Phenotypic plasticity of sun and shade ecotypes of Stellaria longipes in response to light quality signaling, gibberellins and auxin.

Stellaria longipes plant communities (ecotypes) occur in several environmentally distinct habitats along the eastern slopes of southern Alberta's Rocky Mountains. One ecotype occurs in a prairie habitat at ∼1000 m elevation where Stellaria plants grow in an environment in which the light is filtered by taller neighbouring vegetation, i.e. sunlight with a low red to far-red (R/FR) ratio. This ecotype exhibits a high degree of phenotypic plasticity by increasing stem elongation in response to the low R/FR ratio light signal. Another Stellaria ecotype occurs nearby at ∼2400 m elevation in a much cooler alpine habitat, one where plants rarely experience low R/FR ratio shade light. Stem elongation of plants is largely regulated by gibberellins (GAs) and auxin, indole-3-acetic acid (IAA). Shoots of the prairie ecotype plants show increased IAA levels under low R/FR ratio light and they also increase their stem growth in response to applied IAA. The alpine ecotype plants show neither response. Plants from both ecotypes produce high levels of growth-active GA1 under low R/FR ratio light, though they differ appreciably in their catabolism of GA1. The alpine ecotype plants exhibit very high levels of GA8, the inactive product of GA1 metabolism, under both normal and low R/FR ratio light. Alpine origin plants may de-activate GA1 by conversion to GA8 via a constitutively high level of expression of the GA2ox gene, thereby maintaining their dwarf phenotype and exhibiting a reduced phenotypic plasticity in terms of shoot elongation. In contrast, prairie plants exhibit a high degree of phenotypic plasticity, using low R/FR ratio light-mediated changes in GA and IAA concentrations to increase shoot elongation, thereby accessing direct sunlight to optimize photosynthesis. There thus appear to be complex adaptation strategies for the two ecotypes, ones which involve modifications in the homeostasis of endogenous hormones.

Light quality-mediated petiole elongation in Arabidopsis during shade avoidance involves cell wall modification by xyloglucan endotransglucosylase/hydrolases.

Some plants can avoid shaded conditions via rapid shoot elongation, thus growing into better lit areas in a canopy. Cell wall-modifying mechanisms promoting this elongation response, therefore, are important regulatory points during shade avoidance. Two major cell wall-modifying protein families are expansins and xyloglucan endotransglucosylase/hydrolases (XTHs). The role of these proteins during shade avoidance was studied in Arabidopsis (Arabidopsis thaliana). In response to two shade cues, low red to far-red light (implying neighbor proximity) and green shade (mimicking dense canopy conditions), Arabidopsis showed classic shade avoidance features: petiole elongation and leaf hyponasty. Measurement of the apoplastic proton flux in green shade-treated petioles revealed a rapid efflux of protons into the apoplast within minutes, unlike white light controls. This apoplastic acidification probably provides the acidic pH required for the optimal activity of cell wall-modifying proteins like expansins and XTHs. Acid-induced extension, expansin susceptibility, and extractable expansin activity were similar in petioles from white light- and shade-treated plants. XTH activity, however, was high in petioles exposed to shade treatments. Five XTH genes (XTH9, -15, -16, -17, and -19) were positively regulated by low red to far-red light conditions, while the latter four and XTH22 showed a significant up-regulation also in response to green shade. Consistently, knockout mutants for two of these XTH genes also had reduced or absent shade avoidance responses to these light signals. These results point toward the cell wall as a vital regulatory point during shade avoidance.

Light irradiance differentially regulates endogenous levels of cytokinins and auxin in alpine and prairie genotypes of Stellaria longipes.

The growth patterns of plants from alpine (sun) and prairie (shade) ecotypes of Stellaria longipes in response to change in light irradiance was investigated and involvement of cytokinins (CKs), auxin (IAA) and abscisic acid (ABA) was studied to examine the mechanism behind phenotypic plasticity of these plants in response to light signalling. Low light irradiance induced shoot growth in plants of both ecotypes, but IAA levels were higher in plants from alpine, but not prairie ecotype. Dynamics of CK profiles in response to changing photosynthetically active radiation were quite different between ecotypes and changes were more pronounced in the plants of alpine ecotype, where opposite patterns in CK accumulation between low and normal light irradiances were observed. The plants of both ecotypes showed similar trends in ABA levels under low light irradiance. Thus, the highly plastic plants of prairie ecotype may have evolved mechanisms to control the growth in response to reduced light irradiance without major alterations in the levels of CKs or IAA. These results demonstrate that within species, plants from open habitats show less growth response to reduced light irradiance than plants from shaded habitats.

The regulation of cell wall extensibility during shade avoidance: a study using two contrasting ecotypes of Stellaria longipes.

Shade avoidance in plants involves rapid shoot elongation to grow toward the light. Cell wall-modifying mechanisms are vital regulatory points for control of these elongation responses. Two protein families involved in cell wall modification are expansins and xyloglucan endotransglucosylase/hydrolases. We used an alpine and a prairie ecotype of Stellaria longipes differing in their response to shade to study the regulation of cell wall extensibility in response to low red to far-red ratio (R/FR), an early neighbor detection signal, and dense canopy shade (green shade: low R/FR, blue, and total light intensity). Alpine plants were nonresponsive to low R/FR, while prairie plants elongated rapidly. These responses reflect adaptation to the dense vegetation of the prairie habitat, unlike the alpine plants, which almost never encounter shade. Under green shade, both ecotypes rapidly elongate, showing that alpine plants can react only to a deep shade treatment. Xyloglucan endotransglucosylase/hydrolase activity was strongly regulated by green shade and low blue light conditions but not by low R/FR. Expansin activity, expressed as acid-induced extension, correlated with growth responses to all light changes. Expansin genes cloned from the internodes of the two ecotypes showed differential regulation in response to the light manipulations. This regulation was ecotype and light signal specific and correlated with the growth responses. Our results imply that elongation responses to shade require the regulation of cell wall extensibility via the control of expansin gene expression. Ecotypic differences demonstrate how responses to environmental stimuli are differently regulated to survive a particular habitat.

Growth and ethylene evolution by shade and sun ecotypes of Stellaria longipes in response to varied light quality and irradiance.

Plants growing in the shade receive both low light irradiance and light enriched in far red (FR) (i.e., light with a low red (R) to FR ratio). In an attempt to uncouple the R/FR ratio effects from light irradiance effects, we utilized Stellaria longipes because this species has two distinct natural population ecotypes, alpine (dwarf) and prairie (tall). The alpine population occupies the open, sun habitat. By contrast, the prairie population grows in the shade of other plants. Both 'sun' and 'shade' ecotypes responded with increased stem elongation responses under low irradiance, relative to growth under 'normal' irradiance, and this increased growth was proportionally similar. However, only the shade ecotype had increased shoot elongation in response to a low R/FR ratio. By contrast, the sun ecotype showed increased stem elongation in response to increasing R/FR ratio. Varying the R/FR ratios had no significant effect on ethylene evolution in either sun or shade ecotype. Under low irradiance, only the sun ecotype showed a significantly changed (decreased) ethylene evolution. We conclude that R/FR ratio and irradiance both regulate growth, and that irradiance can also influence ethylene evolution of the sun ecotype. By contrast, R/FR ratio and irradiance, while having profound influences on growth of the shade ecotype, do not appear to regulate these growth changes via effects on ethylene production.

Involvement of gibberellins in the stem elongation of sun and shade ecotypes of Stellaria longipes that is induced by low light irradiance.

Plants from two ecotypes of Stellaria longipes, alpine (an open, sunny habitat) and prairie (where adjacent plants provide a shaded habitat), were grown under normal and reduced levels of photosynthetically active radiation (PAR). Growth under low PAR is significantly promoted in both ecotypes. When quantified by the stable isotope dilution method, endogenous gibberellins (GAs) (GA1, GA8, GA20, GA19) were significantly elevated under low PAR in both 'sun' and 'shade' ecotypes, as was GA53 in the shade ecotype. Changes in endogenous GA1 levels were significantly correlated with stem growth during a 28 d growth cycle and with relative growth rate (RGR) for height under low PAR for both ecotypes. Interestingly, under low irradiance PAR, changes (both increases and decreases) in GA8, the 2beta-hydroxylated 'inactive' catabolite of GA1, closely parallel bidaily stem growth changes for both ecotypes. Because the significantly greater stem elongation of both ecotypes in response to low irradiance PAR is associated with significant increases in the endogenous levels of five GAs (GA53, GA19, GA1, GA8) in the early 13-hydroxylation GA biosynthesis pathway (measured at days 7,14 and 21), we conclude that the low irradiance PAR has very likely induced an overall increase in GA biosynthesis.

Isolation and characterization of PHYC gene from Stellaria longipes: differential expression regulated by different red/far-red light ratios and photoperiods.

We have cloned and characterized the phytochrome C ( PHYC) gene from Stellaria longipes. The PHYC gene is composed of a 110-bp 5'-untranslated leader sequence, a 3,342-bp coding region, and a 351-bp 3'-untranslated sequence. The Stellaria PHYC contains three long introns within the coding region at conserved locations as in most angiosperm PHY genes. DNA blot analysis indicates that the Stellaria genome contains a single copy of PHYC. Stellaria PHYC shares 60%, 58%, and 57% deduced amino acid identities with rice, Sorghum, and Arabidopsis PHYC, respectively. Phylogenetic analysis indicates that Stellaria PHYC is located in the dicot branch, but is divergent from Arabidopsis PHYC. The Stellaria PHYC is constitutively expressed in different plant organs, though the level of PHYC gene transcript in roots is slightly higher than in flowers, leaves, and stems. When 2-week old seedlings grown in the dark were exposed to constant white light, PHYC mRNA quickly accumulates within 1-12 h. When plants grown in darkness for 7 days were exposed to different red/far-red light (R/FR) ratios, the levels of PHYC mRNA at R/FR = 0.7 are much lower than under R/FR = 3.5. The levels of PHYC mRNA under short-day (SD) photoperiod are higher than under long-day (LD) photoperiod. Plants under SD conditions do not elongate, and are only about 1.7 cm tall at 19 days. In contrast, plants under LD conditions elongate with an average height of 21.2 cm at 19 days. The plants do not flower under SD conditions, but do so at 18-19 days under LD conditions. These results indicate that under SD conditions the high level of PHYC mRNA may inhibit stem elongation and flower initiation. In contrast, under LD conditions the high level of PHYC mRNA may promote stem elongation and flowering.

Differential regulation of 1-aminocyclopropane-1-carboxylate synthase gene family and its role in phenotypic plasticity in Stellaria longipes.

Using degenerate oligonucleotides that correspond to conserved amino acid residues of known 1-aminocyclopropane-1-carboxylic acid (ACC) synthases, we cloned a genomic fragment that encodes ACC synthase in Stellaria longipes. Southern analysis suggests that ACC synthase is encoded by a small gene family comprising about 4 members. We isolated four unique ACC synthase cDNA clones under different growth conditions from alpine and prairie ecotypes of S. longipes. Northern analyses suggest that ACC synthase genes are differentially and synergistically regulated by photoperiod and temperature. Such differential regulation of ACC synthase genes positively correlate with the levels of ACC and ethylene. Since ethylene has previously been shown to partly control the stem elongation plasticity in S. longipes, we propose that differential regulation of ACC synthase genes may represent one of the underlying molecular mechanisms of phenotypic plasticity in S. longipes.

gamma-Aminobutyric acid stimulates ethylene biosynthesis in sunflower.

gamma-Aminobutyric acid (GABA), a nonprotein amino acid, is often accumulated in plants following environmental stimuli that can also cause ethylene production. We have investigated the relationship between GABA and ethylene production in excised sunflower (Helianthus annuus L.) tissues. Exogenous GABA causes up to a 14-fold increase in the ethylene production rate after about 12 h. Cotyledons fed with [14C]GABA did not release substantial amounts of radioactive ethylene despite its chemical similarity to 1-aminocyclopropane-1-carboxylic acid (ACC), indicating that GABA is not likely to be an alternative precursor for ethylene. GABA causes increases in ACC synthase mRNA accumulation, ACC levels, ACC oxidase mRNA levels, and in vitro ACC oxidase activity. In the presence of aminoethoxyvinylglycine or alpha-aminoisobutyric acid, GABA did not stimulate ethylene production. We therefore conclude that GABA stimulates ethylene biosynthesis mainly by promoting ACC synthase transcript abundance. Possible roles of GABA as a signal transducer are suggested.

Light- and temperature-entrained circadian regulation of activity and mRNA accumulation of 1-aminocyclopropane-1-carboxylic acid oxidase in Stellaria longipes.

Stem and leaf tissues of Stellaria longipes Goldie (prairie ecotype) exhibit circadian rhythmicity in the activity and mRNA abundance for 1-aminocyclopropane-1-carboxylic acid oxidase (EC 1.4.3). The steady-state mRNA levels and enzymatic activity levels fluctuated with a period of approximately 24 h and reached their maxima by the middle of the light phase and minima by the middle of the dark phase. The oscillations showed damping under constant light, constant dark and constant temperature conditions, indicating that the rhythm is entrained by an external signal. The results indicate that light/dark cycles have greater entraining effects than temperature cycles. A 15-min red light pulse, but not a blue light pulse, could reset rhythm in continuous darkness, suggesting the possible role of a red-light signal transduction pathway in the circadian regulation of 1-aminocyclopropane-1-carboxylic acid oxidase.

Molecular cloning of a cDNA encoding cytochrome c of Stellaria longipes (Caryophyllaceae)--and the evolutionary implications.

A cDNA clone encoding cytochrome c of Stellaria longipes was isolated and characterized. The nucleotide and predicted amino acid sequences were highly similar to those from other plant, fungal, and animal species. No significant polymorphism was observed among different genotypes (ecotypes). The S. longipes genome contained a low copy number of nucleotide sequences of cytochrome c. The gene expression of cytochrome c exhibited a certain degree of tissue specificity, with more transcripts in leaf than in stem. A phylogenetic analysis of the cytochrome c amino acid sequences revealed an unusual aspect of plant cytochrome c evolution.

Triose phosphate isomerase of Stellaria longipes (Caryophyllaceae).

A cDNA encoding triose phosphate isomerase (TPI) of Stellaria longipes has been isolated and sequenced. The TPI of S. longipes exhibits high similarity to the TPIs from other species at both the nucleotide and amino acid levels. Southern blot analysis showed that the S. longipes genome contained multiple TPI-like nucleotide sequences and only a low degree of polymorphism was observed among genotypes of different habitats. The levels of TPI mRNA, detected by hybridization to the TPI cDNA, appeared similar between the genotypes. However, different genotypes showed variations in the total TPI activity, reflecting the possible ecological effect on the TPI gene expression. Northern blot hybridization to the cDNA also indicated a higher level of TPI mRNA in leaves than in roots, suggesting tissue-specific expression of TPI gene(s). Phylogenetic analysis of the TPI amino acid sequences from 16 taxa suggested that the TPI from S. longipes was more closely related to cytosolic TPIs from other plants than it was to TPIs from prokaryotes.

Chloroplast DNA inheritance in theStellaria longipes complex (Caryophyllaceae).

Inheritance of chloroplast DNA (cpDNA) was examined in F1 progenies derived from three crosses and three corresponding reciprocal crosses betweenStellaria porsildii andS. longifolia. Chloroplast DNA restriction fragments were analyzed using methods of nonradioactive digoxigenin-11-dUTP labeling and chemiluminescent detection with Lumi-Phos 530. Distinct interspecific restriction fragment polymorphisms were identified and used to demonstrate the mode of cpDNA inheritance. Mode of cpDNA inheritance differed among crosses. Two crosses in whichS. porsildii, SP2920-21, was the maternal parent exhibited three different types of plastids, maternal, paternal and biparental, among the F1 hybrids, suggesting a biparental cpDNA inheritance and plastid sorting-out inStellaria.

EVOLUTION OF PHENOTYPIC PLASTICITY IN THE STELLARIA LONGIPES COMPLEX: COMPARISONS AMONG CYTOTYPES AND HABITATS.

Variation in the amount and pattern of plasticity was studied in three cytotypes (4x, 6x, and 8x) of Stellaria longipes and diploids of its suspected progenitor S. longifolia. All 13 traits considered showed plasticity. There were significant differences among cytotypes and habitats in plasticity for many traits. Overall, the diploids, S. longifolia, were most plastic, and the three cytotypes of S. longipes did not differ in amount of plasticity. Stellaria longifolia showed divergence from S. longipes in the pattern of plasticity as well. In general, cytotypes with more similar chromosome numbers had the same pattern of plasticity for more traits. Individuals from tundra populations differed in their pattern of plasticity from those of montane, boreal, and prairie origin, which were more similar to one another. Differences in plasticity among cytotypes were due primarily to divergence in amount, while differences among habitats were most often accounted for by divergent patterns of plasticity. We conclude that both polyploidy and natural selection have affected the evolution of plastic responses in this species complex. Analysis of the correlation between pairs of traits provided evidence that the pattern and amount of plasticity operate independently of one another and may be evolving separately.