Specific DNA Alterations Associated with the Environmental Induction of Heritable Changes in Flax

Several flax varieties have been shown to undergo environmentally induced heritable changes resulting in stable lines termed genotrophs. The most notable of these is the variety Stormont Cirrus, also termed "plastic" or Pl. A number of morphological, biochemical and genetic differences are associated with environmental induction of heritable changes in flax. We have studied 5S rDNA alterations as a model system for understanding environmental induction of heritable changes in flax. This paper reports the isolation of a flax 5S rRNA gene variant which identifies genotroph specific restriction fragment length polymorphisms (RFLPs) in flax. Restriction fragment patterns for several enzymes were observed in both large and small genotrophs which consistently differed from the progenitor, Stormont Cirrus. Identical RFLP profiles for all restriction endonucleases tested were observed in four small genotrophs produced from separate environmental induction experiments. Comparison between Stormont Cirrus and these small genotrophs showed at least six differing bands in addition to several high molecular weight polymorphisms. Genetic data indicate that the polymorphisms were all produced from a repetitive 5S rRNA gene cluster at a single chromosomal locus. Similar, but not identical, polymorphisms are also detected in other flax varieties and Linum species suggesting that the induced variation is related to that which occurs naturally. The results are evidence that a specific set of DNA alterations occur in association with the induction of heritable changes in flax. This is the first genetic marker which is altered to an identical state in one type of genotroph. The results are discussed with respect to mechanisms for environmentally induced heritable change in plants.     

RFLP and RAPD mapping in flax (Linum usitatissimum)

A map of flax (Linum usitatissimum) using restriction fragment length polymorphisms (RFLPs) and random amplified polymorphic DNAs (RAPDs), and comprising 15 linkage groups containing 94 markers, has been developed covering about 1000 cM. The mapping populations were the F₂ populations from two crosses between diverse cultivars. From one cross, CI1303 and Stormont Cirrus, 20 RFLP and 520 RAPD markers were analyzed. Thirteen RFLP and 80 RAPD markers were on the 15 linkage groups, in addition to one sequence-tagged site (STS). Seven polymorphic RAPD markers were found to have unusual segregation patterns. RAPDs were expressed as dominant markers, but for these markers a prevalence of the progeny lacked a band rather than the expected one-fourth ratio. However, these exceptions may be related to the instability of the genome of Stormont Cirrus in which stable and heritable genomic changes can be induced by environmental factors. The current map could be used for the identification of markers linked to loci controlling the ability to generate heritable changes in response to environmental growth conditions, and to develop anchor loci with STSs for a more general application. Received: 20 March 1999 / Accepted: 16 December 1999     

Variation in the isozymes of flax (Linum usitatissimum) genotrophs

Peroxidase, esterase, and acid phosphatase isozymes of environmentally induced L and S genotrophs, nuclear DNA reversion types, and the orginal plastic (Pl) type of the flax variety Stormont Cirrus have been compared by polyacrylamide gel electrophoresis. Differences were observed in particular line was not correlated with the nuclear DNA amount. The relationship between the isozyme pattern and the phenotypes of the lines in which they are expressed is discussed.     

Pre-harvest retting of flax: effects of water stress on uptake and efficacy of glyphosate

The effects of water stress on the uptake, translocation and efficacy of glyphosate in flax were investigated in relation to pre-harvest retting. Glyphosate (applied at a rate equivalent to 1.44 kg a.e. ha^-1 at 0, 10, 20 or 30 days after the start of flowering) caused little desiccation of flax grown in pots under restricted watering. Glyphosate application to well-watered plants caused the moisture content to decline from an initial value of 70 – 80% to approximately 40% at 3 wk after spraying. Glyphosate was applied 2 wk after the mid-point of flowering to flax grown in soil with moisture contents of 35, 31, 26, 22, 16 or 12%. Soil moisture levels (16% and 12%) which restricted evapotranspiration also reduced the efficacy of glyphosate but did not affect uptake of ¹⁴C-glyphosate. Translocation of ¹⁴C-glyphosate out of treated leaves was reduced only in the most severely stressed plants (12% soil moisture). Experiments with young plants (4 wk old) confirmed that water stress slightly reduced downward translocation of glyphosate. When the herbicide was applied to young plants under conditions which minimised differences in translocation, 10.8 μg glyphosate was sufficient to desiccate unstressed plants but 108 μg had little effect on stressed plants. This indicates that, in addition to any reduction in translocation which occurs during drought, water stress may reduce the susceptibility of flax to glyphosate. Thus only relief of plant water stress by irrigation is likely to improve response of the flax crop to glyphosate.     

Labile DNA sequences in flax identified by combined sample representational difference analysis (csRDA)

Flax (Linum usitatissimum) has a genome in which changes have been associated with environmental factors. The inbred flax variety, Stormont Cirrus (Pl), served as the parent, and several lines (termed genotrophs) were derived from this parent. The phenotypes of the genotrophs were stable in a number of different growth environments, unlike the original Pl line in which changes associated with environmental factors continued to occur. These genotrophs differed from the original line in a number of characteristics, but the only known phenotypic characteristic that is shared by all the genotrophs and different from the parental, Pl, line is the lack of changes associated with the original environmental factors. However, some of these genotrophs have changed in both phenotype and nuclear DNA subsequent to their original growth and differentiation from Pl. Representational difference analysis (RDA) has been used to identify differences between Pl and all the genotrophs in an attempt to identify the loci controlling these aspects of plasticity. Subtractions between Pl DNA as a tester (target) and one of the genotrophs (individual RDA) or a mixture of different types of genotroph (L6, S6, C2, and LH) DNAs as a driver were done (combined sample RDA; csRDA). In addition, contrary RDA, where of the genotroph DNA was used as a tester and Pl DNA as a driver, was also executed. Three difference clones (163-4-2, 123-5-2, and 163-13), from 74 primary clones obtained after three rounds of subtractions with Pl DNA as tester were further characterized. In addition, 2 difference products (213-r1 and 213-r9) were characterized from contrary RDA. The clones 163-4-2 and 163-13 from the csRDA showed polymorphisms between Pl and all the genotrophs when PCR was done with primers derived from sequences of the clones, but only the clone 163-13 polymorphism was confirmed by Southern blot analysis. Four of 5 clones (163-4-2, 123-5-2, 163-13 and 213-r9) that have been characterized appear to be associated with structural changes in the DNA. From the contrary csRDA, it was observed that no clones could be recovered from subtractions between a mixture of genotrophs as a tester and Pl as a driver, and several possible explanations have been proposed.     

Transpiration does not control growth and nutrient supply in the amphibious plant Mentha aquatica

Mentha aquatica L. was grown at different nutrient availabilities in water and in air at 60% RH. The plants were kept at 600 mmol m^?3 free CO₂ dissolved in water (40 times air equilibrium) and at 30 mmol m^?3 CO₂ in air to ensure CO₂ saturation of growth in both environments. We quantified the transpiration-independent water transport from root to shoot in submerged plants relative to the transpiration stream in emergent plants and tested the importance of transpiration in sustaining nutrient flux and shoot growth. The acropetal water flow was substantial in submerged Mentha aquatica, reaching 14% of the transpiration stream in emergent plants. The transpiration-independent mass flow of water from the roots, measured by means of tritiated water, was diverted to leaves and adventitious shoots in active growth. The plants grew well and at the same rates in water and air, but nutrient fluxes to the shoot were greater in plants grown in air than in those that were submerged when they were rooted in fertile sediments. Restricted O₂ supply to the roots of submerged plants may account for the smaller nutrient concentrations, though these exceeded the levels required to saturate growth. In hydroponics, the root medium was aerated and circulated between submerged and emergent plants to minimize differences in medium chemistry, and here the two growth forms behaved similarly and could fully exploit nutrient enrichment. It is concluded that the lack of transpiration from leaf surfaces in a vapour-saturated atmosphere, or under water, is not likely to constrain the transfer of nutrients from root to shoot in herbaceous plants. Nutrient deficiency under these environmental conditions is more likely to derive from restricted development and function of the roots in waterlogged anoxic soils or from low porewater concentrations of nutrients.     

Effect of heterogeneous distribution of nutrients on root growth, ABA content and drought resistance of wheat plants

Heterogeneous distribution of nutrients influenced morphology of wheat roots; they were shorter and had more laterals at the site of high nutrient content. The roots outside the nutrient-rich patch were longer and penetrated deeper in the soil than those of the plants supplied with the same quantity of nutrients but having them homogeneously distributed. The ABA content of roots sampled from the site of high nutrient content was greater than in those elsewhere. It is suggested that this might be important for inhibition of their extension growth and stimulation of laterals in the nutrient rich patch. Heterogeneously distributed nutrients beneficially influenced accumulation of ¹⁵N and assimilation of CO₂ by the plants, enabling an increased supply of nutrients to the parts of the root system beyond the nutrient rich patch and sustained their rapid extension. Enhanced penetration of roots into the deeper soil layers could contribute to the observed increase in drought resistance of plants grown with localised placement of fertilisers under field conditions.     

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.     

A theoretical explanation of the Piper-Steenbjerg effect

The relation between plant yield and plant nutrient concentration is sometimes found to be negative, a phenomenon called the Piper-Steenbjerg (PS) effect. A model was used to examine the underlying causes of the PS effect, and the conditions under which it is most likely to occur. The model uses the nutrient productivity concept for plant growth and a nutrient uptake equation in which root growth rate and external nutrient concentration determine the uptake rate. The study suggests that the PS effect occurs when the fast growth of plants grown in an initially higher nutrient medium eventually leads to a more rapid depletion of external nutrients than the slow growth of plants grown in an initially lower nutrient medium. The fast growth of plants combined with a rapid decrease of nutrient uptake leads to a fall in plant nutrient concentration. When these large plants with very low nutrient concentrations are compared with the smaller, slow-growing plants, a PS effect may be found depending on the time at which the plants are harvested, and on the range of initial values of the external nutrient content. When it occurs, the effect is greatest when the depletion volume per unit new root (V_d) is lowest, and when the mobility of nutrients in the medium is highest (α=1). The results are sufficiently general to apply to a variety of nutrients, plant species and growth media.     

Resource availability affects differentially the levels of gallotannins and condensed tannins in Ceratonia siliqua

Young potted seedlings of the Mediterranean evergreen sclerophyll Ceratonia siliqua were grown in the field under two nutrient and water regimes during the spring growth period. As expected, plants receiving additional nutrients accumulated more above and below ground biomass while the reverse was true for water stressed plants. In accordance with the growth–differentiation balance hypothesis, total leaf phenolics and tannins (astringency) decreased under high nutrients and increased under water stress, with the effects being more pronounced in young leaves. However, the responses of the two tannin types to resource availability were not similar. Only condensed tannins were decreased by nutrient addition while both condensed and gallotannins were increased by water stress. This non-homogeneous behaviour may reflect the different biosynthetic origins of the two tannin types.     

The Effect of Wind and a Reduced Supply of Phosphorus and Nitrogen on the Growth and Water Relations of Festuca arundinacea Schreb

Festuca arundinaceae was grown at high and low wind-speed attwo levels of either soil phosphorus or soil nitrogen. At increasedwind-speed, mean relative growth rate and leaf extension ratewere reduced when plants were grown with high nutrient concentrationsand further reduced when phosphorus or nitrogen stress was imposedon the plants. Transpiration was increased at high wind-speedexcept under conditions of phosphorus stress, where the ratewas actually decreased. Relationships between water stress,wind and nutrient status are discussed, especially in relationto the possible role of phosphorus stress in causing sclerophylly. Festuca arundinacea Schreb., relative growth rate, water stress, wind, nitrogen, phosphorus, sclerophylly     

Differences in chemical composition of plants grown at constant relative growth rates with stable mineral nutrition

Summary Leaf chemistry of a willow clone (Salix aquatica Smith) differed significantly when grown at constant relative growth rates depending upon the relative availability of nutrients and light. Concentration of amino acids and nitrate were high in plants grown with a relative surplus of nutrients. Concentrations of starch, tannin, and lignin, on the other hand, were high in plants grown with a relative surplus of carbon. Photosynthetic rates, expressed per unit leaf area, were similar when plants were grown under high light conditions, regardless of nutrient availability. Dark respiration was much higher in plants supplied with abundant nutrients than in those with a more limited supply, reflecting differences in nitrogen concentration of the tissue. The experimental approach allows plants to be grown to a standard size with differing, but highly uniform chemistry. Plants grown in such a manner may provide good experimental material to evaluate interactions between herbivores or pathogens and their hosts.     

The effects of CO2 and nutrient enrichment on photosynthesis and growth of Poa annua in two consecutive generations

We studied short- and long-term growth responses of Poa annua L. (Gramineae) at ambient and elevated (ambient +200???mol?mol^?1) atmospheric CO₂. In experiment 1 we compared plant growth during the early, vegetative and final, reproductive growth phases. Plant growth in elevated CO₂ was significantly enhanced during the early phase, but this was reversed in the reproductive phase. Seed mass and percentage germination were significantly reduced in elevated CO₂. Experiment 2 tested for the impact of transgenerational and nutrient effects on the response of Poa annua to elevated CO₂. Plants were grown at ambient and elevated CO₂ for one or two consecutive generations at three soil nutrient levels. Leaf photosynthesis was significantly higher at elevated CO₂, but was also affected by both soil nutrient status and plant generation. Plants grown at elevated CO₂ and under conditions of low nutrient availability showed photosynthetic acclimation after 12?weeks of growth but not after 6?weeks. First-generation growth remained unaffected by elevated CO₂, while second-generation plants produced significantly more tillers and flowers when grown in elevated CO₂ compared to ambient conditions. This effect was strongest at low nutrient availability. Average above- and belowground biomass after 12?weeks of growth was enhanced in elevated CO₂ during both generations, but more so during plant generation 2. This study demonstrates the importance of temporal/maternal effects in plant responses to elevated CO₂.     

The effect of phosphorus and nitrogen deficiency on growth of seedlings of spring barley in dependence on irradiance: Growth analysis

Seedlings of spring barley,Hordeum vulgare L. cv. Mirena, were grown in a controlled environment chamber at high (HI: 122 Wm^?2) and low (LI: 28 Wm^?2) irradiance in the complete Richter’s nutrient solution (R) or in solution lacking either phosphorus (R -P) or nitrogen (R -N). The experiment was terminated 15 days after sowing when plants (R-N) at HI ceased to grow. At that time the dry mass of one plant was 449.8 mg, 145.7 mg and 116.8mg at HI and 203.4 mg, 110.1 mg and 91.0 at LI for R, (R-P) and (R-N), respectively. Deficiency of P and especially N reduced the size of loaf area more under HI than under LI conditions. Specific dry mass of leaves was the highest in R-N plants. The values of relative growth rate and assimilation rate are presented. Interaction of the effects of deficiency of mineral nutrients and irradiance during cultivation should be analyzed in further experiments for determination of optimum conditions for utilization of mineral nutrients.     

Iron-Stress Induced Redox Activity in Tomato (Lycopersicum esculentum Mill.) Is Localized on the Plasma Membrane

Tomato plants (Lycopersicum esculentum Mill.) were grown for 21-days in a complete hydroponic nutrient solution including Fe³⁺-ethylenediamine-di(o-hydroxyphenylacetate) and subsequently switched to nutrient solution withholding Fe for 8 days to induce Fe stress. The roots of Fe-stressed plants reduced chelated Fe at rates sevenfold higher than roots of plants grown under Fe-sufficient conditions. The response in intact Fe-deficient roots was localized to root hairs, which developed on secondary roots during the period of Fe stress. Plasma membranes (PM) isolated by aqueous two-phase partitioning from tomato roots grown under Fe stress exhibited a 94% increase in rates of NADH-dependent Fe³⁺-citrate reduction compared to PM isolated from roots of Fe-sufficient plants. Optimal detection of the reductase activity required the presence of detergent indicating structural latency. In contrast, NADPH-dependent Fe³⁺-citrate reduction was not significantly different in root PM isolated from Fe-deficient versus Fe-sufficient plants and proceeded at substantially lower rates than NADH-dependent reduction. Mg²⁺-ATPase activity was increased 22% in PM from roots of Fe-deficient plants compared to PM isolated from roots of Fe-sufficient plants. The results localized the increase in Fe reductase activity in roots grown under Fe stress to the PM.     

Arbuscular mycorrhizal adaptation,spore germination,root colonization,and host plant growth and mineral acquisition at low pH

Arbuscular mycorrhizal (AM) fungi colonize plant roots and often enhance host plant growth and mineral acquisition, particularly for plants grown under low nutrient and mineral stress conditions. Information about AM fungi and mycorrhizal ( +AM) host plant responses at low pH ( < 5) is limited. Acaulospora are widely reported in acid soil, and Gigaspora sp. appear to be more common in acid soils than Glomus sp. Spores of some AM fungi are more tolerant to acid conditions and high Al than others; t Acaulospora sp., Gigaspora sp., and Glomus manihotis are particularly tolerant. Root colonization is generally less in low than in high pH soils. Percentage root colonization is generally not related to dry matter (DM) produced. Maximum enhancement of plant growth in acid soil varies with AM fungal isolate and soil pH, indicating adaptation of AM isolates to edaphic conditions. Acquisition of many mineral nutrients other than P and Zn is enhanced by +AM plants in acid soil, and the minerals whose concentration is enhanced are those commonly deficient in acid soils (Ca, Mg, and K). Some AM fungal isolates are effective in overcoming soil acidity factors, especially Al toxicity, that restrict plant growth at low pH.     

Nutrient Relations of Groundsel (Senecio vulgaris) Infected by Rust (Puccinia lagenophorae) at a Range of Nutrient Concentrations I. Concentrations, Contents and Distribution of N, P and K

Groundsel (Senecio vulgaris L.), healthy or infected with therust fungus Puccinia lagenophorae was grown in sand and fedwith a complete nutrient medium diluted to give a range of concentrations.Analysis of bulked, dried tissues of the plant showed that undernutrient-rich conditions rust infection resulted in increasedconcentrations of total (Kjeldahl) nitrogen and potassium buthad little effect on phosphorus concentration. Thus, despitereduced dry weight growth, total plant nitrogen contents wereno less in rusted than control plants. Although total contentsof phosphorus and potassium were reduced by rust, effects wereprobably related to loss of these nutrients in fungal spores. Interactions between rust infection and nutrient supply weresignificant but differed between nutrients: rust caused increasednitrogen concentrations only under nutrient-rich conditionsbut increased phosphorus concentrations only when nutrient supplywas limited. Increased concentrations were not confined to infectedtissues. Mechanisms underlying rust-nutrient interactions appearto be complex and to depend inter alia on the partitioning andrecycling of the particular nutrient within the plant. Rust-inducedincreases in potassium concentration occurred under both highand low nutrient conditions but were confined to infected tissues.Potassium accumulation in nutrient deficient conditions wasprobably due to increased transpirational flux into infectedtissues, but under nutrient-rich conditions reduced potassiumexport appeared to assume greater significance. The possible implications of the changed nutrient relationsfor the wider interactions of rust-infected plants in naturalvegetation are discussed. Senecio vulgaris, Puccinia lagenophorae, rust infection, nutrient deficiency, nutrient content, nutrient concentration, nutrient distribution