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Identification and characterization of plant Haspin kinase as a histone H3 threonine kinase.

Kurihara D, Matsunaga S, Omura T, Higashiyama T, Fukui K - BMC Plant Biol. (2011)

Bottom Line: Overexpression of a kinase domain mutant of AtHaspin decreased the size of the root meristem, which delayed root growth.Our results indicated that the Haspin kinase is a histone H3 threonine kinase in A. thaliana.Further analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed new light on the regulation of chromosome segregation in cell division during plant growth and development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan.

ABSTRACT

Background: Haspin kinases are mitotic kinases that are well-conserved from yeast to human. Human Haspin is a histone H3 Thr3 kinase that has important roles in chromosome cohesion during mitosis. Moreover, phosphorylation of histone H3 at Thr3 by Haspin in fission yeast, Xenopus, and human is required for accumulation of Aurora B on the centromere, and the subsequent activation of Aurora B kinase activity for accurate chromosome alignment and segregation. Although extensive analyses of Haspin have been carried out in yeast and animals, the function of Haspin in organogenesis remains unclear.

Results: Here, we identified a Haspin kinase, designated AtHaspin, in Arabidopsis thaliana. The purified AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. Live imaging of AtHaspin-tdTomato and GFP-α-tubulin in BY-2 cells showed that AtHaspin-tdTomato localized on chromosomes during prometaphase and metaphase, and around the cell plate during cytokinesis. This localization of AtHaspin overlapped with that of phosphorylated Thr3 and Thr11 of histone H3 in BY-2 cells. AtHaspin-GFP driven by the native promoter was expressed in root meristems, shoot meristems, floral meristems, and throughout the whole embryo at stages of high cell division. Overexpression of a kinase domain mutant of AtHaspin decreased the size of the root meristem, which delayed root growth.

Conclusions: Our results indicated that the Haspin kinase is a histone H3 threonine kinase in A. thaliana. AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. The expression and dominant-negative analysis showed that AtHaspin may have a role in mitotic cell division during plant growth. Further analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed new light on the regulation of chromosome segregation in cell division during plant growth and development.

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Expression patterns of AtHaspin-GFP in Arabidopsis. (A) Expressions of AtHaspin, AtAURs, and AtCycB1;3 during mitotic cell cycle in synchronized Arabidopsis cultured cells. Expression data were obtained from publicly available microarray data [30]. Figure shows expressions of genes after removal of the DNA synthesis inhibitor, aphidicolin. (B) Total RNA was extracted from 3-day-old seedlings (3), roots (R), young leaves (YL), leaves (L), stems (S), flower buds (FB), flowers (F), siliques (Si), and genomic DNA (G). Expression was monitored by RT-PCR. Number of PCR cycles is shown in parentheses after gene names. GAPDH was used as an internal control. (C-N) Expression of AtHaspin-GFP in root tip (C), lateral root (D), shoot meristem and leaf primordia (E-H), leaf primordia and first true leaves (F, G), leaf primordia and second true leaves (H), inflorescence meristem and floral meristem in cauline leaves (I), floral meristem (J), ovules in closed flowers (K), one-cell stage embryo (L), four-cell stage embryo (M), heart stage embryo (N), and torpedo stage embryo (O). Scale bars: 100 μm (C, D, F, H, I, J), 50 μm (E), 30 μm (G, K), 10 μm (L, M), and 20 μm (N, O).
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Figure 5: Expression patterns of AtHaspin-GFP in Arabidopsis. (A) Expressions of AtHaspin, AtAURs, and AtCycB1;3 during mitotic cell cycle in synchronized Arabidopsis cultured cells. Expression data were obtained from publicly available microarray data [30]. Figure shows expressions of genes after removal of the DNA synthesis inhibitor, aphidicolin. (B) Total RNA was extracted from 3-day-old seedlings (3), roots (R), young leaves (YL), leaves (L), stems (S), flower buds (FB), flowers (F), siliques (Si), and genomic DNA (G). Expression was monitored by RT-PCR. Number of PCR cycles is shown in parentheses after gene names. GAPDH was used as an internal control. (C-N) Expression of AtHaspin-GFP in root tip (C), lateral root (D), shoot meristem and leaf primordia (E-H), leaf primordia and first true leaves (F, G), leaf primordia and second true leaves (H), inflorescence meristem and floral meristem in cauline leaves (I), floral meristem (J), ovules in closed flowers (K), one-cell stage embryo (L), four-cell stage embryo (M), heart stage embryo (N), and torpedo stage embryo (O). Scale bars: 100 μm (C, D, F, H, I, J), 50 μm (E), 30 μm (G, K), 10 μm (L, M), and 20 μm (N, O).

Mentions: According to the microarray data publically available at Genevestigator [30], AtHaspin is expressed at relatively high levels in the root tips and shoot apex. We plotted the RNA profiles using published microarray datasets [31]. Expression of AtHaspin was activated at 8 to 16 h after removal of the DNA synthesis inhibitor, aphidicolin (Figure 5A). This profile of AtHaspin expression was very similar to those of AtAURs and mitotic AtCycB1;3, indicating that AtHaspin is a mitotic-specific kinase. RT-PCR analyses showed that AtHaspin was expressed in multiple tissues (Figure 5B). Although Haspin was first identified as a testis-specific gene in mice [17,18], this expression profile indicated that AtHaspin is not a reproduction-specific gene in A. thaliana. AtHaspin showed relatively high expression in flower buds and flowers with high cell division activity.


Identification and characterization of plant Haspin kinase as a histone H3 threonine kinase.

Kurihara D, Matsunaga S, Omura T, Higashiyama T, Fukui K - BMC Plant Biol. (2011)

Expression patterns of AtHaspin-GFP in Arabidopsis. (A) Expressions of AtHaspin, AtAURs, and AtCycB1;3 during mitotic cell cycle in synchronized Arabidopsis cultured cells. Expression data were obtained from publicly available microarray data [30]. Figure shows expressions of genes after removal of the DNA synthesis inhibitor, aphidicolin. (B) Total RNA was extracted from 3-day-old seedlings (3), roots (R), young leaves (YL), leaves (L), stems (S), flower buds (FB), flowers (F), siliques (Si), and genomic DNA (G). Expression was monitored by RT-PCR. Number of PCR cycles is shown in parentheses after gene names. GAPDH was used as an internal control. (C-N) Expression of AtHaspin-GFP in root tip (C), lateral root (D), shoot meristem and leaf primordia (E-H), leaf primordia and first true leaves (F, G), leaf primordia and second true leaves (H), inflorescence meristem and floral meristem in cauline leaves (I), floral meristem (J), ovules in closed flowers (K), one-cell stage embryo (L), four-cell stage embryo (M), heart stage embryo (N), and torpedo stage embryo (O). Scale bars: 100 μm (C, D, F, H, I, J), 50 μm (E), 30 μm (G, K), 10 μm (L, M), and 20 μm (N, O).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Expression patterns of AtHaspin-GFP in Arabidopsis. (A) Expressions of AtHaspin, AtAURs, and AtCycB1;3 during mitotic cell cycle in synchronized Arabidopsis cultured cells. Expression data were obtained from publicly available microarray data [30]. Figure shows expressions of genes after removal of the DNA synthesis inhibitor, aphidicolin. (B) Total RNA was extracted from 3-day-old seedlings (3), roots (R), young leaves (YL), leaves (L), stems (S), flower buds (FB), flowers (F), siliques (Si), and genomic DNA (G). Expression was monitored by RT-PCR. Number of PCR cycles is shown in parentheses after gene names. GAPDH was used as an internal control. (C-N) Expression of AtHaspin-GFP in root tip (C), lateral root (D), shoot meristem and leaf primordia (E-H), leaf primordia and first true leaves (F, G), leaf primordia and second true leaves (H), inflorescence meristem and floral meristem in cauline leaves (I), floral meristem (J), ovules in closed flowers (K), one-cell stage embryo (L), four-cell stage embryo (M), heart stage embryo (N), and torpedo stage embryo (O). Scale bars: 100 μm (C, D, F, H, I, J), 50 μm (E), 30 μm (G, K), 10 μm (L, M), and 20 μm (N, O).
Mentions: According to the microarray data publically available at Genevestigator [30], AtHaspin is expressed at relatively high levels in the root tips and shoot apex. We plotted the RNA profiles using published microarray datasets [31]. Expression of AtHaspin was activated at 8 to 16 h after removal of the DNA synthesis inhibitor, aphidicolin (Figure 5A). This profile of AtHaspin expression was very similar to those of AtAURs and mitotic AtCycB1;3, indicating that AtHaspin is a mitotic-specific kinase. RT-PCR analyses showed that AtHaspin was expressed in multiple tissues (Figure 5B). Although Haspin was first identified as a testis-specific gene in mice [17,18], this expression profile indicated that AtHaspin is not a reproduction-specific gene in A. thaliana. AtHaspin showed relatively high expression in flower buds and flowers with high cell division activity.

Bottom Line: Overexpression of a kinase domain mutant of AtHaspin decreased the size of the root meristem, which delayed root growth.Our results indicated that the Haspin kinase is a histone H3 threonine kinase in A. thaliana.Further analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed new light on the regulation of chromosome segregation in cell division during plant growth and development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan.

ABSTRACT

Background: Haspin kinases are mitotic kinases that are well-conserved from yeast to human. Human Haspin is a histone H3 Thr3 kinase that has important roles in chromosome cohesion during mitosis. Moreover, phosphorylation of histone H3 at Thr3 by Haspin in fission yeast, Xenopus, and human is required for accumulation of Aurora B on the centromere, and the subsequent activation of Aurora B kinase activity for accurate chromosome alignment and segregation. Although extensive analyses of Haspin have been carried out in yeast and animals, the function of Haspin in organogenesis remains unclear.

Results: Here, we identified a Haspin kinase, designated AtHaspin, in Arabidopsis thaliana. The purified AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. Live imaging of AtHaspin-tdTomato and GFP-α-tubulin in BY-2 cells showed that AtHaspin-tdTomato localized on chromosomes during prometaphase and metaphase, and around the cell plate during cytokinesis. This localization of AtHaspin overlapped with that of phosphorylated Thr3 and Thr11 of histone H3 in BY-2 cells. AtHaspin-GFP driven by the native promoter was expressed in root meristems, shoot meristems, floral meristems, and throughout the whole embryo at stages of high cell division. Overexpression of a kinase domain mutant of AtHaspin decreased the size of the root meristem, which delayed root growth.

Conclusions: Our results indicated that the Haspin kinase is a histone H3 threonine kinase in A. thaliana. AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. The expression and dominant-negative analysis showed that AtHaspin may have a role in mitotic cell division during plant growth. Further analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed new light on the regulation of chromosome segregation in cell division during plant growth and development.

Show MeSH
Related in: MedlinePlus