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Phloem unloading in developing walnut fruit is symplasmic in the seed pericarp and apoplasmic in the fleshy pericarp.

The sieve element-companion cell (SE-CC) complex of the sepal bundles feeding the fleshy pericarp of developing walnut (Juglans regia L.) fruit is structurally symplasmically isolated, but the SE-CC complex of the minor ventral carpellary bundles located in the seed pericarp and feeding the seed is structurally symplasmically connected to its adjacent parenchyma cells. 14C-autoradiography indicated that the phloem of both the sepal and carpellary bundles was functional for unloading. Confocal laser scanning microscopy imaging of carboxyfluorescein unloading showed that the dye is confined to the phloem strands of the sepal bundles in the fleshy pericarp, but released from the phloem strands of the minor ventral carpellary bundles into the surrounding parenchyma cells in the seed pericarp. A 60-kDa acid invertase was immunolocalized to the cell wall of SE-CC complex and parenchyma cells in both the fleshy and seed pericarp. These data provide clear evidence for an apoplasmic phloem unloading pathway in the fleshy pericarp and a predominant symplasmic phloem unloading pathway parallel with a possible apoplasmic path as suggested by the presence of the extracellular invertase in the seed pericarp. A model of complex phloem unloading pathways in developing walnut fruit has been proposed.

Carbon Radioisotopes↗

Detection of expansin proteins and activity during tomato fruit ontogeny.

Expansins are plant proteins that have the capacity to induce extension in isolated cell walls and are thought to mediate pH-dependent cell expansion. J.K.C. Rose, H.H. Lee, and A.B. Bennett ([1997] Proc Natl Acad Sci USA 94: 5955-5960) reported the identification of an expansin gene (LeExp1) that is specifically expressed in ripening tomato (Lycopersicon esculentum) fruit where cell wall disassembly, but not cell expansion, is prominent. Expansin expression during fruit ontogeny was examined using antibodies raised to recombinant LeExp1 or a cell elongation-related expansin from cucumber (CsExp1). The LeExp1 antiserum detected expansins in extracts from ripe, but not preripe tomato fruit, in agreement with the pattern of LeExp1 mRNA accumulation. In contrast, antibodies to CsExp1 cross-reacted with expansins in early fruit development and the onset of ripening, but not at a later ripening stage. These data suggest that ripening-related and expansion-related expansin proteins have distinct antigenic epitopes despite overall high sequence identity. Expansin proteins were detected in a range of fruit species and showed considerable variation in abundance; however, appreciable levels of expansin were not present in fruit of the rin or Nr tomato mutants that exhibit delayed and reduced softening. LeExp1 protein accumulation was ethylene-regulated and matched the previously described expression of mRNA, suggesting that expression is not regulated at the level of translation. We report the first detection of expansin activity in several stages of fruit development and while characteristic creep activity was detected in young and developing tomato fruit and in ripe pear, avocado, and pepper, creep activity in ripe tomato showed qualitative differences, suggesting both hydrolytic and expansin activities.

Cell Wall↗

Molecular characterization of a WEE1 gene homologue in tomato (Lycopersicon esculentum Mill.).

Early fruit development in tomato (Lycopersicon esculentum Mill.) proceeds in two distinct phases of growth that comprise cell division and cell expansion, respectively. In pericarp and the jelly like locular tissue of tomato fruit, the transition between cell division to cell expansion is characterized by the arrest of mitotic activity, numerous rounds of nuclear DNA endoreduplication and the inhibition of Cyclin-Dependent Kinase A (CDKA) activity. To investigate whether the WEE1 kinase may play a role during the endoreduplication process, we isolated and characterized the tomato homologue for WEE1. The LeWEE1 gene consisted of 10 exons with a predicted 510 amino acid-long protein. The accumulation of the corresponding transcripts was associated with mitotically active organs: developing fruits, seeds and roots. Interestingly, LeWEE1was expressed in the jelly like locular tissue concomitant with endoreduplication during fruit development. Using tobacco BY-2 synchronized cells, we showed that the WEE1 gene expression is cell-cycle regulated with a maximum transcript accumulation at S phase. Our data indicate the putative dual contribution of LeWEE1 in the classical cell cycle and the endocycle.

Amino Acid Sequence↗

Targeted disruption of the Myxococcus xanthus orotidine 5'-monophosphate decarboxylase gene: effects on growth and fruiting-body development.

The Myxococcus xanthus gene coding for orotidine 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23) was cloned. The M. xanthus uraA gene efficiently complemented an Escherichia coli OMP decarboxylase mutant, permitting it to grow in the absence of uracil. Electroporation of M. xanthus with a circular plasmid carrying a selectable uraA::kan gene disruption resulted in homologous recombination at the chromosomal uraA locus. Chromosomal integration of the gene disruption plasmid created heterozygous (uraA+/uraA::kan) tandem duplications. These tandem duplications were unstable and segregated auxotrophic uraA::kan daughters at frequencies of 2 x 10(-4) to 8 x 10(-4) per viable cell. Rare uraA::kan segregants were easily obtained by selecting for resistance to the toxic analog 5-fluoroorotic acid. Our experiments suggest that the cloned uraA gene could facilitate the use of gene duplications in the genetic analysis of M. xanthus development. The uraA mutants could utilize uracil, uridine, or uridine 5'-phosphate for growth, indicating that M. xanthus has pyrimidine salvage pathways. During multicellular development, uraA::kan gene disruption mutants sporulated to wild-type levels but formed smaller and more numerous aggregates than did their uraA+ parent, regardless of whether uracil was added to the medium. Pyrimidine deprivation of uraA mutants, under conditions that otherwise supported vegetative growth, failed to induce fruiting-body development or sporulation.

Cloning, Molecular↗

A shift of Phloem unloading from symplasmic to apoplasmic pathway is involved in developmental onset of ripening in grape berry.

It remains unclear whether the phloem unloading pathway alters to adapt to developmental transition in fleshy fruits that accumulate high level of soluble sugars. Using a combination of electron microscopy, transport of the phloem-mobile symplasmic tracer carboxyfluorescein, movement of the companion cell-expressed and the green fluorescent protein-tagged viral movement protein, and assays of the sucrose cleavage enzymes, the pathway of phloem unloading was studied in the berries of a hybrid grape (Vitis vinifera x Vitis labrusca). Structural investigations showed that the sieve element-companion cell complex is apparently symplasmically connected through plasmodesmata with surrounding parenchyma cells throughout fruit development, though a small portion of plasmodesmata are apparently blocked in the ripening stage. Both carboxyfluorescein and the green fluorescent protein-tagged viral movement protein were released from the functional phloem strands during the early and middle stages of fruit development, whereas the two symplasmic tracers were confined to the phloem strands during the late stage. This reveals a shift of phloem unloading from symplasmic to apoplasmic pathway during fruit development. The turning point of the phloem unloading pathways was further shown to be at or just before onset of ripening, an important developmental checkpoint of grape berry. In addition, the levels of both the expression and activities of cell wall acid invertase increased around the onset of ripening and reached a high level in the late stage, providing further evidence for an operation of the apoplasmic unloading pathway after onset of ripening. These data demonstrate clearly the occurrence of an adaptive shift of phloem unloading pathway to developmental transition from growing phase to ripening in grape berry.

Carbohydrate Metabolism↗

Nitrogen nutrition and metabolic interconversions of nitrogenous solutes in developing cowpea fruits.

Budgets for import and utilization of ureide, amides, and a range of amino acids were constructed for the developing first-formed fruit of symbiotically dependent cowpea (Vigna unguiculata [L.] Walp. cv Vita 3). Data on fruit total N economy, and analyses of the xylem and phloem streams serving the fruit, were used to predict the input of various solutes while the compositions of the soluble and protein pools of pod, seed coat, and embryo were used to estimate the net consumption of compounds. Ureides and amides provided virtually all of the fruit's N requirements for net synthesis of amino compounds supplied inadequately from the parent plant. Xylem was the principal source of ureide to the pod, while phloem was the major source of amides to pod and seed. All fruit parts showed in vitro activity of urease (EC 3.5.1.5), allantoinase (EC 3.5.2.5), asparaginase (EC 3.5.11), ammonia-assimilating enzymes and aspartate and alanine aminotransferases (EC 2.61.1 and EC 2.6.1.1.2). Asparagine:pyruvate aminotransferase (EC 2.6.1.14) was recovered only from the pod. The pod was initially the major site for processing and incorporating N; later seed coats and finally embryos became predominant. Ureides were broken down mainly in the pod and seed coat. Amide metabolism occurred in all fruit organs, but principally in the embryo during much of seed growth. Seed coats released N to embryos mainly as histidine, arginine, glutamine, and asparagine, hardly at all as ureide. Amino compounds delivered in noticeably deficient amounts to the fruit were arginine, histidine, glycine, glutamate, and aspartate, while seeds received insufficient arginine, histidine, serine, glycine, and alanine. Quantitatively based schemes are proposed depicting the principal metabolic transformation accompanying N-flow between seed compartments during development.

Journal Article↗

Up-regulation of genes encoding novel extracellular proteins during fruit set in pea.

The transition from the carpel of the flower to a developing fruit is a poorly characterized process despite its agricultural importance. We have identified two genes, GIC19 and GIC4, which are expressed after induction of pea (Pisum sativum L.) fruit set either by exogenous gibberellins or by pollination. GIC19 expression is temporally and spatially regulated, with transcripts mainly found in growing carpels and young fruit. Similar to GIC19, GIC4 expression is developmentally regulated during carpel and fruit development. However, GIC4 transcripts are found in other growing tissues throughout the plant. Analysis of their sequences and localization of fusion proteins with GFP indicate that both GIC19 and GIC4 are extracellular proteins. While GIC19 is a small proline-rich protein with no overall homology to other reported proteins. GIC4 belongs to a novel family of proteins. Our results reinforce a model of gibberellin mode of action during pea fruit set and development involving enhanced synthesis of extracellular proteins and secretory activity to provide materials and energy for cell growth.

Amino Acid Sequence↗

Global mutational analysis of NtrC-like activators in Myxococcus xanthus: identifying activator mutants defective for motility and fruiting body development.

The multicellular developmental cycle of Myxococcus xanthus requires large-scale changes in gene transcription, and recent findings indicate that NtrC-like activators play a prominent role in regulating these changes. In this study, we made insertions in 28 uncharacterized ntrC-like activator (nla) genes and found that eight of these insertions cause developmental defects. Hence, these results are consistent with the idea that M. xanthus uses a series of different NtrC-like activators during fruiting body development. Four of the eight developmental mutants we identified have motility defects. The nla1, nla19, and nla23 mutants show S-motility defects, while the nla24 mutant shows defects in both S-motility and A-motility. During development, aggregation of the nla1, nla19, and nla23 mutants is delayed slightly and the nla24 mutant shows no signs of aggregation or sporulation. The nla4, nla6, nla18, and nla28 mutants have no appreciable loss in motility, but they fail to aggregate and to sporulate normally. The nla18 mutant belongs to a special class of developmental mutants whose defects can be rescued when they are codeveloped with wild-type cells, suggesting that nla18 fails to produce a cell-cell signal required for development. The three remaining activator mutants, nla4, nla6, and nla28, appear to have complex developmental phenotypes that include deficiencies in cell-cell developmental signals.

Bacterial Proteins↗

Diurnal mRNA fluctuations of nuclear and plastid genes in developing tomato fruits.

Steady-state transcript levels of nuclear (rbcS, cab) and plastid (rbcL, psbA) encoded photosynthesis-specific genes were determined at noon and 05.00 h in different developmental stages of tomato fruits (7-35 days after anthesis). Small alterations are observed in mRNA levels for the small subunit (ssu) and large subunit (lsu) of RuBPC/Oase and the QB-binding protein of photosystem II at these two time-points, while significant steady-state transcript level fluctuations are detectable for the light harvesting complex protein. LHCP II transcripts accumulate during the day, and decline to low levels during the night. In contrast, the LHC II protein levels remain constant during the same period of development. A detailed analysis of transcript levels of the nuclear and plastid genes at 4-h intervals throughout a 38-h period demonstrates that LHCP II mRNA accumulation is highest at noon and lowest at 04.00 h. The transcripts of the ssu and lsu of RuBPC/Oase, photosystem I and II reaction center proteins, as well as the beta-subunit of the mitochondrial ATPase and the beta-subunit of tubulin, accumulate during the night and decrease to low levels in the afternoon. The transcript levels of the genes examined in this study fluctuate with certain periodicities. We suggest that gene expression in developing tomato fruits is at least partially controlled by diurnal rhythms, which are therefore also operational in other organs, besides leaves, of higher plants.

Cell Nucleus↗

Changes in the Level of Peptidase Activities in Pea Ovaries during Senescence and Fruit Set Induced by Gibberellic Acid.

The activities and changes in the levels of exopeptidase and endopeptidase activities were characterized in unpollinated ovaries of Pisum sativum L. cv Alaska during senescence and early fruit development induced by gibberellic acid (GA(3)). Two aminopeptidases and one iminopeptidase were electrophoretically separated. These peptidases were sensitive to inhibitors of sulfhydryl proteases. Carboxypeptidase activity was inhibited by phenylmethyl sulfonyl fluoride. An azocasein-degrading endopeptidase, sensitive to thiol protease inhibitors, was also found. An increase in the specific activity of aminopeptidase during both fruit development and ovary senescence was observed. In contrast, the specific activity of carboxypeptidase and endopeptidase increased only during senescence of the ovary. Changes in exopeptidase activity in senescing ovaries could be mainly the consequence of a greater stability to proteolysis while the rise in endopeptidase activity appeared to be due to new or increased synthesis of the enzyme. These results suggest that endopeptidase, and not amino or carboxypeptidase, plays a key role in the senescence of pea ovaries and that the changes in unpollinated ovaries leading to ovary senescence or fruit development can be controlled by gibberellins.

Journal Article↗

Occurrence of a major protein associated with fruiting body development in Neurospora and related Ascomycetes.

Electrophoretic and immunological analysis of fruiting body (perithecial) extracts demonstrates the occurrence of a major phase-specific perithecial protein in all Neurospora species and in the closely related Gelasinospora cerealis and Sordariafimicola. The perithecial proteins from these different species fall into a number of groups with different electrophoretic mobilities. They appear to be immunologically closely related but not identical to one another even within the same genus, with only partial identity exhibited between the heterothallic and pseudohomothallic Neurospora on the one hand and the homothallic Neurospora on the other hand. In immunological analysis of fruiting body extracts of the other Ascomycetes, Podospora anserina, Cochliobolus maydis, and Aspergillus nidulans, and of ascus extracts of Saccharomyces cerevisiae, no crossreaction with the Neurospora perithecial protein was found.

Ascomycota↗

Expression of CycD3 is transiently increased by pollination and N-(2-chloro-4-pyridyl)-N'-phenylurea in ovaries of Lagenaria leucantha.

Lagenaria leucantha is an important vegetable crop and a potential model for the study of fruit development. To study the function of D cyclins in fruit development, full-length cDNA clones for two D cyclin genes were isolated from young ovaries of Lagenaria leucantha. They were classified as D3 cyclins by sequence similarities and phylogenetic analysis, and nominated LlCycD3;1 and LlCycD3;2, respectively. The deduced amino acid sequence of both LlCycD3 genes contained a retinoblastoma-binding motif and a PEST-destruction motif. Unpollinated ovaries failed to develop and eventually aborted. N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) induced parthenocarpic fruit significantly larger than pollinated ones. In unpollinated ovaries, the expression of both LlCycD3 genes was abundant at anthesis and then suddenly decreased, concomitant with the cessation of cell division. Pollination/fertilization induced an activation of the cell cycle accompanied by a large increase in the transcript levels of LlCycD3;1 and LlCycD3;2 in young fruits. Treating ovaries with CPPU also reactivated cell division and transcription of CycD3 genes and the effect was more rapid and pronounced than after pollination/fertilization.

Amino Acid Sequence↗

A fruit-specific phospho enolpyruvate carboxylase is related to rapid growth of tomato fruit.

Malic and citric acids accumulate in cherry tomato (Lycopersicon esculentum Mill.) fruit during the period of rapid growth, from the end of cell division to the onset of ripening. The involvement of phospho enolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in organic acid accumulation and tomato fruit development was investigated. Two PEPCases, named LYCes;Ppc1 and LYCes;Ppc2 and mapped to chromosomes 12 and 7, respectively, were shown to be differentially expressed during tomato fruit development. LYCes;Ppc1 mRNA was present in all fruit tissues and in all other plant organs examined. In contrast, LYCes;Ppc2 was strongly and specifically expressed in fruit from the end of cell division to ripening. No LYCes;Ppc2 expression was detected by northern blot in other plant tissues. In fruit, the increase in LYCes;Ppc2 mRNA was closely followed by an increase in fruit PEPCase protein and activity, and was coincident with the increased accumulation of malate and citrate during the initial period of rapid growth rate, from 8 to 20 days post anthesis. Localization of LYCes;Ppc2 mRNA in young tomato fruit by in situ hybridization revealed that LYCes;Ppc2 is preferentially expressed in large cells of the pericarp and in enlarging cells of the gel surrounding the seeds. Examination of the kinetic and regulatory properties of the PEPCases of growing and ripening fruit further showed that PEPCase in growing fruit is less sensitive to low pH and malate inhibition, indicating a high phosphorylation state and/or the presence of a PEPCase isoform with these characteristics. Taken together, these results indicate that in developing tomato fruit PEPCase is probably important in permitting the synthesis of organic acids to provide the turgor pressure necessary for cell expansion.

Chromosome Mapping↗

Sorogen elongation and side branching during fruiting body development in Polysphondylium pallidum.

We report a cinematographic analysis of culmination in fruiting bodies of Polysphondylium pallidum. During the final stages of aggregation, sorogen elongation is slow. An increase in the rate of sorogen elongation occurs when the growing stalk contacts the substratum, lifting the cell mass into the air. The elongation process slows and finally stops as differentation of the terminal spore mass occurs. During the rapid elongation phase, variations in the rate of elongation of the main cell mass are correlated with the process of side branching. Side-branch elongation resembles elongation of the main sorogen with the rate of elongation increasing as the side-branch stalk contacts the main stalk and slowing as spore differentiation occurs. The morphological events of branching have been studied in detail and models to explain this process are discussed.

Morphogenesis↗

Novel insight into vascular, stress, and auxin-dependent and -independent gene expression programs in strawberry, a non-climacteric fruit.

Using cDNA microarrays, a comprehensive investigation of gene expression was carried out in strawberry (Fragaria x ananassa) fruit to understand the flow of events associated with its maturation and non-climacteric ripening. We detected key processes and novel genes not previously associated with fruit development and ripening, related to vascular development, oxidative stress, and auxin response. Microarray analysis during fruit development and in receptacle and seed (achene) tissues established an interesting parallelism in gene expression between the transdifferentiation of tracheary elements in Zinnia elegans and strawberry. One of the genes, CAD, common to both systems and encoding the lignin-related protein cinnamyl alcohol dehydrogenase, was immunolocalized to immature xylem cells of the vascular bundles in the strawberry receptacle. To examine the importance of oxidative stress in ripening, gene expression was compared between fruit treated on-vine with a free radical generator and non-treated fruit. Of 46 genes induced, 20 were also ripening regulated. This might suggest that active gene expression is induced to cope with oxidative stress conditions during ripening or that the strawberry ripening transcriptional program is an oxidative stress-induced process. To gain insight into the hormonal control of non-climacteric fruit ripening, an additional microarray experiment was conducted comparing gene expression in fruit treated exogenously with auxin and control fruit. Novel auxin-dependent genes and processes were identified in addition to transcriptional programs acting independent of auxin mainly related to cell wall metabolism and stress response.

Alcohol Oxidoreductases↗

Tomato fruit cell wall : I. Use of purified tomato polygalacturonase and pectinmethylesterase to identify developmental changes in pectins.

Cell wall isolation procedures were evaluated to determine their effect on the total pectin content and the degree of methylesterification of tomato (Lycopersicon esculentum L.) fruit cell walls. Water homogenates liberate substantial amounts of buffer soluble uronic acid, 5.2 milligrams uronic acid/100 milligrams wall. Solubilization appears to be a consequence of autohydrolysis mediated by polygalacturonase II, isoenzymes A and B, since the uronic acid release from the wall residue can be suppressed by homogenization in the presence of 50% ethanol followed by heating. The extent of methylesterification in heat-inactivated cell walls, 94 mole%, was significantly greater than with water homogenates, 56 mole%. The results suggest that autohydrolysis, mediated by cell wall-associated enzymes, accounts for the solubilization of tomato fruit pectin in vitro. Endogenous enzymes also account for a decrease in the methylesterification during the cell wall preparation. The heat-inactivated cell wall preparation was superior to the other methods studied since it reduces beta-elimination during heating and inactivates constitutive enzymes that may modify pectin structure. This heat-inactivated cell wall preparation was used in subsequent enzymatic analysis of the pectin structure. Purified tomato fruit polygalacturonase and partially purified pectinmethylesterase were used to assess changes in constitutive substrates during tomato fruit ripening. Polygalacturonase treatment of heat-inactivated cell walls from mature green and breaker stages released 14% of the uronic acid. The extent of the release of polyuronides by polygalacturonase was fruit development stage dependent. At the turning stage, 21% of the pectin fraction was released, a value which increased to a maximum of 28% of the uronides at the red ripe stage. Pretreatment of the walls with purified tomato pectinesterase rendered walls from all ripening stages equally susceptible to polygalacturonase. Quantitatively, the release of uronides by polygalacturonase from all pectinesterase treated cell walls was equivalent to polygalacturonase treatment of walls at the ripe stage. Uronide polymers released by polygalacturonase contain galacturonic acid, rhamnose, galactose, arabinose, xylose, and glucose. As a function of development, an increase in the release of galacturonic acid and rhamnose was observed (40 and 6% of these polymers at the mature green stage to 54 and 15% at the red ripe stage, respectively). The amount of galactose and arabinose released by exogenous polygalacturonase decreased during development (41 and 11% from walls of mature green fruit to 11 and 6% at the red ripe stage, respectively). Minor amounts of glucose and xylose released from the wall by exogenous polygalacturonase (4-7%) remained constant throughout fruit development.

Journal Article↗

Influence of Auxin and Gibberellin on in Vivo Protein Synthesis during Early Pea Fruit Growth.

Developing pea fruits (Pisum sativum L.) offer a unique opportunity to study growth and development in a tissue that is responsive to both gibberellins (GAs) and auxin (4-chloroindole-3-acetic acid[4-CI-IAA]). To begin a molecular analysis of the interaction of GAs and auxins in pea fruit development, in vivo labeling with [35S]methionine coupled with two-dimensional gel electrophoresis were used to characterize de novo synthesis of proteins during gibberellic acid (GA3)-, 4-CI-indoleacetic acid-, and seed-induced pea pericarp growth. The most significant and reproducible polypeptide changes were observed between molecular weights of 20 and 60. Comparing about 250 de novo synthesized proteins revealed that seed removal changed the pattern substantially. We identified one class of polypeptides that was uniquely seed induced and five classes that were affected by hormone treatment. The latter included 4-CI-IAA-induced, GA3-induced, GA3- and 4-CI-IAA-induced, 4-CI-IAA-repressed, and GA3- and 4-CI-IAA-repressed polypeptides. Similar patterns of protein expression were associated with both hormone treatments; however, changes unique to GA3 or 4-CI-IAA treatment also indicate that the effects of GA3 and 4-CI-IAA on this process are not equivalent. In general, application of 4-CI-IAA plus GA3 replaced the seed effects on pericarp protein synthesis, supporting our hypothesis that both hormones are involved in pea pericarp development.

Journal Article↗

Response of leaf ontogeny and photosynthetic activity to reproductive growth in cotton.

This study was conducted to determine if reproductive growth in cotton (Gossypium hirsutum L.) affects concurrent leaf development. Apparent photosynthesis (AP), stomatal conductance (Cs), soluble protein (SP), ribulose bisphosphate carboxylase (RuBisCO), and chlorophyll (Chl) were monitored in four main-stem cotton leaves which emerged at approximately 2 week intervals. The leaf which emerged during vegetative growth (48 days after planting) had higher AP, SP, and RuBisCO levels than that present in any leaves which emerged during fruit development. The last leaf studied (89 days after planting) was still present after boll maturation was completed and exhibited a rejuvenation in AP, SP, RuBisCO, and Chl starting at 30 days after leaf emergence. At 96 days after planting, the P700 Chl a-protein complex (PSI) was virtually absent from the leaves that emerged at 48 and 62 days after planting. The light harvesting Chl a/b complex was still present in these leaves, indicating greater degradation of PSI. The data emphasize the influence of developing fruit on concurrently developing leaves, an effect which was alleviated after boll maturation was completed. The declining AP per unit leaf area and smaller leaf size at the top of the plant results in a reduced photosynthetic potential of successively later emerging leaves. This reduction in leaf AP is consistent with earlier reported seasonal canopy photosynthesis patterns.

Journal Article↗