Phosphate Starvation Inducible Metabolism in Lycopersicon esculentum: I. Excretion of Acid Phosphatase by Tomato Plants and Suspension-Cultured Cells

Both tomato (Lycopersicon esculentum cv VF 36) plants and suspension cultured cells show phosphate starvation inducible (psi) excretion of acid phosphatase (Apase). Apase excretion in vitro was proportional to the level of exogenous orthophosphate (Pi). Intracellular Apase activity remained the same in both Pi-starved and sufficient cells, while Apase excreted by the starved cells increased by as much as six times over unstressed control cells on a dry weight basis. At peak induction, 50% of total Apase was excreted. Ten day old tomato seedlings grown without Pi showed slight growth reduction versus unstressed control plants. The Pi-depleted roots showed psi enhancement of Apase activity. Severely starved seedlings (17 days) reached only one-third of the biomass of unstressed control plants but, because of a combination of psi Apase excretion by roots and a shift in biomass to this organ, they excreted 5.5 times the Apase activity of the unstressed control. Observed psi Apase excretion may be part of a phosphate starvation rescue system in plants. The utility of the visible indicator dye 5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine as a phenotypic marker for plant Apase excretion is demonstrated.     

Phosphate Starvation Inducible Metabolism in Lycopersicon esculentum: III. Changes in Protein Secretion under Nutrient Stress

Phosphate starvation increased the secretion of at least six proteins by suspension cultured tomato (Lycopersicon esculentum L. and L. pennellii) cells. Cells exhibited a biphasic response to phosphate (Pi) starvation. The early phase involved enhanced secretion of three proteins in response to transfer to a Pi-depleted media, while biomass accumulation continued at the same rate as in the Pi-sufficient cells. Severe starvation, defined as inhibition of biomass accumulation, induced enhanced secretion of three additional proteins. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, media proteins were immunoblotted with antibodies reacting specifically to oligosaccharides processed by the Golgi apparatus. Binding patterns showed that the enhancement in secretion during both phases of starvation was Golgi-mediated. Cells undergoing severe starvation had a respiration rate approximately twice that of unstressed cells and secreted 4.4 times more protein into the media per unit biomass. These data suggest overlapping Pi starvation-specific and global stress responses in plant cells. Under these conditions, Golgi-mediated protein secretion is enhanced. We present evidence for phosphate starvation inducible enhancement of Pi uptake. Secreted proteins specific for N and Fe starvation are also identified.     

Effect of Phosphorus Nutrition on the Cellular Distribution of Acid Phosphatase in the Leaves of Lycopersicon esculentum L.

A histochemical study using light microscopy has been made ofthe distribution of acid phosphatase (EC 3.1.3.2 [EC] ) activity intransverse sections of fully expanded leaves of Lycopersiconesculentum grown in phosphate-deficient or sufficient media.Leaf tissues were prepared by two methods and were embeddedin paraffin wax. The location of acid phosphatase activity inleaf sections was determined by trapping orthophosphate releasedfrom p-nitrophenyl phosphate with lead acetate and subsequentlyconverting the lead phosphate to optically dense lead sulphide.In leaf sections from control tissue lead sulphide depositswere larpely confined to the spongy mesophyll cells. Whereasthe staining of the palisade cells was limited and of a granularnature, the staining of the spongy mesophyll cells was heavierand coincident with the outline of the individual cells. Moreover,the minor veins were more heavily stained than the surroundingmesophyll cells. Sections of phosphorus-deficient tissues wereheavily stained in both the palisade and spongy mesophyll layersand heavy deposits of lead sulphide were present in the regionsof the minor veins. It is suggested that the enhanced acid phosphataseactivity of the mesophyll cells in fully expanded leaves couldbe involved in the remobilization of phosphate within phosphorus-deficientplants, or be part of a phosphate transporting system, concentratingthe intracellular phosphate from the limiting supply in thesolution bathing the mesophyll cells. Lycopersicon esculentum L., tomato, acid phosphatase, phosphorus nutrition     

磷酸饥饿时番茄幼苗酸性磷酸酶活性的变化与Pi吸收的关系

磷酸饥饿时,番茄幼苗根部及地上部酸性磷酸酶活性均显著增强,根部细胞表面酸性磷酸酶及根部外泌的酸性磷酸酶活性亦明显提高。动力学分析表明,磷酸饥饿提高了番茄幼苗根部的酸性磷酸酶对其底物的亲和力。另外,磷酸饥饿对番茄幼苗根部酸性磷酸酶活性的最适ｐＨ值没有影响。钼酸对番茄幼苗根部酸性磷酸酶活性有强烈的抑制作用,对番茄幼苗Ｐｉ吸收速率也有十分明显的抑制效果。以上结果表明,磷酸饥饿时,番茄幼苗Ｐｉ吸收的适应性变化可能与根部酸性磷酸酶特别是根部细胞表面酸性磷酸酶及其外泌酸性磷酸酶的参与密切关联。     

Relationship between the Changes of Acid Phosphatase Activities and Pi -uptake of Tomato Seedlings during Phosphate Starvation

The acid phosphatase activities from roots and both stems and leaves of tomato seedlings all in-creased markedly under phosphate starvation. Phosphate starvation also increased the activities of acid phos-phatase from cell surface of, and released by roots of tomato seedlings. The kinetic analysis of acid phos-phatase of roots of tomato seedlings revealed that phosphate starvation increased the affinity of the enzyme to its substrate. The results also revealed that phosphate starvation had no effect on the optimum pH (pH 4.93) of the acid phosphatase of roots of tomato seedlings. It was also found that molybdate strongly inhibited not only the activities of acid phosphatase but also Pi- uptake rates of tomato seedlings.     

Regulation of Phosphate Metabolism in NEUROSPORA CRASSA: Identification of the Structural Gene for Repressible Acid Phosphatase

A mutant of Neurospora crassa with an altered repressible acid phosphatase has been isolated. The enzyme is much more thermolabile than that of wild type, and has an increased Michaelis constant. Tests of allelic interactions (in partial diploids) and in vitro mixing experiments were consistent with the mutation being in the structural gene for the enzyme. This gene, pho-3, was found to be located in the right arm of Linkage Group IV (LGIV). Thus, pho-3 and the structural gene for repressible alkaline phosphatase, pho-2 (LG V), map in separate linkage groups and cannot be part of the same operon. Neither of these structural genes is linked to the known regulatory genes, nuc-1 (LG I), nuc-2 (LG II), and preg (LG II).     

An Inborn Error of Potassium Metabolism in the Tomato, Lycopersicon esculentum

A mutant of the tomato (Lycopersicon esculentum Mill.) was obtained through treatment of the seed with ethyl methane sulfonate. Its chief distinguishing characteristic is the requirement for an extraordinarily high K concentration in the medium (20 mm) if it is to develop without pronounced K deficiency symptoms, while the wild type grows without any such symptoms at 0.1 to 0.2 mm K. The lesion of the K-inefficient mutant does not lie in its inability to absorb and translocate K.     

Anatomy and Phenylpropanoid Metabolism in the Incompatible Interaction of Lycopersicon esculentum and Cuscuta reflexa

A time-dependent correlation of anatomical and chemical defence reactions was shown during the incompatible reaction of tomato against the phanerogamic parasite Cuscuta reflexa. Microscopical analysis of the infection sites at the tomato stem revealed the elongation of epidermal, hypodermal and collenchymatic cells beneath the parasitic prehaustorium. After 9–11 days of infection the elongated cells had collapsed forming a visible brownish plaque at the tomato stem followed by a scalariform tissue with lignified and suberized cell walls. Concomitantly, an enhanced accumulation of soluble phenolic compounds (chlorogenic acid and an unidentified hydroxycinnamic acid derivative), as well as a stimulation of peroxidases, was observed. In contrast, PAL activity was not increased. Whereas the stimulation of phenylpropanoid metabolism could also be induced by artificial wounding, the described anatomical changes were only observed during attack of Cuscuta.     

Characterization of the subtilase gene family in tomato (Lycopersicon esculentum Mill.)

The gene family of subtilisin-like serine proteases (subtilases, SBTs) in tomato (Lycopersicon esculentum Mill.) comprises at least 15 members, 12 of which have been characterized in this study. Sequence comparison revealed that tomato subtilases fall into 5 distinct subfamilies. Single genes were shown to exist for LeSBT1, LeSBT2 and tmp, while 5 and 6 genes were found in the LeSBT3/4 and P69 subfamilies, respectively. With the exception of tmp, tomato subtilase genes were found to lack introns. Expression of subtilase genes was confirmed at the mRNA level by northern blot analysis and/or by primer extension experiments. For each of the 5 subtilase subfamilies, a distinctive pattern of expression was observed in tomato organs. At least one of the subtilases was found to be expressed in each organ analysed. Structural features evident from deduced amino acid sequences are discussed with reference to the related mammalian proprotein convertases.     

Long-Term Phosphate Starvation and Respiratory Metabolism in Suspension-Cultured Catharanthus roseus Cells

In order to clarify the metabolic adaptation of respiratorypathways in plants to limited levels of P_i, the effects of long-termstarvation of P_i on the activities of various enzymes relatedto respiratory metabolism were examined in suspension-culturedCatharanthus roseus cells. When the activities were expressedas units per g fresh weight, only those of phosphoenolpyruvate-hydrolyzing(PEP-hydrolyzing) enzyme (which may possibly be equivalent tothe acid phosphatase activity derived from vacuoles) and PEPcarboxylase were higher in the Pⁱ-starved cells than in controlcells. Activities of other enzymes in the Pⁱ-starved cells werelower than or similar to those of the control cells. Time-coursestudies indicated that PEP-hydrolyzing activity was inducibleby starvation of Pⁱ. However, in contrast to the results reportedby Duff et al. [(1989a) Plant Physiol. 90: 1275.], fluctuationsin the activity of PP¹:fructose-6-phosphate 1-phosphotransferaseduring starvation of Pⁱ were similar to those in levels of phosphofructokinaseand 6-phosphogluconate dehydrogenase. These data suggest thatthe concept of the phosphate starvation-inducible ‘bypasses’,which are engineered via the coarse control (i.e., induction)of specified enzymes and were proposed initially by Duff etal. in Brassica nigra cells, is not directly applicable to Catharanthusroseus cells in suspension. Tracer experiments using [U-¹⁴C]glutamineindicated that a significant proportion of respiratory substratescould be supplied from the enlarged pool of amino acids duringstarvation of Pⁱ. These assumptions are supported by the observedfluctuations in levels of free amino acids and of protein inP¹-fed and P¹-deficient Catharanthus roseus cells. ¹Part 41 in the series ‘Metabolic Regulation in PlantCell Cultrue’ ²Present Address: Morinaga Mild Industry, 5-1-83, Higashihara,Zamma-shi, Kanagawa, 228 Japan     

Metabolism of Indole-3-Acetic Acid by Pericarp Discs from Immature and Mature Tomato (Lycopersicon esculentum Mill)

[1′-¹⁴C, ¹³C₆]Indole-3-acetic acid was infiltrated into immature pericarp discs from fruits of tomato (Lycopersicon esculentum Mill., cv Moneymaker). After a 24-h incubation period the discs were extracted with methanol and the partially purified extract was analyzed by reversed-phase high-performance liquid chromatography-radiocounting. Five metabolite peaks (1-5) were detected and subsequently analyzed by combined high-performance liquid chromatography-frit-fast atom bombardment-mass spectrometry. The metabolite 4 fraction was found to contain [¹³C₆]-indole-3-acetylaspartic acid, and analysis of metabolite 5 identified [¹³C₆]indole-3-acetyl-β-d-glucose. The other metabolites could not be identified, but alkaline hydrolysis studies and gel permeation chromatography indicated that metabolites 1 and 3 were both amide conjugates with a molecular weight of approximately 600. Studies with radiolabeled indole-3-acetic acid, indole-3-acetylaspartic acid, and indole-3-acetyl-β-d-glucose demonstrated that in immature pericarp indole-3-acetic acid is deactivated primarily via metabolism to indole-3-acetylaspartic acid, which is further converted to metabolites 1, 2, and 3. In mature, pink pericarp discs, indole-3-acetic acid is converted more extensively to its glucosyl conjugate. Conjugation of indole-3-acetic acid to indole-3-acetylaspartic acid appears to be dependent upon protein synthesis because it is inhibited by cycloheximide. In contrast, cycloheximide has little effect on the further conversion of indole-3-acetylaspartic acid to metabolites 1, 2, and 3.     

Interaction of Auxin-TIBA in Lycopersicon esculentum

[5-³H] indoleacetic acid (IAA) was applied to the apical budof young tomato plants (Lycopersicon esculentum Mill.) treatedor not with 2,3,5-triiodobenzoic acid (TIBA). Chromatographicanalysis showed that treatment with TIBA increased the quantityof breakdown products in the apical 1.5 mm segment of the stem.Autoradiographs of ultra-thin sections of this segment wereprepared after treatment with glutaraldehyde. The inhibitionof polar auxin transport by TIBA caused an increase in the densityof labelling of all the cellular types in the shoot apex. TheTIBA treatment provoked a modification of the structure of theshoot apex in which the greatest increases in density of labellingwere detected in the superficial tunical layer and in the pithmeristem. The increase in radioactivity of the superficial tunicallayer could be explained by the presence of polyphenols in thevacuoles. For the pith meristem, the TIBA treatment intensifiedthe high labelling previously observed at this level in thecontrol plants. This could indicate a role for auxin in cellularelongation. It could also point to the importance of the pithmeristem in the functioning of the shoot apex. Key words: [³H] IAA, Lycopersicon esculentum, TIBA     

Properties and Activity Changes of Chlorogenic Acid:Glucaric Acid Caffeoyltransferase From Tomato (Lycopersicon esculentum)

A novel acyltransferase from cotyledons of tomato (Lycopersicon esculentum Mill.), which catalyzes the transfer of caffeic acid from chlorogenic acid (5-O-caffeoylquinic acid) to glucaric and galactaric acids, was purified with a 2400-fold enrichment and a 4% recovery. The enzyme showed specific activities (theoretical V_max per milligram of protein) of 625 nanokatals (caffeoylglucaric acid formation) and 310 nanokatals (caffeoylgalactaric acid formation). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis it gave an apparent M_r of 40,000, identical to the value obtained by gel filtration column chromatography. Highest activity was found at pH 5.7, which was constant over a range of 20 to 120 millimolar K-phosphate. The isoelectric point of the enzyme was at pH 5.75. The reaction temperature optimum was at 38°C and the apparent energy of activation was calculated to be 57 kilojoules per mole. The apparent K_m values were 0.4 millimolar for glucaric acid, 1.7 millimolar for galactaric acid, and with both acceptors as second substrates 20 millimolar for chlorogenic acid. The relative ratio of the V_max/K_m values for glucaric acid and galactaric acid was found to be 100:12. Substrate-competition experiments support the conclusion that one single enzyme is responsible for both the glucaric and galactaric acid ester formation with marked preference for glucaric acid. It is proposed that the enzyme be called chlorogenic acid:glucaric acid O-caffeoyltransferase (EC 2.3.1.-). The three caffeic acid-dependent enzyme activities involved in the formation of the glucaric and galactaric acid esters, the chlorogenic acid:glucaric acid caffeoyltransferase as the key activity as well as the caffeic acid:CoA ligase and the caffeoyl-CoA:quinic acid caffeoyltransferase as the preceding activities, were determined. The time course of changes in these activities were followed during development of the seedling in the cotyledons and growth of the young plant in the first and second leaf. The results from tomato seedlings suggest a sequential appearance of these enzymes.     

Characterization of Two Inducible Phosphate Transport Systems in Rhizobium tropici

Rhizobium tropici forms nitrogen-fixing nodules on the roots of the common bean (Phaseolus vulgaris). Like other legume-Rhizobium symbioses, the bean-R. tropici association is sensitive to the availability of phosphate (P_i). To better understand phosphorus movement between the bacteroid and the host plant, P_i transport was characterized in R. tropici. We observed two P_i transport systems, a high-affinity system and a low-affinity system. To facilitate the study of these transport systems, a Tn5B22 transposon mutant lacking expression of the high-affinity transport system was isolated and used to characterize the low-affinity transport system in the absence of the high-affinity system. The K_m and V_max values for the low-affinity system were estimated to be 34 ± 3 μM P_i and 118 ± 8 nmol of P_i · min⁻¹ · mg (dry weight) of cells⁻¹, respectively, and the K_m and V_max values for the high-affinity system were 0.45 ± 0.01 μM P_i and 86 ± 5 nmol of P_i · min⁻¹ · mg (dry weight) of cells⁻¹, respectively. Both systems were inducible by P_i starvation and were also shock sensitive, which indicated that there was a periplasmic binding-protein component. Neither transport system appeared to be sensitive to the proton motive force dissipator carbonyl cyanide m-chlorophenylhydrazone, but P_i transport through both systems was eliminated by the ATPase inhibitor N,N′-dicyclohexylcarbodiimide; the P_i transport rate was correlated with the intracellular ATP concentration. Also, P_i movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the wild-type strain or the mutant. These properties suggest that both P_i transport systems are ABC type systems. Analysis of the transposon insertion site revealed that the interrupted gene exhibited a high level of homology with kdpE, which in several bacteria encodes a cytoplasmic response regulator that governs responses to low potassium contents and/or changes in medium osmolarity.     

Sucrose Phosphate Synthase, Sucrose Synthase, and Invertase Activities in Developing Fruit of Lycopersicon esculentum Mill. and the Sucrose Accumulating Lycopersicon hirsutum Humb. and Bonpl

The green-fruited Lycopersicon hirsutum Humb. and Bonpl. accumulated sucrose to concentrations of about 118 micromoles per gram fresh weight during the final stages of development. In comparison, Lycopersicon esculentum Mill. cultivars contained less than 15 micromoles per gram fresh weight of sucrose at the ripe stage. Glucose and fructose levels remained relatively constant throughout development in L. hirsutum at 22 to 50 micromoles per gram fresh weight each. Starch content was low even at early stages of development, and declined further with development. Soluble acid invertase (EC 3.2. 1.26) activity declined concomitant with the rise in sucrose content. Acid invertase activity, which was solubilized in 1 molar NaCl (presumably cell-wall bound), remained constant throughout development (about 3 micromoles of reducing sugars (per gram fresh weight) per hour. Sucrose phosphate synthase (EC 2.4.1.14) activity was present at about 5 micromoles of sucrose (per gram fresh weight) per hour even at early stages of development, and increased sharply to about 40 micromoles of sucrose (per gram fresh weight) per hour at the final stages of development studied, parallel to the rise in sucrose content. In comparison, sucrose phosphate synthase activity in L. esculentum remained low throughout development. The possible roles of the sucrose metabolizing enzymes in determining sucrose accumulation are discussed.     

番茄离体培养过程中器官发生的细胞组织学观察

对番茄下胚轴、子叶、茎段、叶片、叶柄不同类型外植体离体培养中有关细胞启动、分裂、分化以及器官发生作了细胞组织学观察。研究结果表明番茄不同类型外植体在同样的培养条件下,愈伤组织生长表现出明显差异,其中下胚轴、子叶诱导产生愈伤组织时,细胞启动最早,生长最快,其分裂方式基本为无丝分裂,未见有丝分裂,因此我们认为以不定芽方式获得转基因植株时,植株的所有性状变化,是否纯属目的基因所为,应该反复考察,不能忽视不定芽产生过程中的种种变化;下胚轴诱导愈伤组织形成时,细胞不规则的无丝分裂少于子叶,故下胚轴离体培养得到的正常芽的比例高于子叶的;番茄离体培养中不定芽通常发生在愈伤组织的周边区,也可起源于维管组织结节周围的形成层状细胞。不定根则由茎中柱鞘处发生。     

Induced androgenesis in tomato (Lycopersicon esculentum Mill). II. Factors affecting induction of androgenesis

The influence on androgenesis of donor plant growth conditions, anther size and developmental stage of the microspore, medium composition and different anther treatments prior to culture was investigated in L. esculentum Mill. cv Roma and its hybrids. Growth conditions of donor plants affected the induction of tomato androgenesis. Anthers isolated from plants grown in the greenhouse during winter at high humidity and in short days possessed higher androgenetic ability than those grown in the field. The physiological state and age of the donor plants also influenced the processes investigated. Regarding the developmental stage of microspores, the period from prophase to telophase II is optimal for tomato anther implantation. More then 20 culture media were tested. Two, based on Murashige and Skoog medium were selected as most favourable for callus induction, organogenesis and regeneration. The effect on callus induction of 2ip in combination with indole-3-acetic acid (IAA) was greater than that of zeatin and IAA. Zeatin promoted entire plant regeneration. A highly significant interaction between genotype and medium was observed. Temperature and gamma ray treatments of anthers enhanced callus production, shoot formation and plant regeneration. Treatments at 4 °C (48 h) and 10 °C (9 days) stimulated these processes. Combined treatment of anthers with 4 Gy and 10 °C for 9 days was the most efficient. Received: 5 September 1997 / Revision recieved: 5 June 1998 / Accepted: 15 June 1998     

乙酰水杨酸在番茄果实发育期间对蔗糖代谢相关酶活性的影响

研究不同浓度乙酰水杨酸(ASA)对番茄品种‘辽园多丽’果实发育期间蔗糖代谢相关酶影响的结果表明:ASA可抑制果实的维管束和胶质胎座中酸性转化酶(AI)和中性转化酶(NI)活性,而提高蔗糖合成酶(SS)与蔗糖磷酸合成酶(SPS)活性;心室隔壁和中果肉中ASA的作用与此相反。ASA促进果实维管束中可溶性糖积累主要通过调控AI和NI活性实现,而在胶质胎座中主要通过调控SS活性实现;在中果肉和心室隔壁中主要通过调控SS和AI活性实现。