BIN4, a Novel Component of the Plant DNA Topoisomerase VI Complex

How plant organs grow to reach their final size is an important but largely unanswered question. Here, we describe an Arabidopsis thaliana mutant, brassinosteroid-insensitive4 (bin4), in which the growth of various organs is dramatically reduced. Small organ size in bin4 is primarily caused by reduced cell expansion associated with defects in increasing ploidy by endoreduplication.

Raising nuclear DNA content in bin4 by colchicine-induced polyploidization partially rescues the cell and organ size phenotype, indicating that BIN4 is directly and specifically required for endoreduplication rather than for subsequent cell expansion. BIN4 encodes a plant-specific, DNA binding protein that acts as a component of the plant DNA topoisomerase VI complex. Loss of BIN4 triggers an ATM- and ATR-dependent DNA damage response in postmitotic cells, and this response coincides with the upregulation of the cyclin B1;1 gene in the same cell types, suggesting a functional link between DNA Biochain damage response and endocycle control.

The control of organ growth is a fundamental question in plant development, but the intrinsic mechanisms that mediate this process remain largely unknown.

  • The final size of a plant organ is defined by genetic programs, but environmental factors such as nutrition, temperature, and light can also impinge on those programs to develop an organ size appropriate to the surrounding growth conditions.
  • Several plant growth regulators, such as auxin, brassinosteroids (BRs), ethylene, and gibberellins, are also known to modulate organ growth.
  • Given that the overall size of an organ depends on both the number and size of its cells, all of these upstream signals must be transduced into mechanisms that regulate cell proliferation and cell expansion.
  • Recent genetic studies have identified a number of genes that influence these processes and have begun to uncover the molecular and cellular bases of organ growth in plants.
  • The primary driver of organ growth is the addition of new cells into a developing organ; thus, a key control exists in determining the duration of cell proliferation during organogenesis.
  • This process is mediated by several independent genetic pathways that either promote or terminate the maintenance of cellular meristematic competence.
  • In addition, many plant cell types undergo massive postmitotic cell expansion, and the resulting increase in mass also contributes to organ growth. Cell expansion is driven by water uptake into vacuoles and cell wall biogenesis; consistent with this, many mutations that result in defects in organ growth occur in genes involved in one or the other of these processes. In addition, another set of mutations that alters the final size of plant cells points to the control of cell expansion by endoreduplication, the amplification of chromosomal DNA without corresponding mitosis .
  • The correlation between ploidy, nuclear DNA content, and cell size has long been reported in Arabidopsis thaliana and in many other plant species , although the molecular details of how cells endoreduplicate and how an increase in ploidy might lead to an increase in cell size still remain elusive. Recent genetic studies have revealed a number of key cell cycle genes involved in the switch from the mitotic cell cycle to the endocycle  and other positive and negative regulators that function during the progression of successive endocycles  or at their termination . It is well established that various checkpoint mechanisms ensure progression through the mitotic cell cycle. It is reasonable to predict that similar mechanisms operate during endocycles, but the existence of such controls has been poorly investigated.

To gain further insights into the molecular and cellular mechanisms that underlie plant organ growth, we have characterized a previously identified mutant in Arabidopsisbrassinosteroid-insensitive4 (bin4), that displays a severe dwarf phenotype .

We show that the primary defect in bin4 is in cell expansion rather than in cell proliferation and that this is associated with a failure in the progression of endocycles in appropriate cell types. Our study clearly demonstrates a close link between cell size and ploidy and provides genetic evidence that cell expansion requires, and is coupled to, an increase in ploidy through endoreduplication. BIN4 encodes a plant-specific, DNA binding protein, and our genetic and in vivo evidence suggests that BIN4 is a new component of the plant DNA topoisomerase VI (topo VI) complex. We also show that the loss of functional BIN4, or other plant topo VI components, activates the expression of several DNA damage response genes. This response is mediated through the two upstream phosphatidylinositol 3-kinase (PI3K)–like protein kinases, ataxia telangiectasia–mutated (ATM) and ATM and Rad3–related (ATR), that are known to initiate various DNA damage repair processes during the mitotic cell cycle. We propose that the structural integrity of the genome is closely monitored during plant endocycles and that the cellular response to damaged DNA leads to an early arrest of endocycles.

The BR-Insensitive Mutant bin4 Has Cell and Organ Size Defects

An extreme dwarf mutant, bin4, was originally identified in a screen for mutants that have reduced sensitivity to BR . Like other BR-insensitive mutants, organ growth is severely compromised in bin4. Twelve-day-old, light-grown bin4-1 seedlings have small cotyledons and true leaves with short petioles . The size of other organs, such as hypocotyls and roots, is also reduced to 15 to 35% of the wild-type level (see Supplemental Table 1 online). The application of exogenous BR or other plant growth regulators, including auxin, gibberellin, and ethylene, does not rescue the small size phenotype in bin4-1 (data not shown).

To examine the cellular basis of these reduced organ growth phenotypes in bin4, we cleared cotyledons from 8-d-old, light-grown seedlings and measured cell number and cell size on the abaxial surface. As shown in  wild-type cotyledons develop many large, heavily interdigitated epidermal cells. We found that the size of this cell type is dramatically reduced in bin4-1, from an average of 5032 ± 2699 μm2 in the wild type to 790 ± 314 μm2 in bin4-1 (n = 50), whereas total estimated cell number per cotyledon surface is comparable between the wild type (1543 cells) and the mutant (1480 cells) .

 

The small cell size phenotype was also observed in hypocotyls  and roots, and the quantification of cell size in these organs revealed that their cell sizes are reduced to ∼25% of wild-type levels . This defect in cell size is not limited to the epidermal cell layer but is also present in cortex and endodermis . Both root hairs and leaf trichomes are composed of a single cell in Arabidopsis, and the reduced cell size in bin4-1 resulted in more pronounced developmental defects in these cell types (i.e., almost complete lack of root hair formation and unbranched, miniature trichomes [).

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