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

doi:10.21203/rs.3.rs-2123601/v1

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Research Article

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

https://doi.org/10.21203/rs.3.rs-2123601/v1

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published 06 Apr, 2023

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Background

Starch hydrolysates are energy sources for plant growth and development, regulate osmotic pressure and transmit signals in response to biological and abiotic stresses. α-amylase (AMY) and 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, mechanisms how AMY and BAM response 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 an important role 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, and MeAMY1, MeAMY2 and MeBAM3 are proved as key genes in response to drought stress according to their expression patterns under drought. The starch content, soluble sugar content, and amylase activity in cassava significantly altered under different levels of drought stress.

Conclusions

These results could provide fundamental knowledge for further investigation of the function of starch metabolism under drought stress in cassava, offering new perspectives for understanding the underlying mechanism for its ability to survive and produce under drought.

cassava

amylase

abiotic stress

bio-informatics

gene expression

Starch hydrolysates are energy sources for plant growth and development, and they can 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. Both α-amylase (EC3.2.1.1, AMY) and β-amylase (EC3.2.1.2, BAM) are members of the glycoside hydrolase family. As endo-amylase, AMY operates on the 1,4-glycosidic links and breaks the 1,4-glycosidic bonds from the molecular inside to hydrolyze 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/). AMY gene family contains a major Alpha-amylase (PF00128) and BAM’s conserved domain is Glyco_hydro_14 (PF01373) [4].

Starch hydrolysis in plants produce soluble sugars and can alleviate the adverse effects of stress-induced carbon consumption when the synthesis of carbs is hindered [5]. Soluble sugar include glucose, fructose, sucrose, trehalose, maltose, etc, plays a significant role in cell osmotic adjustment acting like an essential osmotic protection agent and compatible solute [6]. A lack of sugar or energy of rice at seeding stage under anoxic circumstances might increase the expression of the RAmy3 subfamily gene [7]. In Arabidopsis leaves, the AtAMY1(At4g25000) gene was triggered by biotic and abiotic stresses and AtAMY1 gene expression was up-regulated, and the corresponding AMY showed high heat tolerance [8]. Arabidopsis AtBAM1 mutant bam1 showed higher tolerance to osmotic and drought stress than control [9, 10]. Arabidopsis AtBAM1 and AtBAM3 gene expression can increase the maltose content in leaves in response to heat stress by protecting the photosynthetic electron transport chain[11, 12]. The AMY3 and BAM1 genes are missing in Arabidopsis double mutants, which show poor root development and vulnerable to osmotic stress. Abscisic acid (ABA) in these mutants contributes in the stress-induced breakdown of starch and regulates the activity of BAM1 and AMY3 during the osmotic stress [13]. Under drought stress, soybean leaves contained less starch and more soluble sugar, and that the expression levels of the genes GmAMY3 and GmBAM1 also altered dramatically [14]. Recent research has shown that VvBAM1 improves plant cold tolerance by controlling starch hydrolysis, promoting reactive oxygen species (ROS), and scavenging in VvBAM1 over-expression transgenic tomato plants [15].

Cassava is widely grown in tropical and subtropical regions because of its starchy root [16, 17], and considered as one of the most drought-tolerant crops. During the whole growth period, cassava is susceptible to a number of stresses and drought is the main factor, which seriously affects the yield of tuberous roots [18, 19]. Its roots have strong water absorption capacity. Leaf stomata are particularly sensitive to environmental humidity changes and they close immediately to reduce water evaporation when drought stress occurred for subjecting low humidity condition [20]. AMY and BAM control the hydrolysis of starch to create soluble sugar, which could has a significant impact on increasing the drought tolerance of cassava. Although AMY and BAM genes have been identified in other plants, only two AMY and BAM genes were cloned and characterized from cassava leaves [21]. The biological role of the AMY and BAM gene families in cassava growth and stress response still unknown. Thus, MeAMY and MeBAM genes from cassava were identified in this study. Characterization, multiple sequence alignment, phylogenetic analysis, conserved structural motif, gene structure analysis, chromosomal distribution, homologs and collinearity analysis, and cis-regulatory element prediction of these two genes were performed. Additionally, members of the AMY and BAM gene families' expression patterns in response to drought stress were also analyzed.

Characterization and subcellular localization of MeAMY and MeBAM genes in cassava

The whole-genome was scanned to identify protein-coding genes containing the specific domain by using both BLASTP and HMMER search. 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 (Fig. 1). The size of MeAMY genes ranged 1227 ~ 2691 bp, and the similarities among sequences ranged from 0.53 to 1.00 (Fig. 1a). The length of MeAMY protein sequence ranged from 408 to 896 aa, and the similarities between protein sequences ranged 0.44 ~ 1.00, while their theoretical pIs ranged from 4.71 to 8.22 (Table 1). The largest molecular mass was MeAMY1.1(101.705kDa) and the smallest one was MeAMY5 (46.962 kDa). The range of aliphatic index was 27.07 ~ 45.39. Proteins were all hydrophilic with a negative GRAVY (− 0.576 ~ − 0.226). The range of instability index of proteins was 27.07 ~ 45.39. MeAMY1.1 ~ MeAMY3.5 were unstable with a instability index which larger than 40, while other four sequences were showed as stable proteins because their instability index were all less than 40.

The size of MeBAM genes ranged 1227 ~ 2691 bp, and the similarities among sequences were 0.41 ~ 1.00 (Fig. 1b). The length of MeBAM protein sequence ranged from 429 to 701 aa, and their similarities among protein sequences were 0.27 ~ 1.00, while their theoretical pIs ranged from 5.3 to 8.92. The largest molecular mass was MeBAM5.1 ~ MeBAM5.5 (79.132 kDa) and the smallest one was MeBAM4.3 (48.937 kDa). The range of aliphatic index was 69.6 ~ 84.41. Proteins were all hydrophilic with a negative GRAVY (− 0.485 ~ − 0.249). The range of instability index of proteins was 31.44 ~ 52.03. MeBAM1, MeBAM2, MeBAM5.1 ~ MeBAM5.6, MeBAM8, MeBAM10.2, and MeBAM10.3 were unstable with a instability index which larger than 40, while other eight sequences were showed as stable proteins.

The subcellular localization of 13 MeAMY and 20 MeBAM family genes was predicted using CELLO v.2.5 and the results were listed in Table 1. MeAMY1 and MeAMY2 located in the cytoplasm, while other MeAMY genes located in the Extracellular. The subcellular localization results for MeBAM was divided into five positions. Among them, MeBAM1 and MeBAM2 were located in chloroplast, MeBAM3 and MeBAM9 located in mitochondria, MeBAM4 ~ MeBAM7 in the cytoplasm, MeBAM8 in the plasma membrane, and MeBAM10 in the nucleus.

Table 1

Characteristics and subcellular localization of the MeAMY and MeBAM proteins
Gene name	protein Accession No.	Length of AA	PI	MW(Da)	GRAVY	Aliphatic index	Instability index	Subcellular localization
MeAMY1.1	XP_021603448.1	896	6.3	101705.99	-0.502	74.08	41.72	Cytoplasmic
MeAMY1.2	XP_021603456.1	812	6.32	91803.7	-0.529	74.45	44.78	Cytoplasmic
MeAMY2.1	XP_021611463.1	896	5.67	101574.29	-0.523	75.29	41.35	Cytoplasmic
MeAMY2.2	XP_021611464.1	812	5.63	91815.24	-0.538	76.49	45.39	Cytoplasmic
MeAMY3.1	XP_021612774.1	408	8.22	46560.08	-0.576	68.85	42.85	Extracellular
MeAMY3.2	XP_021612775.1	408	8.22	46560.08	-0.576	68.85	42.85	Extracellular
MeAMY3.3	XP_021612776.1	408	8.22	46560.08	-0.576	68.85	42.85	Extracellular
MeAMY3.4	XP_021612777.1	408	8.22	46560.08	-0.576	68.85	42.85	Extracellular
MeAMY3.5	XP_021612778.1	408	8.22	46560.08	-0.576	68.85	42.85	Extracellular
MeAMY4	XP_021625997.1	429	5.1	48230.1	-0.311	84.13	31.49	Extracellular
MeAMY5	XP_021631425.1	424	4.71	46962.8	-0.226	83.99	29.81	Extracellular
MeAMY6.1	XP_021601526.1	762	4.89	84901.46	-0.358	76.19	31.03	Extracellular
MeAMY6.2	XP_021601463.1	430	4.92	48130.31	-0.279	79.63	27.07	Extracellular
MeBAM1	XP_021605553.1	569	8.59	64184.29	-0.303	73.88	42.52	Chloroplast
MeBAM2	XP_021608087.1	581	5.67	64970.59	-0.388	71.2	41.1	Chloroplast
MeBAM3	XP_021608216.1	535	6.04	59219.83	-0.344	76.02	37.5	Mitochondrial
MeBAM4.1	XP_021611624.1	545	5.69	61669.84	-0.281	75.69	37.54	Cytoplasmic
MeBAM4.2	XP_021611626.1	517	5.59	58563.38	-0.315	77.72	35.62	Cytoplasmic
MeBAM4.3	XP_021611627.1	429	5.52	48937.51	-0.346	75.94	32.57	Cytoplasmic
MeBAM5.1	XP_021613139.1	701	5.63	79132.18	-0.47	76.5	40.8	Cytoplasmic
MeBAM5.2	XP_021613140.1	701	5.63	79132.18	-0.47	76.5	40.8	Cytoplasmic
MeBAM5.3	XP_021613141.1	701	5.63	79132.18	-0.47	76.5	40.8	Cytoplasmic
MeBAM5.4	XP_021613142.1	701	5.63	79132.18	-0.47	76.5	40.8	Cytoplasmic
MeBAM5.5	XP_021613143.1	701	5.63	79132.18	-0.47	76.5	40.8	Cytoplasmic
MeBAM5.6	XP_021613144.1	697	5.63	78691.58	-0.485	75.97	40.7	Cytoplasmic
MeBAM6.1	XP_021630045.1	594	6.04	67689.21	-0.432	84.41	37.18	Cytoplasmic
MeBAM6.2	XP_021630046.1	521	5.3	59078.17	-0.413	82.59	31.44	Cytoplasmic
MeBAM7	XP_021594996.1	582	6.05	64837.42	-0.42	69.6	36.57	Cytoplasmic
MeBAM8	XP_021595073.1	522	8.92	59418.2	-0.249	82.2	52.03	Plasma Membrane
MeBAM9	XP_021594434.1	546	8.7	61339.7	-0.459	70.04	34.79	Mitochondrial
MeBAM10.1	XP_021594905.1	691	5.51	77653.08	-0.446	71.74	40.13	Nuclear
MeBAM10.2	XP_021594906.1	689	5.44	77325.77	-0.402	73.5	40.35	Nuclear
MeBAM10.3	XP_021594907.1	686	5.5	77103.67	-0.394	73.67	39.9	Nuclear

Analysis of multiple sequence alignment, gene structure, conserved motifs, and alternative splicing of MeAMY and MeBAM

Similar conserved areas were identified of MeAMY and MeBAM protein sequences by comparing the domains of the AtAMY1 and AtBAM1 proteins. Two carbohydrate binding sites and three catalytic residues were found in MeAMY gene family (Fig. S1a), and these amino acid sites were perfectly conserved. Additionally, the MeBAM gene 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, respectivelya9(Fig. S1b). The MeBAM gene family contained 20 carbohydrate binding sites, and two of them had highly conserved amino acids, while 18 carbohydrate binding sites had amino acid mutations. The amino acids in the flexible loop and inner loop showed poor conservation, and there were a large number of mutations.

To better understand the structural evolution of MeAMY and MeBAM, the unrooted evolutionary trees were constructed based on the protein sequences for MeAMY and MeBAM, respectively. Generally, MeAMY and MeBAM that clustered in the same group showed similar gene structures (Fig. 2). For MeAMY, phylogenetic analysis classified 13 MeAMY into three clusters, termed I ~ III, and a total of 15 conserved motifs of MeAMY were identified using MEME software (Fig. 2a). Cluster I contain four MeAMY genes which are alternative splicing of one gene, and they shared the same conserved motifs. The CDS and UTR of MeAMY3.3 and MeAMY3.5 were arranged in the same order. And MeAMY3.1, MeAMY3.2, and MeAMY3.4 were also arranged in the same order, but they had a longer UTR than others at the 5' end (Fig. 2b). 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 unique to Cluster II. Cluster III contains MeAMY4, MeAMY5, MeAMY6.1, and MeAM6.2. MeAMY6.1 has the largest conservative motif and it looks like repeat motifs, while motif 3, 9, and 10 were missing in the repeat (Fig. 2a). The CDS of MeAMY6.1 also repeated and a CDS sequence was also found missing in the gene structure diagram (Fig. 2b). In addition, the 5' and 3' ends of MeAMY4 have the longest UTR in cluster III.

MeBAM gene family can be divided into five clusters (Fig. 2c). The distribution of conserved motifs at the 5' end of the BAM genes in cluster I was inconsistent, and their positions and arrangements of CDS and UTR were also different (Fig. 2d). MeBAM7 contains two motif 9. In cluster II, MeBAM6.1 and MeBAM6.2 belonged to alternative splicing of the same gene and can be treated as alternate promoter (AP). At the 5' end of these two genes, MeBAM6.1 had a motif 9 and a longer CDS sequence with a size of 219 bp than MeBAM6.2. The conserved motifs of MeBAM10.1 ~ MeBAM10.3 genes in cluster III were consistent and belonged to alternate terminator (AT). The gene structure of UTR and CDS sequences of their 3' end were different. In cluster IV, MeBAM4.1, MeBAM4.2, and MeBAM4.3 belonged to AP. The conserved motif of MeBAM4.3 at 5' end had no motif 9 and CDS missing than MeBAM4.1 and MeBAM4.2. The conserved motifs of the six genes in cluster V have the same arrangement and distribution, which CDS are all consistent. MeBAM5.6 lacked a 12 bp CDS sequence, while the UTR sequence at the 5' end are different.

Chromosomal location and collinearity analysis of MeAMY and MeBAM gene families

The location information of MeAMY and MeBAM gene families were obtained from the GFF (Generic Feature Forma) file of cassava genome (Fig. 3). The MeAMY and MeBAM genes were found in eight chromosomes and most of them are distributed at the end of these chromosomes. In the MeAMY gene family, only MeAMY2 is located in the ‘+’ strand, with opposite orientation to the other MeAMY genes. MeBAM1, MeBAM4, MeBAM5, MeBAM7, and MeBAM10 are located in the ‘+’ strand, while MeBAM2, MeBAM3, MeBAM6, MeBAM8, and MeBAM9 are located in the ‘−’ strand.

In order to reveal the expansion and evolution mechanism of MeAMY and MeBAM gene families, the potential gene duplication events in cassava genome were analyzed. Combined with the Arabidopsis genome and the rubber tree genome of euphorbiaceae plants, the collinearity of coding genes was analyzed (Fig. 4, Table S1). There were linear relationships between MeAMY1 and MeAMY2, and MeBAM2 and MeBAM7 genes in cassava genome. MeBAM4 and MeBAM5 were tandem repeat genes. There were 8 pairs of genes with collinearity between cassava and Arabidopsis genome, including 3 pairs of AMY genes and 5 pairs of BAM genes. There were 23 pairs of genes, 10 AMY genes and 13 BAM genes, in collinearity between cassava and rubber tree (Hevea brasiliensis) genome.

Calculation of the gene Ka/Ks value is an effective method to evaluate homologous protein sequence variations in species or taxa with unknown evolutionary status. Based on the collinearity analysis results of MeAMY and MeBAM families in cassava, the Ka/Ks values of homologous gene pairs were calculated. The results showed that the Ka/Ks values of the flanking homologous genes of those three pairs of MeAMY and MeBAM gene families listed in Table 2 ranged from 0.181848632 to 0.30120533. The ratios of these duplicates were all < 1, suggesting that their divergence was driven by purifying selection.

Table 2

homologous gene ka/ks values of MeAMY and MeBAM gene families
Seq-1	Seq-2	ka	ks	Ka/ks
MeAMY1.1	MeAMY2.1	0.080411456	0.266965581	0.30120533
MeBAM2	MeBAM7	0.072314402	0.39766261	0.181848632
MeBAM4.3	MeBAM5.6	0.231696907	1.021698702	0.226776159

Phylogenetic analysis of MeAMY and MeBAM gene families

The AMY and BAM protein sequences of other species were downloaded from NCBI database, and nine rice BAM Protein Sequences 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. A total of 54 AMY proteins from 12 species were used to construct the phylogenetic tree and could be classified into eight groups, including monocotyledonous plants (Group A ~ C) and dicotyledonous plants (Group D ~ H) (Fig. 5). The clustering results of monocotyledonous plants and dicotyledonous plants in the eight groups were significantly different. It is speculated that there are similar groupings in rubber trees and castors (Ricinus communis), and all the cassava AMYs were classified into Group A, B, and C and closely related with other Euphorbiaceae plants. Group D, G, and H are the AMY protein sequences of rice, among which Group G and H have only one rice sequence, Group F is the AMY protein sequence of wheat, and two AMY protein sequences of Group E are from rice and wheat, respectively.

The BAM protein sequences can be divided into nine groups (Fig. 6), and each group contains the BAM protein sequences of monocots and dicots, indicating that some BAM protein sequences in monocots and dicots have similar origins and evolutionary relationships. The BAM protein sequences of Arabidopsis, rice, and cassava were evenly distributed in all groups, while the BAM protein sequences of rubber trees and castors, and Jatropha (Jatropha curcas) were closely related to MeBAM among the five groups. It was speculated that these proteins might come from the same ancestors. The evolution occurred when euphorbiaceae species evolved into different plant lineages, and then evolved independently in each genus.

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

In order to understand the role of MeAMY and MeBAM gene families in plant resistance to stress, the cis-acting elements in the promoter regions of MeAMY and MeBAM genes were analyzed (Fig. 7). Remove core promoter elements CAAT-box, TATA-box, A-box, and AT ~ TATA-box, the cis-acting elements in the promoter regions of MeAMY and MeBAM genes were classified according to Yue method [1].

The number of cis-acting elements responding to abiotic stress is the largest and most widely distributed among all the cis-acting elements, such as MYB, TGACG-motif, ERE, MYC, as-1, ARE. In the promoter region of MeAMY gene, the number of MYB elements was the largest (Fig. 8 and Fig. 9). Nine MYB elements were enriched in MeAMY6.1 and seven ERE elements were enriched in both MeAMY1.1 and MeAMY1.2, while six MYC elements were enriched in MeAMY4 (Fig. 8). In the promoter region of MeBAM gene, the number of ABRE elements was up to 14 and 13 in MeBAM2 and MeBAM7, respectively. In addition, seven MYB e and six MYC elements were enriched in MeBAM5.1 ~ MeBAM5.6. ERE elements are enriched in the promoter regions of MeBAM1, MeBAM2, MeBAM4.1 ~ MeBAM4.2 and MeBAM6.1 ~ MeBAM6.2 genes (Fig. 9). On the other hand, the number of WUN-motif and CGTCA-motif are highest among the cis-acting elements induced by biotic, while TGA-box is the lowest. In the promoter region of MeAMY gene, 76.9% of the sequences contain 2 ~ 3 CGTCA-motifs, and TGA-box only exists in MeAMY6.1. In the promoter region of MeBAM gene, 80% of the sequences contain WUN-motifs, of which five WUN-motifs in MeBAM4.3, while there is only one AT-rich sequence in each of MeAMY6.1, MeAMY7, and MeAMY9.

The promoter regions of MeAMY and MeBAM genes also contain a large number of cis-acting elements related to light response, such as Box 4 and G-box (Fig. 7). Box 4 is the most abundant element, containing 50 elements in the promoter region of MeAMY gene, and 61.52% of the sequences have this element, which enriched in MeAMY1.1 ~ MeAMY1.2, MeAMY2.1 ~ MeAMY2.2 and MeAMY5. And 90% of MeBAM genes contain 91 Box 4 elements, which are mainly enriched in MeBAM1, MeBAM2, MeBAM4.1, MeBAM4.2, and MeBAM4.3. In addition, there are 92.3% of MeAMY promoter region sequences have G-Box elements, while 80% of sequences contain G-Box elements, which are mainly enriched in MeBAM2 and MeBAM7. GA-motif was only found in MeAMY4.

Furthermore, abundant O2-site and CAT-box were also found in the promoter region of MeAMY and MeBAM(Fig. 8 and Fig. 9), while few GCN4-motif, AC-I and MSA-like were found. GCN4-motif associated with the expression of storage protein gene were only found in MeAMY6.2. AC-I were found in MeAMY4 and correlates with vascular tissue gene expression. MSA-like is involved in cell cycle regulation and only exists in MeBAM2. The circadian cis-acting element is found in MeBAM9.

Gene ontology classification analysis of MeAMY and MeBAM genes

GO annotation of MeAMY and MeBAM proteins were separated into two categories: biological processes and molecular functions (Fig. 10). All proteins were involved in plant metabolic process. 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). Their molecular functions were primarily enriched in amylase activity, hydrolase activity, and catalytic activity, according to GO enrichment analysis (Fig. 11a). All MeAMY and MeBAM proteins were concentrated in carbohydrate metabolic process, polysaccharide catabolic process, polysaccharide metabolic process, macromolecule catabolic process, and organic substance catabolic process in biological processes (Fig. 11b).

Expression analysis of MeAMY and MeBAM gene families

The expression patterns of MeAMY and MeBAM gene families were analyzed using RNA-seq data from various cassava tissues (Fig. 12). MeAMY2, MeAM3, and MeBAM3 were found high transcription abundance in all tissues, while MeAMY5 was only detected in 100-day-old stems. MeBAM2, MeBAM6, and MeBAM9 had the highest transcription abundance in 100-day-old stems, while MeBAM2 and MeBAM7 both displayed higher transcription abundance in leaves and MeAMY6 was highly expressed in roots. MeAMY1 and MeBAM2 both had high transcription abundance in 180-day-old roots, while MeAMY1 was highly expressed in 300-day-old roots, MeAMY6 was highly expressed in 300 and 340-day-old roots, and MeBAM7 was highly expressed in 140, 180, and 220-day-old roots.

qRT-PCR was used to analyze the expression of MeAMY and MeBAM genes in cassava during the seedling stage without drought stress (CK). All of the genes were detected in all samples (Fig. 13). MeAMY5 and MeBAM6 showed low expression levels in all tissues, while MeBAM3 showed higher expressions. MeBAM2 showed high expression in leaves and stems, while MeBAM7 showed high expression only in stems and MeBAM9 was mainly expressed in leaves.

The expression patterns of MeAMY and MeBAM genes in cassava under different drought stress were also analyzed. Compare to CK, the expression levels of MeAMY1, MeAMY2, MeAMY4, MeBAM1, MeBAM3, MeBAM5, MeBAM8, and MeBAM10 genes in leaves were considerably up-regulated in the moderate drought (MD) treatment, whereas MeAMY3, MeAMY5, and MeBAM9 genes showed down-regulated (Fig. 14a). The expressions of MeAMY1, MeAMY2, and MeBAM3 genes in leaves of the severe drought treatment (HD) were significantly higher than in the MD treatment, while the expression 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 level (Fig. 14b). MeAMY5 in MD treatment increased significantly and had the highest expression level among all genes, and then decreased in HD treatment in stem, whereas MeAMY2 and MeBAM3 in HD treatment had the highest expression levels. MeAMY5 showed similar trend in root as in stem (Fig. 14c). The expression of MeAMY1, MeBAM6, and MeBAM7 genes in roots showed a downward trend among all treatments, while the expression of MeAMY2 and MeBAM10 was significantly increased. 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.

Variation of carbohydrate contents and amylase activity in cassava

The soluble sugar content and amylase activity in cassava leaves under HD treatment was higher than MD and CK, while its starch content was lower than CK and close to MD (Fig. 15a). Carbohydrates and amylase activity in stems varied similar with leaves (Fig. 15b). In roots, the soluble sugar content of HD treatment was significant higher than MD and CK, while its starch content was lower than CK and slightly higher than MD (Fig. 15c). In contrast, the amylase activity of HD treatment was significant lower than MD and CK.

The AMY and BAM genes have been identified and thoroughly analyzed in Arabidopsis and rice, play important roles in plant growth, development, and stress response [10, 22–24]. In the current study, six MeAMY genes and ten MeBAM genes were identified in the cassava genome, and 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, 23, 25, 26]. MeAMY1and MeAMY2 are on the same branch of the evolutionary tree (Fig. 2a) and both of them formed into two transcripts due to alternative splicing. The conserved motifs and sequence alignment analysis of two transcripts of MeAMY1 showed that they had different CDS sequences at the 5' end due to different translation initiation sites (Fig. 2b). Despite having relatively high similarity (0.95) between the protein sequences translated by these two transcripts, their physicochemical characteristics varied (Table 1). The sequence length of the transcripts produced by MeAMY2 are consistent to those produced by MeAMY1, and their alternative splicing methods are also the same. In the gene structure, their CDS are arranged similarly, located in cytoplasm, and exhibit collinearity (Table 2), which indicated that they may have similar functions. Due to alternative splicing, MeAMY3 in the MeAMY gene family produces five transcripts. The nucleotide sequences and amino acid sequences of these five transcripts are consistent, according to results of the analysis of sequence similarity and conserved motif (Fig. 1a). These five transcripts are divided into two different gene structures due to the difference in the 5' UTR of these genes, which control gene expression and will be affected by the variety of the 5'UTR [27]. MeAMY6 also consists of two transcripts and has a high degree of 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. Although the latter portion (431 ~ 762 aa) of MeAMY6.1 has several CDS and motifs with the former portion, it cannot form a domain, leading to proteins functionally differentiated encoded by MeAMY6.1 and MeAMY6.2. GO annotation also proved that MeAMY6 gene had translation regulation activity (Fig. 10).

In the MeBAM gene family, MeBAM4 and MeBAM6 have the same alternative splicing. Differences among transcripts are mostly in the 5' UTR and CDS. MeBAM5 forms 6 transcripts due to variations in the 5'UTR, and the CDS sequence of MeBAM5.6 is absent (Fig. 2d). MeBAM10 gene forms into three transcripts and they varied with the last exon, which has the most obvious effect on the protein instability index (Table 1). The gene structure of MeBAM10 gene showed that there were large differences in the 3' UTR of the three transcripts, and these differences may affect their post transcriptional regulation mechanism. The 3'UTR can control mRNA expression and translation, regulate mRNA stability through the rich AU element, and regulate protein-protein interactions [28]. Thus, the composition of conserved motifs in MeAMY and MeBAM gene family sequences are not the same. Some motifs only exist in a small number of gene family members cause differences between the gene family members, and these differences may be related to the functional differentiation of MeAMY and MeBAM families during evolution. Additionally, alternative splicing is another important reason for the polymorphism of MeAMY and MeBAM gene transcripts and protein structure and function.

There was a collinear relationship between three pairs of genes in the MeAMY and MeBAM families. MeBAM4 and MeBAM5 were located adjacent to the same chromosome and treated as the result of tandem replication (Fig. 3) according to the definition of tandem replication events by Recep Vatansever et al [29]. Sequence alignment analysis revealed that the amino acids of the five carbohydrate binding sites in these two gene sequences varied, which may lead to changes in their ability binding the substrate, ending different catalytic efficiencies of the substrate. MeAMY1 and MeAMY2, MeBAM2 and MeBAM7 are located on the collinear blocks of different chromosomes, respectively (Fig. 4) and belong to the product of fragment replication. There is no amino acid mutation and their carbohydrate binding sites and catalytic residues are exceedingly conservative. The ka/ks values of three sets of paralogous homologous genes were smaller than 1(Table 2), indicating that purification selection occurred and the protein sequence's stability was somewhat preserved [30]. Comparing to synonymous substitution, nonsynonymous substitution has a lower adaptability and accumulates more slowly throughout evolution.

The phylogenetic tree of the AMY proteins was dived into 8 groups, three groups were dicotyledonous plants and five groups were monocotyledonous plants (Fig. 5), which is consistent with previous studies [1]. The rubber and castor AMY protein sequences are close to that of the cassava MeAMY family, while 10 pairs of AMY genes are collinear in cassava and rubber (Fig. 4), demonstrating a closer evolutionary relationship between these gene family members. The AMY genes of cassava and Arabidopsis in Group A and Group B have collinearity (Fig. 5), and they have the same carbohydrate binding sites and catalytic residues (Fig. S1a). MeAMY3 and AtAMY2 are belong to Group A, while MeAMY1, MeAMY2, and AtAMY3 are belong to Group B. MeAMY4, MeAMY5, and MeAMY6 are all found on the same branch as AtAMY1. MeAMY2 and MeAMY3 had relatively high transcription abundance in roots during the whole growth period of cassava, indicating that these genes were crucial for the development of cassava roots (Fig. 12). Both AtAMY1 (AEE84990.1) and AtAMY2 (AEE358001) are secretory proteins and AtAMY3(AEE34983.1) is a special redox-regulated chloroplast α-amylase. Normal starch degradation was observed in the single, double, and triple mutant Arabidopsis leaves of these three genes, which indicated that α-amylase is not necessary during the transient starch decomposition process in Arabidopsis leaves [31]. AtAMY3 on Group B is found in the chloroplast, it can act on the starch granule surface to release oligosaccharides when other starch degrading enzymes are not present [32].

MeBAM protein and BAM proteins from rubber, castor, and jatropha in the Euphorbiaceae have a tight evolutionary connection (Fig. 6). MeBAM2, MeBA7, and AtBAM1 were found in Group 7 of the phylogenetic tree, while MeBAM4 and AtBAM2 were classified into Group 2 and MeBAM9 and AtBAM3 were found in Group 6, respectively. The light-responding elements Box 4 and G-Box related to the circadian cycle were enriched in MeBAM2, MeBAM7, and MeBAM9 genes and showed higher expression in cassava leaves during the seedling stage (Fig. 13). AtBAM1 was found highly expressed in guard cells of leaves and the activity of the β-amylase encoded by AtBAM1 is only active under reducing conditions when high temperature, drought, and osmotic stress occurred [10]. The purified protein of β-amylase encoded by AtBAM2 has very weak activity in vitro, while potassium ion can dramatically improve the activity of AtBAM2 and exhibit synergistic effects [33]. During the night, AtBAM3 is primarily in charge of starch breakdown in mesophyll cells [34]. The β-amylase encoded by AtBAM4 lacks of catalytic activity due to only one amino acid mutation, glutamic acid (E) was changed to arginine (R), at the catalytic active site Glu-380[35]. MeBAM8 shared the same catalytic active site as AtBAM4, thus, the β-amylase encoded by MeBAM8 may also lack of catalytic activity (Fig.S1B). Both MeBAM5 and MeBAM10 have a BZR1/BES1 domain at their N-termini as AtBAM7 and AtBAM8. Although β-amylases encoded by AtBAM7 and AtBAM8 have very low catalytic activity, they play important roles in the bud growth and development by acting as transcription factors [36, 37]. However, the GO annotation findings failed to adequately define the transcription factor function of these two genes, further research to understand the function of these proteins is needed. In addition, MeBAM1 located at Group 5 without AtBAM (Fig. 6), and Matthias Thalmann et al [38] suggested that the formation of this branch is before the radiation of seed plants.

Under stress conditions, transcription factors combine with the cis-acting elements in the promoter region of stress-responsive genes to promote the transcription and expression of stress-resistant genes, thus making regulatory response [39]. In current study, the MeAMY and MeBAM gene families included a range of stress response cis-acting elements and the largest number of MYB-related elements. MYB transcription factors are crucial for secondary metabolism, cell differentiation, stress response, and plant growth and development [40]. MeAMY and MeBAM genes primarily responded to drought stress in leaves and roots (Fig. 14a and Fig. 14c), and severe drought stress will affect the expression of MeAMY and MeBAM genes. The most of the MeAMY and MeBAM genes in cassava leaves were triggered by drought stress, and their expression levels were significantly increased. Only a small portion of the gene expression in the cassava stems was up-regulated. The majority of the MeAMY and MeBAM genes in cassava roots showed the highest expression level under moderate drought among three water treatments. Under drought stress, MeAMY2 gene expression was stably up-regulated in roots, stems, and leaves (Fig. 14), while MeAMY1 and MeBAM3 gene expression was stably up-regulated in stems and leaves. Thus, these genes can be treated as key genes within the MeAMY and MeBAM gene families that response to drought tolerance in cassava.

Metabolites of carbohydrates in plant leaves can act as osmotic protecting agents, thus maintaining the water balance in cells enables plants to adapt to stress [13, 41–43]. The total enzyme activity of α-amylase and β-amylase increased only in cassava leaves and stems. During drought stress, the starch content of cassava roots, stems, and leaves dropped while the soluble sugar content rose (Fig. 15), suggesting that starch in cassava roots, stems, and leaves was degraded when drought stress occurred, which is consistent with previous researches [13, 14, 34, 44, 45].

However, there was a downward trend in the activity of the enzyme amylase in roots (Fig. 15c), and similar phenomena also found in tobacco (Nicotiana tabacum) and rape (Brassica napus) [41, 46]. The fact that starch hydrolysis involves a number of synergistic amylase (starch phosphorylase, debranching enzyme) reactions suggests that there may be more enzymes involved in the starch degradation caused by stress [44, 47]. Leaves and young stems are more likely to accumulate transient starch and respond quickly to drought stress and the main starch hydrolase in chloroplasts is β-amylase [13, 35]. Another explanation is that the root system contains more maltose and soluble sugar due to the breakdown of starch, high content of maltose inhibits the total enzyme activity of α-amylase and β-amylase [45]. 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 root during stress can provide energy for the rapid recovery of plant shoots after the stress is lifted.

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. MeBAM5 and MeBAM10 may function as transcription factors, according to domain analysis.The promoter regions of MeAMY and MeBAM genes contain a large number of cis-acting elements related to abiotic stress, and MeAMY1, MeAMY2, and MeBAM3 are key genes in response to drought stress. Starch content, soluble sugar content, and amylase activity in cassava roots, stems, and leaves varied when drought stress occurred. These results could provide fundamental knowledge for further investigation of the function of starch metabolism under drought stress in cassava, offering new perspectives for understanding the underlying mechanism for its ability to survive and produce under drought.

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 genes in cassava database were characterized by TBtools-BLAST using the AMY and BAM genes from Arabidopsis and rice as queries [48]. The local Hidden Markov Models of all the known AMY and BAM genes 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 genes in cassava. And protein sequences with consistency ≥ 60% and E value ≤ 1e-5 were selected as queries. The pFAM database (https://pfam.xfam.org/) and SMART (http://smart.embl-heidelberg.de/) database were searched to confirm the complete conserved domains of these selected queries [49, 50]. The target MeAMY and MeBAM genes were identified by doing BLAST analyses on all candidate genes. The cassava protein sequence containing the Alpha-amylase domain (PF00128) was used as the MeAMY gene family sequence, and the protein sequence containing the Glyco_hydro_14 domain (PF01373) was used as the MeBAM gene family sequence.

Protein sequence characterization and its subcellular localization prediction

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) [51]. The optimum widths of the motifs were between 10 and 100, the number of motifs was 15, and the other parameters were used as default settings. Structure features of MeAMY and MeBAM genes were obtained by the cassava genome GFF file. Similarity and composition analysis of MeAMY and MeBAM sequences were done using softwares DNAMAN and BioEdit, respectively. Subcellular localization of MeAMY and MeBAM was predicted by CELLO v. 2.5 (http://cello.life.nctu.edu.tw/) [52].

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

Multiple sequence alignment of MeAMY and MeBAM nucleotides was performed using DNAMAN, the alignment sequence is displayed using jalview (http://www.Jalview.org/). Un-rooted trees were constructed using the maximum likelihood method within MEGA-X software. The phylogenetic tree of AMY and BAM protein sequences of cassava and other species was 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 [53].The collinearity map and chromosome mapping of homologous genes were plotted on chromosomes, and the ratio of homologous genes Ka (nonsynonymous substitution rate) and Ks (synonymous substitution rate) was 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 database and New PLACE database was used to predict the conserved cis-elements in these promoter sequences [54, 55].

Expression and gene ontology classification analysis of MeAMY and MeBAM genes based on RNA-seq data

The expression patterns of MeAMY and MeBAM genes was analyzed based on the raw RNA sequencing data including ten different tissue types (study accession: SRP228273, Table S4)[56], 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 performed the log2(TPM + 1) conversion before visualization. A heat map was created using ggplot2 in R 4.2.1. Gene ontology annotations of MeAMY and MeBAM genes were performed using the omicshare (https://www.omicshare.com/tools).

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 in Guangxi University in 2021 and cassava was planted in cuboid box (40× 40×50 cm) with soil on October. And two cassava seedlings were planted in each box with normal watering and well management after planting. The drought treatment was set as two levels: 70 ~ 80% of soil water content as control (CK), 45 ~ 55% as moderate drought (MD), and 20 ~ 30% as severe drought (HD), and operated on 60 days of after planting. Each treatment contained 6 boxes 12 shoots of cassava and the drought treatment lasted for a week. Soil moisture recorder (LUGE-L99-TWS-1) was used to monitor the moisture content in the soil. After drought treatment, cassava stems (0–10 cm from the top of stem), leaves, and roots samples were collected and 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 crushed for further determinations of starch and sugar contents.

Determination of soluble sugar, starch content and activities of amylase

The determination of contents of soluble sugar and starch in root, stem and leaf of cassava were described by Loewus method [57]. Determination of activities of α-amylase and β-amylase in plant samples using the 3,5-dinitrosalicylic acid (DNS) colorimetric method and details were well described by Bernfeld [58].

RNA extraction and qRT-PCR assays

Total RNA was extracted using the FastPure® Plant Total RNA Isolation Kit (Vazyme, China) 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 NCBI-Primer-BLAST tool was used to design the primers (Table S5), and the product range was limited to 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 (10µM), 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. After 42 cycles, melting curve analysis was performed ranging from 60 to 95℃. The relative expression levels were calculated with the formula 2^−ΔΔCT [59]. Three independent biological replicates were applied for all treatments. Statistical analysis was calculated using IBM SPSS 25.0.

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;

Acknowledgements

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

Authors' 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 provided suggestions for experiments. All authors read and approved the final manuscript.

Funding

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

Availability of data and materials

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 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA578024/). Genomic data for Hevea brasiliensis (ASM165405v1) and Arabidopsis thaliana (TAIR10.1) were obtained from NCBI (https://www.ncbi.nlm.nih.gov/genome/).

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 and collected from Guangxi South Subtropical Agricultural Sciences Research Institute (Chongzuo 532406, China). The study is conducted with plant material complies with relevant institutional, national, and international guidelines and legislation.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no conflict of interest.

Author details

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

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

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

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

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

Yue C, Cao H, Lin H, Hu J, Ye Y, Li J, et al. Expression patterns of alpha-amylase and beta-amylase genes provide insights into the molecular mechanisms underlying the responses of tea plants (Camellia sinensis) to stress and postharvest processing treatments. Planta. 2019; 250(1):281-298.
2. Dietze MC, Sala A, Carbone MS, Czimczik CI, Mantooth JA, Richardson AD, Vargas R. Nonstructural Carbon in Woody Plants. Annu Rev Plant Biol. 2014; 65(1):667-687.
3. Preiss J, Levi C. Starch Biosynthesis and Degradation - ScienceDirect. Carbohydrates: Structure and Function. 1980:371-423.
4. Drula E, Garron ML, Dogan S, Lombard V, Henrissat B, Terrapon N. The carbohydrate-active enzyme database: functions and literature. Nucleic Acids Res. 2022; 50(D1): D571-D577.
5. Dong S, Beckles DM. Dynamic changes in the starch-sugar interconversion within plant source and sink tissues promote a better abiotic stress response. J. Plant Physiol. 2019; 234-235:80-93.
6. Krasensky J, Jonak C. Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. J Exp Bot. 2012, 63(4):1593-1608.
7. Hwang YS, Thomas BR, Rodriguez RL. Differential expression of rice alpha-amylase genes during seedling development under anoxia.Plant Mol Biol. 1999; 40(6):911-920.
8. Doyle EA, Lane AM, Sides JM, Mudgett MB, Monroe JD. An alpha-amylase (At4g25000) in Arabidopsis leaves is secreted and induced by biotic and abiotic stress. Plant Cell Environ. 2007; 30(4):388-398.
9. Prasch CM, Ott KV, Bauer H, Ache P, Hedrich R, Sonnewald U. ss-amylase1 mutant Arabidopsis plants show improved drought tolerance due to reduced starch breakdown in guard cells. J Exp Bot. 2015; 66(19):6059-6067.
Valerio C, Costa A, Marri L, Issakidis-Bourguet E, Pupillo P, Trost P, et al. Thioredoxin-regulated beta-amylase (BAM1) triggers diurnal starch degradation in guard cells, and in mesophyll cells under osmotic stress. J Exp Bot. 2011; 62(2):545-555.
Kaplan F, Guy CL. RNA interference of Arabidopsis beta-amylase8 prevents maltose accumulation upon cold shock and increases sensitivity of PSII photochemical efficiency to freezing stress. Plant J. 2005; 44(5):730-743.
Kaplan F, Guy CL. beta-Amylase induction and the protective role of maltose during temperature shock. Plant Physiol. 2004; 135(3):1674-1684.
Thalmann M, Pazmino D, Seung D, Horrer D, Nigro A, Meier T, et al. Regulation of Leaf Starch Degradation by Abscisic Acid Is Important for Osmotic Stress Tolerance in Plants. Plant Cell. 2016; 28(8):1860-1878.
Du Y, Zhao Q, Chen L, Yao X, Zhang W, Zhang B, et al. Effect of drought stress on sugar metabolism in leaves and roots of soybean seedlings. Plant Physiol Biochem. 2020; 146:1-12.
Liang G, He H, Nai G, Feng L, Li Y, Zhou Q, Ma Z, Yue Y, Chen B, Mao J. Genome-wide identification of BAM genes in grapevine (Vitis vinifera L.) and ectopic expression of VvBAM1 modulating soluble sugar levels to improve low-temperature tolerance in tomato. BMC Plant Biol. 2021; 21(1):156.
Li S, Cui Y, Zhou Y, Luo Z, Liu J, Zhao M. The industrial applications of cassava: current status, opportunities and prospects. J Sci Food Agric. 2017; 97(8):2282-2290.
Rawel HM, Kroll J. The importance of Cassava (Manihot esculenta Crantz) as the main staple food in tropical countries. Dtsch Lebensm-Rundsch. 2003; 99(3):102-111.
Yu XY, Yao Y, Hong YH, Hou PY, Li CX, Xia ZQ, et al. Differential expression of the Hsf family in cassava under biotic and abiotic stresses. Genome. 2019; 62(8):563-569.
Okogbenin E, Setter TL, Ferguson M, Mutegi R, Ceballos H, Olasanmi B, et al. Phenotypic approaches to drought in cassava: review. Front Physiol. 2013; 4:93.
El-Sharkawy MA. Cassava biology and physiology. Plant Mol Biol. 2004; 56(4):481-501.
Lengbamroung P, Vichukit V, Visser R, Nakasathien S. Cloning and Molecular Characterization of α- and β- Amylase Genes from Cassava (Manihot esculenta Crantz). The Kasetsart Journal. 2005; 39(3):446-454.
Koide T, Ohnishi Y, Horinouchi S. Characterization of recombinant beta-amylases from Oryza sativa. Biosci Biotechnol Biochem. 2011; 75(4):793-796.
Stanley D, Fitzgerald AM, Farnden K, Macrae EA, Macrae K. Characterisation of putative α-amylases from apple (Malus domestica) and Arabidopsis thaliana. Biologia - Section Cellular and Molecular Biology. 2002; 11:137-148.
Mitsui T, Yamaguchi J, Akazawa T. Physicochemical and serological characterization of rice alpha-amylase isoforms and identification of their corresponding genes. Plant Physiol. 1996; 110(4):1395-1404.
Zhang DL, Wang Y, Jia BC, Tian XQ, Chu J, Yin HB, et al. Genome-Wide Identification and Expression Analysis of the beta-Amylase Gene Family in Chenopodium quinoa. DNA Cell Biol. 2021; 40(7):936-948.
Gong X, Westcott S, Zhang XQ, Yan G, Lance R, Zhang G, et al. Discovery of novel Bmy1 alleles increasing beta-amylase activity in Chinese landraces and Tibetan wild barley for improvement of malting quality via MAS. PLoS One. 2013; 8(9): e72875.
Srivastava AK, Lu Y, Zinta G, Lang Z, Zhu JK. UTR-Dependent Control of Gene Expression in Plants. Trends Plant Sci. 2018; 23(3):248-259.
Mayr C. Evolution and Biological Roles of Alternative 3'UTRs. Trends Cell Biol. 2016; 26(3):227-237.
Vatansever R, Koc I, Ozyigit II, Sen U, Uras ME, Anjum NA, et al. Genome-wide identification and expression analysis of sulfate transporter (SULTR) genes in potato (Solanum tuberosum L.). Planta. 2016; 244(6):1167-1183.
Zhang Z, Li J, Zhao XQ, Wang J, Wong GK, Yu J. KaKs_Calculator: calculating Ka and Ks through model selection and model averaging. Genom Proteomics Bioinformatics. 2006; 4(4):259-263.
Yu TS, Zeeman SC, Smith SM. α-Amylase Is Not Required for Breakdown of Transitory Starch in Arabidopsis Leaves. J Biol Chem. 2005; 280(11):9773-9779.
Streb S, Delatte T, Umhang M, Eicke S, Schorderet M, Reinhardt D, et al. Starch granule biosynthesis in Arabidopsis is abolished by removal of all debranching enzymes but restored by the subsequent removal of an endoamylase. Plant Cell. 2008; 20(12):3448-3466.
Monroe JD, Breault JS, Pope LE, Torres CE, Gebrejesus TB, Berndsen CE, et al. Arabidopsis beta-Amylase2 Is a K (+)-Requiring, Catalytic Tetramer with Sigmoidal Kinetics. Plant Physiol. 2017; 175(4):1525-1535.
Monroe JD, Storm AR, Badley EM, Lehman MD, Platt SM, Saunders LK, et al. beta-Amylase1 and beta-amylase3 are plastidic starch hydrolases in Arabidopsis That Seem to Be Adapted for Different Thermal, pH, and stress conditions. Plant Physiol. 2014; 166(4):1748-1763.
Fulton DC, Stettler M, Mettler T, Vaughan CK, Li J, Francisco P, et al. Beta-AMYLASE4, a noncatalytic protein required for starch breakdown, acts upstream of three active beta-amylases in Arabidopsis chloroplasts. Plant Cell. 2008; 20(4):1040-1058.
Soyk S, Simkova K, Zurcher E, Luginbuhl L, Brand LH, Vaughan CK, et al. The Enzyme-Like Domain of Arabidopsis Nuclear beta-Amylases Is Critical for DNA Sequence Recognition and Transcriptional Activation. Plant Cell. 2014; 26(4):1746-1763.
Reinhold H, Soyk S, Simkova K, Hostettler C, Marafino J, Mainiero S, et al. beta-amylase-like proteins function as transcription factors in Arabidopsis, controlling shoot growth and development. Plant Cell. 2011; 23(4):1391-1403.
Thalmann M, Coiro M, Meier T, Wicker T, Zeeman SC, Santelia D. The evolution of functional complexity within the beta-amylase gene family in land plants. BMC Evol Biol. 2019;19(1):66.
Yamaguchi-Shinozaki K, Shinozaki K. Organization of cis-acting regulatory elements in osmotic- and cold-stress-responsive promoters. Trends Plant Sci. 2005; 10(2):88-94.
Wang X, Niu Y, Zheng Y. Multiple Functions of MYB Transcription Factors in Abiotic Stress Responses. Int J Mol Sci. 2021; 22(11).
Lv Y, Luo D, Jia Z, Cheng Y, Zou X. The β- Amylase Gene Family in Brassica Napus: Genome Wide Analysis and Expression Profiles in Response to Abiotic Stresses. Agronomy. 2020;10.1855
Kerepesi, IlDiko, Galiba, Gabor. Osmotic and Salt Stress-Induced Alteration in Soluble Carbohydrate Content in Wheat Seedlings. Crop Sci. 2000; 40(2):482.
Liu T, Staden JV: Partitioning of carbohydrates in salt-sensitive and salt-tolerant soybean callus cultures under salinity stress and its subsequent relief. Plant Growth Regul. 2001; 33(1):13-17.
Thalmann M, Santelia D. Starch as a determinant of plant fitness under abiotic stress. New Phytol. 2017; 214(3):943-951.
Zhao L, Yang T, Xing C, Dong H, Qi K, Gao J, et al. The beta-amylase PbrBAM3 from pear (Pyrus betulaefolia) regulates soluble sugar accumulation and ROS homeostasis in response to cold stress. Plant Sci. 2019; 287:110184.
Peng T, Zhu X, Duan N, Liu JH. PtrBAM1, a beta-amylase-coding gene of Poncirus trifoliata, is a CBF regulon member with function in cold tolerance by modulating soluble sugar levels. Plant Cell Environ. 2014; 37(12):2754-2767.
Streb S, Zeeman SC. Starch metabolism in Arabidopsis. Arabidopsis Book. 2012;10: e160.
Chen C, Chen H, Zhang Y, Thomas HR, Frank MH,et al. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol Plant. 2020; 13(8):1194-1202.
Letunic I, Khedkar S, Bork P. SMART: recent updates, new developments and status in 2020. Nucleic Acids Res. 2021; 49(D1): D458-D460.
Mistry J, Chuguransky S, Williams L, Qureshi M, Salazar GA, Sonnhammer E, et al. Pfam: The protein families database in 2021. Nucleic Acids Res. 2021; 49(D1): D412-D419.
Bailey TL, Johnson J, Grant CE, Noble WS. The MEME Suite. Nucleic Acids Res. 2015; 43(W1): W39-W49.
Yu CS, Lin CJ, Hwang JK. Predicting subcellular localization of proteins for Gram-negative bacteria by support vector machines based on n-peptide compositions. Protein Sci. 2004; 13(5):1402-1406.
Wang Y, Tang H, Debarry JD, Tan X, Li J, Wang X, et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012, 40(7): e49.
Magali, Lescot, Patrice, Déhais, Gert, Thijs, et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002; 30(1):325-327.
Higo K, Ugawa Y, Iwamoto M, Korenaga T. Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res. 1999; 27(1):297-300.
Ding Z, Fu L, Tie W, Yan Y, Wu C, Dai J, et al. Highly dynamic, coordinated, and stage-specific profiles are revealed by a multi-omics integrative analysis during tuberous root development in cassava. J Exp Bot. 2020; 71(22):7003-7017.
Loewus, F. A. Improvement in antrone method for determination of carbohydrates. Anal Chem. 1952; 24(1):219.
McKee LS. Measuring Enzyme Kinetics of Glycoside Hydrolases Using the 3,5-Dinitrosalicylic Acid Assay. Methods Mol Biol. 2017; 1588:27-36.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001; 25(4):402-408.

No competing interests reported.

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

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Journal Publication

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Abstract

Background

Results

Conclusions

Figures

Background

Results

Variation of carbohydrate contents and amylase activity in cassava

Discussion

Conclusion

Methods

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

Protein sequence characterization and its subcellular localization prediction

Plant materials and drought treatments

Determination of soluble sugar, starch content and activities of amylase

RNA extraction and qRT-PCR assays

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1