Arbuscular Mycorrhizae Alter Photosynthetic Responses to Drought in Seedlings of Artemisia tridentata.

The establishment of Artemisia tridentata, a keystone species of the sagebrush steppe, is often limited by summer drought. Symbioses with arbuscular mycorrhizal fungi (AMF) can help plants to cope with drought. We investigated this possible effect on A. tridentata seedlings inoculated with native AMF and exposed to drought in greenhouse and field settings. In greenhouse experiments, AMF colonization increased intrinsic water use efficiency under water stress and delayed the decrease in photosynthesis caused by drought, or this decrease occurred at a lower soil water content. In the field, we evaluated the effect of AMF inoculation on colonization, leaf water potential, survival, and inflorescence development. Inoculation increased AMF colonization, and the seedlings experienced water stress, as evidenced by water potentials between -2 and -4 MPa and reduced stomatal conductance. However, survival remained high, and no differences in water potentials or survival occurred between treatments. Only the percentage of plants with inflorescence was higher in inoculated than non-inoculated seedlings. Overall, the greenhouse results support that AMF colonization enhances drought tolerance in A. tridentata seedlings. Yet, the significance of these results in increasing survival in nature remains to be tested under more severe drought than the plants experienced in our field experiment.

Herbivory Amplifies Adverse Effects of Drought on Seedling Recruitment in a Keystone Species of Western North American Rangelands.

Biotic interactions can affect a plant's ability to withstand drought. Such an effect may impact the restoration of the imperiled western North American sagebrush steppe, where seedlings are exposed to summer drought. This study investigated the impact of herbivory on seedlings' drought tolerance for a keystone species in this steppe, the shrub Artemisia tridentata. Herbivory effects were investigated in two field experiments where seedlings were without tree protectors or within plastic or metal-mesh tree protectors. Treatment effects were statistically evaluated on herbivory, survival, leaf water potential, and inflorescence development. Herbivory occurrence was 80% higher in seedlings without protectors. This damage occurred in early spring and was likely caused by ground squirrels. Most plants recovered, but herbivory was associated with higher mortality during the summer when seedlings experienced water potentials between -2.5 and -7 MPa. However, there were no differences in water potential between treatments, suggesting that the browsed plants were less tolerant of the low water potentials experienced. Twenty months after outplanting, the survival of plants without protectors was 40 to 60% lower than those with protectors. The percentage of live plants developing inflorescences was approximately threefold higher in plants with protectors. Overall, spring herbivory amplified susceptibility to drought and delayed reproductive development.

Intraspecific variation mediates density dependence in a genetically diverse plant species.

Interactions between neighboring plants are critical for biodiversity maintenance in plant populations and communities. Intraspecific trait variation and genome duplication are common in plant species and can drive eco-evolutionary dynamics through genotype-mediated plant-plant interactions. However, few studies have examined how species-wide intraspecific variation may alter interactions between neighboring plants. We investigate how subspecies and ploidy variation in a genetically diverse species, big sagebrush (Artemisia tridentata), can alter the demographic outcomes of plant interactions. Using a replicated, long-term common garden experiment that represents range-wide diversity of A. tridentata, we ask how intraspecific variation, environment, and stand age mediate neighbor effects on plant growth and survival. Spatially explicit models revealed that ploidy variation and subspecies identity can mediate plant-plant interactions but that the effect size varied in time and across experimental sites. We found that demographic impacts of neighbor effects were strongest during early stages of stand development and in sites with greater growth rates. Within subspecies, tetraploid populations showed greater tolerance to neighbor crowding compared to their diploid variants. Our findings provide evidence that intraspecific variation related to genome size and subspecies identity impacts spatial demography in a genetically diverse plant species. Accounting for intraspecific variation in studies of conspecific density dependence will improve our understanding of how local populations will respond to novel genotypes and biotic interaction regimes. As introduction of novel genotypes into local populations becomes more common, quantifying demographic processes in genetically diverse populations will help predict long-term consequences of plant-plant interactions.

Consequences of inoculation with native arbuscular mycorrhizal fungi for root colonization and survival of Artemisia tridentata ssp. wyomingensis seedlings after transplanting.

In arid environments, the propagule density of arbuscular mycorrhizal fungi (AMF) may limit the extent of the plant-AMF symbiosis. Inoculation of seedlings with AMF could alleviate this problem, but the success of this practice largely depends on the ability of the inoculum to multiply and colonize the growing root system after transplanting. These phenomena were investigated in Artemisia tridentata ssp. wyomingensis (Wyoming big sagebrush) seedlings inoculated with native AMF. Seedlings were first grown in a greenhouse in soil without AMF (non-inoculated seedlings) or with AMF (inoculated seedlings). In spring and fall, 3-month-old seedlings were transplanted outdoors to 24-L pots containing soil from a sagebrush habitat (spring and fall mesocosm experiments) or to a recently burned sagebrush habitat (spring and fall field experiments). Five or 8 months after transplanting, colonization was about twofold higher in inoculated than non-inoculated seedlings, except for the spring field experiment. In the mesocosm experiments, inoculation increased survival during the summer by 24 % (p = 0.011). In the field experiments, increased AMF colonization was associated with increases in survival during cold and dry periods; 1 year after transplanting, survival of inoculated seedlings was 27 % higher than that of non-inoculated ones (p < 0.001). To investigate possible mechanisms by which AMF increased survival, we analyzed water use efficiency (WUE) based on foliar (13)C/(12)C isotope ratios (δ (13)C). A positive correlation between AMF colonization and δ (13)C values was observed in the spring mesocosm experiment. In contrast, inoculation did not affect the δ (13)C values of fall transplanted seedlings that were collected the subsequent spring. The effectiveness of AMF inoculation on enhancing colonization and reducing seedling mortality varied among the different experiments, but average effects were estimated by meta-analyses. Several months after transplanting, average AMF colonization was in proportion 84 % higher in inoculated than non-inoculated seedlings (p = 0.0042), while the average risk of seedling mortality was 42 % lower in inoculated than non-inoculated seedlings (p = 0.047). These results indicate that inoculation can increase AMF colonization over the background levels occurring in the soil, leading to higher rates of survival.

Assessing the diversity of arbuscular mycorrhizal fungi in semiarid shrublands dominated by Artemisia tridentata ssp. wyomingensis.

Variation in the abiotic environment and host plant preferences can affect the composition of arbuscular mycorrhizal (AMF) assemblages. This study analyzed the AMF taxa present in soil and seedlings of Artemisia tridentata ssp. wyomingensis collected from sagebrush steppe communities in southwestern Idaho, USA. Our aims were to determine the AMF diversity within and among these communities and the extent to which preferential AMF-plant associations develop during seedling establishment. Mycorrhizae were identified using molecular methods following DNA extraction from field and pot culture samples. The extracted DNA was amplified using Glomeromycota specific primers, and identification of AMF was based on phylogenetic analysis of sequences from the large subunit-D2 rDNA region. The phylogenetic analyses revealed seven phylotypes, two within the Claroideoglomeraceae and five within the Glomeraceae. Four phylotypes clustered with known species including Claroideoglomus claroideum, Rhizophagus irregularis, Glomus microaggregatum, and Funneliformis mosseae. The other three phylotypes were similar to several published sequences not included in the phylogenetic analysis, but all of these were from uncultured and unnamed glomeromycetes. Pairwise distance analysis revealed some phylotypes with high genetic variation. The most diverse was the phylotype that included R. irregularis, which contained sequences showing pairwise differences up to 12 %. Most of the diversity in AMF sequences occurred within sites. The smaller genetic differentiation detected among sites was correlated with differences in soil texture. In addition, multiplication in pot cultures led to differentiation of AMF communities. Comparison of sequences obtained from the soil with those from A. tridentata roots revealed no significant differences between the AMF present in these samples. Overall, the sites sampled were dominated by cosmopolitan AMF taxa, and young seedlings of A. tridentata ssp. wyomingensis were colonized in relation to the abundance of these taxa in the soil.

Homoploid hybrid speciation in a rare endemic Castilleja from Idaho (Castilleja christii, Orobanchaceae).

PREMISE OF THE STUDY: Hybridization is an important evolutionary force in the history of angiosperms; however, there are few examples of stabilized species derived through homoploid hybrid speciation. Homoploid hybrid species are generally detected via the presence of genetic additivity of parental markers, novel ecological and spatial distinctions, and novel morphological traits, all of which may aid in the successful establishment of hybrid species from parental types. Speciation and diversification within the genus Castilleja (Orobanchaceae) has been attributed to high levels of hybridization and polyploidy, though currently there are no examples of homoploid hybrid speciation within the genus. We employed multiple lines of evidence to examine a putative hybrid origin in C. christii, a rare endemic, known only from 80 hectares at the summit of Mt. Harrison (Cassia Co., Idaho). • METHODS: We used granule-bound starch synthase II (waxy) sequences and 26 morphological characters to address hybridization between C. christii and widespread congeners C. miniata and/or C. linariifolia in an area of sympatry. Chromosomes of C. christii were also counted for the first time. • KEY RESULTS: All 230 direct-sequenced C. christii individuals had the additive genomes of both C. miniata and C. linariifolia. Castilleja christii shares traits with both parents but also has floral characters that are unique and transgressive. Cytological counts indicated that all three taxa are diploid. • CONCLUSIONS: We conclude that C. christii is a stabilized homoploid hybrid derivative of C. linariifolia and C. miniata and is likely following an independent evolutionary trajectory from its progenitors.

Changes in the expression of carbohydrate metabolism genes during three phases of bud dormancy in leafy spurge.

Underground adventitious buds of leafy spurge (Euphorbia esula) undergo three well-defined phases of dormancy, para-, endo-, and ecodormancy. In this study, relationships among genes involved in carbohydrate metabolism and bud dormancy were examined after paradormancy release (growth induction) by decapitation and in response to seasonal signals. Real-time PCR was used to determine the expression levels of carbohydrate metabolism genes at different phases of bud dormancy. Among differentially-regulated genes, expression of a specific Euphorbia esula beta-amylase gene (Ee-BAM1) increased 100-fold after growth induction and 16,000-fold from July (paradormancy) to December (ecodormancy). Sequence data analysis indicated that two genes, Ee-BAM1 and Ee-BAM2, could encode this beta-amylase. However, real-time PCR using gene-specific primer pairs only amplified Ee-BAM1, indicating that Ee-BAM2 is either specific to other organs or not abundant. The deduced amino acid sequences of these two genes are very similar at the N-terminal but differ at the C-terminal. Both contain a nearly identical, predicted 48-amino acid plastid transit peptide. Immunoblot analyses identified a 29 kD (mature Ee-BAM1 after cleavage of the transit peptide) and a 35 kD (unprocessed EeBAM1) protein. Both 35 and 29 kD proteins were constitutively expressed in growth-induced and seasonal samples. Immunolocalization indicated that Ee-BAM1 is in the cytosol of cells at the shoot tip of the bud. Ee-BAM1 also surrounds the amyloplasts in mature cells toward the base of the bud. These observations suggests that Ee-BAM1 may have dual functions; serving as reserve protein in the cytosol and as a degrading enzyme at the surface of amyloplasts.

Environmental regulation of dormancy loss in seeds of Lomatium dissectum (Apiaceae).

BACKGROUND AND AIMS: Lomatium dissectum (Apiaceae) is a perennial, herbaceous plant of wide distribution in Western North America. At the time of dispersal, L. dissectum seeds are dormant and have under-developed embryos. The aims of this work were to determine the requirements for dormancy break and germination, to characterize the type of seed dormancy, and to determine the effect of dehydration after embryo growth on seed viability and secondary dormancy. METHODS: The temperature requirements for embryo growth and germination were investigated under growth chamber and field conditions. The effect of GA(3) on embryo growth was also analysed to determine the specific type of seed dormancy. The effect of dehydration on seed viability and induction of secondary dormancy were tested in seeds where embryos had elongated about 4-fold their initial length. Most experiments examining the nature of seed dormancy were conducted with seeds collected at one site in two different years. To characterize the degree of variation in dormancy-breaking requirements among seed populations, the stratification requirements of seeds collected at eight different sites were compared. KEY RESULTS: Embryo growth prior to and during germination occurred at temperatures between 3 and 6 degrees C and was negligible at stratification temperatures of 0.5 and 9.1 degrees C. Seeds buried in the field and exposed to natural winter conditions showed similar trends. Interruption of the cold stratification period by 8 weeks of dehydration decreased seed viability by about 30 % and induced secondary dormancy in the remaining viable seeds. Comparison of the cold stratification requirements of different seed populations indicates that seeds collected from moist habitats have longer cold stratification requirements that those from semiarid environments. CONCLUSIONS: Seeds of L. dissectum have deep complex morphophysiological dormancy. The requirements for dormancy break and germination reflect an adaptation to trigger germination in late winter.

Potential roles for autophosphorylation, kinase activity, and abundance of a CDK-activating kinase (Ee;CDKF;1) during growth in leafy spurge.

Leafy spurge (Euphorbia esula L.) is a deep-rooted perennial weed that propagates both by seeds and underground adventitious buds located on the crown and roots. To enhance our understanding of growth and development during seed germination and vegetative propagation, a leafy spurge gene (Accession No. AF230740) encoding a CDK-activating kinase (Ee;CDKF;1) involved in cell-cycle progression was identified, and its function was confirmed based on its ability to rescue a yeast temperature-sensitive CAK mutant (GF2351) and through in vitro kinase assays. Site-directed mutagenesis of Ee;CDKF;1 indicated that two threonine residues (Thr291 and Thr296) were mutually responsible for intra-molecular autophosphorylation and for phosphorylating its substrate protein, cyclin-dependent kinase (CDK). Polyclonal antibodies generated against the Ee;CDKF;1 protein or against a phosphorylated Ee;CDKF;1 peptide [NERYGSL(pT)SC] were used to examine abundance and phosphorylation of CDKF;1 during seed germination and bud growth. The levels of CDKF;1 were lower in dry or imbibed seeds than in germinating seeds or seedlings. Differences in CDKF;1 were also observed during adventitious bud development; small buds appeared to have greater levels of CDKF;1 than large buds. Similar patterns of CDKF;1 expression were detected with either the polyclonal antibody developed using the CDKF;1 protein or the phosphorylated peptide. These results indicated that Thr291 is constitutively phosphorylated in vivo and associated with Ee;CDKF;1 activity. Our results further suggest that a certain level of CDKF;1 activity is maintained in most tissues and may be an important phenomenon for enzymes that regulate early steps in cell-cycle signaling pathways.

Immunolocalization of beta-D-glucans, pectins, and arabinogalactan-proteins during intrusive growth and elongation of nonarticulated laticifers in Asclepias speciosa Torr.

Nonarticulated laticifers are latex-containing cells that elongate indefinitely and grow intrusively between the walls of meristematic cells. To identify biochemical mechanisms involved in the growth of nonarticulated laticifers, we have analyzed the distribution of various polysaccharides and proteoglycans in walls of meristematic cells in contact with laticifers, nonadjacent to laticifers, and in laticifer walls. In the shoot apex of Asclepias speciosa, the levels of callose and a (1-->4)-beta-galactan epitope are lower in meristematic walls in contact with laticifers than in nonadjacent walls. In contrast, we did not detect a decline in xyloglucan, homogalacturonan, and arabinogalactan-protein epitopes upon contact of meristematic cells with laticifers. Laticifer elongation is also associated with the development of a homogalacturonan-rich middle lamella between laticifers and their neighboring cells. Furthermore, laticifers lay down walls that differ from those of their surrounding cells. This is particularly evident for epitopes in rhamnogalacturonan I. A (1-->5)-alpha-arabinan epitope in this pectin is more abundant in laticifers than meristematic cells, while the opposite is observed for a (1-->4)-beta-galactan epitope. Also, different cell wall components exhibit distinct distribution patterns within laticifer walls. The (1-->5)-alpha-arabinan epitope is distributed throughout the laticifer walls while certain homogalacturonan and arabinogalactan-protein epitopes are preferentially located in particular regions of laticifer walls. Taken together, our results indicate that laticifer penetration causes changes in the walls of meristematic cells and that there are differences in wall composition within laticifer walls and between laticifers and their surrounding cells.