From isotopic fingerprints to functional diagnosis in Italian red chicory: linking δ¹³C and δ¹⁵N to photosynthetic performance across geography and genotype

Stable carbon and nitrogen isotopes are widely applied to infer plant water-use efficiency and nutrient dynamics; however, their physiological interpretation remains uncertain when isotopic signals are not supported by functional measurements. Here, we combine elemental and isotopic analysis (EA-IRMS; δ¹³C, δ¹⁵N, %C, %N and C/N ratio) with fast chlorophyll a fluorescence assessed in vivo through the JIP-test (Handy-PEA) to resolve the physiological meaning of isotopic variability across two genotypes and three geographical contexts of typical Italian red chicory.
In December 2024, leaves and roots of two Cichorium intybus cultivars (“Rosso precoce di Chioggia” and “Rosso precoce di Treviso”) were sampled across three sites, resulting in four genotype–site combinations. Plants were collected at their areas of origin, as defined by Protected Geographical Indication (PGI, Chioggia and Treviso), and outside the PGI area in Massenzatica (Ferrara, Italy), where both cultivars are cultivated in a sandy coastal soil. Isotopic and elemental analyses revealed a pronounced site-dependent differentiation. Leaf and root δ¹³C values varied among sites, indicating substantial long-term differences in C discrimination, related to the balance between transpiration and CO₂ assimilation, which seemed more favourable in Chioggia. δ¹⁵N further discriminated sites and cultivars, highlighting marked differences in N dynamics and internal allocation, although without clear attribution to specific sources.
To assess whether isotopic shifts reflected adaptive regulation or functional impairment, chlorophyll fluorescence transients (OJIP) were analysed using JIP-test parameters. All samples exhibited the typical polyphasic OJIP pattern, yet clear differences emerged between cultivars and sites. Across the entire induction curve, the Treviso PGI transient were consistently higher at the J and I steps than the other samples. This pattern was associated with increased light absorption and energy dissipation per photosystem II reaction centre (ABS/RC, DI₀/RC), reduced electron transport efficiency (ET₀/RC), and lower probabilities of electron transfer to photosystem I end acceptors (ψ(RE₀), δ(RE₀)), indicating a tendency to over-reduce the electron transport chain, probably driven by downstream limitations in electron utilisation.
Overall, our results demonstrate that a more negative δ¹³C, while implicating a better water use efficiency, does not necessarily reflect an overall better plant adaptation, which could be affected by  some biochemical photosynthetic constraints. Integrating EA-IRMS with JIP-test fluorescence analysis provides a robust framework to discriminate between stomatal regulation and biochemical limitation, improving the mechanistic interpretation of isotopic markers for geographical fingerprinting and genotypic differentiation in climate-sensitive agroecosystems.

 

This research was allowed by phD fellowship granted by EUROPEAN SOCIAL FUND P L U S - The ESF+ 2021-2027
Programme of the Regione Emilia Romagna