Desensitization of the Perception System for Chitin Fragments in Tomato Cells

Suspension-cultured tomato (Lycopersicon esculentum) cells react to stimulation by chitin fragments with a rapid, transient alkalinization of the growth medium, but behave refractory to a second treatment with the same stimulus (G. Felix, M. Regenass, T. Boller [1993] Plant J 4: 307–316). We analyzed this phenomenon and found that chitin fragments caused desensitization in a time- and concentration-dependent manner. Partially desensitized cells exhibited a clear shift toward lower sensitivity of the perception system. The ability of chitin oligomers to induce desensitization depended on the degree of polymerization (DP), with DP5 ≈ DP4 ≫ DP3 ≫ DP2 > DP1. This correlates with the ability of these oligomers to induce the alkalinization response and to compete for the high-affinity binding site on tomato cells and microsomal membranes, indicating that the alkalinization response and the desensitization process are mediated by the same receptor. The dose required for half-maximal desensitization was about 20 times lower than the dose required for half-maximal alkalinization; desensitization could therefore be used as a highly sensitive bioassay for chitin fragments and chitin-related stimuli such as lipochitooligosaccharides (nodulation factors) from Rhizobium leguminosarum. Desensitization was not associated with increased inactivation of the stimulus or with a disappearance of high-affinity binding sites from the cell surface, and thus appears to be caused by an intermediate step in signal transduction.

Wound Signaling in Tomato Plants1 : Evidence That ABA Is Not a Primary Signal for Defense Gene Activation

The effects of abscisic acid (ABA) on the accumulation of proteinase inhibitors I (Inh I) and II (Inh II) in young, excised tomato (Lycopersicon esculentum L.) plants were investigated. When supplied to excised plants through the cut stems, 100 μm ABA induced the activation of the ABA-responsive le4 gene. However, under the same conditions of assay, ABA at concentrations of up to 100 μm induced only low levels of proteinase-inhibitor proteins or mRNAs, compared with levels induced by systemin or jasmonic acid over the 24 h following treatment. In addition, ABA only weakly induced the accumulation of mRNAs of several other wound-response proteins. Assays of the ABA concentrations in leaves following wounding indicated that the ABA levels increased preferentially near the wound site, suggesting that ABA may have accumulated because of desiccation. The evidence suggests that ABA is not a component of the wound-inducible signal transduction pathway leading to defense gene activation but is likely involved in the general maintenance of a healthy plant physiology that facilitates a normal wound response.

Effects of Xylem pH on Transpiration from Wild-Type and flacca Tomato Leaves1 : A Vital Role for Abscisic Acid in Preventing Excessive Water Loss Even from Well-Watered Plants

The pH of xylem sap from tomato (Lycopersicon esculentum) plants increased from pH 5.0 to 8.0 as the soil dried. Detached wild-type but not flacca leaves exhibited reduced transpiration rates when the artificial xylem sap (AS) pH was increased. When a well-watered concentration of abscisic acid (0.03 μm) was provided in the AS, the wild-type transpirational response to pH was restored to flacca leaves. Transpiration from flacca but not from wild-type leaves actually increased in some cases when the pH of the AS was increased from 6.75 to 7.75, demonstrating an absolute requirement for abscisic acid in preventing stomatal opening and excessive water loss from plants growing in many different environments.

Tomato Fructokinases Exhibit Differential Expression and Substrate Regulation1

Two divergent genes encoding fructokinase, Frk1 and Frk2, have been previously shown to be expressed in tomato (Lycopersicon esculentum L.) and have now been further characterized with regard to their spatial expression and the enzymic properties of the encoded proteins. Frk1 and Frk2 mRNA levels were coordinately induced by exogenous sugar, indicating that both belong to the growing class of sugar-regulated genes. However, in situ hybridization indicated that Frk1 and Frk2 were expressed in a spatially distinct manner, with Frk2 mRNA primarily localized in cells of the fruit pericarp, which store starch, and Frk1 mRNA distributed ubiquitously in pericarp tissue. To evaluate the biochemical characteristics of the products of the Frk1 and Frk2 genes, each cDNA was expressed in a mutant yeast (Saccharomyces cerevisiae) line defective in hexose phosphorylation and unable to grow on glucose or fructose (Fru). Both Frk1 and Frk2 proteins expressed in yeast conferred the ability to grow on Fru and exhibited fructokinase activity in vitro. Although both Frk1 and Frk2 both utilized Fru as a substrate, only Frk2 activity was inhibited at high Fru concentrations. These results indicate that Frk2 can be distinguished from Frk1 by its sensitivity to substrate inhibition and by its temporal and spatial pattern of expression, which suggests that it plays a primary role in plant cells specialized for starch storage.

Tomato Flower Abnormalities Induced by Low Temperatures Are Associated with Changes of Expression of MADS-Box Genes1

Flower and fruit development in tomato (Lycopersicon esculentum Mill.) were severely affected when plants were grown at low temperatures, displaying homeotic and meristic transformations and alterations in the fusion pattern of the organs. Most of these homeotic transformations modified the identity of stamens and carpels, giving rise to intermediate organs. Complete homeotic transformations were rarely found and always affected organs of the reproductive whorls. Meristic transformations were also commonly observed in the reproductive whorls, which developed with an excessive number of organs. Scanning electron microscopy revealed that meristic transformations take place very early in the development of the flower and are related to a significant increase in the floral meristem size. However, homeotic transformations should occur later during the development of the organ primordia. Steady-state levels of transcripts corresponding to tomato MADS-box genes TM4, TM5, TM6, and TAG1 were greatly increased by low temperatures and could be related to these flower abnormalities. Moreover, in situ hybridization analyses showed that low temperatures also altered the stage-specific expression of TM4.

Evidence for 1-(Malonylamino)cyclopropane-1-Carboxylic Acid Being the Major Conjugate of Aminocyclopropane-1-Carboxylic Acid in Tomato Fruit1

Tomato (Lycopersicon esculentum Miller) fruit discs fed with [2,3-14C]1-aminocyclopropane-1-carboxylic acid (ACC) formed 1-malonyl-ACC (MACC) as the major conjugate of ACC in fruit throughout all ripening stages, from immature-green through the red-ripe stage. Another conjugate of ACC, γ-glutamyl-ACC (GACC), was formed only in mature-green fruit in an amount about 10% of that of MACC; conjugation of ACC into GACC was not detected in fruits at other ripening stages. No GACC formation was observed from etiolated mung bean (Vigna radiata [L.] Wilczek) hypocotyls, etiolated common vetch (Vicia sativum L.) epicotyls, or pea (Pisum sativum L.) root tips, etiolated epicotyls, and green stem tissue, where active conversion of ACC into MACC was observed. GACC was, however, formed in vitro in extracts from fruit of all ripening stages. GACC formation in an extract from red fruit at pH 7.15 was only about 3% of that at pH 8.0, the pH at which most assays were run. Our present in vivo data support the previous contention that MACC is the major conjugate of ACC in plant tissues, whereas GACC is a minor, if any, conjugate of ACC. Thus, our data do not support the proposal that GACC formation could be more important than MACC formation in tomato fruit.

Regulation of Tomato Fruit Polygalacturonase mRNA Accumulation by Ethylene: A Re-Examination1

Polygalacturonase (PG) is the major enzyme responsible for pectin disassembly in ripening fruit. Despite extensive research on the factors regulating PG gene expression in fruit, there is conflicting evidence regarding the role of ethylene in mediating its expression. Transgenic tomato (Lycopersicon esculentum) fruits in which endogenous ethylene production was suppressed by the expression of an antisense 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene were used to re-examine the role of ethylene in regulating the accumulation of PG mRNA, enzyme activity, and protein during fruit ripening. Treatment of transgenic antisense ACC synthase mature green fruit with ethylene at concentrations as low as 0.1 to 1 μL/L for 24 h induced PG mRNA accumulation, and this accumulation was higher at concentrations of ethylene up to 100 μL/L. Neither PG enzyme activity nor PG protein accumulated during this 24-h period of ethylene treatment, indicating that translation lags at least 24 h behind the accumulation of PG mRNA, even at high ethylene concentrations. When examined at concentrations of 10 μL/L, PG mRNA accumulated within 6 h of ethylene treatment, indicating that the PG gene responds rapidly to ethylene. Treatment of transgenic tomato fruit with a low level of ethylene (0.1 μL/L) for up to 6 d induced levels of PG mRNA, enzyme activity, and protein after 6 d, which were comparable to levels observed in ripening wild-type fruit. A similar level of internal ethylene (0.15 μL/L) was measured in transgenic antisense ACC synthase fruit that were held for 28 d after harvest. In these fruit PG mRNA, enzyme activity, and protein were detected. Collectively, these results suggest that PG mRNA accumulation is ethylene regulated, and that the low threshold levels of ethylene required to promote PG mRNA accumulation may be exceeded, even in transgenic antisense ACC synthase tomato fruit.

Cloning of a Tobacco Apoplasmic Invertase Inhibitor1 : Proof of Function of the Recombinant Protein and Expression Analysis during Plant Development

Higher plants express several isoforms of vacuolar and cell wall invertases (CWI), some of which are inactivated by inhibitory proteins at certain stages of plant development. We have purified an apoplasmic inhibitor (INH) of tobacco (Nicotiana tabacum) CWI to homogeneity. Based on sequences from tryptic fragments, we have isolated a full-length INH-encoding cDNA clone (Nt-inh1) via a reverse transcriptase-polymerase chain reaction. Southern-blot analysis revealed that INH is encoded by a single- or low-copy gene. Comparison with expressed sequence tag clones from Arabidopsis thaliana and Citrus unshiu indicated the presence of Nt-inh1-related proteins in other plants. The recombinant Nt-inh1-encoded protein inhibits CWI from tobacco and Chenopodium rubrum suspension-cultured cells and vacuolar invertase from tomato (Lycopersicon esculentum) fruit, whereas yeast invertase is not affected. However, only in the homologous system is the inhibition modulated by the concentration of Suc as previously shown for INH isolated from tobacco cells. Highly specific binding of INH to CWI could be shown by affinity chromatography of a total cell wall protein fraction on immobilized recombinant Nt-inh1 protein. RNA-blot analysis of relative transcript ratios for Nt-inh1 and CWI in different parts of adult tobacco plants revealed that the expression of both proteins is not always coordinate.

ADP-Glucose Pyrophosphorylase Is Localized to Both the Cytoplasm and Plastids in Developing Pericarp of Tomato Fruit1

The intracellular location of ADP-glucose pyrophosphorylase (AGP) in developing pericarp of tomato (Lycopersicon esculentum Mill) has been investigated by immunolocalization. With the use of a highly specific anti-tomato fruit AGP antibody, the enzyme was localized in cytoplasm as well as plastids at both the light and electron microscope levels. The immunogold particles in plastids were localized in the stroma and at the surface of the starch granule, whereas those in the cytoplasm occurred in cluster-like patterns. Contrary to the fruit, the labeling in tomato leaf cells occurred exclusively in the chloroplasts. These data demonstrate that AGP is localized to both the cytoplasm and plastids in developing pericarp cells of tomato.

Polypeptide signaling for plant defensive genes exhibits analogies to defense signaling in animals.

The activation of plant defensive genes in leaves of tomato plants in response to herbivore damage or mechanical wounding is mediated by a mobile 18-amino acid polypeptide signal called systemin. Systemin is derived from a larger, 200-amino acid precursor called prosystemin, similar to polypeptide hormones and soluble growth factors in animals. Systemin activates a lipid-based signaling cascade, also analogous to signaling systems found in animals. In plants, linolenic acid is released from membranes and is converted to the oxylipins phytodienoic acid and jasmonic acid through the octadecanoid pathway. Plant oxylipins are structural analogs of animal prostaglandins which are derived from arachidonic acid in response to various signals, including polypeptide factors. Constitutive overexpression of the prosystemin gene in transgenic tomato plants resulted in the overproduction of prosystemin and the abnormal release of systemin, conferring a constitutive overproduction of several systemic wound-response proteins (SWRPs). The data indicate that systemin is a master signal for defense against attacking herbivores. The same defensive proteins induced by wounding are synthesized in response to oligosaccharide elicitors that are generated in leaf cells in response to pathogen attacks. Inhibitors of the octadecanoid pathway, and a mutation that interrupts this pathway, block the induction of SWRPs by wounding, systemin, and oligosaccharide elicitors, indicating that the octadecanoid pathway is essential for the activation of defense genes by all of these signals. The tomato mutant line that is functionally deficient in the octadecanoid pathway is highly susceptible to attacks by Manduca sexta larvae. The similarities between the defense signaling pathway in tomato leaves and those of the defense signaling pathways of macrophages and mast cells of animals suggests that both the plant and animal pathways may have evolved from a common ancestral origin.

Plant virus DNA replication processes in Agrobacterium: insight into the origins of geminiviruses?

Agrobacterium tumefaciens, a bacterial plant pathogen, when transformed with plasmid constructs containing greater than unit length DNA of tomato leaf curl geminivirus accumulates viral replicative form DNAs indistinguishable from those produced in infected plants. The accumulation of the viral DNA species depends on the presence of two origins of replication in the DNA constructs and is drastically reduced by introducing mutations into the viral replication-associated protein (Rep or C1) ORF, indicating that an active viral replication process is occurring in the bacterial cell. The accumulation of these viral DNA species is not affected by mutations or deletions in the other viral open reading frames. The observation that geminivirus DNA replication functions are supported by the bacterial cellular machinery provides evidence for the theory that these circular single-stranded DNA viruses have evolved from prokaryotic episomal replicons.

The N gene of tobacco confers resistance to tobacco mosaic virus in transgenic tomato.

It has been proposed that cloned plant disease resistance genes could be transferred from resistant to susceptible plant species to control important crop plant diseases. The recently cloned N gene of tobacco confers resistance to the viral pathogen, tobacco mosaic virus. We generated transgenic tomato plants bearing the N gene and demonstrate that N confers a hypersensitive response and effectively localizes tobacco mosaic virus to sites of inoculation in transgenic tomato, as it does in tobacco. The ability to reconstruct the N-mediated resistance response to tobacco mosaic virus in tomato demonstrates the utility of using isolated resistance genes to protect crop plants from diseases, and it demonstrates that all the components necessary for N-mediated resistance are conserved in tomato.

Preferential expression of an ammonium transporter and of two putative nitrate transporters in root hairs of tomato.

Root hairs as specialized epidermal cells represent part of the outermost interface between a plant and its soil environment. They make up to 70% of the root surface and, therefore, are likely to contribute significantly to nutrient uptake. To study uptake systems for mineral nitrogen, three genes homologous to Arabidopsis nitrate and ammonium transporters (AtNrt1 and AtAmt1) were isolated from a root hair-specific tomato cDNA library. Accumulation of LeNrt1-1, LeNrt1-2, and LeAmt1 transcripts was root-specific, with no detectable transcripts in stems or leaves. Expression was root cell type-specific and regulated by nitrogen availability. LeNrt1-2 mRNA accumulation was restricted to root hairs that had been exposed to nitrate. In contrast, LeNrt1-1 transcripts were detected in root hairs as well as other root tissues under all nitrogen treatments applied. Analogous to LeNrt1-1, the gene LeAmt1 was expressed under all nitrogen conditions tested, and root hair-specific mRNA accumulation was highest following exposure to ammonium. Expression of LeAMT1 in an ammonium uptake-deficient yeast strain restored growth on low ammonium medium, confirming its involvement in ammonium transport. Root hair specificity and characteristics of substrate regulation suggest an important role of the three genes in uptake of mineral nitrogen.

Primary structure and expression of a pathogen-induced protease (PR-P69) in tomato plants: Similarity of functional domains to subtilisin-like endoproteases.

A 69-kDa proteinase (P69), a member of the pathogenesis-related proteins, is induced and accumulates in tomato (Lycopersicon esculentum) plants as a consequence of pathogen attack. We have used the polymerase chain reaction to identify and clone a cDNA from tomato plants that represent the pathogenesis-related P69 proteinase. The nucleotide sequence analysis revealed that P69 is synthesized in a preproenzyme form, a 745-amino acid polypeptide with a 22-amino acid signal peptide, a 92-amino acid propolypeptide, and a 631-amino acid mature polypeptide. Within the mature region the most salient feature was the presence of domains homologous to the subtilisin serine protease family. The amino acid sequences surrounding Asp-146, His-203, and Ser-532 of P69 are closely related to the catalytic sites (catalytic triad) of the subtilisin-like proteases. Northern blot analysis revealed that the 2.4-kb P69 mRNA accumulates abundantly in leaves and stem tissues from viroid-infected plants, whereas the mRNA levels in tissues from healthy plants were undetectable. Our results indicate that P69, a secreted calcium-activated endopeptidase, is a plant pathogenesis-related subtilisin-like proteinase that may collaborate with other defensive proteins in a general mechanism of active defense against attacking pathogens.

Rep protein of tomato yellow leaf curl geminivirus has an ATPase activity required for viral DNA replication.

The Rep protein of geminiviruses is the sole viral protein required for their DNA replication. The amino acid sequence of Rep protein contains an NTP binding consensus motif (P-loop). Here we show that purified Rep protein of tomato yellow leaf curl virus expressed in Escherichia coli exhibits an ATPase activity in vitro. Amino acid exchanges in the P-loop sequence of Rep causes a substantial decrease or loss of the ATPase activity. In vivo, mutant viruses carrying these Rep mutations do not replicate in plant cells. These results show that ATP binding by the Rep protein of geminiviruses is required for its function in viral DNA replication.