Physiological Response and Transcriptome Analysis of Cotton Leaves under Low Temperature Stress at the Two-leaf Stage

Xiaoman WANG, Jianbing ZENG, Qiongshan WANG, Songbo XIA, Xiaogang WANG, Jiaohai ZHANG, Youchang ZHANG, Shu BIE*

1. Institute of Cash Crops, Hubei Academy of Agricultural Sciences, Wuhan 430064, China; 2. Key Laboratory of Cotton Biology and Breeding in the Middle Reaches of the Yangtze River, Wuhan 430064, China; 3. Hubei Agricultural Science and Technology Innovation Center, Wuhan 430064, China; 4. Agricultural and Rural Bureau of Macheng City, Hubei Province, Macheng 438300, China

Abstract [Objectives] To investigate the effect of low temperature treatment on cotton leaves at the two-leaf stage. [Methods] Two cotton varieties (CN01 and SJB016 (low temperature-tolerant)) were used as the trial materials. They were treated at 25 (CK) and 12 ℃ (low temperature) for 0, 3, 6, 12, 24, 48 and 72 h, respectively. Then, the changes in the contents of MDA, SS and Pro in the cotton leaves were analyzed. Based on the analysis results, RNA-seq verification was performed. [Results] Two cotton varieties (CN01 and SJB016 (low temperature-tolerant)) were used as the trial materials. They were treated at 25 (CK) and 12 ℃ (low temperature) for 0, 3, 6, 12, 24, 48 and 72 h, respectively. Then, the changes in the contents of MDA, SS and Pro in the cotton leaves were analyzed. Based on the analysis results, RNA-seq verification was performed. [Conclusions] These genes may play an important role in improving the cold resistance of cotton.

Key words Cotton leaf, Physiological response, Transcriptome, Gene expression, Low temperature stress

1 Introduction

Cotton is a thermophilic and drought-tolerant crop, with optimum growth temperature of 28-30 ℃. Too high or too low temperature will adversely affect the growth, development, yield and quality of cotton. In spring, the temperature may drop sharply while rising due to the intrusion of cold air, causing damage to crops. In particular, coldness in late spring will lead to rotting and death of seedlings. Coldness in both early and late spring will affect the yield of winter wheat, and that in early spring will reduce the yield of winter wheat by more than 19%. Low temperature stress will significantly reduce the germination rate, germination vigor and germination index of cotton seeds, leading to a reduction in cotton production.

In order to adapt to the new characteristics of the development of the cotton industry, the Hubei Academy of Agricultural Sciences has been reforming the traditional cotton planting method and breeding and promoting cotton varieties suitable for mechanized harvesting. Based on early-maturing and yield formation-consistent varieties suitable for direct seeding and close planting, the cotton line CN01 suitable for mechanized harvesting is screened out. It belongs to the infinite fruit branch type, has moderate tightness, strong lodging resistance, vigorous growth in the seedling stage, strong boll-forming ability, smooth boll-spitting, concentrated boll-spitting, concentrated growth, and is suitable for mechanized harvesting. It has been demonstrated in Hubei Province for 4 years, and the yield has reached 3 955 kg/ha under the condition of mechanized harvesting.

CN01 and a more cold-resistant cotton line SJB016 were used as the trial materials, and their different physiological and biochemical responses and transcriptional regulation mechanisms under low temperature stress were studied. Through the differential expression analysis between the two cotton lines, differentially expressed genes and metabolic pathways related to low temperature stress response were screened. This is of guiding significance to improve the low temperature resistance of machine-picked cotton, and lays the foundation for the optimization and improvement of machine-picked cotton varieties.

2 Materials and methods

2.1 Materials

CN01 and SJB016 were provided by the Institute of Cash Crops of Hubei Academy of Agricultural Sciences. They were both suitable for mechanized harvesting.

2.2 Material treatment

Delinting was completed with concentrated sulfuric acid. The cotton seeds to be delinted were put in a beaker, added a small amount of concentrated sulfuric acid and stirred with a glass rod for 3 min. The delinted seeds were transferred into a mesh bag, rinsed with running water to remove sulfuric acid, dried in the sun for 2-3 d, 5-6 h a day, and flipped frequently. The drying was even and completed until the seeds made a sound when they were shaken.

For cultivation of cotton at the two-leaf stage, plump cotton seeds were selected, and nursed in nutrient bags (10 cm×10 cm) filled with culture substrate (nutrient soil∶vermiculite=3∶1) under the conditions of 25 ℃, RH 75% and 16h/8h (day/night).

In the experiment on the effect of low temperature stress on cotton leaves at the two-leaf stage, two treatments [25 ℃ (CK) and 12 ℃ (low temperature treatment)] and seven time gradients (0, 3, 6, 12, 24, 48 and 72 h) were designed. There were two replicates for each treatment, and for each replicate, there were six uniformly growing cotton plants. From each cotton seedling, two leaves were collected and marked as subsequent test materials. They were put it into liquid nitrogen quickly, and then transferred to -80 ℃ refrigerator for storage.

2.3 Methods

With reference to Cai Yongping, the content of malondialdehyde (MDA) was determined with the thiobarbituric acid colorimetric method, and the content of soluble sugars (SS) was determined by the anthrone-sulfuric acid method. The proline content was determined with the kit of Nanjing Jiancheng Bioengineering Institute. The total RNA from cotton leaves was extracted with the kit of Tiangen Biotech (Beijing) Co., Ltd., and centrifuged at room temperature. RNA reverse transcription and fluorescence quantitative PCR referred to the literature [4].

3 Results and analysis

3.1 Effect of low temperature stress on the MDA content of cotton leaves

The effect of low temperature stress on the MDA content of cotton leaves is shown in Fig.1 and Fig.2. Compared with CK, the MDA content of SJB016 treated by low temperature increased slightly, and at 6 h, the difference reached the maximum, increased by 24.82% (Fig.1). In comparison to CK, the MD content of CN01 increased first and then declined after low temperature treatment, and the difference also reached the maximum at 6 h, increased by 43.91%. The MDA content of both CN01 and SJB016 increased most greatly at 6 h, and the increase of CN01 was greater than that of SJB016.

Note: * represents a significant difference between the CK and treatment groups at the 0.05 level, and ** represents a significant difference between the CK and treatment groups at the 0.01 level. The same below.

Fig.2 Effect of low temperature stress on leaf MDA content of CN01

3.2 Effect of low temperature stress on the soluble sugar content of cotton leaves

The effect of low temperature stress on the SS content of cotton leaves is shown in Fig.3 and Fig.4. As can be seen from Fig.3, the SS content in SJB016 cotton leaves was maintained at a stable level under normal circumstances; and in the first 3 h of the low temperature treatment, the SS content was basically the same as that of CK; when the treatment reached 6 h, the SS content increased sharply, increased by 65.29% compared with CK; at 12 h, the SS content was close to that of CK; and then as treatment time increased, the SS content of low temperature treatment tended to increase. As can be seen from Fig.4, the SS content of CN01 was maintained at a stable level under normal circumstances; the SS content of low temperature treatment was always higher than that of CK, and the growth rate accelerated after 48 h; and the SS content increased by 45.35% at 6 h.

Fig.3 Effect of low temperature stress on leaf soluble sugar content of SJB016

Fig.4 Effect of low temperature stress on leaf soluble sugar content of CN01

3.3 Effect of low temperature stress on the proline content of cotton leaves

The effect of low temperature stress on the proline content of cotton leaves is shown in Fig.5 and Fig.6. As can be seen from Fig.5, the pro contents of SJB016 at 3, 6, 12, 24, 48 and 72 h were higher than those of CK, increased by 11.93%, 6.36%, 0.00% (0.000 23%), 6.43%, 15.17% and 11.56%, respectively. In terms of Pro content, as the time of low temperature stress increased, the plants accumulated more proline to cope with changes. At 3, 6, 12, 24, 48 and 72 h, the Pro contents in the leaves of CN01 were higher than those in CK, increased by 2.1%, 14.62%, 17.23%, 31.82%, 31.48% and 57.49%, respectively (Fig.6). With the extension of the low temperature stress time, the stress on CN01 intensified.

Fig.5 Effect of low temperature stress on leaf proline content of SJB016

Fig.6 Effect of low temperature stress on leaf proline content of CN01

3.4 Analysis of transcriptome sequencing results

Based on the changes in the MDA, SS and Pro contents of cotton leaves, it could be concluded preliminarily that the physiological indexes of cotton leaves changed more greatly after 6 h of low temperature treatment, so the samples treated at low temperature for 6 h were selected for transcriptome sequencing research.

3.4.1

Screening of differentially expressed genes. Differentially expressed genes were screened at

padj

<0.05, and the quantity and expression level distribution of differentially expressed genes obtained is shown in Fig.7. The total number of differentially expressed genes in SJB016 between control and low temperature treatment was 6 815, of which 3 990 were up-regulated and 2 825 were down-regulated. The total number of differentially expressed genes in CN01 between control and low temperature treatment was 4 900, including 3 547 up-regulated and 1 353 down-regulated. The total number of differentially expressed genes shared by the two cotton lines was 3 288. After low temperature stress, the differentially expressed genes shared by CN01 and SJB016 both accounted for more than 1/2 of the total, indicating that the response of these genes to low temperature stress is conserved in different lines and the specifically expressed genes determine the cold tolerance of plants.

Note: CCL, leaves of CN01 at normal temperature; LCL, leaves of CN01 at low temperature stress for 6 h; CSL, leaves of SLB016 at normal temperature; LSL, leaves of SJB016 at low temperature stress for 6 h. The same in Fig.8B.

3.4.2

Analysis of differential transcription factors. Responses of cotton to low temperature stress are regulated by multiple genes, among which, transcription factors play an extremely important role. By analyzing the expression of differential transcription factors between the control group and the treatment group, it was found that (Fig.8) there were 124 transcription factors differentially expressed in CN01, and there were 235 transcription factors differentially expressed in SJB016. These transcription factors mainly belonged to 25 transcription factor families, and the differentially expressed genes were concentrated in AP2-EREBP, bHIH, MYB, WRKY and other families. There were more transcription factors in SJB016 that responded to low temperature stress. The number of differentially expressed genes from the WPKY family reached 28 in SJB016, while there was only one in CN01.

Note: A. Number of differentially expressed genes in differentially expressed transcription factor families; B. Expression abundance of differentially expressed genes in AP2-KREBP, bHLH, MYB and WRKY transcription factor families.

3.4.3

Function annotation of differentially expressed genes. (i) GO annotation. The GO annotation results of the differentially expressed genes in CN01 between control and treatment are shown in Fig.9. The differentially expressed genes were annotated to 3 241 gene functions, including 169 enrichment functions. Among them, 116 functions were clustered in the broad category of biological process, and 5 functions were clustered in the category of molecular function. A total of 30 GO terms with the most significant enrichment were selected and made into a histogram of GO enrichment for differentially expressed genes. Among them, 20 belonged to biological processes, in which single-organism metabolic process (GO: 0044710) enriched the most differentially expressed genes, with a total of 891. The remaining 10 GO terms belonged to molecular functions. Among them, intramolecular oxidoreductase activity (GO: 0016860) enriched the most differentially expressed genes (22).

Fig.9 GO functional classification of differentially expressed genes in CN01

Fig.10 GO functional classification of differentially expressed genes in SJB016

The results of GO annotation for the differentially expressed genes of SJB016 between control and treatment are shown in Fig.10. The differentially expressed genes were annotated to 3 434 gene functions, including 193 enrichment functions. Among them, 148 were clustered to the category of biological process, and 45 were classified to the category of molecular function. Total 30 GO terms with the most significant enrichment were selected and made into differentially expressed gene GO enrichment histogram. Among them, 21 were biological processes, with biological process (Go: 0008150) enriching the most differentially expressed genes (3 854). The remaining 9 terms were molecular functions.

(ii) KEGG annotation. Fig.11 shows the KEGG annotation results of the differentially expressed genes of CN01 between control and treatment. The differentially expressed genes between CCL and LCL were annotated to 122 metabolic pathways. The pathways with the most significant enrichment were selected and made into enrichment scatter plot. The enrichment pathways included carbon metabolism, glyoxylic acid and dicarboxylic acid metabolism, circadian rhythm-plants, pentose phosphate pathways,

etc

. Among them, the number of differentially expressed genes enriched in metabolic pathways was the largest.The KEGG annotation results of the differentially expressed genes of SJB016 between control and treatment are shown in Fig.12. A total of 121 metabolic pathways were annotated for the differentially expressed genes between CSL and LSL. The pathways with the most significant enrichment were selected and made into enrichment scatter plot. It shows that the enrichment pathways included were pentose phosphate pathway, unsaturated fatty acid biosynthesis, glycolysis/gluconeogenesis, fatty acid metabolism,

etc

. Among them, the number of differentially expressed genes enriched in the biosynthesis of secondary metabolites was the largest.Taking Corrected

P

<0.05 as an indicator, the enriched metabolic pathways in CN01 and SJB016 were analyzed. It was found that carbon metabolism, glyoxylic acid and dicarboxylic acid metabolism, pentose phosphate pathway, glycolysis/gluconeogenesis, ascorbic acid and metabolism are metabolic pathways shared by the two cotton lines, and they are conservative metabolic pathway in the two cotton lines that make responses to low temperature stress. Genes related to photosynthesis and plant hormones were expressed differently in CN01 and SJB016.

(iii) Analysis of differentially expressed genes related to photosynthesis. RNA-seq sequencing results show that the differentially expressed genes were mainly distributed in PS I, PS II, cytochrome b6/f complex, photosynthetic electron transporter and F-type ATPase. The number of differentially expressed genes contained in CN01 was 3, 18, 3, 7, 4, respectively, which were all up-regulated. In SJB016, the number of differentially expressed genes was 10, 15, 4, 6 and 4, respectively. The distribution of differentially expressed genes in CN01 and SJB016 was compared. The results show that the common differentially expressed genes were PS II (up-regulated) (D2, cp43, PsbS, PsbZ), PS I (up-regulated) (PsaA), cytochrome b6/f complex (up-regulated) (PetC), photosynthetic electron transporter (up-regulated) (FNR), and F-type ATPase (up-regulated) (β, b). The expression of PsbP and PsbQ increased in CN01, while decreased in SJB016, showing the opposite trend. As components of the oxygen-evolving complex, the effect of PsbP and PsbQ on plant photosynthesis should be verified in the follow-up.

Fig.11 KEGG pathway classification of differentially expressed genes in CN01

Fig.12 KEGG pathway classification of differentially expressed genes in SJB016

(iv) Analysis of differentially expressed genes related to plant hormones. The RNA-seq sequencing mainly analyzed the differential expression of 8 kinds of plant hormone-related genes, auxin (IAA), cytokinin (CTK), gibberellin (GA), abscisic acid (ABA), ethylene (EY), brassinolide, jasmonic acid (JA) and salicylic acid (SA). In CN01, there were 50 differentially expressed genes related to IAA, among which 47 were up-regulated and 3 were down-regulated; there were 2 differentially expressed genes related to CTK, both of which were up-regulated; there were 5 differentially expressed genes related to GA, among which 3 were up-regulated and 2 were down-regulated; 20 differentially expressed genes were related to ABA, among which 19 were up-regulated; 10 differentially expressed genes were related to EY, among which 8 were up-regulated; there were 4 brassinolide-related differentially expressed genes, with one down-regulated; 5 differentially expressed genes were related to JA, with one down-regulated; and there was no significant difference in SA-related genes. The differences were mainly concentrated in genes related to IAA and ABA. In SJB016, 61 differentially expressed genes were found to be related to IAA, and among them, 51 were up-regulated and 10 were down-regulated; 8 differentially expressed genes were related to CTK, and among them, 5 were up-regulated and 3 were down-regulated; there were 6 differentially expressed genes related to GA, with five up-regulated and one down-regulated; the number of differentially expressed genes related to ABA was 26, among which, 19 were up-regulated and 7 were down-regulated; there were 21 differentially expressed genes related to EY, with 17 up-regulated; 4 differentially expressed genes were found to be related to brassinolide; 5 differentially expressed genes were found to be related to JA, with one down-regulated; and no significant difference was observed in SA-related genes.

3.5 qRT-PCR verification of differentially expressed genes

To verify the accuracy of the transcriptome sequencing results, 13 differentially expressed genes were randomly selected. The functions of these genes were UDP-glucose 4-epimerase, GEPI48 protochlorophyll-dependent translocation component, gibberellin 2-β-dioxygenase, flavanone 3-dioxygenase methionine γ-lyase, ferulyl-CoA ortho-hydroxylase, alcohol dehydrogenase, NAD (P) H-quinone oxidoreductase subunit J, PSII CP43 reaction center protein, thioredoxin, NAD-dependent isoform of malic enzyme (59 kDa) and cytochrome P450. The differential gene expression results detected by qPT-PCR and the transcriptome sequencing results were compared. It is found that the differential gene expression of the two results is similar (Fig.13, Fig.14). The expression correlation coefficients (

R

) of CN01 and Yuan 23 were 0.874 4 and 0.718 6, respectively, and the

P

values were 0.699 and 0.395 8, respectively, showing a significant correlation.

4 Conclusions and discussion

4.1 Effect of low temperature stress on the membrane lipid peroxidation of cotton leaves

In the face of adversity damage, plants have a series of defense mechanisms. Among them, SOD, POD, CAT, AsAPOD and other important components in the enzymatic defense system play an irreplaceable role in plant resistance, but the increase in MDA content will affect the activity of these antioxidant enzymes. Adding MDA directly to the cell-free extract of spinach can weaken the activity of SOD, POD and CAT. The changes of MDA content of CN01 and SJB016 cotton leaves under low temperature stress were compared, and it was found that compared with the control, the MDA content first increased and then decreased. The difference in MDA content between CN01 and SJB016 after 6 h of low temperature treatment reached the maximum (

P

<0.01). The sharp increase in MDA content suggested that the low temperature of 12 ℃ caused damage to the cotton leaves, but as the treatment time increased, the MDA content gradually decreased, and gradually became the same as the control. It shows that when plants are suddenly subjected to low temperature stress, the degree of membrane lipid peroxidation in their bodies deepens. But since it is not extremely low temperature, as time increases, the defense system and the active oxygen scavenging system in the plants begin to work, stimulating the expression of some cold resistance genes in plants, thereby enhancing the cold resistance of plants. The MDA content in the plants also begins to decrease, gradually close to the control. The results of this study are the same as those of Zheng Guohua

et

al

.who studied the change trend of MDA content in loquat leaves under low temperature stress. The MDA content in loquat leaves treated at 7 ℃ for 24 h first increased and then decreased, and that in loquat leaves treated at -7 ℃ for 24 h showed an increasing trend.

Fig.13 Comparison of qRT-PCR and transcriptome sequencing results of CN01

Fig.14 Comparison of qRT-PCR and transcriptome sequencing results of SJB016

4.2 Effect of low temperature stress on the contents of osmotic adjustment substances in cotton leaves

Under low temperature stress, plants often accumulate a large amount of soluble sugars. Common ones are sucrose, glucose, fructose, seaweed polysaccharides and so on. Zhu Zheng

et

al

.studied the effect of low temperature on tea leaves. It is found that the longer the low temperature stress, the more the soluble sugar accumulated. After 10 d of treatment, the soluble sugar content showed a double increase. The soluble sugar content of petunia H strain treated at low temperature for 2 h increased significantly. Then, with the extension of treatment time, the soluble sugar content increased slightly, but was always higher than the control.Compared with the control, the soluble sugar content in CN01 treated at low temperature always showed an upward trend, and the rising rate increased after 48 h, and was always higher than the control. This is the same as the change trend of soluble sugar content in tea leaves and petunia under low temperature stress. The soluble sugar content of SJB016 treated at low temperature increased sharply within 6 h, and then declined, close to that of CN01 at 24 h. The rising rate was accelerated. During the experiment, the soluble sugar content of cotton line subjected to low temperature stress was higher than that of the control, indicating that the accumulation of soluble sugar plays an important role in the resistance of cotton to low temperature stress. Wang Xiaoxuan

et

al

.studied the changes in soluble sugar content of tomato seedlings with different cold tolerances under low temperature stress. It was found that the cold tolerance of tomato varieties treated with low temperature was significantly positively correlated with the soluble sugar content.Proline has high solubility, is non-toxic, and can reduce the freezing point of cells. It is an important cold resistance regulator to prevent cells from dehydration and death. Under low temperature stress, plants resist the damage of the external environment by increasing the content of proline. During low temperature stress, the changes of proline content in different plants are not exactly the same. Yelenoskyfound that the proline content of

Citrus

spp. did not change significantly when facing low temperature stress. Li Yujun

et

al

.found that the accumulation of proline in soybean was negatively correlated with its cold resistance. Wu Huifound that after being under low temperature stress, highly cold-tolerant cotton varieties accumulate more proline than cold-sensitive cotton varieties.After low temperature treatment, the proline contents in CN01 and SJB016 were higher than the control. The increase in proline content of CN01 was higher than that of SJB016. The cold tolerance of SJB016 concluded from the germination test of this study was higher than that of CN01, which is contrary to the results about cold-tolerant varieties that had more proline accumulation after low temperature stress concluded by Wu Hui

et

al

. The possible reasons are as follows. First, the effect of low temperature stress on the germination rate of cotton and the cotton at two-leaf stage is not completely synchronized. Second, the identification of cotton cold resistance requires comprehensive consideration of multiple indicators. A discrepancy of an index with previous studies cannot completely represent the poor cold tolerance of the species.

4.3 Effect of low temperature stress on the photosystem of cotton leaves

Low temperature has a huge impact on plant photosynthesis. It can almost affect the normal function of all the main components involved in photosynthesis, including electronic transmission, Calvin cycle, and the degree of opening of the stomata. Compared with the PS I system, the phenomenon of light suppression in PS II is more common. Low temperature stress will cause the PS II reaction center to inactivate, affecting the absorption of light energy, causing the photoinhibition of PS II. In order to cope with the damage of adversity to PS II system and prevent it from affecting photosynthesis of plants, damage and repair are carried out at the same time, and the key role is the rapid turnover of D1 protein. The relative rate of damage and repair directly determines the degree of photoinhibition of PS II.The expression of photosynthesis-related genes in CN01 and SJB016 changes under low temperature stress. In CN01, the expression of the extrinsic protein PsbQ of the PS II complex was up-regulated, while in SJB016, the expression of this gene was down-regulated. A studyreported that the presence of PsbQ has an important impact on the structure and function of PS II oxygen-evolving center. The PS II complex with PsbQ participation is the best functional form of PSII in cyanobacteria. An Feifei

et

al

.found that low temperature stress reduced the expression of oxygen-evolving complexes in the PA II system in cassava leaves. Chen

et

al

.found after 3 h of high temperature stress, the PS II in wheat showed a rapid and strong response, and PsbQ was significantly reduced. In summary, adversity stress will cause the decrease of PsbQ expression.

4.4 Relationship between plant growth regulators and low temperature stress

The content of plant growth regulators in plants is very small, but they play an important role in regulating the growth and development of plants, and also play an important role in the cold resistance of plants.Yue Dan

et

al

.found that the decreased content of gibberellin and the increased content of abscisic acid can improve the cold resistance of apricot trees. As the cold resistance strengthens, the ratio of ABA/GA3 also rises, which is similar to the finding of Rikin

et

al

. The cold resistance of plants is positively correlated with the value of ABA/GA3, which is mainly because that the increase in ABA content increases the ratio. The sequencing results show that the expression of most of the differentially expressed genes related to ABA was up-regulated, suggesting that in order to deal with low temperature stress, both CN01 and SJB016 increased the expression of ABA gene.Yuan

et

al

.found that low temperature stress reduced the content of auxin in amomum seedlings. The expression of

GH

3 that encodes auxin amide synthase was significantly increased. The expression of

GH

3 will reduce free auxin. Du

et

al

.found that the free auxin (IAA) content of rice treated with low temperature increased, the expression of genes related to auxin synthesis was up-regulated, and the gene encoding GH3 protein was down-regulated. The sequencing results of this study show that a large number of differentially expressed genes related to auxin were expressed in CN01 and SJB016 treated with low temperature. Most of the genes were up-regulated and some genes were down-regulated. Specific effect on gene expression, auxin and cold resistance needs to be verified by subsequent experiments.Shi

et

al

.found that while suffering from low temperature stress, the auxin content in the body of

Arabidopsis

was significantly reduced; mutant

Arabidopsis

with overproduction of ethylene exhibited low temperature sensitivity; and

Arabidopsis

with ethylene inhibitors showed increased resistance to low temperature.

4.5 Response of transcription factors to low temperature stress

When subjected to low temperature stress, the transcription factors in the plants will respond to changes, in order to improve the survival rate of plants under low temperature stress. This study found that after low temperature treatment, the differentially expressed transcription factors of CN01 and SJB016 were mainly concentrated in AP2-EREBP, bHLH, MYB and WRKY and other transcription factor families, and the differentially expressed genes were all down-regulated.Chen

et

al

.found that low temperature stress significantly increased the expression of

AhERF

4 in peanuts. Overexpression of the transcription factor CaPF1 in pepper can improve the cold tolerance of

Pinus

strobus

. In this study, the transcription factors of AP2-EREBP family were down-regulated, thus improving the tolerance of cotton to low temperature stress through a negative feedback regulation mechanism.Plant MYB transcription factors are closely related to a variety of biological processes in plants. Changes in the expression of MYB transcription factors can regulate a variety of biological processes and help plants grow normally. Liu Xiaoyue

et

al

.found that the

MYB

gene in low-temperature-tolerant rice was up-regulated at multiple time points. In low-temperature-sensitive plants, the expression patterns of

MYB

gene are numerous. It shows that plants can improve their adaptability to low temperature stress by regulating the expression pattern of

MYB

gene. This study found that there were obvious differences in the expression of MYB family transcription factors in CN01 and SJB016 treated at low temperature, and the number of down-regulated genes in SJB016 was larger than that in CN01. It shows that down-regulation of

MYB

gene can improve the tolerance of CN01 and SJB016 to low temperature.WRKY is a larger transcription factor family in plants, and it participates in various biological processes. The expression of more than 1/2 of the WRKY transcription factors in poplars changed under low temperature stress.

AtWRKY

34 can negatively regulate the CBF-mediated low temperature response pathway. This study found that after low temperature stress treatment, the

WRKY

genes were significantly down-regulated in CN01 and SJB016, but more genes were differentially expressed in SJB016. This should be related to the different tolerance of different cotton lines to low temperature stress.