Out of the light and into the dark: post-illumination respiratory metabolism


Photosynthesis and respiration are often presented as two related, yet separate, physiological processes regulating the carbon (C) balance of cells, leaves, plants and ecosystems. When the two processes are presented in this simplified way, photosynthesis and respiration balance each other, suggesting the interaction is limited to the exchange of the resulting products, which then become the substrate for the other process (Fig. 1). By displaying the required input of solar energy to drive photosynthesis, the simplification can regrettably result in students and researchers mistakenly believing that daytime photosynthesis is balanced by night respiration, not realizing that respiration is a continuous process (i.e. respiration continues in the light). This simplified cyclical exchange hides the myriad ways respiration and photosynthesis interact as intertwined, co-regulated and co-dependent parts of cellular function. Fortunately, scientists from many different disciplines recognize this as a frontier and are now making great progress, both independently and through collaboration, to elucidate and clarify the combined photosynthetic and respiratory function in leaves (Atkin et al., 2010). However, the metabolic interactions between photosynthetic and respiratory metabolism remain difficult to study and challenging to understand. In this issue of New Phytologist, Igor Florez-Sarasa et al. (pp. 136–148) present the results of an innovative and detailed study of the effects of previously light conditions on ‘dark’ respiratory activity and metabolite pools, clearly demonstrating the inter-dependence of the processes and helping to advance our knowledge.

Figure 1.

Simple depiction of the relationship between photosynthesis and respiration, suggesting that with the input of light to drive photosynthesis, the activity of the chloroplast and mitochondrion are balanced with the products of one reaction becoming the substrates of the other. While this depiction may be heuristically useful in some situations, it hides the myriad ways light affects respiration and the degree to which the biochemistry and physiology of both organelles are intertwined.

This work clearly demonstrates the utility of linking targeted metabolite profiling to respiratory activity and moves our thinking forward.

It is known that respiratory function in illuminated leaves is significantly altered in the light, with the tricarboxylic acid (TCA) cycle in particular showing a major re-organization (Tcherkez et al., 2009; Nunes-Nesi et al., 2011). Anabolic processes, nitrogen (N) metabolism and photorespiration may all benefit, and even normal photosynthetic metabolism appears to be dependent on the occurrence of the (altered) respiratory activity during illumination (Nunes-Nesi et al., 2008; Foyer et al., 2011; Araújo et al., 2012). What is less clear are the consequences of this light-driven re-organization on the relationships among respiratory rates, respiratory electron partitioning and metabolites levels following illumination – the focus of the Florez-Sarasa et al.’s contribution. Certainly, a predictive mechanistic understanding of leaf C balance will benefit from knowledge of the biochemical and physiological regulation of respiration during the transition from light-to-dark.

From a very practical point of view, experimentalists can and should ask what the appropriate conditions are for measuring respiratory rates, or perhaps more accurately how to interpret measurements of respiratory activity made under different conditions (e.g. different times of day or lengths of time in the dark). Most researchers are aware of light enhanced dark respiration (LEDR), a phenomenon known to result in higher respiration rates following illumination (Atkin et al., 2000; Padmasree et al., 2002), and avoid making measurements during this time. The implied, but previously untested, assumption is that similar to net rates of C exchange, light-responsive enzymes and metabolites affecting this respiratory flux and electron partitioning would also have returned to dark levels in this time. Florez-Sarasa et al. test these assumptions by combining measurements of various respiratory metabolites and transcript levels with oxygen isotope fractionation in the dark and chlorophyll fluorescence and reveal some surprising relationships.

Enduring effects of illumination on ‘dark’ respiration

Florez-Sarasa et al. investigated the extent to which previous light conditions affect the overall status of metabolites and gene transcript levels during dark respiration, and the impact of a 30-min dark adaption. Consistent with the simple cyclical linking of photosynthesis and respiration, a 2-h high-light exposure resulted in a greater accumulation of photosynthetic products (respiratory substrates) heading into a dark measurement period, and thus higher rates of respiration measured 30 min later (after the subsidence of LEDR). Fluxes through both the alternative and the cytochrome pathway are increased but the relative partitioning is not. Long-term effects of light are similar with higher growth irradiance conditions resulting in higher rates of respiration yet no change in electron partitioning. Importantly the long-term effects of growth irradiance reveal that respiration rates measured after 30 min of dark adaptation are similar to rates measured after 7 h of darkness, suggesting reliable measurements of respiration rate can be measured any time of day.

Looking beyond the flux rates (oxygen (O2) consumption) however, reveals a different story. Both the increased metabolite levels, and transcript levels of two important respiratory genes (NDA1 and AOX1a), were maintained in plants subjected to high-light treatment even after the plants were kept in darkness for 30 min. A second experiment demonstrated the impacts of the antecedent light level on metabolite pools, finding that the general metabolite profile of plants grown under low light was similar to that after an additional 30 min of darkness, whereas the levels of most metabolites observed in plants grown under moderate light were maintained after 30 min of darkness. So while net respiratory rates, and respiratory electron partitioning are similar between dark-adapted leaves and leaves measured at midnight, the metabolite profile at night was markedly different from that of dark-adapted leaves, most of them showing strongly decreased levels at night.

Delving a bit deeper into the specific metabolite responses reveals several provocative findings that point to possible mechanisms underpinning the observed responses. For example, Florez-Sarasa et al. observed that several sugars and TCA cycle intermediate levels were higher following a 2-h treatment with high-light, or growth in moderate light conditions compared to plants grown in low light. This, in addition to a pronounced increase in levels of several amino acids (phenylalanine, alanine and isoleucine), suggests a greater requirement for respiratory C metabolites for amino acid synthesis in the light. Furthermore, glycine levels increased significantly after high-light treatment but less so under moderate growth light levels. These results suggest a large increase in photorespiratory activity and a limitation in the conversion of glycine to serine caused by high light, clearly linking the activities of the chloroplasts with enzyme systems found in the mitochondria.

After 30 min of darkness, the levels of most metabolites induced by the high-light treatment and moderate growth intensity remained high, although a decrease in the levels of a few amino acids was observed after the dark treatment. The latter could be due to the absence of photosynthetic N assimilation, which would decrease amino acid synthesis (Nunes-Nesi et al., 2007; Tcherkez et al., 2008). Importantly, changes in sugars and glycine levels did not correlate with respiratory rates, while variations in the levels of several amino acids and organic acids did. While this is consistent with recent findings (Zell et al., 2010), Florez-Sarasa et al. correctly point out that further analyses of diurnal respiration time courses in parallel with metabolite profiling are needed to investigate the limiting substrates for respiration, especially under field conditions.

Implications and the way forward

We suggest the findings of Florez-Sarasa et al. can be gathered into three important categories. First, there are clear linkages between chloroplastic activity (including photorespiration, N metabolism and C fixation), and dark respiration, which are revealed through light–dark transitions. Second, there is an interesting lack of correlation between carbohydrates or glycine and respiratory flux, suggesting the consumption of these substrates may not in themselves be controlling respiration. Instead, other amino acids (valine, glutamine, beta-alanine, tryptophan and ornithine), and organic acids (lactic, glutamic, fumaric, 2-oxoglutaric, and glutaric acids) are more strongly correlated with respiration rates, raising the question of their role in regulating respiratory activity. Third, that while estimates of respiratory flux may be accurately derived by briefly dark adapting sunlit leaves, the interpretation of these fluxes and linking them to other traits like metabolite levels requires an appreciation of both the antecedent light conditions and the duration of darkness.

This work clearly demonstrates the utility of linking targeted metabolite profiling to respiratory activity and moves our thinking forward. In combination with isotopic labeling and fluxomics (e.g. Tcherkez et al., 2009), these approaches give hope of attaining the goal of elucidating both the mechanisms regulating plant respiration and the metabolic state of leaves under various environmental conditions or in various physiological states. With this in mind, it seems likely that the challenges that remain include distinguishing the role of metabolite flux from that of the resultant metabolite pool sizes, and incorporating the effect of other biotic and abiotic variables that challenge respiration either directly (e.g. temperature or growth), or indirectly (e.g. nutrient availability, atmospheric CO2, or response to disease), in order to clarify the general regulation of metabolic activity. Quantifying the flux rates of various respiratory metabolites under combinations of environmental conditions could lead to a much-needed revision of our conceptual understanding of respiration and its control. The work of Florez-Sarasa et al. demonstrates that it’s time to move beyond the simple conceptual model of a cyclic exchange of products (Fig. 1). The current state of knowledge developing from the greater community of active researchers indicates we need to consider a more useful conceptual framework that includes the transition between two states: a metabolic organization previously considered typical of dark respiration, and a light-altered state that considers the combined activity and regulation of respiration with photosynthesis, photorespiration, N metabolism, and perhaps response to the production of reactive oxygen species (Fig. 2).

Figure 2.

Respiration contributes to overall cellular metabolism during both light and dark periods. The specific function and products of mitochondrial respiration change during light-to-dark or dark-to-light transitions, but the co-regulation of these cycles remains difficult to determine. In the light, respiration (specifically the TCA cycle) is thought to re-organize and contribute to the processing of photorespiratory products, N metabolism and amino acid formation. Metabolic profiling reveals that despite a lack of change in respiratory flux (O2 consumption in the dark) in short-term dark-adapted leaves vs midnight measured (7 h post-illumination), metabolite pools are significantly different, suggesting the interpretation of respiratory activity requires caution and a more complete understanding of the interactions between chloroplast and mitochondrial function.