1. Introduction
Plastids compose a metabolically varying group of organelles, which originated from endosymbiosis of an ancient cyanobacterium [
1]. The chloroplast is the most conventional and best studied type of plastid, which is responsible for photosynthesis in all photosynthetic eukaryotes [
2]. In addition, many non-photosynthetic plastids in plants perform other critical metabolic functions. For instance, the amyloplast, as one of the high differentiated plastids, is related to starch synthesis and storage in sink tissues, which is important for starch crops, such as cassava and potato. Starch granules that are synthesized within the amyloplast compartment are affected not only by enzyme activities required for sucrose metabolism and starch synthesis [
3], but also by amyloplast division processes. Chloroplast division processes and mechanisms are well researched and understood. Several proteins, including FtsZ (filamenting temperature-sensitive Z) [
4,
5,
6,
7], ARC6 (accumulation and replication of chloroplasts 6) [
8,
9], ARC5 (accumulation and replication of chloroplasts 5) [
10], PDV1 (plastid division 1), and PDV2 (plastid division 2) [
11] form at least three contractile components in the plastids division site as follows: first assembled FtsZ ring (Z ring), ARC5 ring, and plastid-diving (PD) ring [
12]. The first assembled Z ring is formed by FtsZ. The FtsZ protein serves as an essential scaffold to recruit the other plastid division related proteins and to generate contractile force by its guanosine triphosphatase (GTPase) for plastid division.
FtsZ genes have been found in various species of Viridiplantae, including many plants and algae, and these genes are clustered into two families, named as FtsZ1 and FtsZ2 [
6,
13,
14]. Genetic analysis in
Arabidopsis has showed that the FtsZ1 and FtsZ2 families have different functions in chloroplast division [
15]. Recent studies have shown that the members of the FtsZ2 family have a closer relationship with their cyanobacterial counterparts than those of the FtsZ1 family, and these studies shown that FtsZ1 has a short N-terminal peptide similar to that of bacterial FtsZs. In comparison to FtsZ1, FtsZ2 lacks the N-terminal peptide [
2,
16]. Previous studies have demonstrated that FtsZ1 was probably evolved by the duplication of FtsZ2 in the evolution of green algae [
17].
The maintenance and divergence of the two FtsZ families (FtsZ1 and FtsZ2) in diverse Viridiplantae suggests that they evolved to have distinct and critical functions in different plastid divisions, such as chloroplast division and amyloplast division. The functions of FtsZ families on chloroplast division have been well researched and understood [
2,
18]. However, the mechanism of amyloplast division has less studied and understood. For instance, it has been previously reported that the transgenic potato resulting from the transformation of the
StFtsZ1 gene under the control of the GBSS promoter has increased StFtsZ1 protein levels with altered pasting properties and phosphate content in tubers, which result in less but larger starch granules [
19]. Yun and Kawagoe reported that the starch granules in the endosperm of
arc5 mutant rice (
Oryza sativa) are smaller in size and have a significantly higher starch gelatinization peak temperature than those in the wild-type rice [
20]. Rice FtsZs play important roles in starch granule synthesis [
21].
Presently, an important consideration in cassava breeding is to increase yield and starch quality by focusing on the expression and regulation of key enzymes in the starch biosynthesis pathway. Amyloplast is the main sink organelle for starch grain synthesis and stores in cassava, and its proliferation directly affects the amount and accumulation of starch granules [
22]. The morphology, size, and number of amyloplasts can be altered by regulating plastid division elements, which probably change the distribution, dispersion, and arrangement of starch granules, thus affecting starch quantity and quality. Most studies on FtsZs have focused on the regulation of chloroplast division [
23]. FtsZs have been shown to mediate the division of the starch-storing amyloplasts in potato [
19], but the mechanism is not completed understood. For example, many studies have shown that plant hormones participate in development of storage organ and improve crop yields (e.g., Cassava storage root and potato tuber); it is unclear the way in regard to the amyloplast division affected by plant hormones.
In this study, three MeFtsZs were cloned from cassava. The phylogenetic and structural analyses of the predicted MeFtsZ proteins were studied. Moreover, the subcellular fate was confirmed through MeFtsZ transient expression experiments in tobacco leaves. The expression profiles of MeFtsZs in cassava plant organs and tissues; or during storage roots development were examined using qRT-PCR. The functions of MeFtsZ2-1 and MeFtsZ2-2 in plastid division were identified. The expressions of MeFtsZs were investigated in response to phytohormones. These results are the basis for further studies on MeFtsZs and their role on the division of cassava starch-storing amyloplasts.
4. Discussion
Three full-length cDNAs of MeFtsZs from cassava were cloned in this study. Alignment analysis of the three
MeFtsZ sequences showed that the two conserved domains (FTSZ-1 and FTSZ-2) reported in the
FtsZ genes were found in all three of the
MeFtsZs [
27]. Phylogenetic analysis showed that MeFtsZ2-1 and MeFtsZ2-2 are highly homologous to each other, sharing 87.84% amino acid identity, and with similar genomic structures of seven exons and six introns. However, MeFtsZ1 is lower percentage of sequence similarity to MeFtsZ2-1, MeFtsZ2-2, and with six exons and five introns (
Figure 2). The different exon-intron structures have been reported in
AtFtsZs from
Arabidopsis [
15]. Phylogenetic analysis showed that the MeFtsZ proteins from cassava were grouped to two clades (FtsZ1 and FtsZ2), in which MeFtsZ1 with a prepeptide was classified into FtsZ1 clade; and, MeFtsZ2-1 and MeFtsZ2-2 without a prepeptide were classified into FtsZ2 clade (
Figure 3). These results are consistent with the findings in other plants [
17,
28]. In plants, FtsZs can be divided into two families according to the presence of the plastid guide peptide, FtsZ1 is located in the plastid inner membrane with the presence of a prepeptide, and the FtsZ2 is located in the plastid outer membrane, with the absence of a prepeptide [
6]. The localization of FtsZ is closely connected to their function. MeFtsZ1, MeFtsZ2-1, and MeFtsZ2-2 could form a ring in tobacco chloroplasts (
Figure 6) suggests that MeFtsZs could be assembled into the “Z ring” and function in the plastid division in cassava plants.
The 3D images of the hypothetical structures of MeFtsZs indicate that all of the three MeFtsZ proteins have an N-terminal GTPase domain and a C-terminal αβ-domain, which are connected by a central α-helix. Furthermore, the two conserved domains, FTSZ-1 and FTSZ-2, are found to be similar among all the three MeFtsZ proteins. The structures of the conserved domains are found to be closely integrated with GTP (
Figure 5). The hypothetical structures of MeFtsZs are consistent with the findings of the previous studies [
29]. Many studies have shown that the bacterial FtsZs undergo in vitro GTP-dependent assembly into homopolymers. The reaction between the FtsZ subunits forms the GTPase active site, and the polymerization of subunits stimulates GTP hydrolysis. This cycle pattern sustains Z-ring constriction and remodeling [
2,
30,
31]. Similarly, plant FtsZs are capable of GTP-dependent homopolymerization and assembly-stimulated GTPase activity, in vitro [
27,
32,
33,
34]. Because, the structures of the cassava MeFtsZs are similar to those in other species, it indicates that they can be assembled into the “Z ring” in the cleavage site of plastid and generate contractile force by the GTPase activity for cassava plastid division.
In Arabidopsis, both deletion and overexpression of
AtFtsZ1 or
AtFtsZ2 cause dose-dependent defects in chloroplast division, and these changes result in a reduction in chloroplast number and an increase in chloroplast size [
6,
35]. It has been suggested that the expression levels of
AtFtsZ1 or
AtFtsZ2 may be critical for their functions in vivo. Thus, the tissue-specific expression patterns of the
MeFtsZ genes may provide a basis for understanding their functions during cassava plant development. The expression analysis by qRT-PCR showed that the
MeFtsZ genes were expressed in all of the tested nine organs or tissues (young and mature leaves, stems, fibrous roots, storage root phloems, storage root xylems, male flower, female flowers, and fruits). The
MeFtsZs were highly expressed in leaves, especially in younger leaves. Subsequent analysis found that the reduced expression levels of
MeFtsZ2-1 and
MeFtsZ2-2 were found in the mature leaves (
Figure 7A). Thus, the
MeFtsZ genes were highly active in the organs that are rich in chloroplasts, and where chloroplasts more actively divide. In non-photosynthetic tissues, the expression patterns of
MeFtsZ genes were similar: highest expression of
MeFtsZ2-1, moderate expression of
MeFtsZ2-2, and lowest expression of
MeFtsZ1 were detected in these tested non-photosynthetic tissues. These data indicated that the three MeFtsZ proteins are more active in cassava leaves for chloroplast division than in non-photosynthetic tissues for plastid division. Amyloplasts originally developed from one type of plastid, and their divisions cause the accumulation of starch in non-photosynthetic organelles in the sink organs. During the process of storage root development and starch accumulation in cassava, the expression levels of
MeFtsZ2-1 and
MeFtsZ2-2 were higher than that of
MeFtsZ1, in which
MeFtsZ2-1 and
MeFtsZ2-2 were comparatively more active at the 135 days after planting when cassava storage roots were in the expanding stage where amyloplasts actively divided and accumulated more starch. These data suggested that more MeFtsZ2-1 and MeFtsZ2-2 might be beneficial for amyloplast division during storage root development in cassava.
In order to identify the function of
MeFtsZ2-1 and
MeFtsZ2-2 in plastid division, these two genes were transformed into
A. thaliana. The results showed that the transgenic plants contained abnormally shape, fewer number, and larger volume chloroplasts (
Figure 10 and
Figure 11). It has been found that maintaining the balance of plastid division-related gene expression is beneficial to the normal division of plastids [
15,
19,
36]. MeFtsZ2-1 and MeFtsZ2-2 are distributed in the chloroplasts in a dot or a filament manner in order to interfere with the normal chloroplast division in transgenic
Arabidopsis (
Figure 11). Our results demonstrate that MeFtsZ2-1 and MeFtsZ2-2 are involved in the plastid division of plants. However, the mechanisms of
MeFtsZ genes regulate the divisions of chloroplasts and amyloplasts in the source organs (leaves) and sink organ (storage roots) need further study.
Chromoplasts are plastids that produce and store pigments, such as carotenoid. Raise the content of carotenoid in cassava storage roots will improve nutritional and health benefits of cassava. Analyzed chromoplasts from 23 landraces that have different carotenoid content showed that chromoplast number and size would affect the amount of carotenoids in cassava storage roots [
37,
38]. Study the expression of
MeFtsZ genes in different carotenoid content of cassava storage roots may help us to further understand the relationship between chromoplast division and
MeFtsZ genes.
Phytohormones play the essential roles in cassava storage root initiation and development [
39]. The contents of IAA and GA were increased at expanding stage, and then slightly reduced at mature stage in the cassava storage roots, while the content of ABA was gradually increased during the cassava storage root development. Thus, it was clear that IAA, GA, and ABA were involved in the phase transition and development of cassava storage roots [
40]. Further, it was found that GA could enhance the mRNA levels of the invertase and sucrose synthase in plants [
41]. ABA signaling transduction could regulate the expression of the starch branching enzyme (SBE) gene in cassava storage roots [
42]. The addition, of SA and MeJA to culture medium could result in raising the number and weight per shingle plant potato micro tuber [
43]. In this study, the expressions of
MeFtsZ genes were regulated by IAA, GA, ABA, SA, and MeJA; whereas, the expression models of the
MeFtsZ genes in response to hormone treatment in cassava seedling roots were different.
MeFtsZs were up regulated and reached a peak after 12 h hormone treatment in aerial organs;
MeFtsZ1 was quickly increased and reached a peak before 6 h in roots. These results indicated that phytohormones were involved in regulating the expressions of
MeFtsZ genes in aerial organs and storage roots of cassava.