A carboxysome-based CO2 concentrating mechanism for C3 crop chloroplasts: advances and the road ahead.

The introduction of the carboxysome-based CO2 concentrating mechanism (CCM) into crop plants has been modelled to significantly increase crop yields. This projection serves as motivation for pursuing this strategy to contribute to global food security. The successful implementation of this engineering challenge is reliant upon the transfer of a microcompartment that encapsulates cyanobacterial Rubisco, known as the carboxysome, alongside active bicarbonate transporters. To date, significant progress has been achieved with respect to understanding various aspects of the cyanobacterial CCM, and more recently, different components of the carboxysome have been successfully introduced into plant chloroplasts. In this Perspective piece, we summarise recent findings and offer new research avenues that will accelerate research in this field to ultimately and successfully introduce the carboxysome into crop plants for increased crop yields.

Rubisco proton production can drive the elevation of CO2 within condensates and carboxysomes.

Membraneless organelles containing the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are a common feature of organisms utilizing CO2 concentrating mechanisms to enhance photosynthetic carbon acquisition. In cyanobacteria and proteobacteria, the Rubisco condensate is encapsulated in a proteinaceous shell, collectively termed a carboxysome, while some algae and hornworts have evolved Rubisco condensates known as pyrenoids. In both cases, CO2 fixation is enhanced compared with the free enzyme. Previous mathematical models have attributed the improved function of carboxysomes to the generation of elevated CO2 within the organelle via a colocalized carbonic anhydrase (CA) and inwardly diffusing HCO3-, which have accumulated in the cytoplasm via dedicated transporters. Here, we present a concept in which we consider the net of two protons produced in every Rubisco carboxylase reaction. We evaluate this in a reaction-diffusion compartment model to investigate functional advantages these protons may provide Rubisco condensates and carboxysomes, prior to the evolution of HCO3- accumulation. Our model highlights that diffusional resistance to reaction species within a condensate allows Rubisco-derived protons to drive the conversion of HCO3- to CO2 via colocalized CA, enhancing both condensate [CO2] and Rubisco rate. Protonation of Rubisco substrate (RuBP) and product (phosphoglycerate) plays an important role in modulating internal pH and CO2 generation. Application of the model to putative evolutionary ancestors, prior to contemporary cellular HCO3- accumulation, revealed photosynthetic enhancements along a logical sequence of advancements, via Rubisco condensation, to fully formed carboxysomes. Our model suggests that evolution of Rubisco condensation could be favored under low CO2 and low light environments.

A cross-scale analysis to understand and quantify the effects of photosynthetic enhancement on crop growth and yield across environments.

Photosynthetic manipulation provides new opportunities for enhancing crop yield. However, understanding and quantifying the importance of individual and multiple manipulations on the seasonal biomass growth and yield performance of target crops across variable production environments is limited. Using a state-of-the-art cross-scale model in the APSIM platform we predicted the impact of altering photosynthesis on the enzyme-limited (Ac ) and electron transport-limited (Aj ) rates, seasonal dynamics in canopy photosynthesis, biomass growth, and yield formation via large multiyear-by-location crop growth simulations. A broad list of promising strategies to improve photosynthesis for C3 wheat and C4 sorghum were simulated. In the top decile of seasonal outcomes, yield gains were predicted to be modest, ranging between 0% and 8%, depending on the manipulation and crop type. We report how photosynthetic enhancement can affect the timing and severity of water and nitrogen stress on the growing crop, resulting in nonintuitive seasonal crop dynamics and yield outcomes. We predicted that strategies enhancing Ac alone generate more consistent but smaller yield gains across all water and nitrogen environments, Aj enhancement alone generates larger gains but is undesirable in more marginal environments. Large increases in both Ac and Aj generate the highest gains across all environments. Yield outcomes of the tested manipulation strategies were predicted and compared for realistic Australian wheat and sorghum production. This study uniquely unpacks complex cross-scale interactions between photosynthesis and seasonal crop dynamics and improves understanding and quantification of the potential impact of photosynthesis traits (or lack of it) for crop improvement research.

The Chlamydomonas reinhardtii chloroplast envelope protein LCIA transports bicarbonate in planta.

LCIA (low CO2-inducible protein A) is a chloroplast envelope protein associated with the CO2-concentrating mechanism of the green alga Chlamydomonas reinhardtii. LCIA is postulated to be a HCO3- channel, but previous studies were unable to show that LCIA was actively transporting bicarbonate in planta. Therefore, LCIA activity was investigated more directly in two heterologous systems: an Escherichia coli mutant (DCAKO) lacking both native carbonic anhydrases and an Arabidopsis mutant (ßca5) missing the plastid carbonic anhydrase ßCA5. Neither DCAKO nor ßca5 can grow in ambient CO2 conditions, as they lack carbonic anhydrase-catalyzed production of the necessary HCO3- concentration for lipid and nucleic acid biosynthesis. Expression of LCIA restored growth in both systems in ambient CO2 conditions, which strongly suggests that LCIA is facilitating HCO3- uptake in each system. To our knowledge, this is the first direct evidence that LCIA moves HCO3- across membranes in bacteria and plants. Furthermore, the ßca5 plant bioassay used in this study is the first system for testing HCO3- transport activity in planta, an experimental breakthrough that will be valuable for future studies aimed at improving the photosynthetic efficiency of crop plants using components from algal CO2-concentrating mechanisms.

Structural Basis for the Allosteric Regulation of the SbtA Bicarbonate Transporter by the PII-like Protein, SbtB, from Cyanobium sp. PCC7001.

Cyanobacteria have evolved a suite of enzymes and inorganic carbon (Ci) transporters that improve photosynthetic performance by increasing the localized concentration of CO2 around the primary CO2-fixating enzyme, Rubisco. This CO2-concentrating mechanism (CCM) is highly regulated, responds to illumination/darkness cycles, and allows cyanobacteria to thrive under limiting Ci conditions. While the transcriptional control of CCM activity is well understood, less is known about how regulatory proteins might allosterically regulate Ci transporters in response to changing conditions. Cyanobacterial sodium-dependent bicarbonate transporters (SbtAs) are inhibited by PII-like regulatory proteins (SbtBs), with the inhibitory effect being modulated by adenylnucleotides. Here, we used isothermal titration calorimetry to show that SbtB from Cyanobium sp. PCC7001 (SbtB7001) binds AMP, ADP, cAMP, and ATP with micromolar-range affinities. X-ray crystal structures of apo and nucleotide-bound SbtB7001 revealed that while AMP, ADP, and cAMP have little effect on the SbtB7001 structure, binding of ATP stabilizes the otherwise flexible T-loop, and that the flexible C-terminal C-loop adopts several distinct conformations. We also show that ATP binding affinity is increased 10-fold in the presence of Ca2+, and we present an X-ray crystal structure of Ca2+ATP:SbtB7001 that shows how this metal ion facilitates additional stabilizing interactions with the apex of the T-loop. We propose that the Ca2+ATP-induced conformational change observed in SbtB7001 is important for allosteric regulation of SbtA activity by SbtB and is consistent with changing adenylnucleotide levels in illumination/darkness cycles.

Progress and challenges of engineering a biophysical CO2-concentrating mechanism into higher plants.

Growth and productivity in important crop plants is limited by the inefficiencies of the C3 photosynthetic pathway. Introducing CO2-concentrating mechanisms (CCMs) into C3 plants could overcome these limitations and lead to increased yields. Many unicellular microautotrophs, such as cyanobacteria and green algae, possess highly efficient biophysical CCMs that increase CO2 concentrations around the primary carboxylase enzyme, Rubisco, to enhance CO2 assimilation rates. Algal and cyanobacterial CCMs utilize distinct molecular components, but share several functional commonalities. Here we outline the recent progress and current challenges of engineering biophysical CCMs into C3 plants. We review the predicted requirements for a functional biophysical CCM based on current knowledge of cyanobacterial and algal CCMs, the molecular engineering tools and research pipelines required to translate our theoretical knowledge into practice, and the current challenges to achieving these goals.

Identification and characterization of a solute carrier, CIA8, involved in inorganic carbon acclimation in Chlamydomonas reinhardtii.

The supply of inorganic carbon (Ci) at the site of fixation by Rubisco is a key parameter for efficient CO2 fixation in aquatic organisms including the green alga, Chlamydomonas reinhardtii. Chlamydomonas reinhardtii cells, when grown on limiting CO2, have a CO2-concentrating mechanism (CCM) that functions to concentrate CO2 at the site of Rubisco. Proteins thought to be involved in inorganic carbon uptake have been identified and localized to the plasma membrane or chloroplast envelope. However, current CCM models suggest that additional molecular components are involved in Ci uptake. In this study, the gene Cia8 was identified in an insertional mutagenesis screen and characterized. The protein encoded by Cia8 belongs to the sodium bile acid symporter subfamily. Transcript levels for this gene were significantly up-regulated when the cells were grown on low CO2. The cia8 mutant exhibited reduced growth and reduced affinity for Ci when grown in limiting CO2 conditions. Prediction programs localize this protein to the chloroplast. Ci uptake and the photosynthetic rate, particularly at high external pH, were reduced in the mutant. The results are consistent with the model that CIA8 is involved in Ci uptake in C. reinhardtii.

[Crossed Aphasia]. / Gekreuzte Aphasie.

Crossed aphasia (CA) is a rare acquired language disorder caused by a right-sided brain lesion in dextrals. Based on a case report, relevant aspects for the diagnosis of CA and differential diagnoses will be outlined. Relevant hypotheses concerning etiology, epidemiology, phenomenology and pathophysiology will be discussed with reference to the literature. The phenomenon of CA has contributed for decades to the development of hypotheses concerning lateralization of cognitive abilities.

Decreased photochemical efficiency of photosystem II following sunlight exposure of shade-grown leaves of avocado: because of, or in spite of, two kinetically distinct xanthophyll cycles?

This study resolved correlations between changes in xanthophyll pigments and photosynthetic properties in attached and detached shade-grown avocado (Persea americana) leaves upon sun exposure. Lutein epoxide (Lx) was deepoxidized to lutein (L), increasing the total pool by ΔL over 5 h, whereas violaxanthin (V) conversion to antheraxanthin (A) and zeaxanthin (Z) ceased after 1 h. During subsequent dark or shade recovery, de novo synthesis of L and Z continued, followed by epoxidation of A and Z but not of L. Light-saturated nonphotochemical quenching (NPQ) was strongly and linearly correlated with decreasing [Lx] and increasing [L] but showed a biphasic correlation with declining [V] and increasing [A+Z] separated when V deepoxidation ceased. When considering [ΔL+Z], the monophasic linear correlation was restored. Photochemical efficiency of photosystem II (PSII) and photosystem (PSI; deduced from the delivery of electrons to PSI in saturating single-turnover flashes) showed a strong correlation in their continuous decline in sunlight and an increase in NPQ capacity. This decrease was also reflected in the initial reduction of the slope of photosynthetic electron transport versus photon flux density. Generally longer, stronger sun exposures enhanced declines in both slope and maximum photosynthetic electron transport rates as well as photochemical efficiency of PSII and PSII/PSI more severely and prevented full recovery. Interestingly, increased NPQ capacity was accompanied by slower relaxation. This was more prominent in detached leaves with closed stomata, indicating that photorespiratory recycling of CO(2) provided little photoprotection to avocado shade leaves. Sun exposure of these shade leaves initiates a continuum of photoprotection, beyond full engagement of the Lx and V cycle in the antenna, but ultimately photoinactivated PSII reaction centers.

Cyanobacterial α-carboxysome carbonic anhydrase is allosterically regulated by the Rubisco substrate RuBP.

Cyanobacterial CO2 concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp. PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial α-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage.

Gymnosperms have increased capacity for electron leakage to oxygen (Mehler and PTOX reactions) in photosynthesis compared with angiosperms.

Oxygen plays an important role in photosynthesis by participating in a number of O2-consuming reactions. O2 inhibits CO2 fixation by stimulating photorespiration, thus reducing plant production. O2 interacts with photosynthetic electron transport in the chloroplasts' thylakoids in two main ways: by accepting electrons from PSI (Mehler reaction); and by accepting electrons from reduced plastoquinone (PQ) mediated by the plastid terminal oxidase (PTOX). In this study, we show, using 101 plant species, that there is a difference in the potential for photosynthetic electron flow to O2 between angiosperms and gymnosperms. We found, from measurements of Chl fluorescence and leaf absorbance at 830 nm, (i) that electron outflow from PSII, as determined by decay kinetics of Chl fluorescence after application of a saturating light pulse, is more rapid in gymnosperms than in angiosperms; (ii) that the reaction center Chl of PSI (P700) is rapidly and highly oxidized in gymnosperms during induction of photosynthesis; and (iii) that these differences are dependent on oxygen. Finally, rates of O2 uptake measured by mass spectrometry in the absence of photorespiration were significantly promoted by illumination in dark-adapted leaves of gymnosperms, but not in those of angiosperms. The light-stimulated O2 uptake was around 10% of the maximum O2 evolution in gymnosperms and 1% in angiosperms. These results suggest that gymnosperms have increased capacity for electron leakage to oxygen in photosynthesis compared with angiosperms. The involvement of the Mehler reaction and PTOX in the electron flow to O2 is discussed.

The cyanobacterial CCM as a source of genes for improving photosynthetic CO2 fixation in crop species.

Crop yields need to nearly double over the next 35 years to keep pace with projected population growth. Improving photosynthesis, via a range of genetic engineering strategies, has been identified as a promising target for crop improvement with regard to increased photosynthetic yield and better water-use efficiency (WUE). One approach is based on integrating components of the highly efficient CO(2)-concentrating mechanism (CCM) present in cyanobacteria (blue-green algae) into the chloroplasts of key C(3) crop plants, particularly wheat and rice. Four progressive phases towards engineering components of the cyanobacterial CCM into C(3) species can be envisaged. The first phase (1a), and simplest, is to consider the transplantation of cyanobacterial bicarbonate transporters to C(3) chloroplasts, by host genomic expression and chloroplast targeting, to raise CO(2) levels in the chloroplast and provide a significant improvement in photosynthetic performance. Mathematical modelling indicates that improvements in photosynthesis as high as 28% could be achieved by introducing both of the single-gene, cyanobacterial bicarbonate transporters, known as BicA and SbtA, into C(3) plant chloroplasts. Part of the first phase (1b) includes the more challenging integration of a functional cyanobacterial carboxysome into the chloroplast by chloroplast genome transformation. The later three phases would be progressively more elaborate, taking longer to engineer other functional components of the cyanobacterial CCM into the chloroplast, and targeting photosynthetic and WUE efficiencies typical of C(4) photosynthesis. These later stages would include the addition of NDH-1-type CO(2) pumps and suppression of carbonic anhydrase and C(3) Rubisco in the chloroplast stroma. We include a score card for assessing the success of physiological modifications gained in phase 1a.

Lutein from deepoxidation of lutein epoxide replaces zeaxanthin to sustain an enhanced capacity for nonphotochemical chlorophyll fluorescence quenching in avocado shade leaves in the dark.

Leaves of avocado (Persea americana) that develop and persist in deep shade canopies have very low rates of photosynthesis but contain high concentrations of lutein epoxide (Lx) that are partially deepoxidized to lutein (L) after 1 h of exposure to 120 to 350 µmol photons m(-2) s(-1), increasing the total L pool by 5% to 10% (ΔL). Deepoxidation of Lx to L was near stoichiometric and similar in kinetics to deepoxidation of violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z). Although the V pool was restored by epoxidation of A and Z overnight, the Lx pool was not. Depending on leaf age and pretreatment, the pool of ΔL persisted for up to 72 h in the dark. Metabolism of ΔL did not involve epoxidation to Lx. These contrasting kinetics enabled us to differentiate three states of the capacity for nonphotochemical chlorophyll fluorescence quenching (NPQ) in attached and detached leaves: ΔpH dependent (NPQ(ΔpH)) before deepoxidation; after deepoxidation in the presence of ΔL, A, and Z (NPQ(ΔLAZ)); and after epoxidation of A+Z but with residual ΔL (NPQ(ΔL)). The capacity of both NPQ(ΔLAZ) and NPQ(ΔL) was similar and 45% larger than NPQ(ΔpH), but dark relaxation of NPQ(ΔLAZ) was slower. The enhanced capacity for NPQ was lost after metabolism of ΔL. The near equivalence of NPQ(ΔLAZ) and NPQ(ΔL) provides compelling evidence that the small dynamic pool ΔL replaces A+Z in avocado to "lock in" enhanced NPQ. The results are discussed in relation to data obtained with other Lx-rich species and in mutants of Arabidopsis (Arabidopsis thaliana) with increased L pools.

The CO2-concentrating mechanism of Synechococcus WH5701 is composed of native and horizontally-acquired components.

The cyanobacterial CO(2)-concentrating mechanism (CCM) is an effective adaptation that increases the carbon dioxide (CO(2)) concentration around the primary photosynthetic enzyme Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (RuBisCO). α-Cyanobacteria (those containing Form1-A RuBisCO within cso-type α-carboxysomes) have a limited CCM composed of a small number of Ci-transporters whereas ß-cyanobacteria (those species containing Form-1B RuBisCO within ccm-type ß-carboxysomes) exhibit a more diverse CCM with a greater variety in Ci-transporter complement and regulation. In the coastal species Synechococcus sp. WH5701 (α-cyanobacteria), the minimal α-cyanobacterial CCM has been supplemented with ß-cyanobacterial Ci transporters through the process of horizontal gene transfer (HGT). These transporters are transcriptionally regulated in response to external Ci-depletion however this change in transcript abundance is not correlated with a physiological induction. WH5701 exhibits identical physiological responses grown at 4% CO(2) (K (1/2) ≈ 31 µM Ci) and after induction with 0.04% CO(2) (K (1/2) ≈ 29 µM Ci). Insensitivity to external Ci concentration is an unusual characteristic of the WH5701 CCM which is a result of evolution by HGT. Our bioinformatic and physiological data support the hypothesis that WH5701 represents a clade of α-cyanobacterial species in transition from the marine/oligotrophic environment to a coastal/freshwater environment.

Engineered Accumulation of Bicarbonate in Plant Chloroplasts: Known Knowns and Known Unknowns.

Heterologous synthesis of a biophysical CO2-concentrating mechanism (CCM) in plant chloroplasts offers significant potential to improve the photosynthetic efficiency of C3 plants and could translate into substantial increases in crop yield. In organisms utilizing a biophysical CCM, this mechanism efficiently surrounds a high turnover rate Rubisco with elevated CO2 concentrations to maximize carboxylation rates. A critical feature of both native biophysical CCMs and one engineered into a C3 plant chloroplast is functional bicarbonate (HCO3 -) transporters and vectorial CO2-to-HCO3 - converters. Engineering strategies aim to locate these transporters and conversion systems to the C3 chloroplast, enabling elevation of HCO3 - concentrations within the chloroplast stroma. Several CCM components have been identified in proteobacteria, cyanobacteria, and microalgae as likely candidates for this approach, yet their successful functional expression in C3 plant chloroplasts remains elusive. Here, we discuss the challenges in expressing and regulating functional HCO3 - transporter, and CO2-to-HCO3 - converter candidates in chloroplast membranes as an essential step in engineering a biophysical CCM within plant chloroplasts. We highlight the broad technical and physiological concerns which must be considered in proposed engineering strategies, and present our current status of both knowledge and knowledge-gaps which will affect successful engineering outcomes.

Plastid signalling to the nucleus and beyond.

Communication between the compartments or organelles of cells is essential for plant growth and development. There is an emerging understanding of signals generated within energy-transducing organelles, such as chloroplasts and mitochondria, and the nuclear genes that respond to them, a process known as retrograde signalling. A recent series of unconnected breakthroughs have given scientists a glimpse inside the 'black box' of organellar signalling thanks to the identification of some of the factors involved in generating and propagating signals to the nucleus and, in some instances, systemically throughout photosynthetic tissues. This review will focus on recent developments in our understanding of retrograde and systemic signals generated by organelles, with an emphasis on chloroplasts.

Improved survival of very high light and oxidative stress is conferred by spontaneous gain-of-function mutations in Chlamydomonas.

Investigations into high light and oxidative stress in photosynthetic organisms have focussed primarily on genetic impairment of different photoprotective functions. There are few reports of "gain-of-function" mutations that provide enhanced resistance to high light and/or oxidative stress without reduced productivity. We have isolated at least four such very high light resistant (VHL(R)) mutations in the green alga, Chlamydomonas reinhardtii, that permit near maximal growth rates at light intensities lethal to wild type. This resistance is not due to an alteration in electron transport rate or quantity and functionality of the two photosystems that could have enhanced photochemical quenching. Nor is it due to reduced excitation pressure by downregulation of the light harvesting antennae or increased nonphotochemical quenching. In fact, photosynthetic activity is unaffected in more than 30 VHL(R) isolates. Instead, the basis of the VHL(R) phenotype is a combination of traits, which appears to be dominated by enhanced capacity to tolerate reactive oxygen species generated by excess light, methylviologen, rose bengal or hydrogen peroxide. This is further evidenced in lower levels of ROS after exposure to very high light in the VHL(R)-S9 mutant. Additionally, the VHL(R) phenotype is associated with increased zeaxanthin accumulation, maintenance of fast synthesis and degradation rates of the D1 protein, and sustained balanced electron flow into and out of PSI under very high light. We conclude that the VHL(R) mutations arose from a selection pressure that favors changes to the regulatory system(s) that coordinates several photoprotective processes amongst which repair of PSII and enhanced detoxification of reactive oxygen species play seminal roles.

Cyanobacterial CO2-concentrating mechanism components: function and prospects for plant metabolic engineering.

Global population growth is projected to outpace plant-breeding improvements in major crop yields within decades. To ensure future food security, multiple creative efforts seek to overcome limitations to crop yield. Perhaps the greatest limitation to increased crop yield is photosynthetic inefficiency, particularly in C3 crop plants. Recently, great strides have been made toward crop improvement by researchers seeking to introduce the cyanobacterial CO2-concentrating mechanism (CCM) into plant chloroplasts. This strategy recognises the C3 chloroplast as lacking a CCM, and being a primordial cyanobacterium at its essence. Hence the collection of solute transporters, enzymes, and physical structures that make cyanobacterial CO2-fixation so efficient are viewed as a natural source of genetic material for C3 chloroplast improvement. Also we highlight recent outstanding research aimed toward the goal of introducing a cyanobacterial CCM into C3 chloroplasts and consider future research directions.

Processes contributing to photoprotection of grapevine leaves illuminated at low temperature.

Photoinactivation of photosystem II (PSII) and energy dissipation at low leaf temperatures were investigated in leaves of glasshouse-grown grapevine (Vitis vinifera L. cv. Riesling). At low temperatures (< 15 degrees C), photosynthetic rates of CO(2) assimilation were reduced. However, despite a significant increase in the amount of light excessive to that required by photosynthesis, grapevine leaves maintained high intrinsic quantum efficiencies of PSII (F(v)/F(m)) and were highly resistant to photoinactivation compared to other species. Non-photochemical energy dissipation involving xanthophylls and fast D1 repair were the main protective processes reducing the 'gross' rate of photoinactivation and the 'net' rate of photoinactivation, respectively. We developed an improved method of energy dissipation analysis that revealed up to 75% of absorbed light is dissipated thermally via pH- and xanthophyll-mediated non-photochemical quenching at low temperatures (5-15 degrees C) and moderate (800 micro mol quanta m(-2) s(-1)) light. Up to 20% of the energy flux contributing to electron transport was dissipated via photorespiration when taking into account temperature-dependent mesophyll conductance; however, this flux used in photorespiration was only a relatively small amount of the total absorbed light energy. Photoreduction of O(2) at photosystem I (PSI) and subsequent superoxide detoxification (water-water cycle) was more sensitive to inhibition by low temperature than photorespiration. Therefore the water-water cycle represents a negligibly small energy sink below 15 degrees C, irrespective of mesophyll conductance.