Quantification of energy-converting protein complexes in plant thylakoid membranes.

Knowledge about the exact abundance and ratio of photosynthetic protein complexes in thylakoid membranes is central to understanding structure-function relationships in energy conversion. Recent modeling approaches for studying light harvesting and electron transport reactions rely on quantitative information on the constituent complexes in thylakoid membranes. Over the last decades several quantitative methods have been established and refined, enabling precise stoichiometric information on the five main energy-converting building blocks in the thylakoid membrane: Light-harvesting complex II (LHCII), Photosystem II (PSII), Photosystem I (PSI), cytochrome b6f complex (cyt b6f complex), and ATPase. This paper summarizes a few quantitative spectroscopic and biochemical methods that are currently available for quantification of plant thylakoid protein complexes. Two new methods are presented for quantification of LHCII and the cyt b6f complex, which agree well with established methods. In addition, recent improvements in mass spectrometry (MS) allow deeper compositional information on thylakoid membranes. The comparison between mass spectrometric and more classical protein quantification methods shows similar quantities of complexes, confirming the potential of thylakoid protein complex quantification by MS. The quantitative information on PSII, PSI, and LHCII reveal that about one third of LHCII must be associated with PSI for a balanced light energy absorption by the two photosystems.

Differential response of the photosynthetic machinery to dehydration in older and younger resurrection plants.

A group of vascular plants called homoiochlorophyllous resurrection plants evolved unique capabilities to protect their photosynthetic machinery against desiccation-induced damage. This study examined whether the ontogenetic status of the resurrection plant Craterostigma pumilum has an impact on how the plant responds to dehydration at the thylakoid membrane level to prepare cells for the desiccated state. Thus, younger plants (<4 months) were compared with their older (>6 months) counterparts. Ultrastructural analysis provided evidence that younger plants suppressed senescence-like programs that are realized in older plants. During dehydration, older plants degrade specific subunits of the photosynthetic apparatus such as the D1 subunit of PSII and subunits of the cytochrome b6f complex. The latter leads to a controlled down-regulation of linear electron transport. In contrast, younger plants increased photoprotective high-energy quenching mechanisms and maintained a high capability to replace damaged D1 subunits. It follows that depending on the ontogenetic state, either more degradation-based or more photoprotective mechanisms are employed during dehydration of Craterostigma pumilum.

Measuring the dynamic response of the thylakoid architecture in plant leaves by electron microscopy.

The performance of the photosynthesis machinery in plants, including light harvesting, electron transport, and protein repair, is controlled by structural changes in the thylakoid membrane system inside the chloroplasts. In particular, the structure of the stacked grana area of thylakoid membranes is highly dynamic, changing in response to different environmental cues such as light intensity. For example, the aqueous thylakoid lumen enclosed by thylakoid membranes in grana has been documented to swell in the presence of light. However, light-induced alteration of the stromal gap in the stacked grana (partition gap) and of the unstacked stroma lamellae has not been well characterized. Light-induced changes in the entire thylakoid membrane system, including the lumen in both stacked and unstacked domains as well as the partition gap, are presented here, and the functional implications are discussed. This structural analysis was made possible by development of a robust semi-automated image analysis method combined with optimized plant tissue fixation techniques for transmission electron microscopy generating quantitative structural results for the analysis of thylakoid ultrastructure. SIGNIFICANCE STATEMENT: A methodical pipeline ranging from optimized leaf tissue preparation for electron microscopy to quantitative image analysis was established. This methodical development was employed to study details of light-induced changes in the plant thylakoid ultrastructure. It was found that the lumen of the entire thylakoid system (stacked and unstacked domains) undergoes light-induced swelling, whereas adjacent membranes on the stroma side in stacked grana thylakoid approach each other.

Protection of the photosynthetic apparatus against dehydration stress in the resurrection plant Craterostigma pumilum.

The group of homoiochlorophyllous resurrection plants evolved the unique capability to survive severe drought stress without dismantling the photosynthetic machinery. This implies that they developed efficient strategies to protect the leaves from reactive oxygen species (ROS) generated by photosynthetic side reactions. These strategies, however, are poorly understood. Here, we performed a detailed study of the photosynthetic machinery in the homoiochlorophyllous resurrection plant Craterostigma pumilum during dehydration and upon recovery from desiccation. During dehydration and rehydration, C. pumilum deactivates and activates partial components of the photosynthetic machinery in a specific order, allowing for coordinated shutdown and subsequent reinstatement of photosynthesis. Early responses to dehydration are the closure of stomata and activation of electron transfer to oxygen accompanied by inactivation of the cytochrome b6 f complex leading to attenuation of the photosynthetic linear electron flux (LEF). The decline in LEF is paralleled by a gradual increase in cyclic electron transport to maintain ATP production. At low water contents, inactivation and supramolecular reorganization of photosystem II becomes apparent, accompanied by functional detachment of light-harvesting complexes and interrupted access to plastoquinone. This well-ordered sequence of alterations in the photosynthetic thylakoid membranes helps prepare the plant for the desiccated state and minimize ROS production.