Heterogeneous expression of stearoyl-acyl carrier protein desaturase genes SAD1 and SAD2 from Linum usitatissimum enhances seed oleic acid accumulation and seedling cold and drought tolerance in Brassica napus

Flax(Linum usitatissimum L.)is a versatile crop and its seeds are a major source of unsaturated fatty acids.Stearoyl-acyl carrier protein desaturase(SAD)is a dehydrogenase enzyme that plays a key role in oleic acid biosynthesis as well as responses to biotic and abiotic stresses.However,the function of SAD orthologs from L.usitatissimum has not been assessed.Here,we found that two LuSAD genes,LuSAD1 and LuSAD2,are present in the genome of L.usitatissimum cultivar‘Longya 10’.Heterogeneous expression of either LuSAD1 or LuSAD2 in Arabidopsis thaliana resulted in higher contents of total fatty acids and oleic acid in the seeds.Interestingly,ectopic expression of LuSAD2 in A.thaliana caused altered plant architecture.Similarly,the overexpression of either LuSAD1 or LuSAD2 in Brassica napus also resulted in increased contents of total fatty acids and oleic acid in the seeds.Furthermore,we demonstrated that either LuSAD1 or LuSAD2 enhances seedling resistance to cold and drought stresses by improving antioxidant enzyme activity and nonenzymatic antioxidant levels,as well as reducing membrane damage.These findings not only broaden our knowledge of the LuSAD functions in plants,but also offer promising targets for improving the quantity and quality of oil,and the abiotic stress tolerance of oil-producing crops,through molecular manipulation.

The UDP-glycosyltransferase OsUGT706D2 positively regulates cold and submergence stress tolerance in rice

In a genome-wide association study,we identified a rice UDP-glycosyltransferase gene,OsUGT706D2,whose transcription was activated in response to cold and submergence stress and to exogenous abscisic acid(ABA).OsUGT706D2 positively regulated the biosynthesis of tricin-4’-O-(syringyl alcohol)ether-7-O-glucoside at both the transcriptional and metabolic levels.OsUGT706D2 mediated cold and submergence tolerance by modulating the expression of stress-responsive genes as well as the abscisic acid(ABA)signaling pathway.Gain of function of OsUGT706D2 increased cold and submergence tolerance and loss of function of OsUGT706D2 reduced cold tolerance.ABA positively regulated OsUGT706D2-mediated cold tolerance but reduced submergence tolerance.These findings suggest the potential use of OsUGT706D2 for improving abiotic stress tolerance in rice.

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.

PHD17 acts as a target of miR1320 to negatively control cold tolerance via JA-activated signaling in rice

Plant Homeo Domain(PHD)proteins are involved in diverse biological processes during plant growth.However,the regulation of PHD genes on rice cold stress response remains largely unknown.Here,we reported that PHD17 negatively regulated cold tolerance in rice seedlings as a cleavage target of miR1320.PHD17 expression was greatly induced by cold stress,and was down-regulated by miR1320 overexpression and up-regulated by miR1320 knockdown.Through 5'RACE and dual luciferase assays,we found that miR1320 targeted and cleaved the 3'UTR region of PHD17.PHD17 was a nuclearlocalized protein and acted as a transcriptional activator in yeast.PHD17 overexpression reduced cold tolerance of rice seedlings,while knockout of PHD17 increased cold tolerance,partially via the CBF cold signaling.By combining transcriptomic and physiological analyses,we demonstrated that PHD17 modulated ROS homeostasis and flavonoid accumulation under cold stress.K-means clustering analysis revealed that differentially expressed genes in PHD17 transgenic lines were significantly enriched in the jasmonic acid(JA)biosynthesis pathway,and expression of JA biosynthesis and signaling genes was verified to be affected by PHD17.Cold stress tests applied with MeJA or IBU(JA synthesis inhibitor)further suggested the involvement of PHD17 in JA-mediated cold signaling.Taken together,our results suggest that PHD17 acts downstream of miR1320 and negatively regulates cold tolerance of rice seedlings through JA-mediated signaling pathway.

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.

Transcription factor CabHLH035 promotes cold resistance and homeostasis of reactive oxygen species in pepper

Plant basic helix-loop-helix(bHLH)transcription factors(TFs)play central roles in various abiotic stresses.However,its role in plant cold resistance is largely unknown.Previously,we characterised CaNAC035 in pepper,which positively regulates tolerance to cold,salt and drought stresses tolerance.Here,we identified CabHLH035,a CaNAC035-interacting protein in pepper.To explore its functions in cold stress tolerance,we silenced the gene in pepper via virus-induced gene silencing(VIGS)and overexpressed the gene in Arabidopsis.The results showed that CabHLH035 expression was induced by cold treatment,and silencing of CabHLH035 decreased cold stress tolerance.Conversely,overexpression of CabHLH035 in Arabidopsis increased cold stress tolerance.To investigate homologs genes of C-repeat binding factor(CBF)pathway proteins and reactive oxygen species(ROS)marker gene expression blocking by CabHLH035,we performed yeast one-hybrid(Y1H),dual luciferase and electrophoretic mobility shift assay experiments.The results showed that CabHLH035 bound to the region upstream of the CaCBF1A and CaAPX promoters.Additionally,CaCBF1A bound to the CaDHN4 promoter.Taken together,our results showed that CabHLH035 plays a crucial role in cold stress tolerance and its potential as a target for breeding cold-resistant crops.The findings provide a basis for studying the functions and regulatory network of cold stress tolerance in pepper.

Physiological Analysis on Mechanisms of Cold-tolerance of Dongxiang Wild Rice(Ⅰ)

[Objective] The research aimed to study the effects of soluble substance concentration in cell and the scavenging activity of active oxygen radicals on the cold-tolerance physiology of Dongxiang wild rice.[Method] Dongxiang wild rice was used as materials to detect and compare the content of soluble sugar,free proline and soluble protein,POD activity,CAT activity,MDA content in the leaves in different growth periods.Using with control of two species of common cultivated rice Xieqingzao and Nongda 288.[Resul...

Evaluation of Cold Tolerance of Sugarcane under Drought Frost Condition

[Objective]The aim was to understand the change characteristics of sugarcane traits and evaluate the cold tolerance of sugarcane varieties under the drought and frost conditions.[Method] The experiment was carried out in Ziyuan County,Guangxi Province where the frost occurred often with 21 domestic and abroad sugarcane varieties（elites）.[Result] There were significant changes in the brix,green leaf number and photosynthetic rate of sugarcane before and after light frost and decreased more in cold-sensitive varieties.However,the correlation was only significant between the damage rate of stem length and internode,percentage of green leaves after heavy frost in relation to sugarcane brix and brix changes after light frost.Further analysis showed that the evaluation for cold tolerance of sugarcane would be more simple and reliable with traits of the damage rate of stem length and percentage of upper green leaves.The evaluation for varieties indicated that if it was not carried out for cold-tolerance identification in breeding program,the percentage of sugarcane varieties with good cold-tolerance will be lower than30%,and higher than 60% with poor cold-tolerance in subtropical and tropical regions.[Conclusion] This study had provided theoretical basis for the cold-resistant evaluation of sugar cane and the breeding of the varieties of cold-resistant.

Study on Change Laws of Cold Tolerance in Wheat under Drought Stress

In order to study the change laws of cold tolerance in wheat under drought stress, 11 wheat varieties planted in Huang-Huai wheat area and one variety representative in middle-lower Yangtze River wheat area were selected, and the their change laws of cold tolerance under normal moisture condition and drought stress condition. The results showed that under drought condition, the cold tolerance of wheat in various growth stages was remarkably lower than that under nondrought condition, and the decreasing amplitudes were the largest in overwintering stage with an average of 4.91 ℃, the smallest in regreening stage, and in the middle in the jointing stage. Under non-drought condition, the cold tolerance of semi- winter varieties in various growth stages was better than that of spring varieties, there were significant differences between different varieties, and the cold tolerance of all varieties in overwintering stage was improved compared with before winter, and rapidly decreased after overwintering. Semiwinter varieties showed cold tolerance remarkably enhanced in overwintering stage, and the cold tolerance of spring varieties was also improved to a certain degree.

Advances in Cold Tolerance Genes and Their Application in Genetic Engineering of Plant for Cold Tolerance

Low temperature is one of the main environmental stress factors influenc- ing plant growth and development and crop yield. Cold tolerance genes and progress of their application in genetic engineering of plant for cold tolerance were reviewed comprehensively and systematically from the aspect of genes that are in- volved in biosynthesis of osmotic substances, genes coding fatty acid desaturation enzymes, antifreeze protein genes, genes coding antioxidant enzymes and so on, aiming at laying the foundation for genetic improvement of cold tolerance and breeding of plants.

Study on Biological Characteristics and Pupa's Cold Tolerance of Liriomyza sativae on Florists cineraria

In this study, Liriomyza sativaes was raised on Florist cinerarias in greenhouse with different temperatures at 15, 20, 25, 30 and 35 ℃, and their corresponding development duration of entire generation were 47.0, 25.0, 15.5, 12.5 and 12.0 d. The beginning growth temperatures of egg, larva, pupa and entire generation of Liriomyza sativae were 8.5, 9.8, 10.2 and 10.4 ℃, and their effective accumulated temperatures were 52.7, 75.8, 158.5 and 262.2 d ·℃, respectively. Liriomyza sativae had 7 generations on Florist cinerarias in Ningxia each year. However, it occurred in the whole year in greenhouse. The length of its development duration in greenhouse decreased in order from cucumber, Callestephus chinensis, Florist cinerarias to pea, but the eclosion rate of its pupa was in the eentrary order. Under the temperature of 10, 0, - 15 and 20 ℃ ,the longer the storage of pupa was,the higher the mortality rate would be. Liriomyza sativae couldn＇t survive in the winter in Ningxia Province in the natural environment.

Response to Low Temperature and Evaluation on Cold Tolerance of Sugarcane at Seedling Stage

This study aimed to screen out cold-tolerant sugarcane cultivars through acomprehensive cold tolerance evaluation. A total of 9 sugarcane cultivars with good agronomic traits, bred by the Yunnan Agricultural University, were selected. They were treated by low temperaturestress （3 ℃）, and the changes of their cold-tolerant physiological and biochemical indices were monitored. The cold tolerance of the sugarcane cultivars was evaluated comprehensively by polar ordination. The results showed that the low temperature stress increased the averagemembrane conductivi- ty, soluble sugar content, MDA content, proline content, soluble protein content, chlorophyll content and peroxidase （POD） activity in sugarcane leaves by 21.21%, 134.1%, 83.60%, 35.47%, 47.72%, 9.07% and 565.2%, respectively, but decreased the superoxide dismutase （SOD） activity in sugarcane leaves by 19.67%. Among the 9 sugarcane cultivars, Dianzhe 03-91 showed the strongest cold tolerance, while Dianzhe 02-39 showed the poorest cold tolerance; the cold tolerance of Dianzhe 08-5, Dianzhe 05-103 and Dianzhe 01-58 was stronger than that of Dianzhe 04- 14, Dianzhe 04-429, Dianzhe 05-522 and Dianzhe 02-227.

The Dynamic Changes in Cold Tolerance of Ground-cover Chrysanthemum Growing in the Open Field during the Overwintering

The ground-cover chrysanthemum （Dendranthemaxgrandiflorum Kitamura） ＇Zixunzhang＇ was used as the experimental material in this study. The physiological and biochemical indexes of leaves, emergent rhizomes and roots from ＇Zixunzhang＇ growing in the open field during the autumn and winter in Beijing area were deter- mined. These indexes showed that root activities decreased slowly with the drop in temperature. The relative water contents of leaves and emergent rhizomes, and the water content of roots decreased. The activities of SOD, CAT and POD from leaves and emergent rhizomes were affected significantly by low temperature, but their change trends were different, which suggested that these 3 enzymes might play an interacted role in the formation of the cold tolerance of ＇Zixunzhang＇ mum, while the changes of the three enzyme activities in roots were little when the tem- perature dropped. With the significant increasing of the content of soluble sugar from leaves, the contents of soluble protein and proline declined overall; and the contents of osmotic regulation substances of emergent rhizomes and roots increased at first and then decreased. The correlation analysis indicated that the cold toler- ance of ground-cover mum ＇Zixunzhang＇ was related closely to relative water con- tents, SOD enzyme activity, soluble protein, soluble sugar and root activity. The morphological observation of leaves, emergent rhizomes and roots also showed that the survival of the ground-cover mum mainly depended on the cold tolerance of roots during the overwintering.

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.

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 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）.

Over-Expression of ICE1 Gene in Transgenic Rice Improves Cold Tolerance

ICE1, an Arabidopsis thaliana transcription factor gene, was cloned by RT-PCR and successfully transformed into rice variety Kenjiandao 10 by the Agrobacterium-mediated transformation method. PCR amplification and Southern blot analysis indicated that ICE1 had been integrated into rice genome. Compared with the non-transgenic plants, the transgenic plants exhibited high resistance to hygromycin B and were consistent with the Mendelian inheritance of a single copy of the transgenic ICE1. Under the low temperature stress, the transgenic plants showed the lower mortality rate and the increased proline content. These results suggest that the Arabidopsis ICE1 is functional in rice and the over-expression of ICE1 improves the tolerance to cold stress in rice.