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A cDNA library was constructed from satsuma mandarin (Citrus unshiu Marc.) fruit tissues during the rapid cell enlargement phase. A total of 950 individual cDNA clones was partially sequenced and compared with GenBank databases for characterizing the gene repertoire expressed during this developmental phase. Among these, 426 cDNA clones (44.8%) showed sequence identity with previously characterized genes with optimized (OPT) scores of ≥200, while 524 clones (55.2%) resulted in low OPT scores (<200) and did not show any significant sequence identity with previously published genes. Based on nucleotide sequence, most clones with OPT scores of ≥200 were assumed to be transcription-, translation-, cell-wall-metabolism-, and stress-response-related genes. Other clones showed homology with published sequences related to housekeeping and stress-response-related genes, including metallothionein-like proteins, late-embryogenesis-abundant (LEA) proteins, and heat-shock proteins. These results suggested that Citrus fruit during rapid cell enlargement were metabolically active and expanding in response to biotic and abiotic stress. For clones with low nucleotide sequence homology, the recurrence was evaluated by aligning nucleotide sequences of each clone and generating contig maps. Expressed sequence tags (ESTs) of 162 clones with OPT scores <200 have not been reported for any other organism. Collectively, randomly sequenced cDNA clones described in this study provided information on types of genes expressed during the rapid cell enlargement phase in Citrus fruit. These genes should be used as candidates for Citrus genome mapping projects.

Young expanding leaves of `Ambersweet' [Citrus reticulata Blanco × C. paradisi Macf. × C. reticulata) × C. sinensis (L) Osb.] with feeding injury by third larval stage of citrus leafminer (Phyllocnistis citrella) were examined by light and electron microscopy for extent of injury and tissue recovery over time. Results confirmed that injury is confined to the epidermal layer, leaving a thin covering over the mine tunnel that consisted of the cuticle and outer cell wall. Wound recovery consisted of two possible responses: the production of callus tissue or the formation of wound periderm. The production of callus tissue developed within 3 days of injury when the uninjured palisade or spongy parenchyma below the injured epidermis produced callus tissue through periclinal or diagonal cell divisions. After 1 month, the entire epidermis was replaced by callus tissue. In the absence of secondary microbial invasion, this callus tissue developed a thick cuticle, followed by development of a covering of platelet wax after 4 months. Alternatively, wound periderm formed if the outer cuticular covering was torn before the cuticle had developed sufficiently to prevent the exposed cells from being desiccated or invaded by fungi, bacteria, or other insects. The wound periderm consisted of a lignified layer of collapsed callus cells, a suberized phellem layer, and a multilayered phelloderm-phellogen. Since there were always cellular collapse or fungi and bacteria associated with wound periderm formation, it was determined to be a secondary effect, not a direct effect of leafminer feeding.

Primary and first-order lateral roots of Panax quinquefolius L. (American ginseng) were collected from plants in an experimental garden during their second year of growth and processed for light and transmission electron microscopy. Roots in primary growth had either a diarch or triarch primary xylem pattern, a pericycle, an endodermis with Casparian bands and subsequently a suberized cell wall, and a cortex of variable thickness with a suberized hypodermal layer. Both root types underwent rapid secondary growth and the primary root particularly formed a fleshy storage organ. The secondary phloem and secondary xylem had abundant parenchyma and few conducting elements. Secretory ducts differentiated in tissue derived from the pericycle and in the secondary phloem. Each schizogenous duct consisted of six to eight epithelial cells, which possessed dense, globular deposits but lacked starch. A phellogen, which produced several layers of suberized phellem, was initiated in the periphery of tissue derived from the pericycle. The results of this study clarify the anatomical localization of secretory duets in roots of this species.

Mechanized shaker harvesting of large acreages of almond [Prunus dulcis (Mill.) Webb.] trees leads to economical use of labor, general cost reduction, and speed of harvest. However, shaking can separate the bark (all tissues external to the vascular cambium) from the wood (all tissues internal to the vascular cambium) and exposes the vascular cambial zone to infection by the fungus Ceratocystis fimbriata Ell. & Halst. leading to mallet wound canker and loss of productivity. Treatment of ethephon caused a significant increase in the cambial strength of both branches and trunks, presumably by ethylene-induced anatomical and biochemical changes in the cambial zone. An increase in the ratio of total tangential area of the groups of ray initials to fusiform initials and the thickness of ray initial cell walls was observed as a response to ethephon treatment. Spraying an ethephon solution (500 μl-liter^-1) to the runoff point on almond trunks caused significant increases in the cambial strength; ethephon, therefore, may be useful in reducing bark injury during shaker harvesting.

Kiwifruit [Actinidia deliciosa (A. Chev) C.F. Liang et A.R. Ferguson] flesh firmness can decline by as much as 94% during fruit ripening. This phenomenon was investigated at the cellular level, with the aim of characterizing changes in the physiological condition and mechanical properties of cells. The tensile strength of kiwifruit outer pericarp tissue was measured, and low-temperature scanning electron microscopy was used to examine the mode of cell failure at fracture surfaces. The propensity with which cells ruptured was determined by incubating tissue discs in hypertonic and hypotonic solutions, and water potentials, osmotic potentials, turgor pressures, and tissue density were measured. An initial rapid reduction in flesh firmness—from 80 to 27 N during 6 weeks of storage at 0C—was related to a reduction in the adhesion between neighboring cells. Following tensile tests, an examination of fracture surfaces indicated that cells from freshly harvested fruit had ruptured, exposing the cell interior. After 6 weeks of storage, neighboring cells separated from each other without breaking open. With 23 additional weeks of storage at 0C, flesh firmness decreased from 27 to 5 N. The final softening stage was associated with an increase in the proportion of cells that separated at the middle lamella and an increase in the plasticity of the cell wall.

Pressure infiltration of `Golden Delicious' and `McIntosh' apples (Malus domestica Borkh.) with polyamides resulted in an immediate increase in firmness. `Golden Delicious' apples were 2.7 N (0.25 mM spermidine) to 6.7 N (1.0 mM spermine) firmer, while `McIntosh' apples were 2.2 N (0.25 mM spermidine) to 5.3 N (1.0 mM spermine) firmer than the water-treated control. During 28 weeks of storage at 0C, the differences between the polyamine-treated and water-treated apples were even larger. Similar results were observed with a 3% Ca treatment, but the Ca treatment reduced the rate of softening to a greater extent than did the polyamine treatments in `Golden Delicious'. Polyamides increased the endogenous levels of the polyamides infiltrated; however, the levels declined rapidly with time in storage. Both polyamine and Ca inhibited the development of chilling injury symptoms (brown core) in `McIntosh'. The influence of polyamines on ethylene production was negligible in both cultivars. The Ca treatment, however, inhibited ethylene evolution in `Golden Delicious'. Polyamides, thus, may affect apple softening through rigidification of cell walls rather than through interactions with ethylene metabolism.

Germination studies indicated that increasing priming duration (-1.0 MPa at 20 °C for 7, 14, or 21 days) increased `Moss Curled' parsley [Petroselinum crispum (Mill.) Nyman ex A.W. Hill] germination rate quadratically and seed moisture content linearly. A histological and anatomical study was conducted to identify and/or quantify principle mericarp organ or tissue volume changes influenced by priming duration. Embryo volume increased as priming duration increased from 7 to 21 days (0.014 to 0.034 mm³), and this was due more to radicle (0.007 to 0.022 mm³) than to cotyledon (0.006 to 0.011 mm³) growth. Concomitant with increased embryo volume was increased volume of the depleted layer (space formation, surrounding the embryo), from 0.038 after 7 days to 0.071 mm³ after 21 days, and increased hydrolysis of central endosperm (a thick-walled endosperm type). In nonprimed mericarps, central endosperm cells constituted 97% of the endosperm volume. The remaining 3% was comprised of 1% depleted layer and 2% distal endosperm (small, thin-walled, and irregularly shaped endosperm cells). During 7 or 21 days of priming, ≈10% or 40%, respectively, of central endosperm cells were hydrolyzed centrifugally around the embryo with a corresponding decrease in volume of central endosperm with thick cell walls. In addition, distal endosperm cells adjacent to the depleted layer, containing reserve materials, were digested of contents following 21 days priming, and sometimes, following 7 days priming. A long priming duration resulted in degradation of pericarp tissues, as indicated visually and by a decline in pericarp volume. We hypothesize that priming duration of parsley primarily influences radicle growth and centrifugal digestion and utilization of central and distal endosperm, resulting in a larger depleted layer required for embryo volume increases. Secondary events influenced by priming duration include cotyledon growth and degradation of pericarp tissues.

Colletotrichum acutatum J. H. Simmonds infects citrus flower petals, causing brownish lesions, young fruit drop, production of persistent calyces, and leaf distortion. This suggests that hormones may be involved in symptom development. To identify the types of hormones, cDNA clones encoding proteins related to ethylene and jasmonate (JA) biosynthesis, indole-3-acetic acid (IAA) regulation, cell-wall modification, signal transduction, or fruit ripening were used to examine differential gene expressions in calamondin (Citrus madurensis Lour) and/or `Valencia' sweet orange (Citrus sinensis Osbeck) after C. acutatum infection. Northern-blot analyses revealed that the genes encoding 1-aminocyclopropane-1-carboxylate (ACC) oxidase and 12-oxophytodienoate required for ethylene and JA biosynthesis, respectively, were highly up-regulated in both citrus species. Both gene transcripts increased markedly in petals, young fruit and stigmas, but not in calyces. The transcripts of the genes encoding IAA glucose transferase and auxin-responsive GH3-like protein, but not IAA amino acid hydrolyase, also markedly increased in both species 5 days after inoculation. The expansin and chitinase genes were slightly up-regulated, whereas the senescence-induced nuclease and ß-galactosidase genes were down-regulated in calamondin. No differential expression of transcripts was detected for the genes encoding expansin, polygalacturonase, and serine-threonine kinase in sweet orange. As compared to the water controls, infection of C. acutatum increased ethylene and IAA levels by 3- and 140-fold. In contrast, abscisic acid (ABA) levels were not significantly changed. Collectively, the results indicate that infection by C. acutatum of citrus flowers triggered differential gene expressions, mainly associated with IAA, ethylene, and JA production and regulation, and increased hormone concentrations, consistent with the hypothesis of the involvement of phytohormones in postbloom fruit drop.

An invertase gene was isolated and its mRNA activity and protein levels were determined during papaya (Carica papaya L.) fruit development. A complete invertase cDNA (AF420223) and a partial sucrose synthase cDNA (AF420224) were isolated from papaya fruit cDNA libraries. The invertase cDNA encoded a predicted polypeptide of 582 residues (MW 65,537 Da), and was 68% and 45% identical with carrot apoplastic and vacuolar invertases, respectively. Key amino acids indicative of an apoplastic invertase were conserved. A full-length gene corresponding to the putative apoplastic invertase cDNA was isolated and was organized into seven exons and six introns. Exon 2 (9 bp long) encoded part of a highly conserved region (NDPNG/A). Invertase mRNA and activity levels increased during fruit maturation and sugar accumulation just before ripening. In contrast, sucrose synthase mRNA levels were high during early fruit growth and low during the fruit sugar accumulation stage. A 73-kDa cell wall extractable protein that cross-reacted with carrot apoplastic invertase antisera substantially increased during papaya fruit maturation and declined in full ripe fruit. The increase in invertase protein levels occurred 2 to 4 weeks before maturity and was markedly higher than the overall increase in enzyme activity at this stage. Subsequently, the increase in enzyme activity was higher than the increase in protein levels between 2 weeks before maturity and fully ripe. The results suggested that mRNA level and invertase activity were related to maturity. The data suggested that the invertase was apoplastic, and that post-translational control of enzyme activity occurred, in which a significant accumulation of invertase occurred before the peak of enzymes activity.

Seasonal alteration of the cytosolic and nuclear Ca²⁺ concentrations of spruce (Picea engelmannii Parry) and brome grass (Bromus inermis Leyss) was investigated by the antimonate precipitation cytochemical technique. Electron microscopic (EM) observations revealed that electron-dense Ca²⁺ antimonate deposits, an indication of Ca²⁺ localization, were seen mainly in the vacuole, the cell wall and the intercellular space in samples of both species, collected on 14 July 1997. Few deposits were found in the cytosol and nuclei, showing a low resting level during summer months. On 8 Aug. 1997 following a decrease in daylength of 1 hour and 12 minutes, Ca²⁺ accumulation was initiated in spruce with increased cytosolic and nuclear Ca²⁺ deposits, but not in brome grass. On 8 Sept. 1997, Ca²⁺ accumulation occurred in the cytosol of brome grass. This followed a drop in ambient temperature to 12 °C. Cytosolic and nuclear Ca²⁺ deposits continued to increase in spruce. Controlled experiments confirmed that it was the low temperature, not shortening daylength, that triggered Ca²⁺ accumulation in brome grass. High cytosolic and nuclear Ca²⁺ concentrations lasted about three months in spruce from early August to early November. However, the high cytosolic and nuclear Ca²⁺ concentrations in brome grass lasted only about 20 days from early September to the end of the month. During winter and spring, both species had low resting cytosolic and nuclear Ca²⁺ concentrations. The relationship between the duration of the high cytosolic and nuclear Ca²⁺ concentrations and the status of the developed dormancy/cold hardiness is discussed in light of current findings.