Secretory structures in plants: Lessons from the Plumbaginaceae on their origin, evolution and roles in stress tolerance.

The Plumbaginaceae (non-core Caryophyllales) is a family well known for species adapted to a wide range of arid and saline habitats. Of its salt-tolerant species, at least 45 are in the genus Limonium; two in each of Aegialitis, Limoniastrum and Myriolimon, and one each in Psylliostachys, Armeria, Ceratostigma, Goniolimon and Plumbago. All the halophytic members of the family have salt glands and salt glands are also common in the closely related Tamaricaceae and Frankeniaceae. The halophytic species of the three families can secrete a range of ions (Na+ , K+ , Ca2+ , Mg2+ , Cl- , HCO3- , SO42- ) and other elements (As, Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn). Salt glands are, however, absent in salt-tolerant members of the sister family Polygonaceae. We describe the structure of the salt glands in the three families and consider whether glands might have arisen as a means to avoid the toxicity of Na+ and/or Cl- or to regulate Ca2+ concentrations with the leaves. We conclude that the establishment of lineages with salt glands took place after the split between the Polygonaceae and its sister group the Plumbaginaceae.

Leaf size as a key determinant of contrasting growth patterns in closely related Limonium (Plumbaginaceae) species.

This study aims to analyze the importance of leaf size on plant growth capacity among an array of closely related Limonium species, and its impact on the underlying determinants of growth reduction under extreme water deficit conditions. To do so, thirteen Balearic Limonium species with contrasting leaf size were grown under long-term well-watered (WW) and severe water-deficit (WD) conditions in a common garden experiment. Fundamental growth traits were measured, including relative growth rate (RGR), net assimilation rate (NAR), leaf area ratio (LAR), leaf mass area (LMA) and leaf mass ratio (LMR). WD promoted small changes in leaf size, and species with larger leaves had higher RGR than species with smaller leaves, irrespective of the water treatment. Most RGR variation across species and treatments was explained by NAR, with comparatively much lower importance of LAR. The factorization of LAR underlying components denoted the importance of LMA in explaining RGR, whereas the impact of LMR on RGR was negligible in Limonium. Further, species with larger leaves had higher water consumption but also higher water use efficiency, especially under WD. Therefore, contrary to general trends in species from dry environments, increased leaf size is linked to increased growth capacity and also increased water use efficiency across closely related Limonium species.

Experimental insights on the function of ancillary pollen and stigma polymorphisms in plants with heteromorphic incompatibility.

Most heterostylous plants possess a reciprocal arrangement of stigmas and anthers (reciprocal herkogamy), heteromorphic self-incompatibility, and ancillary polymorphisms of pollen and stigmas. The topographical complementarity hypothesis proposes that ancillary polymorphisms function in the rejection of incompatible pollen thus promoting disassortative pollination. Here, we test this hypothesis by investigating patterns of pollen transfer and capture in populations of dimorphic Armeria maritima and A. pubigera and distylous Limonium vulgare (Plumbaginaceae), and by studying pollen adherence and germination patterns in A. maritima following controlled hand-pollinations. Armeria lacks reciprocal herkogamy allowing the evaluation of the extent to which ancillary polymorphisms affect the composition of pollen loads. We compared the amounts of compatible and incompatible pollen on stigmas in natural populations and calculated the proficiencies of pollen transfer for each mating type. We detected disassortative pollination in each species, and mating types did not differ in compatible pollen capture, although cob stigmas captured more incompatible pollen. Controlled hand-pollinations revealed the failure of incompatible pollen to adhere and germinate on stigmas. Our results provided evidence that, while structural in nature, pollen-stigma dimorphisms are tightly associated with heteromorphic incompatibility and likely function to promote disassortative pollination, especially in the absence of reciprocal herkogamy.

K(+) accumulation in the cytoplasm and nucleus of the salt gland cells of Limonium bicolor accompanies increased rates of salt secretion under NaCl treatment using NanoSIMS.

Recretohalophytes with specialized salt-secreting structures (salt glands) can secrete excess salts from plant, while discriminating between Na(+) and K(+). K(+)/Na(+) ratio plays an important role in plant salt tolerance, but the distribution and role of K(+) in the salt gland cells is poorly understood. In this article, the in situ subcellular localization of K and Na in the salt gland of the recretohalophyte Limonium bicolor Kuntze is described. Samples were prepared by high-pressure freezing (HPF), freeze substitution (FS) and analyzed using NanoSIMS. The salt gland of L. bicolor consists of sixteen cells. Higher signal strength of Na(+) was located in the apoplast of salt gland cells. Compared with control, 200 mM NaCl treatment led to higher signal strength of K(+) and Na(+) in both cytoplasm and nucleus of salt gland cells although K(+)/Na(+) ratio in both cytoplasm and nucleus were slightly reduced by NaCl. Moreover, the rate of Na(+) secretion per salt gland of L. bicolor treated with 200 mM NaCl was five times that of controls. These results suggest that K(+) accumulation both in the cytoplasm and nucleus of salt gland cells under salinity may play an important role in salt secretion, although the exact mechanism is unknown.

Taxonomic complexity in the halophyte Limonium vulgare and related taxa (Plumbaginaceae): insights from analysis of morphological, reproductive and karyological data.

BACKGROUND AND AIMS: Limonium is a well-known example of a group of plants that is taxonomically complex due to certain biological characteristics that hamper species' delineation. The closely related polyploid species Limonium vulgare Mill., L. humile Mill. and L. narbonense Mill. are defined species and can be used for studying patterns of morphological and reproductive variation. The first two taxa are usually found in Atlantic Europe and the third in the Mediterranean region, but a number of intermediate morphological forms may be present alongside typical examples of these species. This study attempts to elucidate morphological, floral and karyological diversity representative of these taxa in the Iberian Peninsula. METHODS: The extent of morphological differentiation was tested through comparison of 197 specimens from both Portugal and Spain using 17 descriptive morphological characters and 19 diagnostic morphometric characters. Analyses of floral morphisms (heterostyly and pollen-stigma dimorphism) and karyological determinations were also conducted. KEY RESULTS AND CONCLUSIONS: Discriminant analysis using morphometric variables reliably assigned individuals in natural populations to their respective groups. In addition, the results provide the first direct evidence that L. narbonense and a new species, LIMONIUM MARITIMUM: Caperta, Cortinhas, Paes, Guara, Espírito-Santo and Erben, SP NOV: , related to L. vulgare are present on Portuguese coasts. Most of these species are found together in mixed populations, especially L. vulgare and L. narbonense. It is hypothesized that taxonomic biodiversity found in sites where distinct species co-occur facilitates the evolutionary processes of hybridization, introgression and apomixis. This study therefore contributes to the elucidation of the taxonomic diversity in L. vulgare-related species and may also help in implementing future conservation programmes to maintain the evolutionary processes generating biodiversity.

Epigenetic rather than genetic factors may explain phenotypic divergence between coastal populations of diploid and tetraploid Limonium spp. (Plumbaginaceae) in Portugal.

BACKGROUND: The genus Limonium Miller comprises annual and perennial halophytes that can produce sexual and/or asexual seeds (apomixis). Genetic and epigenetic (DNA methylation) variation patterns were investigated in populations of three phenotypically similar putative sexual diploid species (L. nydeggeri, L. ovalifolium, L. lanceolatum), one sexual tetraploid species (L. vulgare) and two apomict tetraploid species thought to be related (L. dodartii, L. multiflorum). The extent of morphological differentiation between these species was assessed using ten diagnostic morphometric characters. RESULTS: A discriminant analysis using the morphometric variables reliably assigns individuals into their respective species groups. We found that only modest genetic and epigenetic differentiation was revealed between species by Methylation Sensitive Amplification Polymorphism (MSAP). However, whilst there was little separation possible between ploidy levels on the basis of genetic profiles, there was clear and pronounced interploidy discrimination on the basis of epigenetic profiles. Here we investigate the relative contribution of genetic and epigenetic factors in explaining the complex phenotypic variability seen in problematic taxonomic groups such as Limonium that operate both apomixis and sexual modes of reproduction. CONCLUSIONS: Our results suggest that epigenetic variation might be one of the drivers of the phenotypic divergence between diploid and tetraploid taxa and discuss that intergenome silencing offers a plausible mechanistic explanation for the observed phenotypic divergence between these microspecies. These results also suggest that epigenetic profiling offer an additional tool to infer ploidy level in stored specimens and that stable epigenetic change may play an important role in apomict evolution and species recognition.

Comparative study of three Plumbago L. species (Plumbaginaceae) by microscopy, UPLC-UV and HPTLC.

This paper presents a comparative study of anatomy of leaves, stems and roots of three species of Plumbago, namely P. auriculata Lam., P. indica L. and P. zeylanica L. by light microscopy. The paper also provides qualitative and quantitative analysis of the naphthoquinone, plumbagin-a major constituent present in these species-using UPLC-UV. Microscopic examinations revealed the presence of distinctive differences in the anatomical features of the leaf, stem and root of the three species, and these can thus be used for identification and authentication of these species. UPLC-UV analysis showed the highest concentration of plumbagin in the roots of P. zeylanica (1.62% w/w) followed by the roots of P. indica (0.97% w/w) and then P. auriculata (0.33-0.53% w/w). In contrast, plumbagin was not detected in the stems and leaves of P. indica and in the leaves of P. auriculata, whereas very low concentrations (<0.02% w/w) of plumbagin were detected in the stems and leaves of P. zeylanica and in the stems of P. auriculata. HPTLC fingerprints of the leaf and root of the three species exhibited distinguishable profiles, while those of the stems were undifferentiated.

Morphological and genetic distinctiveness of metallicolous and non-metallicolous populations of Armeria maritima s.l. (Plumbaginaceae) in Poland.

Patterns of morphological, genetic and epigenetic variation (DNA methylation pattern) were investigated in metallicolous (M) and non-metallicolous (NM) populations of Armeria maritima. A morphological study was carried out using plants from six natural populations grown in a greenhouse. Morphological variation was assessed using seven traits. On the basis of this study, three representative populations were selected for molecular analyses using metAFLP to study sequence- and methylation-based DNA variation. Only one morphological trait (length of outer involucral bracts) was common to both metallicolous populations studied; however, the level of variation was sufficient to differentiate between M and NM populations. Molecular analyses showed the existence of naturally occurring epigenetic variation in A. maritima populations, as well as structuring into distinct between and within population components. We show that patterns of population genetic structure differed depending on the information used in the study. Analysis of sequence-based information data demonstrates the presence of three well-defined and genetically differentiated populations. Methylation-based data show that two major groups of individuals are present, corresponding to the division into M and NM populations. These results were confirmed using different analytical approaches, which suggest that the DNA methylation pattern is similar in both M populations. We hypothesise that epigenetic processes may be involved in microevolution leading to development of M populations in A. maritima.

Effect of salt stress on seed germination and seedling growth of three salinity plants.

Seeds of three halophyte species included Limonium sinense Kuntze, Glycine soja sieb. and Sorghum sudanense Stapf. were used to investigate the effects of different salinity concentrations on their germination percentage, germination energy, germination index, relative germination rate, relative salt-injury rate, radicle length and hypocotyl length. Results showed that seeds of G. soja can germinate well and rapidly at lower level of salt (< 200 mmol L(-1)), 50 mmol L(-1) salt concentrations is better to S. sudanense, suitable salt concentrations was beneficial to germinate of S. sudanense seeds. Seeds of L. sinense and S. sudanense can germinate at higher level of salt (400 mmol L(-1)). Three plant seeds had different physiological mechanism for germination under salt stress, radicle hypocotyl ratio of L. sinense and S. sudanense increased with increasing salt suggested that they had the higher tolerance in shoot growth under NaCl stress, which is perhaps the reason for their wide utilisation for saline soil rehabilitation.