Cloning and characterization of Na+/H+ antiporter (LfNHX1) gene from a halophyte grass Leptochloa fusca for drought and salt tolerance

Abiotic stresses such as salinity and drought have adverse effects on plants. In the present study, a Na⁺/H⁺ antiporter gene homologue (LfNHX1) has been cloned from a local halophyte grass (Leptochloa fusca). The LfNHX1 cDNA contains an open reading frame of 1,623 bp that encodes a polypeptide chain of 540 amino acid residues. LfNHX1 protein sequence showed high similarity with NHX1 homologs reported from other halophyte plants. Amino acid and nucleotide sequence similarity, protein topology modeling and the presence of conserved functional domains in the LfNHX1 protein sequence classified it as a vacuolar NHX1 homolog. The overexpression of LfNHX1 gene under CaMV35S promoter conferred salt and drought tolerance in tobacco plants. Under drought stress, transgenic plants showed higher relative water contents, photosynthetic rate, stomatal conductance and membrane stability index as compared to wild type plants. More negative value of leaf osmotic potential was also observed in transgenic plants when compared with wild type control plants. Transgenic plants showed better germination and root growth at 2 mg L^?1 Basta herbicide and three levels (100, 200 and 250 mM) of sodium chloride. These results showed that LfNHX1 is a potential candidate gene for enhancing drought and salt tolerance in crops.     

Over-expression of an FT-homologous gene of apple induces early flowering in annual and perennial plants

The protein encoded by the FLOWERING LOCUS T (FT) gene from Arabidopsis thaliana seems to be the long-searched florigen, and over-expression of FT orthologues resulted in accelerated flower development in annual and perennial plants. In the present study, we isolated two allelic mRNA sequences of an FT-homologous gene from apple, which was designated as MdFT1. Using a SSR motif this gene was mapped on LG 12 of apple. Over-expression of MdFT1 in Arabidopsis and the commercially important tree species poplar and apple itself using the CaMV 35S or the Arabidopsis Suc2 promoter resulted in significant accelerated flowering compared with wild-type plants. Transgenic T₀ plants of Arabidopsis flowered 4–6 days on average earlier than wild-type Arabidopsis under LD conditions. Under short-day conditions Suc2::MdFT1 plants of the T₁-generation flowered after 66 ± 18 days, while wild-type plants flowered about 22 days later. All transgenic Arabidopsis plants showed a normal habit except for the early flowering phenotype. Early flowering was detected 6–10 months after transformation in transgenic polar clones containing MdFT1 driven by the CaMV 35S, whereas plants of the transgenic apple clone T780 set up its first flowers during in vitro cultivation. Based on our results we conclude that MdFT1 is responsible for inducing flowering and that the function of the apple FT1 gene is conserved in annual herbaceous species as well as perennial woody species. Furthermore, we discuss the role of MdFT1 in flower development with regard to the findings of genetic studies on apple.     

Complete chloroplast genome of cultivated flowering cherry, <Emphasis Type="Italic">Prunus</Emphasis> ×<Emphasis Type="Italic">yedoensis</Emphasis> ‘Somei-yoshino’ in comparison with wild <Emphasis Type="Italic">Prunus yedoensis</Emphasis> Matsum. (Rosaceae)

Prunus ×yedoensis Matsum. ‘Somei-yoshino’ is the most common and widespread cultivar of the ornamental flowering cherries. We hereby report its complete chloroplast (cp) genome sequences generated by whole-genome next-generation sequencing approach. The cp genome size was 157,792 bp in length consisting of four regions; large single-copy region (85,914 bp), small single-copy region (19,120 bp), and a pair of inverted repeat regions (26,379 bp). The genome contained a total of 131 genes, including 86 coding genes, 8 rRNA genes, and 37 tRNA genes. A total of 92 simple sequence repeats (SSRs) were detected within the cp genome. Its molecular features were compared with the complete cp genome of wild P. yedoensis, which occurs rarely in natural habitats of Mt. Halla in Jeju Island, Korea, displaying nearly indistinguishable morphology as P. ×yedoensis ‘Somei-yoshino’. Although both cp genomes were structured highly alike, the sequence variations between them were revealed in several single-nucleotide polymorphisms (SNPs). Using additional individuals of wild and cultivated flowering cherries, PCR amplification confirmed that those SNPs were phylogenetically informative, providing distinction between wild and cultivated flowering cherries. In future study, the SNPs and SSRs reported in this study could be used to identify wild individuals from morphologically identical cultivars of flowering cherries and also to conserve the genetic diversity of wild flowering cherries in Jeju Island.     

The Fast-track breeding approach can be improved by heat-induced expression of the FLOWERING LOCUS T genes from poplar (Populus trichocarpa) in apple (Malus × domestica Borkh.)

The Fast-track breeding approach in apple is based on the utilization of the BpMADS4 gene from Betula pendula. However, this approach has several disadvantages which could be solved using other flowering inducing genes and inducible promoters. The FLOWERING LOCUS T genes (PtFT1 and PtFT2) from poplar (Populus trichocarpa) driven by the heat-inducible Gmhsp 17.5-E (HSP)-promoter from soybean (Glycine max) were transferred into apple (Malus × domestica Borkh.) cv. ‘Pinova’ in order to induce flowering. Seven transgenic apple lines were obtained. All transgenic apple lines micrografted onto ‘Golden Delicious’ seedlings used as rootstocks were transferred to the greenhouse. Six out of seven transgenic lines developed flowers after a heat treatment at 42 °C for 1 h daily over a period of 28 days. The transgenic line T836 failed to flower. Flower morphology and pollen vitality of transgenic lines appeared normal. Transgenic plants were successfully used for hybridizations. Pollen from Malus ×robusta 5 applied to flowers of transgenic plants resulted in fruit formation. Heat induced PtFT1, respectively PtFT2 over-expressing rootstocks did not cause flowering in micrografted non-transgenic ‘Pinova’ scions. The mRNA of the PtFT genes was transported from transgenic rootstocks to non-transgenic scions only in one case. As a balance between plant development and flowering is important for the production of early flowering plants usable for a fast-track breeding program the new approach based on heat-induced flowering could be a refinement of the fast breeding program using the possibility of turning-on-turning-off flowering in physiological well developed plants.     

Genome-type-specific variation of the 19th intron sequence within the RNA polymerase I largest subunit gene in the genus Oryza

Rice PolA1 gene, encoding for the largest subunit of RNA polymerase I, spans ca. 15 kb containing 21 exons and presents as a single-copy-per-haploid genome. The genus Oryza comprises 22 wild species and 9 recognized genome types: AA, BB, CC, EE, FF, GG, BBCC, CCDD, and HHJJ. We analyzed sequences of the 19th intron (PI19) within PolA1 genes in 17 Oryza species. The AA species, containing two cultivated species, showed similar length of PI19 to that of CC species (287–296 bp). The longer PI19s were found in BB (502 bp) and FF (349 bp) species, although EE (217 bp) and GG (222 bp) species had shorter sequences. The size differences of the PI19s are particularly useful to discriminate between diploid (BB and CC) and allotetraploid (BBCC) species using simple PCR analysis. The evolutionary relationship among seven genomes was inferred based on the comparison of their PI19 sequences.     

Genetic Diversity in Exon 2 of the Major Histocompatibility Complex Class II DQB1 Locus in Nigerian Goats

The DQB1 locus is located in the major histocompatibility complex (MHC) class II region and involved in immune response. We identified 20 polymorphic sites in a 228 bp fragment of exon 2, one of the most critical regions of the MHC DQB1 gene, in 60 Nigerian goats. Four sites are located in the peptide binding region, and 10 amino acid substitutions are peculiar to Nigerian goats, compared with published sequences. A significantly higher ratio of nonsynonymous/synonymous substitutions (d _N/d _S) suggests that allelic sequence evolution is driven by balancing selection (P < 0.01). In silico functional analysis using PANTHER predicted that substitution P56R, with a subPSEC score of ?4.00629 (P_deleterious = 0.73229), is harmful to protein function. The phylogenetic tree from consensus sequences placed the two northern breeds closer to each other than either was to the southern goats. This first report of sequence diversity at the DQB1 locus for any African goat breed may be useful in the search for disease-resistant genotypes.     

In vitro propagation of Dendrobium aphyllum (Orchidaceae)—seed germination to flowering

This communication describes asymbiotic seed germination, protocorm development, micropropagation and flowering in in vitro and hardened seedlings of Dendrobium aphyllum (Roxb.) C.E.C. Fischer. Effects of four culture media viz., Murashige and Skoog (MS); Phytamax (Sigma Chemical Co. USA; PM); Mitra et al. (M) and Knudson ‘C’ (KC), 6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D), peptone and activated charcoal were studied on seed germination and protocorm development. Maximum germination (97 %) was recorded in PM basal medium. Peptone (2.0 gl^?1) remarkably enhanced germination percentage (100 %), vigorous growth, high survival and subsequent development of protocorms, while in activated charcoal the response was not encouraging. BAP improved germination percentage, however, 2,4-D showed noticeably low seed germination. The morphogenetic response of protocorms and nodal segments of in vitro raised seedlings varied depending on type of explants and concentrations and combinations of plant growth regulators used. Stout root system was induced in 1/2PM + 0.5 mgl^?1 IAA. Approximately 10 % of the in vitro raised plants (4–5 cm) with 3–4 leaves flowered in vitro irrespective of flowering season. The well-rooted plants showed 80 % survival under green house conditions and flowering was noticed after 5–6 months in 10 % of hardened plants.     

Molecular cytogenetic analysis of the Vigna species distributed in Korea

Vigna plants distributed in Korea were analyzed by molecular cytogenetic fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH) and rDNA ITS/NTS sequences. FISH revealed that variable 45S rRNA gene loci (one to four) were localized on the terminal regions of chromosomes, while two conserved 5S rRNA gene loci from all species examined, except for rice bean (single locus), were detected. FISH and GISH showed the characteristic organization of rRNA gene loci and genomic homology on the chromosomes, indicating their cytogenetic relatationships. ITS sequence revealed that there was considerable variation in length (190–207 bp in ITS1, 205–221 bp in ITS2) and nucleotide composition (7–67 bp). The 5S rRNA gene unit comprised coding region (118 bp) and extensive sequence heterogeneity (97–221 bp). Phylogenetic analysis of the ITS and NTS sequences demonstrated that the Vigna species are divided into two groups: angularis (V. angularis, V. umbellata, V. nakashimae and V. nipponensis) and unguiculata (V. unguiculata, V. sesquipedalis and V. vexillata). Sequence data also showed that mung bean was closer to the angularis group.     

The reproductive biology of two understory plants in the Atlantic rain forest, Brazil

Outcrossing and sexual reproduction of most flowering plants depends on pollinators. Plant traits likely to be involved in pollinator attraction include flower color, shape, and size. Furthermore, plant or flower density and the temporal flowering pattern may have an effect on reproduction. In this study, we examine the pollination ecology, breeding system, female reproductive output, and germination of two tropical understory species, Stenostephanus lobeliiformis (Acanthaceae) and Besleria melancholica (Gesneriaceae), which differ in these traits. Pollinator observations revealed that the dense flowering S. lobeliiformis with pinkish flowers received a higher diversity of pollinators, but visitor frequency measured as visits per flower per hour was much less (0.1 h^?1) than that to B. melancholica, which has a smaller floral display of dull-colored flowers (1.5 h^?1). Pollination experiments revealed that S. lobeliiformis but not B. melancholica is pollen-limited. In addition, both species are partially self-incompatible and depend on pollinators for outcrossing. Natural fruit set of open-pollinated unmanipulated flowers (control treatment) in both species is 22–26 %. Germination studies indicated inbreeding depression in S. lobeliiformis. We conclude that the pollination ecology of these species is influenced by a broad set of traits and that very different combinations of these traits can be successful in terms of reproduction.     

Use of PCR-RFLP for the discrimination of Japanese Porphyra and Pyropia species (Bangiales, Rhodophyta)

In order to distinguish 18 Porphyra and Pyropia species, the present study employed polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis using mitochondrial DNA related to the ATP synthase F0 subunit 6 (ATP6) gene and partial mitochondrial DNA including trnC, rps11, sdh3, trnG, trnN, trnP, and rns. The two primer sets on the mitochondrial DNA used in this study were able to amplify single fragments with PCR in 16 Japanese and 2 non-Japanese Porphyra and Pyropia species. Lengths of partial mitochondrial DNA related to ATP6 gene and trnC–rns ranged from 664 bp (Py. dentata and Py. haitanensis) to 677 bp (Py. lacerata and Py. kurogii) and from 1,292 bp (Py. seriata) to 1,343 bp (Py. kurogii and Py. moriensis), respectively. All 18 species were successfully distinguished using a combination of five restriction enzymes (TaqI, SspI, AciI, Cfr13I, and AluI). It was therefore concluded that PCR-RFLP analysis is a valuable tool for discrimination of wild strains of Porphyra and Pyropia species for potential use in mariculture.     

Comparison of the internal transcribed spacer region (ITS) of the ribosomal RNA genes in wild and cultivated two and six-rowed barleys (Hordeum vulgare L.)

The ITS region of the ribosomal RNA genes from two and six-rowed cultivated barley (Hordeum vulgare subsp. distichon and H. v. subsp. hexastichon, respectively), and its two and six-rowed wild relatives (H. v. subsp. spontaneum and H. v. subsp. agriocrithon, respectively) was isolated and sequenced. The entire ITS region is 598 bp in the two-rowed taxa (H. v. subsp. distichon and H. v. subsp. spontaneum) and 599 bp in the six-rowed ones (agriochriton and hexastichon). The ITS1 is 217 bp in the six-rowed barleys (H. v. subsp. agriochriton and H. v. subsp. hexastichon) and 218 bp in the two-rowed barleys (H. v. subsp. distichon and H. v. subsp. spontaneum). The 5.8S region is 163 bp in all studied H. vulgare taxa. The ITS2 region is 217 bp in the two-rowed barleys (H. v. subsp. distichon and H. v. subsp. spontaneum) and 219 bp in the six rowed ones (H. v. subsp. hexastichon and H. v. subsp. agriochriton). The ITS sequence data of the studied taxa and that of three other wild Hordeum species (H. murinum, H. marinum and H. chilense) were aligned and a phylogeny tree was reconstructed using the Lasergene Program. H. v. subsp. spontaneum was appeared as the ancestor of all other H. vulgare taxa.     

Cranberry flowering times and climate change in southern Massachusetts

Plants in wild and agricultural settings are being affected by the warmer temperatures associated with climate change. Here we examine the degree to which the iconic New England cranberry, Vaccinium macrocarpon, is exhibiting signs of altered flowering phenology. Using contemporary records from commercial cranberry bogs in southeastern Massachusetts in the United States, we found that cranberry plants are responsive to temperature. Flowering is approximately 2 days earlier for each 1 °C increase in May temperature. We also investigated the relationship between cranberry flowering and flight dates of the bog copper, Lycaena epixanthe—a butterfly dependent upon cranberry plants in its larval stage. Cranberry flowering and bog copper emergence were found to be changing disproportionately over time, suggesting a potential ecological mismatch. The pattern of advanced cranberry flowering over time coupled with increased temperature has implications not only for the relationship between cranberry plants and their insect associates but also for agricultural crops in general and for the commercial cranberry industry.     

Phenological Sensitivity of Early and Late Flowering Species Under Seasonal Warming and Altered Precipitation in a Seminatural Temperate Grassland Ecosystem

Shifts in flowering phenology of plants are indicators of climate change. The great majority of existing phenological studies refer solely to gradual warming. However, knowledge on how flowering phenology responds to changes in seasonal variation of warming and precipitation regimes is missing. We report the onset of 22 early (flowering before/within May) and 23 late flowering (flowering after May) species in response to manipulated seasonal warming (equal to + 1.2°C; last 100-year summer/winter warming), additional winter rainfall, and modified precipitation variability (including a 1000-year extreme drought event followed by heavy rainfall) over the growing season in two consecutive years for a species-rich temperate grassland ecosystem. The average onset of flowering (over 2 years) was significantly advanced 3.1 days by winter warming and 1.5 days by summer warming compared to control. Early flowering species responded to seasonal warming in both years, while late-flowering species responded in only 1 year to summer warming. The average onset of early flowering species was significantly advanced, 4.9 days by winter warming and 2.3 days by summer warming. Species-specific analysis showed that even within the early flowering community there were divergences. A positive correlation between plant height and shift in flowering onset was detected under winter warming (R² = 0.20, p = 0.005). The average onsets of early and late flowering community were affected by neither winter rain nor growing season precipitation variability. Seasonal differences in warming, and particularly winter warming, might alter community dynamics among early and late flowering species which can cause shifts in the seasonal performances of temperate ecosystems.     

The possible origin of thick stem in Chinese wild soybean (Glycine soja)

Thicker, erect stem and enlarged seeds are characteristic of the domestication of cultivated soybeans (Glycine max) from its progenitor, wild soybean (G. soja). Wild soybeans have different stem thicknesses but the thick stem as defined here appears in a small number of small-seeded wild soybeans (≤2.0 g/100-seeds) in China. However, little attention has been paid to this phenomenon in considering the origin of thick stem in wild soybean. Here, we addressed this question through the study of a mixed field of wild, semi-wild and cultivated soybeans. Thick-stemmed samples had lower sensitivity to light period, higher mean genetic diversity (H _e = 0.090, H = 0.535) and higher mean multilocus outcrossing rate (t _m = 9.77 %), while thin-stemmed plants were the opposite (H _e = 0.029, H = 0.416) and lower mean outcrossing rate (t _m = 5.88 %). F statistics calculations indicated that there was genetic differentiation between the thin and thick stems. UPGM cluster analysis showed that not only were thick-stemmed wild soybeans genetically different from thin-stemmed ones, but they were also genetically closer to semi-wild soybean, to varying degrees completely dependent on seed size. These data strongly implied that the plants with thick stems had more complicated genetic backgrounds than the thin-stemmed ones, and that they were related to cultivated soybeans. This study suggests that if plants have distinctly thick stems (an average 2.5-fold thicker than other thin-stemmed plants) or stems similar to semi-wild plants and/or near to local soybeans in a natural wild population adjacent to farmlands, such plants could be cultivar-introgressive offspring.     

Regeneration of plants through somatic embryogenesis in Thymus hyemalis Lange,a potential medicinal and aromatic plant

A protocol for somatic embryogenesis was developed for Thymus hyemalis, a wild species in the Mediterranean region. First, the effects of explant type, plant growth regulators [kinetin (KIN) and 2,4-dichlorophenoxyacetic acid (2,4-D)], and genotype on callus induction were tested. For callus induction, the node was the best explant; Murashige and Skoog (MS) medium supplemented with 1.8 μM 2,4-D and 0.5 μM KIN was the best medium, and the genotype had a highly significant effect. To induce production of somatic embryos, the effects of KIN, 6-benzylaminopurine (BAP), and naphthalene acetic acid (NAA) were evaluated. After 5 wk of culture in the dark, MS medium supplemented with 4.44 μM BAP, 0.54 μM NAA, and 4.65 μM KIN gave the highest percentage (85%) of embryogenic callus and the highest number of somatic embryos (27.00) per 45 mg of callus. For germination and plant recovery, somatic embryos were transferred to MS medium without plant growth regulators and plantlet conversion from developed somatic embryos was 90%. In vitro plants with adequate growth and sufficient root systems were subsequently transplanted into a mixture of peat and vermiculite (2:1?v/v) under greenhouse conditions. The survival rate of the plantlets under ex vitro conditions was 80%.