Open Access

Analysis of the myosins encoded in the recently completed Arabidopsis thaliana genome sequence

Genome Biology20012:research0024.1

DOI: 10.1186/gb-2001-2-7-research0024

Received: 3 March 2001

Accepted: 21 May 2001

Published: 3 July 2001

Abstract

Background

Three types of molecular motors play an important role in the organization, dynamics and transport processes associated with the cytoskeleton. The myosin family of molecular motors move cargo on actin filaments, whereas kinesin and dynein motors move cargo along microtubules. These motors have been highly characterized in non-plant systems and information is becoming available about plant motors. The actin cytoskeleton in plants has been shown to be involved in processes such as transportation, signaling, cell division, cytoplasmic streaming and morphogenesis. The role of myosin in these processes has been established in a few cases but many questions remain to be answered about the number, types and roles of myosins in plants.

Results

Using the motor domain of an Arabidopsis myosin we identified 17 myosin sequences in the Arabidopsis genome. Phylogenetic analysis of the Arabidopsis myosins with non-plant and plant myosins revealed that all the Arabidopsis myosins and other plant myosins fall into two groups - class VIII and class XI. These groups contain exclusively plant or algal myosins with no animal or fungal myosins. Exon/intron data suggest that the myosins are highly conserved and that some may be a result of gene duplication.

Conclusions

Plant myosins are unlike myosins from any other organisms except algae. As a percentage of the total gene number, the number of myosins is small overall in Arabidopsis compared with the other sequenced eukaryotic genomes. There are, however, a large number of class XI myosins. The function of each myosin has yet to be determined.

Background

Movement of eukaryotic cells, intracellular transport, signaling, cell division and cell shape are functions of the cytoskeleton [1,2,3,4]. The cytoskeleton is made up of three types of filaments: actin filaments, intermediate filaments and microtubules. Three groups of proteins called molecular motors utilize energy from the hydrolysis of ATP to move in association with the cytoskeleton: kinesins, dyneins and myosins [1,5,6]. Kinesins and dyneins move along microtubules [5,7] and actin is utilized by myosin for motility [8,9].

Molecular motors in non-plant systems have been extensively characterized but less is known about the presence and functions of these motors in plant cells. Using antibodies to mouse dynein, two 400 kDa proteins were identified in tobacco pollen during pollen germination [10] suggesting the presence of dynein in pollen tubes. To date, no report has been published on the presence of dynein at the molecular level. Using animal dynein sequences to search the Arabidopsis database TAIR (The Arabidopsis Information Resource) [11], no sequences similar to heavy or intermediate chains were found. However, some sequences showing similarity to light chains are present in the database. Kinesins have been identified in Arabidopsis and other plant systems [12,13,14,15,16] and their movement along microtubules has been analyzed [16,17,18,19]. Kinesins are a superfamily of molecular motors containing at least nine subfamilies [7,20]. Plant kinesins are represented in all but two of the families. Using the amino-acid sequence of the motor domain of a plant kinesin, a search of the Arabidopsis genome yielded 61 kinesin-like proteins [21]. This is the largest number of kinesins in an organism per thousand genes compared to yeast, Drosophila melanogaster and Caenorhabditis elegans.

Phylogenetic analysis of known myosins in various organisms has resulted in the classification of myosins into several groups. The Myosin Home Page (MHP) [22] has a phylogenetic tree with 143 myosins classified into 17 classes. However, an analysis of the myosin superfamily in Drosophila, concluded that two new mammalian myosins and a Drosophila myosin make up a new class of myosins, class XVIII [23]. These myosins have a unique amino-terminal PDZ domain. The classes have been named according to the order in which each class was first discovered except for myosins I and II. Myosin II is the conventional myosin, which was discovered 60 years ago [8]. The next myosin identified was myosin I and then in order of class name. Myosins have three domains in common; a motor domain that interacts with actin and binds ATP, a neck domain that binds light chains or calmodulin and a tail domain. The tail domain varies by class. Phylogenetic analysis is often based on the motor domain of the myosins. However, using the full-length sequence results in nearly the same grouping, indicating that the heads and tails have evolved together [23,24,25,26]. A study using the head (motor domain), neck and tail domains separately for phylogenetic analysis or the head and neck/tail showed that this is generally true [27]. The neck domain consists of one or more helical sequences termed the IQ motif, which has the consensus sequence IQXXXRGXXXR [28]. The IQ motif binds the conventional myosin II light chains and calmodulin or calmodulin-like proteins in other myosins [29]. Unlike most calmodulin-binding proteins, myosins bind calmodulin in the absence of Ca2+.

As actin is utilized by myosin for motility, the possible functions of myosin in plants are closely linked to the functions of actin. The actin cytoskeleton has been shown to be involved in many processes in plants including transportation, signaling, cell division, cytoplasmic streaming and morphogenesis [2,3]. Much of the cytoplasmic streaming work has been done in algal cells and the direct involvement of actin and myosin has been shown [30,31]. Genetic, biochemical and cell biological studies with trichomes during the past four years have provided interesting insights into the role of the cytoskeleton in trichome morphogenesis. These studies indicate that actin and the microtubule cytoskeleton play a pivotal role in cell expansion and branching during trichome development [32].

Localization studies and visualization of the actin cytoskeleton in live cells with an actin-binding protein tagged with green fluorescent protein (GFP) indicate that the organization of F-actin changes during trichome morphogenesis [33,34]. Chemicals that promote depolymerization or stabilization of the actin cytoskeleton did not effect branching but produced distorted trichomes. The morphology of these trichomes is similar to that observed in a 'distorted' class of mutants, suggesting that at least some of the affected genes are likely to code for proteins involved in actin organization/dynamics (for example myosins, actin-depolymerizing factors, actin-binding proteins). There is also evidence that the actin cytoskeleton is involved in mitosis and during separation of daughter cells after the successful segregation of chromosomes into daughter nuclei [3]. The actin cytoskeleton is also involved in pollen tube growth, and calcium regulation has also been shown to be involved [35,36].

Myosins have been identified in plants both biochemically [37,38,39,40] and at the molecular level [41,42,43]. Immunological detection of myosins using antibodies against animal myosin identified proteins of various sizes from different plants [44,45,46]. Immunofluorescence studies localized myosin to the surface of organelles, the vegetative nuclei and generative cells in pollen grains and tubes [39], to the active streaming lanes and cortical surface in pollen tubes [40] and, more recently, to plasmodesmata in root tissues [38,47]. Motility assays [48] and ATPase assays [48,49,50] using myosin-like proteins isolated from plants have also demonstrated the presence of myosins in plants.

Since 1993, five partial or full-length myosins from Arabidopsis have been characterized at the molecular level. Using PCR-based approaches, Knight and Kendrick-Jones [43] cloned a myosin they called ATM (Arabidopsis thaliana myosin), Kinkema and Schiefelbein [41] cloned the myosin MYA1 and Kinkema et al. [42] cloned another full-length myosin, ATM2, and two partial length myosins MYA2 and MYA3. Kinekema et al. [42] also identified three PCR products that coded for unique myosin motor domain sequence. Phylogenetic analysis using these myosins indicated that the ATM myosins were a unique class and they were named class VIII. The MYA myosins are somewhat related to class V myosins but as other analyses have been done, these myosins were also assigned to a new class, class XI [8,42].

Myosins have been identified in Zea mays, two of which belonged to class XI and one to class VIII [51]. PCR fragments for fern myosins have been reported [52,53] and sequences are available for myosins from Helianthus annuus (0. Vugrek and D. Menzel, unpublished data). Two fern (Anemia phyllitidis) PCR products and the H. annuus myosins also fall either into class VIII or class XI myosins [22,42]. Two algal myosins are also members of the class XI myosins, one from Chara corallina and one from Chlamydomonas reinhardtii [22,54]. A third class of myosins (XIII) is composed of two algal myosins from Acetabularia cliftonii. No animal myosins are in any of these classes and no plant myosins are in any other myosin class. However, the cellular slime mold Dictyostelium discoideum has one myosin (Dd MyoJ), which is alternatively grouped with class V or class XI [27].

Other organisms have myosins from more than two classes. The yeast Saccharomyces cerevisiae has five myosins in three different classes. Caenorhabditis elegans has myosins in seven classes and Drosophila melanogaster in nine. Do plants have only two classes of myosins? How many myosins are in a plant genome? What are the similarities and differences between plant and non-plant myosins that might help establish a function for the myosins? Until the recent completion of the sequencing of the Arabidopsis genome [55], answers to these questions were not known. It is now possible to determine how many myosins are in the Arabidopsis genome and to see if any plant myosins fall into other myosin classes. As the myosin motor domain is highly conserved, the sequence from one myosin motor can be used to search a database for all other myosins. We used the motor domain from MYA1 to search the Arabidopsis database [11] for sequences with similarity to this domain. We identified 17 Arabidopsis myosins, including the 5 reported myosins, in the Arabidopsis genome. Phylogenetic analysis using non-plant and plant myosins showed that all 17 fall into either myosin class VIII or XI. Only 4 are in class VIII and 13 in class XI. An analysis of their exon/intron junctions and sequence similarities indicates that all myosins are highly conserved and some may represent gene duplication events.

Results

Identification of Arabidopsismyosins

Using the amino-acid sequence of the conserved motor domain of the plant myosin MYA1 [41], databases were searched using BLASTP and TBLASTN at TAIR [11]. Other searches using the amino-acid sequence of motor domains from representatives of other classes of myosins were also done but they did not reveal any other myosin sequences. Sixteen unique sequences were obtained that contain a myosin motor domain as identified by the SMART (Smart Modular Architecture Research Tool) program [56]. The sequences obtained in this search were compared to the Munich Information Center for Protein Sequences (MIPS) [57] list of myosin domains in Arabidopsis. MIPS lists 16 Arabidopsis sequences showing myosin domains. A check of these showed that 13 of the sequences were myosins identified in our search and one was a myosin not available in the NCBI (National Center for Biotechnology Information) protein database [58]. Two are not full-length myosins. One is a putative helicase (At1g26370) with no myosin motor domain and one is a possible pseudogene (At1g42680) with only 162 amino acids that have some similarity to the myosin motor domain. MIPS does not list three myosins identified in our search (At XIG, At XIF and At XI-I). Table 1 lists the myosins by names as given in the phylogenetic tree constructed by Hodge and Cope [59] and as assigned by us. There are a total of 17 myosin genes in Arabidopsis. In comparison, S. cerevisiae, Schizosaccharomyces pombe, C. elegans and D. melanogaster have 5, 5, 20 and 13 myosins, respectively (Figure 1) [60,61]. Arabidopsis has the lowest percentage (0.068%) of myosin genes out of the total number of genes, as compared to S. cerevisiae and S. pombe with 0.080% and 0.093%, respectively, C. elegans with 0.11% and D. melanogaster with 0.096% (see Figure 1).
Figure 1

The numbers of myosins in eukaryotic sequenced genomes. The number of myosins in each organism is on the left (red column) and the number per thousand for each organism is on the right (blue column). At, Arabidopsis thaliana; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe.

Table 1

Myosin-like proteins in Arabidopsis

Name

Number of

Protein ID

Gene code

Old name

Class

Domains

Reference

 

amino acids

      

1. At ATM

1166

479413

AT3g19960

(ATM1)*

VIII

MD,CC,IQ

[43]

  

11994771

 

MZE19.1

  

AtDB, MIPS

2. At ATM2

1111

9759501

AT5g54280

MDK4.10

VIII

MD,CC,IQ

AtDB, MIPS

 

1101

499045

 

ATM2/AtMYOS1

  

[42]

3. At VIIIA

1085

5734787

AT1g50360

F14I3.6

VIII

MD,CC,IQ

AtDB, MIPS

4. At VIIIB

1126

3269298

AT4g27370

M4I22.180

VIII

MD,CC,IQ

AtDB, MIPS

5. At MYA1

1520

1076348

AT1g17580

(AtMYA1)*

XI

MD,CC,IQ

[41]

 

1599

8778462

 

F1L3.28

  

AtDB, MIPS

6. At MYA2

1505

2129653

AT5g43900

F6B6.4

XI

MD,IQ

AtDB, MIPS

 

1515

8953751

 

(AtMYA2)*

  

[42]

7. At XIA

1730

2494118

AT1g04600

T1G11.15

XI

MD,CC,IQ

AtDB, MIPS

8. At XIB

1519

3142302

AT1g04160

F20D22.7

XI

MD,IQ

AtDB, MIPS

9. At XIC

1572

3063460

AT1g08730

F22O13.22

XI

MD,CC,IQ

AtDB, MIPS

10. At XID

1611

2924770

AT2g33240

F25I18.2

XI

MD,CC,IQ

AtDB, MIPS

11. At XIE

1529

3776579

AT1g54560

T22H22.1

XI

MD,CC,IQ

AtDB, MIPS

12. At XIF

1556§

4887746

AT2g31900

F20M17.6

XI

MD,IQ

AtDB, MIPS

13. At XIG

1502

4512706

AT2g20290

F11A3.16

XI

MD,CC,IQ

AtDB, MIPS

14. At XIH

1452§

4218127

AT4g28710

F16A16.180

XI

MD,CC,IQ

AtDB, MIPS

15. At XI-I

1374

4455334

AT4g33200

F4I10.130

XI

MD,CC,IQ

AtDB, MIPS

16. At XIJ

1242

11276963

AT3g58160

F9D24.70

XI

MD,CC,IQ

AtDB, MIPS

 

963

602328

 

(AtMYOS3)*,

  

[42]

 

998

629533

 

(AtMYA3)*

  

[42]

17. At XIK

1544

 

AT5g20490

F7C8.80

XI

MD,CC,IQ

MIPS

*Name as reported in the literature. Number of amino acids previously reported for partial sequence. Number of amino acids predicted by NCBI. §Edited by authors for full-length sequence: AtDB, Arabidopsis database; MIPS, Munich Information Center for Protein Sequences; MD, motor domain; CC, coiled-coil region; IQ, putative calmodulin-binding motif.

Only 5 of the 17 Arabidopsis myosins have been reported in the literature [41,42,43]. The other 12 are sequences obtained from the Arabidopsis database sequenced as part of the Arabidopsis Genome Sequencing Project. These sequences are, therefore, predicted sequences that have not been verified by complete cDNAs. The average sequence length of the Arabidopsis myosins is 1,400 residues, with the shortest sequence prediction being 1,085 (At VIIIA) amino acids and the longest 1,730 (At XIA). Some of the intron/exon predictions may not be correct, which could reduce or increase the size of the predicted proteins and so the sizes may change as more characterization is done for each myosin. A case in point is the cDNA that was isolated by Kinkema and Schiefelbein [41] for At MYA1 (At MYA1) which codes for 1,520 amino acids, whereas the predicted protein has 1,599 because of differences in intron prediction.

Using the Arabidopsis Sequence Map Overview of TAIR [62], the location of each myosin was determined (Figure 2). The myosin genes are distributed throughout the Arabidopsis genome. The chromosome lengths are based on the centimorgan (cM) scale as shown on the TAIR Map Overview [62]. The maps reported with the announcement of the Arabidopsis genome sequence show somewhat different lengths than the TAIR maps [55].
Figure 2

Location of myosins on the Arabidopsis chromosomes. Roman numerals represent chromosome numbers. Large numbers indicate chromosome length in cM. Small blue numbers are the myosin numbers from Table 1.

Phylogenetic analysis

All Arabidopsis myosins and a selection of myosins from other organisms representing each of the myosin classes were aligned using the motor domain sequence as determined by the SMART program [56]. The alignment was done in Megalign by the CLUSTAL method and a phylogenetic tree was generated using the Bootstrap (100 replicates) method with a heuristic search of the PAUP 4.0b6 program (Figure 3). The Arabidopsis myosins all group into two classes along with other plant myosins - class VIII and class XI. No animal myosins group with the plant myosins and no plant myosins group into any of the animal myosins. An algal (Chara corallina) myosin, Cc ccm, does group with the plant class XI myosins but is on a separate branch from any other class XI myosin (Figure 3). The D. discoideum myosin Dd myoJ did not fall into a class with any of the plant myosins. In fact, three D. discoideum myosins (Dd myoI, Dd myoJ, and Dd myoM) did not fall into any of the classes (Figure 3). The phylogenetic trees of Hodge and Cope and the tree on the myosin home page [22,59] show the Dd myoI branching from class VII myosins. A heuristic search without bootstrapping also showed the Dd myoI myosin as a branch from class VII and domain analysis shows that Dd myoI has the MyTH4 domain found in other class VII myosins. Other phylogenetic anaylses have placed Dd myoJ as a branch off class XI myosins from plants [22,59]. However, the phylogenetic tree generated from full-length sequences of plant myosins and Dd myoJ (see below) also shows that Dd myoJ is separate from the plant myosins.
Figure 3

Phylogenetic tree. Alignment of the motor domain of representative myosins and all Arabidopsis myosins was done in Megalign by the CLUSTAL method and a phylogenetic tree was generated using the bootstrap method with a heuristic search of the PAUP 4.0b6 program. The myosin groups, as defined by Hodge and Cope [59] and Yamashita et al. [23], are identified on the right in roman numerals. Myosins from the following organisms were used: Ac, Acanthamoeba castellani; Acl, Acetabularia cliftoni; At, Arabidopsis thaliana; Cc, Chara corallina, Ha, Helianthus annuus; Zm, Zea mays; Bt, Bos taurus; Mm, Mus musculus; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Rn, Rattus norvegicus; Sc, Saccharomyces cerevisiae; Hs, Homo sapiens; Dd, Dictyostelium discoideum; Lp, Limulus polyphemus; En, Emericella nidulans; Pg, Pyricularia grisea; Pf, Plasmodium falciparum; and Tg, Toxoplasma gondii. The number at the branches indicates the number of times the dichotomy was supported out of 100 bootstrap tries.

Myosins from another alga, Acetabularia cliftonii, are classified into a separate group (XIII) and one myosin each from the fungi Emericella nidulans and Pyricularia grisea are also assigned to a separate class (XVII). A second alignment was done using the full-length sequences for all Arabidopsis and other known full-length plant myosins with a human heavy-chain myosin (Hs Ib) as an outgroup. The two classes of plant myosins are clearly seen (Figure 4). Among the class XI myosins the similarity ranges from 40-85% (full length) and 61-91% (motor domain). The similarity between the class VIII myosins ranges from 50-83% (full length) and 64-92% (motor domain). When class VIII myosins are compared to class XI myosins the similarity only ranges from 22-29% (full-length) and 35-42% (motor domain). Thirteen Arabidopsis myosins group into class XI. Two subgroups branch off in this class with three outliers (Figure 4). One subgroup consists of two pairs of Arabidopsis myosins, At XIB/At MYA2 and At XIG/At XIH, which are most similar to the sunflower myosin Hahamy4 and then another pair of Arabidopsis myosins, At XID/At XIA. The other subgroup consists of the Arabidopsis myosin pair At XIC/At XIE and two unpaired Arabidopsis myosins, At XIK and At MYA1, that are most closely related to sunflower myosins Hahamy2 and Hahamy5 and to the maize myosin ZmMYO1. At XIJ, AT XIF and At XI-I are on separate branches that group with the other class XI myosins but not within the two subgroups. There are four class VIII Arabidopsis myosins that form two pairs, At ATM/At VIIIA and At VIIIB/At ATM2. The first pair group with class VIII myosins from Z. mays and H. annuus whereas the second pair are on a separate branch.
Figure 4

Phylogenetic tree for plant myosins. Alignment of the full-length Arabidopsis myosins, other full-length plant myosins available in the NCBI database and Dd myoJ was done in Megalign by the CLUSTAL method and a phylogenetic tree was generated using the bootstrap method with a heuristic search of the PAUP 4.0b4a (PPC) program. A human myosin (Hs 1b) was used as an outgroup. At, Arabidopsis thaliana; Dd, Dictyostelium discoideum; Ha, Helianthus annuus; Zm, Zea mays. The number at the branches indicates the number of times the dichotomy was supported out of 100 bootstrap tries.

Characterization of the Arabidopsismyosins

Figure 5 shows schematic diagrams of each myosin. The motor domain in all cases is in the amino-terminal region. The motor domain starts at about 50-55 residues for the class XI myosins whereas the class VIII myosins have a longer amino-terminal region before the motor domain (99-159 residues). The IQ domains usually follow right after the motor domain but are separated slightly from the motor domain in At XID, At XI-I, and At XIK. There are three or four IQ domains in class VIII myosins and five or six in class XI, except for At XIK, which has only four. There are coiled-coil domains, that differ in length and number, in all the myosins. They often follow directly after the IQ domains, but in some cases there is intervening sequence. Based on the presence of the coiled-coil domains, the Arabidopsis myosins are probably dimeric [26]. The class XI myosins are much longer than the class VIII myosins with the difference being in the length of the carboxy-terminal region following the conserved domains found in myosins.
Figure 5

Schematic diagram of Arabidopsis myosins. The numbers refer to the number in Table 1. The motor domain, IQ domains, and coiled-coil domains are as indicated in the key. The first four myosins are in class VIII and the following 13 are in class XI. The bar represents 100 amino acids.

Besides the motor, IQ and coiled-coil domains, other domains have been identified in myosins from classes other than the plant classes VIII and XI. These include SH3 domains (Src homology 3 domains, that bind to target proteins), MYTH4 (a domain of unknown function found in a few classes of myosins), a zinc-binding domain, a pleckstrin homology domain, FERM/talin (band 4.1/ezrin/radixin/moesin), GPA-rich domains and a protein kinase domain [8,22,26]. These domains are involved in protein interactions and presumably give specificity to the action of the myosin. Except for the IQ and coiled-coil domains, the SMART program used to identify the motor domain of the myosin sequences did not identify any domains other than a few with scores less significant than the required threshold.

Myosins have 131 highly conserved residues spread throughout the motor domain that define a core consensus sequence [26]. Comparison of an alignment of Arabidopsis myosin motor domains to these conserved sequences shows a great deal of conservation among them (data not shown). One example is the ATP-binding site which consists of GESGAGKT (179-187 in Dictyostelium myosin II, DmyoII) and NxNSSR-FGK (233-241, DmyoII). With the exception of only one residue these are conserved in all 17 Arabidopsis myosins. The conformational state of myosin changes with ATP hydrolysis and a very conserved region implicated in this process has the conserved sequence LDIxGFExFxxN(S/G)(F/L)EQxxINxxNExLQQxF (453-482, DmyoII) [26]. The plant sequences are very conserved through this region. The sequence in this region is LDIYGFExFxxNSFEQxCINE(K/R)LQQHF (the first x is S in all but one myosin, the fourth x is F in all but one myosin). Cope et al. [26] suggest that release of the γ-phosphate of ATP may be through a hole in the structure centered around an absolutely conserved arginine residue (residue 654, DmyoII) which is also absolutely conserved in all Arabidopsis myosins. The presence of these highly conserved residues in plant myosins suggests that they are capable of motor function. In fact, in vitro motility studies with a purified myosin from Chara (myosin XI, Cc ccm in Figure 3) have confirmed that it is indeed an actin-based motor [54]. A loop present in the motor domain called the HCM (mutations in this loop cause hypertrophic cardiomyopathy) is the location of a phosphorylatable serine (S) or threonine (T) in certain amoeboid myosin I molecules and myosin VI molecules. This S or T residue is 16 residues upstream from the conserved DALAK sequence. The enzyme activity of the amoeboid myosins depends on phosphorylation of this site, but although phosphorylation of the myosin VI T residue has been demonstrated, the regulation of enzyme activity has not [8,63]. Most other myosins have a constitutively negatively charged amino acid, either aspartic acid (D) or glutamic acid (E) at this site. This site has been named the TEDS rule site on the basis of these amino acids [8]. The Arabidopsis and other plant myosins all have aspartic acid, glutamic acid or glycine residue at this site, suggesting that they are not regulated by phosphorylation at this site. However, three residues upstream (19 from DALAK), all the class XI myosins have a threonine residue.

The site for each predicted or actual intron was located and is shown schematically in Figure 6. The intron locations were determined from the information at MIPS [57]. The length of each exon and the domain(s) they code for are shown in Tables 2 and 3 for class VIII and class XI myosins, respectively. The exons vary in length from 12 to greater than 672 nucleotides (the length of the beginning and last exons for each gene are not known as the predicted sizes include only the protein-coding nucleotides) with an average of 122 nucleotides. The four class VIII myosins have seven exons of the same length in the same order within the myosin motor domain (Table 2). The motor domain starts in the third exon of each class VIII myosin. The start of the IQ domains and the coiled-coil domains is more variable except for the At ATM2/At VIIIB pair. The class XI myosins also have many exons that are of the same length and in the same order but that differ from the class VIII pattern (Table 3). The exons coding for the motor domain sequence are most conserved in length. Most class XI myosins motor domains start in the third exon and end in the twentieth. Six of the class XI myosins have an intron after the start codon. Most differences in exon length are in the carboxy-terminal regions (Figure 6 and Table 3). However, even in the carboxy-terminal region there are some exon lengths conserved between some or all of the myosins. The two XI myosins with the closest similarity are At XIB and At MYA2. A Clustal alignment at Pole Bio-Informatique Lyonnais [64] showed 83.88% identity, 8.19% strong similarity and 2.36% weak similarity between these two myosins. Their motor domains are 91.6% identical. Twenty-three of their introns are at the same location in the motor domain area and then following a few different size exons, there are similar sized exons again. They are located on chromosomes I and V, respectively. It is possible that this pair is a result of gene duplication. Class VIII myosins At ATM and At VIIIA have 13 exons of the same length. Their full-length sequences are 79% identical with another 6.72% strongly similar and 3.52% weakly similar. Their motor domains have 93% similarity. At ATM is on chromosome III whereas At VIIIA is on chromosome I. This again may have resulted from a gene duplication. Analysis of the total Arabidopsis genome revealed that a whole genome duplication occurred, followed by subsequent gene loss and extensive local gene duplications [55]. The duplicated segments represent 58% of the Arabidopsis genome. The S. cerevisiae genome has also had a complete ancient genome duplication and 30% of the genes form duplicate pairs. Duplicated genes account for 48% of the total genes of C. elegans and Drosophila [60].
Figure 6

Location of the introns. The numbers refer to the number in Table 1. Arrowheads indicate the location of each intron along the length of the myosin. The bar represents 100 amino acids.

Table 2

Analysis of exon sizes in class VIII myosins and the domain coded by each exon

 

At ATM

At VIIIA

At ATM2

At VIIIB

Number

Size

Domain

Size

Domain

Size

Domain

Size

Domain

1

339

N

315

N

159

N

333

N

2

102

N

132

N

102

N

118

N

3

144

N,M

144

N,M

144

N,M

131

N,M

4

151

M

151

M

151

M

155

M

5

28

M

28

M

25

M

169

M

6

166

M

158

M

129

M

64

M

7

64

M

104

M

64

M

99

M

8

14

M

139

M

99

M

104

M

9

84

M

119

M

104

M

139

M

10

104

M

153

M

139

M

119

M

11

139

M

90

M

119

M

153

M

12

119

M

78

M

153

M

90

M

13

153

M

159

M

90

M

78

M

14

90

M

207

M

78

M

159

M

15

78

M

144

M

159

M

186

M

16

159

M

114

M

186

M

206

M

17

207

M

130

M,I

342

M

136

M

18

206

M

147

I

244

M,I

130

M,I

19

136

M

68

C

116

I

108

I

20

130

M,I

595

C,T

213

I,C

140

I,C

21

147

I

83

T

480

C,T

189

C

22

68

I,C

    

375

C,T

23

672

C,T

      

N, amino-terminal sequence; M, motor domain; I, IQ domain; C, coiled-coil domain; T, tail domain. The size of the first and last exons in each gene reflects only the size of the coding region.

Table 3

Analysis of exon sizes in class XI myosins and the domain coded by each exon

 

At XIG

At XIH

At MYA2

At XIB

At XID

At XIA

At XIF

No.

Size

Domain

Size

Domain

Size

Domain

Size

Domain

Size

Domain

Size

Domain

Size

Domain

1

36

N

3

N

3

N

3

N

3

N

3

N

3

N

2

126

N

139

N

129

N

129

N

171

N

126

N

129

N

3

144

N,M

131

N,M

144

N,M

144

N,M

144

N,M

144

N,M

144

N,M

4

146

M

146

M

146

M

146

M

146

M

146

M

146

M

5

157

M

157

M

157

M

160

M

157

M

157

M

157

M

6

59

M

59

M

59

M

59

M

59

M

59

M

59

M

7

160

M

160

M

160

M

160

M

160

M

160

M

160

M

8

150

M

150

M

150

M

150

M

150

M

150

M

150

M

9

134

M

137

M

137

M

136

M

137

M

137

M

137

M

10

147

M

147

M

147

M

147

M

147

M

147

M

147

M

11

102

M

102

M

102

M

102

M

102

M

102

M

102

M

12

58

M

58

M

58

M

58

M

58

M

58

M

58

M

13

102

M

102

M

102

M

102

M

102

M

102

M

102

M

14

38

M

38

M

38

M

38

M

38

M

38

M

38

M

15

127

M

127

M

127

M

127

M

127

M

127

M

127

M

16

171

M

171

M

168

M

168

M

171

M

171

M

171

M

17

132

M

132

M

132

M

132

M

132

M

132

M

132

M

18

110

M

110

M

110

M

110

M

110

M

110

M

107

M

19

61

M

82

M

61

M

61

M

61

M

61

M

61

M

20

178

M,I

178

M,I

178

M,I

178

M,I

178

M,I

178

M,I

178

M,I

21

194

I

206

I

206

I

206

I

251

I

206

I

206

I

22

120

I

120

I

120

I,C

120

I,C

120

I,C

120

I,C

120

I,C

23

99

U

99

U

99

C

99

C

99

C

99

C

99

C

24

213

C

213

C

213

C

213

C

213

C

288

C

216

C

25

140

C,T

140

C

140

C

140

C

153

C

153

C

140

C

26

112

T

94

C,T

12

C

115

C

54

C

150

C

102

C

27

45

T

168

T

45

C,T

45

C,T

203

C

165

C

109

C,T

28

84

T

144

T

63

T

51

T

94

C,T

140

C

45

T

29

198

T

201

T

171

T

171

T

60

T

115

C,T

60

T

30

144

T

138

T

153

T

150

T

78

T

21

T

171

T

31

162

T

71

T

201

T

192

T

182

T

78

T

156

T

32

111

T

46

T

129

T

129

T

187

T

171

T

207

T

33

71

T

57

T

71

T

71

T

177

T

153

T

150

T

34

100

T

57

T

97

T

97

T

78

T

177

T

71

T

35

57

T

81

T

57

T

57

T

50

T

291

T

100

T

36

57

T

83

T

57

T

57

T

97

T

71

T

57

T

37

81

T

112

T

81

T

164

T

57

T

100

T

57

T

38

65

T

  

83

T

169

T

57

T

57

T

81

T

39

118

T

  

112

T

  

81

T

57

T

83

T

40

        

77

T

81

T

133

T

41

        

115

T

77

T

  

42

          

115

T

  
 

At XIC

At XIE

At XIJ

At MYA1

At XI-I

At XIK

  

No.

Size

Domain

Size

Domain

Size

Domain

Size

Domain

Size

Domain

Size

Domain

  

1

52

N

12

N

126

N

180

N,M

144

N

55

N

  

2

104

N

129

N

144

N,M

138

M

126

N,M

119

N

  

3

144

N,M

144

N,M

146

M

146

M

146

M

144

N,M

  

4

146

M

146

M

157

M

157

M

157

M

146

M

  

5

157

M

157

M

59

M

92

M

59

M

157

M

  

6

59

M

59

M

160

M

160

M

156

M

110

M

  

7

160

M

160

M

150

M

150

M

150

M

160

M

  

8

150

M

150

M

137

M

137

M

137

M

150

M

  

9

137

M

137

M

147

M

147

M

147

M

137

M

  

10

147

M

147

M

102

M

102

M

102

M

111

M

  

11

102

M

102

M

58

M

58

M

58

M

102

M

  

12

58

M

58

M

102

M

102

M

102

M

58

M

  

13

242

M

102

M

38

M

38

M

38

M

102

M

  

14

127

M

38

M

127

M

127

M

131

M

38

M

  

15

171

M

127

M

168

M

171

M

122

M

127

M

  

16

132

M

171

M

132

M

132

M

36

M

159

M

  

17

110

M

132

M

110

M

110

M

132

M

108

M

  

18

61

M

110

M

61

M

61

M

110

M

110

M

  

19

178

M,I

61

M

178

M,I

313

M

61

M

61

M

  

20

206

I

178

MI

206

I

206

M,I

178

M,I

178

M

  

21

120

I

206

I

120

I,C

120

I

206

I

239

I

  

22

99

C

120

I,C

651

C

99

I,C

120

I,C

120

I

  

23

222

C

99

C

140

C,T

219

C

99

C

99

C

  

24

140

C

222

C

257

T

140

C

222

C

222

C

  

25

112

C,T

140

C

53

T

139

C,T

140

C

140

C

  

26

48

T

112

C,T

  

51

T

100

C,T

118

C

  

27

255

T

48

T

  

51

T

51

T

51

C,T

  

28

156

T

255

T

  

171

T

171

T

72

T

  

29

207

T

156

T

  

156

T

63

T

171

T

  

30

144

T

195

T

  

210

T

177

T

156

T

  

31

71

T

144

T

  

147

T

71

T

207

T

  

32

100

T

71

T

  

71

T

100

T

138

T

  

33

57

T

157

T

  

100

T

81

T

75

T

  

34

57

T

57

T

  

114

T

83

T

81

T

  

35

81

T

81

T

  

85

T

151

T

57

T

  

36

83

T

83

T

  

76

T

  

57

T

  

37

124

T

124

T

  

124

T

  

81

T

  

38

          

83

T

  

39

          

136

T

  

40

              

41

              

42

              

N, Amino-terminal sequence; M, motor domain; I, IQ domain; C, coiled-coil domain; U, undefined; T, tail domain. The size of the first and last exons in each gene reflects only the size of the coding region.

If the gene pairs are the result of duplication, it is interesting to note that while exon lengths have been conserved, intron lengths have not. The intron lengths are shown in Table 4. No pattern can be seen in intron lengths between any of the myosins. The average intron length is 131 nucleotides with the shortest intron at 47 nucleotides and the longest at 860. At XI-I has the highest average, 272 nucleotides. It contains the 860-nucleotide intron and three others that are over 500 nucleotides. In a study of 998 introns only 3.3% of the introns were longer than 500 nucleotides with sizes ranging from 59 to 1242 nucleotides [65]. This makes At XI-I unusual in having four out of 33 introns (12%) longer than 500 nucleotides. Only two other myosins had an intron over 500 nucleotides. Of the total 557 splice sites that were identified in the Arabidopsis myosins only six (a little more than 1%) were over 500 nucleotides with four out of the six being in one myosin. Hunt et al. found that a SV40 small-t intron only 66 nucleotides in length was spliced efficiently in tobacco cells [66]. Several of the introns in the myosins are between 66 and 70 nucleotides and so may be long enough to be spliced. Only one is in a cloned myosin known to be spliced at that site (At XIJ). There is also a predicted intron of only 47 nucleotides in length (At XID) which is thought to be too short for efficient splicing. Brown et al. [65] found three introns less than 66 nucleotides in length in known expressed proteins, but none of them was less than 59 nucleotides. Until the expression of At XID is studied, no conclusion can be made as to the validity of this intron prediction. The significance of the range and variability of intron length is not known. In Arabidopsis, in general, the range is even greater (47-6,442) [11].
Table 4

Intron size and sequence of 5' and 3' splice sites

 

At ATM

At VIIIA

At ATM2

At VIIIB

No.

Size

5' site

3' site

Size

5' site

3' site

Size

5' site

3' site

Size

5' site

3' site

1

137

AG GTATTC

TTTAG AT

107

AG GTATTG

TAGAG GC

310

AG GTAATT

TTCAG AA

179

AG GTAAAT

GCCAG AA

2

84

AA GTAAGT

AACAG GT

88

AA GTAAGT

AACAG GT

95

AT GTGAGT

CAAAG GT

81

AA GTTCTT

AGTAG CA

3

124

AT GTAAGT

GCTAG AC

126

AT GTAAAT

GCTAG AC

91

AT GTGAGT

TACAG AG

84

TA GTAAGT

TTTAG AG

4

109

CG GTGGGT

TCCAG AT

92

AG GTTGGA

TTCAG TC

113

AG GTGAGG

AGAAG AG

226

GA GTGAAA

CTTAG TC

5

247

AG GTTAGT

TCCAG CG

302

AG GTTAGT

TCCAG TG

121

AG GTACGG

TATAG AG

159

TC GTGAGT

TGCAG GG

6

114

TT GTAAGC

TACAG GG

643

TT GTAAGC

GACAG GG

152

TT GTGAGA

CACAG GT

194

TT GTAAGA

AGTAG TC

7

103

CT GTAAGT

TGCAG TT

89

AG GTAACT

TTCAG GA

205

TT GTAAGT

GGTAG TC

196

AG GTAACA

TGCAG AG

8

101

AG GTAGCT

AACAG TC

201

AA GTATGG

TCCAG GT

151

AG GTAACA

TGTAG AG

100

TG GTACTT

TATAG GA

9

376

AG GTATGG

TGCAG AG

170

AG GTAGGC

ACCAG GC

102

TG GTAATT

TGCAG GA

98

AG GTAGAG

TACAG CT

10

101

AG GTAATT

TGCAG GA

135

AT GTATGC

TGCAG AA

78

AG GTAGAA

TACAG CT

97

TG GTTTGT

TTCAG GC

11

295

AA GTAAGC

TTCAG GT

114

AG CTAACG

TCCAG GA

94

AG GTAATG

TTAAG GT

75

AG GTTCGT

TTTAG GA

12

326

AG GTATAT

TTCAG GC

207

AG GTAATG

TGCAG AA

89

AG GTTAGT

TTCAG AA

123

TG GTGATC

TTCAG GA

13

197

AT GTATGT

TGCAG AA

146

TG GTAATA

CTCAG GT

82

AG GTGGTT

CTCAG GA

139

TG GTAAGT

TGCAG AA

14

136

AG GTAAAG

TTCAG GA

192

AG GTTGGG

TTCAG GG

95

AG GTAATT

AGCAG AA

126

AG GTCAGT

AATAG GT

15

160

AG GTATAT

TGCAG AA

211

AG GTCGTT

TGGAG AA

125

AG GTCAGT

TACAG GT

111

TG GTGACA

TACAG GC

16

122

AG GTAACA

ATCAG GT

86

TG GTACTT

TGCAG AT

87

AG GTAAAG

TACAG GG

104

TG GTTTGG

AGTAG AT

17

228

AG GTGAGT

TCCAG AG

85

TA GTATTG

TTCAG TT

87

AA GTAAGC

CATAG AT

82

AT GTAAGT

GATAG AT

18

87

AG GTGACA

TGCAG AT

103

TG GTAAAA

TGTAG CA

82

TG GTAAGC

TGCAG CG

109

TA GTAATC

TACAG AT

19

77

AG GTATAA

TGCAG AT

88

TG GTCCTC

TGTAG TG

82

AG GTACTT

TTCAG GA

85

TA GTAAAT

TGTAG TG

20

112

AT GTATAA

TTCAG TT

83

AG GTGGTT

TTGAG AC

88

AG GTCAAA

TGCAG AT

70

GC GTCTCT

TTGAG GT

21

250

AG GTAAAA

TGCAG CA

      

80

AG GTAAGT

TGCAG AT

22

111

AG GTAAAA

CGCAG AC

         
 

At XIG

At XIH

At MYA2

At XIB

No.

Size

5' site

3' site

Size

5' site

3' site

Size

5' site

3' site

Size

5' site

3' site

1

168

TG GTTATT

TTCAG CG

365

AT GTGAGA

TGCAG GC

330

TG GTAAGA

TACAG GT

618

TG GTAAAA

TGCAG GT

2

103

CG GTATGT

TTCAG GT

135

CA GTTTGA

TAAAG TT

100

AT GTATGT

TTCAG GT

127

AA GTATGT

CACAG GT

3

92

AT GTGAGT

ACTAG AC

137

AG GTGAGT

TCCAG AC

74

AT GTGAGT

TTCAG AC

143

AT GTGAGT

TTCAG AC

4

90

AG GTGCTT

TATAG AC

96

AG GTGCCT

GGTAG AC

102

AG GTAATT

TGCAG AC

87

AG GTAATT

TGCAG AC

5

105

AG GTAACT

TGCAG TC

98

AG GTTATC

TGCAG TC

300

AG GTGAAA

TTCAG TC

201

AG GTGAAA

TACAG TC

6

120

AG GTGAAT

TGCAG TC

123

AG GTGTAT

TGCAG TC

76

AG GTAACC

TATAG TC

101

AG GTAAGG

TATAG TC

7

274

AG GTACAT

GACAG GA

289

AG GTACAT

ATCAG GA

125

AA GTAAGT

TACAG GA

93

AA GTAAGT

TTCAG GA

8

76

AG GTAGTT

GTCAG GA

83

AG GTAACT

GTCAG GA

95

AG GTAGTT

TTCAG GA

81

AG GTACCT

TTTAG GA

9

115

AT GTGTGT

TGCAG GT

101

TA GTGAGT

GTCAG GT

103

AG GTAAAT

TCCAG CT

89

AT GTAAAT

TGCAG GT

10

111

TG GTATGT

TGTAG GA

107

TG GTATGT

TTCAG GA

111

TG GTGGGT

TGCAG GC

125

TG GTGAGT

TGCAG GC

11

300

AG GTGCAT

TTCAG TT

284

AG GTGCTT

TGCAG TT

355

AG GTGCTT

TGCAG TT

417

AG GTGCTT

TGCAG TT

12

84

AG GTTTGT

GGCAG CA

88

AG GTTTGT

GGCAG CA

91

AG GTTTGA

TGCAG CA

91

AG GTTTTG

TGCAG CA

13

97

AG GTAACT

TTCAG AA

80

AG GTTAGT

CTCAG AA

234

GA GTCTGT

TTCAG AA

243

AG GTTATC

TTCAG AA

14

82

TG GTAAGC

TGCAG CA

87

TG GTATGA

TGCAG CA

153

TG GTGAGT

TGCAG CA

123

AG GTGAGT

TGCAG CA

15

99

AT GTGAGT

TTCAG GT

104

TA GTGAGT

TTCAG GT

117

AT GTGAGT

TCCAG GT

121

AT GTGAGC

TCCAG GT

16

85

AG GTGCAG

TGCAG CA

82

AG GTGCAG

TGCAG CA

87

AG GTAAGT

TTCAG CA

91

AG GTGAGT

TGCAG CA

17

92

GG GTGAGA

TTTAG GG

87

GG GTGGGA

TTCAG GG

91

GG GTGCGA

TTTAG GG

98

GG GTGCGA

CACAG GG

18

86

AG GTATGC

GCTAG TT

79

AG GTTCCC

TCTAG TA

77

AA GTAAGA

AATAG CT

88

AA GTAAGA

ACTAG TT

19

75

AG GTACTT

CACAG AT

113

AA GTACGT

TCCAG AT

87

AG GTAATT

TGTAG AT

93

AG GTAATT

TGTAG AT

20

99

AG GTATCT

AACAG GT

86

AG GTACTT

TGTAG GT

117

AG GTATTT

GTCAG GT

88

AG GTATTT

TTCAG GT

21

147

AG GTGGAG

CAGAG CC

147

AG GTGCTG

TACAG AG

159

AG GTACAC

TATAG AC

170

AG GTATGA

TACAG AC

22

130

CG GTGTGC

TGCAG GA

296

TG GTGAGC

TGCAG GC

122

TG GTGAGA

CCTAG GC

150

AG GTGAGA

CACAG GC

23

117

GG GTCAGA

TGTAG GT

120

GG GTAAGT

TTTAG AC

125

GG GTGTGA

TGCAG AC

105

GG GTGAGT

TGCAG AC

24

107

AG GTAGGG

TGCAG TC

119

AG GTAGGA

TTCAG TC

150

AG GTTTGT

TACAG AG

120

AG GTGGGT

TGCAG GG

25

99

AA GTATTC

TGCAG TC

94

GA GTACCC

TGCAG AC

89

TG GTATCC

TCCAG GC

87

AG GTACTG

TGCAG GC

26

84

AG GTAGAC

TTTAG AA

392

CA GTTAAG

AGGAG AA

89

AG GTAGAA

TGTAG AA

90

AG GTAGAA

TGCAG AA

27

85

CA GTGTAA

TGCAG GG

133

AG GTACTG

ATCAG GA

104

AT GTATAT

TCCAG GA

106

TA GTAGGG

TTCAG GA

28

152

AT GTATGT

TGAAG AG

89

TG GTATAT

ACCAG GG

82

TT GTATGT

TGCAG AT

82

TT GTACTG

TGCAG GA

29

85

AG GTACTA

TTTAG GA

105

AG GTCAGC

TCTAG GC

181

AG GTAATT

TTCAG AA

316

TG GTAAAT

TTCAG AA

30

97

AG GTATAT

AACAG GG

73

TT GTATGG

TTCAG GT

103

TG GTTTGT

ACCAG AG

86

TG GTATTT

ACCAG AG

31

83

AG GTGACA

TCTAG GC

81

AG GTGAGA

TGTAG CC

95

AG GTTCCT

TTCAG GC

158

TG GTTTCA

TTCAG GC

32

78

TT GTATGT

TACAG GT

150

TT GTAAAA

TGCAG TA

85

AT GTAAGG

TCCAG GT

77

AT GTAAGG

TACAG GT

33

91

AG GTGAGA

TGCAG CC

128

TG GTATGT

AACAG GT

78

AG GTAAGT

TACAG TC

169

AG GTAAAT

AATAG CC

34

81

AG GTAATC

GATAG TA

100

CT GTGAGT

TGCAG AT

95

AA GTAAAA

GGCAG TA

74

AA GTAAGT

TGCAG TA

35

104

TG GTATGT

AACAG CT

92

AT GTATGC

AACAG GT

165

AG GTATGT

TGCAG GT

90

TG GTATGT

ATCAG GT

36

88

CA GTAAGT

CTCAG AA

101

AG GTAACA

CTTAG CA

88

CG GTAAGG

TACAG GT

83

CG GTAAAG

TACAG AT

37

89

AT GTAAGC

AATAG GT

   

103

AA GTACCT

TGCAG GT

86

AG GTAACT

AATAG AC

38

108

AG GTAAGT

CACAG CA

   

156

AG GTGAAA

GACAG CA

   
 

At XID

At XIA

At XIF

At XIC

No.

Size

5' site

3' site

Size

5' site

3' site

Size

5' site

3' site

Size

5' site

3' site

1

228

TG GTACGA

ATCAG GC

430

TG GTACGA

TGCAG GC

89

TG GTAAGC

GTTAG GG

143

AG GTTAGT

TGTAG GT

2

47

AG GTACCT

TGTAG GT

215

CG GTAAGA

CTTAG GT

169

CA GTAAGA

TACAG GT

93

AG GTCCAG

TATAG GT

3

173

AT GTACGC

TACAG AC

134

TA GTAAGC

TCCAG AC

100

TA GTCAGT

CGCAG AC

82

AT GTTTTG

GACAG AC

4

89

AG GTAATC

TTTAG AA

91

AG GTAACT

TTCAG GA

81

TG GTAAAA

ACTAG GG

95

AG GTGAGT

CTCAG GG

5

109

AG GTAGAT

TGCAG TC

112

AG GTAATG

TGCAG TC

71

AG GTGAGT

TATAG TC

93

AA GTAATG

TCCAG TC

6

90

AG GTGGAA

TGCAG TC

93

AG GTGGAG

TGCAG TC

96

AG GTGGTG

GACAG TC

83

AG GTGAAG

CTCAG TC

7

117

AG GTAAAC

TTCAG GA

101

AG GTAAGC

TTCAG GA

84

AG GTAAGT

TTCAG GA

72

AG GTACGT

AGCAG GA

8

68

AG GTACCT

TGTAG GA

66

AG GTACTT

TGTAG GA

76

TG GTTTGT

TTTAG GA

101

AG GTCAGT

AACAG GA

9

84

AT GTATAT

GGTAG GT

86

TA GTAAAT

TGCAG GT

79

TG GTATCT

CGTAG GT

174

AT GTAAAA

TTCAG GT

10

90

GG GTAGGT

CCCAG GC

80

TG GTAGAT

TTAAG GA

264

TG GTATGT

GACAG GA

74

TG GTAAGT

TCTAG TA

11

309

AG FTFCTT

TGCAG TT

297

AG GTGCTT

TGCAG TT

79

AG GTAGAC

CAAAG TT

76

AG GTAAAT

TGCAG TT

12

93

AG GTTGGA

TACAG CA

74

AG GTTGGA

TACAG CA

72

AG GTAGAA

TGCAG CA

71

AG GTATTG

TTCAG CA

13

113

AG GTAAGT

GTCAG AA

99

AG GTTAGT

GTCAG AA

97

AG GTATAA

TTCAG AA

84

TG GTAAAG

TTCAG CA

14

86

TG GTAATG

TACAG TA

84

TG GTAATG

TGCAG CA

106

TG GTAAGT

TGCAG CA

74

AA GTAGGT

TCCAG GT

15

105

AT GTTAGT

TTCAG GT

82

AT GTTAGT

TCCAG GT

78

AT GTGAGA

TCCAG GT

154

AG GTAGGG

TGCAG CT

16

78

AG GTCTAC

TACAG CA

214

AG GTCTGA

TACAG CA

70

AG GTAAGC

CCCAG CA

135

GT GTAAGT

TCTAG GG

17

102

GG GTAAGC

CTCAG GG

105

GG GTAAGC

TTCAG GG

90

GA GTAAGC

AACAG GG

92

AG GTAAGT

AACAG CT

18

111

AG GTAGAT

TATAG CT

128

AG GTAGCT

AATAG CT

102

GG GTAAAA

GACAG AT

120

AG GTAACG

TGCAG AT

19

152

AG GTGCGT

CACAG AT

202

AG GTGCAG

CATAG AT

101

AG GTATGT

TTCAG AT

114

AG GTGAGC

TGTAG GA

20

92

AG GTAATA

TTCAG GA

83

AT GTTATA

TTTAG GT

175

AG GTTTTT

TGTAG CA

88

AG GTTTAG

GGCAG GC

21

69

TC GTATCT

CACAG AG

113

AA GTAAGT

CGCAG AG

292

AG GTACTA

AACAG AG

296

TG GTACAA

TTCAG GC

22

280

TG GTGACT

TCCAG GC

256

TG GTAATC

TTCAG GC

148

TG GTAAGT

CAAAG GC

79

GG GTATTT

TATAG GG

23

86

GG GTACAC

TGCAG AT

126

GG GTACAC

TGCAG AT

73

AG GTATTG

ATTAG GC

114

AG GTACTT

AACAG GT

24

72

AG GTAAGG

CTAAG GA

122

AG GTTAGT

AAAAG GT

68

AG GTAAGT

TGTAG GT

105

AG GTAAGA

ATCAG GA

25

120

CC GTCATT

CGTAG GC

114

AG GTAAGA

CTTAG GC

86

AG GTATAC

TCCAG AT

96

AG GTAAAC

TACAG AG

26

432

AC GTAACA

TACAG GA

117

AG GTAATC

CTTAG GC

176

AG GTACGG

ATCAG CC

92

TG GTAAAT

ATCAG GA

27

118

AG GTTATC

TTTAG GC

87

TA GTTAGT

AACAG GA

84

AG GTGCAA

TGCAG AA

88

AG GTTGGC

CTCAG AC

28

77

AG GTGTCA

TCAAG AA

120

AG GTTTTG

TTTAG GC

70

AG GTACGA

TTCAG GA

113

AG GTGATG

ATTAG AG

29

96

AT GTAAGT

TACAG GA

79

CG GTAAAT

TGCAG CC

121

AG GTATTA

GACAG GA

87

AG GTATGC

AATAG GC

30

86

AT GTATGT

TGCAG GA

105

AG GTAAGT

TACAG GA

93

AG GTAATA

AGAAG GG

85

AT GTGAGT

TTTAG GT

31

78

AA GTTTAA

CTCAG AA

88

TA GTATGT

AGCAG GA

75

AA GTAAGC

TGTAG GG

103

AG GTTTTT

AACAG CC

32

121

AG GTAACA

TTTAG GG

164

AG GTAACC

TTCAG AA

93

AT GTTAGT

AACAG GC

70

AG GTATCT

TTCAG TA

33

360

AG GTAGAA

CTGAG GA

147

TG GTAACG

TTTAG GG

85

AT GTAAAA

TCCAG GT

79

TG GTAACC

TACAG GT

34

109

AC GTAAGA

CTCAG AA

92

TG GTATAC

TTCAG AG

82

AG GTACAA

GGCAG TT

148

CG GTAAGT

GACAG GT

35

97

AG GTAAAA

TGCAG CC

67

AC GTAAGA

TTCAG GT

97

AG GTAGGC

TACAG GC

97

AC GTAAGT

AATAG GT

36

87

AT GTAAGT

TGCAG TT

97

TG GTTATT

TGCAG TC

84

TG GTATAG

TACAG GT

74

AG GTTGTT

TGCAG CA

37

98

TG GTCAGT

TCCAG GT

76

AG GTAAAA

TGCAG TT

230

CG GTAAAG

CTCAG GT

   

38

125

CG GTAACT

CTCAG GC

78

TG GTTTGT

TTCAG GT

123

AG GTAAGT

AATAG GT

   

39

84

AC GTATGT

TGCAG GT

206

CG GTAAGT

GTCAG GT

70

AG GTACGC

TTCAG CA

   

40

91

AG GTATTG

CTCAG CA

79

AG GTACAT

TGCAG GT

      

41

   

84

AG GTACTG

AACAG CA

      
 

At XIE

At XIJ

At MYA1

At XI-I

No.

Size

5' site

3' site

Size

5' site

3' site

Size

5' site

3' site

Size

5' site

3' site

1

111

CA GTGACT

TGCAG GG

120

AT GTAAA

GTCAG GT

330

TG GTAAGA

TACAG GT

134

AG GTCTGA

AAAAG CT

2

86

AG GTGAGT

TGTAG AT

117

AT GTAAGA

GACAG AC

100

AT GTATGT

TTCAG GT

860

AT GTGAAC

TTCAG AC

3

80

AT GTTAGT

GACAG AC

85

AG GTGATT

AACAG GG

74

AT GTGAGT

TTCAG AC

95

AG GTGATC

CCCAG AG

4

80

AG GTGCTC

TTCAG GG

292

AA GTAAGT

TACAG TC

102

AG GTAATT

TGCAG AC

181

AA GTAAGA

TGCAG TC

5

116

AA GTATGA

GGCAG TC

135

AG GTAAAC

TACAG CC

300

AG GTGAAA

TTCAG TC

241

AG GTGGGT

TTCAG CC

6

85

AG GTGAAA

GTCAG AT

72

AG GTAGGT

TGCAG GA

76

AG GTAACC

TATAG TC

149

AT GTAATT

CTTAG GA

7

75

AG GTATAC

ACTAG CA

88

AG GTTTGC

TTCAG GA

25

AA GTAAGT

TACAG GA

90

AG GTATAA

ATCAG GA

8

79

AG GTAAGC

AACAG GA

67

AT GTAATA

TTTAG GT

95

AG GTAGTT

TTCAG GA

91

AA GTACAT

ATCAG GT

9

76

AT GTAAGT

TTTAG GT

91

TG GTAAAT

TCCAG GT

103

AG GTAAAT

TCCAG CT

94

TG GTTTGC

GTCAG GC

10

101

TG GTAAGT

TGCAG GT

315

AG GTGATG

TGCAG TT

111

TG GTGGGT

TGCAG GC

135

AG GTTAGC

TGCAG TT

11

86

AG GTAAGG

TGCAG TT

81

AG GTATGA

TACAG CA

355

AG GTGCTT

TGCAG TT

83

AG GTAATA

TTCAG CA

12

88

AG GTAATT

TTCAG CA

440

AG GTTTGT

TGCAG AA

91

AG GTTTGA

TGCAG CA

717

AG GTCGTT

TGCAG AA

13

115

AG GTTATT

AGCAG AA

110

TG GTATAA

TGCAG CA

234

GA GTCTGT

TTCAG AA

85

TG GTACAA

TGCAG CA

14

91

TG GTAATA

TTCAG CA

88

AT GTAAGT

TTCAG GT

153

TG GTGAGT

TGCAG CA

98

AA GTCTTG

TGAAG CC

15

103

AA GTAAGT

TTCAG GT

138

AG GTGACT

TGCAG CT

117

AT GTGAGT

TCCAG GT

127

AG GTAGAG

TTTAG CA

16

70

AG GTAGAT

GATAG TT

75

GG GTCTGT

TGCAG GG

87

AG GTAAGT

TTCAG CA

547

GG GTTAGT

GATAG CC

17

107

GT GTAAGT

TGTAG GG

106

GA GTATGT

ATCAG GT

91

GG GTGCGA

TTTAG GG

302

AG GTACGA

TGCAG CA

18

85

AA GTAAGT

AACAG CT

154

AG GTAAAG

TGCAG AT

77

AA GTAAGA

AATAG CT

95

AG GTATGG

CACAG CT

19

92

AG GTTTTT

TGCAG GT

99

AG GTGAGG

TTTAG GA

87

AG GTAATT

TGTAG AT

269

AG GTTCCT

GCAAG GA

20

157

AG GTGAAC

TATAG GA

99

AG GTTCTA

TGCAG GC

117

AG GTATTT

GTCAG GT

180

AG GTACTT

TTTAG GC

21

88

AG GTTTTA

TGCAG GC

119

AG GTATTG

TATAG GC

159

AG GTACAC

TATAG AC

96

AG GTATGA

TGCAG GT

22

184

TG GTACGT

TTCAG GC

134

AG GTAATG

TTCAG GC

122

TG GTGAGA

CCTAG GC

80

GA GTATGT

TACAG AC

23

90

GG GTATTT

GTCAG GT

130

AG GTATTA

TCCAG GT

125

GG GTGTGA

TGCAG AC

701

AG GTAATT

CACAG AA

24

164

AG GTACTC

AACAG GC

197

AG GTCAGT

TGCAG GA

150

AG GTTTGT

TACAG AG

88

AG GTTTGT

TTCAG TC

25

125

AG GTAAGT

GTCAG GC

   

89

TG GTATCC

TCCAG GC

277

AA GTATGT

AGCAG AA

26

95

AG GTACGG

AACAG GT

   

89

AG GTAGAA

TGTAG AA

620

TT GTAAGT

ATCAG GA

27

101

TG GTAAGT

ATCAG GA

   

104

AT GTATAT

TCCAG GA

220

AG GTGATC

TGCAG AG

28

91

AG GTTTGT

TTCAG AC

   

82

TT GTATGT

TGCAG AT

129

AT GTGAGT

ACCAG GG

29

85

AG GTGTGT

TCTAG AG

   

181

AG GTAATT

TTCAG AA

466

AG GTGAGA

GATAG GT

30

90

AG GTATAT

AATAG GC

   

103

TG GTTTGT

ACCAG AG

89

AG GTAAAT

TTCAG TC

31

86

AC GTGAGT

CTTAG GT

   

95

AG GTTCCT

TTCAG GC

399

AG GTACAC

TATAG GT

32

79

AG GTCTGT

TACAG TC

   

85

AT GTAAGG

TCCAG GT

88

AG GTGAGT

TGTAG GT

33

92

AG GTACAT

TGCAG GT

   

78

AG GTAAGT

TACAG TC

326

AG GTATTA

TGCAG CA

34

78

CG GTAAGT

TGCAG GT

   

95

AA GTAAAA

GGCAG TA

   

35

80

AC GTAAGT

GATAG GT

   

165

AG GTATGT

TGCAG GT

   

36

99

AG GTTAGT

GGCAG TA

   

88

CG GTAAGG

TACAG GT

   

37

      

103

AA GTACCT

TGCAG GT

   

38

      

156

AG GTGAAA

GACAG CA

   
 

At XIK

 

No.

Size

5' site

3' site

No.

Size

5' site

3' site

No.

Size

5' site

3' site

 

1

237

AA GTGAGT

CCCAG TC

14

157

TG GTAGGC

TGCAG TA

27

98

CG GTAAGG

CACAG GA

 

2

269

CC GTAAGT

TTCAG GT

15

87

AG GTATAA

ATCAG GC

28

110

AG GTATCA

TGCAG GA

 

3

105

AT GTAAGT

CGCAG AC

16

319

AG GTATGC

TTCAG GT

29

118

AA GTAAGT

ACCAG GT

 

4

102

AG GTTATT

GGTAG GG

17

148

AC GTAATT

TTAAG GG

30

99

AA GTAAGA

AATAG GG

 

5

115

TG GTGAGG

GAGAG GC

18

150

AA GTAAGT

TGCAG TT

31

276

AG GTAATT

TATAG GC

 

6

356

AG GTACGT

TGCAG AC

19

87

AA GTAAGC

TCCAG TT

32

90

TG GTAAAA

TACAG GC

 

7

105

AG GTATTG

TGTAG GA

20

193

AG GTATCT

TGGAG TT

33

110

TA GTTTCA

GTGAG TG

 

8

85

AG GTCAGT

ATCAG GA

21

125

AG GTAATT

TTTAG GC

34

91

AA GTAAGC

TACAG TA

 

9

84

AG GTATGT

AAAG GT

22

84

AG GTTCGG

ATCAG GC

35

93

TG GTAAAA

TTCAG GT

 

10

229

GC GTTAGC

TTCAG GC

23

74

GA GTAAGT

TATAG TC

36

94

CG GTATTT

TTCAG GT

 

11

81

AG GTAAAG

CTCAG CT

24

121

AG GTATGT

TACAG GC

37

79

AT GTATGT

CATAG GT

 

12

87

AG GTCCGT

AACAG CA

25

202

AG GTTCGT

TTCAG AC

38

81

AG GTAACC

CGCAG CA

 

13

91

AG GTGTCC

TTCAG AA

26

97

CG GTGCCT

TTCAG AG

     

The consensus nucleotide sequences for the 5' and 3' splice sites are A-2G-1 G+1T+2A+3A+4G+5T+6 and T-5G-4C-3A-2G-1G+1T+2, respectively [65]. The most conserved sequences are the 5' consensus G (100%) T (99%) at the +1, +2 positions, respectively, and the 3' A(100%) G(100%) at the -2, -1 positions, respectively. The splice sites in the reported myosins and the predicted myosins (Table 4) all contain the 5' GT and 3' AG sequences. The sequences in the Arabidopsis myosins upstream and downstream of these two very conserved sites varied as a reflection of the less conserved nature of these nucleotides (Table 4). However, these predicted sites at the 5' and 3' splice sites need to be confirmed experimentally.

Discussion

Only two classes of myosins are present in Arabidopsis. A study of myosins in lily and tobacco pollen tubes using antibodies to three animal-type myosins IA and IB, II and V suggested the presence of three types of myosins in these plants [40]. However, no type I, II or V myosins have been found in any plant and only two types (VIII and XI) have been identified. Class XI are somewhat similar to class V myosins [42] and this may explain the reaction with the type V antibody. Possibly the other reactions were due to similarities in the myosin motor domain. Phylogenetic analysis of Arabidopsis myosins along with other plant myosins suggests that most class XI myosins (except three) fall into two subgroups (Figure 4).

The Arabidopsis myosins have anywhere from three to six IQ domains. The IQ domain in non-plant myosins has been shown to bind to calmodulin in a calcium-independent manner. The regulation of myosin action is thought to be due to calmodulin interaction. In plants, two myosin heavy chains have been shown to associate with calmodulin [37,67]. A myosin-containing protein fraction from tobacco BY2 cells was used in motility assays with F-actin. Concentrations of Ca2+ higher than 10-6 M caused a significant reduction in F-actin sliding [37]. Another study with myosin isolated from lily pollen, also demonstrated a co-precipitation of myosin and calmodulin and a similar effect of Ca2+concentration [67]. Not only did concentrations above 10-6M cause inhibition of myosin activity, but the effects of concentrations higher than 10-5 M were not reversible upon Ca2+ removal. These studies provide evidence that plant myosins bind calmodulin in the absence of Ca2+ and are active when calmodulin is bound and inactivated when the Ca2+ concentration is increased. They also found that when the myosin fraction was pretreated with CaCl2 calmodulin did not bind the myosin, suggesting that calmodulin dissociates from myosin at high concentrations of Ca2+. The myosins in the above studies have not been cloned, and binding to specific IQ domains has not been established. However, the presence of IQ domains in Arabidopsis and other plant myosins suggests that these are the sites of Ca2+ regulation. It would be interesting to investigate the possible phosphorylation of the threonine residue which is three residues upstream from the TEDS rule site in class XI myosins and to see if enzyme activity is regulated by phosphorylation of this residue.

Myosins are involved in a wide range of cellular functions. They have been shown to be involved in movement, translocation, cell division, organelle transport, G-protein-linked signal cascade and maintenance of structure within cells [26]. Insight into the function of plant myosins has been gained by studies in algae. Cytoplasmic streaming is responsible for movement of organelles and vesicles and of generative cells and vegetative nuclei in pollen tubes. Physiological studies in Chara have shown that an increase in Ca2+ concentration causes cytoplasmic streaming to stop [68]. A myosin isolated from the alga Chara corallina was shown to be responsible for cytoplasmic streaming [30,69,70]. The myosin was cloned and characterized and found to be a class XI myosin related to the Arabidopsis MYA myosins [54].

Myosins in plants have also been shown to be involved in cytoplasmic streaming. Using immunofluorescence, myosin was localized to vesicles, organelles and generative cells and vegetative nuclei in grass pollen tubes [39]. A myosin isolated from lily pollen has been shown to be responsible for cytoplasmic streaming in pollen tubes and two myosins were identified in tobacco cell cultures that are also thought to participate in cytoplasmic streaming [37,71]. Antibodies to the myosins recognized a protein in vegetative cells as well as pollen tubes. Liu et al. [51] suggest that class XI myosins are likely candidates for transport of large vesicles because of the number of IQ domains (5-6). Previous studies showed that translocational step size produced by a myosin motor is proportional to the number of IQ domains and the larger the step the faster or more efficiently they are able to transport vesicles [9]. However, the kinetic properties of the motor domain are also involved in speed and there is a wide range of movement speeds for myosin II molecules [2,72,73].

An antibody specific to a Z. mays class XI myosin was used to localize this myosin in fractions of maize proteins and maize root tip cells [51]. The nuclear/cell wall fraction and the plastid fraction contained relatively small amounts of antigen while the mitochondrial fraction and the low density membrane fraction had most of the antigen. The root tip cells showed particulate staining in the cytoplasm, but neither the vacuole membrane nor plasma membrane were stained, although in some cells the staining was too bright to distinguish if the plasma membrane was stained or not. There are 13 class XI myosins in Arabidopsis that could be involved in vesicle and organelle transport. The large number could reflect redundancy of function or differential expression. Patterns of expression were different for the cloned Z. mays and Arabidopsis myosins that have been analyzed [42,51].

Immunolocalization studies have also detected myosin associated with plasmodesmata. Plasmodesmata are interconnections between contiguous plant cells that allow direct cell-to-cell transport of ions and proteins. A recent study using an antibody to a cloned class VIII Arabidopsis myosin ATM1 (At ATM) localized this myosin to the plasmodesmata and the plasma membrane regions involved in the assembly of new cell walls [47]. Earlier work suggested that actin was involved in regulation of plasmodesmal transport [74]. Other studies using antibodies to animal myosins in root tissues of Allium cepa, Z. mays and Hordeum vulagare have also indicated the presence of myosin in the plasmodesmata [38]. However, immunolocalization studies with antibodies to animal myosins need to be interpreted with caution as there are no plant myosins that group with animal myosins.

The recent work by Reichelt et al. [47] is more convincing because they used antibody to plant myosin. The myosin was localized mainly to the transverse walls with some punctate labeling of the longitudinal walls. During cell division the anti-class-VIII myosin staining remains confined to the transverse cell walls and is strongest in the newly formed cell wall. Immunogold electron microscopy showed labeling of class VIII myosin associated with the plasma membrane and plasmodesmata. These studies suggest that class VIII myosins may be involved in new cell wall formation and transport in the plasmodesmata. Reichelt et al. [47] suggest that myosin VIII could act to bring islands of membrane plate material together or could trigger exocytosis of new cell wall material, or alternatively as an anchor for actin along the transverse walls. The role of myosin in the plasmodesmata was studied further by pretreating tissue with 2,3-butanedione 2-moxoxime (BDM), an inhibitor of actin-myosin motility. The pretreatment resulted in a strong constriction of the neck region of plasmodesmata [38]. Myosin VIII in the plasmodesmata could be a part of a gating complex that is thought to control the opening of the plasmodesma neck [74]. There are four class VIII myosins in Arabidopsis that could be involved in these types of functions.

A recent study of the effect of BDM on the distribution of myosins, F-actin, microtubules and cortical endoplasmic reticulum (ER) suggests that myosins may link together microtubules and actin filaments involved in structural interactions [75]. This study used antibody to myosin II from animals and Arabidopsis myosin VIII for immunofluorescence studies. BDM treatment disrupted normal cellular distributions of maize myosins and the characteristic distribution of F-actin was also affected. Myosin may participate in the intracellular distribution of actin filaments as was proposed for myosin XV [76]. Microtubule arrangements in cortical root cells were altered, as was the normal ER network. Post-mitotic cell growth was inhibited by BDM, specifically in the transition zone and the apical parts of the elongation region. The study suggested that actin fibers and microtubules interact together via myosins and that myosin-based contractility of the actin cytoskeleton is essential for the developmental progression of root cells [75]. However, BDM has only been shown to inhibit a few myosins in vitro [77] and is known to be a nonspecific inhibitor; so these results must be viewed with caution.

Conclusions

As the classification system of myosins now stands, plant myosins fall only into two classes - class VIII and class XI. All animal cells examined contain at least one myosin II gene and usually multiple myosin I genes [8], but this is not true for Arabidopsis specifically and possibly for all plants. Also, no animal myosins of type VIII or XI have been identified. Plant and animal cells have some common tasks such as vesicular and organelle movement, but plant cells are unique in many ways and the presence of specific plant myosins is probably a reflection of that uniqueness.

There are 4 class VIII and 13 class XI Arabidopsis myosins. The large number of myosins in class XI could be the result of gene duplication or specialization of function in different tissues or different life cycle times. This work identifies the Arabidopsis myosins, their domains and gene intron/exon structure. The task ahead is to analyze the protein products biochemically and try to establish the function of each myosin.

Materials and methods

Using the conserved motor domain of the plant myosin At MYA1 [41] database searches were performed using BLASTP and TBLASTN at TAIR [11]. The sequences were evaluated for the presence of a myosin motor domain using the SMART program [56]. All sequences with a myosin domain had BLASTP scores greater than 100 and E values less than 10-20. The motor domains of representative myosins from other groups were also used to search the Arabidopsis domain but the searches did not reveal any new myosin genes. The SMART program also identified the IQ and coiled-coil domains and the location of the domains. The sequences found at TAIR were checked against the MIPS database [57]. Sequences identified at MIPS as myosins but not at TAIR were evaluated as above. The sizes of the exons/introns were determined using the exon/intron data for each myosin sequence using the MIPS predictions for myosins not previously cloned. Two sequences (At XIF, At XIH) were edited by comparing the upstream genome sequence translation to conserved sequences present in the other myosins but missing in the predicted sequences.

Sequences of myosins other than the Arabidopsis myosins for phylogenetic analysis were obtained from MHP [22] or NCBI [58]. The names are as in the tree of Hodge and Cope [59]. The motor domain sequences were determined using the SMART program [56]. The motor domain sequences were used for alignment of the plant and non-plant myosins using the Megalign program. The alignment was saved as a PAUP file and the phylogenetic analysis was done using PAUP 4.0b4a (PPC). We performed a bootstrap analysis with 100 replicates using the heuristic method. Full-length sequences were used for analysis of the plant myosins using the same methods as above.

Declarations

Acknowledgements

This work was supported in part by grants from the National Science Foundation (MCB-0079938) and NASA to A.S.N.R. We thank Jun Wen for help with the phylogenetic analysis. We thank the anonymous reviewers for their useful suggestions.

Authors’ Affiliations

(1)
Department of Biology and Program in Cell and Molecular Biology, Colorado State University

References

  1. Hirokawa N: Microtubule organization and dynamics dependent on microtubule-associated proteins. Curr Opin Cell Biol. 1994, 6: 74-81.View ArticleGoogle Scholar
  2. Williamson RE: Organelle movements along actin filaments and microtubules. Plant Physiol. 1986, 82: 631-634.View ArticleGoogle Scholar
  3. Volkmann D, Baluska F: Actin cytoskeleton in plants: from transport networks to signaling networks. Microsc Res Tech. 1999, 47: 135-154. 10.1002/(SICI)1097-0029(19991015)47:2<135::AID-JEMT6>3.0.CO;2-1.View ArticleGoogle Scholar
  4. Reddy ASN: Molecular motors and their functions plants. Intl Rev Cytol Cell Biol. 2001, 204: 97-178.View ArticleGoogle Scholar
  5. Vallee RB, Sheptner HS: Motor proteins of cytoplasmic microtubules. Annu Rev Biochem. 1990, 59: 909-932.View ArticleGoogle Scholar
  6. Langford GM: Actin- and microtubule-dependent organelle motors: interrelationships between the two motility systems. Curr Opin Cell Biol. 1995, 7: 82-88.View ArticleGoogle Scholar
  7. Goldstein LSB, Philip AV: The road less traveled: emerging principles of kinesin motor utilization. Annu Rev Cell Dev Biol. 1999, 15: 141-183.View ArticleGoogle Scholar
  8. Sellers JR: Myosins: a diverse superfamily. Biochim Biophys Acta. 2000, 1496: 3-22.View ArticleGoogle Scholar
  9. Mermall V, Post PL, Mooseker MS: Unconventional myosins in cell movement, membrane traffic, and signal transduction. Science. 1998, 279: 527-533.View ArticleGoogle Scholar
  10. Moscatelli A, Del Casino C, Lozzi L, Cai G, Scali M, Tiezzi A, Cresti M: High molecular weight polypeptides related to dynein heavy chains in Nicotiana tabacum pollen tubes. J Cell Sci. 1995, 108: 1117-1125.Google Scholar
  11. The Arabidopsis Information Resource. [http://www.Arabidopsis.org/]
  12. Mitsui H, Yamaguchi-Shinozaki K, Shinozaki K, Nishikawa K, Takahashi H: Identification of a gene family (kat) encoding kinesin-like proteins in Arabidopsis thaliana and the characterization of secondary structure of KatA. Mol Gen Genet. 1993, 238: 362-368.View ArticleGoogle Scholar
  13. Reddy ASN, Safadi F, Narasimhulu SB, Golovkin M, Hu X: A novel plant calmodulin-binding protein with a kinesin heavy chain motor domain. J Biol Chem. 1996, 271: 7052-7060.View ArticleGoogle Scholar
  14. Reddy ASN, Narasimhulu SB, Safadi F, Golovkin M: A plant kinesin heavy chain-like protein is a calmodulin-binding protein. Plant J. 1996, 10: 9-21.View ArticleGoogle Scholar
  15. Abdel-Ghany SE, Reddy ASN: A novel calcium/calmodulin-regulated kinesin-like protein is highly conserved between monocots and dicots. DNA Cell Biol. 2000, 19: 567-578.View ArticleGoogle Scholar
  16. Asada T, Kuriyama R, Shibaoka H: TKRP125, a kinesin-related protein involved in the centrosome-independent organization of the cytokinetic apparatus in tobacco BY-2 cells. J Cell Sci. 1997, 110: 179-189.Google Scholar
  17. Liu B, Cyr RJ, Palevitz BA: A kinesin-like protein, KatAp, in the cells of Arabidopsis and other plants. Plant Cell. 1996, 8: 119-132.View ArticleGoogle Scholar
  18. Song H, Golovkin M, Reddy ASN, Endow SA: In vitro motility of AtKCBP, a calmodulin-binding kinesin-like protein of Arabidopsis. Proc Natl Acad Sci USA. 1997, 94: 322-327.View ArticleGoogle Scholar
  19. Lee Y-RJ, Liu B: Identification of a phragmoplast-associated kinesin-related protein in higher plants. Curr Biol. 2000, 10: 797-800.View ArticleGoogle Scholar
  20. Kim AJ, Endow SA: A kinesin family tree. J Cell Sci. 2000, 113: 3681-3682.Google Scholar
  21. Reddy ASN, Day IS: Kinesin-like proteins in Arabidopsis: a comparative analysis among eukaryotes. BMC Genomics. 2001, Google Scholar
  22. The Myosin Home Page. [http://www.mrc-lmb.cam.ac.uk/myosin/myosin.html]
  23. Yamashita RA, Sellers JR, Anderson JB: Identification and analysis of the myosin superfamily in Drosophila: a database approach. J Muscle Res Cell Motil. 2000, 21: 491-505.View ArticleGoogle Scholar
  24. Goodson HV, Spudich JA: Molecular evolution of the myosin family: relationships derived from comparisons of amino acid sequences. Proc Natl Acad Sci USA. 1993, 90: 659-663.View ArticleGoogle Scholar
  25. Soldati T, Geissler H, Schwarz EC: How many is enough? Exploring the myosin repertoire in the model eukaryote Dictyostelium discoideum. Cell Biochem Biophys. 1999, 30: 389-411.View ArticleGoogle Scholar
  26. Cope MJ, Whisstock J, Rayment I: Conservation within the myosin motor domain: implications for structure and function. Structure. 2000, 4: 969-986.View ArticleGoogle Scholar
  27. Korn ED: Coevolution of head, neck, and tail domains of myosin heavy chains. Proc Natl Acad Sci USA. 2000, 97: 12559-12564.View ArticleGoogle Scholar
  28. Cheney RE, Mooseker MS: Unconventional myosins. Curr Opin Cell Biol. 1992, 4: 27-35.View ArticleGoogle Scholar
  29. Rhoads AR, Friedberg F: Sequence motifs for calmodulin recognition. FASEB J. 1997, 11: 331-340.Google Scholar
  30. Yamamoto K, Hamada S, Kashiyama T: Myosins from plants. Cell Mol Life Sci. 1999, 56: 227-232.View ArticleGoogle Scholar
  31. Shimmen T, Yokota E: Physiological and biochemical aspects of cytoplasmic streaming. Int Rev Cytol. 1994, 155: 97-140.View ArticleGoogle Scholar
  32. Reddy ASN, Day IS: The role of the cytoskeleton and a molecular motor in trichome morphogenesis. Trends Plant Sci. 2000, 5: 503-505.View ArticleGoogle Scholar
  33. Szymanski DB, Marks DM, Wick SM: Organized F-actin is essential for normal trichome morphogenesis in Arabidopsis. Plant Cell. 1999, 11: 2331-2348.View ArticleGoogle Scholar
  34. Mathur J, Spielhofer P, Kost B, Chua N: The actin cytoskeleton is required to elaborate and maintain spatial patterning during trichome cell morphogenesis in Arabidopsis thaliana. Development. 1999, 126: 5559-5568.Google Scholar
  35. Pierson ES, Cresti M: Cytoskeleton and cytoplasmic organization of pollen and pollen tubes. Intn Rev Cytol. 1992, 140: 73-125.View ArticleGoogle Scholar
  36. Pierson ES, Miller DD, Callaham DA, Shipley AM, Rivers BA, Cresti M, Hepler PK: Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media. Plant Cell. 1994, 6: 1815-1828.View ArticleGoogle Scholar
  37. Yokota E, Yukawa C, Muto S, Sonobe S, Shimmen T: Biochemical and immunocytochemical characterization of two types of myosins in cultured tobacco bright yellow-2 cells. Plant Physiol. 1999, 121: 525-534.View ArticleGoogle Scholar
  38. Radford JE, White RG: Localization of a myosin-like protein to plasmodesmata. Plant J. 1998, 14: 743-750. 10.1046/j.1365-313x.1998.00162.x.View ArticleGoogle Scholar
  39. Heslop-Harrison J, Heslop-Harrison Y: Myosin associated with the surface of organelles, vegetative nuclei and generative cells in angiosperm pollen grains and tubes. J Cell Sci. 1989, 94: 319-325.Google Scholar
  40. Miller DD, Scordilis SP, Hepler PK: Identification and localization of three classes of myosins in pollen tubes of Lilium longiflorum and Nicotiana alata. J Cell Sci. 1995, 108: 2549-2563.Google Scholar
  41. Kinkema M, Schiefelbein J: A myosin from a higher plant has structural similarities to class V myosins. J Mol Biol. 1994, 239: 591-597. 10.1006/jmbi.1994.1400.View ArticleGoogle Scholar
  42. Kinkema M, Wang H, Schiefelbein J: Molecular analysis of the myosin gene family in Arabidopsis thaliana. Plant Mol Biol. 1994, 26: 1139-1153.View ArticleGoogle Scholar
  43. Knight AE, Kendrick-Jones J: A myosin-like protein from a higher plant. J Mol Biol. 1993, 231: 148-54. 10.1006/jmbi.1993.1266.View ArticleGoogle Scholar
  44. Parke J, Miller C, Anderton BH: Higher plant myosin heavy-chain identified using a monoclonal antibody. Eur J Cell Biol. 1996, 41: 9-13.Google Scholar
  45. Qiao L, Grolig F, Jablonsky PP, Williamson RE: Myosin heavy chain: Detection by immunoblotting in higher plants and localization by immunofluorescence in the alga Chara. Cell Biol Int Rep. 1989, 13: 107-117.View ArticleGoogle Scholar
  46. Tang XJ, Hepler PK, Scordilis SP: Immunochemical and immunocytochemical identification of a myosin heavy chain polypeptide in Nicotiana pollen tubes. J Cell Sci. 1989, 92: 569-574.Google Scholar
  47. Reichelt S, Knight AE, Hodge TP, Baluska F, Samaj J, Volkmann D, Kendrick-Jones J: Characterization of the unconventional myosin VIII in plant cells and its localization at the post-cytokinetic cell wall. Plant J. 1999, 19: 555-567. 10.1046/j.1365-313X.1999.00553.x.View ArticleGoogle Scholar
  48. Kohno T, Okagaki T, Kohama K, Shimment T: Pollen tube extract supports the movement of actin filaments in vitro. Protoplasma. 1991, 161: 75-77.View ArticleGoogle Scholar
  49. Vahey M, Titus M, Trautwein R, Scordilis S: Tomato actin and myosin: Contractile proteins from a higher land plant. Cell Motil. 1982, 2: 131-148.View ArticleGoogle Scholar
  50. Ma Y-Z, Yen L-F: Actin and myosin in pea tendrils. Plant Physiol. 1989, 89: 586-589.View ArticleGoogle Scholar
  51. Liu L, Zhou J, Pesacreta TC: Maize myosins: diversity, localization, and function. Cell Motil Cytoskeleton. 2001, 48: 130-148. 10.1002/1097-0169(200102)48:2<130::AID-CM1004>3.3.CO;2-P.View ArticleGoogle Scholar
  52. Plazinski J, Elliott J, Hurley UA, Burch J, Arioli T, Williamson RE: Myosins from angiosperms, ferns, and algae amplification of gene fragments with versatile PCR primers and detection of protein products with a monoclonal antibody to a conserved head epitope. Protoplasma. 1997, 196: 78-86.View ArticleGoogle Scholar
  53. Moepps Y, Conrad S, Schraudolf H: PCR-dependent amplification and sequence characterization of partial cDNAs encoding myosin-like proteins in Anemia phyllitidis (L.) Sw. and Arabidopsis thaliana (L.) Heynh. Plant Mol Biol. 1993, 21: 1077-1083.View ArticleGoogle Scholar
  54. Kashiyama T, Kimura N, Mimura T, Yamamoto K: Cloning and characterization of a myosin from characean alga, the fastest motor protein in the world. J Biochem (Tokyo). 2000, 127: 1065-1070.View ArticleGoogle Scholar
  55. Arabidopsis Genome Initiative: Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature. 2000, 408: 796-815. 10.1038/35048692.View ArticleGoogle Scholar
  56. Simple Modular Architecture Research Tool. [http://smart.embl-heidelberg.de/]
  57. Munich Information Center for Protein Sequences. [http://www.mips.biochem.mpg.de]
  58. National Center for Biotechnology Information, Entrez, Protein. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein]
  59. Hodge T, Cope MJ: A myosin family tree. J Cell Sci. 2000, 113: 3353-3354.Google Scholar
  60. Rubin GM, Yandell MD, Wortman JR, Gabor Miklos GL, Nelson CR, Hariharan IK, Fortini ME, Li PW, Apweiler R, Fleischmann W, et al: Comparative genomics of the eukaryotes. Science. 2000, 287: 2204-2215. 10.1126/science.287.5461.2204.View ArticleGoogle Scholar
  61. Goldstein LS, Gunawardena S: Flying through the cytoskeletal genome. J Cell Biol. 2000, 150: F63-8.View ArticleGoogle Scholar
  62. Arabidopsis Sequence Map Overview. [http://www.Arabidopsis.org/cgi-bin/maps/Schrom]
  63. Brzeska H, Korn ED: Regulation of class I and class II myosins by heavy chain phosphorylation. J Biol Chem. 1996, 271: 16983-16986.View ArticleGoogle Scholar
  64. Pole Bio-Informatique Lyonnais. [http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_clustalw.html]
  65. Brown JWS, Smith P, Simpson CG: Arabidopsis consensus intron sequences. Plant Mol Biol. 1996, 32: 531-535.View ArticleGoogle Scholar
  66. Hunt AG, Morgen BD, Chu NM, Chua N-H: The SV40 small t is accurately and efficiently spliced in tobacco cells. Plant Mol Biol. 1991, 16: 375-379.View ArticleGoogle Scholar
  67. Yokota E, Muto S, Shimmen T: Inhibitory regulation of higher-plant myosin by Ca2+ ions. Plant Physiol. 1999, 119: 231-240.View ArticleGoogle Scholar
  68. Hayama T, Shimmen T, Tazawa M: Participation of Ca2+ in cessation of cytoplasmic streaming induced by membrane excitation in Characeae internodal cells. Protoplasma. 1979, 99: 305-321.View ArticleGoogle Scholar
  69. Yamamoto K, Kikuyama M, Sutoh-Yamamoto N, Kamitsubo E: Purification of actin based motor protein from Chara corallina. Proc Japn Acad. 1994, 70: 175-180.View ArticleGoogle Scholar
  70. Yamamoto K, Kikuyama M, Sutoh-Yamamoto N, Kamitsubo E, Katayama E: Myosin from Alga Chara: unique structure revealed by electron microscopy. J Mol Biol. 1995, 254: 109-112. 10.1006/jmbi.1995.0603.View ArticleGoogle Scholar
  71. Yokota E, Shimmen T: Isolation and characterization of plant myosin from pollen tubes of lily. Protoplasma. 1994, 177: 153-162.View ArticleGoogle Scholar
  72. Canepari M, Rossi R, Pellegrino M, Bottinelli R, Schiaffino S, Reggiani C: Functional diversity between orthologous myosins with minimal sequence diversity. J Muscle Res Cell Motil. 2000, 21: 375-382.View ArticleGoogle Scholar
  73. Sellers JR, Goodson HV, Wang F: A myosin family reunion. J Muscle Res Cell Motil. 1996, 17: 7-22.View ArticleGoogle Scholar
  74. Ding B, Kwon MO, Warnberg L: Evidence that actin filaments are involved in controlling the permeability of plasmodesmata in tobacco mesophyll. Plant J. 1996, 10: 157-164.View ArticleGoogle Scholar
  75. Samaj J, Peters M, Volkmann D, Baluska F: Effects of myosin ATPase inhibitor 2,3-butanedione 2-monoxime on distributions of myosins, F-actin, microtubules, and cortical endoplasmic reticulum in maize root apices. Plant Cell Physiol. 2000, 41: 571-582.View ArticleGoogle Scholar
  76. Liang Y, Wang A, Belyantseva IA, Anderson DW, Probst FJ, Barber TD, Miller W, Touchman JW, Jin L, Sullivan SL, et al: Characterization of the human and mouse unconventional myosin XV genes responsible for hereditary deafness DFNB3 and shaker 2. Genomics. 1999, 61: 243-58. 10.1006/geno.1999.5976.View ArticleGoogle Scholar
  77. Cramer LP, Mitchison TJ: Myosin is involved in postmitotic cell spreading. J Cell Biol. 1995, 131: 179-189.View ArticleGoogle Scholar

Copyright

© Reddy and Day, licensee BioMed Central Ltd 2001