Adaptation of Mycobacteria to Growth Conditions: A Theoretical Analysis of Changes in Gene Expression Revealed by Microarrays

Background

Microarray analysis is a powerful technique for investigating changes in gene expression. Currently, results (r-values) are interpreted empirically as either unchanged or up- or down-regulated. We now present a mathematical framework, which relates r-values to the macromolecular properties of population-average cells. The theory is illustrated by the analysis of published data for two species; namely, Mycobacterium bovis BCG Pasteur and Mycobacterium smegmatis mc² 155. Each species was grown in a chemostat at two different growth rates. Application of the theory reveals the growth rate dependent changes in the mycobacterial proteomes.

Principal Findings

The r-value r _(i) of any ORF (ORF_(i)) encoding protein p _(i) was shown to be equal to the ratio of the concentrations of p _(i) and so directly proportional to the ratio of the numbers of copies of p _(i) per population-average cells of the two cultures. The proportionality constant can be obtained from the ratios DNA: RNA: protein. Several subgroups of ORFs were identified because they shared a particular r-value. Histograms of the number of ORFs versus the expression ratio were simulated by combining the particular r-values of several subgroups of ORFs. The largest subgroup was ORF_(j) (r _(j)  = 1.00± SD) which was estimated to comprise respectively 59% and 49% of ORFs of M. bovis BCG Pasteur and M. smegmatis mc² 155. The standard deviations reflect the properties of the cDNA preparations investigated.

Significance

The analysis provided a quantitative view of growth rate dependent changes in the proteomes of the mycobacteria studied. The majority of the ORFs were found to be constitutively expressed. In contrast, the protein compositions of the outer permeability barriers and cytoplasmic membranes were found to be dependent on growth rate; thus illustrating the response of bacteria to their environment. The theoretical approach applies to any cultivatable bacterium under a wide range of growth conditions.     

Differential sensitivity of four Lemnaceae species to zinc sulphate

Lemnaceae are currently the only freshwater plants required for regulatory toxicity testing of pesticides and other chemicals. Toxicological protocols allow for the use of different Lemnaceae species in tests. However, few studies have compared the relative sensitivity of individual duckweed species. Zinc is an essential plant nutrient but is also a common pollutant in aquatic environments and elevated levels are phytotoxic. This study shows that four species of Lemnaceae differ in their relative sensitivities to zinc sulphate, a commonly used reference chemical. Comparative zinc sensitivity, in order, from most tolerant to most sensitive was: Landoltia punctata > Lemna minor > Wolffia brasiliensis > Lemna gibba. Zinc sensitivity was also endpoint dependant. EC₅₀ values typically increased in order of: specific biomass growth rate < specific frond number growth rate < chlorophyll absorbance. However, specific frond number growth rate was the most sensitive endpoint for L. punctata. Unlike the other species, L. punctata displayed no significant colony disintegration. Lemna species and L. punctata appear to be employing distinct response strategies when exposed to zinc. L. gibba and L. minor produce and release young, single fronds which are severely affected by zinc. In contrast, L. punctata produces fewer fronds, which are not released and form large colonies of high biomass that are relatively zinc tolerant.     

Short term exposure of Lemna minor and Lemna gibba to mercury, cadmium and chromium

The effects of mercury (Hg), cadmium (Cd) and chromium (Cr) in concentrations ranging from 0.02 to 20 mg L^?1 applied for 24 h were assessed in Lemna minor and Lemna gibba by measuring changes in protein concentration, ascorbic acid, phenolics, malondialdehyde (MDA), hydrogen peroxide (H₂O₂), the activity of guaiacol peroxidase (G-POX) and catalase (CAT). Ascorbic acid, phenolics, catalase and guaiacol peroxidase played a key role in the antioxidative response of L. gibba. Inadequate activity of antioxidant enzymes in the L. minor resulted in MDA and H₂O₂ accumulation. In both used species, Hg treatment decreased protein content and increased CAT and G-POX activity, but decreased MDA and H₂O₂ levels. Cadmium and chromium had opposite impacts on two used Lemna species on almost all observed parameters. Enhanced antioxidative responses of L. gibba to lower concentrations of Hg, Cd and Cr indicated greater abiotic stress tolerance than L. minor.     

Gibberellin and CCC Effects on Flowering and Growth in the Long-day Plant Lemna gibba G3

The application of gibberellic acid (GA₃) to the non-rosette long-day plant Lemna gibba G3 at concentrations from 0.1 to 100 mg/l did not induce flowering on short days and inhibited flowering on long days at concentrations of 1 mg/l and higher. On both short and long days GA₃ concentrations above 1 mg/l caused a decrease in frond size and fresh and dry weight, but an increase in the rate of frond production and thus an increase in the # VF (number of vegetative fronds). Identical results were obtained when gibberellin A₇ was used instead of GA₃.     

Influence of nutrients on the development of gibbosity in fronds of the duckweed Lemna gibba L.

The duckweeds Lemna gibba L. and Lemna minor L. only grew wellin undisturbed culture under axenic conditions in low lightintensity when provided with a suitable energy source such asglucose. In media containing N0₃-N gibbosity (a convex ventralsurface) was induced in the presence of the chelating agentethylene-diamine-di-o-hydroxyphenylacetic acid (EDDHA). In nutrientsolutions containing NO₃-N as the only N source, but withoutEDDHA, L. gibba occasionally exhibited gibbosity in culturesolutions of 40 cm³ volumes. More fronds were induced to exhibitgibbosity when the volume of the culture medium was increasedfrom 40 cm³ to 200 cm³. Gibbosity was never induced in L. minor,neither was it induced in L. gibba in media containing NH₄-N,even in the presence of NO₃-N. There was no direct correlationbetween the occurrence of gibbosity and frond growth rate, butgibbosity occurred only when there was good frond growth. In the absence of a sugar, frond growth was enhanced by bubblingair through the culture solution in the light. Increasing theCO₂ concentration in the air up to 1% enhanced growth and inducedgibbosity. Carbon dioxide did not induce gibbosity in mediacontaining NH₄-N. Key words: Ammonium-N, carbon dioxide, gibbosity, Lemna, nitrate-N     

Growth and nitrate uptake properties of plants grown at different relative rates of nitrogen supply. I. Growth of Pisum and Lemna in relation to nitrogen

Abstract The relations between growth and internal nitrogen concentrations were investigated in nonnodulated Pisum sativum L. cv. Marma and Lemna gibba L. grown at relative rates of nitrate-N additions (R_A) varying from 0.03 to 0.27 d ¹(Pisum) and 0.05 to 0.40 d ¹ (Lemna). At R_A≤0.21 d ¹(Pisum) and ≤0.30 d ¹ (Lemna), the relative growth rate (RGR) correlated well with R_A whereas higher R_A was not met by any further increawse in growth rate. The tissue nitrogen concentrations at growth-limiting R_A increased linearly with RGR. The slope of these lines indicate a maximum nitrogen productivity (amount of biomass formed per unit nitrogen and time) of 14.4 g DW g ¹ Nd ¹ for Pisum and 15.9 g DW g ¹ N d ¹ for Lemna. Extrapolation of the plots to RGR=0 yielded intercepts of 10–15 mg N g^?1 DW for Pisum tissue, whereas for Lemna the intercepts were closer to the origin than for Pisum. These intercepts formally define a fraction of the total plant nitrogen that appears not to be active in production of new biomass, her termed ‘non-growth nitrogen’. The partitioning of nitrogen as well as biomass to the roots increased at low R_A, and is discussed in relation to activity of shoots and roots, respectively.     

Interaction of 8-Hydroxyquinoline with Soil Environment Mediates Its Ecological Function

Background

Allelopathic functions of plant-released chemicals are often studied through growth bioassays assuming that these chemicals will directly impact plant growth. This overlooks the role of soil factors in mediating allelopathic activities of chemicals, particularly non-volatiles. Here we examined the allelopathic potential of 8-hydroxyquinoline (HQ), a chemical reported to be exuded from the roots of Centaurea diffusa.

Methodology/Principal Findings

Growth bioassays and HQ recovery experiments were performed in HQ-treated soils (non-sterile, sterile, organic matter-enriched and glucose-amended) and untreated control soil. Root growth of either Brassica campestris or Phalaris minor was not affected in HQ-treated non-sterile soil. Soil modifications (organic matter and glucose amendments) could not enhance the recovery of HQ in soil, which further supports the observation that HQ is not likely to be an allelopathic compound. Hydroxyquinoline-treated soil had lower values for the CO₂ release compared to untreated non-sterile soil. Soil sterilization significantly influenced the organic matter content, PO₄-P and total organic nitrogen levels.

Conclusion/Significance

Here, we concluded that evaluation of the effect of a chemical on plant growth is not enough in evaluating the ecological role of a chemical in plant-plant interactions. Interaction of the chemical with soil factors largely determines the impact of HQ on plant growth.     

Analysis of chloroplast <Emphasis Type="Italic">rpS16</Emphasis> intron sequences in Lemnaceae

Chloroplast rpS16 gene intron sequences were determined and characterized for twenty-five Lemnaceae accessions representing nine duckweed species. For each Lemnaceae species nucleotide substitutions and for Lemna minor, Lemna aequinoctialis, Wolffia arrhiza different indels were detected. Most of indels were found for Wolffia arrhiza and Lemna aequinoctialis. The analyses of intraspecific polymorphism resulted in identification of several gaplotypes in Lemna gibba and Lemna trisulca. Lemnaceae phylogenetic relationship based on rpS16 intron variability data has revealed significant differences between Lemna aequinoctialis and other Lemna species. Genetic distance values corroborated competence of Landoltia punctata separations from Spirodela into an independent generic taxon. The acceptability of rpS16 intron sequences for phylogenetic studies in Lemnaceae was shown.     

Influence of Giving Salicylic Acid for Different Time Periods on Flowering and Growth in the Long-Day Plant Lemna gibba G3

When the long-day plant Lemna gibba L., strain G3 is grown under continuous light on ammonium-free half-strength Hutner's medium (NH₄⁺-free 0.5 H medium) growth is excellent, but flowering is severely inhibited and often is zero. Addition of 10 micromolar salicylic acid (SA) to NH₄⁺-free 0.5 H medium quickly reverses this inhibition and leads to optimal flowering. The SA treatment also leads to a considerable reduction in the growth rate and increase in frond gibbosity. Removal of SA from the medium quickly leads to an increase in the growth rate and a large decrease in flowering. Thus, for maximal effectiveness SA must be present in the medium for the entire experiment, and the effect of SA is clearly not inductive.     

Morphogenesis of Asymmetry and Symmetry in Lemna perpusilla Torr

The determination of which of the two reproductive pockets ofLemna perpusilla will produce the first daughter frond undernon-flowering conditions, or the flower under inductive conditions,is related to asymmetric gradients within the mother frond whichmay be the result of the asymmetric position of the axillaryfrond. Treatments that encourage vigorous growth of axillaryfronds may overcome the normal situation of asymmetry and causea morphological symmetry which is qualitatively different fromthe symmetry induced by treatments (TIBA) which prevent thedevelopment of axillary fronds. The origin and maintenance ofasymmetry in Lemna are discussed. Lemna perpusilla Torr., morphogenesis, asymmetry of fronds, tri-iodobenzoic acid     

Allelopathic activity of luteolin 7-O-β-glucuronide isolated from Chrysanthemum morifolium L.

Two flavones, luteolin 7-O-β-glucuronide and diosmetin 7-O-β-glucuronide, were isolated and identified from Chrysanthemum morifolium L. v. Ramat leaves. Identification techniques included HPLC DAD, MS, ¹H and ¹³C NMR spectroscopy. At concentrations of 0.2 and 2.0 mM, luteolin 7-O-β-glucuronide significantly reduced the frond number and chlorophyll content of Lemna gibba plants, but did not significantly affect dry weight. At a concentration of 0.2 mM diosmetin 7-O-β-glucuronide had no significant effect on frond number, dry weight or chlorophyll concentration of L. gibba. These results indicate that an ortho-3′,4′-dihydroxy arrangement of the B-flavonoid ring in the luteolin compound is probably responsible for allelopathic activity.     

Growth Cycles in Lemna gibba Cultures and Their Effects on Growth Rate and Ultrastructure

A long-established axenic culture of Lemna gibba G3 was maintained in exponential growth phase under controlled conditions. Weekly analyses for 2 years showed that the individual plants of the Lemna gibba clone fluctuated between two forms. One extreme consisted of plants light in weight, small in size, and with a high relative growth rate (RGR), the other of heavy, large, and more slowly growing plants. At intervals plants having intermediate characteristics dominated in the stock culture. Indication of an annual growth-cycle was also found. The magnitude of growth response (weight, RGR, area, and dry matter content) after treatment with abscisic acid (ABA), 6-benzylaminopurine (BAP), and a combination of the two was different for low-weight and heavy plants. The heavy plants were more sensitive to ABA and BAP treatment than the low-weight ones. The accumulation of starch was least in small untreated plants and greatest in ABA treated plants. Large electron transparent globules were found in the chloroplasts of the ABA treated plants and in heavy plants regardless of how they had been treated. The different physiological and ultrastructural characteristics of the two forms of Lemna plants probably reflect an ageing-rejuvenation cycle. Emphasis is placed on the importance of this cycle when Lemna is used as a model plant in physiological experiments.     

Adaptation of Lemna paucicostata to Sublethal Methionine Deprivation

During initial exposure to 40 nanomolar propargylglycine (PAG), Lemna paucicostata colonies undergo abnormal fragmentation and a lag in frond emergence, most severe at 24 to 48 hours. Thereafter, frond emergence resumes and the frond/colony ratio rises. Such `adapted' plants withstand subculture into the same concentration of PAG without fragmentation or decreases in frond emergence, and display enhanced tolerance to higher concentrations. Adaptation is not dependent upon outgrowth of a few preexisting especially tolerant plants. Exogenous methionine prevents these events and overcomes the PAG-induced lag in frond emergence even after it is underway. These changes in frond emergence are not reflected in the rates of protein and wet weight accumulation which decrease by about 25% during the first 24 hours and continue unchanged thereafter. Cystathionine γ-synthase activity rapidly decreases to 9% of control during the first 12 hours of exposure to 40 nanomolar PAG but thereafter climbs to 12% of control. Studies of the uptake and internal concentration of PAG during these events are reported.

Exposure to a combination of 36 micromolar lysine plus 3 micromolar threonine is an alternative means to bring about sublethal methionine deprivation. Thus exposed, Lemna undergoes an analogous sequence of effects on morphology and growth which are preventable by exogenous methionine and which lead to an adapted state. Cystathionine γ-synthase specific activity in plants adapted to 36 micromolar lysine plus 3 micromolar threonine is 1.8 times control. However, addition of PAG showed that under these conditions enzyme activity can be decreased to as little as 54% of control without affecting the growth rate. Together these results suggest that adaptation is related to methionine limitation and that the plants adjust, in part, by increasing the steady-state concentrations of cystathionine γ-synthase and other enzymes in the methionine pathway.

     

Do root hydraulic properties change during the early vegetative stage of plant development in barley (Hordeum vulgare)?

Background and Aims

As annual crops develop, transpirational water loss increases substantially. This increase has to be matched by an increase in water uptake through the root system. The aim of this study was to assess the contributions of changes in intrinsic root hydraulic conductivity (Lp, water uptake per unit root surface area, driving force and time), driving force and root surface area to developmental increases in root water uptake.

Methods

Hydroponically grown barley plants were analysed during four windows of their vegetative stage of development, when they were 9–13, 14–18, 19–23 and 24–28 d old. Hydraulic conductivity was determined for individual roots (Lp) and for entire root systems (Lp_r). Osmotic Lp of individual seminal and adventitious roots and osmotic Lp_r of the root system were determined in exudation experiments. Hydrostatic Lp of individual roots was determined by root pressure probe analyses, and hydrostatic Lp_r of the root system was derived from analyses of transpiring plants.

Key Results

Although osmotic and hydrostatic Lp and Lp_r values increased initially during development and were correlated positively with plant transpiration rate, their overall developmental increases (about 2-fold) were small compared with increases in transpirational water loss and root surface area (about 10- to 40-fold). The water potential gradient driving water uptake in transpiring plants more than doubled during development, and potentially contributed to the increases in plant water flow. Osmotic Lp_r of entire root systems and hydrostatic Lp_r of transpiring plants were similar, suggesting that the main radial transport path in roots was the cell-to-cell path at all developmental stages.

Conclusions

Increase in the surface area of root system, and not changes in intrinsic root hydraulic properties, is the main means through which barley plants grown hydroponically sustain an increase in transpirational water loss during their vegetative development.     

Influence of plant maturity, shoot reproduction and sex on vegetative growth in the dioecious plant Urtica dioica

Background and Aims

Stinging nettle (Urtica dioica) is a herbaceous, dioecious perennial that is widely distributed around the world, reproduces both sexually and asexually, and is characterized by rapid growth. This work was aimed at evaluating the effects of plant maturity, shoot reproduction and sex on the growth of leaves and shoots.

Methods

Growth rates of apical shoots, together with foliar levels of phytohormones (cytokinins, auxins, absicisic acid, jasmonic acid and salicylic acid) and other indicators of leaf physiology (water contents, photosynthetic pigments, α-tocopherol and F_v/F_m ratios) were measured in juvenile and mature plants, with a distinction made between reproductive and non-reproductive shoots in both males and females. Vegetative growth rates were not only evaluated in field-grown plants, but also in cuttings obtained from these plants. All measurements were performed during an active vegetative growth phase in autumn, a few months after mature plants reproduced during spring and summer.

Key Results

Vegetative growth rates in mature plants were drastically reduced compared with juvenile ones (48 % and 78 % for number of leaves and leaf biomass produced per day, respectively), which was associated with a loss of photosynthetic pigments (up to 24 % and 48 % for chlorophylls and carotenoids, respectively) and increases of α-tocopherol (up to 2·7-fold), while endogenous levels of phytohormones did not differ between mature and juvenile plants. Reductions in vegetative growth were particularly evident in reproductive shoots of mature plants, and occurred similarly in both males and females.

Conclusions

It is concluded that (a) plant maturity reduces vegetative growth in U. dioica, (b) effects of plant maturity are evident both in reproductive and non-reproductive shoots, but particularly in the former, and (c) these changes occur similarly in both male and female plants.     

Callus induction and regeneration in<Emphasis Type="Italic"> Spirodela</Emphasis> and<Emphasis Type="Italic"> Lemna</Emphasis>

The development of tissue culture systems in duckweeds has, to date, been limited to species of the genus Lemna. We report here the establishment of an efficient tissue culture cycle (callus induction, callus growth and plant regeneration) for Spirodela oligorrhiza Hegelm SP, Spirodela punctata 8717 and Lemna gibba var. Hurfeish. Significant differences were found among the three duckweed species pertaining to carbohydrate and phytohormone requirements for callus induction, callus growth and frond regeneration. In vitro incubation with poorly assimilated carbohydrates such as galactose (S. oligorrhiza SP and L. gibba var. Hurfeish) and sorbitol (S. punctata 8717) as sole carbon source yielded high levels of callus induction on phytohormone-supplemented medium. Sorbitol is required for optimal callus growth of S. oligorrhiza SP and S. punctata 8717, while sucrose is required for callus growth of L. gibba var. Hurfeish. Sucrose either alone (S. oligorrhiza SP, L. gibba var. Hurfeish) or in addition to sorbitol (S. punctata 8717) is required for frond regeneration.Abbreviations ABA: (±)-Abscisic acid - BA: N⁶-Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - Dicamba: 3,6-Dichloro-2-methoxybenzoic acid - 2iP: N⁶-(2-Isopentenyl)adenine - NAA: -Naphthaleneacetic acid - PCA: p-Chlorophenoxy acetic acid - Picloram: 4-Amino-3,5,6-trichloropicolinic acid - TDZ: Thidiazuron Communicated by A. AltmanJ. Li and M. Jain contributed equally to the research reported in this article.     

Lack of Correlation between Senescence and Stomatal Aperture in Lemna gibba G3

Stomata on the frond of Lemna gibba G3 were found to be non-functional;they remain open regardless of treatments that lead to eitherpromotion or inhibition of frond senescence. These findingsare not in accordance with the notion that leaf senescence isprimarily mediated through the stomatal behavior. ¹Present address: Department of Botany, North Carolina StateUniversity, Raleigh, North Carolina 27695, U.S.A. (Received July 13, 1989; Accepted May 8, 1990)     

Isolation and Identification of Nicotinic Acid as a Flower-Inducing Factor in Lemna

Extracts of flowering plants of the long-day plant Lemna gibbaG3 and the short-day plants Lemna paucicostata 151 and 381 weretested on L. paucicostata 151 for flower-inducing activity.Crude extracts failed to show any activity but after severalpurification steps three fractions with flower-inducing activitywere obtained. One fraction obtained from all three plants wasshown to contain nicotinic acid by mass spectroscopic and NMRspectroscopic analyses. These results raise the possibilitythat nicotinic acid may act to influence the flowering processin Lemna. (Received August 28, 1985; Accepted October 29, 1985)