Changes in Tomato Leaves Induced by NaCl Stress: Leaf Organization and Cell Ultrastructure

The alterations of organization of leaf tissues and cell ultrastructure as a consequence of salt stress (75 and 150 mM NaCl) were studied in two tomato (Lycopersicum esculentum Mill.) cultivars showing different salinity tolerance. The salinity brought changes in cell shape, volume of intercellular spaces and chloroplast number, shape and size. These characteristics were specific in each cultivar. The ultrastructural changes were also different in the two tomato cultivars studied and the most important ones were in the number and size of starch granules in chloroplasts, the number of electron-dense corpuscules in the cytoplasm, the structure of mitochondria, and number of plastoglobuli. This revised version was published online in July 2006 with corrections to the Cover Date.     

Heat Stress and Spermidine: Effect on Chlorophyll Fluorescence in Tomato Plants

Two tomato (Lycopersicon esculentum L.) cultivars: Robin (tolerant) and Roma (sensitive to heat stress) were studied. Chlorophyll fluorescence induction parameters (F_v/F_p, A_max, and Rfd) at 25 °C showed that the PS2 activity was similar for both cultivars. The parameters, measured at 38 °C, decreased in both cultivars, but more in cv. Roma. Exogenous application of 4 mM spermidine improved the plant heat-resistance in both cultivars, and especially in cv. Roma. Analysis of chlorophyll fluorescence changes during linear increase in temperature showed that cv. Robin plants have higher ability to hardening and higher resistance to thermal damage of the pigment-protein complexes structure and the activity of PS2 than cv. Roma.     

低钾胁迫对番茄叶片活性氧及抗氧化酶系的影响

以2种不同低钾耐性大果番茄(钾敏感型番茄081018和耐低钾型番茄081034)为材料,比较低钾处理下2种番茄叶片中活性氧产生及抗氧化酶系活性和相关基因表达差异,明确植物叶片对低钾胁迫的响应机制.结果显示:(1)钾敏感型番茄在低钾胁迫时,叶片中各种保护酶(SOD及其同工酶、POD、CAT、APX)活性随处理时间延长呈下降趋势,同时活性氧(O2-、H2O2)和MDA含量急剧增加;耐低钾型番茄在低钾胁迫条件下,其各类保护酶活性均比对照水平有所升高,而且O2-、H2O2和MDA的含量增加也较少.(2)钾敏感型番茄在低钾胁迫时叶片内Cu/Zn-SOD、CAT和APX基因的相对表达量均有下降趋势,而同期耐低钾型番茄在低钾胁迫时Cu/Zn-SOD、CAT和APX的表达却明显增加,这与其对应的酶活性变化趋势同步.研究表明,低钾胁迫使耐低钾型番茄具有较高保护酶基因表达量,产生较高的保护酶活性,可降低活性氧的破坏作用,防止膜渗透性增加,使之对低钾的适应性较强,而钾敏感番茄品系则相反.     

湖南安息香属植物的叶片比较解剖学研究

利用光学显微镜和扫描电镜对湖南产安息香属10种植物叶表皮微形态和解剖结构进行了观察和测量。结果表明:(1)表皮均由单层细胞构成,细胞形状为多边形和无规则形,垂周壁式样为平直、弓形、波状;(2)气孔器大小及密度在种间的差异明显;(3)气孔外拱盖内缘主要为波状和浅波状,气孔器外围角质纹饰以越南安息香的鸟巢状和栓叶安息香的碗状为显著区别特征;(4)老鸹铃、垂珠花和白花龙的气孔器保卫细胞两极有"T"形加厚;(5)栓叶安息香和大果安息香叶上表皮由大型细胞组成;(6)叶肉分化为栅栏组织和海绵组织,二者的厚度及比值在种间有明显的差异,其中越南安息香栅栏组织分化为2层;(7)大果安息香和芬芳安息香的主脉维管组织构成一圈封闭的环状结构,而其他种则为开放的半圆形结构;(8)叶片及主脉的厚度在种间差异明显。安息香属植物的表皮微形态及解剖结构特征比较稳定,可作为种间鉴定和分类的重要依据。     

Foliar Spraying with Zineb Increases Fruit Productivity and Alleviates Oxidative Stress in Two Tomato Cultivars

The effects of foliar spraying of the dithiocarbamate zineb on two cultivars of tomato grown in the field in a site with high ozone concentrations were studied by means of biomass assessment, antioxidant enzyme assays, lipid peroxidation, and chlorophyll fluorescence measurements. Zineb prevented the peroxidation of membrane lipids and decreased the activity of scavenging enzymes, which suggests that plants sprayed with zineb are subjected to lower oxidative stress than controls. The beneficial effects of zineb protection is the utilization of a larger fraction of absorbed radiant energy in photosynthesis and a larger fruit yield in plants of both cultivars.     

Tomato genome is comprised largely of fast-evolving, low copy-number sequences

Summary Fifty random clones (350–2300 bp), derived from sheared, nuclear DNA, were studied via Southern analysis in order to make deductions about the organization and evolution of the tomato genome. Thirty-four of the clones were mapped genetically and determined to represent points on 11 of the 12 tomato chromosomes. Under moderate stringency conditions (80% homology required) 44% of the clones were classified as single copy. Under higher stringency, the majority of the clones (78%) behaved as single copy. Most of the remaining clones belonged to multicopy families containing 2–20 copies, while a few contained moderately or highly repeated sequences (10% at moderate stringency, 4% at high stringency). Divergence rates of sequences homologous to the 50 random genomic clones were compared with those corresponding to 20 previously described cDNA (coding sequence) clones. Rates were measured by probing each clone (random genomics and cDNAs) onto filters containing DNA from various species from the family Solanaceae (including potato, Datura, petunia and tobacco) as well as one species (watermelon) from another plant family, Cucurbitaceae. Under moderate stringency conditions, the majority of the random clones (single copy and repetitive) failed to detect homologous sequences in the more distantly related species, whereas approximately 90% of the 20 coding sequences analyzed could still be detected in all solanaceous species. The most highly repeated sequences appear to be the fastest evolving and homologous copies could be detected only in species most closely related to tomato. Dispersion of repetitive sequences, as opposed to tandem clustering, appears to be the rule for the tomato genome. None of the repetitive sequences discovered by this random sampling of the genome were tandemly arranged — a finding consistent with the notion that the tomato genome contains only a small fraction of satellite DNA. This study, along with a companion paper (Ganal et al. 1988), provides the first general sketch of the tomato genome at the molecular level and indicates that it is comprised largely of single copy sequences and these sequences, together with repetitive sequences are evolving at a rate faster than the coding portion of the genome. The small genome and paucity of highly repetitive DNA are favourable attributes with respect to the possibilities of conducting chromosome walking experiments in tomato and the fact that coding regions are well conserved among solanaceous species may be useful for distinguishing clones that contain coding regions from those that do not.     

Comparison of Two Methods for Preparing Mitochondria from Tomato Leaves to Study the Ascorbate-Glutathione Cycle Activity

Mitochondria were purified from tomato leaves by a differential centrifugation method and a Percoll density-gradient method. A comparative study on the ascorbate-glutathione cycle activity revealed that there was no difference in the specific activity of the antioxidative enzymes and ascorbate and glutathione concentrations between mitochondria prepared by these two methods. However, the contents of dehydroascorbic acid and oxidised glutathione in Percoll-separated organelles were 260 and 210 %, respectively, of those found in mitochondria purified by the differential centrifugation method.     

Antioxidant Systems in Ripening Tomato Fruits

Two cultivars of tomato (Lycopersicon esculentum Mill.), Selection-7 (shelf life 7–8 d) and ARTH-3 (shelf life 14–15 d) were analyzed for oxidative stress and the antioxidant enzyme system at different stages of fruit ripening. The results presented here suggest that during the early stages of fruit ripening, efficient antioxidant system protects the tomato fruits against the damaging effect of progressive oxidative stress. At later stages, however, oxidative damage occurs due to decreased activities of the ROS scavenging enzymes.     

Effect of Glycinebetaine on Chloroplast Ultrastructure, Chlorophyll and Protein Content, and RuBPCO Activities in Tomato Grown under Drought or Salinity

The effect of foliar-applied glycinebetaine (GB) on chloroplast ultrastructure, the amount of chlorophyll and proteins and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) activity in stressed tomato leaves were analysed. Initial, and total activity and activation state state of RuBPCO were also measured. RuBPCO activities, chloroplast area, and the number of plastoglobuli remained unaffected, while the relative area of starch granules increased in GB-treated, salt-stressed tomato leaflets. Under drought-stress, the relative area of plastoglobuli increased upon GB application. The primary effect of GB was, however, the increased protein and chlorophyll content.     

Expression of Poplar Chitinase in Tomato Leads to Inhibition of Development in Colorado Potato Beetle

The previously described poplar chitinase, WIN6, is induced during infestation by gypsy moth (Lymantria dispar L.) larvae, thus suggesting a role in defense against insect pests. To test this hypothesis, we produced tomato seedlings infected with a recombinant potato virus X (PVX), which produces WIN6, and tested its insecticidal properties on Colorado potato beetle [CPB; Leptinotarsa decemlineata (Say)], which is a serious pest of tomatoes and other crops. The advantage of PVX is that plant material is ready for insect bioassay within 3–4 weeks of constructing the recombinant virus. Considering that production of transgenic tomato seedlings using Agrobacterium takes at least 6 months, this hastens the rate at which genes can be examined. Upon insect bioassay, only 47% CPB neonates feeding on leaves containing >0.3% w/w WIN6 developed to 2nd instar while 93% of controls reached 2nd instar. To our knowledge this is the first plant chitinase that retards development of an insect pest. Revisions requested 12 December 2005; Revisions received 18 January 2006     

Application of Alcohol Dehydrogenase Loci in Testing F1 Hybridity of Tomato

Expression of alcohol dehydrogenase loci was used to estimate hybridity of Lycopersicon esculentum Mill. in 1012 seeds. The banding patterns were obtained by means of vertical block electrophoresis in polyacrylamide gels. It was established that qualitative variation of locus Adh-2 can be applied to prove hybridity of F₁ tomato seeds. This genetic marker is indicative for hybrids with fertile maternal line or with position male sterility line and not only for maternal line with pollen male sterility. This revised version was published online in July 2006 with corrections to the Cover Date.     

Changes in ripening-related processes in tomato conditioned by the alc mutant

Summary The alc mutation affects the ripening and storability of tomato fruit. The alteration of fruit color in alc lines is due to a reduction in total pigment and a reduction in lycopene relative to total carotinoids. Polygalacturonase (PG) activity is reduced to less than 5% of normal, and the isozymes PG2a and PG2b are absent in alc fruit. The level of anti-PG precipitable proteins is also reduced to less than 5% of normal. Total polyA + mRNA is not significantly reduced in ripening alc fruit, but hybridization of polyA + mRNA to different ripening-related cDNA clones showed that specific mRNAs are present at reduced levels in the mutant. Specific mRNA levels were reduced to 10%–80% of normal levels, depending on the cDNA clone used as the probe. PG mRNA was present at 5%–10% of the normal level.All effects of alc on fruit ripening are relived in the line Alcobaca-red, which arose spontaneously from the original alc line, Alcobaca. The Alcobaca-red trait segregates as a single dominant trait at or very near the alc locus, and it is probably the result of a reverse mutation at the alc locus.The chromosomal locations of regions homologous to 5 ripening-related cDNA probes were determined. Regions homologous to 4 of these probes map to chromosomes other than chromosome 10, indicating that the effects of alc are transactive. A cDNA clone for PG was homologous to only one chromosomal region. This region is located on chromosome 10, which is also the chromosome on which alc and nor are located.     

Tomato yellow leaf curl virus DNA in callus cultures derived from infected tomato leaves

Callus cultures were induced from leaves of a tomato plant infected with tomato yellow leaf curl virus (TYLCV) and analyzed for viral DNA presence during successive subcultures. No TYLCV DNA was detected in calli sampled after eight months of culture. Considerable differences in the presence of TYLCV DNA were found within sectors of a callus culture and between different callus cultures, throughout the entire eight months period. Infected calli which were cultured at sub-optimal temperature (15°C) retained the viral DNA longer than at 25 °C. The results suggested that TYLCV disappearance during callus culture was due to a disruption of some of the cell-to-cell connections, resulting in islands of infected cells in the midst of uninfected tissue and/or to the competition between the rate of cell division and that of viral DNA replication.Abbreviations BA benzyladenine - CMV cucumber mosaic virus - NAA naphthaleneacetic acid - TMV tobacco mosaic virus - TYLCV tomato yellow leaf curl virus     

Effect of Short-Term Salinity on Lipid Metabolism and Ion Accumulation in Tomato Roots

To examine the ion accumulation and membrane lipid metabolism in response to salinity we compared two tomato cvs. Pera and Hellfrucht Fruhstamm (HF), considered to be salt-tolerant and sensitive respectively. Na⁺ and K⁺ accumulation was significantly higher in roots of cv. Pera after 24 h and 72 h of 100 mM NaCl. While in cv. HF, a temporary increase in K⁺ accumulation at 24 h was accompanied by a sustained increase in Na⁺ content. Both cultivars enhanced incorporation of [³²P]orthophosphate into phosphatidylinositol 4,5-bisphosphate at 24 h and 72 h of NaCl. In parallel to the increase of phosphatidylinositol 4,5-bisphosphate a decrease in phosphorylation of phosphatidic acid and phosphatidylcholine were observed in the sensitive cv. HF. Structural and signal lipid changes in response to salinity were more evident in the sensitive cv. HF. Salt tolerant cv. Pera accumulated Na⁺ ions in the roots without considerable modifications in lipid metabolism. This revised version was published online in July 2006 with corrections to the Cover Date.     

Changes in Mulberry Leaf Metabolism in Response to Water Stress

A series of experiments were conducted to characterize the water stress-induced changes in the activities of RuBP carboxylase (RuBPCO) and sucrose phosphate synthase (SPS), photosystem 2 activity, and contents of chlorophylls, carotenoids, starch, sucrose, amino acids, free proline, proteins and nucleic acids in mulberry (Morus alba L. cv. K-2) leaves. Water stress progressively reduced the activities of RuBPCO and SPS in the leaf extracts, the chlorophyll content, and PS2 activity in isolated chloroplasts. Plants exposed to drought showed lower content of starch and sucrose but higher total sugar content than control plants. While the soluble protein content decreased under water stress, the amino acid content increased. Proline accumulation (2.5-fold) was noticed in stressed leaves. A reduction in the contents of DNA and RNA was observed. Reduced nitrogen content was associated with the reduction in nitrate reductase activity. SDS-PAGE protein profile showed few additional proteins (78 and 92 kDa) in the water stressed plants compared to control plants.     

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.     

Characterization of the level,target sites and inheritance of cytosine methylation in tomato nuclear DNA

The tomato nuclear genome was determined to have a G+C content of 37% which is among the lowest reported for any plant species. Non-coding regions have a G+C content even lower (32% average) whereas coding regions are considerably richer in G+C (46%).5-methyl cytosine was the only modified base detected and on average 23% of the cytosine residues are methylated. Immature tissues and protoplasts have significantly lower levels of cytosine methylation (average 20%) than mature tissues (average 25%). Mature pollen has an intermediate level of methylation (22%). Seeds gave the highest value (27%), suggesting de novo methylation after pollination and during seed development.Based on isoschizomer studies we estimate 55% of the CpG target sites (detected by Msp I/Hpa II) and 85% of the CpNpG target sites (detected by Bst NI/Eco RI)are methylated. Unmethylated target sites (both CpG and CpNpG) are not randomly distributed throughout the genome, but frequently occur in clusters. These clusters resemble CpG islands recently reported in maize and tobacco.The low G+C content and high levels of cytosine methylation in tomato may be due to previous transitions of 5mCT. This is supported by the fact that G+C levels are lowest in non-coding portions of the genome in which selection is relaxed and thus transitions are more likely to be tolerated. This hypothesis is also supported by the general deficiency of methylation target sites in the tomato genome, especially in non-coding regions.Using methylation isoschizomers and RFLP analysis we have also determined that polymorphism between plants, for cytosine methylation at allelic sites, is common in tomato. Comparing DNA from two tomato species, 20% of the polymorphisms detected by Bst NI/Eco RII could be attributed to differential methylation at the CpNpG target sites. With Msp I/Hpa II, 50% of the polymorphisms were attributable to methylation (CpG and CpNpG sites). Moreover, these polymorphisms were demonstrated to be inherited in a mendelian fashion and to co-segregate with the methylation target site and thus do not represent variation for transacting factors that might be involved in methylation of DNA. The potential role of heritable methylation polymorphism in evolution of gene regulation and in RFLP studies is discussed.     

Efficiency and Stability of High Molecular Weight DNA Transformation: an Analysis in Tomato

The efficiency of the binary bacterial artificial chromosome (BIBAC) vector for Agrobacterium-mediated stable transfer of high molecular weight DNA into plants was tested in tomato. Several variables affecting transformation efficiency were examined including insert size, Agrobacterium genetic background, and the presence of additional copies of the virG, virE1 and virE2 genes. It was found that a helper plasmid containing extra copies of virG was an absolute requirement for obtaining tomato transformants with the BIBAC. MOG101 with the virG helper plasmid was found to be the most efficient strain for transfer of high molecular weight DNA (150kb). Selected high molecular weight DNA transformants were advanced several generations (up to the R4) to assess T-DNA stability. This analysis showed that the T-DNA was stably maintained and inherited through several meioses regardless of whether it was in the hemizygous or homozygous state. Expression of a selectable marker gene within the T-DNA was also examined through several generations and no gene silencing was observed. Thus, the BIBAC is a useful system for transfer of large DNA fragments into the plant genome.     

Influence of Brassinosteroids on Antioxidant Enzymes Activity in Tomato Under Different Temperatures

The effect of brassinosteroids (BRs) on catalase (EC 1.11.1.6), peroxidase (EC 1.11.1.7) and superoxide dismutase (SOD, EC 1.15.1.1) activity in tomato leaf discs was analyzed at 25 and 40 °C. Tomato leaf discs were preincubated for 24 h in Petri dishes with 24-epibrassinolide (EBR) or a polyhydroxylated spirostanic analogue of brassinosteroids (MH5). Both concentrations (10.60 and 2.12 nM) of EBR and MH5 stimulated the activity of SOD at 25 and 40 °C, the MH5-stimulated increase of this enzyme activity was greater. Peroxidase activity was unaffected at 25 °C, while at 40 °C this activity was enhanced by both compounds. The changes in catalase activity markedly depended on the structure BRs, doses and temperature. The results suggest a possible role of EBR and MH5 in the reduction of cell damage produced by heat stress due to induction of enzymatic antioxidants.