Arabidopsis RETINOBLASTOMA-RELATED Is Required for Stem Cell Maintenance, Cell Differentiation, and Lateral Organ Production

Several genes involved in the regulation of postembryonic organ initiation and growth have been identified. However, it remains largely unclear how developmental cues connect to the cell cycle. RETINOBLASTOMA RELATED (RBR) is a plant homolog of the tumor suppressor Retinoblastoma (pRb), which is a key regulator of the cell cycle. Using inducible RNA interference (RNAi) against Arabidopsis thaliana RBR (RBRi), we reduced RBR expression levels at different stages of plant development. Conditional reduction or loss of RBR function disrupted cell division patterns, promoted context-dependent cell proliferation, and negatively influenced establishment of cell differentiation. Several lineages of toti- and pluripotent cells, including shoot apical meristem stem cells, meristemoid mother cells, and procambial cells, failed to produce appropriately differentiated cells. Meristem activity was altered, leading to a disruption of the CLAVATA-WUSCHEL feedback loop and inhibition of lateral organ formation. Release of RBR from RNAi downregulation restored meristem activity. Gene profiling analyses soon after RBRi induction revealed that a change in RBR homeostasis is perceived as a stress, even before genes regulated by RBR-E2F become deregulated. The results establish RBR as a key cell cycle regulator required for coordination of cell division, differentiation, and cell homeostasis.     

miR396-targeted AtGRF transcription factors are required for coordination of cell division and differentiation during leaf development in Arabidopsis

In plants, cell proliferation and polarized cell differentiation along the adaxial-abaxial axis in the primordium is critical for leaf morphogenesis, while the temporal-spatial relationships between these two processes remain largely unexplored. Here, it is reported that microRNA396 (miR396)-targeted Arabidopsis growth-regulating factors (AtGRFs) are required for leaf adaxial-abaxial polarity in Arabidopsis. Reduction of the expression of AtGRF genes by transgenic miR396 overexpression in leaf polarity mutants asymmetric leaves1 (as1) and as2 resulted in plants with enhanced leaf adaxial-abaxial defects, as a consequence of reduced cell proliferation. Moreover, transgenic miR396 overexpression markedly decreased the cell division activity and the expression of cell cycle-related genes, but resulted in an increased percentage of leaf cells with a higher ploidy level, indicating that miR396 negatively regulates cell proliferation by controlling entry into the mitotic cell cycle. miR396 is mainly expressed in the leaf cells arrested for cell division, coinciding with its roles in cell cycle regulation. These results together suggest that cell division activity mediated by miR396-targeted AtGRFs is important for polarized cell differentiation along the adaxial-abaxial axis during leaf morphogenesis in Arabidopsis.     

The E2FC-DPB Transcription Factor Controls Cell Division,Endoreplication and Lateral Root Formation in a SCFSKP2A-Dependent Manner

Cell division is a highly regulated process that has to be coordinated with cell specification and differentiation for proper development and growth of the plants. Cell cycle regulation is carried out by key proteins that control cell cycle entry, progression and exit. This regulation is controlled at different stages such as gene expression, posttranslational modification of proteins and specific proteolysis. The G₁/S and the G₂/M transitions are critical checkpoints of the cell cycle that are controlled, among others, by the activity of cyclin-dependent kinases (CDK). Different CDK activities, still to be fully identified, impinge on the retinoblastoma (RBR)/E2F/DP pathway as well as on the programmed proteolysis pathway. The specific degradation of proteins through the ubiquitin pathway in plants, highly controlled in time and space, is emerging as a powerful mechanism to regulate the levels and the activity of several proteins, including many cell cycle regulators.Key Words: cell cycle, endoreplication, E2F, DP, Ubiquitin, SCF, SKP2, lateral root, Arabidopsis     

Endoreplication Controls Cell Fate Maintenance

Cell-fate specification is typically thought to precede and determine cell-cycle regulation during differentiation. Here we show that endoreplication, also known as endoreduplication, a specialized cell-cycle variant often associated with cell differentiation but also frequently occurring in malignant cells, plays a role in maintaining cell fate. For our study we have used Arabidopsis trichomes as a model system and have manipulated endoreplication levels via mutants of cell-cycle regulators and overexpression of cell-cycle inhibitors under a trichome-specific promoter. Strikingly, a reduction of endoreplication resulted in reduced trichome numbers and caused trichomes to lose their identity. Live observations of young Arabidopsis leaves revealed that dedifferentiating trichomes re-entered mitosis and were re-integrated into the epidermal pavement-cell layer, acquiring the typical characteristics of the surrounding epidermal cells. Conversely, when we promoted endoreplication in glabrous patterning mutants, trichome fate could be restored, demonstrating that endoreplication is an important determinant of cell identity. Our data lead to a new model of cell-fate control and tissue integrity during development by revealing a cell-fate quality control system at the tissue level.     

Balance between cell division and differentiation during plant development

The processes which make possible that a cell gives rise to two daughter cells define the cell division cycle. In individual cells, this is strictly controlled both in time and space. In multicellular organisms extra layers of regulation impinge on the balance between cell proliferation and cell differentiation within particular ontogenic programs. In contrast to animals, organogenesis in plants is a post-embryonic process that requires developmentally programmed reversion of sets of cells from different differentiated states to a pluripotent state followed by regulated proliferation and progression through distinct differentiation patterns. This implies a fine coupling of cell division control, cell cycle arrest and reactivation, endoreplication and differentiation. The emerging view is that cell cycle regulators, in addition to controlling cell division, also function as targets for maintaining cell homeostasis during development. The mechanisms and cross talk among different cell cycle regulatory pathways are discussed here in the context of a developing plant.     

The balance between cell division and endoreplication depends on E2FC-DPB, transcription factors regulated by the ubiquitin-SCFSKP2A pathway in Arabidopsis

The balance between cell proliferation, cell cycle arrest, and differentiation needed to maintain the organogenetic program depends on the coordination of gene expression, posttranslational modification, and specific proteolysis of cell cycle regulators. The G1/S and G2/M transitions are critical checkpoints controlled, in part, by cyclin-dependent kinases in the retinoblastoma (RBR)/E2F/DP pathway. Arabidopsis thaliana DPB is regulated by phosphorylation and targeted to proteasome-mediated proteolysis by the SCF(SKP2A) complex. In addition, DPB interacts in vivo with E2FC, because ectopic coexpression of E2FC and DPB produces severe developmental defects. To understand E2FC/DPB heterodimer function, we analyzed the effect of reducing E2FC mRNA levels with RNA interference. The e2fc-R plants developed organs with more but smaller cells and showed increased cell cycle marker gene expression and increased proliferative activity in developing leaves, meristems, and pericycle cells. This last feature produces plants with more lateral roots, consistent with an E2FC role in restricting lateral root initiation. The e2fc-R plants also show marked reductions in ploidy levels of mature leaves. These results indicate that the transition from cell division to the endocycle is sensitive to different pathways, E2FC/DPB being one of them. Our results show that E2FC/DPB is a key factor in controlling the balance between cell proliferation and the switch to the endocycle program.     

Novel functions of plant cyclin-dependent kinase inhibitors, ICK1/KRP1, can act non-cell-autonomously and inhibit entry into mitosis

In animals, cyclin-dependent kinase inhibitors (CKIs) are important regulators of cell cycle progression. Recently, putative CKIs were also identified in plants, and in previous studies, Arabidopsis thaliana plants misexpressing CKIs were found to have reduced endoreplication levels and decreased numbers of cells consistent with a function of CKIs in blocking the G1-S cell cycle transition. Here, we demonstrate that at least one inhibitor from Arabidopsis, ICK1/KRP1, can also block entry into mitosis but allows S-phase progression causing endoreplication. Our data suggest that plant CKIs act in a concentration-dependent manner and have an important function in cell proliferation as well as in cell cycle exit and in turning from a mitotic to an endoreplicating cell cycle mode. Endoreplication is usually associated with terminal differentiation; we observed, however, that cell fate specification proceeded independently from ICK1/KRP1-induced endoreplication. Strikingly, we found that endoreplicated cells were able to reenter mitosis, emphasizing the high degree of flexibility of plant cells during development. Moreover, we show that in contrast with animal CDK inhibitors, ICK1/KRP1 can move between cells. On the one hand, this challenges plant cell cycle control with keeping CKIs locally controlled, and on the other hand this provides a possibility of linking cell cycle control in single cells with the supracellular organization of a tissue or an organ.     

The tobacco A-type cyclin, Nicta;CYCA3;2, at the nexus of cell division and differentiation

Although most of the components of the cell cycle machinery are conserved in all eukaryotes, plants differ strikingly from animals by the absence of a homolog of E-type cyclin, an important regulator involved in G1/S-checkpoint control in animals. By contrast, plants contain a complex range of A-type cyclins, with no fewer than 10 members in Arabidopsis. We previously identified the tobacco A-type cyclin Nicta;CYCA3;2 as an early G1/S-activated gene. Here, we show that antisense expression of Nicta;CYCA3;2 in tobacco plants induces defects in embryo formation and impairs callus formation from leaf explants. The green fluorescent protein (GFP)-Nicta;CYCA3;2 fusion protein was localized in the nucleoplasm. Transgenic tobacco plants overproducing GFP-Nicta;CYCA3;2 could not be regenerated from leaf disc transformation, whereas some transgenic Arabidopsis plants were obtained by the floral-dip transformation method. Arabidopsis plants that overproduce GFP-Nicta;CYCA3;2 showed reduced cell differentiation and endoreplication and a dramatically modified morphology. Calli regenerated from leaf explants of these transgenic Arabidopsis plants were defective in shoot and root regeneration. We propose that Nicta;CYCA3;2 has important functions, analogous to those of cyclin E in animals, in the control of plant cell division and differentiation.     

Arabidopsis E2FA stimulates proliferation and endocycle separately through RBR-bound and RBR-free complexes

Post-embryonic growth in plants depends on the continuous supply of undifferentiated cells within meristems. Proliferating cells maintain their competence for division by active repression of differentiation and the associated endocycle entry. We show by upregulation and downregulation of E2FA that it is required for maintaining proliferation, as well as for endocycle entry. While E2FB-RBR1 (retinoblastoma-related protein 1) complexes are reduced after sucrose addition or at elevated CYCD3;1 levels, E2FA maintains a stable complex with RBR1 in proliferating cells. Chromatin immunoprecipitation shows that RBR1 binds in the proximity of E2F promoter elements in CCS52A1 and CSS52A2 genes, central regulators for the switch from proliferation to endocycles. Overexpression of a truncated E2FA mutant (E2FA(ΔRB)) lacking the RBR1-binding domain interferes with RBR1 recruitment to promoters through E2FA, leading to decreased meristem size in roots, premature cell expansion and hyperactivated endocycle in leaves. E2F target genes, including CCS52A1 and CCS52A2, are upregulated in E2FA(ΔRB) and e2fa knockout lines. These data suggest that E2FA in complex with RBR1 forms a repressor complex in proliferating cells to inhibit premature differentiation and endocycle entry. Thus, E2FA regulates organ growth via two distinct, sequentially operating pathways.     

利用流式细胞仪研究拟南芥叶发育过程中细胞周期的调控

叶的形态建成依赖于细胞不断地分裂增殖和不同类型细胞的特化。在叶发育早期,叶细胞主要通过旺盛的有丝分裂来增加原基中细胞的数目。随着叶片的生长,叶细胞自顶部向基部逐渐退出有丝分裂进入内复制来增加细胞的倍性,同时伴随细胞的扩展和分化。本文介绍利用流式细胞仪研究双子叶模式植物拟南芥叶发育过程中细胞周期调控的方法和具体研究实例。我们发现至少存在3种类型的细胞周期异常的拟南芥叶发育突变体。此外,我们还介绍利用流式细胞仪测定DNA复制效率的方法。     

S6K1 and E2FB are in mutually antagonistic regulatory links controlling cell growth and proliferation in Arabidopsis

Plant development is dependent on the coordination between growth and cell proliferation. The nutrient sensing TOR kinase and its downstream target, the 40S ribosomal S6 Kinase, are central controllers of cell growth that were also shown to determine cell size by inhibiting the onset of mitosis in yeast and animal cells. We have shown that the Arabidopsis S6 Kinase1 inhibits cell proliferation through the RBR-E2FB complex. S6K1 interacts with RBR via its N-terminal RBR binding motif, promotes its nuclear localization and consequent RBR-dependent repression of cell cycle genes through E2FB. Here we show that S6K1 and E2FB are in a mutually antagonistic relationship both in their protein abundance and in their activity. We propose that this double inhibitory regulatory connection between S6K1 and E2FB forms a regulatory switch that might be important to determine whether cells divide or grow.     

The APC/C subunit 10 plays an essential role in cell proliferation during leaf development

The largest E3 ubiquitin-ligase complex, known as anaphase-promoting complex/cyclosome (APC/C), regulates the proteolysis of cell cycle regulators such as CYCLIN B and SECURIN that are essential for sister-chromatid separation and exit from mitosis. Despite its importance, the role of APC/C in plant cells and the regulation of its activity during cell division remain poorly understood. Here, the Arabidopsis thaliana APC/C subunit APC10 was characterized and shown to functionally complement an apc10 yeast mutant. The APC10 protein was located in specific nuclear bodies, most probably resulting from its association with the proteasome complex. An apc10 Arabidopsis knockout mutant strongly impaired female gametogenesis. Surprisingly, constitutive overexpression of APC10 enhanced leaf size. Through kinematic analysis, the increased leaf size was found to be due to enhanced rates of cell division during the early stages of leaf development and, at the molecular level, by increased APC/C activity as measured by an amplification of the proteolysis rate of the mitotic cyclin, CYCB1;1.     

Cell cycling and cell enlargement in developing leaves of Arabidopsis.

Cell cycling plays an important role in plant development, including: (1) organ morphogenesis, (2) cell proliferation within tissues, and (3) cell differentiation. In this study we use a cyclin::beta-glucuronidase reporter construct to characterize spatial and temporal patterns of cell cycling at each of these levels during wild-type development in the model genetic organism Arabidopsis thaliana (Columbia). We show that a key morphogenetic event in leaf development, blade formation, is highly correlated with localized cell cycling at the primordium margin. However, tissue layers are established by a more diffuse distribution of cycling cells that does not directly involve the marginal zone. During leaf expansion, tissue proliferation shows a strong longitudinal gradient, with basiplastic polarity. Tissue layers differ in pattern of proliferative cell divisions: cell cycling of palisade mesophyll precursors is prolonged in comparison to that of pavement cells of the adjacent epidermal layers, and cells exit the cycle at different characteristic sizes. Cell divisions directly related to formation of stomates and of vascular tissue from their respective precursors occur throughout the period of leaf extension, so that differing tissue patterns reflect superposition of cycling related to cell differentiation on more general tissue proliferation. Our results indicate that cell cycling related to leaf morphogenesis, tissue-specific patterns of cell proliferation, and cell differentiation occurs concurrently during leaf development and suggest that unique regulatory pathways may operate at each level.