Mark L. Brenner

Sucrose synthase, starch accumulation, and tomato fruit sink strength

Contrasting evidence has accumulated regarding the role of acid invertase and sucrose synthase in tomato fruit sink establishment and maintenance. In this work the relationships among the activities of sucrose synthase and acid invertase, Lycopersicon esculentum Mill cv UC-82B fruit growth, and starch accumulation were analyzed in fruit at 0 to 39 d after anthesis. Sucrose synthase, but not acid invertase, was found to be positively correlated with tomato fruit relative growth rate and with starch content in the pericarp tissue. A similar association between sucrose synthase activity and starch accumulation was also evident in the basal portion of the stem. Heat-shock treatments, which inhibited the increase in sucrose synthase activity at the beginning of the light period and had no effect on acid invertase activity, were used to examine the importance of sucrose synthase in relation to sucrose metabolism and starch synthesis. After the heat-shock treatment, concomitantly with the suppressed sucrose synthase activity relative to the controls, there was a reduction in sucrose cleavage and starch accumulation. These data substantiate the conclusion that, during the early phases of tomato fruit development, sucrose synthase rather than acid invertase is the dominant enzyme in metabolizing imported sucrose, which in turn plays a part in regulating the import of sucrose into the fruit.

The Role of Hormones in Photosynthate Partitioning and Seed Filling

The movement of photoassimilates from sites of synthesis in leaf tissue (source) to the sites of net accumulation in a different tissue (sink) potentially can be regulated at numerous points. Regulation of the net flow of photoassimilates is an integrated process. It is generally accepted that the concentration gradient of photoassimilates between the source and sink is the primary determinant of the current rate of transport and pattern of partitioning (14, 19, 60). However, close examination of the various components involved in the overall process of partitioning indicates that endogenous plant hormones may serve as modulators of many of the specific rate limiting components. This chapter will focus on the involvement of plant hormones as natural regulators of partitioning of photoassimilates especially to developing seeds.

Regulatable endogenous production of cytokinins up to toxic levels in transgenic plants and plant tissues

The effects of expressing a chimeric gene consisting of a soybean heat shock gene promoter and a sequence that encodes an enzyme catalyzing the synthesis of a potent phytohormone, the cytokinin iPMP, have been analyzed in transgenic tobacco plants. The production of cytokinin endogenously produced several effects previously undocumented. The differentiation of shoots independent of exogenous cytokinin from heat-treated transgenic plant leaf explants demonstrates that long-term heat treatments do not interfere with complex developmental processes. This extends the potential usefulness of heat shock gene promoters to conditionally express genes during windows of development that span several weeks.

A one-step enzymatic assay for sucrose with sucrose phosphorylase.

A one-step, enzymatic assay for sucrose using sucrose phosphorylase is described. Sucrose phosphorylase, which is now commercially available, was isolated from Leuconostoc mesenteroides strain B-1200 and partially purified by ammonium sulfate precipitation. Samples containing 5 to 80 nmol of sucrose are mixed with potassium phosphate, NAD, sucrose phosphorylase, and two commercial enzymes, phosphoglucomutase and NAD-accepting glucose-6-phosphate dehydrogenase. After 30 min incubation at room temperature, absorbance at 340 nm is proportional to initial sucrose content. A 20-fold molar excess of glucose or a twofold excess of fructose have no effect on the assay, while a fourfold excess of fructose interferes with the assay by decreasing absorbance ca. 20%. This assay was designed to provide a rapid method for determining sucrose in studies of sugar transport by plants. To test the assay, corn pedicel extracts were assayed enzymatically and by high-pressure liquid chromatography. Estimates of sucrose content made by the two methods were equivalent, and exogenous addition of sucrose to these samples resulted in the expected increase in apparent sucrose content.

Temporal and Spatial Expression Pattern of Sucrose Synthase during Tomato Fruit Development

Sucrose synthase is proposed to play an important role in the early stages of tomato fruit (Lycopersicon esculentum Mill.) growth. In this work, the temporal and spatial expression patterns of sucrose synthase during tomato fruit development were investigated. Fruit contained the majority of the sucrose synthase protein and mRNA relative to other organs. Only trace levels of sucrose synthase protein and mRNA were detected in the stem, petiole, and roots. Sucrose synthase mRNA was detected in pistils prior to anthesis, reached peak levels in fruit 5 to 7 d after anthesis (DAA), and was not detectable after 35 DAA. Sucrose synthase protein levels reached a maximum at 20 to 25 DAA and then declined to nondetectable levels after 45 DAA. The lack of coordination between protein and mRNA levels suggests that sucrose synthase expression may be controlled at the levels of both transcription and translation. Sucrose synthase mRNA was differentially localized in the fruit, being most abundant in the mesocarp cells adjacent to the placenta, the columella, and the cells surrounding the vascular bundle. Except around the vascular tissue, the localization of sucrose synthase mRNA positively correlates with starch granule accumulation at the cellular level.

Characterization of Conditioning Factors that Increase Colony Formation from «Black Mexican Sweet Corn» Protoplasts

Summary Colony-formation frequency of protoplasts isolated from suspension cultures of «Black Mexican Sweet (BMS) corn» ( Zea mays L.) is increased about ten-fold when conditioned medium (CM) isolated from BMS suspension cultures is supplied to protoplasts plated at 50,000 protoplasts/ml. The objective of this study was to characterize the factor(s) (CMF) from conditioned medium responsible for the increased colony formation. Colony-formation frequency was a linear function of the amount of CM, indicating that either a single CMF is present in CM or that several CMF are present but each acts independently. Removal of polyvalent cations stabilized CMF at elevated temperatures. CMF was most stable at pH 5, losing activity rapidly at pH = 8 or pH = 2. Based on its partitioning between water and ethyl acetate or butanol, and its solubilities in mixtures of water and isopropanol, CMF is very hydrophilic. Neither anion-exchange (NO 3 - form, in H 2 O) nor cation-exchange (Na + form, in H 2 O) resins at pH 5 retained CMF, suggesting that CMF is uncharged. Ultrafiltration showed that the molecular weight of CMF is slightly less than 1350. Subsequent gel filtration with Biogel P-2 and Biogel P-6 resolved CMF into a major peak with an apparent molecular weight of ca. 1200. Thus, CM appears to contain a single major CMF, which has characteristics similar to that of an oligosaccharide. Although the final structure of CMF is unresolved, CMF is chemically unlike the reported conditioning factor(s) of either Hordeum vulgare anther cultures or of dicot suspension cultures, or the cell-division-inducing phytohormones typically used in the culture of protoplasts.

4-chloroindole-3-acetic and indole-3-acetic acids in Pisum sativum

Abstract Endogenous IAA and 4-chloroindole-3-acetic acid (4-C1-IAA) were analysed in vegetative and reproductive tissues of the garden pea (Pisum sativum) using GC-MS selected ion monitoring in the presence of stable-isotope labelled internal standards. In fruit collected 3–8 days after anthesis (DAA) conjugates of both auxins were more abundant than the free hormones. Auxin levels (ng g−1 fr. wt) in the seeds were higher by 1–2 orders of magnitude than in the pericarps. However, as seeds are small at this stage, the pericarp nevertheless contains a substantial fraction of the overall quantity of IAA and 4-C1-IAA. While, in young fruit tissues, IAA was more abundant than 4-C1-IAA, the opposite was true for seeds at the ‘table-ready’ stage and for roots of nine-day-old seedlings. Our data suggest that both IAA and 4-C1-IAA are required to coordinate the vegetative and reproductive growth of pea plants.

The effect of conditioned medium on colony formation from ‘black mexican sweet’ corn protoplasts

Abstract Protoplasts from suspension cultures of ‘Black Mexican Sweet’ corn ( Zea mays L.) exhibited increased colony formation frequency when cultured in the presence of conditioned medium isolated from Black Mexican Sweet (BMS) cultures. The increase in colony formation frequency of protoplasts plated at 5 × 10 4 protoplasts/ml was used as a bioassay for detecting conditioning factors and to investigate the response of protoplasts to conditioned medium. Conditioned medium was stable when stored frozen at −14°C, retained 80% activity when stored for 10 days at 24°C and was inactivated by heating to 100°C for 10 min. The most active isolates of conditioned medium were recovered during the exponential growth phase of the suspension cultures. Colony formation frequencies were greatest when conditioned medium was added to protoplasts within 12 h of plating and was not removed for 9 days thereafter. Addition of conditioned medium to protoplasts increased cell division frequency and did not increase protoplast viability or cell wall formation above the control cultures. Most of the conditioning activity was due to factors that were rejected by a 500 molecular weight (MW) cutoff ultrafiltration membrane. A mixture of threonine, glutamate, cysteine and methionine (0.25 μM each), which were the only amino acids detected in conditioned medium, and proline (1 mM) increased colony formation but to lower levels than 25% conditioned medium. Our results indicate that BMS culture cells produce conditioning factors that increase and sustain cell division from BMS protoplasts and that free amino acids are not the active factors in conditioned medium.

Identification of three C20-gibberellins: GA97 (2β-hydroxy-GA53), GA98 (2β-hydroxy-GA44) and GA99 (2β-hydroxy-GA19)

Abstract Three new C 20 -gibberellins, GA 97 (2β-hydroxy-GA 53 ), GA 98 (2β-hydroxy-GA 44 ) and GA 99 (2β-hydroxy-GA 19 ), have all been isolated from spinach, GA 97 also from tomato root cultures and pea pods, and GA 98 from maize pollen. The structures of these compounds were established by GC-mass spectrometric comparisons of the trimethylsilylated methyl esters with authentic samples prepared from gibberellic acid (GA 3 ).

Energetics of the response of maize coleoptile tissue to indoleacetic acid : Characterization by flow calorimetry as a function of time, IAA concentration, and pH.

Indole-3-acetic acid (IAA) promotes an increase in steady-state heat production by corn (Zea mays L.) coleoptile tissue; this increase is associated with an elevation in aerobic respiration rates. A detailed time dependence of the exothermic response to IAA was obtained using flow calorimetry. The latent period and magnitude of response were evaluated as a function of IAA concentration and pH. The data indicate that more than one response may occur. The optimal change in heat production was produced by an IAA concentration of 3·10-5 M. It was initiated within 5 min after the start of the IAA treatment, and reached a magnitude in excess of 25% of the tissues basal heat production. Concentrations of IAA greater than 1·10-4 M resulted in diminished response(s), but the effect was strongly pH dependent. Several possibilities for the increased heat production triggered by IAA are discussed.