Limiting Carbon Source Research Articles

Cover Picture: Proteomics 8/2009

AbstractIn this issue of Proteomics you will find the following highlighted articles:Are you sick or are you just getting old: Does it matter?We all joke about the hazards of aging: various systems that break down, some you didn't even know you had. But then there's the alternative of not aging. Hmmm. Now what if aging were a disease? If so, it is worse than any cold I've had. Zürbig et al. have found that the proteome of the aging kidney has many markers in common with chronic kidney disease. The degree of match among small peptide markers ranged from 4% to 22% for IgA nephropathy to diabetic nephropathy, respectively. From these data they developed an age estimating scale that revealed some individuals had kidneys apparently “older” than their bodies. If these findings hold up, they could offer new approaches to diagnosis and therapy of chronic kidney diseases.Zürbig, P. et al., Proteomics 2009, 9, 2108‐2117.Sharing your niche with an unrelated speciesAnyone who's ever lived with a roommate knows the pain of dividing up the refrigerator space and the cleaning duties as well as the rent. Is it based on number of people, the size of bedrooms, or size of biceps? Many “free‐living” bacteria share their living space with other species in stable consortia to which each member contributes. Bobadilla Fazzini et al. use proteomic and other tools to examine the changes resulting from shifts in limiting carbon sources. Their system is a continuous culture of 9:1 Pseudomonas reinekei (MT1): Achromobacter xylosoxidans (MT3), cultured from a contaminated stream and able to grow on 4‐chlorosalicylate, an intermediate in the degradation of toxic furans and dioxins. MT1 OprF, the outer membrane protein and homolog of E. coli OmpA, is a “slow porin” that contributes to toxin resistance. After a shift in carbon sources, MT1 OprF was up‐regulated ∼11‐fold in mixed culture vs. pure culture.Bobadilla Fazzini, R. A. et al., Proteomics 2009, 9, 2273‐2285.Heart to heart: Biomarkers for MACEMace is a spice, not an herb. It is a badge of office and a weapon (albeit now of a defensive sort). It is also an acronym for a Major Adverse Cardiac Event, otherwise known as a big heart attack, something you want to know is coming and to prevent. So what to do? Biomarkers to the rescue. Currently the FDA has approved one prospective test: the CardioMPO™ ELISA test for myeloperoxidase. The MPO marker is >60% accurate in predicting a MACE over 30 days and 6 months. Zhou et al. propose an alternative statistical method for evaluating a panel of mass spectrometry markers. An improved preprocessing procedure utilizes low‐level signal processing and spectrum cleanup routines followed by partial least squares logistic regression and support vector machine classifier to select the markers. The prediction is done by an improved genetic algorithm with local optimization. Using seven markers yields >75% accuracy.Zhou, X. et al., Proteomics 2009, 9, 2286‐2294.

Stable repeated-batch production of recombinant dye-decolorizing peroxidase (rDyP) from Aspergillus oryzae

Recombinant Aspergillus oryzae expressing a dye-decolorizing peroxidase gene (dyp) was cultivated for repeated-batch production of recombinant dye-decolorizing peroxidase (rDyP) using maltose as a carbon source. High-level rDyP activity in limitation of carbon and nitrogen sources was maintained stably for 26 cycles of repeated 1-d batches of A. oryzae pellets without any additional pH control. Cultures maintained at 4 degrees C for 20 d resumed rDyP production following a single day of incubation. One liter filtrated crude rDyP containing 4600 U rDyP decolorized 5.07 g RBBR at the apparent decolorization rate of 17.7 mg l(-1) min(-1).

Comparative Analysis of the Physiological and Structural Properties of a Medium Chain Length Polyhydroxyalkanoate Depolymerase from Pseudomonas putida KT2442

AbstractMedium‐chain‐length polyhydroxyalkanoates (mcl‐PHAs) produced by Pseudomonas putida KT2442 can be chiral 3‐hydroxyalkanoic acids catabolized by the intracellular PhaZ depolymerase when the bacteria are cultured under carbon limitation. PhaZ is an intracellular depolymerase and is attached to PHA granules. In this study, the PhaZ expression was analyzed by Western blot and Real Time RT‐PCR assays. The PhaZ expression depends on the carbon source and nitrogen limitation conditions. The substrate recognition of the binding surface was altered using directed mutagenesis experiments. A structural comparison between intracellular and extracellular PHA depolymerases was carried out using PhaZ from P. putida KT2442 and P. fluorescens GK13, respectively. Both share the presence of a core displaying an α/β‐hydrolase fold, but the former would possess an additional “lid” domain that suggests some kind of interfacial activation and that may account for differences in substrate specificity for both proteins.

Effects of internal recycling time mode and hydraulic retention time on biological nitrogen and phosphorus removal in a sequencing anoxic/anaerobic membrane bioreactor process

This study investigated the effects of internal recycling time mode and hydraulic retention time (HRT) on nutrient removal in the sequencing anoxic/anaerobic membrane bioreactor process. Denitrification and phosphorus release were reciprocally dependent on the anoxic/anaerobic time ratio (Ax/An). As Ax/An increased, nitrogen removal rate increased but phosphorus removal rate decreased. The increasing Ax/An provided the longer denitrification period so that the organic substrate were consumed more for denitrification rather than phosphorus release in the limited condition of readily biodegradable substrate. Decreasing HRT increased both nitrogen and phosphorus removal efficiency because as HRT decreased, food-to-microorganism loading ratio increased and thus enhanced the biological capacity and activity of denitrifying bacteria. This could be verified from the observation mixed liquor suspended solids concentration and specific denitrification rate. The change of Ax/An and HRT affected phosphorus removal more than nitrogen removal due to the limitation of favourable carbon source for phosphorus accumulating organisms.

Nitrification and denitrification in biological activated carbon filter for treating high ammonia source water

Since the ammonia in the effluent of the traditional water purification process could not meet the supply demand, the advanced treatment of a high concentration of NH4+-N micro-polluted source water by biological activated carbon filter (BACF) was tested. The filter was operated in the downflow manner and the results showed that the removing rate of NH4+-N was related to the influent concentration of NH4+-N. Its removing rate could be higher than 95% when influent concentration was under 1.0 mg/L. It could also decrease with the increasing influent concentration when the NH4+-N concentration was in the range from 1.5 to 4.9 mg/L and the dissolved oxygen (DO) in the influent was under 10 mg/L, and the minimum removing rate could be 30%. The key factor of restricting nitrification in BACF was the influent DO. When the influent NH4+-N concentration was high, the DO in water was almost depleted entirely by the nitrifying and hetetrophic bacteria in the depth of 0.4 m filter and the filter layer was divided into aerobic and anoxic zones. The nitrification and degradation of organic matters existed in the aerobic zone, while the denitrification occurred in the anoxic zone. Due to the limited carbon source, the denitrification could not be carried out properly, which led to the accumulation of the denitrification intermediates such as NO2−. In addition to the denitrification bacteria, the nitrification and the heterotrophic bacteria existed in the anoxic zone.

Physiology and genetic traits of reverse osmosis membrane biofilms: a case study with Pseudomonas aeruginosa

Biofilm formation of Pseudomonas aeruginosa on the surface of a reverse osmosis (RO) membrane was studied using a synthetic wastewater medium to simulate conditions relevant to reclamation of secondary wastewater effluent. P. aeruginosa biofilm physiology and spatial activity were analyzed following growth on the membrane using a short-life green fluorescent protein derivative expressed in a growth-dependent manner. As a consequence of the limiting carbon source prevailing in the suspended culture of the RO unit, a higher distribution of active cells was observed in the biofilm close to the membrane surface, likely due to the higher nutrient levels induced by concentration polarization effects. The faster growth of the RO-sessile cells compared to the planktonic cells in the RO unit was reflected by the transcriptome of the two cultures analyzed with DNA microarrays. In contrast to the findings recently reported in gene expression studies of P. aeruginosa biofilms, in the RO system, genes related to stress, adaptation, chemotaxis and resistance to antibacterial agents were induced in the planktonic cells. In agreement with the findings of previous P. aeruginosa biofilm studies, motility- and attachment-related genes were repressed in the RO P. aeruginosa biofilm. Supported by the microarray data, an increase in both motility and chemotaxis phenotypes was observed in the suspended cells. The increase in nutrient concentration in close proximity to the membrane is suggested to enhance biofouling by chemotaxis response of the suspended cells and their swimming toward the membrane surface.

Production of melanin bleaching enzyme of fungal origin and its application in cosmetics

We screened wild fungal isolates for melanolytic activity and found thatSporotrichum pruinosum was the most promising of the very limited number of fungi that decolourised synthetic melanin. We used a submerged aerobic process to produce a skin depigmentation enzyme by this strain, and found that in the medium the presence of Mn2+ ions was necessary, the limitation of carbon source was beneficial, and Zn2+ ions were inhibitory. Cultivation in a stirred bioreactor required immobilization of mycelium and use of low stirring velocity. A partially purified enzyme was prepared and tested for depigmentation of human skin corneocytes and whole epidermis of phototypes III and V. This is the first study demonstrating the effective enzymatic degradation of the skin melanin rather than inhibition of its synthesis. This opens the possibility of using melanolytic enzymes in cosmetic skin lightening.

Molecular cloning, characterization and regulation of a peroxiredoxin gene from Schizosaccharomyces pombe

A gene encoding a putative peroxiredoxin (Prx) of the fission yeast Schizosaccharomyces pombe was characterized and its regulation was studied. The full length of the prx gene was introduced into the shuttle vector pRS316 after PCR amplification, resulting in the recombinant plasmid pPrx10. The determined DNA sequence carries 1,327 bp encoding a putative Prx with a molecular mass of 19,510 Da. Prx activity was significantly increased in the S. pombe cells harboring pPrx10. The accelerated growth was observed in the S. pombe/pPrx10 cells, implying the involvement of the cloned gene in the yeast growth. To study transcriptional regulation of the prx gene, the prx-lacZ fusion gene was constructed using the yeast-E. coli shuttle vector YEp367R, and named pPrxup10. The synthesis of beta-galactosidase from the fusion gene was enhanced under carbon source-limited conditions and nitrogen starvation. Under the same growth conditions, the prx mRNA levels of the wild-type yeast cells were increased. The prx mRNA level was markedly decreased in the Pap1-negative mutant, compared with that in the wild-type yeast, suggesting that the basal expression of the prx gene is mediated by a transcription factor, Pap1. The reactive oxygen species (ROS) level was diminished in the S. pombe/pPrx10 cells than in the control cells. The extra copies of the prx gene were able to resist elevation of ROS level under limited carbon source condition and menadione treatment. In brief, the S. pombe Prx is linked with the yeast growth and up-regulated by metabolic oxidative stress on a transcriptional level. The Prx protein is partly responsible for maintaining low ROS level under normal and stressful growth conditions in the fission yeast.

Enhancement of 1,4-Dihydroxy-2-Naphthoic Acid Production by Propionibacterium freudenreichii ET-3 Fed-Batch Culture

The production of 1,4-dihydroxy-2-naphthoic acid (DHNA) was investigated using a fed-batch culture of Propionibacterium freudenreichii ET-3. DHNA is a precursor of menaquinone (MK) and is transformed to MK by combination with an isoprenoid unit. We found that ET-3 stopped MK production and increased DHNA production in an anaerobic fed-batch culture by maintaining the lactose concentration at approximately zero. The maximum DHNA concentration observed in the anaerobic fed-batch culture was markedly higher than the maximum DHNA concentration observed in an anaerobic batch culture. Moreover, MK or DHNA production was affected by the lactose feeding rate; this suggests that lactose metabolism participates in the syntheses of these products. On the other hand, accumulation of propionate was found to inhibit DHNA production in the fed-batch culture. Based on the fact that ET-3 increases DHNA production in an aerobic culture by consuming propionate, we carried out a cultivation experiment in which an anaerobic fed-batch culture was switched to an anaerobic batch culture and found that the DHNA production was increased to a greater extent than the DHNA production in an anaerobic fed-batch culture. These results suggest that DHNA production by ET-3 is markedly influenced by carbon source limitation and the oxygen supply.

Effective inhibition of lytic development of bacteriophages λ, P1 and T4 by starvation of their host, Escherichia coli

BackgroundBacteriophage infections of bacterial cultures cause serious problems in genetic engineering and biotechnology. They are dangerous not only because of direct effects on the currently infected cultures, i.e. their devastation, but also due to a high probability of spreading the phage progeny throughout a whole laboratory or plant, which causes a real danger for further cultivations. Therefore, a simple method for quick inhibition of phage development after detection of bacterial culture infection should be very useful.ResultsHere, we demonstrate that depletion of a carbon source from the culture medium, which provokes starvation of bacterial cells, results in rapid inhibition of lytic development of three Escherichia coli phages, λ, P1 and T4. Since the effect was similar for three different phages, it seems that it may be a general phenomenon. Moreover, similar effects were observed in flask cultures and in chemostats.ConclusionBacteriophage lytic development can be inhibited efficiently by carbon source limitation in bacterial cultures. Thus, if bacteriophage contamination is detected, starvation procedures may be recommended to alleviate deleterious effects of phage infection on the culture. We believe that this strategy, in combination with the use of automated and sensitive bacteriophage biosensors, may be employed in the fermentation laboratory practice to control phage outbreaks in bioprocesses more effectively.

Transcript and proteomic analyses of wild-type and gpa2 mutant Saccharomyces cerevisiae strains suggest a role for glycolytic carbon source sensing in pseudohyphal differentiation

In response to limited nitrogen and abundant carbon sources, diploid Saccharomyces cerevisiae strains undergo a filamentous transition in cell growth as part of pseudohyphal differentiation. Use of the disaccharide maltose as the principal carbon source, in contrast to the preferred nutrient monosaccharide glucose, has been shown to induce a hyper-filamentous growth phenotype in a strain deficient for GPA2 which codes for a Galpha protein component that interacts with the glucose-sensing receptor Gpr1p to regulate filamentous growth. In this report, we compare the global transcript and proteomic profiles of wild-type and Gpa2p deficient diploid yeast strains grown on both rich and nitrogen starved maltose media. We find that deletion of GPA2 results in significantly different transcript and protein profiles when switching from rich to nitrogen starvation media. The results are discussed with a focus on the genes associated with carbon utilization, or regulation thereof, and a model for the contribution of carbon sensing/metabolism-based signal transduction to pseudohyphal differentiation is proposed.

Inorganic carbon limitations on nitrification: Experimental assessment and modelling

Nitrification is a two-step process that involves two different biomass populations: ammonia oxidising biomass (AOB) and nitrite oxidising biomass (NOB). Both populations are autotrophic (i.e. their carbon source is inorganic). Therefore, a deficit of this substrate should result in a decrease of the process rate. Recent technology advances such as the SHARON ® process have brought new scenarios in biological nitrogen removal where these limitations should be considered. Hence, this work examines the inorganic carbon limitation using respirometric and titrimetric techniques. For this aim, the nitrification rate was measured at different total inorganic carbon (TIC) concentrations. The experimental results obtained show that AOB was limited at TIC concentrations lower than 3 mmol C L −1. At the same time, no carbon source limitation for NOB was observed in spite of the low TIC concentrations attained (lower than 0.1 mmol C L −1). The AOB limitation could be successfully modelled using Monod, Tessier and sigmoidal kinetics. The best fit was obtained with sigmoidal kinetics. However, unexpected biomass activity (oxygen consumption) was observed despite a very low TIC concentration (lower than 0.1 mmol C L −1).

Obtaining highly enriched cultures of Candidatus Accumulibacter phosphates through alternating carbon sources

Candidatus Accumulibacter Phosphatis is widely considered to be a polyphosphate accumulating organism (PAO) of prime importance in enhanced biological phosphorus removal (EBPR) systems. This organism has yet to be isolated, despite many attempts. Previous studies on the biochemical and physiological aspects of this organism, as well as its response to different EBPR operational conditions, have generally relied on the use of mixed culture enrichments. One frequent problem in obtaining highly enriched cultures of this organism is the proliferation of glycogen accumulating organisms (GAO) that can compete with PAOs for limited carbon sources under similar operational conditions. In this study, Candidatus Accumulibacter Phosphatis has been enriched in a lab-scale bioreactor to a level greater than 90% as quantified by fluorescence in situ hyrbridisation (FISH). This is the highest enrichment of this organism that has been reported thus far, and was obtained by alternating the sole carbon source in the feed between acetate and propionate every one to two sludge ages, and operating the bioreactor within a pH range of 7.0–8.0. Simultaneously, the presence of two known groups of GAOs was eliminated under these operational conditions. Excellent phosphorus removal performance and stability were maintained in this system, where the phosphorous concentration in the effluent was below 0.2 mg/L for more than 7 months. When a disturbance was introduced to this system by adding sludge from an enriched GAO culture, Candidatus Accumulibacter Phosphatis once again became highly enriched, while the GAOs were out-competed. This feeding strategy is recommended for future studies focused on describing the physiology and biochemistry of Accumulibacter, where a highly-enriched culture of this organism is of high importance.

The extracellular proteome of Bacillus licheniformis grown in different media and under different nutrient starvation conditions

The now finished genome sequence of Bacillus licheniformis DSM 13 allows the prediction of the genes involved in protein secretion into the extracellular environment as well as the prediction of the proteins which are translocated. From the sequence 296 proteins were predicted to contain an N-terminal signal peptide directing most of them to the Sec system, the main transport system in Gram-positive bacteria. Using 2-DE the extracellular proteome of B. licheniformis grown in different media was studied. From the approximately 200 spots visible on the gels, 89 were identified that either contain an N-terminal signal sequence or are known to be secreted by other mechanisms than the Sec pathway. The extracellular proteome of B. licheniformis includes proteins from different functional classes, like enzymes for the degradation of various macromolecules, proteins involved in cell wall turnover, flagellum- and phage-related proteins and some proteins of yet unknown function. Protein secretion is highest during stationary growth phase. Furthermore, cells grown in complex medium secrete considerably higher protein amounts than cells grown in minimal medium. Limitation of phosphate, carbon and nitrogen sources results in the secretion of specific proteins that may be involved in counteracting the starvation.

Spore number control and breeding in Saccharomyces cerevisiae: a key role for a self-organizing system.

Spindle pole bodies (SPBs) provide a structural basis for genome inheritance and spore formation during meiosis in yeast. Upon carbon source limitation during sporulation, the number of haploid spores formed per cell is reduced. We show that precise spore number control (SNC) fulfills two functions. SNC maximizes the production of spores (1–4) that are formed by a single cell. This is regulated by the concentration of three structural meiotic SPB components, which is dependent on available amounts of carbon source. Using experiments and computer simulation, we show that the molecular mechanism relies on a self-organizing system, which is able to generate particular patterns (different numbers of spores) in dependency on one single stimulus (gradually increasing amounts of SPB constituents). We also show that SNC enhances intratetrad mating, whereby maximal amounts of germinated spores are able to return to a diploid lifestyle without intermediary mitotic division. This is beneficial for the immediate fitness of the population of postmeiotic cells.

Production of polysaccharides from Ganoderma lucidum (CCRC 36041) under limitations of nutrients

This research studied the production of polysaccharides from Ganoderma lucidum under the various limitations of nutrients, including carbon-source, nitrogen-source, phosphate-source, magnesium-source, and dissolved oxygen. The different responses of polysaccharide production were observed under different limitations of nutrients. The concentration of polysaccharides was from 1.79 g/L decreasing to 0.91 g/L when the concentration of glucose was from 60 g/L decreasing to 20 g/L. The highest specific polysaccharide production was found at 60 g/L glucose media with 0.299 g/g-cell, and the lowest molecular weight was found in carbon-source limitation. Under nitrogen-source limitation the concentration of cells was low, but both polysaccharide production (1.61 g/L) and specific polysaccharide production (0.492 g/g-cell) were the highest. The lowest molecular weight of polysaccharides was found under nitrogen-source limitation. Both the phosphate-source and magnesium-source limitations showed low cell growth. With the phosphate-source limitation both low polysaccharide production and a lower molecular weight of polysaccharides was found. In the magnesium-source limitation low polysaccharide production, but a higher molecular weight of polysaccharides, was found. For the factor of oxygen supply the best polysaccharide production was found with sufficient oxygen for the first 5 days’ cultivation and then, after changing to oxygen limitation for another 5 days’ cultivation. On the other hand, the highest molecular weight of polysaccharides was found from the beginning with oxygen limitation throughout the process of fermentation.

Effect of cycle time on fungal morphology, broth rheology, and recombinant enzyme productivity during pulsed addition of limiting carbon source

For many years, high broth viscosity has remained a key challenge in large-scale filamentous fungal fermentations. In previous studies, we showed that broth viscosity could be reduced by pulsed addition of limiting carbon during fed-batch fermentation. The objective in this study was to determine how changing the frequency of pulsed substrate addition affects fungal morphology, broth rheology, and recombinant enzyme productivity. To accomplish this, a series of duplicate fed-batch fermentations were performed in 20-L fermentors with a recombinant glucoamylase producing strain of Aspergillus oryzae. The total cycle time for substrate pulsing was varied over a wide range (30-2,700 s), with substrate added only during the first 30% of each cycle. As a control, a fermentation was conducted with continuous substrate feeding, and in all fermentations the same total amount of substrate was added. Results show that the total biomass concentration remained relatively unaltered, while a substantial decrease in the mean projected area of fungal elements (i.e., average size) was observed with increasing cycle time. This led to reduced broth viscosity and increased oxygen uptake rate. However, high values of cycle time (i.e., 900-2,700 s) showed a significant increase in fungal conidia formation and significantly reduced recombinant enzyme productivity, suggesting that the fungi channeled substrate to storage compounds rather than to recombinant protein. In addition to explaining the effect of cycle time on fermentation performance, these results may aid in explaining the discrepancies observed on scale-up to larger fermentors.

Starvation for Different Nutrients in Escherichia coli Results in Differential Modulation of RpoS Levels and Stability

Levels of RpoS increase upon glucose starvation in Escherichia coli, which leads to the transcription of genes whose products combat a variety of stresses. RpoS stability is a key level of control in this process, as SprE (RssB)-mediated degradation is inhibited under glucose starvation. Starvation for ammonia or phosphate also results in increased stress resistance and induction of RpoS-dependent genes. However, we demonstrate that RpoS levels following ammonia starvation are only slightly increased compared to growing cells and are 10-fold below the levels observed under glucose or phosphate limitation. This difference is largely due to regulated proteolysis of RpoS, as its stability in ammonia-starved cells is intermediate between that in logarithmic-phase cells and glucose-starved cells. Use of an rpoS construct that is devoid of the gene's native transcriptional and translational control regions reveals that stability differences are sufficient to explain the different levels of RpoS observed in logarithmic phase, ammonia starvation, and glucose starvation. Under phosphate starvation, however, rpoS translation is increased. The cellular response to nutrient limitation is much more complex than previously appreciated, as there is not simply one response that is activated by starvation for any essential nutrient. Our data support the hypothesis that SprE activity is the key level at which ammonia and glucose starvation signals are transmitted to RpoS, and they suggest that carbon source and/or energy limitation are necessary for full inactivation of the SprE pathway.

Mathematical modeling of regulatory mechanisms in yeast colony development

In the present study, yeast colony development serves as a model system to study growth of fungal populations with negligible nutrient and signal transport within the mycelium. Mathematical simulations address the question whether colony development is governed by diffusional limitation of nutrients. A hybrid one-dimensional cellular automaton model was developed that describes growth of discrete cells based upon microscopic interaction rules in a continuous field of nutrient and messenger. The model is scaled for the geometry of the experimental setup, cell size, growth- and substrate uptake rates. Therefore, calculated cell density profiles and nutrient distributions can be compared to experimental results and the model assumptions can be verified. In the physiologically relevant parameter range, simulations show an exponentially declining cell density along the median axis of the colonies in case of a diffusion limited growth scenario. These results are in good agreement with cell density profiles obtained in cultivations of the yeast Candida boidinii with glucose as the limiting carbon source but stand in contrast to the constant cell density profile estimated for Yarrowia lipolytica grown under the same conditions. While from the comparison of experimental results and simulations a diffusion limited growth mechanism is proposed for glucose limited C. boidinii colonies, this hypothesis is rejected for the growth of Y. lipolytica. As an alternative, a quorum sensing model was developed that can explain the evolution of constant cell density profiles based on the effect of a not further characterized unstable or volatile messenger.

A comparison of the combined effect of water stress and crop load on fruit growth during different phenological stages in young peach trees.

SummaryThe combined effect of fruit load and water stress on fruit water content and dry-matter accumulation was analysed for three phenological stages of fruit growth. Irrigation treatments were no irrigation during Stage I (NI-SI), Stage II (NI-SII), or Stage III (NI-SIII) compared with a fully irrigated control. Three thinning treatments were imposed within each irrigation treatment resulting in fruit loads ranging from low to high. Fruit harvests at the end of Stage I, II and III were used to determine total tree fruit fresh and dry matter after each stage of fruit development. Fruit water accumulation was highly sensitive to the effect of water stress at high fruit loads in all fruit developmental phases, but reductions in fruit water content were more apparent during Stages II and III than during Stage I. On the other hand, fruit dry-matter accumulation was relatively insensitive to water stress at any fruit load level and developmental stage. However, reductions in dry-matter accumulation were obtained during Stage III from those trees that were not irrigated during Stage I (NISI). Since these reductions occurred only for mid-to-high fruit load conditions, the decreases in fruit growth during Stage III appeared to be related to a carbon source limitation. The possible reasons for this source limitation are discussed.