Improved monitoring of cryptogam gas-exchange and volatile emissions during desiccation-rehydration cycles with a within-chamber hydration method.

Desiccation-rehydration studies in cryptogams constitute an important tool to understand the relation of key physiological traits with species stress tolerance and environmental adaptability. Real-time monitoring of responses has been limited by the design of commercial or custom measuring cuvettes and difficulties in experimental manipulation. We developed a within-chamber rehydration method that allows to rewater the samples rapidly, without the need to open the chamber and take out the sample for manual rehydration by the investigator. Data is collected in real-time and simultaneously with an infrared gas-analyzer (LICOR-7000), a chlorophyll fluorometer (Maxi Imaging-PAM) and a proton transfer reaction time-of-flight mass-spectrometer (PTR-TOF-MS) for volatile organic compound emissions. The system was tested on four cryptogam species with contrasting ecological distributions. No major errors or kinetics disruptions were found during system testing and measurements. Our within-chamber rehydration method improved accuracy, as measurement periods were not lacking, and repeatability of the protocol by reducing error variance in sample manipulation. This method provides an improved technique to conduct desiccation-rehydration measurements, contributing to the standardization and accuracy of current existing methodologies. A close real-time and simultaneous monitoring of photosynthesis, chlorophyll fluorescence and volatile organic compound emission data, offers a novel perspective in the analysis of the cryptogam stress responses that is yet to be fully explored.

Stomatal VPD Response: There Is More to the Story Than ABA.

Guard cells shrink and close stomatal pores when air humidity decreases (i.e. when the difference between the vapor pressures of leaf and atmosphere [VPD] increases). The role of abscisic acid (ABA) in VPD-induced stomatal closure has been studied using ABA-related mutants that respond to VPD in some studies and not in others. The importance of ABA biosynthesis in guard cells versus vasculature for whole-plant stomatal regulation is unclear as well. Here, we show that Arabidopsis (Arabidopsis thaliana) lines carrying mutations in different steps of ABA biosynthesis as well as pea (Pisum sativum) wilty and tomato (Solanum lycopersicum) flacca ABA-deficient mutants had higher stomatal conductance compared with wild-type plants. To characterize the role of ABA production in different cells, we generated transgenic plants where ABA biosynthesis was rescued in guard cells or phloem companion cells of an ABA-deficient mutant. In both cases, the whole-plant stomatal conductance, stunted growth phenotype, and leaf ABA level were restored to wild-type values, pointing to the redundancy of ABA sources and to the effectiveness of leaf ABA transport. All ABA-deficient lines closed their stomata rapidly and extensively in response to high VPD, whereas plants with mutated protein kinase OST1 showed stunted VPD-induced responses. Another strongly ABA-insensitive mutant, defective in the six ABA PYR/RCAR receptors, responded to changes in VPD in both directions strongly and symmetrically, indicating that its VPD-induced closure could be passive hydraulic. We discuss that both the VPD-induced passive hydraulic stomatal closure and the stomatal VPD regulation of ABA-deficient mutants may be conditional on the initial pretreatment stomatal conductance.

Photosystem II cycle and alternative electron flow in leaves.

Sunflower (Helianthus annuus L.) and tobacco (Nicotiana tabacum L.) were grown in the laboratory and leaves were taken from field-grown birch trees (Betula pendula Roth). Chlorophyll fluorescence, CO2 uptake and O2 evolution were measured and electron transport rates were calculated, J(C) from the CO2 uptake rate considering ribulose-1,5-bisphosphate (RuBP) carboxylation and oxygenation, J(O) from the O2 evolution rate, and J(F) from Chl fluorescence parameters. Mesophyll diffusion resistance, r(md), used for the calculation of J(C), was determined such that the in vivo Rubisco kinetic curve with respect to the carboxylation site CO2 concentration became a rectangular hyperbola with Km(CO2) of 10 microM at 22.5 degrees C. In sunflower, in the absence of external O2, J(O) = 1.07 J(C) when absorbed photon flux density (PAD) was varied, showing that the O2-independent components of the alternative electron flow to acceptors other than CO2 made up 7% of J(C). Under saturating light, J(F), however, was 20-30% faster than J(C), and J(F)-J(C) depended little on CO2 and O2 concentrations. The inter-relationship between J(F)-J(C) and non-photochemical quenching (NPQ) was variable, dependent on the CO2 concentration. We conclude that the relatively fast electron flow J(F)-J(C) appearing at light saturation of photosynthesis contains a minor component coupled with proton translocation, serving for nitrite, oxaloacetate and oxygen reduction, and a major component that is mostly cyclic electron transport around PSII. The rate of the PSII cycle is sufficient to release the excess excitation pressure on PSII significantly. Although the O2-dependent Mehler-type alternative electron flow appeared to be under the detection threshold, its importance is discussed considering the documented enhancement of photosynthesis by oxygen.