L.H.W. van der Plas

The genetics of plant metabolism

Variation for metabolite composition and content is often observed in plants. However, it is poorly understood to what extent this variation has a genetic basis. Here, we describe the genetic analysis of natural variation in the metabolite composition in Arabidopsis thaliana. Instead of focusing on specific metabolites, we have applied empirical untargeted metabolomics using liquid chromatography–time of flight mass spectrometry (LC-QTOF MS). This uncovered many qualitative and quantitative differences in metabolite accumulation between A. thaliana accessions. Only 13.4% of the mass peaks were detected in all 14 accessions analyzed. Quantitative trait locus (QTL) analysis of more than 2,000 mass peaks, detected in a recombinant inbred line (RIL) population derived from the two most divergent accessions, enabled the identification of QTLs for about 75% of the mass signals. More than one-third of the signals were not detected in either parent, indicating the large potential for modification of metabolic composition through classical breeding.

Internal browning in pear fruit (Pyrus communis L. cv Conference) may be a result of a limited availability of energy and antioxidants

Storage of pears (Pyrus communis) under hypoxia, especially in the presence of increased CO2 partial pressures, can lead to development of brown core. Disorder development, concentrations of ascorbic acid (AA) and adenosine triphosphate (ATP), and respiration were examined under various O-2 (0-21 kPa) and CO2 (0 and 5 kPa) atmospheres during 31 days of storage. ATP production was estimated using the respiration data. Hypoxia increased brown core incidence, decreased AA and ATP concentrations, and lowered ATP production. AA concentrations decreased before brown core became visible. Adding CO2 to the storage atmosphere increased the severity of brown core. CO2 addition also decreased AA levels by about 46% at O-2 partial pressures of 2.5 kPa and higher. CO2, however, had variable effects on ATP production. No brown core was found in fruit kept at 0 kPa O-2 with or without CO2, nor decreased AA levels. These results support the hypothesis that brown core initiation is a consequence of membrane damage caused by a combination of oxygen free radical action and a lack of maintenance energy. This combination may lead to decompartmentation of intracellular structures and the initiation of brown pigmentation, visible in pears with disorders

PPO activity and polyphenol content are not limiting factors during brown core development in pears (Pyrus communis L. cv. conference)

Summary Brown core is a disorder in pears that is frequently observed in the Conference cultivar. The brown colour is the result of an enzymatic oxidation of polyphenol substances to o -quinones, a reaction catalysed by polyphenoloxidases (PPO). In a later stage pears can form cavities in and nearby the core. The goal of our research was to understand and predict the development of core browning. Pears from orchards in Spain and The Netherlands were compared to observe climatic influences on the development of brown core. PPO activity and total polyphenol content are not affected by harvest date, storage atmosphere and orchard, and both do not seem to be limiting during development of brown core. An observed decrease of tyrosinase activity might be the consequence of the decreased viability of brown tissue. We hypothesize that core browning is induced by decompartmentation of intracellular membrane structures.

Changes in gene expression during programmed cell death in tomato cell suspensions

To identify genes involved in plant programmed cell death (PCD), changes in gene expression during PCD in a model system of suspension-cultured tomato cells were studied. In this system, cell death is triggered by treatment with camptothecin, an inhibitor of topoisomerase I. Cell death was accompanied by internucleosomal DNA degradation, indicating that the cell death process shares similarities with apoptosis in animals. Tomato homologues of DAD1 and HSR203, two genes that have been implicated in PCD, were isolated. During camptothecin-induced PCD tomato DAD1 mRNA levels roughly halve, while tomato HSR203 mRNA levels increase 5-fold. A differential display approach was used to identify novel genes that show changes in expression levels during camptothecin-induced PCD. This resulted in isolation of two up-regulated (CTU1 and CTU2) and four down-regulated (CTD1, CTD2, CTD4, and CTD5) cDNA clones. CTU1 shows high homology to various gluthatione S-transferases, whereas CTU2 is as yet unidentified. CTD1 is highly similar to Aux/IAA early-auxin-responsive genes. CTD2 corresponds to the tomato RSI-1 gene, CTD4 is an unknown clone, and CTD5 shows limited homology with a proline-rich protein from maize. Addition of the calcium channel blocker lanthanum chloride prevented camptothecin-induced cell death. The effect of lanthanum chloride on camptothecin-induced gene expression was studied to discriminate between putative cell death genes and general stress genes. The possible role of the various predicted gene products in plant PCD is discussed.

Effect of Growth Temperature on Erucic Acid Levels in Seeds and Microspore-derived Embryos of Oilseed Rape, Brassica napus L.

Summary Effect of growth temperature on the fatty acid composition of triglycerides was compared for developing seeds and microspore-derived embryos of rapeseed. Plants were grown under standard conditions and subsequently seed development was allowed to take place at 15 or 25 °C. The composition of seed oil of the low erucic acid cultivar Aurora and a high erucic acid Gulle-derived line were not affected by growth temperature. The cultivar Reston showed a 30 % reduction in erucic acid level in seeds from plants grown at 25 °C as compared with seeds from plants grown at 15 °C. Seeds from plants that were transferred once during development from 15° to 25° or vice versa , after two thirds of their development had been completed, showed an oil composition similar to seeds that developed completely at the initial temperature. Seeds transferred before or during the period of maximum lipid synthesis showed oil compositions that were similar to oil formed at the final temperature or intermediate between those of seeds completely grown at 15 °C or 25 °C, respectively. In microspore-derived embryos (MDEs) similar responses to temperature in the level of erucic acid could be observed. However, absolute levels of erucic acid in MDEs of both Reston and Gulle were 30–50 % lower than in seed oil. Results are discussed in relation to possible regulatory mechanisms of temperature-induced changes in oil composition.

S-Carvone inhibits phenylalanine ammonia lyase (PAL) activity and suberization during wound healing of potato tubers.

Summary The periderm formation of wound tissue of potato tuber was inhibited temporarily after exposure to S-carvone. In wound tissue of control tubers a suberized cell layer was visible after 4 days whereas in S-carvone treated tissue this was observed only after 14 days in most of the tubers. At day 21 all the S-carvone treated tubers had formed suberin but only 10 % of the tubers had developed a (begin of a) cambium layer. The appearance of suberin was related to the activity of phenylalanine ammonia lyase (PAL); in control and S-carvone treated tissue, maximum PAL activity preceded the appareance of the first suberized cell layers by 2 and 5 days, respectively. The specific activity reached the same level in both control and S-carvone treated wound tissue. A short term treatment (2, 4 and 7 days) of tuber wound tissue with S-carvone delayed suberin formation. All the treated tubers showed suberization about 4 days after removal of S-carvone and this was closely related to the increase in PAL-activity. After the S-carvone treatment, the glutathione metabolism increased: after an initial decrease, as observed in the control, the total glutathione content increased fourfold after 9 days and remained high during the treatment. However, the length of the wound healing period before the start of a S-carvone treatment was of great influence on the increase of the glutathione metabolism and on the induction of glutathione reductase.

A quantitative comparison of respiration in cells and isolated mitochondria from Petunia hybrida suspension cultures. A high yield isolation procedure.

Abstract Mitochondria were isolated from small amounts (2-10g FW) of Petunia hybrida cell suspensions. A high yield was obtained (about 32%) as measured by fumarase activity. The quality of the mitochondria was comparable to that of other plant sources. RC values of about 2.2 could be reached and various substrates were oxidized. The respiration of cells and isolated mitochondria followed the same course during a batch cycle. Like whole cells, mitochondria showed a rapid increase in the capacity of the cytochrome pathway and the CN-resistant, alternative pathway during the first part of the batch cycle, which was followed by a decline. The engagement of the alternative pathway of cells showed the same course in time but was far from complete. In mitochondria, the alternative pathway was fully engaged during the whole batch cycle. Respiration of cells and isolated mitochondria was compared by correcting mitochondrial respiration for yield. In the late logarithmic phase the respiration rate of the cytochrome pathway of mitochondria (in state 3) appeared to be higher than that of cells; in the early logarithmic and stationary phase it was equal. By uncoupling respiration with FCCP it was shown that substrate was not limiting the in vivo respiration during the whole batch cycle, and that ADP was limiting only in the late logarithmic phase. The mechanism by which respiration is adapted to varying growth conditions is discussed.

Anthraquinone glycosylation and hydrolysis in Morinda citrifolia cell suspensions: Regulation and function

Summary Morinda citrifolia cells grown in suspension are able to accumulate significant amounts of anthraqui-nones (AQ) when grown in a low-auxin medium. As most of these anthraquinones are stored as glycosides, we studied the nature of the sugar moiety and several aspects of the glycosylation and hydrolysis reactions involved in the synthesis and degradation of these secondary metabolites. The sugar moiety of anthraquinones produced by Morinda cell suspensions appeared to be the disaccharide primverose (6-O-β-D-Xylopyranosyl-D-glucose). Extracts of AQ-producing cells contained also free primverose that was probably formed during the extraction procedure by the action of hydrolytic enzymes. This hydrolytic capacity was also present in non-AQ-producing, 2,4-D grown Morinda cells. These non-producing cells were also used for feeding experiments in which the capacity of Morinda cells to process added AQ (aglucons and glycosides) was tested. Addition of the aglucon alizarin (in the presence of DMSO) resulted in uptake and glycosylation; extracts of these cells also contained primverose, indicating that the normal glycosylation machinery was also active in these non-producing cells. Addition of AQ glycosides (a mixture of alizarin-primveroside and lucidin-primveroside) also resulted in uptake and processing of the added metabolites. Successive hydrolysis and glycosylation reactions lead to cells which contain a mixture of AQ aglucons and AQ glycosides after a few days of incubation. Apparently the mechanism for hydrolysis and glycosylation of AQ is also present or rapidly induced in cells that do not produce these compounds themselves, indicating that glycosylation of AQ and subsequent storage in the vacuoles probably is not a crucial step in the regulation of the production of these compounds. The significance of the presence of this hydrolysis and glycosylation machinery for the handling of e.g. xenobiotics is discussed.

ABA reduces respiration and sugar metabolism in developing carrot (Daucus carota L.) embryoids.

Summary Addition of abscisic acid (ABA) to developing carrot embryoids affects respiration and carbohydrate metabolism. Non-treated embryoids have a high level of respiration expressed per gram protein and consume al most all of their endogenous carbohydrates during the ten day culture period. In contrast, embryoids grown with either 1.9 or 38 µM ABA, have a lowered respiration rate and maintain their carbohydrate contents at 20 % of the DW. Embryoids acquire complete desiccation tolerance, when they are treated with 38 µM ABA, whereas only 65 % of the embryoids survive desiccation with 1.9 µM ABA. The reduced respiration of the developing embryoids might result in lower free radical levels after dehydration, in this way preventing a subsequent viability loss. We suggest that there is a relation between viability loss due to desiccation and respiration rate, although the latter is not the only factor involved.

The triose-hexose phosphate cycle and the sucrose cycle in carrot (Daucus carota L.) cell suspensions are controlled by respiration and PPi: fructose-6-phosphate phosphotransferase.

Summary Short-term labelling was applied to two different lines of Daucus carota L. cell suspensions by feeding [1- 13 C]-glucose. The A10-line, containing 10 % proembryogenic masses (PEMs) and 90 % large, vacuolated cells (VACs), showed a 2 times higher label exchange from C-1 to C-6 carbons within sucrose and hexoses than the A+ -line, containing 80 % PEMs. This label exchange is known to be caused by cycling of carbon from hexose phosphates to triose phosphates and vice versa, in which ATP-dependent phosphofructokinase (PFK, EC 2.7.1.11) catalyses the glycolytic reaction and PP i -dependent phosphofructokinase (PFP, EC 2.7.1.90) the gluconeogenetic reaction. The ratio of extractable PFP/PFK was 3 times higher in the A10-line compared to the A+-line. However, PEMs and VACs from one line showed identical PFP/PFK ratios and identical label exchange. It is concluded that the level of PFP is genetically determined and that this level influences the amount of label exchange from C-1 to C-6 carbons in hexoses and sucrose in Daucus cells. High levels of the reversible enzyme PFP might give plants the advantage to respond adequately to quickly changing demands for substrates for either glycolytic or gluconeogenetic reactions. Both triose-hexose phosphate cycling and respiration were higher when suspensions were aerated with 100% O 2 instead of 6% O 2 . It is concluded that high respiratory activity stimulated both the flow of hexose phosphates into the respiratory pathway and the back-flow from triose to hexose phosphates. However, total labelled sucrose was at least two times higher at 6% O 2 than at 100% O 2 , indicating that more hexose phosphates were available for sucrose synthesis at 6% O 2 .