Chilling-sensitive Research Articles

Genetic linkage mapping and quantitative trait locus (QTL) analysis of sweet basil (Ocimum basilicum L.) to identify genomic regions associated with cold tolerance and major volatiles.

Chilling sensitivity is one of the greatest challenges affecting the marketability and profitability of sweet basil (Ocimum basilicum L.) in the US and worldwide. Currently, there are no sweet basils commercially available with significant chilling tolerance and traditional aroma profiles. This study was conducted to identify quantitative trait loci (QTLs) responsible for chilling tolerance and aroma compounds in a biparental mapping population, including the Rutgers advanced breeding line that served as a chilling tolerant parent, 'CB15', the chilling sensitive parent, 'Rutgers Obsession DMR' and 200 F2 individuals. Chilling tolerance was assessed by percent necrosis using machine learning and aroma profiling was evaluated using gas chromatography (GC) mass spectrometry (MS). Single nucleotide polymorphism (SNP) markers were generated from genomic sequences derived from double digestion restriction-site associated DNA sequencing (ddRADseq) and converted to genotype data using a reference genome alignment. A genetic linkage map was constructed and five statistically significant QTLs were identified in response to chilling temperatures with possible interactions between QTLs. The QTL on LG24 (qCH24) demonstrated the largest effect for chilling response and was significant in all three replicates. No QTLs were identified for linalool, as the population did not segregate sufficiently to detect this trait. Two significant QTLs were identified for estragole (also known as methyl chavicol) with only qEST1 on LG1 being significant in the multiple-QTL model (MQM). QEUC26 was identified as a significant QTL for eucalyptol (also known as 1,8-cineole) on LG26. These QTLs may represent key mechanisms for chilling tolerance and aroma in basil, providing critical knowledge for future investigation of these phenotypic traits and molecular breeding.

A major gene for chilling tolerance variation in Indica rice codes for a kinase OsCTK1 that phosphorylates multiple substrates under cold.

Rice is susceptible to chilling stress. Identifying chilling tolerance genes and their mechanisms are key to improve rice performance. Here, we performed a genome-wide association study to identify regulatory genes for chilling tolerance in rice. One major gene for chilling tolerance variation in Indica rice was identified as a casein kinase gene OsCTK1. Its function and natural variation are investigated at the physiological and molecular level by its mutants and transgenic plants. Potential substrates of OsCTK1 were identified by phosphoproteomic analysis, protein-protein interaction assay, in vitro kinase assay, and mutant characterization. OsCTK1 positively regulates rice chilling tolerance. Three of its putative substrates, acidic ribosomal protein OsP3B, cyclic nucleotide-gated ion channel OsCNGC9, and dual-specific mitogen-activated protein kinase phosphatase OsMKP1, are each involved in chilling tolerance. In addition, a natural OsCTK1 chilling-tolerant (CT) variant exhibited a higher kinase activity and conferred greater chilling tolerance compared with a chilling-sensitive (CS) variant. The CT variant is more prevalent in CT accessions and is distributed more frequently in higher latitude compared with the CS variant. This study thus enables a better understanding of chilling tolerance mechanisms and provides gene variants for genetic improvement of chilling tolerance in rice.

Different responses of banana classical AGP genes and cell wall AGP components to low-temperature between chilling sensitive and tolerant cultivars.

Seventeen classical MaAGPs and 9 MbAGPs were identified and analyzed. MaAGP1/2/6/9/16/17, the antigens of JIM13 and LM2 antibodies are likely to be involved in banana chilling tolerance. Classical arabinogalactan proteins (AGPs) belong to glycosylphosphatidylinositol-anchored proteins, which are proved to be involved in signaling and cell wall metabolism upon stresses. However, rare information is available on the roles of classical AGPs in low temperature (LT) tolerance. Cultivation of banana in tropical and subtropical region is seriously threatened by LT stress. In the present study, 17 classical MaAGPs and nine MbAGPs in banana A and B genome were identified and characterized, respectively. Great diversity was present among different classical MaAGP/MbAGP members while five members (AGP3/6/11/13/14) showed 100% identity between these two gene families. We further investigated different responses of classical AGPs to LT between a chilling sensitive (CS) and tolerant (CT) banana cultivars. In addition, different changes in the temporal and spatial distribution of cell wall AGP components under LTs between these two cultivars were compared using immunofluorescence labeling. Seven classical MbAGPs were upregulated by LT(s) in the CT cultivar. Classical MaAGP4/6 was induced by LT(s) in both cultivars while MaAGP1/2/9/16/17 only in the CT cultivar. Moreover, these genes showed significantly higher transcription abundance in the CT cultivar than the CS one under LT(s) except classical MaAGP4. Similar results were observed with the epitopes of JIM13 and LM2 antibodies. The antigens of these antibodies and classical MaAGP1/2/6/9/16/17 might be related to LT tolerance of banana. These results provide additional information about plant classical AGPs and their involvement in LT tolerance, as well as their potential as candidate genes to be targeted when breeding CT banana.

HSP90 Contributes to chs3-2D-Mediated Autoimmunity.

Plants employ multi-layered immune system to fight against pathogen infections. Different receptors are able to detect the invasion activities of pathogens, transduce signals to downstream components, and activate defense responses. Among those receptors, nucleotide-binding domain leucine-rich repeat containing proteins (NLRs) are the major intracellular ones. CHILLING SENSITIVE 3 (CHS3) is an Arabidopsis NLR with an additional Lin-11, Isl-1 and Mec-3 (LIM) domain at its C terminus. The gain-of-function mutant, chs3-2D, exhibiting severe dwarfism and constitutively activated defense responses, was selected as a genetic background in this study for a forward genetic screen. A mutant allele of hsp90.2 was isolated as a partial suppressor of chs3-2D, suggesting that HSP90 is required for CHS3-mediated defense signaling. In addition, HSP90 is also required for the autoimmunity of the Dominant Negative (DN)-SNIPER1 and gain-of-function ADR1-L2 D484V transgenic lines, suggesting a broad role for HSP90 in NLR-mediated defense. Overall, our work indicates a larger contribution of HSP90 not only at the sensor, but also the helper NLR levels.

Stronger Spring Phenological Advance in Future Warming Scenarios for Temperate Species With a Lower Chilling Sensitivity.

Spring warming could induce earlier leaf-out or flowering of temperate plant species, and decreased chilling in winter has a delaying effect on spring phenology. However, the relative contribution of the decreased chilling and increased forcing on spring phenological change is unclear. Here, we analyzed the experimental data for 14 temperate woody species in Beijing, China and quantified the forcing requirements (FR) of spring phenology and chilling sensitivity (the ratio of the FR at the low chilling condition to the FR at the high chilling condition) for each species. Furthermore, using species-specific functions between the amount of chilling and FR, we established a phenological model to simulate the annual onset dates of spring events during the past 69 years (1952–2020) and in the future (2021–2099) under RCP 4.5 and RCP 8.5 climate scenarios. We also developed a novel method to quantitatively split the predicted phenological change into the effects caused by changes in forcing and those caused by changes in chilling. The results show that the FR of spring events decreased with the increase in the amount of chilling, and this relationship could be described as an exponential decay function. The basic FR (the FR at the high chilling condition) and chilling sensitivity varied greatly among species. In the 1952–2020 period, the advancing effect of increased forcing was stronger than the effect of chilling, leading to earlier spring events with a mean trend of −1.96 days/decade. In future climate scenarios, the spring phenology of temperate species would continue to advance but will be limited by the decreased chilling. The species with lower chilling sensitivities showed stronger trends than those with high chilling sensitivities. Our results suggested that the delaying effect of declining chilling could only slow down the spring phenological advance to a certain extent in the future.

Lipidomics reveals the difference of membrane lipid catabolism between chilling injury sensitive and non-sensitive green bell pepper in response to chilling

Changes in membrane lipid composition play multiple roles in the response of pepper to chilling injury (CI). A membrane lipidomic approach was used to elucidate the response of a harvested peppers of a chilling-sensitive (CS) cultivar, ‘129′ and a chilling-tolerant (CT) cultivar, ‘130′ to chilling temperatures during storage. Changes in the lipid composition of CT fruit were proportionally smaller relative to the changes in CS fruit. Lipidomic analysis revealed that CI in CS fruit is associated with a decrease in lysophosphatidic acid, and an increase in phosphatidic acid, triacylglycerols, and steryl esters. Furthermore, a lower level of electrolyte leakage, malondialdehyde, lipoxygenase and phospholipases D activity was observed in CT fruit, indicating that CT fruit were able to maintain membrane function despite exposure to the low temperature. Overall, results indicate that CI in pepper is associated with extensive alterations in membrane lipid metabolism and that CT fruit can avoid CI by maintaining membrane lipid homeostasis.

The truncated TNL receptor TN2-mediated immune responses require ADR1 function.

The loss of function of exocyst subunit EXO70B1 leads to autoimmunity, which is dependent on TIR-NBS2 (TN2), a truncated intracellular nucleotide-binding and leucine-rich repeat receptor (NLR). However, how TN2 triggers plant immunity and whether typical NLRs are required in TN2-activated resistance remain unclear. Through the CRISPR/Cas9 gene editing system and knockout analysis, we found that the spontaneous cell death and enhanced resistance in exo70B1-3 were independent of the full-length NLR SOC3 and its closest homolog SOC3-LIKE 1 (SOC3-L1). Additionally, knocking out SOC3-L1 or TN2 did not suppress the chilling sensitivity conferred by chilling sensitive 1-2 (chs1-2). The ACTIVATED DISEASE RESISTANCE 1 (ADR1) family and the N REQUIREMENT GENE 1 (NRG1) family have evolved as helper NLRs for many typical NLRs. Through CRISPR/Cas9 gene editing methods, we discovered that the autoimmunity of exo70B1-3 fully relied on ADR1s, but not NRG1s, and ADR1s contributed to the upregulation of TN2 transcript levels in exo70B1-3. Furthermore, overexpression of TN2 also led to ADR1-dependent autoimmune responses. Taken together, our genetic analysis highlights that the truncated TNL protein TN2-triggered immune responses require ADR1s as helper NLRs to activate downstream signaling, revealing the importance and complexity of ADR1s in plant immunity regulation.

Comparative transcriptome analysis of leaves during early stages of chilling stress in two different chilling-tolerant brown-fiber cotton cultivars.

Chilling stress generates significant inhibition of normal growth and development of cotton plants and lead to severe reduction of fiber quality and yield. Currently, little is known for the molecular mechanism of brown-fiber cotton (BFC) to respond to chilling stress. Herein, RNA-sequencing (RNA-seq)-based comparative analysis of leaves under 4°C treatment in two different-tolerant BFC cultivars, chilling-sensitive (CS) XC20 and chilling-tolerant (CT) Z1612, was performed to investigate the response mechanism. A total of 72650 unigenes were identified with eight commonly used databases. Venn diagram analysis identified 1194 differentially expressed genes (DEGs) with significant up-regulation in all comparison groups. Furthermore, enrichment analyses of COG and KEGG, as well as qRT-PCR validation, indicated that 279 genes were discovered as up-regulated DEGs (UDEGs) with constant significant increased expression in CT cultivar Z1612 groups at the dimensions of both each comparison group and treatment time, locating in the enriched pathways of signal transduction, protein and carbohydrate metabolism, and cell component. Moreover, the comprehensive analyses of gene expression, physiological index and intracellular metabolite detections, and ascorbate antioxidative metabolism measurement validated the functional contributions of these identified candidate genes and pathways to chilling stress. Together, this study for the first time report the candidate key genes and metabolic pathways responding to chilling stress in BFC, and provide the effective reference for understanding the regulatory mechanism of low temperature adaptation in cotton.

Chilling sensitivity of four near-isogenic fruit-color genotypes of summer squash (Cucurbita pepo, Cucurbitaceae) and its association with tocopherol content

• Fruit color genes B and D affected chilling sensitivity of summer squash. • Yellow B/B fruit were more chilling sensitive than green b/b fruit. • Gene D (enhanced pigmentation) alleviated chilling sensitivity caused by gene B . • Exocarp tocopherol content accorded with chilling tolerance of the genotypes. • Low-tocopherol B/B d/d fruit were prone to chilling injury. Accessions of Cucurbita pepo vary in the sensitivity of their young fruit (summer squash) to chilling injury (CI). The dominant gene B ( Bicolor ), which confers yellow fruit coloration, is known to increase susceptibility of summer squash to CI as compared with green, b/b , fruit. Another dominant gene, D ( Dark stem ), confers dark plant stems and somewhat enhanced coloration of the young fruit. However, the effect of this gene on chilling sensitivity of summer squash is unknown. Freshly harvested fruit of vegetable marrow-type summer squash from four near-isogenic genotypes, B/B D/D , B/B d/d , b/b D/D , and b/b d/d , were stored for 14 d at three temperature regimes, 4–5, 8–9, and 11–12 °C. Storage at 4–5 °C resulted in CI development in the fruit of all four genotypes. The yellow, B/B genotypes suffered injury also at 8–9 °C, especially the fruit of the light yellow, B/B d/d genotype that showed slight CI symptoms even at 11–12 °C. Chilling tolerance of the genotypes was in accordance with the total tocopherol contents of their exocarp, being highest in the green-skinned b/b accessions and lowest in the light yellow B/B d/d genotype. Relatively higher tocopherol content and lower CI severity were observed in the fruit of the enhanced-yellow B/B D/D genotype. The dominant D allele can partially alleviate the CI sensitivity of summer squash that is conferred by the dominant B allele.

Specific Composition of Lipid Phases Allows Retaining an Optimal Thylakoid Membrane Fluidity in Plant Response to Low-Temperature Treatment.

Thylakoid membranes isolated from leaves of two plant species, the chilling tolerant (CT) pea and chilling sensitive (CS) runner bean, were assessed for the composition of lipids, carotenoids as well as for the arrangement of photosynthetic complexes. The response to stress conditions was investigated in dark-chilled and subsequently photo-activated detached leaves of pea and bean. Thylakoids of both species have a similar level of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), but different sulfoquinovosyldiacylglycerol to phosphatidylglycerol (PG) ratio. In pea thylakoid fraction, the MGDG, DGDG and PG, have a higher double bond index (DBI), whereas bean thylakoids contain higher levels of high melting point PG. Furthermore, the lutein to the β-carotene ratio is higher in bean thylakoids. Smaller protein/lipid ratio in pea than in bean thylakoids suggests different lipid-protein interactions in both species. The differences between species are also reflected by the course of temperature-dependent plots of chlorophyll fluorescence pointing various temperatures of the lipid phase transitions of pea and bean thylakoids. Our results showed higher fluidity of the thylakoid membrane network in pea than in bean in optimal temperature conditions. Dark-chilling decreases the photochemical activity and induces significant degradation of MGDG in bean but not in pea leaves. Similarly, substantial changes in the arrangement of photosynthetic complexes with increase in LHCII phosphorylation and disturbances of the thylakoid structure take place in bean thylakoids only. Changes in the physical properties of bean thylakoids are manifested by the conversion of a three-phase temperature-dependent plot to a one-phase plot. Subsequent photo-activation of chilled bean leaves caused a partial restoration of the photochemistry and of membrane physical properties, but not of the photosynthetic complexes arrangement nor the thylakoid network structure. Summarizing, the composition of the thylakoid lipid matrix of CT pea allows retaining the optimal fluidity of its chloroplast membranes under low temperatures. In contrast, the fluidity of CS bean thylakoids is drastically changed, leading to the reorganization of the supramolecular structure of the photosynthetic complexes and finally results in structural remodeling of the CS bean thylakoid network.

Genome-wide analyses of banana fasciclin-like AGP genes and their differential expression under low-temperature stress in chilling sensitive and tolerant cultivars.

Thirty MaFLAs vary in their molecular features. MaFLA14/18/27/29 are likely to be involved in banana chilling tolerance by facilitating the cold signaling pathway and enhancing the cell wall biosynthesis. Although several studies have identified the molecular functions of individual fasciclin-like arabinogalactan protein (FLA) genes in plant growth and development, little information is available on their involvement in plant tolerance to low-temperature (LT) stress, and the related underlying mechanism is far from clear. In this study, the different expression of FLAs of banana (Musa acuminata) (MaFLAs) in the chilling-sensitive (CS) and chilling-tolerant (CT) banana cultivars under natural LT was investigated. Based on the latest banana genome database, a genome-wide identification of this gene family was done and the molecular features were analyzed. Thirty MaFLAs were distributed in 10 out of 11 chromosomes and these clustered into four major phylogenetic groups based on shared gene structure. Twenty-four MaFLAs contained N-terminal signal, 19 possessed predicted glycosylphosphatidylinositol (GPI), while 16 had both. Most MaFLAs were downregulated by LT stress. However, MaFLA14/18/29 were upregulated by LT in both cultivars with higher expression level recorded in the CT cultivar. Interestingly, MaFLA27 was significantly upregulated in the CT cultivar, but the opposite occurred for the CS cultivar. MaFLA27 possessed only N-terminal signal, MaFLA18 contained only GPI anchor, MaFLA29 possessed both, while MaFLA14 had neither. Thus, it was suggested that the accumulation of these FLAs in banana under LT could improve banana chilling tolerance through facilitating cold signal pathway and thereafter enhancing biosynthesis of plant cell wall components. The results provide background information of MaFLAs, suggest their involvement in plant chilling tolerance and their potential as candidate genes to be targeted when breeding CT banana.

Regulation of carbon metabolism in two maize sister lines contrasted for chilling tolerance.

Maize can grow in cool temperate climates but is often exposed to spring chilling temperatures that can affect early seedling growth. Here, we used two sister double-haploid lines displaying a contrasted tolerance to chilling to identify major determinants of long-term chilling tolerance. The chilling-sensitive (CS) and the chilling-tolerant (CT) lines were grown at 14 °C day/10 °C night for 60 d. CS plants displayed a strong reduction in growth and aerial biomass compared with CT plants. Photosynthetic efficiency was affected with an increase in energy dissipation in both lines. Chilling tolerance in CT plants was associated with higher chlorophyll content, glucose-6-phosphate dehydrogenase activity, and higher sucrose to starch ratio. Few changes in cell wall composition were observed in both genotypes. There was no obvious correlation between nucleotide sugar content and cell wall polysaccharide composition. Our findings suggest that the central starch-sucrose metabolism is one major determinant of the response to low temperature, and its modulation accounts for the ability of CT plants to cope with low temperature. This modulation seemed to be linked to a strong alteration in the biosynthesis of nucleotide sugars that, at a high level, could reflect the remobilization of carbon in response to chilling.

Differential down-regulation of zeaxanthin epoxidation in two rice (Oryza sativa L.) cultivars with different chilling sensitivities

When the leaf segments of rice (Oryza sativa L.) plants were subjected to chilling in the moderate light, zeaxanthin (Zx) formation was faster in a chilling-tolerant Dongjin-byeo (DJ) than in a chilling-sensitive IR841. Although the rate of Zx formation was accelerated by the treatment of 5 mM salicylaldoxime, an inhibitor of Zx epoxidase (ZE), there was almost no changes in DJ. A similar result was observed when leaf segments were treated with 50 mM sodium fluoride, a potent inhibitor of chloroplast phosphatase. The slow Zx epoxidation in IR841 during light-chilling was confirmed in leaf segments treated with 10 mM dithiothreitol, an inhibitor of violaxanthin de-epoxidase (VDE). However, the differences between the two cultivars were not observed at 25oC. These results suggest that compared with IR841 the higher rate of Zx formation in DJ is not due to the higher VDE activity in DJ but is due to more rapid down-regulation of ZE in DJ, possibly by its phosphorylation. Compared with DJ, IR841 accumulated more superoxide with PSI inactivation during light-chilling, which eliminates the possibility of increased ZE down-regulation in DJ leaves by photo-oxidation. In vitro study with alkaline phosphatase supports the idea of down-regulation of ZE by phosphorylation under light-chilling condition. We propose that this reversible down-regulation of Zx epoxidation possibly by the phosphorylation of ZE is an important regulation mechanism of violaxanthin cycle that confers chilling tolerance of a rice cultivar under chilling stress in the light with moderate intensities.

Autoimmunity conferred by chs3-2D relies on CSA1, its adjacent TNL-encoding neighbour.

Plant innate immunity depends on the function of a large number of intracellular immune receptor proteins, the majority of which are structurally similar to mammalian nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) proteins. CHILLING SENSITIVE 3 (CHS3) encodes an atypical Toll/Interleukin 1 Receptor (TIR)-type NLR protein with an additional Lin-11, Isl-1 and Mec-3 (LIM) domain at its C-terminus. The gain-of-function mutant allele chs3-2D exhibits severe dwarfism and constitutively activated defense responses, including enhanced resistance to virulent pathogens, high defence marker gene expression, and salicylic acid accumulation. To search for novel regulators involved in CHS3-mediated immune signaling, we conducted suppressor screens in the chs3-2D and chs3-2D pad4-1 genetic backgrounds. Alleles of sag101 and eds1-90 were isolated as complete suppressors of chs3-2D, and alleles of sgt1b were isolated as partial suppressors of chs3-2D pad4-1. These mutants suggest that SAG101, EDS1-90, and SGT1b are all positive regulators of CHS3-mediated defense signaling. Additionally, the TIR-type NLR-encoding CSA1 locus located genomically adjacent to CHS3 was found to be fully required for chs3-2D-mediated autoimmunity. CSA1 is located 3.9 kb upstream of CHS3 and is transcribed in the opposite direction. Altogether, these data illustrate the distinct genetic requirements for CHS3-mediated defense signaling.

华南地区3种具有不同入侵性的近缘植物对低温胁迫的敏感性

Chilling is an important constraint factor to the growing development and geographic distribution of plant species.Ipomoea cairica( L.) Sweet is one of the most notorious invasive weeds in South China,while it seems to be not invasive outside South China. Ipomoea nil( L.) Choisy( slightly invasive or non-invasive) and Ipomoea triloba L.( non-invasive)are two of the I. cairica's closely related species that are also originated from the tropical America. In the present study,we compared the chilling sensitivities of these three vine plants by testing their ecophysiological responses to low-temperature treatments( 15 ℃,10 ℃) in a hydroponic experiment,so as to illustrate the potential connections between the chilling sensitivity and invasiveness. We hypothesized that( i) the three alien plants were all sensitive to chilling stress and( ii)the chilling sensitivity of the non-invasive( or slightly invasive) plants I. triloba and I. nil were higher than that of the serious invasive species I. cairica.Cuttings of I. cairica,I. nil and I. triloba collected from selected habitats in South China were planted in water to let the root grow for one week,and then cultivated in Hoagland nutrient solution for 4 weeks. Similarly sized plants of each species were placed in the light incubators with different temperatures( 28 ℃,15 ℃,10 ℃) for 8 days. The chlorophyll fluorescence technique was used to monitor the photosynthetic apparatus of these Ipomoea plants every other day. After harvest the shoot biomass and root length of new growth for all plants were calculated. The accumulation of H 2 O 2, malodialdehyde( MDA),proline and soluble carbohydrate in the leaves,as well as the root metabolic activity and membrane integrity were also tested as indications of physiological performance after different temperature treatments. The chlorophyll fluorescence results showed that photosystem Ⅱ( PS Ⅱ) of all three Ipomoea species was partly damaged by chilling( 15 ℃,resulted in increased initial fluorescence level( F 0),but decreased electron transport rate( ETR) and maximum photochemical efficiency( F v / F m)),especially for I. triloba,which always was most sensitive. There was also higher non-photochemical quenching( NPQ) under chilling stress in all three species,indicating increased thermal dissipation in PS Ⅱ. Compared to the control( 28 ℃),all plants under chilling stress accumulated significantly more proline and soluble carbohydrate( P 0. 01),both of which could improve their chilling tolerance. There were significantly less shoot biomass and root length of new growth,and higher levels of cell membrane integrity in roots under the 15 ℃ and 10 ℃ treatment for all three species( P0. 05),and that should be partly attributed to the accumulation of H 2 O 2 and MDA,which were observed in all of the plants under chilling stress. These results clearly showed that at 15 ℃ there had been significant stress effects in these Ipomoea species. The first hypothesis of this research,i. e. the three species have high chilling sensitivities,was thus supported. As for the other aspect,when comparing the response intensities of the three Ipomoea species to chilling treatment,it is found that at the 10 ℃ treatment only I. cairica has continued to grow,while no growth was observed in I. nil and I. triloba during the periods of chilling stress,indicating that I. cairica has the highest chilling tolerances among the three species. The second hypothesis of this study was thus also supported. Besides,among the three Ipomoea species,I. triloba seems to be the most sensitive one to chilling as it usually had the highest physiological response intensities to chilling stress. The results from this study implied that the comparatively higher chilling tolerance of I. cairica in comparison to I. nil and I. triloba should be one of the reasons that in South China it is more invasive than the latter two species. The chilling sensitivity is an important key factor to the invasiveness of alien plants. Besides,the relatively high chilling sensitivity of I. cairica should be one of the most important reasons that it has not invaded other areas than South China. Therefore,it is necessary to pay more attention to the potential range expansion of I. cairica under the background of global warming.

Effect of thermal injury on embryos of banded coral shrimp (Stenopus hispidus) under hypothermal conditions

Cryopreservation technology regarding banded coral shrimp (Stenopus hispidus) embryos is important as it could improve cultivation and preservation of the species. The development of this technology is to reduce collections of this species from the wild, thus preventing damage to coral reefs. This study investigated the tolerance of different developmental stages of S. hispidus embryos in response to low temperature in the presence or absence of cryoprotectant. Embryos undergoing three stages of embryonic development (eye-formation, heart beat and pre-hatch stage) were selected and exposed to 5, 0 and −5°C in cryoprotectant solutions of 1 or 2M methanol for 1, 2, 4, 6, 8, 16 and 32h. Embryo survival was evaluated based on their hatching percentage. In experiments on the effect of different concentrations of methanol on chilling sensitivity of embryos, it was determined that methanol at 1M methanol reduced the chilling sensitivities of embryos most effectively when compared to the other tested concentrations. Experiments regarding the chilling sensitivity of embryos in different developmental stages indicated that pre-hatch stage embryos were more resistant to subzero temperatures than early stage embryos; they tolerated the 32h exposure at 5 and 0°C without a loss in survival. The study also indicated that late stage embryos are considered to be resistant to chilling, and that pre-late stage embryos are better candidate for cryopreservation.

Closure of plasmodesmata in maize (Zea mays) at low temperature: a new mechanism for inhibition of photosynthesis

Photosynthesis is one of the processes most susceptible to low-temperature inhibition in maize, a tropical C4 crop not yet fully adapted to a temperate climate. C4 photosynthesis relies on symplasmic exchange of large amounts of photosynthetic intermediates between Kranz mesophyll (KMS) and bundle sheath (BS) cells. The aim of this study was to test the hypothesis that the slowing of maize photosynthesis at low temperature is related to ultrastructural changes in the plasmodesmata between KM and BS as well as BS and vascular parenchyma (VP) cells. Chilling-tolerant (CT) KW 1074 and chilling-sensitive (CS) CM 109 maize (Zea mays) lines were studied. The effect of moderate chilling (14 °C) on the rate of photosynthesis, photosynthate transport kinetics, and the ultrastructure of plasmodesmata linking the KMS, BS and VP cells were analysed. Additionally, the accumulation of callose and calreticulin was studied by the immunogold method. Chilling inhibited photosynthesis, photosynthate transfer to the phloem and photosynthate export from leaves in both lines. This inhibition was reversible upon cessation of chilling in the CT line but irreversible in the CS line. Simultaneously to physiological changes, chilling induced swelling of the sphincters of plasmodesmata linking KMS and BS cells and a decreased lumen of the cytoplasmic sleeve of plasmodesmata at the BS/VP interface in the CS line but not in the CT line. Accumulation of calreticulin, which occurred near the neck region of the closed plasmodesmata was observed after just 4 h of chilling and over-accumulation of callose at the KMS/BS and BS/VP interfaces occurred after 28 h of chilling. Stronger chilling sensitivity of the CM 109 maize line compared with the KW 1074 line, shown by decreased photosynthesis and assimilate export from a leaf, is related to changes in the ultrastructure of leaf plasmodesmata at low temperature. The chain of reactions to chilling is likely to include calreticulin action resulting in rapid and efficient closure of the plasmodesmata at both KMS/BS and BS/VP interfaces. Callose deposition in a leaf was a secondary effect of chilling.

Salicylic acid-induced enhancement of cold tolerance through activation of antioxidative capacity in watermelon

The purpose of this study is to determine suitable temperature for selecting germplasm with cold tolerance and to investigate the effects of exogenous SA on cold tolerance in watermelon. We found that low temperature (10 °C) was the most suitable temperature to identify cold tolerance germplasm in watermelon. Moreover, we classified the capacities of four different watermelon germplasms, of which No. 9 was the germplasm with strongest cold tolerance and No. 2 was the one with chilling sensitiveness. The suitable exogenous SA (1.0 mmol/L) enhanced cold tolerance in watermelon through activations of antioxidative capacity, such as, G-POD, ASA-POD, CAT, SOD and GR. The activities of antioxidative enzymes were more significantly increased in cold tolerance germplasm No. 9 than that in chilling sensitive germplasm No. 2 in watermelon after being treated with suitable exogenous SA (1.0 mmol/L), although they were also been enhanced in No. 2. However, higher concentration of exogenous SA decreased the activity of antioxidative enzymes, consequently, weakened the capacity of cold tolerance in watermelon.

Chilling response of plants: importance of galactolipase, free fatty acids and free radicals*

The chilling response of plants is complex and based on the interplay of two important metabolic processes--lipolytic degradation of membrane lipids and a set of oxidative reactions leading to lipid peroxidation and membrane damage evoked in chilling-sensitive (CS) plants subjected to low temperature and light. The effects of chilling of detached leaves and intact plants differ and are often neglected during experiments. In closely-related species, the activity of several constitutive enzymes (i.e. superoxide dismutase, ascorbate peroxidase and glutathione reductase) appears to be higher in chilling-tolerant (CT) than in CS species; while in several native, closely-related CS species, lipid acyl hydrolase (galactolipase) activity is higher than in CT species. Moreover, in chilling-insensitive (CI) plants, galactolipase activity is very low and is neither activated by detachment of leaves nor under stress conditions in growing plants. Dark and low-temperature treatments of detached leaves of CS species and post-chilling recovery of growing plants in the light activate galactolipase, which is responsible for the release of free fatty acids (FFA), the main substrates of peroxidation by lipoxygenase and free radicals. In several CS species, increased galactolipase activity is an important factor contributing to chilling susceptibility. Thus, it seems likely that enhancement of chilling tolerance may be achieved by genetically suppressing galactolipase in order to reduce both the degradation of chloroplast lipids and the level of released FFA, and thereby avoiding the deleterious action of their peroxidation products on plant tissues.

Contrasting effect of dark-chilling on chloroplast structure and arrangement of chlorophyll-protein complexes in pea and tomato: plants with a different susceptibility to non-freezing temperature.

The effect of dark-chilling and subsequent photoactivation on chloroplast structure and arrangements of chlorophyll-protein complexes in thylakoid membranes was studied in chilling-tolerant (CT) pea and in chilling-sensitive (CS) tomato. Dark-chilling did not influence chlorophyll content and Chl a/b ratio in thylakoids of both species. A decline of Chl a fluorescence intensity and an increase of the ratio of fluorescence intensities of PSI and PSII at 120 K was observed after dark-chilling in thylakoids isolated from tomato, but not from pea leaves. Chilling of pea leaves induced an increase of the relative contribution of LHCII and PSII fluorescence. A substantial decrease of the LHCII/PSII fluorescence accompanied by an increase of that from LHCI/PSI was observed in thylakoids from chilled tomato leaves; both were attenuated by photoactivation. Chlorophyll fluorescence of bright grana discs in chloroplasts from dark-chilled leaves, detected by confocal laser scanning microscopy, was more condensed in pea but significantly dispersed in tomato, compared with control samples. The chloroplast images from transmission-electron microscopy revealed that dark-chilling induced an increase of the degree of grana stacking only in pea chloroplasts. Analyses of O-J-D-I-P fluorescence induction curves in leaves of CS tomato before and after recovery from chilling indicate changes in electron transport rates at acceptor- and donor side of PS II and an increase in antenna size. In CT pea leaves these effects were absent, except for a small but irreversible effect on PSII activity and antenna size. Thus, the differences in chloroplast structure between CS and CT plants, induced by dark-chilling are a consequence of different thylakoid supercomplexes rearrangements.