Dramatic changes in leaf development of the native <Emphasis Type="Italic">Capsicum chinense</Emphasis> from the Seychelles at temperatures below 24°C

When a pepper cultivar (Capsicum chinense cv. Seychelles-2, Sy-2) native to the Seychelles was grown in Japan, all seedlings showed seasonal developmental abnormalities such as development of abnormally shaped leaves. Other pepper cultivars grew well in all seasons while the growth of cv. Sy-2 was stunted. In this study, we first examined the effects of various changes in temperature and photoperiod on the cv. Sy-2 phenotype. The results showed that temperatures lower than 24°C led to the formation of abnormal leaves. Second, morphological and anatomical analyses of cotyledons and true leaves developed at 28 and 20°C were conducted. The narrower and thicker cotyledons developed at 20°C had fewer palisade cells in the leaf-length direction, and more cells in the leaf-thickness direction. True leaves developed at 20°C were irregularly shaped, thicker and had smaller leaf area. In addition, true leaves developed at 20°C had fewer palisade cells in the leaf-length and leaf-width directions and had more cells in the leaf-thickness direction. Furthermore, abnormal periclinal cell divisions in the mesophyll and/or epidermal cell layers were observed during leaf blade development at 20°C. These results suggest that the observed changes in cell proliferation and abnormal periclinal cell divisions were related, at least in part, to abnormal leaf development of cv. Sy-2 at temperatures below 24°C.     

Defense response of a pepper cultivar cv. Sy-2 is induced at temperatures below 24°C

Temperature is one of the most important environmental factors that influence plant growth and development. Recent studies imply that plants show various responses to non-extreme ambient temperatures. Previously, we have found that a pepper cultivar cv. Sy-2 (Capsicum chinense) shows developmental defects at temperatures below 24°C. In this study, to gain new insights into the temperature sensitivity of cv. Sy-2, temperature-sensitive genes were screened using microarray techniques. At restrictive temperature of 20°C, almost one-fourth of the 411 up-regulated genes were defense related or predicted to be defense related. Further expression analyses of several defense-related genes showed that defense-related genes in cv. Sy-2 were constitutively expressed at temperatures below 24°C. Moreover, accumulation of high level of salicylic acid (SA) in cv. Sy-2 grown at 20°C suggests that the defense response is activated in the absence of pathogens. To confirm that the defense response is induced in cv. Sy-2 below 24°C, we evaluated the resistance to biotrophic bacterial pathogen Xanthomonas campestris pv. vesicatoria and necrotrophic fungal pathogen Cercospora capsici. Cv. Sy-2 showed enhanced resistance to X. campestris pv. vesicatoria, but not to C. capsici.     

Genetic Relationships Within and Between Capsicum Species

Genetic relationships were estimated among 24 accessions belonging to 11 species of Capsicum, using 2,760 RAPD markers based on touch-down polymerase chain reactions (Td-RAPD-PCR). These markers were implemented in analyses of principal coordinates, unweighted pair group mean average, and 2,000 bootstrap replications. The accessions were divided into four groups, corresponding to previously described Capsicum complexes: C. annuum complex (C_A), C. baccatum complex (C_B), C. pubescens complex (C_P), and C. chacoense accessions (C_A/_B). Their overall mean genetic similarity index was 0.487 ± 0.082, ranging from 0.88 to 0.32, based on Jaccard’s coefficient. The highest genetic variation was observed among the accessions in C_P; the accessions in C_B had a low level of variation as judged from the standard deviations of the genetic similarity indices. Based on the Td-RAPD-PCR markers, the 24 accessions were divided into four major groups, three of which corresponded to the three distinct Capsicum complexes. Accessions of C. chacoense were found to be equally related to complexes C_A, C_B, and C_P.     

Screening Genetic Resources of Capsicum Peppers in Their Primary Center of Diversity in Bolivia and Peru

For most crops, like Capsicum, their diversity remains under-researched for traits of interest for food, nutrition and other purposes. A small investment in screening this diversity for a wide range of traits is likely to reveal many traditional varieties with distinguished values. One objective of this study was to demonstrate, with Capsicum as model crop, the application of indicators of phenotypic and geographic diversity as effective criteria for selecting promising genebank accessions for multiple uses from crop centers of diversity. A second objective was to evaluate the expression of biochemical and agromorphological properties of the selected Capsicum accessions in different conditions. Four steps were involved: 1) Develop the necessary diversity by expanding genebank collections in Bolivia and Peru; 2) Establish representative subsets of ~100 accessions for biochemical screening of Capsicum fruits; 3) Select promising accessions for different uses after screening; and 4) Examine how these promising accessions express biochemical and agromorphological properties when grown in different environmental conditions. The Peruvian Capsicum collection now contains 712 accessions encompassing all five domesticated species (C. annuum, C. chinense, C. frutescens, C. baccatum, and C. pubescens). The collection in Bolivia now contains 487 accessions, representing all five domesticates plus four wild taxa (C. baccatum var. baccatum, C. caballeroi, C. cardenasii, and C. eximium). Following the biochemical screening, 44 Bolivian and 39 Peruvian accessions were selected as promising, representing wide variation in levels of antioxidant capacity, capsaicinoids, fat, flavonoids, polyphenols, quercetins, tocopherols, and color. In Peru, 23 promising accessions performed well in different environments, while each of the promising Bolivian accessions only performed well in a certain environment. Differences in Capsicum diversity and local contexts led to distinct outcomes in each country. In Peru, mild landraces with high values in health-related attributes were of interest to entrepreneurs. In Bolivia, wild Capsicum have high commercial demand.     

Genetic and enzymatic characterization of conditional lethal mutants of the yeast Schizosaccharomyces pombe with a temperature-sensitive DNA ligase.

The DNA ligase activities of wild type and temperature-sensitive lethal cdc 17 mutants of Schizosaccharomyces pombe have been studied by measuring effects on the conversion of relaxed DNA circles containing a single nick to a closed circular form. Such assays have revealed that all cdc 17 mutants have a thermosensitive DNA ligase deficiency, that this deficiency cosegregates 2:2 with their temperature-sensitive cdc-lethality in three tetrads derived from a cross against wild type, and that genetic reversion of the temperature-sensitive cdc^? phenotype is accompanied by a restoration of DNA ligase activity; all of which implies that the temperature-sensitive cdc^? phenotype of cdc 17 mutants is due to a single nuclear mutation causing a DNA ligase deficiency. Both wild type and mutant enzymes have been partially purified by chromatography in heparin/agarose columns. The wild-type enzyme is completely stable in vitro at both permissive (25 °C) and restrictive (35 °C) temperatures, whereas that of two different mutants, though completely stable at 25 °C, is rapidly inactivated at 35 °C, implying that their mutations are located in the structural gene for DNA ligase.     

A versatile PCR marker for pungency in Capsicum spp.

Pungency in Capsicum spp. is an important quality trait for pepper breeding. The perception of pungency in pepper is due to the presence of a group of compounds named capsaicinoids, only found within the Capsicum genus. How pungency is controlled at genetic and molecular levels has not been completely elucidated. The use of molecular markers to assess pungency trait is required for molecular breeding, despite the difficulty of development of universal markers for this trait. In this work, a DNA sequence possibly related to pungency with a high similarity to Pun1 locus was studied, and sequence analysis of this homolog revealed a 15?bp deletion in non-pungent pepper accessions. An allele-specific pair of primers was designed and specific fragments of 479?bp from non-pungent and 494?bp from pungent accessions were obtained. Polymorphism of this marker, named MAP1, was tested in a wide range of accessions, belonging to several Capsicum species, including pungent and non-pungent accessions of C. annuum L., and pungent accessions of C. chinense, C. baccatum, C. frutescens, C. pubescens, C. galapagoense, C. eximium, C. tovarii, C. cardenasii, and C. chacoense. All these Capsicum accessions were correctly discriminated. The marker suitability to assess pungency in domesticated and wild Capsicum species was demonstrated, and therefore it will be very useful in marker assisted selection (MAS). Moreover, MAP1 was located in a saturated pepper linkage map and its possible relationship with the Pun1 locus has been discussed. Among the available markers for this complex quality trait, the marker developed in this study is the most universal so far.     

Efficient markerless gene replacement in Corynebacterium glutamicum using a new temperature-sensitive plasmid

Random chemical mutation of a Corynebacterium glutamicum-Escherichia coli shuttle vector derived from plasmid pCGR2 was done using hydroxylamine. It brought about amino acid substitutions G109D and E180K within the replicase superfamily domain of the plasmid's RepA protein and rendered the plasmid highly unstable, especially at higher incubation temperatures. Colony formation of C. glutamicum was consequently completely inhibited at 37 °C but not at 25 °C. G109 is a semi-conserved residue mutation which resulted in major temperature sensitivity. E180 on the other hand is not conserved even among RepA proteins of closely related C. glutamicum pCG1 family plasmids and its independent mutation caused relatively moderate plasmid instability. Nonetheless, simultaneous mutation of both residues was required to achieve temperature-sensitive colony formation. This new pCGR2-derived temperature-sensitive plasmid enabled highly efficient chromosomal integration in a variety of C. glutamicum wild-type strains, proving its usefulness in gene disruption studies. Based on this, an efficient markerless gene replacement system was demonstrated using a selection system incorporating the temperature-sensitive replicon and Bacillus subtilis sacB selection marker, a system hitherto not used in this bacterium. Single-crossover integrants were accurately selected by temperature-dependent manner and 93% of the colonies obtained by the subsequent sucrose selection were successful double-crossover recombinants.     

Mutation of the gene for the second-largest subunit of RNA polymerase I prolongs the period length of the circadian conidiation rhythm in Neurospora crassa

The period length of the circadian conidiation rhythm was examined in a mutant strain of Neurospora crassa, un-18, that is temperature sensitive for mycelial growth. The un-18 mutant showed a temperature-sensitive phenotype with respect to both mycelial growth and the period length of the conidiation rhythm. Below 22°?C, the un-18 mutation did not affect the period length, but at temperatures between 22°?C and 32°?C, the period length of the un-18 mutant was ～2 h longer than that of the wild-type strain. The un-18 ⁺ gene was cloned and was found to encode the second-largest subunit of RNA polymerase I, which is involved in the synthesis of rRNA. These results indicate that a defect in ribosome synthesis, which must result in a lower rate of protein synthesis, lengthens the period of the circadian conidiation rhythm in Neurospora.     

SwoHp, a nucleoside diphosphate kinase, is essential in Aspergillus nidulans

The temperature-sensitive swoH1 mutant of Aspergillus nidulans was previously identified in a screen for mutants with defects in polar growth. In the present work, we found that the swoH1 mutant swelled, lysed, and did not produce conidia during extended incubation at the restrictive temperature. When shifted from the permissive to the restrictive temperature, swoH1 showed the temperature-sensitive swelling phenotype only after 8 h at the higher temperature. The swoH gene was mapped to chromosome II and cloned by complementation of the temperature-sensitive phenotype. The sequence showed that swoH encodes a homologue of nucleoside diphosphate kinases (NDKs) from other organisms. Deletion experiments showed that the swoH gene is essential. A hemagglutinin-SwoHp fusion complemented the mutant phenotype, and the purified fusion protein possessed phosphate transferase activity in thin-layer chromatography assays. Sequencing of the mutant allele showed a predicted V83F change. Structural modeling suggested that the swoH1 mutation would lead to perturbation of the NDK active site. Crude cell extracts from the swoH1 mutant grown at the permissive temperature had ~20% of the NDK activity seen in the wild type and did not show any decrease in activity when assayed at higher temperatures. Though the data are not conclusive, the lack of temperature-sensitive NDK activity in the swoH1 mutant raises the intriguing possibility that the SwoH NDK is required for growth at elevated temperatures rather than for polarity maintenance.     

Species-Dependent Phenotypes of Replication-Temperature-Sensitive trfA Mutants of Plasmid RK2: a Codon-Neutral Base Substitution Stimulates Temperature Sensitivity by Leading to Reduced Levels of trfA Expression

TrfA is the only plasmid-encoded protein required for initiation of replication of the broad-host-range plasmid RK2. Here we describe the isolation of four trfA mutants temperature sensitive for replication in Pseudomonas aeruginosa. One of the mutations led to substitution of arginine 247 with cysteine. This mutant has been previously described to be temperature sensitive for replication, but poorly functional, in Escherichia coli. The remaining three mutants were identical, and each of them carried two mutations, one leading to substitution of arginine 163 with cysteine (mutation 163C) and the other a codon-neutral mutation changing the codon for glycine 235 from GGC to GGU (mutation 235). Neither of the two mutations caused a temperature-sensitive phenotype alone in P. aeruginosa, and the effect of the neutral mutation was caused by its ability to strongly reduce the trfA expression level. The double mutant and mutant 163C could not be stably maintained in E. coli, but mutant 235 could be established and, surprisingly, displayed a temperature-sensitive phenotype in this host. Mutation 235 strongly reduced the trfA expression level also in E. coli. The glycine 85 codon in trfA mRNA is GGU, and a change of this to GGC did not significantly affect expression. In addition, we found that wild-type trfA was expressed at much lower levels in E. coli than in P. aeruginosa, indicating that this level is a key parameter in the determination of the temperature-sensitive phenotypes in different species. The E. coli lacZ gene was translationally fused at the 3′ end and internally in trfA, in both cases leading to elimination of the effect of mutation 235 on expression. We therefore propose that this mutation acts through an effect on mRNA structure or stability.     

Submergence tolerance in relation to variable floodwater conditions in rice

Flash floods adversely affect rice productivity in vast areas of rainfed lowlands in South and Southeast Asia and tropical Africa. Tolerant landraces that withstand submergence for 1–2 weeks were identified; however, incorporation of tolerance into modern high-yielding varieties through conventional breeding methods has been slow because of the complexity of both the tolerance phenotype and floodwater conditions, and the ensuing discrepancies encountered upon phenotyping in different environments. Designing an effective phenotyping strategy requires a thorough understanding of the specific floodwater characteristics that most likely affect survival during flooding. We investigated the implications of floodwater temperature and light penetration, caused by artificial shading, seasonal variation, or water turbidity, for seedling survival after submergence. Three field experiments were conducted using rice genotypes contrasting in their tolerance of submergence: FR13A and Kusuma (tolerant); Gangasiuli (intermediate); Sabita, CRK-2-6 and Raghukunwar (elongating/avoiding types); and IR42 (sensitive). We tested the hypotheses that warmer floodwater decreases plant survival and that turbid water augment plant mortality by causing effects similar to those caused by shading, by reducing light penetration. Plants survive better when water is cooler, and survival decreased at about 8% per unit increase in water temperature above 26 °C. Lower intensity of light and warmer temperatures seem to reduce biomass and increase mortality under flooding. An increase in the concentrations of O₂ and CO₂ and a decrease in water pH did not improve survival in clear unshaded water. Turbid floodwater was more damaging to rice as plant mortality increased as the percentage of silt increased, and the effects of water turbidity cannot be explained by the reduction in light penetration alone. Even the most tolerant rice cultivar, FR13A, experienced higher mortality when flooded with turbid floodwater. Correlation studies revealed that cultivars with the capacity to maintain higher biomass, higher chlorophyll, and non-structural carbohydrate concentrations after submergence had higher survival. These findings help to understand the variation observed in submergence tolerance when screening is done under different environments. The study could have implications for designing proper screening strategies and assessing the damage submergence causes across different rice-growing regions.     

Low temperature resistance in saplings and ramets of Polylepis sericea in the Venezuelan Andes

The frequent occurrence of all year-round below zero temperatures in tropical high mountains constitutes a most stressful climatic factor that plants have to confront. Polylepis forests are found well above the continuous forest line and are distributed throughout the Andean range. These trees require particular traits to overcome functional limitations imposed on them at such altitudes. Considering seedling and sapling stages as filter phases in stressful environments, some functional aspects of the regeneration of Polylepis sericea, a species associated to rock outcrops in the Venezuelan Andes, were studied. We characterized microclimatic conditions within a forest, in a forest gap and surrounding open páramo and determined low temperature resistance mechanisms in seedlings, saplings and ramets. Conditions in the forest understory were more stable compared to the forest gaps and open surrounding páramo. Minimum temperatures close to the ground were 3.6 °C lower in the open páramo compared to the forest understory. Maximum temperatures were 9.0 °C higher in the open páramo. Ice nucleation and injury temperatures occurred between ?6 and ?8 °C for both ramets and saplings, an evidence of frost avoidance to low nighttime temperatures. In this particular forest, this resistance ability is determinant in their island-like distribution in very specific less severe temperature habitats.     

Analysis of genetic diversity and population structure in <Emphasis Type="Italic">Capsicum</Emphasis> landraces from North Eastern India using TE-AFLP markers

North eastern (NE) India harbours a precious germplasm repository of Capsicum in the form of various landraces. The present study was undertaken to characterise the extent of genetic variation present in different Capsicum landraces from north eastern India. A set of 171 Capsicum accessions were characterised using three-endonuclease amplified fragment length polymorphism (AFLP) markers. Out of 416 bands obtained from six primer combinations, 254 (61 %) were polymorphic. The pairwise genetic dissimilarity among accessions ranged from 0.03 to 0.97. Cluster analysis based on neighbour joining showed two major clusters. Cluster I contained most of the bhut jolokia accessions whereas cluster II contained all of the Capsicum annuum genotypes. Similar grouping was observed with population STRUCTURE analysis as well as principle coordinate analysis. Analysis of molecular variance (AMOVA) revealed 45 and 54 % variation among and within populations, respectively. This information on population structure analysis and molecular characterisation will be helpful for effective utilisation of this germplasm in Capsicum improvement programs.     

Response of Antarctic, temperate, and tropical microalgae to temperature stress

The global temperature increase has significant implications on the survival of microalgae which form the basis of all aquatic food webs. The aim of this study was to compare the response of similar taxa of microalgae from the Antarctic (Chlamydomonas UMACC 229, Chlorella UMACC 237, and Navicula glaciei UMACC 231), temperate (Chlamydomonas augustae UMACC 247, Chlorella vulgaris UMACC 248, and Navicula incerta UMACC 249), and tropical (C. augustae UMACC 246, C. vulgaris UMACC 001, and Amphiprora UMACC 239) regions to changing temperature. The Antarctic, temperate, and tropical strains were grown over specific temperature ranges of 4 °C to 30 °C, 4 °C to 32 °C, and 13 °C to 38 °C, respectively. The three Antarctic strains survived at temperatures much higher than their ambient regime. In comparison, the tropical strains are already growing at their upper temperature limits. The three Chlorella strains from different regions are eurythermal, with a large overlap on tolerance ranging from 4 °C to 38 °C. The specific growth rate (μ) of the Antarctic Navicula decreased (<0.34 day^?1) at temperatures above 4 °C, showing it to be sensitive to temperature increase. If further warming of Earth occurs, N. glaciei UMACC 231 is likely to have the most deleterious consequences than the other two Antarctic microalgae studied. The percentage of polyunsaturated fatty acids (PUFA) decreased with increasing temperature in the Antarctic Navicula. As temperature increases, the growth and nutritional value of this commonly occurring diatom in the Antarctic may decrease, with consequences for the aquatic food web. Of the three Chlamydomonas strains, only the Antarctic strain produced predominantly PUFA, especially 16:3 (48.4–57.2 % total fatty acids).     

<Emphasis Type="Italic">Capsicum</Emphasis> Use in Cambodia: The Continental Region of Southeast Asia Is Not Related to the Dispersal Route of <Emphasis Type="Italic">C. frutescens</Emphasis> in the Ryukyu Islands

Capsicum Use in Cambodia: The Continental Region of Southeast Asia Is Not Related to the Dispersal Route of C. frutescens in the Ryukyu Islands. The local nomenclature and use of Capsicum by Khmer and other ethnic groups in Cambodia and the distribution of the diagnostic ShDH-B isozyme pattern of C. frutescens were studied. People in Cambodia use Capsicum in various ways, not only as a condiment but also as a vegetable, a medicine, and a colorant, and in popular beliefs, agricultural rituals, taboos, and rice malt. The findings showed that the ShDH-B phenotype may not have occurred as a mutation in Asia but in the Americas and then was introduced to Asia. Also, the ShDH-B phenotype is distributed in the insular regions of Southeast and East Asia and Oceania, but seems not to be distributed in the continental region of Southeast Asia. One possible hypothesis is that C. frutescens possessing the ShDH-B phenotype was introduced directly from the Americas via Oceania to the Philippines, and it thereafter dispersed into the insular regions.     

Temperature responses of tropical to warm temperateCladophora species in relation to their distribution in the North Atlantic Ocean

The relationship between distribution boundaries and temperature responses of some North AtlanticCladophora species (Chlorophyta) was experimentally examined under various regimes of temperature, light and daylength. Experimentally determined critical temperature intervals, in which survival, growth or reproduction was limited, were compared with annual temperature regimes (monthly means and extremes) at sites inside and outside distribution boundaries. The species tested belonged to two phytogeographic groups: (1) the tropical West Atlantic group (C. submarina: isolate from Curaçao) and (2) the amphiatlantic tropical to warm temperate group (C. prolifera: isolate from Corsica;C. coelothrix: isolates from Brittany and Curaçao; andC. laetevirens: isolates from deep and shallow water in Corsica and from Brittany). In accordance with distribution from tropical to warm temperate regions, each of the species grew well between 20–30°C and reproduction and growth were limited at and below 15°C. The upper survival limit in long days was <35°C in all species but high or maximum growth rates occurred at 30°C.C. prolifera, restricted to the tropical margins, had the most limited survival at 35°C. Experimental evidence suggests thatC. submarina is restricted to the Caribbean and excluded from the more northerly American mainland and Gulf of Mexico coasts by sporadic low winter temperatures in the nearshore waters, when cold northerly weather penetrates far south every few years. Experimental evidence suggests thatC. prolifera, C. coelothrix andC. laetevirens are restricted to their northern European boundaries by summer temperatures too low for sufficient growth and/or reproduction. Their progressively more northerly located boundaries were accounted for by differences in growth rates over the critical 10–15°C interval.C. prolifera andC. coelothrix are excluded or restricted in distribution on North Sea coasts by lethal winter temperatures, again differences in cold tolerance accounting for differences in their distribution patterns. On the American coast, species were probably restricted by lethal winter temperatures in the nearshore and, in some cases, by the absence of suitable hard substrates in the more equable offshore waters. Isolates from two points along the European coast (Brittany, Corsica) ofC. laetevirens showed no marked differences in their temperature tolerance but the Caribbean and European isolates ofC. coelothrix differed markedly in their tolerance to low temperatures, the lethal limit of the Caribbean isolate lying more than 5°C higher (at ca 5°C).     

Isolation and Characterization of an Escherichia coli K-12 Mutant with a Temperature-Sensitive RecA− Phenotype

A mutation that causes a temperature-sensitive RecA(-) phenotype was identified in a derivative of a PolA(-) strain that failed to grow at high temperature. The mutant allele (recA200) was shown to be linked to cysC, conferred a sharply temperature-sensitive, ultraviolet-sensitive Rec(-) phenotype in the range 35 to 42 C, and in crosses failed to show complementation at 42 C with Hfr's that transferred recA(-). Double mutants that carried both recA200 and polA were examined for ability to grow and synthesize DNA at restrictive temperatures.     

Effects of temperature on growth of Vallisneria americana in a sub-tropical estuarine environment

The submersed aquatic vegetation (SAV) species Vallisneria americana Michx. (tape grass) is a valuable resource in the Caloosahatchee estuary and in many other aquatic systems. Given the variable nature of freshwater inflows and environmental conditions in the Caloosahatchee, it is necessary to understand how tape grass will respond to high and low salinity conditions at different light and temperature levels. Specifically, quantitative information is needed as input to modeling tools that can be applied to predict growth and survival of tape grass under a range of environmental conditions present in the estuary. We determined growth rates for small and medium sized tape grass plants obtained from the Caloosahatchee estuary, southwest coastal Florida, USA in freshwater (0.5 psu) under high (331 μE m^?2 s^?1) and low light (42 μE m^?2 s^?1) and at 10 psu under high light conditions. We ran six treatments at five temperatures spanning 13–32 °C for 8–9 weeks. The optimum temperature for growth was roughly 28 °C, with a minimum threshold temperature of 13 °C and a maximum threshold temperature of 38 °C. Plants grew fastest in freshwater, at high light and temperatures greater than 20 °C. The slowest growth rates were observed at 13 °C regardless of salinity, light or plant size. Our results suggest that tape grass growth is strongly influenced by water temperature and that additional stressors such as low light and elevated salinity can reduce the range of temperature tolerance, especially at colder water temperatures.