We revisit the relationship between plant water use efficiency and carbon isotope signatures (delta(13)C) of plant material. Based on the definitions of intrinsic, instantaneous and integrated water use efficiency, we discuss the implications for interpreting delta(13)C data from leaf to landscape levels, and across diurnal to decadal timescales. Previous studies have often applied a simplified, linear relationship between delta(13)C, ratios of intercellular to ambient CO(2) mole fraction (C (i)/C (a)), and water use efficiency. In contrast, photosynthetic (13)C discrimination (Delta) is sensitive to the ratio of the chloroplast to ambient CO(2) mole fraction, C (c)/C (a) (rather than C (i)/C (a)) and, consequently, to mesophyll conductance. Because mesophyll conductance may differ between species and over time, it is not possible to determine C (c)/C (a) from the same gas exchange measurements as C (i)/C (a). On the other hand, water use efficiency at the leaf level depends on evaporative demand, which does not directly affect Delta. Water use efficiency and Delta can thus vary independently, making it difficult to obtain trends in water use efficiency from delta(13)C data. As an alternative approach, we offer a model available at http://carbonisotopes.googlepages.com to explore how water use efficiency and (13)C discrimination are related across leaf and canopy scales. The model provides a tool to investigate whether trends in Delta indicate changes in leaf functional traits and/or environmental conditions during leaf growth, and how they are associated with trends in plant water use efficiency. The model can be used, for example, to examine whether trends in delta(13)C signatures obtained from tree rings imply changes in tree water use efficiency in response to atmospheric CO(2) increase. This is crucial for predicting how plants may respond to future climate change.

Non-photosynthetic, or heterotrophic, tissues in C3 plants tend to be enriched in 13C compared with the leaves that supply them with photosynthate. This isotopic pattern has been observed for woody stems, roots, seeds and fruits, emerging leaves, and parasitic plants incapable of net CO2 fixation. Unlike in C3 plants, roots of herbaceous C4 plants are generally not 13C-enriched compared with leaves. We review six hypotheses aimed at explaining this isotopic pattern in C3 plants: (1) variation in biochemical composition of heterotrophic tissues compared with leaves; (2) seasonal separation of growth of leaves and heterotrophic tissues, with corresponding variation in photosynthetic discrimination against 13C; (3) differential use of day v. night sucrose between leaves and sink tissues, with day sucrose being relatively 13C-depleted and night sucrose 13C-enriched; (4) isotopic fractionation during dark respiration; (5) carbon fixation by PEP carboxylase; and (6) developmental variation in photosynthetic discrimination against 13C during leaf expansion. Although hypotheses (1) and (2) may contribute to the general pattern, they cannot explain all observations. Some evidence exists in support of hypotheses (3) through to (6), although for hypothesis (6) it is largely circumstantial. Hypothesis (3) provides a promising avenue for future research. Direct tests of these hypotheses should be carried out to provide insight into the mechanisms causing within-plant variation in carbon isotope composition.

Crassulacean acid metabolism (CAM) is a photosynthetic adaptation that facilitates the uptake of CO(2) at night and thereby optimizes the water-use efficiency of carbon assimilation in plants growing in arid habitats. A number of CAM species have been exploited agronomically in marginal habitats, displaying annual above-ground productivities comparable with those of the most water-use efficient C(3) or C(4) crops but with only 20% of the water required for cultivation. Such attributes highlight the potential of CAM plants for carbon sequestration and as feed stocks for bioenergy production on marginal and degraded lands. This review highlights the metabolic and morphological features of CAM that contribute towards high biomass production in water-limited environments. The temporal separation of carboxylation processes that underpins CAM provides flexibility for modulating carbon gain over the day and night, and poses fundamental questions in terms of circadian control of metabolism, growth, and productivity. The advantages conferred by a high water-storage capacitance, which translate into an ability to buffer fluctuations in environmental water availability, must be traded against diffusive (stomatal plus internal) constraints imposed by succulent CAM tissues on CO(2) supply to the cellular sites of carbon assimilation. The practicalities for maximizing CAM biomass and carbon sequestration need to be informed by underlying molecular, physiological, and ecological processes. Recent progress in developing genetic models for CAM are outlined and discussed in light of the need to achieve a systems-level understanding that spans the molecular controls over the pathway through to the agronomic performance of CAM and provision of ecosystem services on marginal lands.

Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO 2 -fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO 2 . Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO 2 . We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1's four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency.pyrenoid | Rubisco | carbon fixation | Chlamydomonas reinhardtii | CO 2 -concentrating mechanism

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