly or only in plants that increase production and transport of N-intensive metabolites in response to stress, such as nicotine in tobacco.^7,10-13

Taken together, our results indicate that sink-source relationships must frequently determine local CHO/N ratios. Many have noted an inverse relationship in entire plants between N availability and the constitutive concentration of CHO-based metabolites, especially polyphenols.^14-16 The relationship between nitrogen availability and phenolic synthesis is regulated at the transcriptional level.^17,18A localized carbon excess relative to nutrient availabilities could result from many causes, including the impact of elevated light levels or CO₂ concentrations on photosynthesis, heterogeneous vascular connections or changing carbohydrate transport as leaves mature.^17,19,20 Since invertase activity is generally influenced by the same factors,³ varying sink strength probably underlies such constitutive variation, which can be ecologically important.^19,21-23

But what of elicitation, or “induction” of CHO-based defenses in response to insects or other stresses? Hunter and Schultz¹⁶ showed that defoliation-induced increases in polyphenols by oak leaves were suppressed by nitrogen fertilization, as did Keinanen et al.²⁴ for birch and Blodgett et al.²⁵ for pine. Our research has linked short-term, local increases in sink strength via enhanced invertase activity to increased CHO accumulation and an increase in the products of the phenylpropanoid pathway.^1,3,4 Like Tuomi et al.²² we also found that this sequence of events is limited to individual branches.¹ Since we now see that increased sink strength is associated with no change or even a decrease in local N concentrations, induced sink strength should increase the local CHO/N ratio in elicited tissues. Tuomi et al.²² and Roitto et al.²⁶ suggested that a decrease in N alone would be enough to cause “induction” of polyphenol production in individual branches. Our evidence leads us to suggest that branch-level “induction” of CHO-based compounds in response to insects or other elicitors arises from an increased CHO/N ratio driven by enhanced sink strength. This is consistent with evidence that induced “defense” is a local phenomenon, constrained by vascular architecture^27-28 and that local nutrient status can influence both constitutive and inducible defenses.^22,27,29,30

Substrates for many putative CHO-based defenses, e.g., flavonoids and tannins, arise from the shikimic acid and phenylpropanoid pathways. These pathways are upregulated by carbon availability and typically respond positively to enhanced carbon supply; they may be a sink for excess reduced carbon and energy.³¹ According to this view, any photosynthetically active tissue should respond to a real or perceived increase in CHO by shunting excess carbon substrates into these pathways. “Dumping” excess CHO may occur in any tissue that constitutes a strong sink, including fruits, insect galls,³² expanding leaves²⁰ or interrupted nutrient supply,³³ and altered “defense” may be an after-effect.

The dynamic interaction between C and N pools in plants and the role of the shikimate and phenylpropanoid pathways as places to sequester excess carbohydrate suggest that we need to re-examine the interpretation of polyphenol accumulation as an active defense. As suggested 30 y ago^34,35 for entire plants, it appears to us that the relationship between N and C availability also determines how phenylpropanoids accumulate at the local level and that sink strength forms the driving force in “induced” responses to elicitors. Phenylpropanoid production may be an adaptive way to dispose of carbohydrates and energy that have accumulated in excess of the tissue’s ability to use them in growth or maintenance when sink strength is increased.

Abbreviations:
C	=	carbon
CHO	=	carbohydrate
JA	=	jasmonate
CWI	=	cell wall invertase
N	=	nitrogen

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.