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Genome-wide characterization and expression analysis of α-amylase and β-amylase genes underlying drought tolerance in cassava

BMC Genomics volume 24, Article number: 190 (2023) Cite this article

Abstract

Background

Starch hydrolysates are energy sources for plant growth and development, regulate osmotic pressure and transmit signals in response to both biological and abiotic stresses. The α-amylase (AMY) and the β-amylase (BAM) are important enzymes that catalyze the hydrolysis of plant starch. Cassava (Manihot esculenta Crantz) is treated as one of the most drought-tolerant crops. However, the mechanisms of how AMY and BAM respond to drought in cassava are still unknown.

Results

Six MeAMY genes and ten MeBAM genes were identified and characterized in the cassava genome. Both MeAMY and MeBAM gene families contain four genes with alternative splicing. Tandem and fragment replications play important roles in the amplification of MeAMY and MeBAM genes. Both MeBAM5 and MeBAM10 have a BZR1/BES1 domain at the N-terminus, which may have transcription factor functions. The promoter regions of MeAMY and MeBAM genes contain a large number of cis-acting elements related to abiotic stress. MeAMY1, MeAMY2, MeAMY5, and MeBAM3 are proven as critical genes in response to drought stress according to their expression patterns under drought. The starch content, soluble sugar content, and amylase activity were significantly altered in cassava under different levels of drought stress.

Conclusions

These results provide fundamental knowledge for not only further exploring the starch metabolism functions of cassava under drought stress but also offering new perspectives for understanding the mechanism of how cassava survives and develops under drought.

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Background

Starch hydrolysates are energy sources for plant growth and development, which also regulate osmotic pressure and transmit signals in response to biological and abiotic stresses [1, 2]. Amylase is the key enzyme during the starch hydrolysis process and both α-amylase (EC3.2.1.1, AMY) and β-amylase (EC3.2.1.2, BAM) are members of the glycoside hydrolase family. As an endo-amylase, AMY catalyzes the hydrolysis of the 1,4-glycosidic links, breaks the 1,4-glycosidic bonds inside the molecular and thus hydrolyzes amylose, amylopectin, or glycogen. BAM is an exo-amylase that progressively removes maltose or glucose from the non-reducing end by acting on the 1,4-glycosidic bond of amylose, amylopectin, and glycogen. Maltose, glucose, and dextrin are the main hydrolysates [1, 3]. AMY and BAM are classified into the glycoside hydrolase family 13 (GH 13) and GH 14 by Cazy (http://www.cazy.org/), respectively, and GH13 includes several isozymes. Their conserved domains can be queried within the Pfam database (http://pfam.xfam.org/). Based on the AMY and BAM gene family members and major structural features of the model plants (rice and Arabidopsis), the dominant conserved domains of the AMY and BAM gene families are found as Alpha-amylase (PF00128) and Glyco_hydro_14 (PF01373), respectively [4].

As the starch hydrolysates in plants, soluble sugar can alleviate the adverse effects of stress-induced carbon consumption when the synthesis of carbs is hindered [5] and also play significant roles in cell osmotic adjustment, acting like the essential osmotic protection agent and compatible solute [6]. The lack of sugar or energy of rice under anoxic circumstances during the seeding stage increased the expression of the Amy3 subfamily gene [7]. In Arabidopsis leaves, the AtAMY1 (At4g25000) gene was up-regulated under biotic and abiotic stresses and the corresponding AMY showed high heat tolerance [8]. The AtBAM1 mutant bam1 showed higher tolerance to osmotic and drought stress than the control [9, 10]. The expressions of AtBAM1 and AtBAM3 genes increased the maltose content in Arabidopsis leaves, which relieved the heat stress to a certain extent by protecting their photosynthetic electron transport chains [11, 12]. Poor root systems of Arabidopsis double mutants under osmotic stress were found, which AMY3 and BAM1 genes were missing [13]. Under drought stress, soybean leaves contained less starch and more soluble sugars than control, while the expression levels of GmAMY3 and GmBAM1 also altered dramatically [14]. Furthermore, overexpression of VvBAM1 improved tomato cold tolerance by modulating starch hydrolysis and scavenging reactive oxygen species (ROS) in plants [15].

Cassava is widely grown in tropical and subtropical regions because of its starchy root and is treated as one of the most drought-tolerant crops [16, 17]. Although AMY and BAM genes have been identified in other plants, only two genes of the MeAMY and MeBAM families were cloned and characterized from cassava leaves [18]. The biological roles of the MeAMY and MeBAM families in cassava under stress are still unknown. Thus, MeAMY and MeBAM genes were identified in the current study. Characterization, multiple sequence alignment, phylogenetic analysis, conserved structural motif, gene structure analysis, chromosomal distribution, homologs, collinearity analysis, and cis-regulatory element prediction of these two genes were performed. Additionally, the expression patterns of the MeAMY and MeBAM families under drought stress were also analyzed.

Results

Characterization and prediction of the subcellular localization of MeAMY and MeBAM genes

To identify protein-coding genes which contain the specific domain, the whole genome was scanned using BLASTP and HMMER. In total, six MeAMY genes and ten MeBAM genes were identified and renamed. Their characteristics and subcellular localization information were all listed in Table 1. Homology analysis was conducted on 13 transcripts of MeAMY and 20 transcripts of MeBAM, respectively (Fig. S1). The size of MeAMY genes ranged from 1227 ~ 2691 bp and the similarities among sequences ranged from 0.53 to 1.00 (Fig. S1a). The length of MeAMY protein sequence ranged from 408 to 896 aa and their similarities ranged from 0.44 ~ 1.00, while their theoretical pIs ranged from 4.71 to 8.22 (Table 1). The range of molecular mass and aliphatic index were 46.560 ~ 101.705 kDa and 27.07 ~ 45.39, respectively. Proteins were all hydrophilic with a negative GRAVY (− 0.576 ~ − 0.226) and their instability index ranged from 27.07 to 45.39. MeAMY1.1 ~ MeAMY3.5 were unstable with an instability index larger than 40, while the other four sequences were shown as stable proteins.

The size of MeBAM genes ranged from 1227 ~ 2691 bp and their similarities were 0.41 ~ 1.00 (Fig. S1b). The length of MeBAM protein sequence ranged from 429 to 701 aa and their similarities were 0.27 ~ 1.00, while their theoretical pIs ranged from 5.3 to 8.92. The range of molecular mass and aliphatic index were 48.937 ~ 79.132 kDa and 69.6 ~ 84.41, respectively. Proteins were all hydrophilic with a negative GRAVY (− 0.485 ~ − 0.249) and their instability index range was 31.44 ~ 52.03. MeBAM1, MeBAM2, MeBAM5.1 ~ MeBAM5.6, MeBAM8, MeBAM10.1, and MeBAM10.2 were unstable, while the other nine sequences were shown as stable proteins.

The prediction of the subcellular localization of 13 MeAMY and 20 MeBAM genes was conducted using CELLO v.2.5 and the results were listed in Table 1. MeAMY1 and MeAMY2 are located in the cytoplasm, while other MeAMY genes located in the Extracellular. MeBAM1 and MeBAM2 were located in chloroplast, while MeBAM3 and MeBAM9 were found in mitochondria, MeBAM4 ~ MeBAM7 in the cytoplasm, MeBAM8 in the plasma membrane, and MeBAM10 in the nucleus.

Table 1 Characteristics and prediction of subcellular localization of the MeAMY and MeBAM proteins
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Structure and alternative splicing of MeAMY and MeBAM genes with analyses of conserved domains and motifs of their proteins

Comparing the domains of the AtAMY1 and AtBAM1 proteins, similar conserved areas of the MeAMY and MeBAM protein sequences were identified. Two carbohydrate binding sites and three catalytic residues were found in MeAMY family (Fig. S2a) and these amino acid sites were perfectly conserved. Additionally, the MeBAM family has two catalytic residues Glu-186 and Glu-380. The amino acid at Glu-186 was highly conserved, while Glu-380 showed poor conservation of amino acids. A mutation at Glu-380 occurred in five different sequences and it was changed to glutamine (Gln), arginine (Arg), and valine (Val) in MeBAM3, in MeBAM8, and in MeBAM10.1 ~ 10.3, respectively (Fig. S2b). The MeBAM contained 20 carbohydrate binding sites and two of them had highly conserved amino acids, while 18 carbohydrate binding sites had amino acid mutations. Both the amino acids in the flexible loop and in the inner loop showed poor conservation with a large number of mutations.

To better understand the structural evolution of MeAMY and MeBAM, the unrooted evolutionary trees were constructed based on their protein sequences. For MeAMY, 13 MeAMY were classified into three clusters I ~ III and a total of 15 conserved motifs of MeAMY were identified using MEME software (Fig. 1a). The cluster I contains five MeAMY genes which are alternative splicing of one gene and they shared the same conserved motifs (Fig. 1b). The four genes in Cluster II shared the same conserved motifs. MeAMY1.1 and MeAMY2.1 belonged to alternate donor site (ADS) and all had a CDS sequence of 252 bp longer than MeAMY1.2 and MeAMY2.2, respectively. Motif 6, 7, 12, 13, and 15 were only observed in Cluster II. Cluster III contains MeAMY4, MeAMY5, MeAMY6.1, and MeAM6.2. MeAMY6.1 has the largest number of conservative motifs and motif 3, 9, and 10 were missing in the repeat, which indicated that MeAMY6.1 is repeat motifs. In addition, the 5’ and 3’ ends of MeAMY4 have the longest UTR in cluster III (Fig. 1a and b).

The MeBAM was divided into five clusters (Fig. 1c). The distribution of conserved motifs at the 5’ end of MeBAM in cluster I was inconsistent. The positions and arrangements of their CDS and UTR were also different (Fig. 1d). In cluster II, MeBAM6.1 and MeBAM6.2 were alternative splicing of the same gene and treated as alternate promoters (AP). The MeBAM10.1 ~ MeBAM10.3 genes in cluster III shared same conserved motifs and belonged to alternate terminators (AT). Their UTR and CDS sequences of the 3’ end were different. In cluster IV, MeBAM4.1, MeBAM4.2, and MeBAM4.3 can be treated as AP. The six genes in cluster V shared same conserved motifs and CDS.

Fig. 1
figure 1

Bioinformatics analyzes of MeAMY and MeBAM gene families. a: Evolutionary tree, conserved motifs, and domains of MeAMY families. b: Gene structure of MeAMY genes family. c: Evolutionary tree, conserved motifs, and domain of MeBAM families. d: Gene structure of MeBAM genes family. The black, red, and blue boxes in a and c represent the conserved domains

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Chromosomal location and collinearity analysis of MeAMY and MeBAM

The MeAMY and MeBAM genes were found in eight chromosomes according to the Generic Feature Forma file of the cassava genome (Fig. S3). For MeAMY, only MeAMY2 was located in the ‘+’ strand, while others were located in the ‘−’ strand. MeBAM1, MeBAM4, MeBAM5, MeBAM7, and MeBAM10 were located in the ‘+’ strand, while the remaining MeBAM genes were located in the ‘−’ strand.

In order to reveal the expansion and evolution mechanisms of MeAMY and MeBAM gene families, the collinearities of coding genes and the potential gene duplication events in cassava genome were analyzed. Combined with the Arabidopsis genome and the rubber tree (Hevea brasiliensis) genome, the collinearities of coding genes among different species were also analyzed (Fig. 2, Table S1). There were linear relationships between MeAMY1 and MeAMY2, MeBAM4 and MeBAM5, and MeBAM2 and MeBAM7, respectively. MeBAM4 and MeBAM5 were tandem repeat genes. The Ka/Ks values of the flanking homologous genes of those three pair genes with linear relationships of the MeAMY and MeBAM families ranged from 0.181848632 to 0.30120533, suggesting that their divergence was driven by purifying selection (Table 2; Fig. 2).

Furthermore, there were eight pairs of genes with collinearity between the cassava genome and the Arabidopsis genome, including three pairs of AMY genes and five pairs of BAM genes. There were 23 pairs of genes, 10 AMY genes and 13 BAM genes, in collinearity between the cassava genome and the rubber tree genome.

Fig. 2
figure 2

The collinear relationships of AMY and BAM genes in Manihot esculenta, Arabidopsis thaliana and Hevea brasiliensis

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Table 2 Homologous gene Ka/Ks values of MeAMY and MeBAM gene families
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Phylogenetic analysis of MeAMY and MeBAM proteins

The AMY and BAM protein sequences of other species were downloaded from NCBI database and nine rice BAM protein sequences were downloaded from Rice Genome Annotation Project (Table S2). A neighbor-joining tree was constructed based on the alignments of proteins of AMY and BAM families from cassava, rice, Arabidopsis, and other species. In total, 54 AMY proteins from 12 species were used to construct the phylogenetic tree and classified into eight groups, including monocotyledonous plants (Group A ~ C) and dicotyledonous plants (Group D ~ H) (Fig. 3a). The cassava AMYs were classified into Group A, B, and C and closely related with rubber trees and castors (Ricinus communis), which were all Euphorbiaceae plants.

The BAM protein sequences were divided into nine groups. Each group contains both the BAM protein sequences of monocots and dicots (Fig. 3b). The BAM protein sequences of Arabidopsis, rice, and cassava were evenly distributed in all groups, while the BAM protein sequences of rubber trees, castors, and Jatropha (Jatropha curcas) were close to MeBAM among all groups.

Fig. 3
figure 3

The phylogenetic tree of AMY (a) and BAM (b) proteins in Manihot esculenta and other species

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Cis-acting elements in the promoter regions of MeAMY and MeBAM genes

In order to understand how the MeAMY and MeBAM gene families respond to stress, the cis-acting elements in their promoter regions were also analyzed (Fig. S4). The core promoter elements CAAT-box, TATA-box, A-box, and AT ~ TATA-box were first removed in the promoter regions of MeAMY and MeBAM genes and cis-acting elements were then classified according to Yue method [1]. The number of cis-acting elements responding to abiotic stress is the largest among all the cis-acting elements, such as MYB, TGACG-motif, ERE, MYC, as-1, and ARE. In the promoter region of MeAMY gene, the number of MYB elements was the largest and 76.9% of the sequences contained 2 ~ 3 CGTCA-motifs (Fig. 4a). There were nine MYB elements enriched in the promoter region of MeAMY6.1. There were seven ERE elements enriched in the promoter regions of both MeAMY1.1 and MeAMY1.2, while six MYC elements were enriched in MeAMY4 (Fig. 4a). For MeBAM, 80% of all the sequences contained WUN-motifs and there were five WUN-motifs enriched in the promoter region of MeBAM4.3 (Fig. 4b). There were 14 ABRE elements and 13 ABRE elements enriched in MeBAM2 and in MeBAM7, respectively. There seven MYB and six MYC elements enriched in MeBAM5.1 ~ MeBAM5.6. ERE elements were enriched in the promoter regions of MeBAM1, MeBAM2, MeBAM4.1, MeBAM4.2, MeBAM4.3, MeBAM6.1, and MeBAM6.2 genes.

Furthermore, the promoter regions of MeAMY and MeBAM genes also contained a large number of cis-acting elements related to light response, such as Box 4 and G-box (Fig. S4). Box 4 was the most abundant element, containing 50 elements in the promoter region of MeAMY gene. And 61.52% of the sequences contained this element, which enriched in MeAMY1.1, MeAMY1.2, MeAMY2.1, MeAMY2.2, and MeAMY5. And 90% of MeBAM genes contained 91 Box 4 elements, which were mainly enriched in MeBAM1, MeBAM2, MeBAM4.1, MeBAM4.2, and MeBAM4.3. In addition, there were 92.3% of MeAMY promoter region sequences included G-box elements, while 80% of MeBAM sequences contained G-box elements, which are mainly enriched in MeBAM2 and MeBAM7.

Fig. 4
figure 4

Distribution of cis-acting elements of MeAMY (a) and MeBAM (b) genes. Number in color blocks represents the number of cis-acting elements

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Gene ontology classification of MeAMY and MeBAM proteins

GO annotation of MeAMY and MeBAM proteins were separated into two categories: biological processes and molecular functions (Fig. 5a). MeAMY proteins were involved in both single-organism and cellular processes, while only MeAMY6 was involved in biological regulation. MeAMY and MeBAM proteins were further classified and then annotated to 111 GO IDs (Table S3). The molecular functions of MeAMY and MeBAM primarily included amylase activity, hydrolase activity, and catalytic activity (Fig. 5b). All MeAMY and MeBAM proteins were involved in carbohydrate metabolic process, polysaccharide catabolic process, polysaccharide metabolic process, macromolecule catabolic process, and organic substance catabolic process (Fig. 5c).

Fig. 5
figure 5

Gene ontology annotations of MeAMY and MeBAM proteins. a: overview of the GO annotations. b: molecular function of the top 20 GO terms. c: biological process of the top 20 GO terms

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Expression analysis of MeAMY and MeBAM

The expression patterns of MeAMY and MeBAM were analyzed using RNA-seq data from various cassava tissues under well-watering conditions (Fig. 6a). MeAMY2, MeAMY3, and MeBAM3 showed high expression levels in all studied tissues. MeBAM2, MeBAM6, and MeBAM9 had the highest expression levels in 100-day-old stems among all collected samples, while both MeBAM2 and MeBAM7 displayed higher expression levels in leaves and MeAMY6 was highly expressed in old roots. Under drought stress, the expression of MeAMY1, MeAMY4, MeAMY6, MeBAM2, MeBAM3, MeBAM5, MeBAM7, MeBAM8, and MeBAM10 genes in cassava leaves were up-regulated, while the expression of MeAMY3 and MeBAM9 genes were down-regulated. The expression levels of MeAMY2, MeBAM2, MeBAM3, MeBAM6, and MeBAM7 under the five days drought treatment were higher than the control and the ten days drought treatment (Fig. 6b).

Fig. 6
figure 6

Expression profiles of MeAMY and MeBAM genes of cassava. a: samples collected under well watering conditions. b: cassava leaves under drought stress. Color scale represents transcripts per million reads (TPM) normalized log2(TPM + 1), where blue indicates low expression and red indicates high expression

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qRT-PCR was used to analyze the expression of MeAMY and MeBAM genes in cassava during the seedling stage without drought stress (CK) and with drought stress. All of the genes were detected in all samples from CK (Fig. S5). MeAMY5 and MeBAM6 showed low expression levels in all tissues, while MeBAM3 showed higher expressions. MeBAM2 showed high expression in both leaves and stems, while MeBAM7 showed high expression only in stems and MeBAM9 was mainly expressed in leaves.

Compared to CK, the expression levels of MeAMY1, MeAMY2, MeAMY4, MeBAM1, MeBAM3, MeBAM5, MeBAM6, MeBAM8, and MeBAM10 genes in leaves were up-regulated in the moderate drought (MD) treatment, whereas MeAMY3, MeAMY5, and MeBAM9 genes showed down-regulated (Fig. 7a). MeAMY5 gene was highly expressed in stem and root under moderate drought (Fig. 7b and c). MeAMY1, MeAMY2, and MeBAM3 genes showed higher expression levels in leaves of the severe drought treatment (HD) than in the MD treatment, while the expression levels of MeAMY4, MeBAM1, MeBAM5, MeBAM6, MeBAM8, and MeBAM10 genes decreased under HD treatment. Expression levels of MeAMY1, MeAMY2, MeAMY4, MeAMY5, MeAMY6, MeBAM3, and MeBAM8 in stems increased with drought stress (Fig. 7b). MeAMY3, MeAMY4, MeAMY5, MeAMY6, MeBAM1, MeBAM2, MeBAM3, MeBAM4, MeBAM5, MeBAM8, and MeBAM9 genes were highly up-regulated in root under the MD treatment, but down-regulated under the HD treatment (Fig. 7c).

Fig. 7
figure 7

Expression patterns of MeAMY and MeBAM genes of cassava under drought stress. a: expression patterns in leaves; b: expression patterns in stems; c: expression patterns in roots. Expression levels were determined using qRT-PCR and calculated using the 2−ΔΔCt method under the control of the actin housekeeping gene. Different letters indicate significant differences among different treatments of cassava (P < 0.05). CK, well watering; MD, the moderate drought; HD, the severe drought treatment

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Variation of carbohydrate contents and amylase activity in cassava

The soluble sugar content and the amylase activity in cassava leaves under HD treatment were higher than MD and CK, while its starch content was lower than CK and close to MD (Fig. 8a). Carbohydrates and amylase activity in stems varied similarly with leaves (Fig. 8b). In roots, the soluble sugar content of HD treatment was higher than MD and CK, while its starch content was lower than CK and slightly higher than MD (Fig. 8c). In contrast, the amylase activity of HD treatment was lower than MD and CK.

Fig. 8
figure 8

Soluble sugar and starch contents and the amylase activities of cassava under different water treatments. Amylase represents the total enzymatic activity of α-amylase and β-amylase. DW, dried weight; FW, fresh weight. CK, well watering; MD, the moderate drought; HD, the severe drought treatment

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Discussion

Previous studies on the AMY and BAM genes in quinoa, apple, barley, tea plants, and other plants revealed that they belonged to the multigene families [1, 19,20,21]. In the current study, six MeAMY genes and ten MeBAM genes were identified in the cassava genome. The composition of conserved motifs in MeAMY and MeBAM protein sequences were different. Some motifs only existed in a small number of gene family members causing differences among them and these differences related to the functional differentiation of MeAMY and MeBAM families during evolution. Additionally, alternative splicing was another crucial reason for the polymorphism of protein structure and function and transcripts of MeAMY and MeBAM. Both MeAMY and MeBAM gene families contain four genes with alternative splicing, MeAMY1 and MeAMY2 were on the same branch of the evolutionary tree (Fig. 1a) and their alternative splicing methods were also the same, ending up with similar transcripts. Their CDS are arranged similarly, located in the cytoplasm, and exhibit collinearity (Table 2), which indicates that they might have similar functions. In addition, MeAMY3 in the MeAMY gene family produced five transcripts (Fig. 1a) and differences of these five transcripts are mainly in the 5’ UTR, leading to different expression patterns of these genes [22]. MeAMY6 included two transcripts, which shared a high sequence similarity (0.99). MeAMY6.2 shares the same protein sequence as the former part (1 ~ 430 aa) of MeAMY6.1 and they both have a complete Alpha-amylase domain. GO annotation also proved that the MeAMY6 had translation regulation activity (Fig. 5a). In the MeBAM gene family, the MeBAM10 gene formed into three transcripts and the last exon varied, which has the most obvious effect on the protein instability index (Table 1). The 3’ UTR of those three transcripts also varied, which control mRNA expression and translation, regulate mRNA stability through the rich AU element, and regulate protein-protein interactions [23].

The collinearity analysis results indicated that purification selection occurred of MeAMY and MeBAM and their protein sequence stabilities were somewhat preserved (Table 2) [24]. MeBAM4 and MeBAM5 were tandem replications (Fig. S3) according to the definition of tandem replication events by Recep Vatansever [25]. The amino acids of the five carbohydrate binding sites in these two gene sequences varied, which may lead to changes in their abilities to bind the substrate, ending up with different catalytic efficiencies of the substrate (Fig. S2). MeAMY1 and MeAMY2, MeBAM2 and MeBAM7 were fragment replication (Fig. 2). There was no amino acid mutation and their carbohydrate binding sites and catalytic residues were exceedingly conservative.

The phylogenetic tree of the AMY proteins was divided into eight groups, including three dicotyledonous-plant groups and five monocotyledonous-plant groups (Fig. 3a), which is consistent with previous studies [1]. All the cassava AMYs were also classified into Group A, B, and C and the same as rubber trees and castors (Ricinus communis), demonstrating closer evolutionary relationships of the AMYs among Euphorbiaceae plants than other families (Fig. 3a). The AMY proteins of cassava and Arabidopsis in Groups A and B had collinearity (Fig. 2), sharing the same carbohydrate binding sites and catalytic residues (Fig. S2a). MeAMY3 and AtAMY2 were clustered into Group A, while MeAMY1, MeAMY2, and AtAMY3 were in Group B. MeAMY4, MeAMY5, and MeAMY6 were all found on the same branch as AtAMY1. Both AtAMY1 (AEE84990.1) and AtAMY2 (AEE35800.1) were secretory proteins and AtAMY3(AEE34983.1) was a special redox-regulated chloroplast α-amylase. Starch degradation could still be observed in the single, double, and triple knock-out mutant Arabidopsis leaves of these three genes, which indicated that α-amylase was not necessary during the transient starch decomposition process in Arabidopsis leaves [26]. Furthermore, AtAMY3 on Group B could act on the starch granule surface to release oligosaccharides when other starch-degrading enzymes were not present [26, 27]. AMY3 was also found participated in the plant response to stress, particularly osmotic stress by mediating starch degradation and controlling the light-induced stomatal opening in guard cells [13].

BAM proteins from cassava, rubber, castor, and jatropha in the Euphorbiaceae have tight evolutionary connections (Fig. 3b). MeBAM2, MeBAM7, and AtBAM1 were found in Group 7, while MeBAM4 and AtBAM2 were classified into Group 2. MeBAM9 and AtBAM3 were classified into Group 6. AtBAM1 was found to be highly expressed in guard cells of leaves and the activity of the β-amylase encoded by AtBAM1 was only active when high temperature, drought, and osmotic stress occurred [10]. Expression profiles of MeBAM2 of cassava leaves were also found up-regulated under drought stress (Figs. 6b and 7a), which suggested that it can be one of the candidates genes response to drought. During the night, AtBAM3 was primarily in charge of starch breakdown within mesophyll cells [28]. Due to glutamic acid was changed to arginine at the catalytic active site Glu-380, the β-amylase encoded by AtBAM4 lacked catalytic activity [29]. MeBAM8 shared the same catalytic active site as AtBAM4. Thus, the β-amylase encoded by MeBAM8 may also lack catalytic activity (Fig. S2b). Both MeBAM5 and MeBAM10 had a BZR1/BES1 domain at their N-termini as AtBAM7 and AtBAM8, which had been proved that played essential roles in bud growth and development by acting as transcription factors [30, 31]. However, the GO annotations did not adequately define transcription factor functions of MeBAM5 and MeBAM10. In addition, MeBAM1 was located in Group 5 with no Arabidopsis BAM (Fig. 3b) and Matthias Thalmann [32] suggested that this branch was formed before the seed plants radiation.

Under stress conditions, transcription factors combined with the cis-acting elements in the promoter region of stress-responsive genes and then promoted the transcription and expression of these genes, thus making a regulatory response [33]. In the current study, the MeAMY and MeBAM gene families included a range of stress response cis-acting elements and the largest number cis-acting elements were MYB-related elements, which is consistent with previous report [1]. MYB transcription factors are crucial for secondary metabolism, cell differentiation, stress response, and plant growth and development [34]. Under drought stress, most of MeAMY and MeBAM genes showed up-regulated expressions in cassava leaves (Fig. 6b), while less genes expressed up-regulated in the stems and roots (Fig. 7b and c). The majority of the MeAMY and MeBAM genes in cassava roots showed the highest expression level under moderate drought among three water treatments. The MeAMY5 gene was highly expressed in stem and root under moderate drought. MeAMY2 gene expression was stably up-regulated in roots, stems, and leaves (Fig. 7), while MeAMY1 and MeBAM3 gene expression was stably up-regulated in stems and leaves. Thus, these genes can be treated as critical genes in response to drought tolerance in cassava.

Metabolites of carbohydrates in plant leaves release soluble sugar, which can act as osmotic protecting agents, thus maintaining the water balance in cells enables plants to adapt to stress [13, 35,36,37]. During drought stress, the total enzyme activity of α-amylase and β-amylase increased in cassava leaves and stems, leading to higher soluble sugar and lower starch contents of cassava roots, stems, and leaves than those of the CK (Fig. 8), which is consistent with previous researches [13, 14, 28, 38, 39]. However, the activity of amylase in cassava roots under drought were decreased with the stress level (Fig. 8c) and similar phenomena were also found in tobacco (Nicotiana tabacum) and rape (Brassica napus) [35, 40]. These can be explained by previous findings. Starch hydrolysis process in roots might involve a number of synergistic amylases reactions [38, 41], leading to high maltose and soluble sugars contents than stem and leaf. And the high content of maltose inhibited the total enzyme activity of α-amylase and β-amylase [39]. In addition, it may also be a self-protection mechanism formed during the evolution of cassava. As the main energy storage organ of cassava, the starch preservation in the root during stress can provide energy for the rapid recovery of plant shoots after the stress is lifted.

Conclusion

Ten MeBAM genes and six MeAMY genes were identified and characterized in the cassava genome. Both MeAMY and MeBAM gene families contain four genes with alternative splicing. According to domain analysis, MeBAM5 and MeBAM10 proteins may function as transcription factors. The promoter regions of MeAMY and MeBAM genes contain a large number of cis-acting elements related to abiotic stress. MeAMY1, MeAMY2, MeAMY5, and MeBAM3 are critical genes in response to drought stress. These results provide fundamental knowledge for not only further exploring the starch metabolism functions of cassava under drought stress but also offering new perspectives for understanding the mechanism of how cassava survives and develops under drought.

Methods

Data resource for AMY and BAM gene families in cassava and other species

Cassava genome data were downloaded from Ensembl Plants Database (Manihot_esculenta_v6, http://plants.ensembl.org/Manihot_esculenta/Info/Index). The protein sequences of AMY and BAM genes of Arabidopsis thaliana and Oryza sativa were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/) and Rice Genome Annotation Project (http://rice.uga.edu/index.shtml)(Table S2), respectively. The AMY and BAM sequences in the cassava database were characterized using the AMY and BAM proteins from Arabidopsis and rice as queries by TBtools-BLAST [42]. The local Hidden Markov Models of all the known AMY and BAM proteins of Arabidopsis and rice were constructed by matching their protein sequence dataset in HMMER (http://hmmer.org/). These models were employed to identify the candidate AMY and BAM proteins in cassava. And protein sequences with consistency ≥ 60% and E value ≤ 1e-5 were selected. The Pfam database (https://pfam.xfam.org/) and SMART (http://smart.embl-heidelberg.de/) database were then applied for searching the complete conserved domains of these selected sequences [43, 44]. The target MeAMY and MeBAM genes were identified by BLAST analyses on all candidate genes. The cassava protein sequences containing the Alpha-amylase domain (PF00128) and the Glyco_hydro_14 domain (PF01373) were treated as the MeAMY gene family sequences and the MeBAM gene family sequences, respectively.

Characterization and subcellular localization prediction of MeAMY and MeBAM protein sequences

The relative molecular mass, instability index, grand average of hydropathicity (GRAVY), and isoelectric point (pI) of the identified MeAMY and MeBAM were predicted by the ExPASy-ProtParam platform (https://www.expasy.org/resources/protparam). Motifs of MeAMY and MeBAM were analyzed with MEME software (http://meme-suite.org/tools/meme) [45]. The optimum width of the motifs was set as 10 ~ 100, the number of motifs was set as 15, and the other parameters were used as default settings. The gene structure features of MeAMY and MeBAM were obtained based on the cassava genome GFF file. Similarity and composition analysis were done using software DNAMAN and BioEdit, respectively. Subcellular localizations of MeAMY and MeBAM were predicted by CELLO v. 2.5 (http://cello.life.nctu.edu.tw/) [46].

Sequence alignment, phylogenetic analysis, and classification of MeAMY and MeBAM protein sequences

Multiple sequence alignment of MeAMY and MeBAM sequences was performed using DNAMAN and the alignment sequences were displayed using jalview (http://www.Jalview.org/). Un-rooted trees were constructed using the maximum likelihood method within MEGA-X software. The phylogenetic trees of AMY and BAM protein sequences of cassava and other species were built using the Neighbor-joining method, where the bootstrap was set to 1000 replicates.

Chromosomal localization and collinearity analysis of MeAMY and MeBAM

The genome-wide protein database of cassava, Arabidopsis, and rubber trees was constructed by TBtools software. MCScanX (https://github.com/wyp1125/MCScanx) was used to analyze gene duplication and collinearity [47]. The collinearity map and chromosome mapping of homologous genes were plotted on chromosomes. The ratio of homologous genes Ka (nonsynonymous substitution rate) and Ks (synonymous substitution rate) was also calculated.

Cis-regulatory elements analysis of MeAMY and MeBAM genes

The upstream regions comprising 2000 bp of translation start sites (initiation codon) of MeAMY and MeBAM sequences were downloaded as the promoter sequences from the Cassava Genome. The PlantCARE and New PLACE databases were used to predict the conserved cis-elements in these promoter sequences [48, 49].

Gene ontology classification and expression analysis of MeAMY and MeBAM genes based on RNA-seq data

Gene ontology annotations of MeAMY and MeBAM genes were performed within the omicshare platform (https://www.omicshare.com/tools). The expression patterns of MeAMY and MeBAM genes were analyzed based on the raw RNA sequencing data, including different tissue types (study accession: SRP228273, SRP076160, and SRP354996, Table S4) [50], which was obtained from NCBI Sequencing Read Archive (https://www.ncbi.nlm.nih.gov/sra/). The RNA-seq data was first standardized in the form of TPM (transcripts per million reads) and then the log2(TPM + 1) conversion was performed before visualization. A heat map was created using ggplot2 in R 4.2.1.

Plant materials and drought treatments

Cassava variety South China 205 (SC205) was used as plant material, which is the most popular cultivated in China because of its stable root yields and provided by Guangxi South Subtropical Agricultural Sciences Research Institute. The study was conducted at Guangxi University in 2021 and cassava was planted in a cuboid box (40 × 40 × 50 cm) with soil in October. Two cassava seedlings were planted in each box with normal watering and well management. The water treatment was set as three levels: 70 ~ 80% of soil water content as control (CK), 45 ~ 55% as moderate drought (MD), and 20 ~ 30% as severe drought (HD). And drought treatments were operated on 60 days after planting and lasted for one week. Each treatment contained six boxes and 12 shoots of cassava. A soil moisture recorder (LUGE-L99-TWS-1) was used to monitor the moisture content in the soil during the drought treatment period. After that, cassava stems (0 ~ 10 cm from the top of the stem), leaves, and roots samples were collected. Each sample was separated into two parts. One part was frozen in liquid nitrogen and stored at -80 °C for molecular and physiological analysis. The other part was oven-dried and ground for further determinations of the starch and sugar contents.

Determination of contents of soluble sugar and starch and activities of amylase of cassava tissues

The protocol for analyzing the contents of soluble sugar and starch in root, stem, and leaf of cassava was described by Loewus [51]. Determination of activities of amylase in plant samples using the 3,5-dinitrosalicylic acid (DNS) colorimetric method and details were well described by Bernfeld [52].

RNA extraction and qRT-PCR assays

Total RNA was extracted using the FastPure® Plant Total RNA Isolation Kit (Vazyme, China) and operated according to the manufacturer’s instructions. The first-strand cDNA was synthesized using a HiScript® II Q Select RT Super Mix for qPCR + gDNA wiper Kit (Vazyme). The housekeeping gene of cassava actin was used as an internal control to normalize the data. The primers were designed using the NCBI-Primer-BLAST tool (Table S5) and the range of the PCR products was set as 80 ~ 200 bp. qRT-PCR was carried out using ChamQ Universal SYBR qPCR Master Mix (Vazyme) in a qTOWER2.2 real-time PCR system (Analytik Jena, Germany) in accordance with the manufacturer’s protocol. The total volume of 10 µL qRT-PCR reaction was used, including 5 µL ChamQ Universal SYBR qPCR Master Mix, 0.4 µL of forward primer (10 µM), 0.4 µL of reverse primer (10 µM), 1 µL of cDNA, and 3.2 µL of ddH2O. The cycling conditions were 95 ℃ for 30 S, followed by 42 cycles at 95 ℃ for 10 S and 60 ℃ for 30 S. Melting curve analysis was then performed ranging from 60 to 95 ℃. The relative expression levels in CK and drought treatments were calculated with the formulas 2−ΔCt and 2−ΔΔCt [53], respectively. Three independent biological replicates were applied for all treatments. Statistical analysis was calculated using IBM SPSS 25.0.

Data Availability

All data used in this study are publicly available and included in supplementary information files. Manihot esculenta Crantz genome sequences were obtained from the Ensembl Plants Database (Manihot_esculenta_v6, http://plants.ensembl.org/Manihot_esculenta/Info/Index), while transcriptomic sequencing data were downloaded from NCBI BioProject PRJNA578024, PRJNA324539, PRJNA796531(https://www.ncbi.nlm.nih.gov/bioproject/). Genomic data for Hevea brasiliensis (ASM165405v1) and Arabidopsis thaliana (TAIR10.1) were obtained from NCBI (https://www.ncbi.nlm.nih.gov/genome/).

Abbreviations

AMY:

α-amylases

BAM:

β-amylases

Glyco_hydro_14:

Glycoside hydrolase family 14

GFF:

Generic Feature Forma

ABA:

Abscisic acid

ROS:

reactive oxygen species

ADS:

Alternate donor site

AP:

Alternate promoter

AT:

Alternate terminator

GRAVY:

Grand averages of hydropathicity

pI:

Isoelectric points

qRT-PCR:

Quantitative real-time polymerase chain reaction

Ka:

nonsynonymous substitution rate

Ks:

synonymous substitution rate

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Acknowledgements

The authors would like to thank Jie Luo, College of Agronomy, Guangxi University provided technical support for qRT-PCR experiments.

Funding

Financial supports were provided by the Guangxi Science and Technology Planning Project (AD20297144).

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Authors and Affiliations

  1. College of Agronomy, Guangxi University, Nanning, 530004, China

    Taiyi Yang, Liangwu Li, Yuanhang Huang & Maogui Wei

  2. Guangxi South Subtropical Agricultural Sciences Research Institute, Chongzuo, 532406, China

    Hengrui Li & Wanling Wei

  3. Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, 530007, China

    Faqian Xiong

  4. State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, 530004, China

    Maogui Wei

  5. Guangxi Key Laboratory of Agro-environment and Agro-products Safety, Guangxi University, Nanning, 530004, Guangxi, China

    Maogui Wei

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Contributions

Maogui Wei, as the corresponding author designed the experiment and revised the manuscript. Taiyi Yang carried out the bioinformatic analysis, experimental treatments, qRT-PCR analysis, and wrote the manuscript. Wanling Wei provides experimental materials. Hengrui Li, Liangwu Li and Yuanhang Huang participated in the experimental treatment. Faqian Xiong designed the experiment and valuable comments for experiments. All authors read and approved the final manuscript.

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Correspondence to Maogui Wei.

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Ethics approval and consent to participate

Cassava stakes (Manihot esculenta Crantz ‘South China 205’) used in this study were not endangered or protected species and provided free of charge from Guangxi South Subtropical Agricultural Sciences Research Institute (Chongzuo 532406, China). The study was conducted with plant material, which complies with relevant institutional, national, and international guidelines and legislation.

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Additional file 1: Table S1.

Collinear gene pairs of AMY and BAM encoding genes among Manihot esculenta, Arabidopsis thaliana, and Hevea brasiliensis.

Additional file 2: Table S2.

Accession numbers for AMY and BAM protein sequences in different plants.

Additional file 3: Table S3.

GO annotation results of MeAMY and MeBAM genes.

Additional file 4: Table S4.

RNA-seq data information of cassava.

Additional file 5: Table S5.

Primers for qRT-PCR.

Additional file 6: Fig. S1.

Similarities of MeAMY (a) and MeBAM (b) gene family sequences.

Additional file 7: Fig. S2.

Alignment analysis of MeAMY and MeBAM protein sequences.

Additional file 8: Fig. S3.

Chromosomal distribution of MeAMY and MeBAM genes.

Additional file 9: Fig. S4.

Predicted cis-elements in the promoter regions of MeAMY (a) and MeBAM (b) genes.

Additional file 10: Fig. S5.

Expression patterns of MeAMY and MeBAM genes in different parts of cassava under well watering condition.

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Yang, T., Li, H., Li, L. et al. Genome-wide characterization and expression analysis of α-amylase and β-amylase genes underlying drought tolerance in cassava. BMC Genomics 24, 190 (2023). https://doi.org/10.1186/s12864-023-09282-9

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