Genome-wide identification and characterization of MeAnns
Using genome-wide retrieval techniques, in all, 12 supposed
Annexin genes were found out in the cassava genome and named
MeAnn1-12. ProtParam was used to characterize the MeAnn physiological and biochemical characteristics.
The results showed that MeAnn lengths ranged from 309 (MeAnn12) to 382 (MeAnn6) AA
(amino acids), with the predicted MW (molecular weights) ranging from 35.26 (MeAnn12)
to 43.78 (MeAnn6) kDa. The GRAVY (grand average of hydropathicity) of the MeAnns ranged
from -0.554 (MeAnn5) to -0.271 (MeAnn7), and the theoretical pI (isoelectric point)
ranged from 5.80 (MeAnn6) to 9.46 (MeAnn9). The II (instability index) of MeAnn proteins
ranged from 24.66 (MeAnn2) to 50.62 (MeAnn6), with six MeAnns (MeAnn2, 3, 7, 8, 11,
and 12) denoted as stable proteins, while the other six MeAnns are unstable proteins.
The AI (aliphatic index) of the MeAnns ranged from 80.14 (MeAnn10) to 100 (MeAnn7)
(Table 1). Signal peptide (SP) analysis showed that only one of the twelve MeAnns
(MeAnn6) has a signal peptide (Table 1). Subcellular localization prediction indicated that the MeAnns are located in one to
three organelles including the cytoplasm (Cytop), mitochondria (Mito), nucleus (Nuc),
chloroplast (Chlo) and plasma membrane (Plasm). Most MeAnns have two subcellular localization
organelles. For example, MeAnn1 is located in the cytoplasm and mitochondria; MeAnns2,
3, 7 and 8 are located in the cytoplasm and nucleus; MeAnn4 is located in the mitochondria
and chloroplast; MeAnn6 is located in the plasma membrane and nucleus; and MeAnn10
is located in the mitochondria and nucleus. MeAnn5 and MeAnn12 are located in three
organelles, including the cytoplasm, mitochondria and nucleus, whereas MeAnn9 and
MeAnn11 are only located in the mitochondria (Table 1).
Location and Distribution of MeAnns on Chromosomes
Using chromosomal localization analysis, the 12 MeAnn genes were assigned to eight chromosomes, including 4, 6, 9, 11, 12, 15, 17, and
18 (Fig. 1). The distribution of MeAnns among the chromosomes is unequal. In particular, chromosomes 4 and 11 harbor 6 MeAnns, with each one containing three genes. By contrast, chromosomes 6, 9, 12, 15, 17
and 18 each harbor only one MeAnn gene (Fig. 1). MeAnn1, 2, 5 and 6 have the same orientation, whereas the other MeAnnsare in the opposite orientation.
Cassava MeAnn Sequence Alignment
To obtain detailed information on each cassava MeAnn protein, the 12 MeAnn proteins were subjected to multiple sequence alignment
(Fig. 2.) All of the MeAnns contain typical annexin domains with four 70-amino acid
repeats (Repeats Ⅰ- Ⅳ). The type Ⅱ Ca2+ binding sites appear in both Repeats Ⅰ and Ⅳ. Only MeAnn7 and 8 have typical G-X-GTD-{38
residues}-E/D sites. The “G-X-GTD” is changed to “G-X-GTN” in MeAnn1-4; to “G-X-GVD”
in MeAnn5 and 6; and to “G-X-GCD” in MeAnn9, 11 and 12. The typical G-X-GTD-{38 residues}-E/D
site is missing from MeAnn10 (Fig. 2). MeAnns 1-4 and 10 contain a phospholipid binding-related
“W” residue in Repeat Ⅰ (Fig. 2). All the MeAnns except MeAnns 5-7 and 10 contain
a peroxidase activity-related heme-binding site (“H” residue) (Fig. 2). MeAnn1, 2,
7 and 8 contain the typical GTP-binding site “DXXG” in Repeat Ⅳ; however, the “D”
residue is changed to “E” in MeAnns 3, 4, 9, 11 and 12; to “N” in MeAnns 5 and 6;
and to “A” in MeAnn10 (the “DXXG” is changed to “AXXXG”) (Fig. 2). MeAnn1, 3-5 and
11 contain an F-actin binding site (IRI) in Repeat Ⅲ; the “IRI” is changed to “IRV”
in MeAnn2, to “VRI” in MeAnn6, to “VYI” in MeAnn7, to “VYV” in MeAnn8, to “IQI” in
MeAnn9 and 12, and to “LEI” in MeAnn10 (Fig. 2). MeAnn10 has only one redox reaction-related
S3 cluster in Repeat Ⅳ, whereas the other 11 MeAnns have two S3 clusters, one each
in Repeats Ⅱ and Ⅳ (Fig. 2).
Phylogenesis Analysis, Gene Structures and Conserved Motifs in cassava MeAnns
To make clear the relationships among theMeAnn members in cassava, a phylogenetic tree consisting of all the annexins from
M. esculeta,
A. thaliana,
O. sativa and
S. lycopersicum was framed using the Neighboring-Joining method. The results showed that the MeAnns
in the phylogenetic tree are classified into six groups (Fig. 3). One MeAnn protein
(MeAnn10) is clustered into Group Ⅰ, three proteins (MeAnn9, 11 and 12) are clustered
into Group Ⅱ, MeAnn5 and 6 are clustered into Group Ⅲ, MeAnn7 and 8 are clustered
into Group Ⅳ, MeAnn3 and 4 are clustered into Group Ⅴ, and MeAnn1 and 2 are clustered
into Group Ⅵ. MeAnn10 is clustered together with two annexins from rice, whereas the
other 11 MeAnns are clustered together with annexins from
Arabidopsis, rice and tomato (Fig. 3).
The gene structures of the annexin members in cassava and
Arabidopsis were determined using the GSDS online tool. The genomic DNA lengths of
MeAnn genes vary from 1586 to 3162 bp, which are longer than their orthologs from
Arabidopsis. However, their CDS lengths vary from 930 to 1149 (bp), which are similar to their
orthologs from
Arabidopsis (Table 1, Fig. 4B). The
MeAnn gene structures have four (
MeAnn10) to seven (
MeAnn4) exons that are interrupted by three to six gene-specific introns with different
lengths. Gene structure analysis revealed that the structures of
MeAnn9, 11 and
12 in Group Ⅱ;
MeAnn5 and
6 in Group Ⅲ; and
MeAnn7 and
8 in Group Ⅳ have six exons, which is similar to their orthologs from
Arabidopsis (Fig. 4A, B). In Group Ⅴ, the structure of
MeAnn3 is similar to its ortholog
AtAnn8, which has six exons; however,
MeAnn4, which is in the same group, has seven exons. In Group Ⅵ,
MeAnn1 and
2 each have five exons, which is similar to
AtAnn2, though it is different from
AtAnn16 and
7 in the same group. Only one gene (
MeAnn10) in Group Ⅰ has four exons in its structure (Fig. 4A, B).
Ten different conserved motifs were identified from MeAnns and AtAnns (Fig. 3 C).
Generally speaking, the members in the same groups share a similar motif structure,
except for the members in Group Ⅲ. All the MeAnns and AtAnns in Groups Ⅳ-Ⅵ contain
all ten motifs. All the MeAnns and AtAnns in Group Ⅱ contain nine motifs, only lacking
motif10. In Group Ⅲ, MeAnn5 consists of seven motifs, lacking motif3, 8 and 9, and
MeAnn6 includes eight motifs, lacking motif3 and 8. By contrast, AtAnn4, which is
also in Group Ⅲ, contains six motifs, lacking motif3 and 8-10. In Group I, MeAnn10
includes seven motifs, lacking motif5, 6 and 8 (Fig. 3A, C).
Presence of hormone- and stress-related cis-acting elements in the MeAnn promoter
To better understand the feasible biological responses of
MeAnns under hormone and abiotic stresses, the 2 kb sequence upstream of the translation
start site of each MeAnn was analyzed by PlantCARE. Twelve hormone response
cis-elements, including an ABRE, ABRE4, as-1, CCTCA motif, ERE, GARE motif, TATC box,
TCA, TCA element, TGA box, TGA element and TGACG motif, were predicted (Fig. 4, Table
S1). Fifteen stress response cis-elements, including an ARE, RDE core, LTR, MBS, Myb,
MYB recognition site, MYB, Myc, MYC, STRE, TC-rich repeats, W box, WRE3, and WUN motif,
were predicted (Fig. 4). MeAnns possess at least 3 hormone response-related cis-elements
and 1 stress response-related cis-element, which indicates that
MeAnn expression might be regulated by these hormone and abiotic stress factors. All twelve
MeAnns contain one to seven EREs, suggesting potential responses to ethylene. One to four
ABREs are present in nine
MeAnns (except
MeAnn9, 10 and
12) and one to two ABRE4s are present in
MeAnn1,
2,
6,
7 and
11, which indicates that these genes might be regulated by ABA. One to three CGTCA motifs
and TGACG motifs are found in three genes (
MeAnn1,
4 and
9), indicating that these genes might respond to MeJA. Four genes (
MeAnn 4,
6,
7 and
11) contain a GARE motif and five genes (
MeAnn1,
4,
10-12) contain a TATC box, figuring that these
MeAnns might be adjusted by gibberellin (GA). Eight genes (except for
MeAnn1,
2,
4 and
12) have TCA or TCA elements that might respond to salicylic acid (SA). The five genes
(
MeAnn4,
5,
8,
9 and
11) with a TGA box or TGA element might respond to auxin. The genes (
MeAnn1,
4 and
9) with an as-1 cis-element may respond to auxin, salicylic acid and methyl jasmonate
(JA). All the genes contain Myb, MYB, or MYB recognition sites, which indicates that
all MeAnns might be regulated by MYB transcript factors. All the
MeAnns except
MeAnn3 have Myc or MYC sites. Nine
MeAnns, except for
MeAnn9,
10 and
12, show ARE motifs, which are cis-acting regulatory elements important for anaerobic
induction.
MeAnn5,
8 and
9 contain a DRE core, which is a cis-acting element related to dehydration, low temperature,
and salt stress responses.
MeAnn1,
8,
9 and
11 contain an LTR motif, which is a cis-acting element involved in low temperature responsiveness.
MeAnn3,
5,
8,
9 and
11 contain an MBS, which is a MYB binding site related to drought-inducibility.
MeAnn4-10 and
12 contain one to four stress-responsive elements (STREs).
MeAnn2,
3 and
12 contain one to two cis-acting responsive elements related to defense and stress responsiveness
(TC-rich repeats). Except for
MeAnn9, all the other
MeAnns have at least one wound-responsive element (WRE3, W box or WUN motif). Among the
hormone-associated cis-elements, ERE is the most abundant and ABRE is second. Among
the stress-related elements, MYB- and MYC-associated cis-elements are the most abundant,
followed by wound-responsive elements.
Analysis of Tissue-Specific Expression Patterns of the 12 MeAnns
To evaluate the tissue specific expression levels of the
MeAnns, RNA-seq data were downloaded from NCBI and analyzed. The gene expression levels
of the 12
MeAnns were analyzed in a variety of the organs or tissues in cassava, such as leaves,
stems, fiber roots, storage roots, midveins, lateral buds, OES, FEC, petioles, RAM,
and SAM. The results, which are shown in a heatmap, indicated that some
MeAnns are expressed in all detected organs or tissues, suggesting that these genes may
have essential biological roles in cassava growth and development. For instance,
MeAnn1,
MeAnn2 and
MeAnn5 exhibited very high expression levels in all the tested organs or tissues. Their
expression in young tissues, such as RAM and SAM, is higher than in mature organs,
such as the leaves and roots. By contrast,
MeAnn12 exhibits very low levels in all of the tested organs or tissues. The other genes
exhibit varying expression patterns (Fig. 6). The expression patterns of
MeAnns in leaves and midveins are similar; aside from
MeAnn4 and
MeAnn12, the other
MeAnns are highly expressed.Moreover,
MeAnn expression patterns in lateral buds and s
hoot apical meristems are similar; aside from
MeAnn12, the other eleven
MeAnns are highly expressed. The
MeAnn expression patterns in fibrous roots are similar to that in root apical meristems,
which show lower expression levels. The
MeAnn expression patterns in stems and petioles are similar. The
MeAnn expression patterns vary in other organs or tissues such as somatic organized embryogenic
structures, friable embryogenic calli and storage roots (Fig. 6).
Examination of MeAnn gene expression under cold, drought and salt stresses
To uncover the changes in MeAnn expressionin response to cold, drought and salt stress, the relative expressions of the 12 MeAnns were analyzed by qRT-PCR under 4℃, 20% PEG6000 and 300 mmol/L NaCl treatments, respectively,
at different time points (from 0 to 48 h). The results indicated varied MeAnn expression profilesafter cold, drought, and salinity treatments (Fig. 7-9). Most obviously, MeAnn5 and 9 were more sensitive to the three stresses (Fig. 7-9).
When cassava seedlings were under 4℃ cold stress, MeAnn5, 6 and 9 were significantly upregulated in leaves; MeAnn1 and 10 were suppressed at all six cold stress stages; MeAnn2 was downregulated at all stages except 9 h; MeAnn3 was downregulated at 9, 12 and 48 h; MeAnn4 was upregulated at 3, 6 and 24 h; MeAnn7 was upregulated at 9 h and downregulated at 12 h; MeAnn11 was only significantly upregulated at 9 h; and MeAnn12 was downregulated at 9 and 12 h (Fig. 7). In shoots, MeAnn1 was downregulated at 9 and 12 h; MeAnn2 was upregulated at 9 and 12 h; MeAnn4 was upregulated at 9 h; MeAnn5 and 6 were upregulated at 9 h; MeAnn7 was downregulated at 24 and 48 h; MeAnn8 was downregulated at 12 h; MeAnn9 was upregulated at 6 h and downregulated at 9, 12 and 24 h; MeAnn10 was upregulated at 3, 9, 12, 24 and 48 h; MeAnn11 was markedly upregulated at 3 and 12 h; and MeAnn12 was upregulated at 6, 24 and 48 h. The differential expression of MeAnn3 was weaker at all the stages under cold stress (Fig. 7). In roots, MeAnn1, 9 and 12 were significantly downregulated under cold stress at all six stages. Moreover, MeAnn2 was upregulated at 12 h; MeAnn7 was upregulated at 12 and 48 h; MeAnn4 was downregulated at 9, 12, 24 and 48 h; MeAnn10 was downregulated at 3, 6, 9 and 24 h; and MeAnn11 was downregulated at 24 h cold stress. MeAnn3, 5, 6 and 8 showed weaker differential expression under cold stress than under control conditions
(Fig. 7).
When the cassava seedlings were under 20% PEG drought stress, in leaves, MeAnn4-6 and 9 were markedly upregulated, MeAnn2 and 10 were downregulated at all time points; MeAnn8 and 11 were upregulated at 3 h; the expression of MeAnn7 was markedly upregulated at 3, 6 and 12 h; MeAnn12 was upregulated at 12 h; and MeAnn1 was significantly downregulated at 9 and 48 h (Fig. 8). In shoots under drought stress,
MeAnn1, 5 and 9 were downregulated at all time points; MeAnn6, 7 and 10 wereupregulated at 3 h; MeAnn1 was downregulated and MeAnn10 was upregulated at 6 h; and all the other genes had weaker differential expression
at the six time points (Fig. 8). In roots, at 3 and 6 h, only MeAnn2 was distinctly downregulated; at 9 h, only MeAnn6 was distinctly upregulated; at 12 h, MeAnn2, 6, 7, 10 and 11 were upregulated and MeAnn9 and 12 were downregulated; and at 24 and 48 h, only MeAnn12 was distinctly downregulated (Fig. 8).
When the cassava seedlings were under 300 mmol/L NaCl stress, in leaves, MeAnn5-7 and 9 were markedly upregulated, MeAnn2 and 10 were downregulated at all time points; MeAnn4 was upregulated from 3-24 h; MeAnn11 expression was markedly upregulated at 24 and 48 h; and MeAnn1 was significantly downregulated at 12 h (Fig. 9). In shoots, all 12 MeAnns had weak differential expression at 3 and 48 h; MeAnn1, 7 and 9 were downregulated at 6 h; MeAnn4 and 12 were upregulated at 9 h; MeAnn1, 3-5, 8 and 9 were significantly downregulated and MeAnn7 and MeAnn10 were upregulated at 12 h; and MeAnn4 was upregulated at 24 h (Fig. 9). In roots, all the MeAnns had slightly lower differential expression compared to the control at 3 h; only
MeAnn1 was upregulated at 6 h; MeAnn4 and 7 were upregulated at 9 h; MeAnn1, 6, 7,and 9-11 were upregulated and MeAnn2 and 8 were downregulated at 12 h; MeAnn1, 7, 9 and 10 were upregulated at 24 h; and MeAnn1 and 9 were upregulated at 48 h (Fig. 9).
Examination of MeAnn gene expression in response to Ca2+ signaling and hormones by qRT-PCR
To reveal the responses of the MeAnns to Ca2+ signaling, the cassava seedlings were treated with different concentrations of CaCl2. The results indicated that MeAnn1, 2 and 10 were downregulated, while MeAnn5 and 9 were upregulated in leaves under all the CaCl2 treatments. MeAnn6 and 7 had similar expression patterns in leaves under CaCl2 treatments. MeAnn1 and 9 had similar expression patterns and were downregulated under all treatments in shoots.
MeAnn3, 4, 8 and 12 were downregulated under 20 mmol/L CaCl2 treatment in shoots. MeAnn1 and 9 as well as MeAnn4 and 12 had the same expression patterns in roots, which were downregulated under CaCl2 treatments. MeAnn2 was upregulated under 10, 20 and 50 mmol/L CaCl2 treatments in roots. MeAnn6 and MeAnn7 were upregulated in roots under 50 mmol/L and 10, 40 and 50 mmol/L CaCl2 treatments, respectively. MeAnn10 and 11 were upregulated under the 40 mmol/L CaCl2 treatment in roots. MeAnn3 and MeAnn8 were downregulated under 40 mmol/L and 20 and 30 mmol/L CaCl2 treatment in roots, respectively (Fig. 10).
In leaves under ABA treatment, MeAnn4 and 11 were upregulated and MeAnn10 and 12 were downregulated. MeAnn4-6 and 9 were upregulated and MeAnn2 and 10 were downregulated by GA and JA treatments. MeAnn4 and 6 were upregulated and MeAnn9, 10 and 12 were downregulated by SA treatments. In shoots, MeAnn9 was downregulated by ABA treatment. MeAnn7 and 8 were upregulated by GA treatment. MeAnn9 and 12 were upregulated by JA treatment. All the MeAnns in shoots were weakly responsive to SA treatment. In roots, MeAnn2 and 9 were downregulated, while MeAnn4, 6 and 8 were upregulated by ABA treatment. MeAnn9 was downregulated by the ABA, GA, JA and SA treatments. MeAnn1 was downregulated by JA treatment, MeAnn2 was downregulated by ABA treatment, MeAnn3 was upregulated by SA treatment, MeAnn4 was upregulated by ABA and SA treatments, MeAnn6 and 8 were upregulated by ABA treatment, and MeAnn11 was downregulated by SA treatment. MeAnn5, 7, 10 and 12 showed slight responses to the four hormones (Fig. 11). Among all the MeAnns, MeAnn9 showed an obvious up- or down-regulated response to most of the hormones in different
tissues.