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1.  Effects of water availability and pest pressures on tea (Camellia sinensis) growth and functional quality 
AoB Plants  2014;6:plt054.
Extreme shifts in water availability linked to global climate change are impacting crops worldwide. This study examines effects of water availability and pest pressures on the growth and functional quality of tea, the world's most consumed beverage after water. Results show that higher water availability and pest pressures significantly increased the growth of new leaves while their effect on tea quality varied with individual secondary metabolites. Findings point to the fascinating dynamics of climate change effects on tea plants with offsetting interactions between rainfall and pest pressures and the need for future climate studies to examine interactive environmental effects.
Extreme shifts in water availability linked to global climate change are impacting crops worldwide. The present study examines the direct and interactive effects of water availability and pest pressures on tea (Camellia sinensis; Theaceae) growth and functional quality. Manipulative greenhouse experiments were used to measure the effects of variable water availability and pest pressures simulated by jasmonic acid (JA) on tea leaf growth and secondary metabolites that determine tea quality. Water treatments were simulated to replicate ideal tea growing conditions and extreme precipitation events in tropical southwestern China, a major centre of tea production. Results show that higher water availability and JA significantly increased the growth of new leaves while their interactive effect was not significant. The effect of water availability and JA on tea quality varied with individual secondary metabolites. Higher water availability significantly increased total methylxanthine concentrations of tea leaves but there was no significant effect of JA treatments or the interaction of water and JA. Water availability, JA treatments or their interactive effects had no effect on the concentrations of epigallocatechin 3-gallate. In contrast, increased water availability resulted in significantly lower concentrations of epicatechin 3-gallate but the effect of JA and the interactive effects of water and JA were not significant. Lastly, higher water availability resulted in significantly higher total phenolic concentrations but there was no significant impact of JA and their interaction. These findings point to the fascinating dynamics of climate change effects on tea plants with offsetting interactions between precipitation and pest pressures within agro-ecosystems, and the need for future climate studies to examine interactive biotic and abiotic effects.
PMCID: PMC3922301  PMID: 24790117
Camellia sinensis; catechins; climate change; herbivory; methylxanthines; precipitation; tea; total phenolic concentrations.
2.  Bark and leaf chlorophyll fluorescence are linked to wood structural changes in Eucalyptus saligna 
AoB Plants  2014;6:plt057.
The efficiency of leaf photosynthesis has been measured using a technique called chlorophyll fluorescence. It is not widely known that in addition to leaf photosynthesis the bark of certain “smooth barked” trees can photosynthesize. In this paper we use chlorophyll fluorescence to measure the efficiency of bark photosynthesis. In this way we are able to compare the amount of wood decay in a tree with bark photosynthetic efficiency using chlorophyll fluorescence. The link between bark photosynthesis and wood decay discovered in this work has not been explored before.
Wood structure and wood anatomy are usually considered to be largely independent of the physiological processes that govern tree growth. This paper reports a statistical relationship between leaf and bark chlorophyll fluorescence and wood density. A relationship between leaf and bark chlorophyll fluorescence and the quantity of wood decay in a tree is also described. There was a statistically significant relationship between the leaf chlorophyll fluorescence parameter Fv/Fm and wood density and the quantity of wood decay in summer, but not in spring or autumn. Leaf chlorophyll fluorescence at 0.05 ms (the O step) could predict the quantity of wood decay in trees in spring. Bark chlorophyll fluorescence could predict wood density in spring using the Fv/Fm parameter, but not in summer or autumn. There was a consistent statistical relationship in spring, summer and autumn between the bark chlorophyll fluorescence parameter Fv/Fm and wood decay. This study indicates a relationship between chlorophyll fluorescence and wood structural changes, particularly with bark chlorenchyma.
PMCID: PMC3922302  PMID: 24790120
Bark; chlorophyll fluorescence; photosynthesis; stress physiology; wood decay; wood structure.
3.  Rapid flooding-induced adventitious root development from preformed primordia in Solanum dulcamara 
AoB Plants  2014;6:plt058.
Flooding strongly affects plant growth, as it leads to low oxygen concentrations in the submerged tissues. Understanding plant responses to flooding may benefit both management of wetland ecosystems and improve progress in creating flood-tolerant crop species. Bittersweet (Solanum dulcamara), a species related to tomato and eggplant, has dormant primordia on the stem that develop into adventitious roots within 3 days of flooding. Changes in gene expression were present within 2 hours and included activation of hypoxia and ethylene signalling genes. Unexpectedly, these early changes in gene expression were closely similar in primordia and adjacent stem tissue, suggesting a dominant general response in tissues during early flooding.
Flooding is a common stress factor in both natural and agricultural systems, and affects plant growth by the slow diffusion rate of gases in water. This results in low oxygen concentrations in submerged tissues, and hence in a decreased respiration rate. Understanding the responses of plants to flooding is essential for the management of wetland ecosystems, and may benefit research to improve the flood tolerance of crop species. This study describes the response to partial submergence of bittersweet (Solanum dulcamara). Bittersweet is a Eurasian species that grows both in dry habitats such as coastal dunes, and in wetlands, and therefore is a suitable model plant for studying responses to a variety of environmental stresses. A further advantage is that the species is closely related to flood-intolerant crops such as tomato and eggplant. The species constitutively develops dormant primordia on the stem, which we show to have a predetermined root identity. We investigated adventitious root growth from these primordia during flooding. The synchronized growth of roots from the primordia was detected after 2–3 days of flooding and was due to a combination of cell division and cell elongation. Gene expression analysis demonstrated that the molecular response to flooding began within 2 h and included activation of hypoxia and ethylene signalling genes. Unexpectedly, these early changes in gene expression were very similar in primordia and adjacent stem tissue, suggesting that there is a dominant general response in tissues during early flooding.
PMCID: PMC3922303  PMID: 24790121
Adventitious roots; cDNA-AFLP; gene expression; partial submergence; root primordia; soil flooding; Solanum dulcamara; waterlogging.
4.  Chloroplast genes as genetic markers for inferring patterns of change, maternal ancestry and phylogenetic relationships among Eleusine species 
AoB Plants  2014;6:plt056.
Phylogenetic relationship between the nine species of Eleusine was investigated based on RFLP of the seven amplified chloroplast genes/intergenic spacers, trnK gene sequence and cpSSR markers. The maternal genome donor (E. indica, 2n=2x=18) of the allotetraploid (2n=4x=36, 2n=2x=38) Eleusine species, and the phylogenetic relationships between cultivated E. coracana (2n=4x=36) and wild species have been successfully resolved. The species-specific markers were also identified. The explicit identification of the maternal parent and that of the immediate wild progenitor of finger millet will be immensely useful for future genetic improvement and biotechnological program(s) of the crop species.
Assessment of phylogenetic relationships is an important component of any successful crop improvement programme, as wild relatives of the crop species often carry agronomically beneficial traits. Since its domestication in East Africa, Eleusine coracana (2n = 4x = 36), a species belonging to the genus Eleusine (x = 8, 9, 10), has held a prominent place in the semi-arid regions of India, Nepal and Africa. The patterns of variation between the cultivated and wild species reported so far and the interpretations based upon them have been considered primarily in terms of nuclear events. We analysed, for the first time, the phylogenetic relationship between finger millet (E. coracana) and its wild relatives by species-specific chloroplast deoxyribonucleic acid (cpDNA) polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and chloroplast simple sequence repeat (cpSSR) markers/sequences. Restriction fragment length polymorphism of the seven amplified chloroplast genes/intergenic spacers (trnK, psbD, psaA, trnH–trnK, trnL–trnF, 16S and trnS–psbC), nucleotide sequencing of the chloroplast trnK gene and chloroplast microsatellite polymorphism were analysed in all nine known species of Eleusine. The RFLP of all seven amplified chloroplast genes/intergenic spacers and trnK gene sequences in the diploid (2n = 16, 18, 20) and allotetraploid (2n = 36, 38) species resulted in well-resolved phylogenetic trees with high bootstrap values. Eleusine coracana, E. africana, E. tristachya, E. indica and E. kigeziensis did not show even a single change in restriction site. Eleusine intermedia and E. floccifolia were also shown to have identical cpDNA fragment patterns. The cpDNA diversity in Eleusine multiflora was found to be more extensive than that of the other eight species. The trnK gene sequence data complemented the results obtained by PCR–RFLP. The maternal lineage of all three allotetraploid species (AABB, AADD) was the same, with E. indica being the maternal diploid progenitor species. The markers specific to certain species were also identified.
PMCID: PMC3924058  PMID: 24790119
cpSSR; Eleusine; PCR–RFLP; phylogeny; Poaceae; trnK gene sequence.
5.  Genetic variation among mainland and island populations of a native perennial grass used in restoration 
AoB Plants  2014;6:plt055.
Knowledge of species-level patterns of genetic diversity can inform and improve protocols when population reintroduction is a restoration objective. We describe the population genetic structure of a geographically widespread species, Elymus glaucus, which is now rare in temperate grasslands as a result of biological invasion and land conversion. Our study contrasts data for mainland and Channel Island locations, and makes recommendations for seed provenance selection in ecological restoration using genetic marker data and considering prior field studies of adaptive divergence
Genetic marker studies can assist restoration practice through selection of seed sources that conserve historical levels of gene diversity and population genetic differentiation. We examined genetic variation and structure within and among mainland and island populations of Elymus glaucus, a perennial bunchgrass species native to western North American grasslands that is targeted for grassland restoration. Island populations of E. glaucus represent sensitive sites and potentially distinctive seed sources for reintroduction, and little is known of their genetic composition. Genetic diversity and structure were estimated using amplified fragment length polymorphism markers for 21 populations and 416 individuals distributed across two coastal California mainland locations and three California Channel Islands. Eight primer combinations resulted in 166 markers, of which 165 (99.4 %) were polymorphic. The number of polymorphic bands was significantly greater among mainland populations relative to island sites, and locally common alleles were present for each sampled island and mainland location. Population structure was high (62.9 %), with most variation (55.8 %) distributed among populations, 7.1 % between mainland and island locations, and the remainder (37.1 %) within populations. Isolation by distance was only apparent among islands. Using marker data to recommend appropriate seed sources for restoration, E. glaucus seeds are best derived within islands with collections representing a large number of individuals from matching environments. Given the limited gene flow and prior evidence of adaptive divergence among populations of this species, regional collections are recommended in all cases to maintain diversity and to avoid long-distance introductions of highly differentiated plant material.
PMCID: PMC3966692  PMID: 24790118
AFLP markers; California Channel Islands; ecological restoration; Elymus glaucus; genetic drift; seed source; self-pollination; spatial genetic structure.
6.  Comparison of the morphogenesis of three genotypes of pea (Pisum sativum) grown in pure stands and wheat-based intercrops 
AoB Plants  2014;6:plu006.
In intercropping systems, plant morphology highly determines the amount of resources captured by each component species. However, morphogenesis of cultivated species has been mainly described in mono-specific growing conditions, although plasticity can occur in multi-specific stands. This paper reports on the variability of the morphogenesis of three pea genotypes grown in pure stands and mixed with wheat. Most morphogenetic parameters of pea were dependent on the genotype. However, there was low variability of pea morphogenesis between sole and mixed stands, except for plant height and branching of the long cycle cultivar.
Cereal–legume intercrops represent a promising way of combining high productivity and agriculture sustainability. The benefits of cereal–legume mixtures are highly affected by species morphology and functioning, which determine the balance between competition and complementarity for resource acquisition. Studying species morphogenesis, which controls plant architecture, is therefore of major interest. The morphogenesis of cultivated species has been mainly described in mono-specific growing conditions, although morphogenetic plasticity can occur in multi-specific stands. The aim of the present study was therefore to characterize the variability of the morphogenesis of pea plants grown either in pure stands or mixed with wheat. This was achieved through a field experiment that included three pea cultivars with contrasting earliness (hr and HR type) and branching patterns. Results show that most of the assessed parameters of pea morphogenesis (phenology, branching, final number of vegetative organs and their kinetics of appearance) were mainly dependent on the considered genotype, which highlights the importance of the choice of cultivars in intercropping systems. There was however a low variability of pea morphogenesis between sole and mixed stands except for plant height and branching of the long-cycle cultivar. The information provided in the present study at stand and plant scale can be used to build up structural–functional models. These models can contribute to improving the understanding of the functioning of cereal–legume intercrops and also to the definition of plant ideotypes adapted to the growth in intercrops.
PMCID: PMC3974333  PMID: 24790127
Morphogenesis; Pisum sativum; plant architecture; plasticity; Triticum aestivum; wheat–pea intercropping.
7.  Growing up or growing out? How soil pH and light affect seedling growth of a relictual rainforest tree 
AoB Plants  2014;6:plu011.
Our aim was to uncover the drivers of seedling growth in a rare rainforest conifer. Wollemia nobilis is limited to canyons, characterized by deeply shaded understories and acid soils. In a glasshouse experiment, we grew seedlings at a range of pH and light levels. Growth increased with increasing light, and was higher at low pH, regardless of light. Number of stems, however, was greatest in lower light. Wollemia nobilis seedlings may vary their architecture - growing up when light is high, and growing out when light is lower. Nevertheless, low light is likely the key limitation of W. nobilis growth in the wild.
Seedling growth rates can have important long-term effects on forest dynamics. Environmental variables such as light availability and edaphic factors can exert a strong influence on seedling growth. In the wild, seedlings of Wollemi pine (Wollemia nobilis) grow on very acid soils (pH ∼4.3) in deeply shaded sites (∼3 % full sunlight). To examine the relative influences of these two factors on the growth of young W. nobilis seedlings, we conducted a glasshouse experiment growing seedlings at two soil pH levels (4.5 and 6.5) under three light levels: low (5 % full sun), medium (15 %) and high (50 %). Stem length and stem diameter were measured, stem number and branch number were counted, and chlorophyll and carotenoid content were analysed. In general, increased plant growth was associated with increased light, and with low pH irrespective of light treatment, and pigment content was higher at low pH. Maximum stem growth occurred in plants grown in the low pH/high light treatment combination. However, stem number was highest in low pH/medium light. We hypothesize that these differences in stem development of W. nobilis among light treatments were due to this species' different recruitment strategies in response to light: greater stem growth at high light and greater investment in multiple stem production at low light. The low light levels in the W. nobilis habitat may be a key limitation on stem growth and hence W. nobilis recruitment from seedling to adult. Light and soil pH are two key factors in the growth of this threatened relictual rainforest species.
PMCID: PMC4004931  PMID: 24790132
Araucariaceae; conifer; conservation; light; rainforest; relictual species; soil pH; threatened species; Wollemi pine.
8.  Learning from nature: the use of non-model species to identify novel acclimations to flooding stress 
AoB Plants  2014;6:plu016.
Floods have a severe impact on plant performance. In general, crops are flood intolerant and are at an increased risk to flooding events due to global climate change. It is a grand challenge to improve agricultural production to feed the increasing human world population despite the increase of flooding stress. Mechanistic understanding of flooding tolerance is mainly achieved with model species such as Oryza sativa and Arabidopsis thaliana. However, wild plants from flood-prone environments have evolved in frequently flooded environments and therefore possess unique traits that facilitate growth and reproduction during and after flooding stress. Flooding research with these non-model wild plants might help us to identify novel adaptive traits that can be applied to improve flooding tolerance of crops.
Excess water in the form of waterlogged soil or deeper submergence (generically termed ‘flooding’) influences plant growth, survival and species distribution in many natural ecosystems. It also has a negative impact on crop growth and yield since many agricultural species are flooding intolerant. The often devastating effect of flooding on plant performance is related to its interference with gas exchange between the plant and its environment. This results in energy deficiency and carbohydrate starvation. In the near future, flooding frequency is expected to increase due to global climate change and the human population is expected to increase to ∼9 billion people by 2050. The need for increased agricultural productivity is self-evident and this will require a better mechanistic understanding of the interaction between plants and abiotic stresses such as flooding. We argue that, in seeking this understanding, we should not restrict the research to model species such as rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana). This is because some stress-tolerance mechanisms are not found in these species. Examples are given of how flooding tolerance is achieved by non-model species of Rumex and Rorippa that have evolved to cope with flooding in natural environments. These findings will add usefully to the spread of resources available to plant breeding programmes aimed at improving flooding tolerance in crop plants.
PMCID: PMC4011469  PMID: 24876298
Abscisic acid; climate change; ethylene; flooding; learning from nature; Rorippa; Rumex; waterlogging.
9.  Molecular basis of floral petaloidy: insights from androecia of Canna indica 
AoB Plants  2014;6:plu015.
Floral organs usually take on the characteristics of petals in ginger plants. In Canna indica, the most ornamental parts of the flowers are considered to be staminode, which means rudimentary and sterile stamen. However, the precise nature of these petaloid organs is yet to be determined. Two floral organ identity genes GLOBOSA (GLO) and AGAMOUS (AG) are isolated from Canna indica. Their expression patterns suggest that petaloid staminodes and labellum are of androecium identity, in agreement with their position within the flower. But the current molecular, morphological and anatomical data, are still not sufficient to explain the distinct morphology observed in staminodes and fertile stamen in Canna indica.
Floral organs that take on the characteristics of petals can occur in all whorls of the monocot order Zingiberales. In Canna indica, the most ornamental or ‘petaloid’ parts of the flowers are of androecial origin and are considered staminodes. However, the precise nature of these petaloid organs is yet to be determined. In order to gain a better understanding of the genetic basis of androecial identity, a molecular investigation of B- and C-class genes was carried out. Two MADS-box genes GLOBOSA (GLO) and AGAMOUS (AG) were isolated from young inflorescences of C. indica by 3′ rapid amplification of cDNA ends polymerase chain reaction (3′-RACE PCR). Sequence characterization and phylogenetic analyses show that CiGLO and CiAG belong to the B- and C-class MADS-box gene family, respectively. CiAG is expressed in petaloid staminodes, the labellum, the fertile stamen and carpels. CiGLO is expressed in petals, petaloid staminodes, the labellum, the fertile stamen and carpels. Expression patterns in mature tissues of CiGLO and CiAG suggest that petaloid staminodes and the labellum are of androecial identity, in agreement with their position within the flower and with described Arabidopsis thaliana expression patterns. Although B- and C-class genes are important components of androecial determination, their expression patterns are not sufficient to explain the distinct morphology observed in staminodes and the fertile stamen in C. indica.
PMCID: PMC4011470  PMID: 24876297
ABC model; Canna indica; floral organ identity; MADS-box gene; phylogenetic analysis; real-time PCR.
10.  Bulk elastic moduli and solute potentials in leaves of freshwater, coastal and marine hydrophytes. Are marine plants more rigid? 
AoB Plants  2014;6:plu014.
The flexibility of plant cell walls is characterized by bulk modulus of elasticity (ϵ); which is an important component of how plants maintain adequate water continent. For example, plants with rigid tissues (high ϵ) that accumulate solutes may better tolerate drought or saline soils. This concept is termed the ‘cell water conservation hypothesis.’ While it is generally held that marine plants have higher ϵ, no study has considered that notion across a number of species residing in marine and coastal habitats. The finding from this study show that aquatic marine plants do maintain rigid tissues with lower osmotic potentials (relative to freshwater plants), and support the tenets of the cell water conservation hypothesis
Bulk modulus of elasticity (ɛ), depicting the flexibility of plant tissues, is recognized as an important component in maintaining internal water balance. Elevated ɛ and comparatively low osmotic potential (Ψπ) may work in concert to effectively maintain vital cellular water content. This concept, termed the ‘cell water conservation hypothesis’, may foster tolerance for lower soil-water potentials in plants while minimizing cell dehydration and shrinkage. Therefore, the accumulation of solutes in marine plants, causing decreases in Ψπ, play an important role in plant–water relations and likely works with higher ɛ to achieve favourable cell volumes. While it is generally held that plants residing in marine systems have higher leaf tissue ɛ, to our knowledge no study has specifically addressed this notion in aquatic and wetland plants residing in marine and freshwater systems. Therefore, we compared ɛ and Ψπ in leaf tissues of 38 freshwater, coastal and marine plant species using data collected in our laboratory, with additional values from the literature. Overall, 8 of the 10 highest ɛ values were observed in marine plants, and 20 of the lowest 25 ɛ values were recorded in freshwater plants. As expected, marine plants often had lower Ψπ, wherein the majority of marine plants were below −1.0 MPa and the majority of freshwater plants were above −1.0 MPa. While there were no differences among habitat type and symplastic water content (θsym), we did observe higher θsym in shrubs when compared with graminoids, and believe that the comparatively low θsym observed in aquatic grasses may be attributed to their tendency to develop aerenchyma that hold apoplastic water. These results, with few exceptions, support the premise that leaf tissues of plants acclimated to marine environments tend to have higher ɛ and lower Ψπ, and agree with the general tenets of the cell water conservation hypothesis.
PMCID: PMC4025192  PMID: 24876296
Bulk elastic modulus; halophytes; hydrophytes; salinity; solute potential; symplastic water content.
11.  Poplar saplings exposed to recurring temperature shifts of different amplitude exhibit differences in leaf gas exchange and growth despite equal mean temperature 
AoB Plants  2014;6:plu018.
Most investigations of plant responses to changes in temperature have focused on a constant increase in temperature. However, changes in fluctuation in temperature, even if the mean temperature is the same, may affect plant growth. We tested the effects of weekly warm and then cool moderate (5°C) and large (10°C) fluctuation in temperature (with the same biweekly temperature sum) on plant growth. We found that, while the ratio of photosynthesis to respiration did not change, fluctuations in temperature did increase biomass accumulation and alter biomass allocation. Our findings suggest that, like mean temperature, fluctuation in temperature can significantly impact plant growth.
Most investigations of plant responses to changes in temperature have focused on a constant increase in mean day/night temperature without considering how differences in temperature cycles can affect physiological processes and growth. To test the effects of changes in growth temperature on foliar carbon balance and plant growth, we repeatedly exposed poplar saplings (Populus deltoides × nigra) to temperature cycles consisting of 5 days of a moderate (M, +5 °C) or extreme (E, +10 °C) increase in temperature followed by 5 days of a moderate (M, −5 °C) or extreme (E, −10 °C) decrease in temperature, with respect to a control treatment (C, 23.4 °C). The temperature treatments had the same mean temperature over each warm and cool cycle and over the entire study. Our goal was to examine the influence of recurring temperature shifts on growth. Net photosynthesis (A) was relatively insensitive to changes in growth temperature (from 20 to 35 °C), suggesting a broad range of optimum temperature for photosynthesis. Leaf respiration (R) exhibited substantial acclimation to temperature, having nearly the same rate at 13 °C as at 33 °C. There was no evidence that preconditioning through temperature cycles affected the response of A or R to treatment temperature fluctuations. Averaged across the complete warm/cool temperature cycle, the A : R ratio did not differ among the temperature treatments. While foliar carbon balance was not affected, the temperature treatments significantly affected growth. Whole-plant biomass was 1.5 times greater in the M treatment relative to the C treatment. Carbon allocation was also affected with shoot volume and biomass greater in the M and E treatments than in the C treatment. Our findings indicate that temperature fluctuations can have important effects on growth, though there were few effects on leaf gas exchange, and can help explain differences in growth that are not correlated with mean growth temperature.
PMCID: PMC4029210  PMID: 24876300
A : R ratio; carbon allocation; leaf proteins; Populus deltoides × nigra.
12.  Influence of root-bed size on the response of tobacco to elevated CO2 as mediated by cytokinins 
AoB Plants  2014;6:plu010.
The effect of elevated CO2 on the growth of tobacco under high light (16 h), continuous water and nutrient supply was investigated. Biomass production depended strongly on the size of the root bed. Inhibition by a small root bed was higher at 700 than at 360 ppm CO2. Relative growth rates showed a head-start of the high-CO2 plants which gave rise to a persistently higher biomass production. Root-bed size and CO2 concentration were mirrored by the quantitative cytokinin patterns of the various plant parts. Amounts of the cytokinins moving from the root to the shoot were higher in high-CO2 plants.
The extent of growth stimulation of C3 plants by elevated CO2 is modulated by environmental factors. Under optimized environmental conditions (high light, continuous water and nutrient supply, and others), we analysed the effect of an elevated CO2 atmosphere (700 ppm, EC) and the importance of root-bed size on the growth of tobacco. Biomass production was consistently higher under EC. However, the stimulation was overridden by root-bed volumes that restricted root growth. Maximum growth and biomass production were obtained at a root bed of 15 L at ambient and elevated CO2 concentrations. Starting with seed germination, the plants were strictly maintained under ambient or elevated CO2 until flowering. Thus, the well-known acclimation effect of growth to enhanced CO2 did not occur. The relative growth rates of EC plants exceeded those of ambient-CO2 plants only during the initial phases of germination and seedling establishment. This was sufficient for a persistently higher absolute biomass production by EC plants in non-limiting root-bed volumes. Both the size of the root bed and the CO2 concentration influenced the quantitative cytokinin patterns, particularly in the meristematic tissues of shoots, but to a smaller extent in stems, leaves and roots. In spite of the generally low cytokinin concentrations in roots, the amounts of cytokinins moving from the root to the shoot were substantially higher in high-CO2 plants. Because the cytokinin patterns of the (xylem) fluid in the stems did not match those of the shoot meristems, it is assumed that cytokinins as long-distance signals from the roots stimulate meristematic activity in the shoot apex and the sink leaves. Subsequently, the meristems are able to synthesize those phytohormones that are required for the cell cycle. Root-borne cytokinins entering the shoot appear to be one of the major control points for the integration of various environmental cues into one signal for optimized growth.
PMCID: PMC4038427  PMID: 24790131
Biomass portioning; C/N ratio; cytokinins; elevated CO2; growth; root-bed volume; tobacco.
13.  Identifying the genes underlying quantitative traits: a rationale for the QTN programme 
AoB Plants  2014;6:plu004.
Identifying quantitative trait nucleotides (QTNs), the genetic polymorphisms linked to phenotypic variation, has become a goal for many plant ecologists and evolutionary biologists in recent years. But what is the true value of this potentially expensive and labor intensive programme of research? In this review we discuss the ways by which the QTN programme can offer unique insight into the ecology and evolution of adaptation in plants. We cite recent noteworthy examples of QTN work and provide recommendations for refocusing efforts to identify and study the genes underlying ecologically important traits.
The goal of identifying the genes or even nucleotides underlying quantitative and adaptive traits has been characterized as the ‘QTN programme’ and has recently come under severe criticism. Part of the reason for this criticism is that much of the QTN programme has asserted that finding the genes and nucleotides for adaptive and quantitative traits is a fundamental goal, without explaining why it is such a hallowed goal. Here we outline motivations for the QTN programme that offer general insight, regardless of whether QTNs are of large or small effect, and that aid our understanding of the mechanistic dynamics of adaptive evolution. We focus on five areas: (i) vertical integration of insight across different levels of biological organization, (ii) genetic parallelism and the role of pleiotropy in shaping evolutionary dynamics, (iii) understanding the forces maintaining genetic variation in populations, (iv) distinguishing between adaptation from standing variation and new mutation, and (v) the role of genomic architecture in facilitating adaptation. We argue that rather than abandoning the QTN programme, we should refocus our efforts on topics where molecular data will be the most effective for testing hypotheses about phenotypic evolution.
PMCID: PMC4038433  PMID: 24790125
Adaptation; ecological genomics; ecologically important traits; genetic variation; phenotypic evolution; population genomics; QTL; QTN; quantitative genetics; vertical integration.
14.  Waterlogging-induced changes in root architecture of germplasm accessions of the tropical forage grass Brachiaria humidicola 
AoB Plants  2014;6:plu017.
Brachiaria humidicola, a tropical forage grass, develops aerenchyma in nodal roots to adapt to waterlogging. A large body of work has focused on the functional role of aerenchyma in nodal roots under waterlogged soil conditions. On the other hand, quantification of responses of lateral roots to waterlogging has been often overlooked in past work. Our data indicated that although waterlogging reduced the overall proportion of lateral roots, its proportion significantly increased in the top 10 cm of the soil. This suggests that soil flooding increases lateral root proliferation of B. humidicola in upper soil layers. This may compensate the reduction of root surface area brought by the restriction of root growth at depths below 30 cm into waterlogged soil.
Waterlogging is one of the major factors limiting the productivity of pastures in the humid tropics. Brachiaria humidicola is a forage grass commonly used in zones prone to temporary waterlogging. Brachiaria humidicola accessions adapt to waterlogging by increasing aerenchyma in nodal roots above constitutive levels to improve oxygenation of root tissues. In some accessions, waterlogging reduces the number of lateral roots developed from main root axes. Waterlogging-induced reduction of lateral roots could be of adaptive value as lateral roots consume oxygen supplied from above ground via their parent root. However, a reduction in lateral root development could also be detrimental by decreasing the surface area for nutrient and water absorption. To examine the impact of waterlogging on lateral root development, an outdoor study was conducted to test differences in vertical root distribution (in terms of dry mass and length) and the proportion of lateral roots to the total root system (sum of nodal and lateral roots) down the soil profile under drained or waterlogged soil conditions. Plant material consisted of 12 B. humidicola accessions from the gene bank of the International Center for Tropical Agriculture, Colombia. Rooting depth was restricted by 21 days of waterlogging and confined to the first 30 cm below the soil surface. Although waterlogging reduced the overall proportion of lateral roots, its proportion significantly increased in the top 10 cm of the soil. This suggests that soil flooding increases lateral root proliferation of B. humidicola in the upper soil layers. This may compensate for the reduction of root surface area brought about by the restriction of root growth at depths below 30 cm. Further work is needed to test the relative efficiency of nodal and lateral roots for nutrient and water uptake under waterlogged soil conditions.
PMCID: PMC4038435  PMID: 24876299
Lateral root proportion; oxygen deficiency; rooting depth; root length; soil flooding; vertical root distribution.
15.  Live cell imaging reveals extensive intracellular cytoplasmic colonization of banana by normally non-cultivable endophytic bacteria 
AoB Plants  2014;6:plu002.
The general understanding about endophytic microorganisms is that they are inhabitants in the free intercellular spaces primarily in roots. This study uncovers extensive cytoplasmic colonization by endophytic bacteria in banana shoot-tissue which prima-facie appeared like ‘Brownian movement’. Live cell imaging on tissue sections, callus, cell suspensions and protoplasts directly and after vital / SYTO-9 staining has brought out two intracellular niches, namely cytoplasmic and periplasmic. Designated as ‘Cytobacts’ and ‘Peribacts’, these organisms appeared to be normally not amenable for cultivation and thus possibly escaped the attention of biologists. The observations here open the way to study these intracellular entities distinct from micro-organelles.
It is generally believed that endophytic microorganisms are intercellular inhabitants present in either cultivable or non-cultivable form primarily as root colonizers. The objective of this study was to determine whether the actively mobile micro-particles observed in the intracellular matrix of fresh tissue sections of banana included endophytic bacteria. Tissue sections (50–100 µm) from apical leaf sheaths of surface-disinfected suckers (cv. Grand Naine) displayed ‘Brownian motion’-reminiscent abundant motile micro-particles under bright-field and phase-contrast (×1000), which appeared similar in size and motility to the pure cultures of endophytes previously isolated from banana. Observations on callus, embryonic cells and protoplasts with intact cell wall/plasma membrane confirmed their cytoplasmic nature. The motility of these entities reduced or ceased upon tissue fixation or staining with safranin/crystal violet (0.5 % w/v), but continued uninterrupted following treatment with actin-disrupting drugs, ruling out the possibility of micro-organelles like peroxisomes. Staining with 2,3,5-triphenyl tetrazolium chloride (TTC) confirmed them to be live bacteria with similar observations after dilute safranin (0.005 %) treatment. Tissue staining with SYTO-9 coupled with epi-fluorescence or confocal laser scanning microscopy showed bacterial colonization along the peri-space between cell wall and plasma membrane initially. SYTO-9 counterstaining on TTC- or safranin-treated tissue and those subjected to enzymatic permeabilization revealed the cytoplasmic bacteria. These included organisms moving freely in the cytoplasm and those adhering to the nuclear envelope or vacuoles and the intravacuolar colonizers. The observations appeared ubiquitous to different genomes and genotypes of banana. Plating the tissue homogenate on nutrient media seldom yielded colony growth. This study, supported largely by live cell video-imaging, demonstrates enormous intracellular colonization in bananas by normally non-cultivable endophytic bacteria in two niches, namely cytoplasmic and periplasmic, designated as ‘Cytobacts’ and ‘Peribacts’, respectively. The integral intracellular association with their clonal perpetuation suggests a mutualistic relationship between endophytes and the host.
PMCID: PMC4038436  PMID: 24790123
Bacterial endophytes; Brownian motion; confocal laser scanning microscopy; epi-fluorescence microscopy; micropropagation; Musa sp.; non-cultivable bacteria; plant tissue culture; triphenyl tetrazolium chloride.
16.  Inbreeding depression does not increase in foreign environments: a field experimental study 
AoB Plants  2014;6:plu009.
Inbreeding depression can lower individual fitness and cause the extinction of populations. As a result, it is of interest to evolutionary biologists and conservationists alike. Studies have shown that inbreeding depression can increase in stressful environments. However, most of these studies do not utilize natural environmental stress. Hereford tested how natural environmental stress from transplanting into foreign habitats influences inbreeding depression. While there was significant inbreeding depression, there was no difference in inbreeding depression between plants in their native environment versus foreign habitats. These results imply that inbreeding depression does not increase when environmental stress reflects natural variation.
Early successional species often disperse to novel environments, and if they are selfing, this dispersal will frequently be carried out by inbred individuals. If inbred immigrants are less likely to successfully establish populations than outbred immigrants, dispersal will be less effective and mating system evolution will favour outcrossing. I performed a reciprocal transplant of inbred and outbred plants grown in native and foreign planting sites to test the hypothesis that inbred immigrants had lower fitness. Inbreeding within populations was estimated with allozyme loci to confirm that the populations were inbred. While inbred and outbred plants had significantly lower fitness in foreign habitats, inbreeding depression was of similar magnitude at native sites and foreign habitats. There was no significant difference between inbred and outbred plants at foreign sites of the native habitat. Populations appear to be highly selfing, yet there is an advantage to outcrossing in both the native environment and foreign environments. The implications of this advantage with respect to mating system evolution may depend on whether novel environments are occupied or unoccupied.
PMCID: PMC4038437  PMID: 24790130
Dispersal; gene flow; inbreeding; inbreeding depression; local adaptation; reciprocal transplant.
17.  Giving drought the cold shoulder: a relationship between drought tolerance and fall dormancy in an agriculturally important crop 
AoB Plants  2014;6:plu012.
Fall dormant/freezing tolerant plants often also exhibit superior tolerance to drought conditions compared to their non-fall dormant/freezing intolerant counterparts. This experiment aimed to investigate this phenomenon in an agriculturally important crop. Seven alfalfa cultivars with varying levels of fall dormancy/freezing tolerance were exposed to a water deficit. The more fall dormant cultivars had superior tolerance to a mild water deficit. Two genes, CAS18 (encodes for a dehydrin like protein) and CorF (encodes for a galactinol synthase), were up regulated in association with this drought tolerance. Both these genes are early response genes, providing clues to the stress signalling pathways involved.
The growth of fall dormant/freezing tolerant plants often surpasses the growth of non-fall dormant/non-freezing tolerant types of the same species under water-limited conditions, while under irrigated conditions non-fall dormant types exhibit superior yield performance. To investigate the mechanism behind this phenomenon, we exposed seven diverse alfalfa (Medicago sativa) cultivars to water-limited and fully watered conditions and measured their shoot growth, shoot water potential and gas exchange parameters and the relative abundance of taproot RNA transcripts associated with chilling stress/freezing tolerance. Fall dormant cultivars had greater shoot growth relative to the fully watered controls under a mild water deficit (a cumulative water deficit of 625 mL pot−1) and did not close their stomata until lower shoot water potentials compared with the more non-fall dormant cultivars. Several gene transcripts previously associated with freezing tolerance increased in abundance when plants were exposed to a mild water deficit. Two transcripts, corF (encodes galactinol synthase) and cas18 (encodes a dehydrin-like protein), increased in abundance in fall dormant cultivars only. Once water deficit stress became severe (a cumulative water deficit of 2530 mL pot−1), the difference between fall dormancy groups disappeared with the exception of the expression of a type 1 sucrose synthase gene, which decreased in fall dormant cultivars. The specific adaptation of fall dormant cultivars to mild water deficit conditions and the increase in abundance of specific genes typically associated with freezing tolerance in these cultivars is further evidence of a link between freezing tolerance/fall dormancy and adaption to drought conditions in this species.
PMCID: PMC4038438  PMID: 24790133
Alfalfa; forage legumes; gene expression; lucerne; moisture stress.
18.  Spatial and temporal functional changes in alpine summit vegetation are driven by increases in shrubs and graminoids 
AoB Plants  2014;6:plu008.
In order to determine the mechanisms that drive changes in plant community composition across spatial and temporal scales, plant functional traits were used to interpret the results of a repeat species survey across a gradient of five alpine summits in south-east Australia. Vegetation changes were strongly affected by the high and increasing proportion of tall shrubs and graminoids, especially at the lower elevation summits. Several significant relationships between the community trait-weighted mean of different traits and elevation may suggest processes such as competition are influencing vegetation preferentially across the elevation gradient, with shrubs and graminoids driving these patterns.
Classical approaches to investigating temporal and spatial changes in community composition offer only partial insight into the ecology that drives species distribution, community patterns and processes, whereas a functional approach can help to determine many of the underlying mechanisms that drive such patterns. Here, we aim to bring these two approaches together to understand such drivers, using an elevation gradient of sites, a repeat species survey and species functional traits. We used data from a repeat vegetation survey on five alpine summits and measured plant height, leaf area, leaf dry matter content and specific leaf area (SLA) for every species recorded in the surveys. We combined species abundances with trait values to produce a community trait-weighted mean (CTWM) for each trait, and then combined survey results with the CTWMs. Across the gradient of summits, more favourable conditions for plant growth (warmer, longer growing season) occurred at the lower elevations. Vegetation composition changes between 2004 and 2011 (according to non-metric multi-dimensional scaling ordination) were strongly affected by the high and increasing abundance of species with high SLA at high elevations. Species life-form categories strongly affected compositional changes and functional composition, with increasing dominance of tall shrubs and graminoids at the lower-elevation summits, and an overall increase in graminoids across the gradient. The CTWM for plant height and leaf dry matter content significantly decreased with elevation, whereas for leaf area and SLA it significantly increased. The significant relationships between CTWM and elevation may suggest specific ecological processes, namely plant competition and local productivity, influencing vegetation preferentially across the elevation gradient, with the dominance of shrubs and graminoids driving the patterns in the CTWMs.
PMCID: PMC4038439  PMID: 24790129
Alpine vegetation; community composition; functional composition; functional traits; GLORIA; Snowy Mountains.
19.  Biaxial tensile tests identify epidermis and hypodermis as the main structural elements of sweet cherry skin 
AoB Plants  2014;6:plu019.
Sweet cherry and other fleshy fruit crack when the surface is exposed to water. Osmotic water uptake is believed to increase fruit volume and hence surface area, thereby subjecting the skin to stress and strain. The objectives were to (1) establish a standardized biaxial tensile test that simulates the in vivo strain of the skin and (2) characterize its mechanical properties. A bulging device was used to pressurize skin segments. Pressure and extent of bulging were monitored. The data demonstrate that (1) epidermis and hypodermis form the structural backbone of the fruit skin and (2) deformation is reversible.
The skin of developing soft and fleshy fruit is subjected to considerable growth stress, and failure of the skin is associated with impaired barrier properties in water transport and pathogen defence. The objectives were to establish a standardized, biaxial tensile test of the skin of soft and fleshy fruit and to use it to characterize and quantify mechanical properties of the sweet cherry (Prunus avium) fruit skin as a model. A segment of the exocarp (ES) comprising cuticle, epidermis, hypodermis and adhering flesh was mounted in the elastometer such that the in vivo strain was maintained. The ES was pressurized from the inner surface and the pressure and extent of associated bulging were recorded. Pressure : strain responses were almost linear up to the point of fracture, indicating that the modulus of elasticity was nearly constant. Abrading the cuticle decreased the fracture strain but had no effect on the fracture pressure. When pressure was held constant, bulging of the ES continued to increase. Strain relaxation upon releasing the pressure was complete and depended on time. Strains in longitudinal and latitudinal directions on the bulging ES did not differ significantly. Exocarp segments that released their in vivo strain before the test had higher fracture strains and lower moduli of elasticity. The results demonstrate that the cherry skin is isotropic in the tangential plane and exhibits elastic and viscoelastic behaviour. The epidermis and hypodermis, but not the cuticle, represent the structural ‘backbone’ in a cherry skin. This test is useful in quantifying the mechanical properties of soft and fleshy fruit of a range of species under standardized conditions.
PMCID: PMC4038440  PMID: 24876301
Biomechanics; fracture; mechanical properties; Prunus avium; rheology; skin; strain; stiffness.
20.  Responses of sap flow, leaf gas exchange and growth of hybrid aspen to elevated atmospheric humidity under field conditions 
AoB Plants  2014;6:plu021.
We demonstrate that higher air humidity mitigates the effect of low soil water availability on broadleaved trees during dry years by reducing stomatal limitation to photosynthesis, allowing higher net photosynthetic rates and supporting higher growth rates. At the same time, rising atmospheric humidity increases sensitivity of canopy conductance to water deficit and reduces the responsiveness of intrinsic water-use efficiency. The results imply that a future rise in atmospheric humidity at high latitudes may be disadvantageous in evenly rainy/humid years and expose trees to higher dehydration risk during weather extremes, although mitigating the impact of soil water deficit under moderate drought.
An increase in average air temperature and frequency of rain events is predicted for higher latitudes by the end of the 21st century, accompanied by a probable rise in air humidity. We currently lack knowledge on how forest trees acclimate to rising air humidity in temperate climates. We analysed the leaf gas exchange, sap flow and growth characteristics of hybrid aspen (Populus tremula × P. tremuloides) trees growing at ambient and artificially elevated air humidity in an experimental forest plantation situated in the hemiboreal vegetation zone. Humidification manipulation did not affect the photosynthetic capacity of plants, but did affect stomatal responses: trees growing at elevated air humidity had higher stomatal conductance at saturating photosynthetically active radiation (gs sat) and lower intrinsic water-use efficiency (IWUE). Reduced stomatal limitation of photosynthesis in trees grown at elevated air humidity allowed slightly higher net photosynthesis and relative current-year height increments than in trees at ambient air humidity. Tree responses suggest a mitigating effect of higher air humidity on trees under mild water stress. At the same time, trees at higher air humidity demonstrated a reduced sensitivity of IWUE to factors inducing stomatal closure and a steeper decline in canopy conductance in response to water deficit, implying higher dehydration risk. Despite the mitigating impact of increased air humidity under moderate drought, a future rise in atmospheric humidity at high latitudes may be disadvantageous for trees during weather extremes and represents a potential threat in hemiboreal forest ecosystems.
PMCID: PMC4052457  PMID: 24887000
Atmospheric humidity; canopy conductance; climate change; net photosynthesis; photosynthetic capacity; relative stomatal limitation; stomatal conductance; water-use efficiency.
21.  Belowground advantages in construction cost facilitate a cryptic plant invasion 
AoB Plants  2014;6:plu020.
Energetic costs of tissue construction were compared in two subspecies of Phragmites australis, the common reed – namely the primary native and introduced lineages in North America. Caplan et al. report that the introduced lineage has lower construction costs than the native under all environmental conditions assessed, driven mainly by its lower cost rhizomes. These results highlight the fact that belowground energetics, which are seldom investigated, can influence the performance advantages that drive many plant invasions. The authors also demonstrate that tissue construction costs in organs not typically assessed can shift with global change, suggesting that they may have increasingly important implications into the future.
The energetic cost of plant organ construction is a functional trait that is useful for understanding carbon investment during growth (e.g. the resource acquisition vs. tissue longevity tradeoff), as well as in response to global change factors like elevated CO2 and N. Despite the enormous importance of roots and rhizomes in acquiring soil resources and responding to global change, construction costs have been studied almost exclusively in leaves. We sought to determine how construction costs of aboveground and belowground organs differed between native and introduced lineages of a geographically widely dispersed wetland plant species (Phragmites australis) under varying levels of CO2 and N. We grew plants under ambient and elevated atmospheric CO2, as well as under two levels of soil nitrogen. We determined construction costs for leaves, stems, rhizomes and roots, as well as for whole plants. Across all treatment conditions, the introduced lineage of Phragmites had a 4.3 % lower mean rhizome construction cost than the native. Whole-plant construction costs were also smaller for the introduced lineage, with the largest difference in sample means (3.3 %) occurring under ambient conditions. In having lower rhizome and plant-scale construction costs, the introduced lineage can recoup its investment in tissue construction more quickly, enabling it to generate additional biomass with the same energetic investment. Our results suggest that introduced Phragmites has had an advantageous tissue investment strategy under historic CO2 and N levels, which has facilitated key rhizome processes, such as clonal spread. We recommend that construction costs for multiple organ types be included in future studies of plant carbon economy, especially those investigating global change.
PMCID: PMC4060782  PMID: 24938305
Carbon dioxide; common reed; construction cost; eutrophication; intraspecific; invasion ecology; Phragmites; plant functional traits; rhizomes; wetlands.
22.  Reprogramming of cassava (Manihot esculenta) microspores towards sporophytic development 
AoB Plants  2014;6:plu022.
Gametes have a unique potential to enter the sporophytic pathway, called androgenesis. The plants produced are usually haploid and recombinant due to the preceding meiosis and they can double their chromosome number to form doubled haploids (DHs), which are completely homozygous. The present study demonstrates that reprogramming occurs in the isolated microspores at tetrad stage as the first step if androgenesis and this is paving the way for the development of an efficient technique for the production of homozygous lines in cassava. This is the first ever detailed report of microspore reprogramming at the tetrad stage and the first report of microspore embryogenesis induction in cassava with detailed evidence.
Gametes have the unique potential to enter the sporophytic pathway, called androgenesis. The plants produced are usually haploid and recombinant due to the preceding meiosis and they can double their chromosome number to form doubled haploids, which are completely homozygous. Availability of the doubled haploids facilitates mapping the genes of agronomically important traits, shortening the time of the breeding process required to produce new hybrids and homozygous varieties, and saving the time and cost for inbreeding. This study aimed to test the feasibility of using isolated and in vitro cultured immature cassava (Manihot esculenta) microspores to reprogramme and initiate sporophytic development. Different culture media and different concentrations of two ion components (Cu2+ and Fe2+) were tested in two genotypes of cassava. External structural changes, nuclear divisions and cellular changes during reprogramming were analysed by scanning electron microscopy, by staining with 4′,6-diamidino-2-phenylindole, and through classical histology and transmission electron microscopy. In two cassava genotypes, different developmental stages of microspores were found to initiate sporophytic cell divisions, that is, with tetrads of TMS 60444 and with mid or late uni-nucleate microspores of SM 1219-9. In the modified NLN medium (NLNS), microspore enlargements were observed. The medium supplemented with either sodium ferrous ethylene-diamine-tetraacetic acid (NaFeEDTA) or CuSO4·5H2O induced sporophytic cell division in both genotypes. A low frequency of the reprogramming and the presence of non-responsive microspores among the responsive ones in tetrads were found to be related to the viability and exine formation of the microspores. The present study clearly demonstrated that reprogramming occurs much faster in isolated microspore culture than in anther culture. This paves the way for the development of an efficient technique for the production of homozygous lines in cassava. This is the first ever detailed report of microspore reprogramming at the tetrad stage and the first report of microspore embryogenesis induction in cassava with detailed evidence.
PMCID: PMC4061485  PMID: 24887001
Androgenesis; DAPI; histology; Manihot esculenta; reprogramming; scanning electron microscopy; transmission electron microscopy.
23.  Flooding and fragment size interact to determine survival and regrowth after fragmentation in two stoloniferous Trifolium species 
AoB Plants  2014;6:plu024.
Clonal plants are common in frequently flooded habitats because of their resilience to disturbance. We investigated if submergence prior to fragmentation of clones of two clover species reduced survival and regrowth of clonal fragments, and if these fitness parameters were different between genotypes from highly disturbed river forelands and less disturbed coastal dune slacks. We found that soil flooding severely decreased survival and regrowth, and that plants from the more disturbance-prone habitat were less negatively affected by fragmentation. However, internode size was, surprisingly, often negatively correlated with survival after fragmentation, but positively correlated with regrowth. Apparently, contrasting selection pressures exist on internode size in stoloniferous species growing in disturbed habitats.
Clonal plants, which reproduce by means of stolons and rhizomes, are common in frequently flooded habitats. Resilience to disturbance is an important trait enabling plants to survive in such highly disturbed habitats. Resource storage is thought to enable clonal plants to resume growth after clonal fragmentation caused by disturbance. Here we investigated if submergence prior to disturbance reduces survival and regrowth of clonal fragments and whether or not genotypes originating from highly disturbed riverine habitats are more resistant to mechanical disturbance than genotypes from less disturbed coastal dune slack habitats. We further tested if variation in survival and regrowth was affected by internode size. Clones from contrasting habitats of two closely related Trifolium species were first genotypically characterized by amplification fragment length polymorphism and then subjected to soil flooding and subsequent clonal fragmentation. These species differ with respect to their abundance in riverine and dune slack habitats, with Trifolium repens mainly occurring in riverine grasslands and Trifolium fragiferum in coastal dune slacks. Soil flooding decreased survival and regrowth by up to 80 %. Plants originating from riverine grasslands were less negatively affected by fragmentation than plants from dune slack habitats. Surprisingly, ramets did not always benefit from being attached to a larger internode, as internode size was often negatively correlated with survival after fragmentation. Regrowth, on the other hand, was generally positively correlated with internode size. These unexpected results indicate that there may be contrasting selection pressures on internode size in stoloniferous species growing in severely disturbed habitats.
PMCID: PMC4062869  PMID: 24887003
Clonal growth; disturbance; fragmentation; soil flooding; Trifolium fragiferum; Trifolium repens.
24.  Differences in proton pumping and Na/H exchange at the leaf cell tonoplast between a halophyte and a glycophyte 
AoB Plants  2014;6:plu023.
The tonoplast Na+/H+-antiporter and the tonoplast H+-pumps are essential components of salt tolerance in plants. We investigated the transport activity of the Na+/H+-antiporter and the H+-pumps in a highly tolerant salt accumulating halophyte, Salicornia dolichostachya, and compared them with activities in the related glycophyte Spinacia oleracea. Our results suggest that S. dolichostachya generates a high tonoplast H+-gradient already at low external salinities. At high external salinities, S. dolichostachya showed improved growth compared to S. oleracea, but H+-pump and Na+/H+-exchange activities were comparable between the species, which might imply that S. dolichostachya more efficiently retains Na+ in the vacuole.
The tonoplast Na+/H+ antiporter and tonoplast H+ pumps are essential components of salt tolerance in plants. The objective of this study was to investigate the transport activity of the tonoplast Na+/H+ antiporter and the tonoplast V-H+-ATPase and V-H+-PPase in a highly tolerant salt-accumulating halophyte, Salicornia dolichostachya, and to compare these transport activities with activities in the related glycophyte Spinacia oleracea. Vacuolar membrane vesicles were isolated by density gradient centrifugation, and the proton transport and hydrolytic activity of both H+ pumps were studied. Furthermore, the Na+/H+-exchange capacity of the vesicles was investigated by 9-amino-6-chloro-2-methoxyacridine fluorescence. Salt treatment induced V-H+-ATPase and V-H+-PPase activity in vesicles derived from S. oleracea, whereas V-H+-ATPase and V-H+-PPase activity in S. dolichostachya was not affected by salt treatment. Na+/H+-exchange capacity followed the same pattern, i.e. induced in response to salt treatment (0 and 200 mM NaCl) in S. oleracea and not influenced by salt treatment (10 and 200 mM NaCl) in S. dolichostachya. Our results suggest that S. dolichostachya already generates a high tonoplast H+ gradient at low external salinities, which is likely to contribute to the high cellular salt accumulation of this species at low external salinities. At high external salinities, S. dolichostachya showed improved growth compared with S. oleracea, but V-H+-ATPase, V-H+-PPase and Na+/H+-exchange activities were comparable between the species, which might imply that S. dolichostachya more efficiently retains Na+ in the vacuole.
PMCID: PMC4064249  PMID: 24887002
Halophyte; membrane transport; NHX1; Salicornia; salt tolerance; vacuole; V-H+-ATPase; V-H+-PPase.
25.  Genome size variation and evolution in allotetraploid Arabidopsis kamchatica and its parents, Arabidopsis lyrata and Arabidopsis halleri 
AoB Plants  2014;6:plu025.
Genome doubling and changes in genome size are fundamental evolutionary processes. Arabidopsis kamchatica has been reported to contain both diploid and tetraploid individuals (2 or 4 copies of every chromosome). We did find genome size differences among populations, and among populations genome size varied 7%. However, all sampled A. kamchatica plants from a wide geographic range were tetraploids. This level of intraspecific genome size variation in A. kamchatica is lower than in other Arabidopsis taxa. Due to its close relationship to A. thaliana, A. kamchatica has the potential to be very useful in the study of polyploidy and genome evolution.
Polyploidization and subsequent changes in genome size are fundamental processes in evolution and diversification. Little is currently known about the extent of genome size variation within taxa and the evolutionary forces acting on this variation. Arabidopsis kamchatica has been reported to contain both diploid and tetraploid individuals. The aim of this study was to determine the genome size of A. kamchatica, whether there is variation in ploidy and/or genome size in A. kamchatica and to study how genome size has evolved. We used propidium iodide flow cytometry to measure 2C DNA content of 73 plants from 25 geographically diverse populations of the putative allotetraploid A. kamchatica and its parents, Arabidopsis lyrata and Arabidopsis halleri. All A. kamchatica plants appear to be tetraploids. The mean 2C DNA content of A. kamchatica was 1.034 pg (1011 Mbp), which is slightly smaller than the sum of its diploid parents (A. lyrata: 0.502 pg; A. halleri: 0.571 pg). Arabidopsis kamchatica appears to have lost ∼37.594 Mbp (3.6 %) of DNA from its 2C genome. Tetraploid A. lyrata from Germany and Austria appears to have lost ∼70.366 Mbp (7.2 %) of DNA from the 2C genome, possibly due to hybridization with A. arenosa, which has a smaller genome than A. lyrata. We did find genome size differences among A. kamchatica populations, which varied up to 7 %. Arabidopsis kamchatica ssp. kawasakiana from Japan appears to have a slightly larger genome than A. kamchatica ssp. kamchatica from North America, perhaps due to multiple allopolyploid origins or hybridization with A. halleri. However, the among-population coefficient of variation in 2C DNA content is lower in A. kamchatica than in other Arabidopsis taxa. Due to its close relationship to A. thaliana, A. kamchatica has the potential to be very useful in the study of polyploidy and genome evolution.
PMCID: PMC4076644  PMID: 24887004
Allotetraploid; Arabidopsis halleri ssp. gemmifera; Arabidopsis kamchatica; Arabidopsis lyrata; C-value; 2C DNA content; flow cytometry; genome size; genome size variation

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