The transcriptome and metabolic pathways of Persea americana under drought and low-temperature stress

Zhang, Y., Luo, M., Yuan X., Yang, Z., Wang, Y., and Zheng, Y. (2023). “The transcriptome and metabolic pathways of Persea americana under drought and low-temperature stress,” BioResources 18(2), 3938-3960.

Abstract

Persea americana Mill. is an important cash crop that contains effective ingredients to reduce cholesterol and protect the cardiovascular system. Presently, the gene regulation mechanism and signal pathway of stress response in P. americana are unclear. To explore the gene expression changes of P. americana under drought and low-temperature stress, the transcripts of P. americana were sequenced under these conditions. The results produced 42,815,960 bp raw reads. Analysis of the related metabolic pathways and differentially expressed genes showed that under drought stress, the gene expression of beta-amylase 3, glyceraldehyde-3-phosphate dehydrogenase and hexokinase were upregulated, while the gene expression of UDP-glycosyltransferase superfamily protein isoform, glucose-1-phosphate adenylyltransferase, and glucose-6-phosphate 1-epimerase were downregulated. Under low-temperature stress, the expression of beta-amylase and shikimate O-hydroxycinnamoyl transferase genes was downregulated. In addition, WRKY, MYB, bHLH, and NAC transcription factors were expressed under drought and low-temperature stress. Finally, the RNA-Seq data were validated using real-time fluorescence quantitative analysis to identify the key genes of P. americana regulated at the transcriptional level under drought and low-temperature stress. This study provides a theoretical basis for the selection of drought-resistant and low-temperature tolerant P. americana varieties.

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The Transcriptome and Metabolic Pathways of Persea americana under Drought and Low-temperature Stress

Yi Zhang,^a,b,1 Maniya Luo,^c,1 Xiaolong Yuan,^b Zhijuan Yang,^a Yi Wang,^b,* and Yuan Zheng ^a,*

DOI: 10.15376/biores.18.2.3938-3960

Keywords: Persea americana; Drought stress; Low-temperature stress; Metabolic pathways; Transcriptome; qRT-PCR

Contact information: a: College of Forestry, Southwest Forestry University, Kunming, 650224, China; b: Laboratory of Forest Plant Cultivation and Utilization, The Key Laboratory of Rare and Endangered Forest Plants of State Forestry Administration, Yunnan Academy of Forestry and Grassland, Kunming, 650201, China; c: National Culture Research Institute of Jinggu Dai and Yi Autonomous County, Puer, 666400, China; ¹ These authors equally contributed to this work;

* Corresponding authors: dog_608@qq.com; zhengyuan_001@126.com

GRAPHICAL ABSTRACT

INTRODUCTION

Persea americana Mill., also known as the ‘alligator pear,’ is a Lauraceae native to Mexico and Central America (Chanderbali et al. 2008; Bhuyan et al. 2019). It is one of the world’s most important tropical/subtropical fruit crops and has high economic value (Kuhn et al. 2019). Its fruit is not only rich in protein and fat-soluble vitamins but also in tocopherols, lutein, and other carotenoids, which are widely used in the pharmaceutical and cosmetic industries (Duarte et al. 2016; Tabeshpour et al. 2017). In addition, the oil extracted from P. americana seeds can be used as an alternative biodiesel source for the biofuel industry (Ge et al. 2021).

Because P. americana fruit benefits human health, there is an increased demand for its functional compounds in the market. Alkhalaf et al. (2019) evaluated the antioxidant, anti-inflammatory, and anti-cancer potential of each extract of P. americana fruit and seed. Tremocoldi et al. (2018) used HPLC-MS/MS to conduct a real-time analysis of phenolic compounds obtained from green extraction of P. americana by-products to determine their contribution to the total biological activity of P. americana by-products. Ibarra-Laclette et al. (2015) used a high-throughput sequencing platform to develop a gene expression map of the P. americana transcriptome. Specifically, they analyzed the expression of genes involved in acyl lipid metabolism, maturation process, and organ specificity, aiding further research on P. americana basic biology. Ge et al. (2019a) used molecular identification methods to study the different geographical sources of P. americana germplasm resources in southern China and conducted a supplementary analysis. Due to its high economic value, P. americana is widely cultivated and developed in some provinces in southern China, including Taiwan, Hainan, Guangxi, and Yunnan (Ge et al. 2017, 2019b). Yunnan Province is in the southwest of China, has a complex terrain, is affected by two Asian summer monsoons, and is extremely sensitive to climate change. According to previous research, extreme weather events (such as floods, low temperatures, and droughts) have often occurred in this province in recent decades (Shi and Chen 2018), so breeding P. americana with strong resistance is a key factor for the success of introduction.

Abiotic stresses such as drought and low temperature affect plant growth, development, and secondary metabolism (Li et al. 2021). Stress reduces net photosynthetic rate, destroys chloroplast structure, reduces reactive oxygen species clearance, and reduces carbohydrate metabolism (Valliyodan and Nguyen 2006; Maestrini et al. 2009; Abdel-Ghany et al. 2020). Moreover, it leads to changes in functional genes and transcription factors (TFs) involved in metabolic and physiological processes during transcription (Wang et al. 2017). Xu et al. (2021) used Illumina sequencing technology to analyze the transcriptome of Salix cupularis Rehd under drought stress and identified 4,289 differentially expressed genes (DEGs) – 2,340 were upregulated, and 1,949 were downregulated. Li et al. (2021) found that Larix kaempferi (Lamb.) Carr had 27 DEGs involved in drought stress. Under drought stress, Prunus mahaleb L is mainly involved in carbohydrate metabolism and hormone signal transduction (Feng et al. 2017). Pugionium cornutum (L.) Gaertn was found to be mainly involved in photosynthesis, nitrogen metabolism, and hormone signal transduction pathways (Wang et al. 2017). Under low-temperature stress, Populus tomentosa Carr is mainly involved in metabolic pathways such as sugar metabolism, antioxidant defense system, hormone signaling and photosynthesis (Yang et al. 2019). Populus simonii Carr is mainly involved in photosynthesis, abscisic acid (ABA) transport, and antioxidant defense system (Song et al. 2013). Wang et al. (2013) identified 1,770 DEGs from the whole genome of Camellia sinensis (L.) Kuntze, of which 1,168 genes were upregulated, mainly involved in carbohydrate metabolism and calcium signaling pathways, and 58 TFs families responded to low-temperature stress. Gong et al. (2018) identified 239 TFs that responded to low-temperature stress from the whole genome of Hevea brasiliensis (Willd. ex A. Juss.) Muell. Arg. Those TFs are involved in flavonoid biosynthesis, phenylalanine biosynthesis and metabolism, plant hormone signal transduction, and starch and sucrose metabolism. Therefore, this study was conducted to investigate the molecular response mechanisms made by P. americana under different stresses.

In this study, NovaSeq technology was used to sequence the participating transcriptome of P. americana and excavate the key genes and TFs related to the metabolic pathway of drought and low-temperature stress; the results were verified by qRT PCR. The results lay the foundation for the in-depth study of P. americana and provide a theoretical basis for breeding drought and low-temperature resistant varieties.

EXPERIMENTAL

Plant Materials

Annual P. americana seedlings with good growth conditions were planted in the greenhouse at the Bailong Campus of Southwest Forestry University (Kunming, China). In line with the material treatment reference (Wang et al. 2014; Wu et al. 2015), the treatment conditions of the control group (PAMZ) were as follows: the relative soil water content was kept at 80% of the field water capacity, and normal watering (five times/d) at room temperature (25 ℃). Drought treatment (PAMG): watering of seedlings at room temperature was terminated after sufficient water has been applied to saturate the soil with water, allowing the relative soil moisture content to fall to 40% of the field holding capacity, and sampling after the leaves had curled. Low temperature treatment: P. americana seedlings were placed in a low temperature incubator at a low temperature of 4 ℃ (PAM4), a low temperature of 15 ℃ (PAM5), and the rest of the incubation conditions were the same. Sampling was carried out after 6 h of normal watering, and the collected leaves were immediately snap-frozen in liquid nitrogen, stored at -80 ℃, and sent to Personalbio (Shanghai, China) for transcriptome sequencing.

RNA Extraction and Sequencing

Total RNA extracted from mature leaves tissue of P. americana was determined under different treatments using the Ultrapure RNA Kit (CWBIO, China). The concentration and quality of RNA were detected by NanoDrop 2000 (NanoDrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA) and 1.5% agarose gel electrophoresis, respectively. The mRNA with polyA structure was enriched by Oligo (dT) magnetic beads and broken into fragments of approximately 300 bp in length by ion interruption. The first strand of cDNA was synthesized with RNA as a template, using 6-base random primers, and reverse transcriptase. The second strand of cDNA was synthesized with the first cDNA as a template. The cDNA fragment with a specific length of 450 bp was recovered for PCR amplification and sequenced using the Illumina NovaSeq platform.

Transcriptome Data Processing, Gene Ontology (GO) Annotation, and KEGG Enrichment Analysis

The obtained raw data were filtered using Cutadapt v1.12 online software to remove joints at the 3’ end and read with average mass fraction below Q20 to obtain Clean Reads, and the resulting Clean Reads were annotated in the following databases: NCBI Non-Redundant Protein Sequences (Nr), eggNOG, Swissport, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO). GO and KEGG enrichment analyses were performed using topGO and clusterprofiler to identify GO terms and KEGG pathways that are significantly enriched for differential genes by calculating P-values (the criterion for significant enrichment is P < 0.05) by the hypergeometric distribution method.

Screening of Differentially Expressed Genes

HTSeq statistics were used to compare to Read Count values on each gene as the original expression of the gene, normalized gene expression using FPKM values (FPKM ＞1), and performed differentially expressed gene analysis by DESeq, screening differentially expressed genes with the following conditions: expression difference fold |log2FoldChange|>1, significant P- value<0.05.

Quantitative Real-Time PCR (qRT-PCR) Analysis

To verify the results of transcriptome sequencing and the expression pattern of target genes, ten differentially expressed genes were randomly selected, and their gene expression was detected by qRT-PCR. Using the transcriptome data, primers were designed (Table 1) with 1-alpha (EF1a) gene as the internal regulator of gene normal expression. In addition, SYBR Green (Invitrogen Beijing) was used to detect the PCR products of specific primers in the ABI 7300 Real-Time PCR System (Applied Biosystems, Foster City, USA). The conditions of the PCR reactions were as follows: denaturation (94 °C, 2min), 40 cycles of quantitative amplification (94 °C – 15s; 60 °C – 15s; 72 °C – 45s), with a final extension step (72 °C, 7min).

Table 1. Primer Sequences

RESULTS AND DISCUSSION

Gene Sequence Assembly and Function Annotation

P. americana leaves after different treatments were analyzed using the platform Illumina NovaSeq platform, and the obtained data were uploaded to NCBI (https://www.ncbi.nlm.nih.gov/sra/SRX19802688). The results showed that the raw reads generated from each set of samples were at least 42,815,960 bp, and the clean reads obtained after filtering were at least 38,591,384 bp, and the total sequence comparison rate with the reference genome (GCA_003546025.1) was higher than 84.88%, Q20> 96.33% and Q30> 90.88%(Table 2). The above data indicated that the obtained P. americana transcriptome data were of high quality and could be analyzed in the next step.

Table 2. Statistical Results

The obtained Unigenes were subjected to BLASTx comparison of the following databases and the results showed (Fig. 1 and Table S1) that 38,178 genes were annotated in the NR database, 17,970 in the GO database, 17,816 in the KEGG database, 37,873 in the eggNOG database and 3,960 in the Swissprot database, with annotation in the NR database being the most numerous.

Fig. 1. Gene function annotation

Analysis of Metabolic Pathways

The results of GO analysis (Fig. 2 and Table S2) show that a total of 17,970 genes were divided into 92 functional groups, which came from three major categories: molecular function (MF), cellular component (CC), and biological process (BP). Most genes annotated for the PAMZ-PAMG comparison group were related to photosystem I and ion transport pathways: a total of 1,354 genes – including 406 MF (30%), 123 CC (9.1%), and 825 BP (60.9%). A total of 1341 genes were annotated for the PAMZ-PAM4 group, including 397 MF (29.6%), 170 (12.7%) CC, and 882 BP (65.8%) – mainly related to the inositol bisdiphosphate tetrakisphosphate diphosphatase activity, inositol diphosphate pentakisphosphate diphosphatase activity, and photosynthesis metabolic pathways. A total of 1,508 genes were annotated for the PAMZ-PAM5 comparison group, including 456 MF (30.2%), 170 CC (11.3%), and 882 BP (58.5%). The gamma-aminobutyric acid transmembrane transporter activity was the latter group’s most enriched metabolic pathway. A total of 1,387 genes were annotated in the PAMG-PAM4 comparison group, including 416 MF (30%), 149 CC (10.7%), and 912 BP (65.8%). The most enriched metabolic pathways for PAMG-PAM4 were xyloglucan: xyloglucosyl transferase activity and hemicellulose metabolic process. For the PAMG-PAM5 comparison group, 1,477 genes were annotated, including 416 MF (28.2%), 149 CC (10.1%), and 912 BP (61.7%). PAMG-PAM15 most enriched metabolic pathway was the xyloglucan metabolic process. A total of 1,581 genes were annotated in the PAM15-PAM4 comparison group, including 469 MF (29.7%), 160 CC (10.1%), and 952 BP (60.2%). The latter group’s most enriched metabolic pathways were inositol bisdiphosphate tetrakisphosphate diphosphatase activity and photosynthesis.

Fig. 2. GO enrichment of P. americana unigenes. The x-coordinate represents the GO term, and the ordinate is the -log10 (p-value) enriched by the GO Term.

The KEGG pathway analysis identified that a total of 23,224 genes were enriched into 10 KEGG metabolic pathways (Fig. 3 and Table S3). In addition, 1,442 DEGs were involved in metabolism, genetic information processing, environmental information processing, cell processes, and biological system pathways. Among those, the three most significantly enriched KEGG metabolic pathways were starch and sucrose metabolism, glycolysis/gluconeogenesis, and flavonoid biosynthesis. Therefore, it is speculated that drought and low-temperature stress impact P. americana gene expression.

Fig. 3. KEGG enrichment of P. americana unigenes. The x-coordinate is KEGG Term, and the ordinate is -log10 (p-value) enriched by KEGG Term.

Identification of Differentially Expressed Genes

Differential analysis of gene expression using DESeq identified a total of 5,171 differential genes (Table 3), of which 800 were identified for PAMG_vs_PAM4 (280 upregulated, 520 downregulated), 797 for PAMG_vs._PAM5 (203 upregulated, 594 downregulated), 991 for PAM5_vs_PAM4 (714 upregulated, 277 downregulated), 850 for PAMZ_vs_PAM5 (271 upregulated, 579 downregulated), 866 for PAMZ_vs_PAMG (556 upregulated, 310 downregulated) and 867 for PAMZ_vs_PAM4 (458 upregulated, 409 downregulated).

Table 3. The Number of Differentially Expressed Genes (DEGs)

Three treatment groups, PAMZ-vs-PAMG, PAMZ-vs-PAM4 and PAMG-vs-PAM4, were selected to produce a Venn diagram (Fig. 4), which showed that the number of shared unique differential genes between the three treatment groups was 27 (22 upregulated and 5 downregulated), further revealing that drought and low temperature stress affects gene expression in P. americana and regulates it through DEGs.

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Fig. 4. Venn diagrams showing the number of differentially expressed genes (DEGs). (a) Venn diagram showing the upregulated DEGs; (b) Venn diagram showing the downregulated DEGs

Transcription Factors with High Transcript Levels

Under drought and low-temperature stress, 5,290 differentially expressed genes were found in P. americana, belonging to 53 TF families, of which the bHLH family was the largest, with 525 participating genes, followed by NAC and ERF, with 406 and 347 genes involved, respectively (Fig. 5). Moreover, C2H2, MYB, and WRKY family members were also involved in different biological stress responses. Taking the normal treatment as the control, at least 28 TF families were identified to be differentially expressed under drought and low-temperature stress. It was found that 25 DEGs of ERF were upregulated under drought and low-temperature stress, MYB was concentrated under drought stress, and WRKY was concentrated under low-temperature stress.

Analysis of Gene Expression in P. americana under Drought And Low-Temperature Stress

The online software heatmap (V1.0.8) was used to draw gene expression heat maps, and to evaluate the effects of drought and low-temperature stress on preliminary analysis of gene expression of the americana metabolic pathway and transcription factor family (Fig. 6 and Table. S4, Table. S5). The results revealed that three metabolic pathways, starch and sucrose metabolism, sugar degradation and sugar metabolic synthesis and flavonoid biosynthesis were the most significantly enriched under drought and low temperature stress, with a total of 19 genes differentially expressed. The expression of Pa0058, Pa0202 and Pa0748 genes, which were significantly enriched in starch and sucrose metabolism and sugar degradation and sugar metabolism anabolic pathways, were upregulated under drought stress, while Pa0001, Pa0068, Pa0249 and Pa0571 genes were downregulated. The expression of Pa0822 gene, which was significantly enriched in flavonoid biosynthesis, was upregulated and Pa0005, Pa0058 and Pa1187 genes were downregulated under low temperature stress, presumably the flavonoid biosynthesis pathway was the main biosynthetic pathway in avocados under low temperature stress.

Fig. 5. Analysis of differentially expressed transcription factor genes in P. americana under drought and cold stress. (a) Distribution of P. americana transcription factor family. (b) PAMZ_vs_PAMG; (c) PAMZ_vs_PAM4. The x-coordinate is a different transcription factor family, and the ordinate is the number of different genes falling into this transcription factor family.

A total of 28 transcription factors in the transcription factor family were highly expressed under drought and low temperature stress, including the bHLH, NAC, ERF, MYB and WRKY transcription factor families. 40 DEGs encoding highly expressed transcription factors were selected for heat map analysis. The results showed that under drought stress, the expression of eight genes encoding ERF (pa0027, pa0035, pa0039, pa0157, pa0822) and NAC (pa0036, pa0168, pa0553) was upregulated, and the expression of genes encoding ERF (pa0149), NAC (pa0005, pa1027) and WRKY (pa0674) was downregulated. Under low-temperature stress, the expression of 5 DEGs of bHLH (pa0131, pa00204, pa0335), WRKY (pa0537, pa0889), and ERF (pa0822) transcription factors were upregulated, of which the expression of bHLH and WRKY transcription factor family was significantly different, and the expression of 3 DEGs of ERF (pa0027, pa0035) and NAC (pa0014) transcription factors was downregulated.

Fig. 6. (a): Heat map of 19 differentially expressed genes; (b): Heat map of transcription factor family gene expression. Horizontally represent genes, and each column represents a sample. Green and pink color marks indicate up-regulation and down-regulation of gene expression, respectively

Validation of Transcriptome Data by qRT-PCR Analyses

To verify the accuracy of the transcriptome data, nine differential genes were randomly selected for qRT-PCR analysis (Fig. 7). The results showed that the expression of Pa0058, Pa0027, Pa0039 and Pa0168 genes were upregulated, Pa0068 and Pa0005 genes were downregulated under drought stress; the expression of Pa0068, Pa0537 and Pa0005 genes were upregulated; and Pa0149 gene expression was downregulated under low temperature stress. The P. americana data indicates that several key genes related to drought and low temperature stress in P. americana were regulated at the transcriptional level. The gene expression levels obtained by qRT-PCR analysis were generally consistent with the trend of transcriptome expression profiling, indicating that the transcriptome sequencing results were reliable.

When plants are under stress, genes’ expression type and expression level will change significantly at the transcriptional level. The response mechanism of plants to drought and low temperature is a complex process in which multiple genes and metabolic pathways participate in regulation and interact with each other (Hwang et al. 2018). To further understand the gene expression of P. americana under drought and low-temperature stress, this study used Illumina high-throughput sequencing technology to sequence the transcriptome of P. americana leaves with different drought and low-temperature treatments. A total of 24,616 genes were obtained, and 5,171 differential genes were screened, including 2,482 upregulated genes and 2,689 downregulated genes. Based on KEGG pathway analysis, it was shown that starch and sucrose metabolism, flavonoid biosynthesis, and transcription factors are critically involved in the response of P. americana to drought and low temperature. Meanwhile, the expression changes of these genes and their putative functions were analyzed to provide new insights into the molecular mechanisms of P. americana related resistance.

Fig. 7. The expression profiles of nine transcripts in P. americana by qRT-PCR. The x-coordinate is a different treatment group, and the ordinate is DEGs expression changes.

Starch is the main carbohydrate in plants, releasing energy, sugars, and secondary metabolites through its own starch reserves under abiotic forcing (Thalmann and Santelia 2017). When subjected to drought and low-temperature stress, plants receive external stress signals for physiological resistance responses. As drought and low-temperature stress intensify, the production of intracellular reactive oxygen species and extracellular electrolyte leakage increase, causing damage to their biomolecules. Therefore, plants alleviate osmotic stress by increasing the content of small molecules or soluble proteins, while reducing the intensity of respiration, slowing down metabolic activity, increasing photosynthesis, and accumulating sugars, thus reducing the negative effects of stress (Thalmann et al. 2016; Zanella et al. 2016; Li et al. 2017). β-amylase (BAM) is the main enzyme of starch catabolism metabolism and is involved in the response of different plants to a variety of abiotic stresses (Sørensen et al. 2012). For example, the expression of the gene encoding BAM was upregulated under drought stress in Millet (Cao et al. 2022), and the gene encoding BAM, VvBAM1, was significantly upregulated under low-temperature stress in tomato (Liang et al. 2021). In the study of Pyrus bretschneideri, three genes encoding BAM were upregulated in expression under both drought and low temperature stresses (Zhao et al. 2019). In the present study, the gene encoding BAM, Pa0058, was upregulated under drought stress and downregulated under low-temperature stress, in general agreement with the results of the former study under drought stress. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme in the glycolytic metabolic pathway and plays an important role in carbon metabolism, plant development, and stress tolerance (Wei et al. 2022). In the present study, Pa0202, the gene encoding GAPDH, was expressed upregulated under both drought and low-temperature stress, which is similar to the results of upregulated of the expression of genes encoding GAPDH in banana under mild and moderate low-temperature stress (Liu et al. 2020), and upregulated of the expression of most genes encoding GAPDH in Triticum aestivum roots under drought stress (Zeng et al. 2016), suggesting that GAPDH is a key factor in plant response to abiotic stresses.

Flavonoids, as important components of plant secondary metabolites, have important roles in microbial signaling, protection against pathogens and pests, UV protection, growth hormone transport regulation, and pigmentation (Han et al. 2019). The flavonoid biosynthetic pathway is one of the best known secondary metabolic pathways in plants, and many key genes have been studied in response to environmental stresses (Saito et al. 2013). Flavanone 3-hydroxylase (F3H) is a key enzyme in the flavonoids biosynthesis pathway and plays an important role in biotic and abiotic stress (Ma et al. 2014). It was reported that the transcript of F3H was significantly enhanced in Reaumuria soongorica under drought stress (Liu et al. 2013), and in the present study, one gene encoding F3H was also significantly enhanced under drought stress, and the results of both studies were generally consistent. Chalcone synthase (CHS) is the key enzyme in the first committed step of the flavonoid biosynthetic pathway and catalyzes the stepwise condensation of 4-coumaroyl-CoA and malonyl-CoA to naringenin chalcone (Deng et al. 2014). In the study of Hybrid Poplar (P. tremula × P. alba), the expression of PtCHS, a gene encoding CHS, gradually increased with increasing time of drought stress (Ahmed et al. 2021), and in the present study, the expression of two genes encoding CHS was upregulated under drought stress, in agreement with the previous study the results were consistent with the previous study. In the study of tobacco, the gene Nitab4.5_0001066g0070 encoding CHS in cultivars Xiangyan7 was downregulated under low-temperature stress (Hu et al. 2022), and in the present study, the gene encoding CHS was downregulated under low-temperature stress, and both results were consistent. The above results suggest that flavonoids have protective effects against drought and low temperature stresses, and different varieties may have different response mechanisms to different stresses.

Transcription factors play an important regulatory role in plant growth, development, metabolism, and other functions (Fan et al. 2021). They regulate transcription through the expression of other transcription factors, synthesizing the corresponding proteins to guide the generation of metabolites and respond to drought and low-temperature stress. In the present work, 53 transcription factor families were identified related to drought and cold stress response through genome-wide analysis, of which bHLH, NAC, ERF, MYB, and WRKY family members were the most representative. At present, a large number of studies have shown that transcription factor families such as the bZIP, AP2/ERE, WRKY, NAC, and MYB are closely related to the expression and regulation of genes under drought and low-temperature stress (Xu et al. 2011; Wang et al. 2020). Among them, the present study found that the expression of 25 DEGs in the ERF family was significantly altered: five DEGs were upregulated, one DEG was downregulated under drought stress, and two DEGs were downregulated under low-temperature stress. For WRKY members, 13 DEGs were upregulated under low-temperature stress. In a study of Ammopiptanthus nanus, Cao et al. (2020) found that DREB responds to low-temperature and drought stress by binding to WRKY. In a study of Salix matsudana leaves, Xu et al. (2021) found that the expression of five genes encoding WRKY transcription factors was upregulated under drought stress. In a study of tea, Wang et al. (2013) found that the expression of five genes encoding WRKY transcription factors was upregulated in three and downregulated in two under low temperature stress. Chen et al. (2014) found that under low-temperature stress, the expression of DEGs encoding AP2/ERF, WRKY, and NAC transcription factors in Populus euphratica was upregulated, indicating that the three transcription factor families play a key role in the low-temperature response of P. euphratica. The results of the transcription factors in this study were generally consistent with those of previous studies, which strongly suggest that ERF and WRKY transcription factors are associated with drought and low temperature in plants and play important roles in drought and low-temperature stresses.

This study preliminarily explored the key genes and transcription factor families of P. americana-related metabolic pathways under drought and low-temperature stress, laying the foundation for further research on biosynthesis, secondary metabolic pathways, and transcriptome. Moreover, it provides a theoretical basis for breeding P. americana varieties with drought and low-temperature resistance. Further functional characterization of DEGs and their pathways are needed because they potentially serve as targets for marker-assisted selection or transgenes to enhance stress tolerance.

CONCLUSIONS

Abiotic stresses such as drought and low temperature limit the acreage of P. americana in Yunnan, China. In this study, transcriptome analysis of P. americana was performed under drought and low-temperature stress. The result of transcriptome analysis showed that the three most significantly enriched KEGG metabolic pathways responded to drought and low temperature were starch and sucrose metabolism, glycolysis/gluconeogenesis, and flavonoid biosynthesis. The gene expression of beta-amylase, glyceraldehyde-3-phosphate dehydrogenase and hexokinase were upregulated under drought stress, while the gene expression of UDP-glycosyltransferase superfamily protein isoform, glucose-1-phosphate adenylyltransferase and glucose-6-phosphate 1-epimerase were downregulated. The gene expression of beta-amylase and shikimate O-hydroxycinnamoyl transferase was downregulated under low-temperature stress. WRKY, MYB, bHLH, and NAC transcription factors also were involved in responding to drought and low-temperature stress.

ACKNOWLEDGMENTS

This work was supported by National Natural Science Foundation of China (No. 32160736, 31860177); Major Project of Agricultural Basic Research Program in Yunnan Province (202101BD070001-020); General Project of Basic Research Program in Yunnan Province (202101AT070218, 202101AT070044); and the Reserve Talents for Young and Middle-aged Academic and Technical Leaders of Yunnan Province (202205AC160044).

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Article submitted: October 26, 2022; Peer review completed: March 18, 2023; Revised version received and accepted: April 3, 2023; Published: April 20, 2023.

DOI: 10.15376/biores.18.2.3938-3960