MIR1868 negatively regulates rice cold tolerance at both the seedling and booting stages

Low temperature causes rice yield losses of up to 30%–40%,therefore increasing its cold tolerance is a breeding target.Few genes in rice are reported to confer cold tolerance at both the vegetative and reproductive stages.This study revealed a rice-specific 24-nt miRNA,miR1868,whose accumulation was suppressed by cold stress.Knockdown of MIR1868 increased seedling survival,pollen fertility,seed setting,and grain yield under cold stress,whereas its overexpression conferred the opposite phenotype.Knockdown of MIR1868 increased reactive oxygen species(ROS)scavenging and soluble sugar content under cold stress by increasing the expression of peroxidase genes and sugar metabolism genes,and its overexpression produced the opposite effect.Thus,MIR1868 negatively regulated rice cold tolerance via ROS scavenging and sugar accumulation.

Effects of Silicon Formulations on Cold Tolerance of Rice Seedlings

To investigate the effects of silicon formulations on the cold tolerance of rice seedlings,Song Japonica 16(not cold tolerant)and Dongnong 427(cold tolerant)rice varieties were used as test materials and four different types of silicon formulations,Si-50-G,Si-60-G,Si-T-G,and Si-E-G,were applied as foliar sprays at the seedling stage,and a control group CK(equal amount of distilled water)was set up.One week after the first silicon spray,two types of rice were subjected to low-temperature stress treatments at day/night temperatures of 12℃/10℃for 2,4,6,and 8 days.The effects of different silicon formulations on the chlorophyll,proline(Pro)and soluble sugar contents as well as superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)activities of rice seedlings under low-temperature stress were compared to find out the effects of silicon formulations on the cold tolerance of rice seedlings.The results showed that silicon formulations could significantly increase the chlorophyll content of rice seedling leaves,with Si-50-G being the most effective,with a significant increment of 40.17%compared to the CK at 2 days of low temperature.Four silicon formulations significantly increased the proline content and soluble sugar content of rice leaves at low temperature for 4-8 days.For Song Japonica 16,the most significant increment in leaf POD activity was observed in Si-E-G treatment at 2,4 and 8 days of temperature stress,with 73.58%,20.95%and 217.24%increases compared to the CK,respectively.For 4 and 6 days of temperature stress,the most significant increase in CAT activity was observed in Si-E-G treatment,with 25.70%and 75.78%increases compared to the CK,respectively.For Dongnong 427,the Si-60-G treatment showed the highest increase in leaf SOD activity for 4 and 8 days of temperature stress,with significant increases of 58.15%and 82.76%compared to the CK,respectively,and the Si-E-G treatment showed the highest increase in leaf POD activity for 2 and 8 days of temperature stress,with significant increases of 97.75%and 245.10%compared to the CK,respectively.It showed that the spraying of silicon formulations could significantly enhance the cold tolerance of rice.This study provided a scientific basis for the rational use of silicon formulations to enhance cold tolerance in rice and had important theoretical and practical significance for ensuring sustainable high and stable rice yields in Heilongjiang Province,as well as for the development of silicon fertilizers.

More than a simple egg:Underlying mechanisms of cold tolerance in avian embryos

Avian embryos,which develop within eggs,exhibit remarkable tolerance to extremely low temperatures.Despite being a common trait among all birds,the mechanisms underlying this cold tolerance in avian embryos remain largely unknown.To gain a better understanding of this phenomenon and the coping mechanisms involved,we reviewed the literature on severe cold tolerance in embryos of both wild and domestic birds.We found that embryos of different bird orders exhibit tolerance to severe cold during their development.In response to cold stress,embryos slow down their heartbeat rates and metabolism.In severe cold temperatures,embryos can suspend these processes,entering a torpid-like state of cardiac arrest.To compensate for these developmental delays,embryos extend their regular incubation periods.Depending on their embryonic age,embryos of all bird species can tolerate acute severe cold regimes;only a few tolerate chronic severe cold regimes.We also discussed various extrinsic and intrinsic factors that affect the tolerance of bird embryos to low temperatures before and after incubation.Cold tolerance appears to be a heritable trait shared by wild and domestic embryos of all bird classes,regardless of egg size or development(altricial/precocial).Driven by environmental variability,cold tolerance in avian embryos is an optimal physiological and ecological strategy to mitigate the adverse effects of cold conditions on their development in response to fluctuating environmental temperatures.

MdWRKY40is directly promotes anthocyanin accumulation and blocks MdMYB15L, the repressor of MdCBF2, which improves cold tolerance in apple

Cold stress is an important factor that limits apple production. In this study, we examined the tissue-cultured plantlets of apple rootstocks ‘M9T337' and ‘60-160', which are resistant and sensitive to cold stress, respectively. The enriched pathways of differentially expressed genes(DEGs) and physiological changes in ‘M9T337' and ‘60-160' plantlets were clearly different after cold stress(1°C) treatment for 48 h, suggesting that they have differential responses to cold stress. The differential expression of WRKY transcription factors in the two plantlets showed that MdWRKY40is and MdWRKY48 are potential regulators of cold tolerance. When we overexpressed MdWRKY40is and MdWRKY48in apple calli, the overexpression of MdWRKY48 had no significant effect on the callus, while MdWRKY40is overexpression promoted anthocyanin accumulation, increased callus cold tolerance, and promoted the expression of anthocyanin structural gene MdDFR and cold-signaling core gene MdCBF2. Yeast one-hybrid screening and electrophoretic mobility shift assays showed that MdWRKY40is could only bind to the MdDFR promoter. Yeast twohybrid screening and bimolecular fluorescence complementation showed that MdWRKY40is interacts with the CBF2inhibitor MdMYB15L through the leucine zipper(LZ). When the LZ of MdWRMY40is was knocked out, MdWRKY40is overexpression in the callus did not affect MdCBF2 expression or callus cold tolerance, indicating that MdWRKY40is acts in the cold signaling pathway by interacting with MdMYB15L. In summary, MdWRKY40is can directly bind to the MdDFR promoter in order to promote anthocyanin accumulation, and it can also interact with MdMYB15L to interfere with its inhibitory effect on MdCBF2, indirectly promoting MdCBF2 expression, and thereby improving cold tolerance.These results provide a new perspective for the cold-resistance mechanism of apple rootstocks and a molecular basis for the screening of cold-resistant rootstocks.

A systematic identification of cold tolerance genes in peanut using yeast functional screening system

Peanut(Arachis hypogaea L.)is a thermophilic crop,and low temperature leads to a significant reduction in annual yields.Despite a few cold tolerant germplasms or cultivars have been discovered and developed,molecular mechanisms governing peanut cold tolerance is poorly understood.Identification of keys genes involved in cold tolerance is the first step to address the underlying mechanism.In this study,we isolated and characterized 157 genes with potentials to confer cold tolerance in peanut by using a yeast functional screening system.GO(Gene ontology)and KEGG(Kyoto encyclopedia of genes and genomes)enrichment analysis of these genes revealed that ribosome and photosynthesis proteins might play essential roles in peanut cold response.Transcriptome results indicated that 60 cold tolerance candidate genes were significantly induced or depressed by low temperature.qRT-PCR analysis demonstrated that several candidate genes could be also regulated by salt or drought stress.Individual overexpression of two UDP-glycosyltransferases(AhUGT2 and AhUGT268)in transgenic yeast cells could enhance their tolerance to multiple abiotic stress.In conclusion,this study advances our understanding of the mechanisms associated with the cold stress responses in peanut,and offers valuable gene resources for genetic improvement of abiotic stress tolerance in crops.

The Comparison of Cold tolerance of Three Species of Evergreen Broad-leaved Woody Plants

[Objectives]This study aimed to select evergreen broad-leaved woody plants with higher ornamental value and stronger cold tolerance for introduction from the south to the north,and to apply them to urban greening,so as to enrich the plant community structure of the landscape in the northern region.[Methods]Three species of evergreen broad-leaved woody plants,i.e.,Ligustrum lucidum,Ilex cornuta and Eriobotrya japonica,were selected as the experimental materials.The morphological performances and the changes of the physiological indexes were observed and measured during the overwintering period in the open field in Beijing.The relationship between the indexes and the low temperature was also analyzed.The strength of cold tolerance of the three species was compared.[Results]The electrical conductivity,the contents of MDA and proline were negatively correlated with the corresponding low temperature.The contents of soluble sugar and soluble protein increased with the dropping temperature,but they had little response to the short-term temperature rise.[Conclusions]Combined with morphological and physiological indexes,it was found that the changes of the contents of proline and soluble sugar among the physiological indexes were closely related to the cold tolerances of the three tree species of broad-leaved woody plants.The cold tolerance of I.cornuta was the strongest,E.japonica was the second,and that of L.lucidum was the worst.

Comparison of Cold Tolerance in Three Ground-cover Chrysanthemum Cultivars during Wintering in the Open Field

[Objectives]Among many chrysanthemum types,the ground-cover chrysanthemum cultivar group has the characteristics of dwarf plant,long flowering period,rich color and strong resistance to stress.This study aimed to screen out cultivars with strong cold tolerance in order that they can be used for planting in urban green space and economic forests in northern cold areas,so as to enrich the plant community structures of landscape in cold regions.[Methods]Three ground-cover chrysanthemum cultivars,i.e.,‘Meiaihuang’,‘Xinhong’and‘Zixunzhang’,were selected as the experimental materials.During the wintering period in the field in Beijing,they were observed and measured for the changes of the morphological manifestations and the physiological indices,and the relationship between the indices and low temperature was analyzed.The cold tolerance of the three ground-cover chrysanthemum cultivars was also comprehensively evaluated.[Results]During the open-field overwintering period,the root activity of the three ground-cover chrysanthemum cultivars increased slowly with cooling,decreased at the freezing low temperature,and then increased again in early spring.With natural cooling,the membrane injury percentages(MIPs)of the leaves and foot shoots increased significantly,and the contents of malondialdehyde(MDA)increased;the contents of soluble sugar(SS)and proline(Pro)gradually accumulated,and the contents of SS and Pro in foot shoots increased significantly in late winter;and the superoxide dismutase(SOD)activity showed a similar trend of rising slowly at first and then increasing significantly.There were correlations between MIPs in the leaves and foot shoots of the three cultivars and other physiological indices:a significant negative correlation with MDA content,and a significant positive correlation with SS content,Pro content and SOD activity.[Conclusions]Combining the morphological manifestations and physiological indices,the cold tolerance of the three ground-cover chrysanthemum cultivars was evaluated by the subordinate function method.The results showed that the cold tolerance of the three ground-cover chrysanthemum cultivars ranked as‘Meiaihuang’>‘Xinhong’>‘Zixunzhang’.The strong cold tolerance of foot shoots and roots was the main reason that the three ground-cover chrysanthemum cultivars could overwinter in the open field.

Effect of Rapid Cold Hardening on the Cold Tolerance of the Larvae of the Rice Stem Borer,Chilo suppressalis(Walker)

The effects of rapid cold hardening （RCH） on the cold tolerance of the last instar larvae of Chilo suppressalis （Walker） were evaluated for the first time. The discriminating temperature, induction, detection, duration and extent of RCH of the larvae in the laboratory were tested, and the supercooling points （SCPs） and the contents of water and lipid of the larvae after RCH treatment were determined, respectively. The results showed that the discriminating temperature of the larvae was about -21℃. Mean survival rates of the larvae which exposed to either 0 or 5℃ for 2 and 4 h before exposure to the discriminating temperature for 2 h were significantly higher than those of the control groups （P 〈 0.05）. Moreover, the highest survival rate appeared in the larvae after 0℃ for 4 h treatment. The protection against low temperature gained by RCH at 0℃ for 4 h was rapidly lost on return to 28℃. Mean survival rates of RCH larvae were significantly higher than those of non-acclimated （NACC） larvae and acclimation （ACC） larvae when they were exposed to the discriminating temperature for 2 or 4 h （P〈 0.05）. Moreover, the rates of NACC, ACC, RCH and ACC ＋ RCH larvae from 2 to 6 h to the discriminating temperature resulted in a significant decline. The values of SCPs and the contents of lipid of the larvae which exposed to either 0 or 5℃ for 2 and 4 h showed no significant difference at 0.05 level compared to those of the control groups. But the contents of water in the larvae were obviously decreased. Therefore, it could be concluded that RCH could enhance cold tolerance and affect partly physiological and biochemical components of the larvae of C. suppressalis, but the underlying mechanisms needs to be further explored.

Genetic Analysis of Cold Tolerance at Seedling Stage and Heat Tolerance at Anthesis in Rice (Oryza sativa L.)

A set of 240 introgression lines derived from the advanced backcross population of a cross between a japonica cultivar,Xiushui 09,and an indica breeding line,IR2061,was developed to dissect QTLs affecting cold tolerance （CT） at seedling stage and heat tolerance （HT） at anthesis.Survival rate of seedlings （SRS） and spikelet fertility （SF）,the index traits of CT and HT,showed significant differences between the two parents under stresses.A total of four QTLs （qSRS1,qSRS7,qSRS11a and qSRS11b） for CT were identified on chromosomes 1,7,11,and the Xiushui 09 alleles increased SRS at all loci except qSRS7.Four QTLs for SF were identified on chromosomes 4,5,6,and 11.These QTLs could be classified into two major types based on their behaviors under normal and stress conditions.The first was QTL expressed only under normal condition;and the second QTL was apparently stress induced and only expressed under stress.Among them,two QTLs （qSF4 and qSF6） which reduced the trait difference between heat stress and normal conditions must have contributed to HT because of their obvious contribution to trait stability,and the IR2061 allele at the qSF6 and the Xiushui 09 allele at the qSF4 improved HT,respectively.No similar QTL was found between CT at seedling stage and HT at anthesis.Therefore,it is possible to breed a new variety with CT and HT by pyramiding the favorable CT-and HT-improved alleles at above loci from Xiushui 09 and IR2061,respectively,through marker-assisted selection （MAS）.

Effect and Mechanism of Cold Tolerant Seed-Coating Agents on the Cold Tolerance of Early Indica Rice Seedlings

To better understand the effect and mechanism of cold tolerant seed-coating agents on the cold tolerance of rice seedlings, the physiological and biochemical effects of four cold tolerant seed-coating agents （HET, YKJ, YKZYJ, and the ABA seed coating agents） on two early indica rice varieties were studied under chilling stress. The results showed that the rice seedlings treated with cold tolerant seed-coating agents under chilling stress maintained dramatically higher root vigor, POD, CAT and SOD activities, and chlorophyll content, had lower MDA content and electrolyte leakage, and accumulated more soluble sugar and free proline, when compared with the control without the treatment, and finally showed lower plant injury rate. It was indicated that the cold tolerant seed coating agent improved the ability of rice seedlings in resisting to chilling stress. YKZYJ was ranked the first in terms of the efficiency in cold tolerance among the four cold tolerant seed-coating agents tested.

Genetic parameters for cold tolerance and body weight of Chinese fleshy prawn,Fenneropenaeus chinensis

The inability of Fenneropenaeus chinensis to tolerate low temperatures is of major economic concern in temperate climates,as it reduces their growing season and leads to over-winter mortality.In this study,the heritability of body weight under low grow-out temperature and cold tolerance in F.chinensis were first investigated and estimated using 88 ful-sib families,which might provide crucial information in Chinese fleshy prawn breeding programs.The heritability for body weight under suitable and low temperature of F.chinensis were both moderate（0.158 0±0.307 5 and 0.132 0±0.026 9 respectively）;the large coefficient of variation（approximately 21%） and moderate estimate of heritability for body weight indicated substantial potential for selective breeding.The heritability estimate for cold tolerance was low（0.019 2±0.023 5）,and showed no significant differences from zero（P〉0.05）.A weak genetic correlation between cold tolerance and body weight was also estimated in the present study,also showing no significant differences from zero（P〉0.05）.Thus,more research needs to be conducted on the more accurate heritability estimate of cold tolerance and genetic correlations between traits in F.chinensis to further improve the achievement of breeding goals.

Identification of Cold Tolerance of Rice Germplasm Resource at Germinating Stage in Cold Region

Total 75 rice varieties （lines） in Heilongjiang Province （or cold region） as germplasm resources were identified for cold tolerance at germinating stage by controlling temperature in artificial incubator. The results showed that the shooting seed rate at the germinating stage could be used as the evaluation index of cold tolerance. The cold tolerance was recorded on 1-9 scale and could be identified by the criteria of five indexes such as highly tolerant （HT）, tolerant （T）, moderately tolerant （MT）, susceptible （S）, highly susceptible （HS）.

Identification of QTLs for Rice Cold Tolerance at Plumule and 3-Leaf-Seedling Stages by Using QTLNetwork Software

A doubled haploid （DH） population consisted of 120 lines, derived from a cross between an indica vadety, TN1, and a japonica variety, Chunjiang 06, was used to identify QTLs controlling rice cold tolerance at the plumule and 3-leaf-seedling stages by using the QTLNetwork software. The percentages of normal plumules after 4~C treatments for 7 d, 9 d, 11 d, and 14 d, as well as the cold stress tolerance index （CSTI） and the withering index （WI） of rice seedling were investigated. A total of five single-effect QTLs, each for percentages of normal plumules after 4℃treatments for 9 d, 11 d and 14 d, and CSTI and WI, respectively were identified. The QTLs for the percentages of normal plumules after low temperature treatments for g d, 11 d and 14 d were on chromosomes 4, 2 and 11, accounting for 14.1%, 17.3% and 21.5% of the phenotypic variation, respectively. QTLs for CSTI and WI were on chromosomes 10 and 1, respectively. Two pairs of epistatic loci were identified, but none of the epistatic loci involved the single-effect QTLs. The RM528-RM340 interval on chromosome 6 interacted with the RM278-RM3919B interval on chromosome 9 for CSTI, and the epistatic interaction accounted for 17.7% of the phenotypic variation. A pair of epistatic loci was identified for WI, the RM246-RM5461 interval on chromosome 1 interacted with the ISA-RM447 interval on chromosome 8, which accounted for 22.6% of the phenotypic variation.

Linkage map construction and QTL mapping for cold tolerance in Oryza rufipogon Griff. at early seedling stage

Cold stress is one of the major restraints for rice production. Cold tolerance is controlled by complex genetic factor. In this study, a backcross inbred lines （BILs） population derived from an inter-specific cross （Oryza sativa L.xO. rufipogon Griff.） was used for genetic linkage map construction and quantitative trait locus （QTL） mapping. A linkage map consisting of 153 markers was constructed, spanning 1 596.8 cM with an average distance of 11.32 cM between the adjacent markers. Phenotypic evaluation of the parents and BILs under （6±1）℃ cold stress revealed that the ability of cold tolerance in BILs at early seedling obeyed a skewed normal and continuous distribution. Fifteen QTLs on chromosomes 6, 7, 8, 11, and 12 were identified using survival percent （SP） and non death percent （NDP） as indicators of cold tolerance, which could explain 5.99 to 40.07% of the phenotypic variance, of which the LOD values ranged from 3.04 to 11.32. Four QTLs on chromosomes 3, 5 and 7 were detected using leaf conductivity （LC） and root conductivity （RC） as indicators of cold tolerance, ranging from 19.54 to 33.53% for the phenotypic variance explained and 2.54 to 6.12 for the LOD values. These results suggested that there might be multi major QTLs in O. rufipogon and some useful genes for cold tolerance have been transferred into cultivated rice, which would be helpful for cloning and utilizing the cold tolerance-responsive genes from wild rice.

Large-scale evaluation of pea(Pisum sativum L.)germplasm for cold tolerance in the field during winter in Qingdao

As a cool-season crop, pea(Pisum sativum L.) can tolerate frost at the vegetative stage but experiences yield loss when freezing stress occurs at the reproductive stage. Cold-tolerance improvement of pea varieties is important for stable yield and expansion of the winter pea planting area. Under natural low-temperature conditions during winter in Qingdao,Shandong, China, we evaluated the cold tolerance of 3672 pea germplasm accessions in the field and categorized them as displaying high resistance(214), moderate resistance(835), or susceptibility(2623). The highly and moderately resistant genotypes were validated in the following year. We found that genotypes from the winter production region showed higher cold tolerance than genotypes from the spring production region. The accessions identified as having high levels of cold tolerance are recommended as potential genetic resources in cold-tolerance breeding of pea.

Detection of QTL for Cold Tolerance at Bud Bursting Stage Using Chromosome Segment Substitution Lines in Rice (Oryza sativa)

Ab The cold tolerance at the bud bursting stage （CTB） was evaluated at 5℃ by using a set of 95 chromosome segment substitution lines （CSSLs） derived from an indica rice 9311 and a japonica rice Nipponbare with a genetic background of 9311. The result showed that six CSSLs had slightly stronger effect on CTB than 9311. Total four quantitative trait loci （QTLs） for CTB were preliminary mapped on chromosomes 5 and 7 by substitution mapping, qCTB-5-1, qCTB-5-2 and qCTB-5-3 were mapped in the region of RM267-RM1237, RM2422-RM6054 and RM3321-RM1054, which were 21.3 cM, 27.4 cM and 12.7 cM in genetic distance on rice chromosome 5, respectively, qCTB-7 was mapped in a 6.8-cM region of RM11-RM2752 on rice chromosome 7.

Effects of variety and chemical regulators on cold tolerance during maize germination

Maize growth and development is affected by low temperature(LT) especially at the early stages of development. To describe the response of different varieties to LT stress and determine an effective method to cope with LT stress, maize hybrids Zhengdan 958(ZD 958) and Danyu 39(DY 39) were planted and grown at 10 and 25°C, respectively. Effects of the chemicals potassium chloride(KCl), gibberellin(GA), 2-diethylaminoethyl-3,4-dichlorophenylether(DCPTA), and all three combined chemicals(KGD) on coping with LT stress were tested by seed priming. The varieties performed significantly different at 10°C. Compared to leaf, root growth was more severely affected by LT stress. Root/leaf ratio is likely a more reliable parameter to evaluate cold tolerance based on its close correlation with leaf malondialdehyde(MDA) content(R=–0.8). GAadvanced seed germination by about 2 days compared with control treatment of water. GAand DCPTA both resulted in lower leaf MDA content and higher leaf and root area, and root/leaf ratio. KCl resulted in the highest evenness of plant height. KGD performed the best in increasing cold tolerance of maize morphologically and physiologically. Strategies to increase maize tolerance of cold stress, such as variety breeding or chemical selection, would increase maize yield especially at high-latitude regions and have great implications for food security.

Mapping a Novel Gene of Cold Tolerance at Booting Stage by Using Near-Isogenic Lines in japonica Rice

Genetic analysis showed that cold tolerance at booting stage of near-isogenic lines （NILs） of Kunmingxiaobaigu was controlled by a gene with large phenotypic variance. One hundred and sixty-four simple sequence repeats （SSR） distributed over 12 chromosomes were used to screen polymorphism between Towata （recurrent parent, RP） and near-isogenic line pool （NILP）, and two SSR markers at the long arm of chromosome 5 showed polymorphism in comparison with RP genome. Of the two markers, RM31 was found possibly linked with the cold tolerance gene at booting stage through one-way ANOVA. Twelve SSR markers around RM31 were then used to detect polymorphism between RP and NIL, and only RM7452 had polymorphism. The gene of cold tolerance at booting stage was further mapped on chromosome 5 between RM7452 and RM31 with genetic distances of 4.8 cM and 8.0 cM, respectively. This gene explained 10.50% of phenotypic variance and 5.10% of phenotypic variance of fully filled grains, and was tentatively designated as Ctb（t）.

Evaluation on Sixty-eight Rice Germplasms in Cold Tolerance at Germination Stage

Sixty-eight rice germplasms were used for cold tolerance evaluation at the germination stage in laboratory. Seeds of rice germplasms germinated at three different temperatures containing low temperature （constant 13°C for 28 days）, alternative temperature （a temperature cycle of 12 h at 20°C and 12 h at 23°C for 14 days） and control （constant 26°C for 7 days）. Analysis of variance revealed that temperature had a significant effect on germination rate, coleoptile length and radicle length. Germination rate was strongly affected by the low temperature and alternative treatments. Stress of low temperature at the germination stage on the tested rice germplasms caused a reduction in final germination rate and the lengths of coleoptile and radicle. The normal and healthy seeds began to germinate within 36 h after imbibition and germination were completed on the 7th day in the control, but delayed under the low and alternative temperature treatments. The low and alternative temperature treatments delayed the growth of coleoptile and radicle compared to the control, and the average lengths of coleoptile and radicule were strongly inhibited with the decreasing temperature in all of the tested germplasms. Taichung, a semi-dwarf, low amylose content and early maturing rice variety, showed a significantly higher germination rate at the three temperature treatments and its coleoptile length was significantly higher than other germplasms.