Class III compensation, represented by KRP2 overexpression, depends on V-ATPase activity in proliferative cells

Abstract

Compensation refers to an increase in cell size when the cell number is significantly decreased due to the mutation or gain of function of a gene that negatively affects the cell cycle. Given the importance of coordinated growth during organogenesis in both animal and plant systems, compensation is important to understand the mechanism of size regulation. In leaves, cell division precedes cell differentiation (which involves cell expansion); therefore, a decrease in cell number triggers enhanced cell expansion (compensated cell expansion; hereafter, CCE). Functional analyses of genes for which a loss or gain of function triggers compensation have increased our understanding of the molecular mechanisms underlying the decrease in cell number. Nevertheless, the mechanisms that induce enhanced cell expansion (the link between cell cycling and expansion), as well as the cellular machinery mediating CCE, have not been characterized. We recently characterized an important pathway involved in cell enlargement in KRP2-overexpressing plants. Here, we discuss the potential role of plant KRPs in triggering enlargement in cells with meristematic features.

Keywords: :

• Arabidopsis
• leaf development
• cell size
• organ-size regulation
• compensation
• KRP2
• V-ATPase
• det3-1

This article refers to:

Plant leaves are common models of size regulation due to their rather simple shape and constant size under controlled growth conditions.^1-4 However, how the size of an organ(ism) is determined remains unclear. Plant leaves, with their relatively simple form and determinate fate, can be used to address this question. In this study, we explored the “key logics” of size regulation in plants by examining compensation.⁵ Although the coordination between cell proliferation and expansion processes during leaf development and its impact on size control is relatively well-characterized, the cellular processes mediating cell enlargement remain unclear.^6-10

Previously, we analyzed 8 different mutant and transgenic lines showing compensation.¹¹ A kinematic analysis of cell size during leaf development demonstrated that compensated cell expansion (CCE) occurs through 3 different mechanisms depending on the cause of the impairment in cell proliferation: an enhanced post-mitotic cell expansion rate (Class I), an extended post-mitotic cell expansion period (Class II), and an increase in size of the dividing cells (Class III).¹¹ Although the molecular nature of the first 2 classes of CCEs remains unclear, the third class, represented by KIP-RELATED PROTEIN 2 (KRP2) overexpressors (o/e), was described in our recent study.¹² We demonstrated that the unusually large cell size in KRP2-overexpressing plants relies on increased vacuolar type H⁺-ATPase (V-ATPase) activity. This finding increased our understanding of the molecular nature of one major pathway mediating CCE in plant leaves.¹²

The Arabidopsis genome is known to possess 7 KRP genes with low sequence similarities and distinct expression patterns.^13,14 Among these genes, the individual overexpression of KRP1, KRP2, and KRP3 results in relatively dwarfed plants with strongly serrated leaves containing considerably fewer but significantly larger cells at maturity.^11,13,14,15 We analyzed the cellular dynamism (i.e., cell number and size) in leaf primordia from 4 d after sowing (DAS) to leaf maturity (30 DAS).¹¹ Interestingly, in all compensation mutants analyzed to date, the actively dividing cells were similar in size to those in wild type, excluding class III observed in KRP2 o/e, in which the size of the dividing cells was 2-fold larger than in wild type.^11-13 Based on the central and often redundant functions of plant KRPs in cell cycle regulation, similarities between plants overproducing KRP1, KRP2, and KRP3 were expected. However, the reason that a gain of function triggers an increase in cell size during the cell cycle is not understood.

After wild-type Arabidopsis plants exit mitosis, most cell types undergo a rapid increase in size (e.g., > 40-fold increase in palisade cells in terms of cell area).^8,11 This increase in cell size is even more striking in compensation exhibiting mutants, including a ~150-fold increase in fas1–5 an3–4 double mutant cell area (7473 ± 885 µm²), if we assume that the size of the dividing cells is ~50 µm².^8,11 Until recently, the mechanisms mediating this enhanced cell expansion ability remained unclear.

KRP2 overproduction inhibits the G2/M phase transition, significantly decreasing cell number. This was proposed to trigger CCE during the post-mitotic stage of leaf development. However, in KRP2 o/e, the dividing cells are 2-fold larger than in wild type and all other compensation mutants analyzed.¹¹ Furthermore, the final cell size in the mature leaves is typically 2.2-fold greater in KRP2 o/e, and varies between 1.4 and 1.8 higher in other mutants, including fas1, an3, and fugu5, compared with wild type.^11,16 Based on these results, KRP2 o/e undergo size inflation compared with other mutants. However, this may not be the case if we consider the original size of the cells in leaf primordia when cells are actively dividing. Based on the findings of Kawade et al.,⁹ the induction of KRP2 overexpression by heat shock during the proliferative stage of leaf development (4 DAS) is required to induce CCE in leaves. In contrast, delayed induction at 7 or 10 DAS produced leaves with significantly smaller cells than in the KRP2 o/e (i.e., 35S::KRP2 lines) or equal in size to wild-type cells, respectively.⁹ This suggests that KRP2 has no direct function in enhancing cell expansion in post-mitotic cells.⁹ In other words, if we assume that increased V-ATPase activity in KRP2 o/e mediates CCE,¹² this increase must occur during the mitotic stage of leaf development. Thus, 1) the large cells in the KRP2 o/e represents the outcome of increased V-ATPase activity during the proliferative stage, 2) the final cell size in the KRP2 o/e is a consequence of 1), 3) the increased cell size in the KRP2 o/e is not triggered by a decrease in cell number because cell enlargement had already occurred and was sustained during the proliferative stage in these lines, and 4) endoreduplication does not contribute to the cell size increase.¹¹ Overall, these features of KRP2 o/e differ from those of other compensation exhibiting mutants such as an3, fas1, and fugu5.^11,17 Since the cellular phenotypes of KRP2 o/e an3 double mutants are additive,⁹ and decreased V-ATPase activity does not suppress CCE in an3; fas1 and fugu5,¹² CCE in KRP2 o/e and other mutant lines differs significantly. Therefore, we propose that the class III compensation observed in KRP2 o/e should be assigned to a sub-category distinct from typical compensation.

Plants are sessile, yet they must cope with continuously fluctuating environments; thus, they show flexible morphogenetic control. Organogenesis can occur independently (to some extent) from cell division, provided that KRP2 o/e can develop organs (although they are abnormal in size and shape and have 90% fewer cells than wild type).^11,12,13,18 In addition, in KRP2 o/e, the cell size remained constant and the cells maintained their ability to divide, indicating that the increased size of the KRP2 o/e was not due to uncoupling between cell cycling and cell growth.^7,11

During normal leaf development, 2 qualitatively different modes (non-cell-autonomous and cell-autonomous modes in an3 and KRP2 o/e, respectively) are involved in the coordination of cell proliferation and post-mitotic cell expansion in leaves.^7,9,19 The fact that decreased V-ATPase activity in det3–1 KRP2 o/e suppressed class III CCE in KRP2 o/e, but not an3, supports the cell-autonomous nature of CCE and the immobile nature of the V-ATPase complex.^9,12,19

A study by Jun et al. indicated that KRP3 overexpression significantly reduced the number of cells in leaves, but that their increased size (~2.2-fold) involved an increase in ploidy, which is different from KRP2 o/e.¹⁴ Although KRPs overproduction results in morphological similarities, their functions are only partially overlapping. Excluding KRP4, for which overexpressing transgenic lines have not been reported, the overproduction of most other KRP genes increases the cell size in tissues with meristematic features, including the shoot apical meristem and dividing cells in young leaf primordia.^11,13,14 This increase can be achieved either by increased endocycling and/or V-ATPase activity (probably through enhanced cell wall synthesis).^12,14 To understand the contribution of these KRPs to cell size regulation, a detailed analysis considering the above processes should be performed.

In tobacco, overproduction of a dominant-negative (DN) form of Arabidopsis cyclin-dependent kinase (CDKA;1) has similar phenotypic effects on leaf development to that of KRP2 o/e.^11,12,13,18 Importantly, the nuclear DNA content in DN tobacco lines was similar to that in control plants.¹⁸ Compensation has been reported in transgenic rice plants overexpressing OsKRP1.²⁰ These observations suggest that class III compensation is a universal phenomenon in monocots and eudicots, and that V-ATPase plays a role in the increased cell size in plant species other than Arabidopsis. Overall, our results may be applicable to the entire plant kingdom.

Abbreviations:
CCE	=	compensated cell expansion
DAS	=	days after sowing
KRP2 o/e	=	KIP-RELATED PROTEIN 2 overexpressors
V-ATPase	=	vacuolar type H+-ATPase

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by a Grant-in-Aid for Young Scientists (B) (to Ferjani A); Scientific Research on Priority Areas (to Tsukaya H); Scientific Research [23248017 and 24114706, to Maeshima M]; the Steel Foundation for Environmental Protection Technology (to Maeshima M); Creative Scientific Research (to Tsukaya H); the Toray Science Foundation (grants to Tsukaya H); Grant-in-Aid for Scientific Research on Innovative Areas (to Ferjani A, Horiguchi G, and Tsukaya H).