Cloning and DNA-binding properties of ethylene response factor,LeERF1 and LeERF2, in tomato

Two new genes, LeERF1 andLeERF2, were isolated from a tomato (Lycopersicon esculentum cv. Lichun) cDNA library. Phylogenetic analysis indicated that they encoded Ethylene Responsive Element Binding Proteins (EREBPs), characterized by a conserved ERF (ethylene response factor) domain of specific binding plant cis-acting elements GCC box. Both LeERF1 and LeERF2 proteins were obtained via prokaryotic expression and purification. Electrophoretic mobility shift assay showed that LeERF1 and LeERF2 protein could bind to the promoter of the NP24 gene coding for pathogenesis-related protein osmotin precursor but not the mutant promoter where its GCC box was deleted. Polyclonal antibodies of LeERF1 and LeERF2 blocked their binding in vitro.Revisions requested 4 January 2005; Revisions received 28 January 2005     

Metabolomic analysis of tomato seed germination

Introduction

Seed germination is inherently related to seed metabolism, which changes throughout its maturation, desiccation and germination processes. The metabolite content of a seed and its ability to germinate are determined by underlying genetic architecture and environmental effects during development.

Objective

This study aimed to assess an integrative approach to explore genetics modulating seed metabolism in different developmental stages and the link between seed metabolic- and germination traits.

Methods

We have utilized gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS) metabolite profiling to characterize tomato seeds during dry and imbibed stages. We describe, for the first time in tomato, the use of a so-called generalized genetical genomics (GGG) model to study the interaction between genetics, environment and seed metabolism using 100 tomato recombinant inbred lines (RILs) derived from a cross between Solanum lycopersicum and Solanum pimpinellifolium.

Results

QTLs were found for over two-thirds of the metabolites within several QTL hotspots. The transition from dry to 6 h imbibed seeds was associated with programmed metabolic switches. Significant correlations varied among individual metabolites and the obtained clusters were significantly enriched for metabolites involved in specific biochemical pathways.

Conclusions

Extensive genetic variation in metabolite abundance was uncovered. Numerous identified genetic regions that coordinate groups of metabolites were detected and these will contain plausible candidate genes. The combined analysis of germination phenotypes and metabolite profiles provides a strong indication for the hypothesis that metabolic composition is related to germination phenotypes and thus to seed performance.

     

Phytochrome A-mediated inhibition of seed germination in tomato

Far-red light (FR) inhibition of seed germination of tomato (Solanum lycopersicum L.) was studied with the phytochrome (phy)-hypersensitive mutants, hp-1w, hp-1w,fri1, a phyA-deficient double mutant, and hp-1w,tri1, a phyB1-deficient double mutant. Seeds of all mutants germinated readily in the dark at 25 degrees C, and the germination was retarded by a single 100-s FR pulse given 1-3 h after sowing. The effect of an FR pulse was red-light reversible in all mutants used. After 24 h where a single FR pulse was no longer effective, prolonged FR exposure or hourly FR pulses suppressed germination in hp-1w and hp-1w,tri1, whereas in hp-1w,fri1 the suppressive effect of FR was almost absent. The effect of the prolonged FR was greater than that of the hourly 3-min FR pulses having equal photon fluence, and was fluencerate dependent. Thus we conclude that the germination inhibition by FR in tomato seed consists of a low-fluence response and a high irradiance response (HIR); the latter is controlled by phyA, but not phyB1. This is the first indication of phyA being involved in the HIR of seed germination inhibition.     

AtNEK6 interacts with ARIA and is involved in ABA response during seed germination

ARIA is an ARM repeat protein that is a positive regulator of ABA response. To identify ARIA-interacting proteins, we conducted yeast two-hybrid screening. One of the positive clones obtained from the screen encoded a protein kinase, AtNEK6, which belongs to the NIMA (Never In Mitosis, gene A)-related kinase family. We analyzed AtNEK6 over-expression (OX) and knockout (KO) lines to investigate its in vivo function. The AtNEK6 OX lines grew slowly, whereas the KO line germinated and grew faster than wild type plants. AtNEK6 also affected ABA and stress responses. During seed germination, AtNEK6 OX lines were hypersensitive to ABA and high osmolarity, whereas its KO line was partially insensitive to ABA and high osmolarity. Previously, AtNEK6 was shown to be involved in epidermal cell morphogenesis. Our results indicate that AtNEK6 is also involved in plant growth regulation and responses to ABA and high osmolarity during the seed germination stage.     

Hormonal regulation of tomato seed germination at a supraoptimal temperature

Germination of tomato cv. New Yorker seed is inhibited at 35°C. This thermoinhibition was partially counteracted by application of GA₄₊₇ alone, the compound applied in combination with ACC or ethephon markedly enhancing the process. The latter compound alone was not able to induce germination at 35 °C. Thermoinhibition of seeds at 35 °C was also counteracted by fluridone, an inhibitor of ABA biosynthesis. At 25 °C, an optimal temperature, ABA inhibited germination of New Yorker seeds. Although another known growth inhibitor MeJA, when applied at an optimal temperature (25 °C), had also a slightly inhibitory effect on germination of those seeds and clearly delayed the process, inhibitors of its biosynthetic pathway (ibuprofen, indoprofen, antypiryne and salicylic acid) did not remove thermoinhibition at 35 °C. An increase in endo-β-mannanase activity after 24 hours of incubation at 35 °C was observed in the seeds incubated in the presence of gibberellins, ACC, ethephon, fluridone used alone and in combinations, but it was not clearly correlated with the effects of these compounds on alleviation of seed germination. However, fluridone present in the same incubation medium at 35 °C with ABA was able to counteract the inhibitory effect of ABA on endo-β-mannanase activity. The results of our study suggest that gibberellins, ethylene (produced from ACC or ethephon) and ABA, but not jasmonates, regulate tomato seed germination at supraoptimal temperatures. Alleviation of thermoinhibition of New Yorker seed germination by plant growth regulators and fluridone is partially associated with their controlling endo-β-mannanase activity.     

Vacuolar H(+)-ATPase is expressed in response to gibberellin during tomato seed germination

Completion of germination (radicle emergence) by gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill.) seeds is dependent upon exogenous GA, because weakening of the endosperm tissue enclosing the radicle tip requires GA. To investigate genes that may be involved in endosperm weakening or embryo growth, differential cDNA display was used to identify mRNAs differentially expressed in gib-1 seeds imbibed in the presence or absence of GA(4+7). Among these was a GA-responsive mRNA encoding the 16-kD hydrophobic subunit c of the V(0) membrane sector of vacuolar H(+)-translocating ATPases (V-ATPase), which we termed LVA-P1. LVA-P1 mRNA expression in gib-1 seeds was dependent on GA and was particularly abundant in the micropylar region prior to radicle emergence. Both GA dependence and tissue localization of LVA-P1 mRNA expression were confirmed directly in individual gib-1 seeds using tissue printing. LVA-P1 mRNA was also expressed in wild-type seeds during development and germination, independent of exogenous GA. Specific antisera detected protein subunits A and B of the cytoplasmic V(1) sector of the V-ATPase holoenzyme complex in gib-1 seeds only in the presence of GA, and expression was localized to the micropylar region. The results suggest that V-ATPase plays a role in GA-regulated germination of tomato seeds.     

Stimulation of lettuce seed germination by ethylene

Ethylene increased the germination of freshly imbibed lettuce (Lactuca sativa L. var. Grand Rapids) seeds. Seeds receiving either red or far-red light or darkness all showed a positive response to the gas. However, ethylene was apparently without effect on dormant seeds, those which failed to germinate after an initial red or far-red treatment. Carbon dioxide, which often acts as a competitive inhibitor of ethylene, failed to clearly reverse ethylene-enhanced seed germination. While light doubled ethylene production from the lettuce seeds, its effect was not mediated by the phytochrome system since both red and far-red light had a similar effect.     

The AtTudor2, a protein with SN-Tudor domains, is involved in control of seed germination in Arabidopsis

The 4SN-Tudor domain protein is an almost ubiquitous eukaryotic protein with four Staphylococcal nuclease domains at the N terminus and a Tudor domain towards the C terminus. It has been found that Tudor-SN protein has multiple roles in governing gene expression during cell growth and development in animals. In plant, although Tudor-SN orthologs have been found in rice, pea and Arabidopsis, and are associated with cytoskeleton, their roles in growth and development are poorly understood. In this study, we investigated the function of Arabidopsis Tudor-SN protein, AtTudor. Our results indicated that the expression of AtTudor2 in seeds was evidently higher than in other tissues. Furthermore, we found that the expression of a key enzyme for GA biosynthesis, AtGA20ox3, was downregulated obviously in AtTudor2 T-DNA insertion mutant and AtTudor1/AtTudor2 RNAi transgenic lines. Together, our results suggest that AtTudor2 is involved in GA biosynthesis and seed germination of Arabidopsis.     

Low temperature seed germination of several tomato genotypes

Environmental stresses at particularly vulnerable stages during crop development may severely diminish productivity. At temperature of 10 °C or below cultivated tomato germinate slowly if at all. In this study, seven tomato genotypes bred at the Research Institute of Vegetable Crops were evaluated for germination time at 10 °C. Analysis identified that one genotype which has L. chilense in its pedigree, germinated most rapidly while four other genotypes germinated slower. After 21 days, four out of five of the genotypes resulted in seed germination from 81 to 98 %.     

Inhibition of seed germination and development of tomato plants in soil infested with Pseudomonas tomato

Infestation of sterilised or natural soil with Pseudomonas tomato at inoculum concentrations of 10² to 10⁹ propagules/ml inhibited germination of seeds and caused damping-off of tomato cv. VF-198, susceptible to bacterial speck. Infestation with saprophytic P. fluorescens at an inoculum concentration of 10⁹ propagules/ml did not cause any damage. Germination of seeds of tomato cv. Rehovot-13, resistant to P. tomato, was not affected in P. tomato-infested natural soil, but was inhibited when tested in P. tomato-infested, sterilised soil. Tomato plants which were symptomless from sowing to the flowering stage when growing in infested soil had 20–30% less foliage than plants growing in uninfested soil.     

Effects of neodymium magneto-priming on seed germination and salinity tolerance in tomato

Earth's biosphere is surrounded by magnetic fields that affect all living organisms. A plant's response to magnetic fields is displayed in terms of its seed's vigor, growth, and yield. Examining seed germination in such magnetic fields is the first step in the investigation of how magnetic fields might be used to enhance plant growth and maximize crop performance. In this study, salinity-sensitive Super Strain-B tomato seeds were primed with the northern and southern poles of neodymium magnets of 150, 200, and 250 mT. The magneto-primed seeds showed a significant increase in germination rate and speed, where the orientation of the magnet was identified as being crucial for germination rate and the orientation of seeds towards the magnet was shown to affect the germination speed. The primed plants exhibited enhanced growth characteristics, including longer shoots and roots, larger leaf area, more root hairs, higher water content, and more tolerance to salinity levels, up to 200 mM NaCl. All magneto-primed plants showed a significant decrease in chlorophyll content, continuous chlorophyll fluorescence yield (Ft), and quantum yield (QY). The salinity treatments decreased all chlorophyll parameters in control plants, significantly, but did not lower such parameters in magneto-primed tomatoes. The results of this study illustrate the positive effects of neodymium magnet on the growth and development of tomato plants in terms of their germination, growth, and salinity tolerance, and negatively affected the chlorophyll content in tomato leaves. © 2023 Bioelectromagnetics Society.     

不同基因型番茄种子萌发期的耐盐性

选用14种不同基因型番茄进行萌芽期NaCl胁迫耐盐性筛选,对相对发芽势和相对发芽率两项指标进行聚类分析,将其划分为耐盐性强（5种）和耐盐性弱（9种）两类,从中选出4种耐盐性和生物性状不同的番茄(耐盐性强：野生醋栗番茄、小果型辽园红玛瑙、大果型红宝石;耐盐性弱：大果型辽园红多丽)分别进行不同种类钠盐以及NaCl、Na⁺、Cl^-两组胁迫试验.结果表明：4种不同基因型番茄对各种盐胁迫响应与NaCl的鉴定结果一致;不同Na⁺盐中碱性盐NaHCO₃对番茄的影响最大,在100 mmol·L^-1 Na⁺浓度下,4种基因型番茄的相对胚芽长度都在8%以下,5种盐对番茄种子萌发的抑制顺序为：NaNO₃2SO₄2PO₄3;NaCl、Na⁺、Cl^-胁迫下,Cl^-对番茄的伤害最小.     

AtAPOSTART1, an Arabidopsis thaliana PH-START domain protein involved in seed germination

The seed in the mature and dry state is metabolically inactive (quiescent) and is thus able to withstand extreme environmental conditions, such as drought and cold. Germination commences when the dry seed, shed from its parent plant, takes up water (imbibition) and ends when the root emerges through the seed coat. During seedling establishment, the reserves stored in the seed are metabolized, whereas the subsequent vegetative and reproductive growth is supported by photosynthesis. Here, we describe the functional characterization of the PH-START protein AtAPO1 (Arabidopsis thaliana APOSTART1), the putative homologue of PpAPO1 (Poa pratensis APOSTART1) in Arabidopsis thaliana. By using translational fusion of the AtAPO1 promoter to the uiaD gene and in situ hybridization analyses, we show that AtAPO1 is expressed in mature embryo sacs and developing embryos. The functional analysis of two at-apostart mutant alleles suggests that AtAPO1 is involved in the control of seed germination.