Identification, Characterization and Expression Analysis Reveal the Potential Function of Annexin Genes in Response to Abiotic stress, Calcium and Hormones in Cassava (Manihot esculeta Crantz)

The calcium (Ca2+)-dependent phospholipid binding protein annexin gene family, which is known to be related to membrane lipid and cytoskeletal components, is involved in a diverse range of biological functions. However, in cassava (Manihot esculenta Crantz), no studies focusing on the roles of annexin genes in response to abiotic stresses, calcium, and hormones have been informed. 12 annexin genes were found and assigned to eight chromosomes in the cassava genome. All of the MeAnns contain a typical annexin domain with four 70-amino acid repeats. The MeAnns are classified into six groups in the phylogenetic tree. In their promoter regions, MeAnns possess at least 3 hormone response-related cis-elements and 1 abiotic stress response-related cis-acting element. MeAnn1, MeAnn2 and MeAnn5 exhibit very high levels of expression in each tested organs or tissues. By contrast, MeAnn12 exhibits very low levels in all the tested organs or tissues. qRT-PCR analysis indicates that both MeAnn5 and MeAnn9 have significantly high expression in leaves after cold, drought, and salt treatments and are highly responsive to CaCl2, GA and JA treatments. MeAnn2 and MeAnn10 are significantly downregulated in leaves by cold, drought and salt stress and negatively respond to CaCl2, GA and JA. The expression patterns of MeAnns under cold, drought, and salt stress are irregular in shoots. In roots, MeAnn1 and MeAnn9 are downregulated by cold, CaCl2 and JA treatments, while their other gene expression patterns are irregular. genes Our results laid All Myb, MYB, or MYB recognition sites, which all MeAnns 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 MeAnn s 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. To identify the annexin members in the cassava genome, the eight known annexins members AtAnn1-8 from A. thaliana were used as probes for BLASTP of the M. esculenta V6.1 database. The MeAnn protein sequences were further analyzed with the Pfam [60]and SMART[61] online tools. The physiological and biochemical characteristics of the MeAnn proteins, such as the molecular weight (MW), theoretical isoelectric point (pI), and hydrophilic mean (GRAVY), were analyzed with the ProtParam online tool [62]. The prediction of MeAnn protein subcellular location was performed with the CELLO2GO online tool[63]. GA μmol/L ABA for the The 30-day SC8 seedlings were individually in and mmol/L CaCl for h for the calcium treatments. The 30-day SC8 seedlings Each treatment three biological Collected cassava leaf, root and stem samples liquid until the follow-up experiments. amino acids; MW: molecular weights; GRAVY: grand average of PL: Protein length; MW: Molecular weight of the amino acid sequence; GRAVY: grand average of hydropathicity; pI: theoretical isoelectric point; II: Instability index; AI: Aliphatic index; S: stable; U: unstable; Cytop: cytoplasm; Mito: mitochondria; Nuc: nucleus; Chlo: chloroplast; and Plasm: plasma membrane; OES: the somatic organized embryogenics; FEC: the friable embryogenic calli; FR: the fibrous roots; SR: storage roots; RAM: the root apical meristems; and SAM: shoot apical meristems.

the foundation for further study of the function of cassava anesxin genes and provided an entry point for understanding the response mechanism of cassava to abiotic stress.

Background
As an evolutionarily conserved group, annexins are calcium (Ca 2+ )-dependent phospholipid binding proteins known to be related to membrane lipid and cytoskeletal components [1,2].The remarkable and special characteristics of these proteins function in membrane organization, vesicle trafficking and the Ca 2+ signaling pathway and are involved in a diverse range of cellular functions [1].Generally, annexins exist in almost all eukaryotes, among which plant annexins represent a monophyletic branch of homologs that evolved from green algae; however, some prokaryotes also have annexins [2].
Annexin multigene families have high diversity numbers across various organisms [3].The primitive variety of annexin proteins date back to 1-1.5 billion years ago in Giardia lambia (unicellular protist) [4].The annexin gene (Ann) was firstly detected in tomato over two decades ago [5], after which it has also been identified in Giardia lamblia [4], Capsicum annuum [6], Nicotiana tabacum [7], Triticum aestivum [8], Arabidopsis thaliana [9,10], Brassica juncea [11] and Oryza sativa [12].Among these, annexin genes in A. thaliana, B. juncea and O. sativa have been identified and well-characterized.So far, studies have shown that these plants have relatively large and diverse annexin families, whereas the original low single-cell plants, such as Micromonas and Ostreococcus,have small families [13,14].In vertebrates, low annexin A1 expression has been predicted to have a role in the induction of chemotherapy in oral cancer patients [15].The annexin gene Ann2 in vertebrates is a multipotent calcium and anionic phospholipid-binding protein that is found to play a considerable role in endocytosis, exocytosis, ion channel conduction, membrane tissues and other processes [16].During nodule formation in Medicago truncatula, annexins perform important functions in the generation and transmission of calcium signals [3].A previous study has indicated that the GbAnn6 interacted with Actin1 toregulating the fiber elongation in cotton [17].AtAnn1 from A. thaliana is significant for regulating H 2 O 2 -induced root calcium signaling [18].
Structurally, almost all annexins have four to eight repeats of a 60-70 amino acid motif [12].It has been reported that annexins consist of four annexin motif repeats, with the conserved GxGT-(38 residues)-D/E calcium binding sequence exist in the first and fourth motif repeats [3].Compared with vertebrates, annexins from plants have a short Nterminal region and lack calcium binding sites (CBS) in their second and third motif repeats [3].Reports has also demonstrated the presence or absence of motifs that play key roles in calcium channels, peroxidases, and ATPase/GTPase activity [19].From the phylogenetic point of view, plant annexins and animal annexins are independent populations [3,19].
In recent studies, plant annexins have been reported to be expressed in many diverse tissues during plant development [9,12].Tissue-specific expression profiles of plant annexin families have been observed in plants such as A. thaliana [9], triticum astivum [20], and O. sativa [12].AtAnn1 is expressed in all examined tissues, but is richest in stems, while AtAnn2 is most abundant in roots [9].Additionally, many studies have reported the functions of annexins in response to abiotic stresses.Abiotic stresses start with stress perception and then lead to the activation of gene expression through signaling pathways, consequently influencing plant physiology, growth and development [21,22].It has been reported that some annexin genes related to membrane organization are involved in abiotic stress tolerance [20].For instance, overexpression of AtAnn1 from A. thaliana inplants enhanced drought tolerance, while AtAnn1 knockout in plants reduced drought tolerance [10].AtAnn1 and AtAnn4 from Arabidopsis can interact with one another and are adjusted to response to stresses such as aridity and salt [23].Expression analysis also revealed differential annexin expression patterns in O. sativa seedling stages under diverse abiotic stresses such as salt, drought, cold and heat [12].
In the tropical and subtropical regions of the world, cassava (Manihot esculenta Crantz) is generally known as an essential food source for at least 500 million people [24,25].
Recently, many studies have focused on increasing its productive yield and understanding the effects of abiotic stresses such drought and infertility [26].In this present study, twelve MeAnn annexin genes from cassava have been identified.The conserved domains, motif distribution, evolutionary relationships, gene structures, and chromosomal localization of the MeAnn genes were inquired into.To identify the possible roles of MeAnns in cassava, the cis-elements on the MeAnn promoters were predicted.The differential expression of MeAnns under abiotic stresses, such as salt, cold and drought stresses, and during calcium ion and hormone signaling were examined by qRT-PCR in cassava.These results will help to ulteriorly reveal the important biological functions of MeAnn genes in cassava.
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).

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 stressrelated 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 ciselements, 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 one to four stress-responsive elements (STREs).MeAnn2, 3 and 12 contain one to two cisacting 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).
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).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.
Previous studies have also demonstrated that plant annexins show dynamic and reversible distribution profiles, with their distributions changing between the membrane and cytoplasm under different stress conditions [43][44][45][46].In this study, the subcellular localization of MeAnns were predicted to have five sites including the cytoplasm, mitochondria, nucleus, chloroplast, and plasma membrane.Here, our results are in partial agreement with previous results.
Sequence analysis of the annexin amino acid sequences from cassava shows that the four repeats and some conserved motifs are present in MeAnns.There are two type II calciumbinding sites in the first and fourth annexin repeats, an IRI motif related to actin binding in the third repeat, and a DXXG motif related to GTPase activity in the fourth repeat.
These results show the consistency of the annexin protein domains among cassava and other plants reported in other studies (Fig. 2) [12].It has been reported that all annexins have a core domain that is composed of four similar approximately 70-amino acid repeats [3].The C-terminal region of the vertebrates annexin contains 4 motif repeats that have a greatly conserved sequence (GxGT-[38 residues]-D/E) for Ca 2+ binding [3].It has been demonstrated that cotton ANXD36 has higher GTPase activity than ATPase activity and is inhibited by calcium due to the presence of the GTPase activity site, such as the DXXG in the fourth repeat [47].All plant annexins belong to soluble proteins and contain greatly conserved calcium-binding domains and diverse N-terminal regions and lack the long Nterminal regions compared to vertebrates [20].Analysis of the full-length MeAnn genomic DNA sequences showed a variety of MeAnn gene structures with different numbers of exons and introns (Fig. 4B).Similar results are also found in other plants; for example, AtAnn1 has two introns, AtAnn6 and 7 have three introns, AtAnn2 has four introns and other AtAnns have five introns [5,6].Previous studies have indicated that multiple annexin genes could be locate on one chromosome.For example, in Arabidopsis, 4 annexin genes are located on chromosome 5 [38]; in rice, two annexins are locate on chromosome 5 and three are located on chromosome 9 [38]; in poplar, three annexin genes are located on chromosome 1 [38]; and in Zea mays, three annexin genes, MeAnn7-9, arelocated on chromosome 6 [39].In cassava, both chromosomes 4 and 11 contain three annexin genes (Fig. 1).Thus, the conserved domain and the DNA structure reveal that MeAnns from M. esculenta belong to the annexin multifamily.
By analyzing the cis-acting elements present in the promoters, the possible biological functions of genes can be roughly inferred.In this study, the results indicate that MeAnn promoter regionspossess at least 3 hormone response-related cis-elements and 1 stress response-related cis-element [Fig.5].The stress-or hormone-related cis-acting elements also have been reported to exist in other annexin gene promoters.In maize annexin gene promoters, numerous of cis-elements, including ABRE, DRE, and LTRE, related to abiotic stress responses were found [39].Hormone-related cis-elements, such as ABA, GA, or auxin recognition sites, and stress-related cis-acting elements, such as MYC or MYB recognition sites, were also found in the promoters of tomato annexins [33].Hormone or stress treatments inducing the expression of annexin genes have been reported in Indian mustard [11], tomato (Solanum pennellii) [32], Arabidopsis [43,48], and alfalfa [49].The expression of annexin genes induced by auxin [50], SA [51], and methyl jasmonate (JA) [11] have also been reported.Here, at least one stress treatment was found to enhance the expression of several cassava annexin genes (Fig. 7-11).For instance, the expression of MeAnn4, MeAnn6, and MeAnn8 in roots as well as MeAnn4 and MeAnn11 in leaves was highly enhanced by ABA treatment (Fig. 11).In cassava leaves, the expression of MeAnn4-7 and MeAnn9 was highly enhanced by GA and JA (Fig. 11), while the expression of MeAnn4 and MeAnn6 was enhanced by SA treatment (Fig. 11).However, it is hard to speculate the exact expression characteristics of a particular gene based only on cis-acting element analysis.For instance, nine MeAnn promoter regions contain putative ABA-responsive elements.However, in this study, only MeAnn4, MeAnn6, MeAnn8 and MeAnn11 were significantly enhanced by ABA (Fig. 5 and Fig. 11).JA-related cis-elements are found in the promoters of three MeAnns, MeAnn1 , 4 and 9.However, the expression of only five MeAnns, MeAnn4-7 and MeAnn9, were highly enhanced by JA (Fig. 5 and Fig. 11).GArelated cis-elements are found in the promoters of seven MeAnns, though the expression of only five MeAnn swere highly enhanced by GA (Fig. 5 and Fig. 11).Eight MeAnns contained SA-related cis elements, but the expression of only three MeAnn genes were highly induced by SA (Fig. 5 and Fig. 11) .Similar results were also reported in tomato annexins [33].Therefore, it is necessary to verify the functions of genes through detailed experiments.
The expression patterns of annexins in plant tissues or organs have been reported in a number of plant species.For instance, in Arabidopsis, AtAnn1 was demonstrated to specifically express in mature pollen and germinated pollen [52,53].In M. trancatula, MtAnn2 has been indicated to express in arbuscule-containing cells in mycorrhizal roots [54].In Solanum lycopersium, SlAnn4 is specifically expressed during fruit ripening, SlAnn6 is specifically expressed in the stigma or ovary, and SlAnn8 is specifically expressed in the stamen [33].In Tintorera red grapes, Annexin D1 was observed to express in fresh and raised grape fruits [55].In soybean , annexin was differentially expressed in suspension-cultured soybean cells [56].In our study, the expression patterns of MeAnns in 11 cassava tissues were detected [Fig.7].The heatmap showed that MeAnn1, MeAnn2 and MeAnn5 exhibit very high levels of expression in all the tested organs or tissues.By contrast, MeAnn12 exhibits very low levels in whole of the detected organs or tissues.In leaves and midveins, all the MeAnns except MeAnn4 and MeAnn12 are highly expressed.In lateral buds and s hoot apical meristems, all the MeAnns except MeAnn12 are highly expressed.The MeAnn expression patterns in fibrous roots are similar to those in root apical meristems, showing that their expression levels are lower than in other tissues.The MeAnn expression patterns in stems and petioles are also similar.By contrast, MeAnn expression patterns are differential in the remaining organs or tissues, such as OES (somatic organized embryogenics), FEC (friable embryogenic callus) and SR (storage roots) (Fig. 6).
Gene expression of annexin genes, in response to abiotic stresses such as drought, salt and cold, have been studied in some plants [27,44,48,57].In M. sativa, the annexin MsAnn2 was observed to respond to salt and drought stresses [48].Arabidopsis transcriptome analysis reported that annexins were upregulated under salt, cold and osmotic stresses [57].This analysis showed that AtAnn1i AtAnn6 and AtAnn8 showed significantly increased expression in Arabidopsis under salt and dehydration stresses [27].
Subsequently, AtAnn1 was intensively studied to reveal its major role in drought stress tolerance [10].AtAnn1 and AtAnn4 were also revealed to interact with one another and in a light-dependent manner to regulate salt and drought stresses [23].The role of AtAnn8 in Arabidopsis salt and dehydration tolerance was also confirmed in a recent study [58].In wild tomato, researchers have demonstrated that SpUSP functions in association with SpAnn2 in drought stress signaling [59].Overexpressing SpAnn2 could enhance the salt and drought tolerance of transgenic tomato plants [34].In rice, OsAnn1 was reported to be associated with heat stress [44], while OsAnn3 was reported to be associated with cold stress [36].In our study, MeAnn5 and MeAnn9 in leaves were significantly upregulated by cold, drought and salt stresses (Fig. 7-9).Interestingly, the MeAnn expression patterns under cold stress were more similar to the expression patterns under CaCl 2 than under drought and salt stresses (Fig. 7-10).MeAnn5, 6 and 9 were significantly upregulated, while MeAnn1 and 10 were downregulated at all six time points during cold stress to cassava leaves.In roots, MeAnn1, 9 and 12 were significantly downregulated under cold stress at all six time points (Fig. 7).MeAnn4-6 and 9 were significantly upregulated, while MeAnn2 and 10 were downregulated under PEG-induced drought stress in leaves (Fig. 8).
MeAnn5-7 and 9 were significantly upregulated, while MeAnn2 and 10 were downregulated under salt stress from 3-48 hours in cassava leaves (Fig. 9).Unlike the MeAnn gene responses to low temperature, no increase was observed in the transcription of any rice annexin genes in response to low temperature treatment, although decreased relative expression of OsAnn2 and OsAnn10 was observed in the untreated control [12].In wheat, Breton et al. (2000) identified that there are four annexins that respond to low temperatures; they also showed their association with the membrane, but that this interaction was calcium-independent [8].Gene expression of Annexin genes in response to drought has also been studied in Arabidopsis, which was also subject to oxidation-driven post-translational regulation [10].Compared with the untreated control, the expression of OsAnn6 and OsAnn7 showed significant upregulation in response to NaCl treatment, while the expression of OsAnn1 and OsAnn10 showed a significant decrease under the same salt treatment [12].The comparative discussion of expression patterns among cassava and other plants indicates that MeAnns from cassava can respond to abiotic stresses, though with specific patterns for each gene, which are similar with other plants.

Conclusions
In this paper, annexin genes were searched in the cassava genome, systematically characterized and examined for their relationships and expression analysis.Twelve MeAnn genes were identified in the cassava genome.All MeAnns contain typical annexin domains.

Identification and Characterization of MeAnns from Cassava
To identify the annexin members in the cassava genome, the eight known annexins members AtAnn1-8 from A. thaliana were used as probes for BLASTP of the M. esculenta V6.1 database.The MeAnn protein sequences were further analyzed with the Pfam [60]and SMART [61] online tools.The physiological and biochemical characteristics of the MeAnn proteins, such as the molecular weight (MW), theoretical isoelectric point (pI), and hydrophilic mean (GRAVY), were analyzed with the ProtParam online tool [62].The prediction of MeAnn protein subcellular location was performed with the CELLO2GO online tool [63].

Analysis of the Location and Distribution of MeAnns on Cassava Chromosomes
The chromosomal localization of each MeAnn gene and the chromosome sizes were obtained from the cassava open genome database (Bioproject: PRJNA234389).Then, the Mapinspect software (Mike Lischke, Berlin, Germany) was utilized to map the chromosomal localizations of the MeAnn genes.The Photoshop software CS (San Jose, CA, USA) was utilized to beautify and enrich the image.

MeAnn Phylogenetics, Structures and Motifs
The annexin protein sequences from cassava, Arabidopsis, rice and tomato were collected and multi-aligned with ClustalW.Then, a phylogenetic tree was produced using the MEGA7.0software and based on the neighbor-joining method [64].The AtAnn and MeAnn gene structureswere determined with the GSDS online software [65].The MeAnn motifs were identified with the MEME Suite 4.12.0 online tool [66].

Analysis of the MeAnn Promoter Cis-Elements
To understand the cis-element distribution in the MeAnn promoters, 2000-bp sequences upstream of the initiation codon (ATG) of each MeAnn genewere downloaded from the public cassava genome database.The cis-elements in the MeAnn gene promoters were identified by searching the PlantCARE database website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).The data were counted and processed with Microsoft Office Excel (One Microsoft Way, Redmond, WA98052-6399) to create tables and graphs.

MeAnn Expression Patterns in Cassava
To study the expression levels of the MeAnns in different organs and tissues of cassava, the expression data for the 12 MeAnns were downloaded from the public RNA-seq data of cassava (Bioproject ID PRJNA324539).The eleven different organs and tissues in cassava included the leaf, petioles, midveins, stems, shoot apical meristems (SAMs), lateral buds, fibrous roots, storage roots, root apical meristems (RAMs), somatic organized embryogenics (OES), and friable embryogenic calli (FEC).The heatmap was created with the OmicShare tools online (http://www.omicshare.com/tools).

Plant Materials and Treatments
The cassava cultivar SC8 was supplied by Cassava Research Center of the Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Science.Thirty-daycultivated SC8 cassava seedlings were individually placed in a 4℃ incubator for cold stress, in 300 mmol/L NaCl solution for salt stress, and in a 20% PEG-simulated dry environment for drought stress treatments.The samples were harvested at the treatment stages of 0, 3, 6, 9, 12 and 24 h, separately.Total RNA isolation and qRT-PCR analysis A RNAplant plus (TIANGEN, China) was used to extract total RNA from cassava.A PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan) was used to synthesize the first cDNA chain.qPCR SYBR Premix Ex Taq II (Takara, Japan) was used to conduct qRT-PCR analysis in a ABI 7900HT Fast Real-Time PCR System (ABI, USA).The relative expression levels of the MeAnn genes were evaluated by the 2 − △△Ct method and normalized by log2 values.The normalized relative expression data were used to construct a heatmap.Three technical replicates were settled.The qRT-PCR primers are listed in Table S2      The stress-and hormone-related cis-acting elements in the cassava MeAnn gene promoters.

4 and 9 )
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, 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 2+ signaling and hormones by qRT-PCRTo reveal the responses of the MeAnns to Ca2+ signaling, the cassava seedlings were treated with different concentrations of CaCl 2 .The results indicated that MeAnn1, 2 and 10 were downregulated, while MeAnn5 and 9 were upregulated in leaves under all the CaCl 2 treatments.MeAnn6 and 7 had similar expression patterns in leaves under CaCl 2 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 CaCl 2 treatment in shoots.MeAnn1 and 9 as well as MeAnn4 and 12 had the same expression patterns in roots, which were downregulated under CaCl 2 treatments.MeAnn2 was upregulated under 10, 20 and 50 mmol/L CaCl 2 treatments in roots.MeAnn6 and MeAnn7 were upregulated in roots under 50 mmol/L and 10, 40 and 50 mmol/L CaCl 2 treatments, respectively.MeAnn10 and 11 were upregulated under the 40 mmol/L CaCl 2 treatment in roots.MeAnn3 and MeAnn8 were downregulated under 40 mmol/L and 20 and 30 mmol/L CaCl 2 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

The
MeAnns were classified into six groups.All the MeAnns contain hormone or stress response-related cis-elements in their promoter regions.MeAnn1, MeAnn2 and MeAnn5 exhibit very high levels of expressions in all the detected organs or tissues.By contrast, MeAnn12 exhibited very low levels in all the detected organs or tissues.Expression pattern analysis under abiotic stresses suggests that MeAnn5 and MeAnn9 have positive responses to abiotic stresses and calcium signaling and are induced by GA and JA in cassava leaves, while MeAnn2 and MeAnn10 have the opposite expression patterns under the same stresses.The expression patterns of MeAnns under abiotic stresses are irregular in shoots.In roots, MeAnn1 and MeAnn9 have the similar expression patterns, as both are downregulated by cold, CaCl 2 and JA treatments, while the remaining genes display irregular expression patterns.These results will promote the subsequent functional investigations and the utilization of MeAnns in cassava and other plants.
The 30-day SC8 seedlings were individually placed in 50 μmol/L IAA solution, 2 mmol/L SA solution, 100 μmol/L GA 3 solution, 100 μmol/L JA solution, and 100 μmol/L ABA solution for 12 h for the hormone treatments.The 30-day SC8 seedlings were individually placed in 10, 20, 30, 40 and 50 mmol/L CaCl 2 for 12 h for the calcium treatments.The 30-day SC8 seedlings treated with H 2 O were employed as blank controls.Each treatment was performed with three biological replicates.Collected cassava leaf, root and stem samples were outright frozen in liquid nitrogen and then saved at -80 ℃ until the follow-up experiments.

Figures Figure 1
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Figure 2 Multiple
Figure 2

Figure 6 Gene
Figure 6

Figure 7 Expression
Figure 7

Figure 8 Expression
Figure 8

Figure 9 Expression
Figure 9

Figure 10 Expression
Figure 10

Table 1 .
. Ann:annexin gene; CBS: calcium binding sites; AA : amino acids; MW: molecular weights; GRAVY: grand average of hydropathicity; PL: Protein length; MW: Molecular weight of the amino acid sequence; GRAVY: grand average of hydropathicity; pI: theoretical isoelectric point; II: Instability index; AI: Aliphatic index; S: All of the authors consent to publish this article.preparation of the studied materials, and qRT-PCR.YS, YY, JL and SF worked on primer design, and technical and informatics analyses of these genes.XH and JG were responsible for the programs and all experiments, critically revised the manuscript, and provided the final approval of the article.A list of the twelve MeAnns identified in this study.