Dissection of enhanced cell expansion processes in leaves triggered by a defect in cell proliferation, with reference to roles of endoreduplication

Leaf development relies on cell proliferation, post-mitotic cell expansion and the coordination of these processes. In several Arabidopsis thaliana mutants impaired in cell proliferation, such as angustifolia3 (an3), leaf cells are larger than normal at their maturity. This phenomenon, which we call compensated cell enlargement, suggests the presence of such coordination in leaf development. To dissect genetically the cell expansion system(s) underlying this compensation seen in the an3 mutant, we isolated and utilized 10 extra-small sisters (xs) mutant lines that show decreased cell size but normal cell numbers in leaves. In the xs single mutants, the palisade cell sizes in mature leaves are about 20-50% smaller than those of wild-type cells. Phenotypes of the palisade cell sizes in all combinations of xs an3 double mutants fall into three classes. In the first class, the compensated cell enlargement was significantly suppressed. Conversely, in the second class, the defective cell expansion conferred by the xs mutations was significantly suppressed by the an3 mutation. The residual xs mutations had effects additive to those of the an3 mutation on cell expansion. The endopolyploidy levels in the first class of mutants were decreased, unaffected or increased, as compared with those in wild-type, suggesting that the abnormally enhanced cell expansion observed in an3 could be mediated, at least in part, by ploidy-independent mechanisms. Altogether, these results clearly showed that a defect in cell proliferation in leaf primordia enhances a part of the network that regulates cell expansion, which is required for normal leaf expansion.     

A Decrease in Ambient Temperature Induces Post-Mitotic Enlargement of Palisade Cells in North American Lake Cress

In order to maintain organs and structures at their appropriate sizes, multicellular organisms orchestrate cell proliferation and post-mitotic cell expansion during morphogenesis. Recent studies using Arabidopsis leaves have shown that compensation, which is defined as post-mitotic cell expansion induced by a decrease in the number of cells during lateral organ development, is one example of such orchestration. Some of the basic molecular mechanisms underlying compensation have been revealed by genetic and chimeric analyses. However, to date, compensation had been observed only in mutants, transgenics, and γ-ray–treated plants, and it was unclear whether it occurs in plants under natural conditions. Here, we illustrate that a shift in ambient temperature could induce compensation in Rorippa aquatica (Brassicaceae), a semi-aquatic plant found in North America. The results suggest that compensation is a universal phenomenon among angiosperms and that the mechanism underlying compensation is shared, in part, between Arabidopsis and R. aquatica.     

Coordination of cell proliferation and cell expansion in the control of leaf size in Arabidopsis thaliana

Size is an important parameter in the characterization of organ morphology and function. To understand the mechanisms that control leaf size, we previously isolated a number of Arabidopsis thaliana mutants with altered leaf size. Because leaf morphogenesis depends on determinate cell proliferation, the size of a mature leaf is controlled by variation in cell size and number. Therefore, leaf-size mutants should be classified according to the effects of the mutations on the cell number and/or size. A group of mutants represented by angustifolia3/grf-interacting factor1 and aintegumenta exhibits an intriguing cellular phenotype termed compensation: when the leaf cell number is decreased due to the mutation, the leaf cell size increases, leading to compensation in leaf area. Several lines of genetic evidence suggest that compensation is probably not a result of the uncoupling of cell division from cell growth. Rather, the evidence suggests an organ-wide mechanism that coordinates cell proliferation with cell expansion during leaf development. Our results provide a key, novel concept that explains how leaf size is controlled at the organ level.     

Loss of 26S Proteasome Function Leads to Increased Cell Size and Decreased Cell Number in Arabidopsis Shoot Organs

Although the final size of plant organs is influenced by environmental cues, it is generally accepted that the primary size determinants are intrinsic factors that regulate and coordinate cell proliferation and cell expansion. Here, we show that optimal proteasome function is required to maintain final shoot organ size in Arabidopsis (Arabidopsis thaliana). Loss of function of the subunit regulatory particle AAA ATPase (RPT2a) causes a weak defect in 26S proteasome activity and leads to an enlargement of leaves, stems, flowers, fruits, seeds, and embryos. These size increases are a result of increased cell expansion that compensates for a reduction in cell number. Increased ploidy levels were found in some but not all enlarged organs, indicating that the cell size increases are not caused by a higher nuclear DNA content. Partial loss of function of the regulatory particle non-ATPase (RPN) subunits RPN10 and RPN12a causes a stronger defect in proteasome function and also results in cell enlargement and decreased cell proliferation. However, the increased cell volumes in rpn10-1 and rpn12a-1 mutants translated into the enlargement of only some, but not all, shoot organs. Collectively, these data show that during Arabidopsis shoot development, the maintenance of optimal proteasome activity levels is important for balancing cell expansion with cell proliferation rates.     

Phytochrome B control of total leaf area and stomatal density affects drought tolerance in rice

We report that phytochrome B (phyB) mutants exhibit improved drought tolerance compared to wild type (WT) rice (Oryza sativa L. cv. Nipponbare). To understand the underlying mechanism by which phyB regulates drought tolerance, we analyzed root growth and water loss from the leaves of phyB mutants. The root system showed no significant difference between the phyB mutants and WT, suggesting that improved drought tolerance has little relation to root growth. However, phyB mutants exhibited reduced total leaf area per plant, which was probably due to a reduction in the total number of cells per leaf caused by enhanced expression of Orysa;KRP1 and Orysa;KRP4 (encoding inhibitors of cyclin-dependent kinase complex activity) in the phyB mutants. In addition, the developed leaves of phyB mutants displayed larger epidermal cells than WT leaves, resulting in reduced stomatal density. phyB deficiency promoted the expression of both putative ERECTA family genes and EXPANSIN family genes involved in cell expansion in leaves, thus causing greater epidermal cell expansion in the phyB mutants. Reduced stomatal density resulted in reduced transpiration per unit leaf area in the phyB mutants. Considering all these findings, we propose that phyB deficiency causes both reduced total leaf area and reduced transpiration per unit leaf area, which explains the reduced water loss and improved drought tolerance of phyB mutants.     

Developmentally controlled farnesylation modulates AtNAP1;1 function in cell proliferation and cell expansion during Arabidopsis leaf development

In multicellular organisms, organogenesis requires tight control and coordination of cell proliferation, cell expansion, and cell differentiation. We have identified Arabidopsis (Arabidopsis thaliana) nucleosome assembly protein 1 (AtNAP1;1) as a component of a regulatory mechanism that connects cell proliferation to cell growth and expansion during Arabidopsis leaf development. Molecular, biochemical, and kinetic studies of AtNAP1;1 gain- or loss-of-function mutants indicate that AtNAP1;1 promotes cell proliferation or cell expansion in a developmental context and as a function of the farnesylation status of the protein. AtNAP1;1 was farnesylated and localized to the nucleus during the cell proliferation phase of leaf development when it promotes cell division. Later in leaf development, nonfarnesylated AtNAP1;1 accumulates in the cytoplasm when it promotes cell expansion. Ectopic expression of nonfarnesylated AtNAP1;1, which localized to the cytoplasm, disrupts this developmental program by promoting unscheduled cell expansion during the proliferation phase.     

拟南芥YUAN1基因调控器官形状和大小

器官形状和大小的控制是一个基本的发育生物学过程, 受细胞分裂和细胞扩展的影响。到目前为止, 人们对植物器官形状和大小的调控机制知之甚少。本实验室前期研究发现了一个种子和器官大小的调控基因DA1, 其编码一个泛素受体。在拟南芥(Arabidopsis thaliana)中, DA1通过抑制细胞的分裂来限制种子和器官的大小。本研究通过激活标签的方法在da1-1突变体背景下筛选到一个叶子形状发生改变的半显性突变体(yuan1-1D)。yuan1-1D形成短而圆的叶片和短的叶柄, 细胞学分析显示, 叶片和叶柄变短的主要原因是细胞的长向扩展降低导致的。YUAN1编码一个含有PHD锌指结构域的蛋白。GFP-YUAN1融合蛋白定位在细胞核内。过量表达YUAN1基因导致叶片和叶柄变短。遗传学分析显示, YUAN1和DA1、ROT3以及ROT4在控制叶片形状和大小方面作用于不同的遗传途径中。因此, 本研究鉴定了一个新的控制器官形状和大小的基因YUAN1, 为阐明植物器官形状和大小调控的分子机制提供了重要线索。     

Brassinosteroids control the proliferation of leaf cells of Arabidopsis thaliana

The growth of leaves in the model plant, Arabidopsis thaliana (L.) Heynh., is determined by the extent of expansion of individual cells and by cell proliferation. Mutants of A. thaliana with known defects in the biosynthesis or perception of brassinosteroids develop small leaves. When the leaves of brassinosteroid-related mutants, det2 (de-etiolated2 = cro1) and dwf1 (dwarf1 = cro2) were compared to wild-type plants, an earlier cessation of leaf expansion was observed; a detailed anatomical analysis further revealed that the mutants had fewer cells per leaf blade. Treatment of the det2 mutants with the brassinosteroid, brassinolide, reversed the mutation and restored the potential for growth to that of the wild type. Restoration of leaf size could not be explained solely on the basis of an increase in individual cell volume, thus suggesting that brassinosteroids play a dual role in regulating cell expansion and proliferation.     

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

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.     

Leaf shape: genetic controls and environmental factors

In recent years, many genes have been identified that are involved in the developmental processes of leaf morphogenesis. Here, I review the mechanisms of leaf shape control in a model plant, Arabidopsis thaliana, focusing on genes that fulfill special roles in leaf development. The lateral, two-dimensional expansion of leaf blades is highly dependent on the determination of the dorsoventrality of the primordia, a defining characteristic of leaves. Having a determinate fate is also a characteristic feature of leaves and is controlled by many factors. Lateral expansion is not only controlled by general regulators of cell cycling, but also by the multi-level regulation of meristematic activities, e.g., specific control of cell proliferation in the leaf-length direction, in leaf margins and in parenchymatous cells. In collaboration with the polarized control of leaf cell elongation, these redundant and specialized regulating systems for cell cycling in leaf lamina may realize the elegantly smooth, flat structure of leaves. The unified, flat shape of leaves is also dependent on the fine integration of cell proliferation and cell enlargement. Interestingly, while a decrease in the number of cells in leaf primordia can trigger a cell volume increase, an increase in the number of cells does not trigger a cell volume decrease. This phenomenon is termed compensation and suggests the existence of some systems for integration between cell cycling and cell enlargement in leaf primordia via cell-cell communication. The environmental adjustment of leaf expansion to light conditions and gravity is also summarized.     

The TMK Subfamily of Receptor-Like Kinases in Arabidopsis Display an Essential Role in Growth and a Reduced Sensitivity to Auxin

Mechanisms that govern the size of plant organs are not well understood but believed to involve both sensing and signaling at the cellular level. We have isolated loss-of-function mutations in the four genes comprising the transmembrane kinase TMK subfamily of receptor-like kinases (RLKs) in Arabidopsis. These TMKs have an extracellular leucine-rich-repeat motif, a single transmembrane region, and a cytoplasmic kinase domain. While single mutants do not display discernable phenotypes, unique double and triple mutant combinations result in a severe reduction in organ size and a substantial retardation in growth. The quadruple mutant displays even greater severity of all phenotypes and is infertile. The kinematic studies of root, hypocotyl, and stamen filament growth reveal that the TMKs specifically control cell expansion. In leaves, TMKs control both cell expansion and cell proliferation. In addition, in the tmk double mutants, roots and hypocotyls show reduced sensitivity to applied auxin, lateral root induction and activation of the auxin response reporter DR5: GUS. Thus, taken together with the structural and biochemical evidence, TMKs appear to orchestrate plant growth by regulation of both cell expansion and cell proliferation, and as a component of auxin signaling.     

Targeted degradation of the cyclin-dependent kinase inhibitor ICK4/KRP6 by RING-type E3 ligases is essential for mitotic cell cycle progression during Arabidopsis gametogenesis

Following meiosis, plant gametophytes develop through two or three rounds of mitosis. Although the ontogeny of gametophyte development has been defined in Arabidopsis thaliana, the molecular mechanisms regulating mitotic cell cycle progression are not well understood. Here, we report that RING-H2 group F 1a (RHF1a) and RHF2a, two RING-finger E3 ligases, play an important role in Arabidopsis gametogenesis. The rhf1a rhf2a double mutants are defective in the formation of male and female gametophytes due to interphase arrest of the mitotic cell cycle at the microspore stage of pollen development and at female gametophyte stage 1 of embryo sac development. We demonstrate that RHF1a directly interacts with and targets a cyclin-dependent kinase inhibitor ICK4/KRP6 (for Interactors of Cdc2 Kinase 4/Kip-related protein 6) for proteasome-mediated degradation. Inactivation of the two redundant RHF genes leads to the accumulation of ICK4/KRP6, and reduction of ICK4/KRP6 expression largely rescues the gametophytic defects in rhf1a rhf2a double mutants, indicating that ICK4/KRP6 is a substrate of the RHF E3 ligases. Interestingly, in situ hybridization showed that ICK4/KRP6 was predominantly expressed in sporophytes during meiosis. Our findings indicate that RHF1a/2a-mediated degradation of the meiosis-accumulated ICK4/KRP6 is essential to ensure the progression of subsequent mitoses to form gametophytes in Arabidopsis.     

Targeted manipulation of leaf form via local growth repression

A classical view is that leaf shape is the result of local promotion of growth linked to cell proliferation. However, an alternative hypothesis is that leaf form is the result of local repression of growth in an otherwise growing system. Here we show that leaf form can indeed be manipulated in a directed fashion by local repression of growth. We show that targeting expression of an inhibitor of a cyclin-dependent kinase (KRP1) to the sinus area of developing leaves of Arabidopsis leads to local growth repression and the formation of organs with extreme lobing, including generation of leaflet-like organs. Directing KRP1 expression to other regions of the leaf using an miRNA target sequence tagging approach also leads to predictable novel leaf forms, and repression of growth in the leaf margin blocks the outgrowth of lobes, leading to a smoother perimeter. In addition, we show that decreased growth around the perimeter and across the leaf abaxial surface leads to a change in 3D form, as predicted by mechanical models of leaf growth. Our analysis provides experimental evidence that local repression of growth influences leaf shape, suggesting that it could be part of the mechanism of morphogenesis in plants in the context of an otherwise growing system.     

The Arabidopsis ARGOS-LIKE gene regulates cell expansion during organ growth

Cell expansion, and its coordination with cell division, plays a critical role in the growth and development of plant organs. However, the genes controlling cell expansion during organogenesis are largely unknown. Here, we demonstrate that a novel Arabidopsis gene, ARGOS-LIKE (ARL), which has some sequence homology to the ARGOS gene, is involved in this process. Reduced expression or overexpression of ARL in Arabidopsis results in smaller or larger cotyledons and leaves as well as other lateral organs, respectively. Anatomical examination of cotyledons and leaves in ARL transgenic plants demonstrates that the alteration in size can be attributed to changes in cell size rather than cell number, indicating that ARL plays a role in cell expansion-dependent organ growth. ARL is upregulated by brassinosteroid (BR) and this induction is impaired in the BR-insensitive mutant bri1, but not in the BR-deficient mutant det2. Ectopic expression of ARL in bri1-119 partially restores cell growth in cotyledons and leaves. Our results suggest that ARL acts downstream of BRI1 and partially mediates BR-related cell expansion signals during organ growth.     

Brassinosteroids control meristem size by promoting cell cycle progression in Arabidopsis roots

Brassinosteroids (BRs) play crucial roles in plant growth and development. Previous studies have shown that BRs promote cell elongation in vegetative organs in several plant species, but their contribution to meristem homeostasis remains unexplored. Our analyses report that both loss- and gain-of-function BR-related mutants in Arabidopsis thaliana have reduced meristem size, indicating that balanced BR signalling is needed for the optimal root growth. In the BR-insensitive bri1-116 mutant, the expression pattern of the cell division markers CYCB1;1, ICK2/KRP2 and KNOLLE revealed that a decreased mitotic activity accounts for the reduced meristem size; accordingly, this defect could be overcome by the overexpression of CYCD3;1. The activity of the quiescent centre (QC) was low in the short roots of bri1-116, as reported by cell type-specific markers and differentiation phenotypes of distal stem cells. Conversely, plants treated with the most active BR, brassinolide, or mutants with enhanced BR signalling, such as bes1-D, show a premature cell cycle exit that results in early differentiation of meristematic cells, which also negatively influence meristem size and overall root growth. In the stem cell niche, BRs promote the QC renewal and differentiation of distal stem cells. Together, our results provide evidence that BRs play a regulatory role in the control of cell-cycle progression and differentiation in the Arabidopsis root meristem.     

RKP, a RING finger E3 ligase induced by BSCTV C4 protein, affects geminivirus infection by regulation of the plant cell cycle

The C4 protein from Curtovirus is known as a major symptom determinant, but the mode of action of the C4 protein remains unclear. To understand the mechanism of involvement of C4 protein in virus–plant interactions, we introduced the C4 gene from Beet severe curly top virus (BSCTV) into Arabidopsis under a conditional expression promoter; the resulting overexpression of BSCTV C4 led to abnormal host cell division. RKP, a RING finger protein, which is a homolog of the human cell cycle regulator KPC1, was discovered to be induced by BSCTV C4 protein. Mutation of RKP reduced the susceptibility to BSCTV in Arabidopsis and impaired BSCTV replication in plant cells. Callus formation is impaired in rkp mutants, indicating a role of RKP in the plant cell cycle. RKP was demonstrated to be a functional ubiquitin E3 ligase and is able to interact with cell-cycle inhibitor ICK/KRP proteins in vitro . Accumulation of the protein ICK2/KRP2 was found increased in the rkp mutant. The above results strengthen the possibility that RKP might regulate the degradation of ICK/KRP proteins. In addition, the protein level of ICK2/KRP2 was decreased upon BSCTV infection. Overexpression of ICK1/KRP1 in Arabidopsis could reduce the susceptibility to BSCTV. In conclusion, we found that RKP is induced by BSCTV C4 and may affect BSCTV infection by regulating the host cell cycle.