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Showing papers in "Functional Plant Biology in 2007"


Journal ArticleDOI
TL;DR: In this paper, the authors present theoretical models describing the influences on stable oxygen isotope analysis of plant organic material (δ18Op), using recently published experimental work to outline strengths and weaknesses in the models.
Abstract: With the development of rapid measurement techniques, stable oxygen isotope analysis of plant tissue is poised to become an important tool in plant physiological, ecological, paleoclimatic and forensic studies. Recent advances in mechanistic understanding have led to the improvement of process-based models that accurately predict variability in the oxygen isotope composition of plant organic material (δ18Op). δ18Op has been shown to reflect the isotope composition of soil water, evaporative enrichment in transpiring leaves, and isotopic exchange between oxygen atoms in organic molecules and local water in the cells in which organic molecules are formed. This review presents current theoretical models describing the influences on δ18Op, using recently published experimental work to outline strengths and weaknesses in the models. The potential and realised applications of the technique are described.

536 citations


Journal ArticleDOI
TL;DR: Results show that a plant's ability to maintain high K+/Na+ ratio (either retention of K+ or preventing Na+ from accumulating in leaves) is a key feature for salt tolerance in barley.
Abstract: A large-scale glasshouse trial, including nearly 70 barley cultivars (5300 plants in total), was conducted over 2 consecutive years to investigate plant physiological responses to salinity. In a parallel set of experiments, plant salt tolerance was assessed by non-invasive microelectrode measurements of net K+ flux from roots of 3-day-old seedlings of each cultivar after 1 h treatment in 80 mm NaCl as described in our previous publication (Chen et al. 2005). K+ flux from the root in response to NaCl treatment was highly (P < 0.001) inversely correlated with relative grain yield, shoot biomass, plant height, net CO2 assimilation, survival rate and thousand-seed weight measured in glasshouse experiments after 4-5 months of salinity treatment. No significant correlation with relative germination rate or tillering was found. In general, 62 out of 69 cultivars followed an inverse relationship between K+ efflux and salt tolerance. In a few cultivars, however, high salt tolerance (measured as grain yield at harvest) was observed for plants showing only modest ability to retain K+ in the root cells. Tissue elemental analysis showed that these plants had a much better ability to prevent Na+ accumulation in plant leaves and, thus, to maintain a higher K+/Na+ ratio. Taken together, our results show that a plant's ability to maintain high K+/Na+ ratio (either retention of K+ or preventing Na+ from accumulating in leaves) is a key feature for salt tolerance in barley.

302 citations


Journal ArticleDOI
TL;DR: Under drought, canopy temperature (CT) was the single-most drought-adaptive trait contributing to a higher performance and can be utilised as a selection criteria to identify high-yielding wheat genotypes or as an important predictor of yield performance under drought.
Abstract: Agronomic and physiological traits associated with drought adaptation were assessed within the Seri/Babax recombinant inbred line population, which was derived from parents similar in height and maturity but divergent in their sensitivity to drought. Field trials under different water regimes were conducted over 3 years in Mexico and under rainfed conditions in Australia. Under drought, canopy temperature (CT) was the single-most drought-adaptive trait contributing to a higher performance (r 2 = 0.74, P < 0.0001), highly heritable (h 2 = 0.65, P < 0.0001) and consistently associated with yield phenotypically (r =− 0.75, P < 0.0001) and genetically (R(g) =− 0.95, P < 0.0001). CT epitomises a mechanism of dehydration avoidance expressed throughout the cycle and across latitudes, which can be utilised as a selection criteria to identify high-yielding wheat genotypes or as an important predictor of yield performance under drought. Early response under drought, suggested by a high association of CT with estimates of biomass at booting (r =− 0.44, P < 0.0001), leaf chlorophyll (r =− 0.22 P < 0.0001) and plant height (r =− 0.64, P < 0.0001), contrast with the small relationships with anthesis and maturity (averaged, r =− 0.10, P < 0.0001), and with osmotic potential (r =− 0.20, P < 0.0001). Results suggest that the ability to extract water from the soil under increasing soil water deficit is a major attribute of drought adaptation. The genetic variation and transgressive segregation suggest further genomic and transcriptomic studies for unravelling the complex relationship between drought adaptation and performance under drought.

234 citations


Journal ArticleDOI
TL;DR: Interest in nutrient loading of seeds is fuelled by its central importance to plant reproductive success and human nutrition and it is speculated that aquaporins concentrated in vascular and surrounding parenchyma cells of legume seed coats could provide a gated pathway of water movement in these tissues.
Abstract: Interest in nutrient loading of seeds is fuelled by its central importance to plant reproductive success and human nutrition. Rates of nutrient loading, imported through the phloem, are regulated by transport and transfer processes located in sources (leaves, stems, reproductive structures), phloem pathway and seed sinks. During the early phases of seed development, most control is likely to be imposed by a low conductive pathway of differentiating phloem cells serving developing seeds. Following the onset of storage product accumulation by seeds, and, depending on nutrient species, dominance of path control gives way to regulation by processes located in sources (nitrogen, sulfur, minor minerals), phloem path (transition elements) or seed sinks (sugars and major mineral elements, such as potassium). Nutrients and accompanying water are imported into maternal seed tissues and unloaded from the conducting sieve elements into an extensive post-phloem symplasmic domain. Nutrients are released from this symplasmic domain into the seed apoplasm by poorly understood membrane transport mechanisms. As seed development progresses, increasing volumes of imported phloem water are recycled back to the parent plant by process(es) yet to be discovered. However, aquaporins concentrated in vascular and surrounding parenchyma cells of legume seed coats could provide a gated pathway of water movement in these tissues. Filial cells, abutting the maternal tissues, take up nutrients from the seed apoplasm by membrane proteins that include sucrose and amino acid/H+ symporters functioning in parallel with non-selective cation channels. Filial demand for nutrients, that comprise the major osmotic species, is integrated with their release and phloem import by a turgor-homeostat mechanism located in maternal seed tissues. It is speculated that turgors of maternal unloading cells are sensed by the cytoskeleton and transduced by calcium signalling cascades.

181 citations


Journal ArticleDOI
John E. Lunn1
TL;DR: This survey shows that the gene families encoding the enzymes of trehalose metabolism are very ancient, pre-dating the divergence of the streptophyte and chlorophyte lineages, and provides a frame of reference for future studies to elucidate the function of tre Halose metabolism in plants.
Abstract: The genomes of Arabidopsis thaliana L., rice (Oryza sativa L.) and poplar (Populus trichocarpa Torr. & A.Gray) contain large families of genes encoding trehalose-phosphate synthase (TPS) and trehalose-phosphatase (TPP). The class I subfamily of TPS genes encodes catalytically active TPS enzymes, and is represented by only one or two genes in most species. A. thaliana is atypical in having four class I TPS genes, three of which (AtTPS2-4) encode unusual short isoforms of TPS that appear to be found only in members of the Brassicaceae family. The class II TPS genes encode TPS-like proteins with a C-terminal TPP-like domain, but there is no experimental evidence that they have any enzymatic activity and their function is unknown. Both classes of TPS gene are represented in the genomes of chlorophyte algae (Ostreococcus species) and non-flowering plants [Physcomitrella patens (Hedw.) Bruch & Schimp.(B.S.G.) and Selaginella moellendorffii (Hieron. in Engl. & Prantl.)]. This survey shows that the gene families encoding the enzymes of trehalose metabolism are very ancient, pre-dating the divergence of the streptophyte and chlorophyte lineages. It also provides a frame of reference for future studies to elucidate the function of trehalose metabolism in plants.

138 citations


Journal ArticleDOI
TL;DR: Methodological advice is offered to delay use of automatically calculated fluorescence parameters, presented by the instrument software, until raw data 'traces' have been carefully inspected to ensure the integrity of findings.
Abstract: The determination of chlorophyll fluorescence emission is a powerful tool for assessing the status of PSII and the allocation of absorbed light to photosynthesis v. photoprotective energy dissipation. The development of field-portable fluorometers has enabled growing numbers of scientists to measure fluorescence emission from plants in diverse field settings. However, the ease of operation of contemporary fluorometers masks the many challenges associated with collecting meaningful and interpretable fluorescence signals from leaves exposed to relevant environmental conditions. Here, we offer methodological advice aimed at, but not limited to, the non-specialist for the proper measurement of fluorescence parameters, with an emphasis on avoiding common errors in the use of fluorescence under field conditions. Chief among our suggestions is (1) to delay use of automatically calculated fluorescence parameters, presented by the instrument software, until raw data 'traces' have been carefully inspected to ensure the integrity of findings, and (2) to combine chlorophyll fluorescence analysis, as a rapid, preliminary method of assessing plant responses to stress, with additional methods of characterising the system of interest (e.g. gas exchange, foliar pigment composition, thylakoid protein composition).

129 citations


Journal ArticleDOI
TL;DR: A model for the truncated Lx cycle in strong light is proposed in which VDE converts Lx to L which then occupies sites L2 and V1 in the light-harvesting antenna complex of PSII (Lhcb), displacing V and Z.
Abstract: Several xanthophyll cycles have been described in photosynthetic organisms. Among them, only two are present in higher plants: the ubiquitous violaxanthin (V) cycle, and the taxonomically restricted lutein epoxide (Lx) cycle, whereas four cycles seem to occur in algae. Although V is synthesised through the β-branch of the carotenoid biosynthetic pathway and Lx is the product of the α-branch; both are co-located in the same sites of the photosynthetic pigment-protein complexes isolated from thylakoids. Both xanthophylls are also de-epoxidised upon light exposure by the same enzyme, violaxanthin de-epoxidase (VDE) leading to the formation of zeaxanthin (Z) and lutein (L) at comparable rates. In contrast with VDE, the reverse reaction presumably catalysed by zeaxanthin epoxidase (ZE), is much slower (or even inactive) with L than with antheraxanthin (A) or Z. Consequently many species lack Lx altogether, and although the presence of Lx shows an irregular taxonomical distribution in unrelated taxa, it has a high fidelity at family level. In those plants which accumulate Lx, variations in ZE activity in vivo mean that a complete Lx-cycle occurs in some (with Lx pools being restored overnight), whereas in others a truncated cycle is observed in which VDE converts Lx into L, but regeneration of Lx by ZE is extremely slow. Accumulation of Lx to high concentrations is found most commonly in old leaves in deeply shaded canopies, and the Lx cycle in these leaves is usually truncated. This seemingly anomalous situation presumably arises because ZE has a low but finite affinity for L, and because deeply shaded leaves are not often exposed to light intensities strong enough to activate VDE. Notably, both in vitro and in vivo studies have recently shown that accumulation of Lx can increase the light harvesting efficiency in the antennae of PSII. We propose a model for the truncated Lx cycle in strong light in which VDE converts Lx to L which then occupies sites L2 and V1 in the light-harvesting antenna complex of PSII (Lhcb), displacing V and Z. There is correlative evidence that this photoconverted L facilitates energy dissipation via non-photochemical quenching and thereby converts a highly efficient light harvesting system to an energy dissipating system with improved capacity to engage photoprotection. Operation of the α- and β-xanthophyll cycles with different L and Z epoxidation kinetics thus allows a combination of rapidly and slowly reversible modulation of light harvesting and photoprotection, with each cycle having distinct effects. Based on the patchy taxonomical distribution of Lx, we propose that the presence of Lx (and the Lx cycle) could be the result of a recurrent mutation in the epoxidase gene that increases its affinity for L, which is conserved whenever it confers an evolutionary advantage.

124 citations


Journal ArticleDOI
TL;DR: The results are consistent with transitory starch accumulation and remobilisation governing the diel rhythm of δ13C in phloem-transported OM and fragmentation fractionation occurring during respiration, which should be considered for assessing ecosystem processes or plant reactions towards environmental constraints.
Abstract: Post-photosynthetic carbon isotope fractionation might alter the isotopic signal imprinted on organic matter (OM) during primary carbon fixation by Rubisco. To characterise the influence of post-photosynthetic processes, we investigated the effect of starch storage and remobilisation on the stable carbon isotope signature (δ13C) of different carbon pools in the Eucalyptus delegatensis R. T. Baker leaf and the potential carbon isotope fractionation associated with phloem transport and respiration. Twig phloem exudate and leaf water-soluble OM showed diel variations in δ13C of up to 2.5 and 2‰, respectively, with 13C enrichment during the night and depletion during the day. Damped diel variation was also evident in bulk lipids of the leaf and in the leaf wax fraction. δ13C of nocturnal phloem exudate OM corresponded with the δ13C of carbon released from starch. There was no change in δ13C of phloem carbon along the trunk. CO2 emitted from trunks and roots was 13C enriched compared with the potential organic substrate, and depleted compared with soil-emitted CO2. The results are consistent with transitory starch accumulation and remobilisation governing the diel rhythm of δ13C in phloem-transported OM and fragmentation fractionation occurring during respiration. When using δ13C of OM or CO2 for assessing ecosystem processes or plant reactions towards environmental constraints, post-photosynthetic discrimination should be considered.

115 citations


Journal ArticleDOI
TL;DR: Cell biology and gene expression studies showed a temporary closure of fibre plasmodesmata (PD) at the rapid phase of elongation, and the duration of the PD closure correlates positively with the final fibre length attained, supporting the view that PD closure may be required for fibres to achieve extended elongation.
Abstract: Higher plants comprise mixtures of some 40 different cell types, and this often complicates the interpretation of data obtained at the tissue level. Studies for a given cell type may provide novel insights into the mechanisms underlying defined cellular and developmental processes. In this regard, the cotton fibre represents an excellent single-cell model to study the control of rapid cell elongation and cellulose synthesis. These single cells, initiated from the ovule epidermis at anthesis, typically elongate to ∼3-5 cm in the tetraploid species before they switch to intensive secondary cell wall cellulose synthesis. By maturity, more than 94% of fibre weight is cellulose. To unravel the mechanisms of fibre elongation and cellulose synthesis, two hypotheses have been examined: (a) that sucrose degradation and utilisation mediated by sucrose synthase (Sus) may play roles in fibre development and (b) that symplastic isolation of the fibre cells may be required for their rapid elongation. Reverse genetic and biochemical analyses have revealed the critical role that Sus plays in fibre initiation and early elongation. Late in development, plasma-membrane and cell wall association of Sus protein seems to be involved in rapid cellulose synthesis. Cell biology and gene expression studies showed a temporary closure of fibre plasmodesmata (PD), probably due to the deposition of callose, at the rapid phase of elongation. The duration of the PD closure correlates positively with the final fibre length attained. These data support the view that PD closure may be required for fibres to achieve extended elongation. The branching of PD towards the secondary cell wall stage is postulated to function as a molecule sieve for tight control of macromolecule trafficking into fibres to sustain intensive cellulose synthesis.

108 citations


Journal ArticleDOI
TL;DR: It is concluded that diurnal changes in transcript levels are integrated, over days, as changes in the levels of enzymes in starchless pgm mutants facilitates an adjustment of metabolism to a mid-term shift in the conditions, while ignoring noise due to diurnalChanges and day-to-day fluctuations.
Abstract: Plants alternate between a net surplus of carbon in the light and a net deficit at night. This is buffered by accumulating starch in the light and degrading it at night. Enough starch is accumulated to support degradation throughout the night, with a small amount remaining at the end of the 24-h diurnal cycle. This review discusses how this balance between the supply and utilisation of carbon is achieved in Arabidopsis. It is important to regulate starch turnover to avoid an acute carbon deficiency. A 2–4 h extension of the night leads to exhaustion of starch, a collapse of sugars, a switch from biosynthesis to catabolism and an acute inhibition of growth by low carbon, which is not immediately reversed when carbon becomes available again. In starchless pgm mutants, where sugars are depleted each night, this leads to a recurring inhibition of growth that is not reversed until 5–6 h into the following light period. Several lines of evidence show that starch accumulation is regulated in response to events that are initiated during periods of low carbon. Starch accumulation is decreased when small amounts of sucrose are included in the growth medium. Sets of sugar-responsive genes were identified by supplying sugars to carbon-starved seedlings, or by illuminating 5-week-old plants in the presence of 350 or 50 ppm [CO2]. Almost all of these genes show large diurnal changes in starchless pgm mutants, which are driven by the depletion of carbon during the night. Many show significant diurnal changes in wild type plants, showing that ‘anticipatory’ changes in signalling pathways occur before acute carbon limitation develops. However, these diurnal changes of transcripts do not lead to immediate changes of enzyme activities. Whereas an extension of the night leads to major changes of transcripts within 4–6 h, changes in enzyme activities require several days. In pgm, enzyme activities and the levels of >150 metabolites resemble those found in wild type plants after several days in the dark. It is concluded that diurnal changes in transcript levels are integrated, over days, as changes in the levels of enzymes. We hypothesise that this facilitates an adjustment of metabolism to a mid-term shift in the conditions, while ignoring noise due to diurnal changes and day-to-day fluctuations. The rapid adjustment of starch synthesis after a period of acute carbon depletion is a consequence of the transient inhibition of growth. This leads to accumulation of sugars when carbon becomes available again, which triggers a large increase in trehalose-6-phosphate. This signal metabolite promotes thioredoxin-dependent post-translational activation of ADP glucose pyrophosphorylase. Mid-term acclimation to a decreased carbon supply may be mediated by a combination of post-translational regulation, longer-term changes in enzyme activities, and a decrease in the rate of growth.

107 citations


Journal ArticleDOI
TL;DR: An extra isoenzyme of glutathione reductase (GR) was induced in the presence of selenite, which confirmed the previous results obtained with Cd and Ni indicating that this GR isoenzymes may have the potential to be a marker for oxidative stress in coffee.
Abstract: Selenium (Se) is an essential element for humans and animals that is required for key antioxidant reactions, but can be toxic at high concentrations. We have investigated the effect of Se in the form of selenite on coffee cell suspension cultures over a 12-day period. The antioxidant defence systems were induced in coffee cells grown in the presence of 0.05 and 0.5 mm sodium selenite (Na2SeO3). Lipid peroxidation and alterations in antioxidant enzymes were the main responses observed, including a severe reduction in ascorbate peroxidase activity, even at 0.05 mm sodium selenite. Ten superoxide dismutase (SOD) isoenzymes were detected and the two major Mn-SOD isoenzymes (bands V and VI) responded more to 0.05 mm selenite. SOD band V exhibited a general decrease in activity after 12 h of treatment with 0.05 mm selenite, whereas band VI exhibited the opposite behavior and increased in activity. An extra isoenzyme of glutathione reductase (GR) was induced in the presence of selenite, which confirmed our previous results obtained with Cd and Ni indicating that this GR isoenzyme may have the potential to be a marker for oxidative stress in coffee.

Journal ArticleDOI
TL;DR: An overview of current models of starch breakdown in leaves is provided and it is now apparent that glucan phosphorylation is required for normal rates of starch mobilisation to occur, although a detailed understanding of this step is still lacking.
Abstract: The aim of this article is to provide an overview of current models of starch breakdown in leaves. We summarise the results of our recent work focusing on Arabidopsis, relating them to other work in the field. Early biochemical studies of starch containing tissues identified numerous enzymes capable of participating in starch degradation. In the non-living endosperms of germinated cereal seeds, starch breakdown proceeds by the combined actions of α-amylase, limit dextrinase (debranching enzyme), β-amylase and α-glucosidase. The activities of these enzymes and the regulation of some of the respective genes on germination have been extensively studied. In living plant cells, additional enzymes are present, such as α-glucan phosphorylase and disproportionating enzyme, and the major pathway of starch breakdown appears to differ from that in the cereal endosperm in some important aspects. For example, reverse-genetic studies of Arabidopsis show that α-amylase and limit-dextrinase play minor roles and are dispensable for starch breakdown in leaves. Current data also casts doubt on the involvement of α-glucosidase. In contrast, several lines of evidence point towards a major role for β-amylase in leaves, which functions together with disproportionating enzyme and isoamylase (debranching enzyme) to produce maltose and glucose. Furthermore, the characterisation of Arabidopsis mutants with elevated leaf starch has contributed to the discovery of previously unknown proteins and metabolic steps in the pathway. In particular, it is now apparent that glucan phosphorylation is required for normal rates of starch mobilisation to occur, although a detailed understanding of this step is still lacking. We use this review to give a background to some of the classical genetic mutants that have contributed to our current knowledge.

Journal ArticleDOI
Ian C. Dodd1
TL;DR: Evaluating the relationship between Jdry and Ψsoil may assist in maintaining export of ABA (and other growth regulators) from the drying part of the root system, to achieve desirable horticultural outcomes during PRD.
Abstract: The effects of different irrigation techniques on leaf xylem ABA concentration ([X-ABA]leaf) were compared in tomato (Lycopersicon esculentum Mill.). During partial rootzone drying (PRD), water was distributed unevenly to the root system such that part was irrigated while the remainder was allowed to dry the soil. During conventional deficit irrigation (DI), plants received the same volume of water as PRD plants, but water was distributed evenly to the entire root system. When the plant root system was allowed to explore two separate soil compartments, DI plants had a higher [X-ABA]leaf than PRD plants with moderate soil drying, but PRD plants had a higher [X-ABA]leaf than DI plants as the soil dried further. The difference in [X-ABA]leaf between the two sets of plants was not because of differences in either whole pot soil water content (θpot) or leaf water potential (Ψleaf). To investigate the contribution of different parts of the root system to [X-ABA]leaf, individual shoots were grafted onto the root systems of two plants grown in two separate pots, so that the graft union had the appearance of an inverted ‘Y’. After sap collection from detached leaves, removal of the shoot below the graft union allowed sap collection from each root system. Again, DI plants had a higher [X-ABA]leaf than PRD plants when the soil was relatively wet, but the opposite occurred as the soil dried. Root xylem ABA concentration ([X-ABA]root) increased exponentially as soil water content (θ) declined. In DI plants, [X-ABA]root from either pot (or the arithmetic mean of [X-ABA]root) accounted for a similar amount of the variation in [X-ABA]leaf. In PRD plants, [X-ABA]root from the watered side underestimated [X-ABA]leaf, whereas [X-ABA]root from the dry side overestimated [X-ABA]leaf. The arithmetic mean of [X-ABA]root best explained the variation in [X-ABA]leaf, implying continued sap flow from the dry part of the root system (Jdry) at soil water potentials (Ψsoil) at which Jdry had ceased in previous studies of PRD plants (Yao et al. 2001). Evaluating the relationship between Jdry and Ψsoil may assist in maintaining export of ABA (and other growth regulators) from the drying part of the root system, to achieve desirable horticultural outcomes during PRD.

Journal ArticleDOI
TL;DR: Results suggested that overexpression of SBPase was an effective method for enhanncing salt tolerance in rice.
Abstract: Activity of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase; EC3.1.3.37) was increased in the transgenic rice cultivar zhonghua11 (Oryza sativa L. ssp. japonica) by overexpressing OsSbp cDNA from the rice cultivar 9311 (Oryza sativa ssp. indica). This genetic engineering enabled the transgenic plants to accumulate SBPase in chloroplasts and resulted in enhanced tolerance of transgenic rice plants to salt stress at the young seedlings stage. Moreover, CO2 assimilation in transgenic rice plants was significantly more tolerant to salt stress than in wild-type plants. The analysis of chlorophyll fluorescence and the activity of SBPase indicated that the enhancement of photosynthesis in salt stress was not related to the function of PSII but to the activity of SBPase. Western-blot analysis showed that salt stress led to the association of SBPase with the thylakoid membranes from the stroma fractions. However, this association was much more prominent in wild-type plants than in transgenic plants. Results suggested that under salt stress, SBPase maintained the activation of ribulose-1,5-bisphosphate carboxylase-oxygenase by providing more regeneration of the acceptor molecule ribulose-1,5-bisphosphate in the soluble stroma and by preventing the sequestration of Rubisco activase to the thylakoid membrane from the soluble stroma, and, thus, enhanced the tolerance of photosynthesis to salt stress. Results suggested that overexpression of SBPase was an effective method for enhanncing salt tolerance in rice.

Journal ArticleDOI
TL;DR: Recovered grapevine plants might be able to achieve a long-term, sustained and tissue-specific accumulation of H2O2 in leaves, which reduces numbers or prevents further infection by Flavescence dorée phytoplasma, without altering the levels of other antioxidant systems and without incurring an increased oxidative risk.
Abstract: In the present work, we compared hydrogen peroxide (H2O2) localisation and the activities/contents of antioxidant enzymes and metabolites in the leaf tissues of grapevine (Vitis vinifera L. cv. Prosecco) plants showing different sanitary status, namely diseased by Flavescence doree, healthy or recovered. Polymerase chain reaction analysis revealed that the pathogen associated with Flavescence doree (proposed as 'Candidatus Phytoplasma vitis') was detected in the leaf tissues of symptomatic plants, but was not observed in either the healthy or recovered plants. Hydrogen peroxide accumulated in the phloem plasmalemma of recovered grapevine leaves, but was not detected in either healthy or diseased material. When compared to diseased or healthy plants, recovered plants had distinctly lower extractable levels of catalase and ascorbate peroxidase, two enzymes primarily involved in the scavenging of excess H2O2 generated in different cell compartments. Among healthy, diseased and recovered leaves there was no significant difference in the amount of 2-thiobarbituric acid-reactive substances, which are assumed to reflect the extent of peroxidative breakdown of membrane lipids. Therefore, it is suggested that recovery from Flavescence doree disease in grapevine might be associated with a long-term, sustained and tissue-specific accumulation of H2O2 in leaves, which reduces numbers or prevents further infection by Flavescence doree phytoplasma. Recovered grapevine plants might be able to achieve such H2O2 accumulation through a selective and presumably stable downregulation of enzymatic H2O2 scavengers, without altering the levels of other antioxidant systems and without incurring an increased oxidative risk.

Journal ArticleDOI
TL;DR: It was found that transition from light to dark conditions caused a rapid increase in JO and xylem [CO2], both in manipulated trees and in an intact tree with no sap manipulation, which resulted in an increased resistance to radial CO2 diffusion during the dark, at least for trees with smaller daytime resistances.
Abstract: Rates of CO2 efflux of stems and branches are highly variable among and within trees and across stands. Scaling factors have only partially succeeded in accounting for the observed variations. In this study, the resistance to radial CO2 diffusion was quantified for tree stems of an eastern cottonwood (Populus deltoides Bartr. ex Marsh.) clone by direct manipulation of the CO2 concentration ([CO2]) of xylem sap under controlled conditions. Tree-specific linear relationships between rates of stem CO2 efflux (JO) and xylem [CO2] were found. The resistance to radial CO2 diffusion differed 6-fold among the trees and influenced the balance between the amount of CO2 retained in the xylem v. that which diffused to the atmosphere. Therefore, we hypothesised that variability in the resistance to radial CO2 diffusion might be an overlooked cause for the inconsistencies and large variations in woody tissue CO2 efflux. It was found that transition from light to dark conditions caused a rapid increase in JO and xylem [CO2], both in manipulated trees and in an intact tree with no sap manipulation. This resulted in an increased resistance to radial CO2 diffusion during the dark, at least for trees with smaller daytime resistances. Stem diameter changes measured in the intact tree supported the idea that higher actual respiration rates occurred at night owing to higher metabolism in relation to an improved water status and higher turgor pressure.

Journal ArticleDOI
TL;DR: The results suggest mannitol, which is known to be a compatible solute and antioxidant, protects photosynthesis against salt-related damage to chloroplasts.
Abstract: In celery, mannitol is a primary photosynthetic product that is associated with celery’s exceptional salt tolerance. Arabidopsis plants transformed with celery’s mannose-6-phosphate reductase (M6PR) gene produce mannitol and grow normally in the absence of stress. Daily analysis of the increase in growth (fresh and dry weight, leaf number, leaf area per plant and specific leaf weight) over a 12-day period showed less effect of salt (100 mm NaCl) on the M2 transformant than wild type (WT). Following a 12-day treatment of WT, M2 and M5 plants with 100 or 200 mm NaCl the total shoot fresh weight, leaf number, and leaf area were significantly greater in transformants than in WT plants. The efficiency of use of energy for photochemistry by PSII was measured daily under growth conditions. In WT plants treated with 100 mm NaCl, the PSII yield begin decreasing after 6 days with a 50% loss in yield after 12 days, indicating a severe loss in PSII efficiency; whereas, there was no effect on the transformants. Under atmospheric levels of CO2, growth with 200 mm NaCl caused an increase in the substomatal levels of CO2 in WT plants but not in transformants. It also caused a marked decrease in carboxylation efficiency under limiting levels of CO2 in WT compared with transformants. When stress was imposed and growth reduced by withholding water for 12 days, which resulted in a similar decrease in relative water content to salt-treated plants, there were no differences among the genotypes in PSII yields or growth. The results suggest mannitol, which is known to be a compatible solute and antioxidant, protects photosynthesis against salt-related damage to chloroplasts.

Journal ArticleDOI
TL;DR: Tomato (Lycopersicon esculentum Mill.) is the only plant species from which four HXK and four FRK genes have been identified and characterised, and differences in location suggest that the cytoplasmic HxK and FRK have distinct roles to play in sugar metabolism.
Abstract: Hexose phosphorylation is an essential step of sugar metabolism. Only two classes of glucose and fructose phosphorylating enzymes, hexokinases (HXK) and fructokinases (FRK), have been found in plants. Tomato (Lycopersicon esculentum Mill.) is the only plant species from which four HXK and four FRK genes have been identified and characterised. One HXK and one FRK isozyme are located within plastids. The other three HXK isozymes are associated with the mitochondria, and the other three FRK isozymes are dispersed in the cytosol. These differences in location suggest that the cytoplasmic HXK and FRK have distinct roles to play in sugar metabolism. The specific roles of each of the HXK and FRK genes have been investigated using transgenic plants with modified expression of the genes. Sugar signalling effects were obtained with modified expression of the mitochondria associated HXK. In contrast, modified expression of the cytosolic FRK affected fructose metabolism rather than sugar signalling. Future research efforts will aim to determining the roles of specific hexose phosphorylating enzymes in tomato plants, the source of the hexose monomers to be phosphorylated, and their intracellular trafficking route.

Journal ArticleDOI
TL;DR: It is concluded that anthocyanin production during winter is likely not associated with diminished photosynthetic capacity, and may simply represent an alternative photoprotective strategy utilised by some species during winter.
Abstract: Leaves of many evergreen species turn red when exposed to high sunlight during winter due to production of photoprotective anthocyanin pigments, while leaves of other species, lacking anthocyanin, remain green. Why some evergreen species synthesise anthocyanin pigments while others do not is currently unknown. Furthermore, the relative photosynthetic performance of anthocyanic (red) and acyanic (green) evergreens has yet to be described. Here we present seasonal ecophysiological data for five red and green broadleaf evergreen species. We hypothesise that species which synthesise anthocyanins in winter leaves correspond to those with the most drastic seasonal photosynthetic declines, as reduced energy sinks increase vulnerability to photoinhibition and need for photoprotection. Our results did not support this hypothesis, as gas exchange measurements showed no difference in mean seasonal photosynthetic capacity between red- and green-leafed species. Consistent with anthocyanin's shading effect, red-leafed species had significantly higher chlorophyll content, lower chlorophyll a/b ratios, and higher maximum light capture efficiency of PSII (Fv/Fm) than green-leafed species during the winter, but not during the summer (when all leaves were green). We conclude that anthocyanin production during winter is likely not associated with diminished photosynthetic capacity, and may simply represent an alternative photoprotective strategy utilised by some species during winter.

Journal ArticleDOI
TL;DR: The results showed that bicarbonate could induce Fe chlorosis by inhibiting the expression of the ferric reductase, the iron transporter and the H+-ATPase genes, probably through alteration of theexpression of Fe efficiency reactions (FER) (or FER-like) transcription factors.
Abstract: Bicarbonate is considered one of the most important factors causing Fe chlorosis in Strategy I plants, mainly on calcareous soils. Most of its negative effects have been attributed to its capacity to buffer a high pH in soils, which can diminish both Fe solubility and root ferric reductase activity. Besides its pH-mediated effects, previous work has shown that bicarbonate can inhibit the induction of enhanced ferric reductase activity in Fe-deficient Strategy I plants. However, to date it is not known whether bicarbonate affects the upregulation of the ferric reductase gene and other genes involved in Fe acquisition. The objective of this work has been to study the effect of bicarbonate on the expression of several Fe acquisition genes in Arabidopsis (Arabidopsis thaliana L.), pea (Pisum sativum L.), tomato (Lycopersicon esculentum Mill.) and cucumber (Cucumis sativus L.) plants. Genes for ferric reductases AtFRO2, PsFRO1, LeFRO1 and CsFRO1; iron transporters AtITR1, PsRIT1, LeIRT1 and CsIRT1; H+-ATPases CsHA1 and CsHA2; and transcription factors AtFIT and LeFER have been examined. The results showed that bicarbonate could induce Fe chlorosis by inhibiting the expression of the ferric reductase, the iron transporter and the H+-ATPase genes, probably through alteration of the expression of Fe efficiency reactions (FER) (or FER-like) transcription factors.

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TL;DR: Data suggest strong developmental, metabolic and genetic control of AsA accumulation in blackcurrant fruit and indicate the potential for breeding high AsA cultivars.
Abstract: Blackcurrant (Ribes nigrum L.) is a widely grown commercial crop valued for its high vitamin C (l-ascorbic acid, AsA) content. In the present study, a systematic analysis of the mechanism of fruit AsA accumulation was undertaken. AsA accumulation occurred during fruit expansion and was associated with high in situ biosynthetic capacity via the l-galactose pathway and low rates of turnover. Cessation of AsA accumulation was associated with reduced biosynthesis and increased turnover. Translocation of AsA from photosynthetic or vegetative tissues contributed little to fruit AsA accumulation. Manipulation of substrate availability by defoliation had no effect on fruit AsA concentration but significantly reduced fruit yields. Supply of the AsA precursor l-galactono-1,4-lactone to intact, attached fruit transiently increased fruit AsA concentration which rapidly returned to control levels after removal of the compound. These data suggest strong developmental, metabolic and genetic control of AsA accumulation in blackcurrant fruit and indicate the potential for breeding high AsA cultivars.

Journal ArticleDOI
TL;DR: Results indicate that the higher rate of photosynthesis in 4×-C11 leaves was not an immediate outcome of chromosome doubling; rather, it was due to adjustment and adaptation during the process of genome stabilisation.
Abstract: Polyploidy affects photosynthesis by causing changes in morphology, anatomy and biochemistry However, in newly developed polyploids, the genome may be unstable In this study, diploid (2×) and synthetic autotetraploids in initial (4×-C0) and 11th generations (4×-C11) of Phlox drummondii Hook were used to study the effects of chromosome doubling and genome stabilisation on leaf photosynthesis and anatomical properties The light-saturated photosynthetic rate on a leaf area basis at 360 µmol CO2 mol–1 air (A360) was highest in 4×-C11 leaves, intermediate in 4×-C0 leaves, and lowest in 2× leaves Rubisco amounts, CO2-saturated photosynthetic rate at 1200 µmol CO2 mol–1 air at PPFD of 1000 µmol m–2 s–1 (A1200, representing the capacity for RuBP regeneration), cumulative surface areas of chloroplasts facing intercellular spaces (Sc), all expressed on a leaf area basis, were all higher in 4× leaves than in 2× leaves, and stomatal conductance (gs) at 360 µmol CO2 mol–1 air was only higher in the 4×-C11 leaves A360 for the 4×-C11 leaves was greater than that in the 4×-C0 leaves despite having similar amounts of Rubisco This was presumably associated with a greater RuBP regeneration capacity, as well as an increase in Sc and gs, which would increase the CO2 concentration of Rubisco These results indicate that the higher rate of photosynthesis in 4×-C11 leaves was not an immediate outcome of chromosome doubling; rather, it was due to adjustment and adaptation during the process of genome stabilisation

Journal ArticleDOI
TL;DR: From screening more than 230 samples of Cleomaceae species, based on a measure of the carbon isotope composition (δ13C) in leaves, it is indicated that this is an interesting family for studying the genetic basis for C4 photosynthesis and its evolution from C3 species.
Abstract: C4 photosynthesis has evolved many times in 18 different families of land plants with great variation in leaf anatomy, ranging from various forms of Kranz anatomy to C4 photosynthesis occurring within a single type of photosynthetic cell. There has been little research on photosynthetic typing in the family Cleomaceae, in which only one C4 species has been identified, Cleome gynandra L. There is recent interest in selecting and developing a C4 species from the family Cleomaceae as a model C4 system, since it is the most closely related to Arabidopsis, a C3 model system (Brown et al. 2005). From screening more than 230 samples of Cleomaceae species, based on a measure of the carbon isotope composition (δ13C) in leaves, we have identified two additional C4 species, C. angustifolia Forssk. (Africa) and C. oxalidea F.Muell. (Australia). Several other species have δ13C values around –17‰ to –19‰, suggesting they are C4-like or intermediate species. Eight species of Cleome were selected for physiological, anatomical and biochemical analyses. These included C. gynandra, a NAD–malic enzyme (NAD–ME) type C4 species, C. paradoxa R.Br., a C3–C4 intermediate species, and 6 others which were characterised as C3 species. Cleome gynandra has C4 features based on low CO2 compensation point (Γ), C4 type δ13C values, Kranz-type leaf anatomy and bundle sheath (BS) ultrastructure, presence of C4 pathway enzymes, and selective immunolocalisation of Rubisco and phosphoenolpyruvate carboxylase. Cleome paradoxa was identified as a C3–C4 intermediate based on its intermediate Γ (27.5 μmol mol–1), ultrastructural features and selective localisation of glycine decarboxylase of the photorespiratory pathway in mitochondria of BS cells. The other six species are C3 plants based on Γ, δ13C values, non-Kranz leaf anatomy, and levels of C4 pathway enzymes (very low or absent) typical of C3 plants. The results indicate that this is an interesting family for studying the genetic basis for C4 photosynthesis and its evolution from C3 species.

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TL;DR: Analysis of mRNA abundance in wildtype and lutein-deficient mutants, lut2 and ccr2, in response to light transitions and herbicide treatments demonstrated that the mRNA abundance of the carotenoid isomerase (CRTISO) and epsilon-cyclase (ϵLCY) can be rate limiting steps in lutenin biosynthesis.
Abstract: Carotenoids are critical for photosynthetic function in chloroplasts, and are essential for the formation of the prolamellar body in the etioplasts of dark-grown (etiolated) seedlings. They are also precursors for plant hormones in both types of plastids. Lutein is one of the most abundant carotenoids found in both plastids. In this study we examine the regulation of lutein biosynthesis and investigate the effect of perturbing carotenoid biosynthesis on the formation of the lattice-like membranous structure of etioplasts, the prolamellar body (PLB). Analysis of mRNA abundance in wildtype and lutein-deficient mutants, lut2 and ccr2, in response to light transitions and herbicide treatments demonstrated that the mRNA abundance of the carotenoid isomerase (CRTISO) and epsilon-cyclase (ϵLCY) can be rate limiting steps in lutein biosynthesis. We show that accumulation of tetra-cis-lycopene and all-trans-lycopene correlates with the abundance of mRNA of several carotenoid biosynthetic genes. Herbicide treatments that inhibit carotenoid biosynthetic enzymes in wildtype and ccr2 etiolated seedlings were used to demonstrate that the loss of the PLB in ccr2 mutants is a result of perturbations in carotenoid accumulation, not indirect secondary effects, as PLB formation could be restored in ccr2 mutants treated with norflurazon.

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TL;DR: The presence of multiple forms of actinidin and varying protein levels in fruit will impact on the ability to breed new kiwifruit varieties with altered actinIDin levels.
Abstract: Actinidin is a cysteine protease found in Actinidia Lindl. (kiwifruit) species that affects the nutraceutical properties, processing characteristics and allergenicity of the fruit. Given the increased consumption of kiwifruit worldwide and the release of new varieties from different Actinidia species, the expression of actinidin mRNA and protein in a range of kiwifruit tissues was examined. Ten different actinidin mRNAs were identified encoding mature proteins of similar molecular weight (~24 kDa), but with predicted pIs ranging from acidic (pI 3.9) to basic (pI 9.3). In A. deliciosa ‘Hayward’ (green-fleshed kiwifruit) and A. chinensis ‘Hort16A’ and EM4 (gold-fleshed kiwifruit), actinidin mRNAs for acidic and basic proteins were expressed at comparable levels throughout ripening. Actinidin mRNA expression was highest in fruit at harvest, expression decreased as fruit ripened and was much lower in the core compared with outer pericarp tissue. Two-dimensional gel electrophoresis, combined with western analysis and liquid chromatography mass spectrometry (LC-MS) identified low levels of a novel basic actinidin protein in ripe A. deliciosa and A. chinensis fruit. Extremely high levels of an acidic actinidin protein were detected in A. deliciosa fruit and EM4, but this acidic protein appeared to be absent in ‘Hort16A’, the most important commercial cultivar of A. chinensis. Analyses on native gels indicated that both the basic and acidic actinidin isoforms in A. deliciosa were active cysteine proteases. Immunolocalisation showed that actinidin was present in small cells, but not large cells in the outer pericarp of mature A. deliciosa fruit at harvest. Within the small cells, actinidin was localised diffusely in the vacuole, associated with the plasma membrane, and in a layer in the plastids near starch granules. The presence of multiple forms of actinidin and varying protein levels in fruit will impact on the ability to breed new kiwifruit varieties with altered actinidin levels.

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TL;DR: In this article, the authors compared the response of C4 grasses to water deficit imposed by the addition of polyethylene glycol to the nutrient solution in which they were grown and found that the reduction of photosynthesis in P. dilatatum under water deficit was not closely related to the activities of the carboxylating enzymes or to chlorophyll a fluorescence.
Abstract: C4 plants are considered to be less sensitive to drought than C3 plants because of their CO2 concentrating mechanism. The C4 grasses, Paspalum dilatatum Poiret (NADP-ME), Cynodon dactylon (L.) Pers (NAD-ME) and Zoysia japonica Steudel (PEPCK) were compared in their response to water deficit imposed by the addition of polyethylene glycol to the nutrient solution in which they were grown. The effects of drought on leaf relative water content (RWC), net photosynthesis, stomatal conductance, carboxylating enzyme activities and chlorophyll a fluorescence were investigated. In C. dactylon the RWC was more sensitive, but the photosynthetic activity was less sensitive, to water deficit than in P. dilatatum and Z. japonica. The decrease of photosynthesis in P. dilatatum under water deficit was not closely related to the activities of the carboxylating enzymes or to chlorophyll a fluorescence. However, decreased activities of ribulose 1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase, in addition to decreased stomatal conductance, may have contributed to the decrease of photosynthesis with drought in C. dactylon and Z. japonica. The different responses to water deficit are discussed in relation to the natural habitats of C4 grasses.

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TL;DR: For most traits, variation was more pronounced at the community than at the species level, except for stomatal density, which varied much more strongly within than between species.
Abstract: Macroecological patterns of leaf traits can be used to assess adaptive responses of plants to environmental stress. Here we present the first such study on a large number of fern species (403) along gradients of elevation (temperature) and humidity. To assess how the representation of traits such as degree of lamina dissection, leaf length, leaf mass per area (LMA), trichome density, venation density, stomatal density, and of adaptive strategies such as poikilohydry vary at the community and species levels in response to changes in humidity and temperature in the Bolivian Andes, we (1) compared whole pteridophyte communities at 14 sites, and (2) analysed intraspecific variation of the morphological traits of 17 fern species along an elevational gradient at 1700-3400 m in humid forest. Among the fern communities of the 14 sites, leaf length decreased with elevation and aridity, LMA increased with elevation, and trichome density and venation density increased with aridity. The study of intraspecific variation among 17 species showed an increase of stomatal density with elevation in six of 11 species (filmy ferns lacked stomata), an increase of specific weight in 15 species, a decrease of trichome density in seven of 10 species (other species lacked hairs), and a decrease of venation density in seven of 10 cases. Some of these trends can be interpreted adaptively: leaf thickness appears to increase in situations with low nutrient availability rather than with low water availability, whereas a dense cover of scales or hairs serves as a protection against insolation or as a vehicle for the absorption of water in poikilohydric species. In arid areas, trichome density increased with elevation, while it decreased with elevation in cloudy and humid regions. For most traits, variation was more pronounced at the community than at the species level, except for stomatal density, which varied much more strongly within than between species. Several of these morphological and anatomical characters can be used to infer palaeoclimatic conditions based on fossil pteridophyte floras.

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TL;DR: The results suggest that, in orthodox seeds, the ascorbate-glutathione cycle plays an important role in the acquisition of tolerance to desiccation, in protein maturation, and in protection from reactive oxygen species.
Abstract: The ascorbate-glutathione system was studied during development and desiccation of seeds of two Acer species differing in desiccation tolerance: Norway maple (Acer platanoides L., orthodox) and sycamore (Acer pseudoplatanus L., recalcitrant). The results showed remarkable differences in the concentration and redox balance of ascorbate and glutathione between these two kinds of seeds during development, and a significant dependence between glutathione content and acquisition of desiccation tolerance in Norway maple seeds. There were relatively small differences between the species in the activities of enzymes of the ascorbate-glutathione cycle: ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (GR, EC 1.6.4.2). At the end of seed maturation, ascorbic acid content and the activities of the above enzymes was about the same in both species The electrophoretic pattern of APX isoenzymes was also similar for both species, and the intensity of the bands decreased at the end of seed maturation in both species. When sycamore seeds were desiccated to a moisture content of less than 26%, there was a marked decrease in seed viability and an increase in the production of reactive oxygen species. During desiccation, Norway maple seeds had a more active defence system, which was reflected in a higher glutathione content, a higher glutathione redox status, a higher ascorbate redox status, and higher activities of APX, MR, DHAR, GR and GPX (glutathione peroxidase). During desiccation, sulfhydryl-to-disulfide transition into proteins was more intense in Norway maple seeds than sycamore seeds. All of these results suggest that, in orthodox seeds, the ascorbate-glutathione cycle plays an important role in the acquisition of tolerance to desiccation, in protein maturation, and in protection from reactive oxygen species.

Journal ArticleDOI
TL;DR: Variation within and among species in nighttime water loss has implications for breeding to improve crop water use efficiency and under ambient conditions nighttime transpiration varied within and amongst species and was 8-33% of maximal daytime transpiration.
Abstract: Incomplete stomatal closure at night can result in substantial water loss at times when photosynthetic carbon gain is not occurring in C3 and C4 plant species. To investigate the magnitude of nighttime water loss for a crop species in the field, measurements of nighttime water loss by tomato (Lycopersicon esculentum Mill. cv. Heinz 8892) were made by three methods: a field-scale lysimeter and two leaf-level instruments, an automated viscous flow porometer and a portable photosynthesis system. The portable photosynthesis system indicated nighttime transpiration of 10% of maximal daytime transpiration and the viscous flow porometer demonstrated partially open stomata. Integrated crop water loss during the dark, non-photosynthetic hours measured on the lysimeter was 3-10.8% of total daily water loss. In the glasshouse, a survey of closely related wild and cultivated tomato species showed that under ambient conditions nighttime transpiration varied within and among species and was 8-33% of maximal daytime transpiration. Implications of such a substantial fraction of total daily crop water use occurring during the night are significant in agronomic, environmental, and economic terms. Further, variation within and among species in nighttime water loss has implications for breeding to improve crop water use efficiency.

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TL;DR: The hypothesis that the transpiration (λEp) of high-coupled canopies, such as olive groves, may be calculated on a daily basis with sufficient precision by the Penman-Monteith 'big leaf' equation is tested by a model of bulk daily canopy conductance (gc) capable of scaling for canopy dimension.
Abstract: We tested the hypothesis that the transpiration (λEp) of high-coupled canopies, such as olive groves, may be calculated on a daily basis with sufficient precision by the Penman–Monteith ‘big leaf’ equation, by a model of bulk daily canopy conductance (gc) capable of scaling for canopy dimension. Given the limited data required, such a model could replace the standard approach (ET0 × Kc) for calculating olive water requirements, enhancing the precision of estimates. We developed a specific model of daily gc for unstressed olive canopies that was calibrated by transpiration measurements obtained by water balance from a 2-year experiment in a mature orchard with λEp ranging from 0.6 (February 1993) to 11.5 (July 1994) MJ m–2 day–1 and where leaf area index (L) changed from 1.25 to 2.5. The model uses the intercepted fraction of daily PAR and a linear function of average daytime temperature. The model was validated with λEp data collected by eddy covariance in a 3-year experiment conducted in a growing orchard that differed in L and cultivar from the one used in the calibration. The gc model, when used in the Penman–Monteith equation, gave very good daily λEp predictions for all seasons during 3 years, ranging from 0.5 (November 1998) to 5.5 (June 2000) MJ m–2 day–1, indicating that the goals of dealing with the dependence of olive gc on L and of simulating the seasonal variations in gc were achieved. A comparison with the Jarvis gc model, calibrated with 2 months of measured gc hourly data, showed that the gc model developed here performed better than the Jarvis model for the 3-year dataset. The exception to this was the period in which the Jarvis model was calibrated. This indicates that (1) the Jarvis model did not account for the seasonal variations in gc of the olive trees; and (2) the spatial and temporal scale assumptions required in the calibration of gc generate seasonal errors in the simulated bulk daily λEp for this crop. The applicability of this bulk gc model is restricted to well watered olive canopies and to the one-layer approach of calculating λEp but it could be adapted to rain-fed canopies in the future.