Isolation of a mRNA encoding a nucleoside diphosphate kinase from tomato that is up-regulated by wounding

A cDNA clone (TAB2) encoding a nucleoside diphosphate (NDP) kinase has been isolated from a tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) cDNA library. The clone is 590 bp long and exhibits a high degree of sequence identity with spinach NDP kinases I and II, Pisum sativum NDP kinase I, Arabidopsis thaliana NDP kinase, Drosophila melanogaster NDP kinase, Dictyostelium discoideum NDP kinase and human Nm 23-H1 and Nm23-H2. Northern analysis has revealed that the mRNA encoded by TAB2 is up-regulated in both leaf and stem tissue in response to wounding. The increase is apparent within 1 h of wounding and is not further elevated by application of ethylene. Southern blot analysis indicates that TAB2 is a member of a small gene family.     

Isolation of two cDNA clones from tomato containing two different superoxide dismutase sequences

A cDNA library was derived from the poly(A)⁺ RNA of young tomato leaves. The library was cloned in a gt11 system and screened by synthetic oligonucleotide probes having sequences that match the codes of conserved regions of amino acid sequences of Cu,Zn superoxide dismutase (SOD) proteins from a wide range of eukaryotic organisms. Two cDNAs were isolated, cloned and sequenced. One of the cDNAs, P31, had a full-size open reading frame of 456 bp with a deduced amino acid sequence having an 80% homology with the deduced amino acid sequence of the cytosolic SOD-2 cDNA of maize. The other cDNA, T10 (extended by T1), had a 651 bp open reading frame that revealed, upon computer translation, 90% homology to the amino acid sequence of mature spinach chloroplast SOD. The 5 end of the reading frame seems to code for a putative transit peptide. This work thus suggests for the first time an amino acid sequence for the transit peptide of chloroplast SOD. Northern hybridizations indicated that each of the P31 and T10 clones hybridized to a blotted poly(A)⁺ RNA species. These two species are differentially expressed in the plant organs: e.g., the species having the T10 sequence was detected in the leaves but not in roots, while the one with the P31 sequence was expressed in both leaves and roots. The cDNA clones P31 and T10 were also hybridized to Southern blots of endonuclease fragmented tomato DNA. The clones hybridized to specific fragments and no cross hybridization between the two clones was revealed under stringent hybridization conditions; the hybridization pattern indicated that, most probably, only one locus is coding for each of the two mRNA species.     

Characterization of a cDNA encoding the protein moiety of a putative arabinogalactan protein from Lycopersicon esculentum

The nucleotide sequence of a cDNA prepared from poly(A)⁺ RNA from Lycopersicon esculentum fruit codes for a protein, M _r 20812, with features representative of the protein core of arabinogalactan proteins. The deduced amino acid sequence resembles that of peptides of arabinogalactan proteins isolated from carrot and rose and is most similar to the sequence of tryptic peptides from Lolium multiflorum (Gleeson et al., Biochem J 264 (1989) 857–862). The similar sequences include a number of Ala-Pro repeats, a feature considered distinctive of arabinogalactan proteins. The amino acid composition is similar to that of the peptide core of the Lolium multiflorum arabinogalactan protein; alanine, serine and proline account for 57% of the polypeptide. The mRNA corresponding to the cDNA sequence was detected in roots, leaves and fruit. The levels of mRNA are reduced in older leaves, in fruit that have commenced ripening and in leaves and fruit that have been wounded.     

Isolation of milligram quantities of nuclear DNA from tomato (Lycopersicon esculentum), A plant containing high levels of polyphenolic compounds

We have developed a protocol for isolating milligram quantities of highly purified DNA from tomato nuclei. The protocol utilizes fresh seedlings or leaves without freezing. Tissues are treated with ethyl ether, thoroughly washed, and placed in a buffer containing the nuclear-stabilizing agent 2-methyl-1,4-pentanediol. Nuclei are liberated from tomato cells by homogenization in a Waring blender. The interaction of nuclear DNA with oxidized polyphenols is inhibited by compounds that adsorb polyphenols or prevent oxidation reactions. Chloroplasts and mitochondria are preferentially eliminated with Triton X-100. Nuclei are concentrated using a Percoll gradient and lysed with SDS. DNA is subsequently purified by RNase and protease digestions and phenol/chloroform extractions. The isolated DNA is essentially free of polyphenols and other major contaminants based upon its lack of coloration, A₂₆₀/A₂₈₀ ratio, digestibility with restriction enzymes, melting profile, and reassociation properties.     

An improvement of tomato protoplast culture for rapid plant regeneration

Tomato mesophyll protoplasts were cultured in TM2 medium containing 5.7 M -naphthaleneacetic acid and 2.4 M benzyladenine and were incubated either in stationary culture or on an orbital shaker at 25–30 strokes per min, in combination with interval addition of fresh medium. The effects of stationary and shaking conditions on the growth of the colonies and their subsequent shoot organogenesis were significantly different. The cultures maintained in stationary condition without adding fresh medium accumulated a thin membranous layer on the medium surface and whitish substance in the medium that seemed to precede cell browning and premature colony death. Mild shaking conditions along with the reduction of colony density by one half by dividing the contents of one dish into two dishes, after adding 2 ml of fresh medium on the 4th day and further addition of fresh medium (0.5 ml) on the 8th day of plating, provided optimal conditions for colony growth and suppressed thin layer and whitish substance accumulation. Ten-day-old colonies raised through this protocol regenerated shoots rapidly (within 19–20 days after initial plating) after transfer to regeneration medium (MS medium with 2.8 M zeatin riboside, 0.06–0.1 M gibberellic acid, 4% sucrose and 1% type VII agarose) directly bypassing the callus phase.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IAA indoleacetic acid - MS Murashige & Skoog (1962) medium - NAA -naphthaleneacetic acid - SPM stroke per min - GLM General Linear Models - 2,4-D 2,4-dichlorophenoxyacetic acid     

Isolation of a 6.2 kb genomic fragment carrying the Adh1 gene of tomato and its expression in transgenic tobacco

An 11 kb Eco RI genomic fragment containing the alcohol dehydrogenase (Adh1) gene was cloned. Cross-hybridization with three Adh2 cDNA clones suggested that the entire coding region of the Adh1 gene was contained on a 6.2 kb Xba I/Hind III subfragment. Using RFLP linkage analysis, the genomic clone was mapped on chromosome 4 between the markers TG 182 and TG 65 in a position corresponding to the Adh1 locus. To further confirm the Adh1 origin of the genomic clone, tobacco plants were transformed with the 6.2 kb Xba I/Hinb III genomic subfragment. Isozyme analysis demonstrated that in transgenic tobacco plants functional tomato specific ADH-1 homodimers were synthesized as well as heterodimers composed of tobacco and tomato subunits.     

Subcellular localization of chorismate-mutase isoenzymes in protoplasts from mesophyll and suspension-cultured cells of Nicotiana silvestris

The subcellular locations of two readily discriminated chorismate-mutase (EC 5.4.99.5) isoenzymes from Nicotiana silvestris Speg. et Comes were determined in protoplasts prepared from both leaf tissue and isogenic suspension-cultured cells. Differential centrifugation was used to obtain fractions containing plastids, a mixture of mitochondria and microbodies, and soluble cytosolic proteins. Isoenzyme CM-1 is sensitive to feedback inhibition by l-tyrosine and comprises the major fraction of total chorismate mutase in suspension-cultured cells. Isoenzyme CM-2 is not inhibited by l-tyrosine and its expression is maximal in organismal (leaf) tissue. Isoenzyme CM-1 is located in the plastid compartment since (i) proplastids contained more CM-1 activity than chloroplasts, (ii) both chloroplast and proplastid fractions possessed the tyrosine-sensitive isoenzyme, and (iii) latency determinations on washed chloroplast preparations confirmed the internal location of a tyrosine-sensitive isoenzyme. Isoenzyme CM-2 is located in the cytosol since (i) the supernatant fractions were heavily enriched for the tyrosineinsensitive activity, and (ii) a relatively greater amount of tyrosine-insensitive enzyme was present in the supernatant fraction derived from organismal tissue.     

The distribution and utilization of calcium by two tomato (Lycopersicon esculentum Mill.) lines differing in calcium efficiency when grown under low-Ca stress

Two lines of tomato (Lycopersicon esculentum Mill.) representing extremes in utilization effciency of absorbed Ca were studied to detect internal differences in Ca transport and distribution and factors responsible for strain differences in susceptibility to low Ca-stress. Differences in efficiency of Ca use were expressed as CaER (mg of dry weight produced for each mg of Ca absorbed by the plant).Ca-efficiency in line 113(E) appeared to be associated with a slow continuous movement of absorbed Ca, allowing for continued growth of the shoot apex and upper lamina under Ca-deficiency conditions. In the inefficient line 67(I), in contrast, Ca was rapidly deposited in the lower leaves with little upward movement in the plant after absorption.Fractionation of tissue Ca into various chemical forms suggested that Ca inefficiency also was associated with higher concentrations of insoluble Ca in the shoot tissue. The efficient line, although sustaining growth at lower levels of Ca, was capable of maintaining a higher ratio of soluble to insoluble Ca in all shoot tissues.Calcium was concentrated in the lower plant tissues of the inefficient strain, limiting its availability for continued shoot growth.Autoradiographs of lines fed⁴⁵Ca during the final 8 days of a 24-day experiment suggested that upward movement was sustained in line 113(E), in spite of vastly reduced transpiration rates and a root system characterized by leakage of K ions from the roots back into the solution.     

Transient increase in locular pressure and occlusion of endocarpic apertures in ripening tomato fruit

Locular pressure was monitored during ripening of tomato (Lycopersicon esculentum Mill.) fruit and the anatomy of the endocarp surface examined using scanning electron microscopy. The manometric pressure of the locule tissue increased from 0 in mature-green fruit to 10 to 50 Pa at the turning or pink stages, and then subsided in ripe fruit. Nonclimacteric fruit containing the ripening inhibitor (rin) mutation showed a similar pattern of internal pressure accumulation during senescence. Build-up of locular tissue pressure occurred in fruit ripening, on or off the plant, as well as in fruit with different susceptibility to cuticle cracking. Apertures ranging from 18-31 μm in width and 33-41 μm in length, with densities ranging from 6.7 to 47.9 apertures · mm⁻² were observed in the endocarp of mature-green fruit. These apertures were progressively occluded during early ripening and were absent in late ripening fruit. Aperture occlusion might result in reduced gas exchange between the locule and external fruit atmosphere, resulting in modification of the locular gas composition.     

cDNA structure and regulatory properties of a family of starvation-induced ribonucleases from tomato

In previous work we have determined the primary structure of two of the five ribonucleases which are induced by phosphate starvation in cultured tomato cells. Here, we present the isolation and characterization of the cDNAs for the extracellular ribonuclease LE and the intracellular, but extravacuolar ribonuclease LX. Structural analysis of these cDNAs together with partial protein-sequencing of vacuolar ribonucleases LV1, LV2 and LV3 revealed a family of very similar ribonucleases. From these data we assume identity between ribonucleases LE and LV3 for which the targeting mechanism has to be shown. Furthermore, RNase LV1 and RNase LV2 might be posttranslational processing products of RNase LX which travel to the vacuoles after splitting off the putative ER retention signal present at RNase LX. Additionally, we show by northern blot analysis that phosphate starvation in plant cells leads to an increase in the steady-state level of this type of enzymes revealing close similarities of the plant response to a limited supply of inorganic phosphate with the PHO regulation in bacteria and fungi.     

Localization of proton-ATPase genes expressed in arbuscular mycorrhizal tomato plants

In arbuscular mycorrhizal symbioses, solutes such as phosphate are transferred to the plant in return for photoassimilates. The uptake mechanism is probably facilitated by a proton gradient generated by proton H⁺-ATPases. We investigated expression of Lycopersicon esculentum Mill. H⁺-ATPases in mycorrhizal and non-mycorrhizal plants to determine if any are specifically regulated in response to colonization. Tissue expression and cellular localization of H⁺-ATPases were determined by RNA gel blot analysis and in situ hybridization of mycorrhizal and non-mycorrhizal roots. LHA1, LHA2, and LHA4 had high levels of expression in roots and were expressed predominantly in epidermal cells. LHA1 and LHA4 were also expressed in cortical cells containing arbuscules. The presence of arbuscules in root sections was correlated with lower levels of expression of these two isoforms in the epidermis. These results suggest that LHA1 and LHA4 expression is decreased in epidermal cells located in regions of the root that contain arbuscules. This provides evidence of differential regulation between molecular mechanisms involved in proton-coupled nutrient transfer either from the soil or fungus to the plant.     

Effect of diazocyclopentadiene on tomato ripening

Diazocyclopentadiene (DACP) in the presence of fluorescent light delayed ripening of tomato fruits treated at the mature green (no visible red) stage. At 25 °C, ripening was delayed 10 days if DACP [185 µl/1 (gas)] was added as a single treatment and longer if DACP was added intermittently at 5-day intervals. The addition of 1000 µl/1 ethylene following DACP and light treatment did not hasten ripening. Little ripening delay was noted for fruit + DACP held in darkness. Tomatoes covered with aluminum foil so as to exclude light but not light-activated DACP, showed ripening inhibition. Apparently, the light-activated product from DACP is stable long enough to diffuse into fruit held in darkness. After an initial inhibition, ethylene production was greatly increased in tomatoes treated with DACP. Tomatoes with or without DACP treatment were held either in air or 5% O₂/95% N₂ for 12 days then treated with ethylene. Treatment with 5% O₂ alone delayed ripening when compared to air alone, however, both groups reached 80% red color by 18 days. DACP treated fruit, whether held in air or 5% O₂, still were green after 18 days and only approached 80% red color after approximately 27 days. Thus, 5% oxygen did not appear to slow the reversal of DACP inhibition of ripening.     

Cloning and analysis of a defender against apoptotic cell death (DAD1) homologue from tomato

A cDNA clone homologous to the human defender against apoptotic cell death (DAD1) gene, which is believed to be a conserved inhibitor of programmed cell death, was isolated from tomato (Lycopersicon esculentum cv. Prisca). The 351 basepairs open reading frame predicted a 116 amino acid protein sequence (LeDAD1) that showed high homology to other DAD1 proteins. Northern analysis revealed that LeDAD1 was constitutively expressed during ripening of wildtype, rin,andNr tomato fruit.     

Structural analysis of a 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase with an N-terminal chorismate mutase-like regulatory domain

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) catalyzes the first step in the biosynthesis of a number of aromatic metabolites. Likely because this reaction is situated at a pivotal biosynthetic gateway, several DAHPS classes distinguished by distinct mechanisms of allosteric regulation have independently evolved. One class of DAHPSs contains a regulatory domain with sequence homology to chorismate mutase-an enzyme further downstream of DAHPS that catalyzes the first committed step in tyrosine/phenylalanine biosynthesis-and is inhibited by chorismate mutase substrate (chorismate) and product (prephenate). Described in this work, structures of the Listeria monocytogenes chorismate/prephenate regulated DAHPS in complex with Mn(2+) and Mn(2+) + phosphoenolpyruvate reveal an unusual quaternary architecture: DAHPS domains assemble as a tetramer, from either side of which chorismate mutase-like (CML) regulatory domains asymmetrically emerge to form a pair of dimers. This domain organization suggests that chorismate/prephenate binding promotes a stable interaction between the discrete regulatory and catalytic domains and supports a mechanism of allosteric inhibition similar to tyrosine/phenylalanine control of a related DAHPS class. We argue that the structural similarity of chorismate mutase enzyme and CML regulatory domain provides a unique opportunity for the design of a multitarget antibacterial.