CESICOP · Elucidating the control of cell size in plants

Elucidating the control of cell size in plants

Background A fascinating challenge in biology is to understand how organ size is determined. In plants, organ size is determined by the total number of cells and the average size of cells within organs. Recent studies have revealed several regulatory molecules of cell proliferation; however, little is known about how cell size is regulated due to a lack of cell-expansion specific mutants. The atkin13a mutant has a specific increase in cell size in petals, suggesting that it can be used to understand how cell size is regulated. Objectives The four objectives of this fellowship were: (1) to define the relationship between KIN13A and 13B during plant development; (2) to define the relationship between excess cell expansion in atkin13 mutants and cell wall integrity sensors; (3) to define the cellular basis of the excess cell expansion in atkin13 mutants; and (4) to define the relationship between the excess cell enlargement in atkin13a mutants and compensated cell enlargement. Results (1) Relationship between KIN13A and 13B during plant development: The Arabidopsis genome has a related protein to AtKIN13A, called AtKIN13B. The KIN13B protein shows over 80 % similarity in the motor domain to KIN13A and a highly conserved C-terminal domain, suggesting that they act redundantly. Therefore, understanding the full biological role of this subfamily of plant kinesins will require generating double mutants lacking both genes. To address this issue, kin13a kin13b double mutant was produced. This is complicated by the fact that both genes are tightly linked on chromosome III, separated only by 200 kb. Amongst more than 2 000 plants in an F2 population from a cross between kin13a and kin13b mutants, we could not find recombinant chromosomes carrying both mutant alleles. These results suggested that KIN13A and KIN13B have a coordinated role for cell expansion during plant development and loss of both KIN13A and 13B function is gametophytic lethal. To gain more insight about the relationship between KIN13A and KIN13B, we observed the phenotype in detail in artificial KIN13A/B knock down plants. Petals with a loss of KIN13B function in a kin13a mutant background showed impaired petal development. The same is true in kin13b mutant with loss of KIN13A function. These result suggested that both KIN13A and KIN13B have a substantial functional redundancy during plant development and specifically loss of both functions leads to impaired petal growth. (2) The relationship between excess cell expansion in atkin13 mutants and cell wall integrity sensors: We have genetically tested the hypothesis that the excessive synthesis of cell-wall material in atkin13 mutants results from the activation of a pathway sensing cell-wall integrity and stimulating additional cell-wall synthesis. We combined the atkin13 mutant with mutations in the1 and wak2. We confirmed double mutant by polymerase chain reaction (PCR)-based genotyping assay in F2 progeny. In the kin13a the1 double mutant, petal size was decreased due to impaired cell expansion. This result strongly suggested that the excess cell expansion in kin13a mutants is mediated by THE1 pathway. On contrast, wak2 mutation did not affect cell enlargement in kin13a background. (3) The cellular basis of the excess cell expansion in atkin13 mutants: To address the hypothesis that the excessive synthesis of cell-wall material in atkin13a mutants results from the activation of a pathway sensing cell-wall integrity and stimulating additional cell-wall synthesis, FTIR and quantitative rea-time (qRT-PCR) analysis were carried out. Preliminary results from dry weight analysis suggested that kin13a mutant petals might have more cell wall materials than WT. FTIR analysis also showed that loss of KIN13A function causes altered cell wall composition. qRT-PCR analysis revealed that accumulation of CesA genes, which encode important components for cellulose synthesis, were significantly decreased in kin13a mutant, suggesting that kin13a mutants have altered cell wall composition. By contrast, accumulation of CesA genes in kin13a the1 double mutant was increased compared to parental kin13a mutant. Together, these results argue that the loss of kin13a function causes decreased CesA gene expression, partly mediated by activation of the THE1 pathway. (4) The relationship between the excess cell enlargement in atkin13a mutants and compensated cell enlargement: The atkin13a mutant shows a strong synergistic interaction with the xs10 mutant that impairs cell expansion, resulting in vastly enlarged petals in the double mutant. To understand the basis of this interaction, the xs10 mutation was mapped and next-generation sequencing was used to identify the mutation. This showed that in xs10 a large chromosomal fragment containing more than 30 annotated genes is deleted. Due to time limitations, it was not possible to define the loss of which of these genes is responsible for the synergistic interaction with atkin13a. Conclusion This fellowship has defined the basis of the cell-size regulation in Arabidopsis at a developmental and genetic level and has shed light on the molecular basis of petal size regulation. Impact The knowledge gained about plant cell size control improves our understanding of an important process in plant science.

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