Identification of cytoskeleton-associated genes expressed during Arabidopsis syncytial endosperm development

Abstract

During the early stages of Arabidopsis seed development, the endosperm is syncytial and proliferates rapidly through multiple rounds of mitosis in the absence of cytokinesis and cell wall formation. This stage of endosperm development is important in determining seed viability and size. To identify genes involved in syncytial endosperm development, we analyzed the endosperm transcriptome, obtained using laser capture microdissection of developing seeds at four days after pollination. Our results support the idea that similar sets of genes are required for conventional somatic mitosis with cytokinesis and syncytial proliferation. Furthermore, we identify cytoskeleton associated genes that may act to facilitate syncytial development thereby providing an important resource for further characterization of the processes involved in syncytial endosperm development.

Seed development begins with double fertilization where the haploid egg cell and the double haploid central cell are both fertilized by haploid sperm cells contributed from a single pollen grain. This generates the diploid embryo and the triploid endosperm, respectively. The embryo and the endosperm grow rapidly in a coordinated manner that is influenced by the surrounding maternal integument tissues that later form the seed coat. During these early stages, maternal resources are used for the rapid cell division and growth that occurs as seed tissues form and develop. In many plant species, including Arabidopsis, the triploid primary endosperm nucleus undergoes several rounds of free-nuclear division, growing rapidly as a syncytium. The organization of microtubule arrays during this early stage of endosperm development is markedly different from those found in vegetative tissues.¹ During the syncytial phase, interphase microtubules emanate from the nucleus into the cytoplasm and this nucleus-based radial microtubule system aids in positioning of nuclei and the designation of cytoplasm into nucleo-cytoplasmic domains. At about six days after pollination, cell wall deposition is initiated by the formation of phragmoplasts at the margins of nucleocytoplasmic domains. Interestingly, interzonal arrays which resemble phragmoplasts form between nuclei after karyokinesis, but they never participate in cell plate formation.² This unusual form of cellular development is conducive to a rapid cellular proliferation; however, the molecular mechanisms that underlie the uncoupling of cell wall formation from mitosis remain elusive.

To gain further insights into the early stages of endosperm biology, we have used laser capture microdissection to obtain RNA from syncytial stage endosperm at four days after pollination. At this stage of development, the endosperm has undergone six to seven rounds of nuclear division (∼200 nucleocytoplasmic domains) but has not cellularized and the embryo is at the globular phase. Endosperm RNA was amplified using two-round in vitro transcription, then labeled and used, along with similarly treated silique amplified RNA, to probe long oligonucleotide arrays (based on the Operon V1.0 Arabidopsis set).³ The procedure identified 2,568 individual loci as being preferentially expressed in the endosperm and further analysis showed approximately 800 of these to be early seed-specific. This data has been further validated by quantitative reverse transcription PCR and by using GUS reporter lines.⁴ Here, we identify endosperm-preferred early seed-specific genes derived from statistically over-represented TAIR gene ontology categories relating to the cytoskeleton. Twenty-three genes that are involved in cytoskeletal organization/ dynamics were identified (Table 1) and further functional analysis will likely provide insight into the intriguing syncytial form of cellular development.

Table 1.

Early seed specific endosperm preferred cytoskeleton-associated genes taken from gene ontology terms provided by the arabidospsis information resource

Locus	Name	Description	Pa	Fold change
Spindle assembly
At2g28620	AtKRP125c	Kinesin-5	0.01	2.6
At2g36200	AtKRP125b	Kinesin-5	0.02	3.1
At3g10310	ATK4 like	Kinesin-14	0.01	3.4
At4g21270	ATK1	Kinesin-14	0.02	2.8
At4g05190	ATK5	Kinesin-14	0.00	4.1
At5g27000	ATK4	Kinesin-14	0.00	6.9
Phragmoplast formation
At1g18370	HIK	Kinesin-14	0.01	2.8
At3g20150	PAKRP1	Kinesin-12	0.01	3.7
At3g23670	PAKRP1L	Kinesin-12	0.00	2.7
At4g14330	PAKRP2	Kinesin-12	0.00	3.4
Uncharacterized kinesins
At1g59540		Kinesin-7	0.00	5.0
At3g63480		Orphan, Ungrouped	0.01	4.1
At2g22610		Kinesin-14	0.03	2.6
At5g02370		Kinesin-13	0.00	3.7
At5g23910		Kinesin-13	0.00	4.2
At1g63640		Kinesin-14	0.00	2.0
At1g55550		Kinesin-4	0.00	5.7
Microtubule-associated proteins
At5g55230	AtmaP65-1	MAP	0.00	2.1
At5g67270	AteB1c	+TIP	0.00	5.7
Actin nucleation/actin based movement
At1g42980	Formin-like protein 12	formin	0.00	5.5
At2g20290	AtXiG	myosin	0.01	4.4
At3g12380	AtARP5	ARP	0.00	2.0
At3g63110	RanGaP1	GAP	0.07	2.0

Three different types of molecular motors have important roles in the dynamics, organization and transport processes associated with the cytoskeleton. The kinesin and dynein motors move cargo along microtubules, whereas the myosin family of molecular motors move cargo on actin filaments. Among the cytoskeleton associated motor proteins, kinesins are particularly well represented in the early seed-specific endosperm preferred list (17 of the 23 genes in Table 1 belong to the kinesin family). The high abundance of kinesins among cytoskeleton-associated proteins is likely a reflection of the large size of the kinesin family in Arabidopsis (61 kinesins).⁵ Ten kinesins with endosperm preferred expression fell into two groups, spindle assembly and phragmoplast formation (Table 1). To our knowledge, the remaining seven kinesins (At1g59540, At3g63480, At2g22610, At5g02370, At5g23910, At1g63640 and At1g55550) have not yet been evaluated for their functions.

Spindle Assembly

Based on their demonstrated (ATK1, ATK5) and predicted (ATK4, ATK4 like, AtKRP125b, AtKRP125c) functions, one group of kinesins is involved mainly in spindle assembly. In Arabidopsis atk1 mutants, accumulation of microtubules was reduced at spindle poles, suggesting a role for ATK1 in spindle assembly. Nevertheless, segregation of chromosomes is not effected in these mutants.⁶ Kinesin ATK5 is required for the coalignment of antiparallel interpolar microtubules which are important for spindle integrity.⁷ AtKRP125b and AtKRP125c are classified as Kinesin-5 proteins which indicates they might form homotetramers which facilitate the sliding of antiparallel microtubules⁸ and are essential for spindle bipolarity. The tobacco orthologue of AtKRP125b/AtKRP125c, TKRP125 decorates microtubules in S, G₂ and mitosis, but is not expressed in G₁ phase.⁹ When cultured tobacco cells were incubated with an antibody against TKRP125, translocation of microtubules within the phragmoplast was inhibited. TKRP125 seems to be involved in diverse aspects of microtubule array formation during the cell cycle and is particularly important during phragmoplast expansion. The endosperm preferred expression of these kinesins suggests that spindle formation and assembly in the endosperm, and conventional cytokinesis are accomplished by similar pathways.

Phragmoplast Formation

The second group of kinesins with early seed specific and endosperm preferred expression are associated with the phragmoplast or its expansion (AtPAKRP1, AtPAKRP1L, AtKRP2, HIK). AtPAKRP1 and AtPAKRP1L localize to phragmoplast microtubule plus ends, and act redundantly in male meiotic cytokinesis.¹⁰ Double mutant plants fail to form the phragmoplast array in the first post-meiotic cytokinesis and consequently cell plates are not formed. AtPAKRP2 accumulate at the phragmoplast in a punctuate pattern, suggesting a role in transport of Golgi derived vesicles to the phragmoplast midzone.¹¹ In tobacco the HINKEL (HIK) orthologue, NACK1 activates a mitogen activated protein kinase (MAPK) cascade upon binding to the corresponding MAP kinase kinase kinase (MAPKKK) and promotes the successive expansion of the phragmoplast from the cell center towards the periphery of the cell.¹² hik mutant embryos exhibit multinucleate cells and incomplete cell walls indicating a role for HIK in cytokinesis.¹³ Furthermore, endosperm cellularization fails largely in hik mutants.¹⁴ Thus, HIK/NACK1 kinesin is involved in phragmoplast expansion in conventional cytokinesis and, consistent with this study, is also required for cellularization in the endosperm. It will be interesting to investigate whether the endosperm of mutants corresponding to the endosperm preferred genes found in this study behave in a similar manner. It is not clear how the interzonal antiparallel microtubule arrays, which resemble phragmoplasts, are maintained, but it seems that these arrays do not recruit protein complexes required for formation of the cell plate.² Analysis of localization patterns of some of the proteins involved in phragmoplast expansion and cell plate formation might be useful to gain insights whether the two arrays differ in the distribution of these proteins.

Microtubule-Associated Proteins

Another class of proteins regulating microtubule dynamics via association with the microtubule lattice is microtubule-associated proteins (MAPs). Although previously annotated as a gene of unknown function, the Endosperm Defective1 gene (EDE1) has recently been shown to encode a MAP.¹⁵ EDE1 is expressed in both embryo and endosperm and is essential for microtubule function during the mitotic and cytokinetic aspects of early seed development. The seed of the ede1 mutant never cellularises, contains a reduced number of enlarged polyploid nuclei, and features an aberrant microtubule cytoskeleton, where the specialized radial microtubule systems and cytokinetic phragmoplasts are absent.¹⁵ This study also indicates that the MAPS, MAP65-1 and AtEB1c, may also play important roles that are specific to early endosperm development. Studies concerning these genes have revealed that MAP65s form 25 nm cross bridges between microtubules.¹⁶ Binding to microtubules depends on the phosphorylation status of AtMAP65-1 and underlies tight cell cycle control. AtMAP65-1 localizes to the spindle midzone and its inactivation is required for transition to anaphase¹⁷ whereas AtEB1c-GFP weakly accumulates in the nucleus and at the PPB, but is highly abundant at spindle and phragmoplast in mitosis;¹⁸ however functional data are not yet available.

Actin nucleation and actin-based movement

Whilst cytokinesis in plants primarily involves microtubule dependent structures and vesicular transport, actin nucleation may also play an important role. Formins are actin-organising proteins that are involved in cell polarity and cytokinesis. Although twenty Arabidopsis genes have strong similarity to formins, only Formin Homology5 (FH5) has been characterized.¹⁹ FH5 is expressed in most vegetative tissues, however during early seed development, expression is confined to the endosperm compartment of the seed. Analysis of the fh5 mutant revealed an endosperm specific phenotype with altered migration of nodules to the chalazal cyst during the syncytial stage of development and delayed cellularisation.¹⁹

Delayed cellularisation in the fh5 mutant is thought to be due to stalling in the expansion of the cell plate due to aberrant assembly of short actin filaments observed during the formation of the plate.¹⁹ However, the lack of an observable phenotype in other tissues of the fh5 mutants and the eventual cellularisation and formation of the chalazal cyst indicate some functional redundancy may be operating within the formin family. Here we identify Formin Homology12 as having endosperm preferred expression and analysis of fh5 and fh12 double mutants might further clarify the role of formins during endosperm development.

Unlike the Ran G-protein activating protein 1 (RanGAP1), TANGLED (TAN) is not included in the TAIR gene ontology group used to generate this study. However, both genes have endosperm preferred expression and are implicated in orienting the cell division plane.^20,21 Mutations in maize TAN1,²² and its Arabidopsis homologue (TAN) exhibit misplaced cell walls as a consequence of inefficient phragmoplast guidance.²⁰ Inducible disruption of RanGAP function in a double mutant of redundant rangap1 and rangap2 also revealed cytokinesis defects.²¹ TAN and RanGAP decorate the cell division plane throughout mitosis.^20,21 In addition, RanGAP1 is also present at the nuclear envelope in prophase and accumulates at the cell plate during cytokinesis. Analysis of endosperm development in tan and rangap mutants might reveal additional roles for these proteins.

Uncharacterized Genes with Strong Endosperm Expression

The cytoskeleton-associated genes identified here are likely to have important roles during early seed development but remain to be characterized. There is also strong additional evidence for the endosperm specific expression of three genes (the kinesins At1g55550 and At5g27000 and the myosin At2g20290) from unpublished data profiling different seed compartments (NCBI GEO series GSE11262 contributed by the Harada Laboratory UC Davis CA, data not shown). The kinesin coded for by At1g55550 clusters with kinesin-4s, which are involved in chromosome segregation and At1g55550 has not been listed among the mitotic kinesins.²³ Whether this observation reflects a more specific role for At1g55550 in competing with and thereby blocking the action of kinesins involved in conventional spindle assembly and/or phragmoplast formation needs to be elucidated in a comprehensive study. The kinesin ATK4 (At5g27000) is reported to be expressed during the mitotic phase in synchronized Arabidopsis cell culture.^23–25 Like ATK1, ATK4-like and ATK5, ATK4 clusters with the kinesin-14s, characterized by their C-terminal motor domain and is therefore likely to be involved in spindle formation and spindle integrity.⁸ ATK4 also contains a calponin homology domain, which mediates interaction with actin.

The actin cytoskeleton seems to play a minor role during conventional mitosis. Although actin filaments are components of all mitotic arrays, their depolymerization does not inhibit cell cycle progression,^26,27 however cell plate orientation might be effected. Neither the myosin encoded by At2g20290, nor the other actin cytoskeleton proteins identified in this study with early seed specific and endosperm preferred expression, have been functionally characterized.

Conclusions

Although some cytoskeletal arrays in the endosperm are unique, the protein complement facilitating transition of cytoskeletal arrays seems to be conserved. Most of the cytoskeletal proteins identified in the present study have already been described for their function in conventional mitosis and cytokinesis in vegetative tissues. Our work providing the syncytial endosperm transcriptome therefore supports the notion that a similar set of genes are required for conventional somatic cell division and syncytial proliferation via mitosis.¹⁴ However, a number of uncharacterized genes do appear to have specific expression during syncytial endosperm development. Analysis of these genes may shed some light on this fascinating type of tissue proliferation and help elucidate the key genes that enable the uncoupling of cytokinesis and cell wall formation from mitosis.

Addendum to: Day RC, Herridge RP, Ambrose BA, Macknight RC. Transcriptome analysis of proliferating Arabidopsis endosperm reveals biological implications for the control of syncytial division, cytokinin signaling and gene expression regulation. Plant Physiol. 2008;148:1964–1984. doi: 10.1104/pp.108.128108.