Amphistomy: stomata patterning inferred from 13C content and leaf-side-specific deposition of epicuticular wax.

BACKGROUND AND AIMS: The benefits and costs of amphistomy (AS) vs. hypostomy (HS) are not fully understood. Here, we quantify benefits of access of CO2 through stomata on the upper (adaxial) leaf surface, using 13C abundance in the adaxial and abaxial epicuticular wax. Additionally, a relationship between the distribution of stomata and epicuticular wax on the opposite leaf sides is studied. METHODS: We suggest that the 13C content of long-chain aliphatic compounds of cuticular wax records the leaf internal CO2 concentration in chloroplasts adjacent to the adaxial and abaxial epidermes. This unique property stems from: (1) wax synthesis being located exclusively in epidermal cells; and (2) ongoing wax renewal over the whole leaf lifespan. Compound-specific and bulk wax 13C abundance (δ) was related to amphistomy level (ASL; as a fraction of adaxial in all stomata) of four AS and five HS species grown under various levels of irradiance. The isotopic polarity of epicuticular wax, i.e. the difference in abaxial and adaxial δ (δab - δad), was used to calculate the leaf dorsiventral CO2 gradient. Leaf-side-specific epicuticular wax deposition (amphiwaxy level) was estimated and related to ASL. KEY RESULTS: In HS species, the CO2 concentration in the adaxial epidermis was lower than in the abaxial one, independently of light conditions. In AS leaves grown in high-light and low-light conditions, the isotopic polarity and CO2 gradient varied in parallel with ASL. The AS leaves grown in high-light conditions increased ASL compared with low light, and δab - δad approached near-zero values. Changes in ASL occurred concomitantly with changes in amphiwaxy level. CONCLUSIONS: Leaf wax isotopic polarity is a newly identified leaf trait, distinguishing between hypo- and amphistomatous species and indicating that increased ASL in sun-exposed AS leaves reduces the CO2 gradient across the leaf mesophyll. Stomata and epicuticular wax deposition follow similar leaf-side patterning.

13 CO2 labelling as a tool for elucidating the mechanism of cuticle development: a case of Clusia rosea.

The plant cuticle is an important plant-atmosphere boundary, the synthesis and maintenance of which represents a significant metabolic cost. Only limited information regarding cuticle dynamics is available. We determined the composition and dynamics of Clusia rosea cuticular waxes and matrix using 13 CO2 labelling, compound-specific and bulk isotope ratio mass spectrometry. Collodion was used for wax collection; gas exchange techniques to test for any collodion effects on living leaves. Cutin matrix (MX) area density did not vary between young and mature leaves and between leaf sides. Only young leaves incorporated new carbon into their MX. Collodion-based sampling discriminated between epicuticular (EW) and intracuticular wax (IW) effectively. Epicuticular differed in composition from IW. The newly synthetised wax was deposited in IW first and later in EW. Both young and mature leaves synthetised IW and EW. The faster dynamics in young leaves were due to lower wax coverage, not a faster synthesis rate. Longer-chain alkanes were deposited preferentially on the abaxial, stomatous leaf side, producing differences between leaf sides in wax composition. We introduce a new, sensitive isotope labelling method and demonstrate that cuticular wax is renewed during leaf ontogeny of C. rosea. We discuss the ecophysiological significance of the new insights.