Involvement of polyamines in the interacting effects of low temperature and mineral supply on Pringlea antiscorbutica (Kerguelen cabbage) seedlings

Pringlea antiscorbutica, which is the sole endemic crucifer in the subantarctic zone, undergoes seedling development in a harsh and cold environment. Since, at the mature stage, this species exhibits several adaptations linked to cold tolerance such as high polyamine levels, potential adaptations and polyamine response were investigated in seedlings. In order to assess the specificity of responses, P. antiscorbutica was compared with Arabidopsis thaliana, which is characterized by a life cycle preventing cold exposure at seedling stage. P. antiscorbutica and A. thaliana seedlings were found to have strikingly contrasted responses to temperature changes and to mineral nutrition. Whereas A. thaliana seedlings showed the typical growth arrest of chilling-sensitive plants, P. antiscorbutica seedlings showed optimal root growth at low temperature (5/10 degrees C) and temperate conditions caused the early arrest of root growth. Cold tolerance was associated with increased levels of polyamines or with maintenance of high levels of polyamines. Comparison of both species showed that polyamine levels could be a significant marker of chilling tolerance in seedlings. Treatments with varying mineral supply showed a positive relationship between root growth rate and variations of agmatine and putrescine endogenous contents in roots of P. antiscorbutica. This may be the first demonstration that, even under conditions of accumulation induced by environmental stress, polyamine levels can still be correlated with developmental processes. Com parison of mineral supply and temperature effects strongly indicated a trade-off of polyamine involvement between development and response to stress.     

Induction of the arginine decarboxylase ADC2 gene provides evidence for the involvement of polyamines in the wound response in Arabidopsis

Polyamines are small ubiquitous molecules that have been involved in nearly all developmental processes, including the stress response. Nevertheless, no direct evidence of a role of polyamines in the wound response has been described. We have studied the expression of genes involved in polyamine biosynthesis in response to mechanical injury. An increase in the expression of the arginine decarboxylase 2 (ADC2) gene in response to mechanical wounding and methyl jasmonate (JA) treatment in Arabidopsis was detected by using DNA microarray and RNA gel-blot analysis. No induction was observed for the ADC1 gene or other genes coding for spermidine and spermine synthases, suggesting that ADC2 is the only gene of polyamine biosynthesis involved in the wounding response mediated by JA. A transient increase in the level of free putrescine followed the increase in the mRNA level for ADC2. A decrease in the level of free spermine, coincident with the increase in putrescine after wounding, was also observed. Abscisic acid effected a strong induction on ADC2 expression and had no effect on ADC1 expression. Wound-induction of ADC2 mRNA was not prevented in the JA-insensitive coi1 mutant. The different pattern of expression of ADC2 gene in wild-type and coi1 mutant might be due to the dual regulation of ADC2 by abscisic acid and JA signaling pathways. This is the first direct evidence of a function of polyamines in the wound-response, and it opens a new aspect of polyamines in plant biology.     

Carbon metabolism in the subantarctic Kerguelen cabbage Pringlea antiscorbutica R. Br.: environmental controls over carbohydrates and proline contents and relation to phenology

The subantarctic Brassicaceae Pringlea antiscorbutica R. Br. (Kerguelen cabbage) was used as a model to study the physiological adaptations of higher plants to the subantarctic environment. ¹³C-nuclear magnetic resonance permitted, in combination with biochemical methods, the identification and quantification of the major solutes in leaves, stem and roots. As characterized in many Brassicaceae, proline was a major solute in all organs of the plants, and its accumulation was mainly controlled by salt stress rather than temperature. Glucose was the major soluble sugar in the leaves, whilst sucrose and starch accumulated in stems and roots. Over a period of 1 year we found strong correlations between (i) glucose content in leaves and irradiance, and (ii) starch content in non-photosynthetic organs and air temperature. The pattern of carbohydrate accumulation indirectly indicated that photosynthetis was sustained throughout the year, even during cold days when the temperature remained near 0 °C. This is consistent with the direct gas exchange measurements showing that photosynthetic capacity is mainly influenced by irradiance and weakly by temperature. Taken together, these characteristics demonstrated that the growth and development cycle occurs without a period of dormancy.     

Temperature-dependent changes of amine levels during early seedling development of the cold-adapted subantarctic crucifer Pringlea antiscorbutica

Pringlea antiscorbutica R. Br., an endemic crucifer from the Kerguelen Archipelago in the subantarctic, has been previously shown to be unable to acclimatize to 25°C when transferred after several months cultivation under cold conditions. Furthermore, the polyamine composition was greatly modified in such high-temperature-treated plants. The development of seedlings of this species was investigated under a regime mimicking the subantarctic summer thermoperiod (5/10°C night/day) and a regime with high temperatures (22/25°C night/day). In parallel, the associated changes in polyamine composition that occurred during the first 6 days of seedling life were determined. Marked acceleration of seedling growth and intense cotyledon greening were observed at day 4 in 5/10°C-grown seedlings but not in 22/25°C-grown seedlings. Seedlings grown at high temperature accumulated agmatine and putrescine, whereas cold-cultivated seedlings maintained high levels of spermidine. Cold-cultivated seedlings accumulated the uncommon long-chain polyamines norspermidine and homospermidine. These seedlings also accumulated free 1,3-diaminopropane, cadaverine, N¹-acetylspermidine, N¹-acetylspermine and bound polyamines, whereas seedlings under high temperature accumulated N¹-acetylputrescine. Aromatic amine metabolism also appeared to be very responsive to temperature: seedlings under a cold regime accumulated free dopamine and bound phenylethylamine and tyramine, whereas seedlings grown at high temperature accumulated free tyramine. The possible relationships between the observed amine patterns and seedling growth under low and high temperature are discussed.     

High flexibility in growth and polyamine composition of the crucifer Pringlea antiscorbutica in relation to environmental conditions

Although polyamine (PA) metabolism in plants responds to abiotic stresses, few studies have investigated this response in plants under natural conditions. Using high-performance liquid chromatography, we studied the amine composition of the subantarctic crucifer Pringlea antiscorbutica R. Br. both in the field (Crozet and Kerguelen Islands) and under controlled temperatures in the laboratory. Plants collected from different sites showed a large variability of amine composition and contents. The aliphatic and acetylated amine composition of leaves allowed to statistically identify two groups. The first group, composed of Kerguelen coastal plants, was characterized by high levels of acetylspermidine and acetylspermine, showing the presence in P. antiscorbutica of this uncommon regulation pathway. PA acetylation may be induced in P. antiscorbutica under conditions of low water availability. The second group, composed of plants from Kerguelen altitude sites and plants from Crozet sites, showed high levels of free spermidine (Spd). In these plants, the ratio Spd/putrescine did not positively correlate with plant size as was found in developmental studies, suggesting that free Spd may be devoted to other aims, such as cold tolerance. Some differences between Crozet and Kerguelen plant responses suggested the possibility that different regulations of amine metabolism could take place in plants from these two islands. Agmatine accumulation pattern was diverse and suggested this amine to be sensitive to combinations of environmental factors. Studies on amine variation patterns in P. antiscorbutica provide insights into the roles of rarely reported amines, such as acetylated amines, in plant metabolic adaptation to abiotic stresses.     

小麦幼苗期水分胁迫所诱导基因表达谱的初步分析

利用抑制差减杂交(Suppression Subtractive Hybridization,SSH)和高密度点阵膜技术研究小麦2叶幼苗期水分胁迫诱导表达基因。通过筛选具有1530个克隆的SSH文库,获得181个阳性克隆。序列同源性比较和功能查询结果发现,83．2％的水分胁迫诱导表达基因分别与不同逆境胁迫条件下表达的基因具有较高的同源性,这些基因在生物体内的功能都是直接或间接对细胞遭受逆境胁迫起保护作用。其中17个EST未找到同源性较高的匹配序列,已经在GenBank注册。用反向Northern、RT-PCR和Northern进一步检验所获得的功能已知EST,初步建立了小麦幼苗期水分胁迫诱导的基因表达谱。     

Inducible overexpression of oat arginine decarboxylase in transgenic tobacco plants

To test the possible interaction of polyamines in plant growth responses, transgenic tobacco plants containing the Avena sativa L. (oat) arginine decarboxylase (ADC) gene under the control of a tetracycline-inducible promoter were generated. Inducible overexpression of oat ADC in transgenic tobacco led to an accumulation of ADC mRNA, increased ADC activity and changes in polyamine levels. Transgenic lines, induced during vegetative stage, displayed different degrees of an altered phenotype, the severity of which was correlated with putrescine content. These phenotypic changes were characterized by short internodes, thin stems and leaves, leaf chlorosis and necrosis, as well as reduced root growth. This is the first report to show altered phenotypes as a consequence of polyamine changes under tetracycline-induction in in vivo conditions. Interestingly, overexpression of oat ADC in tobacco resulted in similar detrimental effects to those observed by ADC activation induced by osmotic stress in the homologous oat leaf system. In the context of the role of specific polyamines in plant growth and development, the present results indicate that activation of the ADC pathway leading to high levels of endogenous putrescine (or its catabolytes) is toxic for the vegetative growth of the plant. In contrast, no visible phenotypic effects were observed in flowering plants following tetracycline induction. Further characterization of the different transgenic lines may shed light on the action of specific polyamines in different plant developmental processes.     

Transcriptional profiles of primary metabolism and signal transduction-related genes in response to water stress in field-grown sunflower genotypes using a thematic cDNA microarray

A sunflower cDNA microarray containing about 800 clones covering major metabolic and signal transduction pathways was used to study gene expression profiles in leaves and embryos of drought-tolerant and -sensitive genotypes subjected to water-deficit stress under field conditions. Using two-step ANOVA normalization and analysis models, we identified 409 differentially expressed genes among genotypes, water treatment and organs. The majority of the cDNA clones differentially expressed under water stress was found to display opposite gene expression profiles in drought-tolerant genotype compared to drought-sensitive genotype. These dissimilarities suggest that the difference between tolerant and non-tolerant plants seems to be associated with changes in qualitative but not quantitative mRNA expression. Comparing leaves and embryos, 82 cDNA clones showing organ-specific variation in gene expression levels were identified in response to water stress across genotypes. Genes related to amino acids and carbohydrates metabolisms, and signal transduction were induced in embryos and repressed in leaves; suggesting that vegetative and reproductive organs respond differentially to water stress. Adaptive mechanisms controlling water deficit tolerance are proposed and discussed.     

Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae

Arginine decarboxylase (ADC) is an important enzyme in the production of putrescine and polyamines in plants. It is encoded by a single or low-copy nuclear gene that lacks introns in sequences studied to date. The rate of Adc amino acid sequence evolution is similar to that of ndhF for the angiosperm family studied. Highly conserved regions provide several target sites for PCR priming and sequencing and aid in nucleotide and amino acid sequence alignment across a range of taxonomic levels, while a variable region provides an increased number of potentially informative characters relative to ndhF for the taxa surveyed. The utility of the Adc gene in plant molecular systematic studies is demonstrated by analysis of its partial nucleotide sequences obtained from 13 representatives of Brassicaceae and 3 outgroup taxa, 2 from the mustard oil clade (order Capparales) and 1 from the related order Malvales. Two copies of the Adc gene, Adc1 and Adc2, are found in all members of the Brassicaceae studied to data except the basal genus Aethionema. The resulting Adc gene tree provides robust phylogenetic data regarding relationships within the complex mustard family, as well as independent support for proposed tribal realignments based on other molecular data sets such as those from chloroplast DNA.      

Differential expression of a chlorophyll a/b binding protein gene and a proline rich protein gene in juvenile and mature phase English ivy (Hedera helix)

Two cDNA clones representing mRNAs which are differentially expressed during in vitro culture of juvenile and mature leaf petioles of English ivy ( Hedera helix L.) were isolated by differential screening. The mRNA represented by clone HW101 is expressed at a higher level in untreated juvenile than in untreated mature in-vitro-cultured petioles. Treatment of petioles with α-naphthaleneacetic acid (NAA) at the initiation of culture decreases HW101 mRNA levels in juvenile but not mature, petioles. In intact plants. HW101 mRNA is expressed at a higher level in juvenile laminae, petioles and stems than in identical tissues of mature plants. DNA sequence analysis indicates that HW1O1 cDNA is significantly similar to a light harvesting chlorophyll a/b binding protein gene ( Lhcb ) of pea. The gene represented by the second clone. HW103, is expressed at a higher level in mature than in juvenile in-vitro-cultured petisoles. Treatment of petioles with NAA at the initiation of culture decreases HW103 mRNA levels in chronologically young mature but not older mature and juvenile petioles. However, expression of the HW103 gene is not detectable in petioles, or in any other vegetative organ tested, immediately after excision. It is, however, expressed in developing seeds. In otherwise intact plants, the HW103 gene is expressed in wounded petioles of mature plants 5 days after wounding but not in wounded petioles of juvenile plants. It is also expressed at a higher level in wounded stems of mature plants than in those of juvenile plants. However, it is not expressed in wounded lamina of either juvenile or mature plants. DNA sequence analysis indicates that HW103 cDNA is similar to a cell wall proline rich protein (PRP) gene of soybean. This is the first report of differential expression of a PRP gene in tissues from juvenile and mature plants. Southern blot analysis of nuclear DNA of H. helix shows that both HW101 and HW103 are members of small gene families.     

Subcellular compartmentation of proline in the leaves of the subantarctic Kerguelen cabbage Pringlea antiscorbutica R. Br. In vivo13C-NMR study

Proline is one of the major solutes accumulated upon salt stress in leaves, stem and roots of the subantarctic Brassicaceae Pringlea antiscorbutica R. Br. (Kerguelen cabbage). Using in vivo ¹³C-NMR techniques, it was possible for the first time to visualize the subcellular compartmentation of proline between cytoplasmic and vacuolar compartments in Pringlea leaves. We observed that this osmolyte accumulated at a 2–3 times higher concentration in the cytoplasm than in the vacuole.     

嫁接对铜胁迫下黄瓜幼苗根系多胺代谢的影响

采用营养液栽培法,研究了嫁接(以黑籽南瓜为砧木)对铜胁迫下黄瓜幼苗根系活力及多胺代谢的影响.结果表明:铜胁迫下黄瓜幼苗根系活力下降,电解质渗漏率升高,而嫁接苗的变化幅度显著小于黄瓜自根苗;铜胁迫下黄瓜嫁接植株根系中除游离态腐胺(Put)含量显著低于自根苗外,结合态和束缚态Put、3种形态亚精胺(Spd)和精胺(Spm)含量均显著高于自根苗,嫁接苗根系中游离态Put含量及腐胺/多胺(Put/PAs)显著低于自根苗;铜胁迫下,嫁接苗根系精氨酸脱羧酶(ADC)、鸟氨酸脱羧酶(ODC)和S-腺苷蛋氨酸脱羧酶(SAMDC)活性高于自根苗,而二胺氧化酶(DAO)和多胺氧化酶(PAO)活性显著低于自根苗.表明嫁接黄瓜幼苗根系PAs的合成增加,降解减少,使PAs含量维持在较高水平,从而提高了黄瓜幼苗抗铜胁迫能力.