Proteomic snapshot of spearmint (Mentha spicata L.) leaf trichomes: A genuine terpenoid factory

Peltate glandular trichomes from Mentha spicata were purified on a Percoll gradient and soluble and membrane proteins were trypsinized and the peptides were separated by nano‐LC fractionation and analyzed by MALDI‐MS/MS. The vast majority of the 1666 proteins identified were housekeeping proteins or involved in the primary metabolism. However, 57 were predicted to be involved in the secondary metabolism. Of these, 21 were involved in the synthesis of phenylpropanoids and phenolics and 32 in terpenoid synthesis. Of the 14 membrane transporters identified, the 11 ATP‐binding cassette transporters provide good material for assessing whether active transport is required for the transfer of monoterpenoid intermediates between cellular compartments and for the secretion of the final products into the subcuticular storage cavity. In conclusion, this proteome analysis of M. spicata peltate trichomes has identified several candidate proteins that might be involved in terpenoid synthesis and transport. The data have been deposited to the ProteomeXchange with identifier PXD000352 ( http://proteomecentral.proteomexchange.org/dataset/PXD000352 ).     

Metabolic engineering of monoterpene biosynthesis: two-step production of (+)-trans-isopiperitenol by tobacco

Monoterpenoid biosynthesis in tobacco was modified by introducing two subsequent enzymatic activities targeted to different cell compartments. A limonene-3-hydroxylase (lim3h) cDNA was isolated from Mentha spicata L. 'Crispa'. This cDNA was used to re-transform a transgenic Nicotiana tabacum'Petit Havana' SR1 (tobacco) line expressing three Citrus limon L. Burm. f. (lemon) monoterpene synthases producing (+)-limonene, gamma-terpinene and (-)-beta-pinene as their main products. The targeting sequences of these synthases indicate that they are probably localized in the plastids, whereas the sequence information of the P450 hydroxylase indicates targeting to the endoplasmatic reticulum. Despite the different location of the enzymes, the introduced P450 hydroxylase proved to be functional in the transgenic plants as it hydroxylated (+)-limonene, resulting in the emission of (+)-trans-isopiperitenol. Some further modifications of the (+)-trans-isopiperitenol were also detected, resulting in the additional emission of 1,3,8-p-menthatriene, 1,5,8-p-menthatriene, p-cymene and isopiperitenone.     

Exploiting wild population diversity and somaclonal variation in the salt marsh grass Distichlis spicata (Poaceae) for marsh creation and restoration

The salt marsh grass Distichlis spicata was regenerated from tissue culture and propagated in a greenhouse. Selected regenerants, along with selections from six wild populations, were grown for two years in a common garden flood-irrigated thrice weekly with tidal creek water. Selected wild and regenerated plants were also planted in a created salt marsh. Significant differences among regenerant and wild population selections were found in several functionally important salt marsh plant characteristics, including potential detritus production, belowground organic matter production, canopy structure, and decomposition rate. A combination of characteristics not found in the wild populations was evident in a regenerated line that exhibited both a high detritus production potential and a high decomposition rate. The amount of variation that occurred among regenerants from one parental line via somaclonal variation was similar to that which occurred among the wild population selections. Results of this study suggest that tissue culture may provide a means of producing marsh grasses with specific characteristics for directing the functional development of newly created salt marshes.     

Effects of salinity on chlorophyll fluorescence and CO<Subscript>2</Subscript> fixation in C<Subscript>4</Subscript> estuarine grasses

The effects of salinity (sea water at 0 ‰ versus 30 ‰) on gross rates of O₂ evolution (J _O2) and net rates of CO₂ uptake (P _N) were measured in the halotolerant estuarine C₄ grasses Spartina patens, S. alterniflora, S. densiflora, and Distichlis spicata in controlled growth environments. Under high irradiance, salinity had no significant effect on the intercellular to ambient CO₂ concentration ratio (C _i/C _a). However, during photosynthesis under limiting irradiance, the maximum quantum efficiency of CO₂ fixation decreased under salinity across species, suggesting there is increased leakage of the CO₂ delivered to the bundle sheath cells by the C₄ pump. Growth under salinity did not affect the maximum intrinsic efficiency of photosystem 2, PS2 (F_V/F_M) in these species, suggesting salinity had no effect on photosynthesis by inactivation of PS2 reaction centers. Under saline conditions and high irradiance, P _N was reduced by 75 % in Spartina patens and S. alterniflora, whereas salinity had no effect on P _N in S. densiflora or D. spicata. This inhibition of P _N in S. patens and S. alterniflora was not due to an effect on stomatal conductance since the ratio of C _i/C _a did not decrease under saline conditions. In growth with and without salt, P _N was saturated at ∼500 μmol(quantum) m⁻² s⁻¹ while J _O2 continued to increase up to full sunlight, indicating that carbon assimilation was not tightly coupled to photochemistry in these halophytic species. This increase in alternative electron flow under high irradiance might be an inherent function in these halophytes for dissipating excess energy.     

The influence of herbivory on plant cover and species composition in the Pryor Mountain Wild Horse Range,USA

We investigated the effects of short- and long-term ungulate grazing on plant species cover and composition in arid lowland and more mesic upland communities of the Pryor Mountain Wild Horse Range (PMWHR). Measurements were taken over two years which differed significantly in growing season precipitation. Interannual differences in plant cover were significantly greater than differences between grazed and ungrazed communities. In the arid lowlands total plant cover decreased from 47% in 1993, a relatively wet year, to 29% in 1994, a relatively dry year. In the more mesic uplands total plant cover decreased from 107% in 1993 to 56% in 1994. The magnitude of change in cover was greatest in the grasses, especially for Pseudoroegneria spicata, the most abundant species in the lowland communities, which decreased from 21% cover in 1993 to 11% in 1994. There was not a consistent effect of herbivory on plant cover across sites, but its effects, particularly on the dominant perennial grasses, were conspicuous at some sites. For instance, in the lowlands cover of P. spicata was 3–12% in long-term grazed sites and 9–28% in short- to long-term ungrazed sites. Our study indicates that abiotic factors (e.g., precipitation) are more likely than grazing to affect abundances of key plant species, and hence ecosystem dynamics, in the PMWHR, and that the effects of herbivory are more localized and more prevalent in the lowland grasses than in the other plant functional groups.     

Females make tough neighbors: sex-specific competitive effects in seedlings of a dioecious grass

If males and females of a species differ in their effect on intraspecific competition then this can have significant ecological and evolutionary consequences because it can lead to size and mortality disparities between the sexes, and thus cause biased population sex ratios. If the degree of sexual dimorphism of competitive effect varies across environments then this variation can generate sex ratio variation within and between populations. In a California population of Distichlis spicata, a dioecious grass species exhibiting extreme within-population sex ratio variation (spatial segregation of the sexes), I evaluated the intraspecific competitive effects of male and female D. spicata seedlings in three soil types. The sex of seedlings was determined using a RAPD-PCR marker co-segregating with female phenotype. Distichlis spicata seedlings, regardless of sex, were six times larger when grown with male versus female conspecific seedlings in soil from microsites where the majority of D. spicata plants are female, and this sexual dimorphism of competitive effect was weaker or did not occur in other soil types. This study suggests that it is not just the higher costs of female versus male reproduction itself that cause spatial segregation of the sexes in D. spicata, but that differences in competitive abilities between the sexes—which occur as early as the seedling stage—can generate sex ratio variation.     

Population structure in Pseudoroegneria spicata (Poaceae: Triticeae) modeled by Bayesian clustering of AFLP genotypes

Pseudoroegneria spicata (Poaceae: Triticeae) is an abundant, allogamous species widely adapted to the temperate, semiarid steppe and open woodland regions of western North America. Amplified fragment length polymorphism (AFLP), model-based Bayesian clustering, and other methods of hypothesis testing were used to investigate genetic diversity and population structure among 565 P. spicata plants from 82 localities representing much of the species distribution. Comparisons with four Asiatic Pseudoroegneria species and two North American Elymus wawawaiensis accessions demonstrate cohesiveness in P. spicata. However, P. spicata genotypes group by locality and geographic region based on genetic distance analysis. Average DNA polymorphism among P. spicata localities was significantly correlated (r = 0.58) with geographical distance. The optimum Bayesian cluster model included 21 P. spicata groups, indicating that dispersal among sampling locations was not sufficient to group genotypes into one unstructured population. Approximately 18.3% of the DNA polymorphism was partitioned among the 21 regional groups, 14.9% among localities within groups, and 66.8% within accessions. Average DNA polymorphism among Bayesian groups was correlated (r = 0.53) with the average geographic distance among Bayesian groups, which partly reflects isolation by distance. However, conspicuous regional boundaries were discernable among several divergent genetic groups.     

Freezing avoidance in Andean giant rosette plants

Abstract Frost avoidance mechanisms were studied in Espeletia spicata and Espeletia timotensis, two Andean giant rosette species. The daily courses of soil, air and tissue temperatures were measured at a site at circa 4000 m. Only the leaves were exposed to subzero temperatures; the apical bud and stem pith tissues were insulated by surrounding tissues. The leaf tissues avoided freezing by supercooling rather than by undergoing active osmotic changes. The temperatures at which ice formed in the tissues (the supercooling points) coincided with injury temperatures indicating that Espeletia tissue does not tolerate any kind of ice formation. For insulated tissue (apical bud, stem pith, roots) the supercooling point was around - 5°C coinciding with the injury temperature. Supercooling points of about –13 to - 16°C were observed for leaves. These results contrast with those reported for Afroalpine giant rosettes which tolerate extracellular freezing. The significance of different adaptive responses of giant rosettes to similar cold tropical environments is discussed.