High throughput procedure utilising chlorophyll fluorescence imaging to phenotype dynamic photosynthesis and photoprotection in leaves under controlled gaseous conditions.

Abstract

Background As yields of major crops such as wheat ( T. aestivum ) have begun to plateau in recent years, there is growing pressure to efficiently phenotype large populations for traits associated with genetic advancement in yield. Photosynthesis encompasses a range of steady state and dynamic traits that are key targets for raising Radiation Use Efficiency (RUE), biomass production and grain yield in crops. Traditional methodologies to assess the full range of responses of photosynthesis, such a leaf gas exchange, are slow and limited to one leaf (or part of a leaf) per instrument. Due to constraints imposed by time, equipment and plant size, photosynthetic data is often collected at one or two phenological stages and in response to limited environmental conditions. Results Here we describe a high throughput procedure utilising chlorophyll fluorescence imaging to phenotype dynamic photosynthesis and photoprotection in excised leaves under controlled gaseous conditions. When measured throughout the day, no significant differences ( P > 0.081) were observed between the responses of excised and intact leaves. Using excised leaves, the response of three cultivars of T. aestivum to a user-defined dynamic lighting regime was examined. Cultivar specific differences were observed for maximum PSII efficiency ( F v '/ F m '- P F q '/ F m '- P = 0.04) under both low and high light. In addition, the rate of induction and relaxation of non-photochemical quenching (NPQ) was also cultivar specific. A specialised imaging chamber was designed and built in-house to maintain gaseous conditions around excised leaf sections. The purpose of this is to manipulate electron sinks such as photorespiration. The stability of carbon dioxide (CO 2 ) and oxygen (O 2 ) was monitored inside the chambers and found to be within ± 4.5% and ± 1% of the mean respectively. To test the chamber, T. aestivum 'Pavon76' leaf sections were measured under at 20 and 200 mmol mol -1 O 2 and ambient [CO 2 ] during a light response curve. The F v '/ F m 'was significantly higher ( P 2 ] for the majority of light intensities while values of NPQ and the proportion of open PSII reaction centers (qP) were significantly lower under > 130 μmol m -2 s -1 photosynthetic photon flux density (PPFD). Conclusions Here we demonstrate the development of a high-throughput (> 500 samples day -1 ) method for phenotyping photosynthetic and photo-protective parameters in a dynamic light environment. The technique exploits chlorophyll fluorescence imaging in a specifically designed chamber, enabling controlled gaseous environment around leaf sections. In addition, we have demonstrated that leaf sections do not different from intact plant material even > 3 h after sampling, thus enabling transportation of material of interest from the field to this laboratory based platform. The methodologies described here allow rapid, custom screening of field material for variation in photosynthetic processes.

Plant phenotyping relevance

葉緑素蛍光イメージングと専用チャンバーを用いた高スループットな光合成・光防御形質の取得法を開発し、葉およびチャンバー条件を検証しているため、植物フェノタイピング手法が中心である。

Code and data availability

The paper's phenotyping pipeline (chlorophyll fluorescence imaging of excised wheat leaves in custom gas-controlled chambers) is supported by a paper-specific public asset: Additional file 2, a ZIP supplement containing the CAD files, printer settings, and construction notes for the custom imaging chambers, explicitly'