Transcriptomic and proteomic studies have improved our knowledge of guard cell function; however, metabolic changes in guard cells remain relatively poorly understood. Here we analysed metabolic changes in guard cell-enriched epidermal fragments from tobacco during light-induced stomatal opening. Increases in sucrose, glucose and fructose were observed during light-induced stomatal opening in the presence of sucrose in the medium while no changes in starch were observed, suggesting that the elevated fructose and glucose levels were a consequence of sucrose rather than starch breakdown. Conversely, reduction in sucrose was observed during light- plus potassium-induced stomatal opening. Concomitant with the decrease in sucrose, we observed an increase in the level as well as in the (13) C enrichment in metabolites of, or associated with, the tricarboxylic acid cycle following incubation of the guard cell-enriched preparations in (13) C-labelled bicarbonate. Collectively, the results obtained support the hypothesis that sucrose is catabolized within guard cells in order to provide carbon skeletons for organic acid production. Furthermore, they provide a qualitative demonstration that CO2 fixation occurs both via ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPcase). The combined data are discussed with respect to current models of guard cell metabolism and function.

We aimed to evaluate the currently used allometric models, as well as to propose a reliable and accurate model using non-destructive measurements of leaf length (L) and/or width (W), for estimating the area of leaves of eight field-grown coffee cultivars. For model construction, a total of 1563 leaves were randomly selected from different levels of the tree canopies and encompassed the full spectrum of measurable leaf sizes (0.3-263 cm 2 ) for each genotype. Power models better fit coffee leaf area (LA) than linear models. To validate the model, an independent data set of 388 leaves was used. We demonstrated that the currently used allometric models are biased, underestimating the area of a coffee leaf. We developed a single power modelbased on two leaf dimensions [LA = 0.6626 (LW) 1.0116 ; standard errors: b 0 = 0.0064, b 1 = 0.0019; R 2 = 0.996] with high precision and accuracy, random dispersion pattern of residuals and also unbiased, irrespective of cultivar and leaf size and shape. Even when the L (but not width) alone was used as the single leaf dimension, the power model developed still predicted with good accuracy the LA but at the expense of some loss of precision, as particularly found for 8% of the leaves sampled with length-to-width ratios below 2.0 or above 3.0.

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Sucrose has long been thought to play an osmolytic role in stomatal opening. However, recent evidence supports the idea that the role of sucrose in this process is primarily energetic. Here we used a combination of stomatal aperture assays and kinetic [U- C]-sucrose isotope labelling experiments to confirm that sucrose is degraded during light-induced stomatal opening and to define the fate of the C released from sucrose breakdown. We additionally show that addition of sucrose to the medium did not enhance light-induced stomatal opening. The isotope experiment showed a consistent C enrichment in fructose and glucose, indicating that during light-induced stomatal opening sucrose is indeed degraded. We also observed a clear C enrichment in glutamate and glutamine (Gln), suggesting a concerted activation of sucrose degradation, glycolysis and the tricarboxylic acid cycle. This is in contrast to the situation for Gln biosynthesis in leaves under light, which has been demonstrated to rely on previously stored C. Our results thus collectively allow us to redraw current models concerning the influence of sucrose during light-induced stomatal opening, in which, instead of being accumulated, sucrose is degraded providing C skeletons for Gln biosynthesis.

As water availability for agriculture decreases, breeding or engineering of crops with improved water use efficiency (WUE) will be necessary. As stomata are responsible for controlling gas exchange across the plant epidermis, metabolic processes influencing solute accumulation in guard cells are potential targets for engineering. In addition to its role as an osmoticum, sucrose breakdown may be required for synthesis of other osmotica or generation of the ATP needed for solute uptake. Thus, alterations in partitioning of sucrose between storage and breakdown may affect stomatal function. In agreement with this hypothesis, potato (Solanum tuberosum) plants expressing an antisense construct targeted against sucrose synthase 3 (SuSy3) exhibited decreased stomatal conductance, a slight reduction in CO2 fixation and increased WUE. Conversely, plants with increased guard cell acid invertase activity caused by the introduction of the SUC2 gene from yeast had increased stomatal conductance, increased CO2 fixation and decreased WUE. 14 CO2 feeding experiments indicated that these effects cannot be attributed to alterations in photosynthetic capacity, and most likely reflect alterations in stomatal function. These results highlight the important role that sucrose breakdown may play in guard cell function and indicate the feasibility of manipulating plant WUE through engineering of guard cell sucrose metabolism.

Summary• Perturbations of the source-sink balances were performed in field-grown coffee (Coffea arabica) trees to investigate the possible role of carbohydrates in feedback regulation of photosynthesis.• Four treatments were applied at the whole-plant level: (i) complete defruiting and maintenance of the full leaf area, (ii) the half crop load and full leaf area, (iii) the full crop load and full leaf area and (iv) the full crop load and half leaf area. Sampling and measurements were performed twice during the phase of dry matter accumulation of fruits. Gas exchange, chlorophyll a fluorescence, carbon isotope labelling and steady-state metabolite measurements were assessed in source leaves.• The average rate of net photosynthetic rate (A) and stomatal conductance (g s ) were larger (> 50%), and carbon isotope composition ratio was lower, in trees with a full crop load and half leaf area than in defruited trees, with individuals of the other two treatments showing intermediate values. However, differences in A seem unlikely to have been caused either by photochemical impairments or a direct end-product-mediated feedback down-regulation of photosynthesis.• It is proposed that the decreased A in defruited coffee trees was independent of carbon metabolism and was rather directly related to a lower CO 2 availability coupled to lower g s .

Coffee is native to shady environments but often grows better and produces higher yields without shade, though at the expense of high fertilization inputs, particularly nitrogen (N). Potted plants were grown under full sunlight and shade (50%) conditions and were fertilized with nutrient solutions containing either 0 or 23 mM N. Measurements were made in southeastern Brazil during winter conditions, when relatively low night temperatures and high diurnal insolation are common. Overall, the net carbon assimilation rate was quite low, which was associated with diffusive, rather than biochemical, constraints. N deficiency led to decreases in the concentrations of chlorophylls (Chl) and total carotenoids as well as in the Chl/N ratio. These conditions also led to qualitative changes in the carotenoid composition, e.g., increased antheraxanthin (A) and zeaxanthin (Z) pools on a Chl basis, particularly at high light, which was linked to increased thermal dissipation of absorbed light. The variable-to-maximum fluorescence ratio at predawn decreased with increasing A+Z pools and decreased linearly with decreasing N. We showed that this ratio was inadequate for assessing photoinhibition under N limitation. Expressed per unit mass, the activities of superoxide dismutase and glutathione reductase were not altered with the treatments. In contrast, ascorbate peroxidase activity was lower in low N plants, particularly under shade, whereas catalase activity was lower in shaded plants than in sun-grown plants, regardless of the N level. Glutamine synthetase activity was greater in sun-grown plants than in shaded individuals at a given N level and decreased with decreasing N application. Our results suggest that the photoprotective and antioxidant capacity per amount of photons absorbed was up-regulated by a low N supply; nevertheless, this capacity, regardless of the light conditions, was not enough to prevent oxidative damage, as judged from the increases in the H(2)O(2) and malondialdehyde concentrations and electrolyte leakage. We demonstrated that N fertilization could adequately protect the coffee plants against photodamage independently of the anticipated positive effects of N on the photosynthetic capacity.

SummaryIsoform 3 of sucrose synthase (SUS3) is highly expressed in guard cells; however, the precise function of SUS3 in this cell type remains to be elucidated.Here, we characterized transgenic Nicotiana tabacum plants overexpressing SUS3 under the control of the stomatal-specific KST1 promoter, and investigated the changes in guard cell metabolism during the dark to light transition.Guard cell-specific SUS3 overexpression led to increased SUS activity, stomatal aperture, stomatal conductance, transpiration rate, net photosynthetic rate and growth. Although only minor changes were observed in the metabolite profile in whole leaves, an increased fructose level and decreased organic acid levels and sucrose to fructose ratio were observed in guard cells of transgenic lines. Furthermore, guard cell sucrose content was lower during lightinduced stomatal opening. In a complementary approach, we incubated guard cell-enriched epidermal fragments in 13 C-NaHCO 3 and followed the redistribution of label during dark to light transitions; this revealed increased labeling in metabolites of, or associated with, the tricarboxylic acid cycle. The results suggest that sucrose breakdown is a mechanism to provide substrate for the provision of organic acids for respiration, and imply that manipulation of guard cell metabolism may represent an effective strategy for plant growth improvement.