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.     

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.     

Genetic Relationship Between Angustifolia3 and Extra-Small Sisters Highlights Novel Mechanisms Controlling Leaf Size

Coordination between cell proliferation and cell expansion is pivotal in leaf size determination. A group of mutants that are impaired in cell proliferation such as the angustifolia3 (an3) has provided a clue to understanding how these cellular processes are coordinated. In these mutants, impaired cell proliferation is accompanied by enhanced cell enlargement. We propose to call this phenomenon “compensated cell enlargement.” Previously, we isolated ten extra-small sisters (xs) mutants that are specifically impaired in post-mitotic cell expansion and found that several xs mutations are able to suppress compensated cell enlargement in an3. Thus, the enhanced cell expansion observed in an3 results from the hyperactivation of post-mitotic cell expansion involving specific members of the XS gene family. These results suggested that cell proliferation process(es) and post-mitotic cell expansion process(es) are somehow linked in an as yet unknown fashion in leaf primordia. In this addendum, we propose possible models for the linking mechanisms that coordinate AN3-dependent cell proliferation and XS-dependent cell expansion in leaf development.Key Words: Arabidopsis, cell expansion, cell proliferation, extra-small sisters (xs), angustifolia3 (an3), compensated cell enlargement, leaf, organ size controlMature leaf size is determined by the final number and size of cells within a leaf. Thus, the spatial and temporal regulation of cell proliferation and cell expansion plays pivotal roles in establishing developmentally programmed leaf size in a reproducible fashion. During leaf development, cell proliferation is maintained in the basal part of the leaf primordium and terminates basipetally.^1,2 Cells that exit cell cycling undergo differentiation and expand enormously.¹ Although many studies have revealed the molecular mechanisms underlying the above processes, the coordination of cell proliferation and cell expansion in the context of organogenesis is not yet understood.In recent years, an interesting phenomenon has been reported in leaves of several mutants or transgenic plants with impaired cell proliferation. These mutants not only have a defect in cell proliferation, but have larger cells than does the wild type, suggesting that the cell proliferation process interacts with the cell expansion process during leaf organogenesis.^3–8 This phenomenon, called “compensation,” has highlighted the existence of a coordination system between cell proliferation and cell expansion in leaf development.^9–13Conceptually, this compensation can be dissected into two processes: the induction process involves the reduction of cell proliferation and the response process directs the enhancement of cell expansion and “compensated cell enlargement.” Recently, we showed that, in the typical compensation-exhibiting mutant angustifolia3 (an3), the expansion of post-mitotic, but not mitotic, cells is specifically enhanced.⁸ Thus, the induction and the response processes should take place separately in proliferating and differentiating cells, respectively. To dissect compensation genetically, with an emphasis on the response process, we isolated 10 mutants, named extra-small sisters (xs), that are specifically impaired in post-mitotic cell expansion.^14–16 We classified xs mutants into three classes based on the effect of each xs mutation on compensated cell enlargement, using an3 as a representative of compensation-exhibiting mutants.¹⁴ As expected, a group of xs mutants (xs1, xs2, xs4 and xs5) completely suppressed compensated cell enlargement in an3 mutants (named the “small-cell” class), whereas the other two classes had either no suppressive or additive effects on cell enlargement.¹⁴ This finding demonstrated that these XS genes act downstream of cell expansion pathways that are regulated by compensation and triggered in an3. How is this relationship established in the context of leaf organogenesis? When considering the above result, one might speculate that, in addition to a promotive role in cell proliferation, AN3 has a role in cell expansion post-mitotic cells. However, AN3 is hardly expressed in differentiating cells, and the overexpression of AN3 has no effects on post-mitotic cell expansion,⁶ suggesting that this possibility is unlikely. Taken together, these results indicate that the AN3-dependent cell proliferation pathway is somehow linked by an intermediary process to post-mitotic cell expansion pathway(s) involving the small-cell class XS.Based on these data, we propose two possible scenarios for the intermediary process, categorized in terms of cell autonomy (Fig. 1). In the non-cell-autonomous case, proliferating cells located in the basal region of the developing leaf may regulate the expansion of differentiating cells located in the upper region of the leaf via unknown cell-cell communications (Fig. 1A and B). In the cell-autonomous case, the activity involved in cell proliferation may be memorized in each cell, and, depending on this memory, each post-mitotic cell determines its own final size (Fig. 1C). Whatever the mechanism, we can assume that regulatory signal(s) would be affected by cell proliferation. Irrespective of cell autonomy, this putative signal acts either positively or negatively on cell expansion. When cell proliferation is impaired by the an3 mutation, the strength of the negative signal would be reduced and become insufficient to prevent differentiating cells from excessive cell expansion. Conversely, if this signal plays a positive role in cell expansion, the signal may be insufficient to positively control cell expansion in the wild type. However, when the cell number is significantly reduced by the an3 mutation, this positive signal(s) would hyperactivate cell expansion pathways. Further analyses of the factors involved in the intermediary process should provide an important insight into signaling mechanisms that control leaf size.Open in a separate windowFigure 1Proposed models for the leaf size control inferred from the analysis of compensated cell enlargement. (A and B) Non-cell-autonomous model. Cells located in the basal part of a leaf primordium would produce signal(s) that inhibit (A) or promote (B) cell expansion of post-mitotic cells present in the apical part of the leaf primordium. (A) If a significant reduction in cell number occurs in an3, the strength of the inhibitory signal would be reduced and cell expansion would be de-repressed, resulting in the abnormal enlargement of leaf cells. (B) When we assume a cell expansion-promoting signal(s), its strength may be insufficient to enhance cell expansion in the wild type. When a significant reduction in cell number occurs in an3, the promoting signal(s) would increase sufficiently to cause compensation. (C) “Cell memory” model. A specific signal reflecting cell proliferation activity in proliferating cells is retained during cell differentiation and affects the magnitude of cell expansion. Inhibitory and stimulatory signal examples are shown in the upper and lower panels, respectively. The relationship between the strength of the signals and the induction of compensation is the same as that described in the non-cell-autonomous model.Before our reports on the actions of an3, fugu and xs mutants,^8,14 compensated cell enlargement was considered to be caused by the uncoupling of cell division and growth. Now, this possibility is clearly ruled out. Cell proliferation and post-mitotic cell expansion are the most basic cellular processes and are each supported by different regulatory networks. The putative signaling systems discussed here provide a new perspective on how developmental programs integrate these networks into a super-network to control organ size.     

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。     

Analysis of leaf development in fugu mutants of Arabidopsis reveals three compensation modes that modulate cell expansion in determinate organs

In multicellular organisms, the coordination of cell proliferation and expansion is fundamental for proper organogenesis, yet the molecular mechanisms involved in this coordination are largely unexplored. In plant leaves, the existence of this coordination is suggested by compensation, in which a decrease in cell number triggers an increase in mature cell size. To elucidate the mechanisms of compensation, we isolated five new Arabidopsis (Arabidopsis thaliana) mutants (fugu1-fugu5) that exhibit compensation. These mutants were characterized together with angustifolia3 (an3), erecta (er), and a KIP-RELATED PROTEIN2 (KRP2) overexpressor, which were previously reported to exhibit compensation. Time-course analyses of leaf development revealed that enhanced cell expansion in fugu2-1, fugu5-1, an3-4, and er-102 mutants is induced postmitotically, indicating that cell enlargement is not caused by the uncoupling of cell division from cell growth. In each of the mutants, either the rate or duration of cell expansion was selectively enhanced. In contrast, we found that enhanced cell expansion in KRP2 overexpressor occurs during cell proliferation. We further demonstrated that enhanced cell expansion occurs in cotyledons with dynamics similar to that in leaves. In contrast, cell expansion was not enhanced in roots even though they exhibit decreased cell numbers. Thus, compensation was confirmed to occur preferentially in determinate organs. Flow cytometric analyses revealed that increases in ploidy level are not always required to trigger compensation, suggesting that compensation is only partially mediated by ploidy-dependent processes. Our results suggest that compensation reflects an organ-wide coordination of cell proliferation and expansion in determinate organs, and involves at least three different expansion pathways.     

Co-Ordination of Cell Division and Tissue Expansion in Sunflower, Tobacco, and Pea Leaves: Dependence or Independence of Both Processes?

Abstract Temporal analyses of cell division and tissue expansion in pea, tobacco, and sunflower leaves reveal that both processes follow similar patterns during leaf development. Relative cell division and relative tissue expansion rates are maximal and constant during early leaf development, but they decline later. In contrast, relative cell expansion rate follows a bell-shaped curve during leaf growth. Cell division and tissue expansion have common responses to temperature, intercepted radiation, and water deficit. As a consequence, final leaf area and cell number remain highly correlated throughout a large range of environmental conditions for these different plant species, indicating that cell division and tissue expansion are co-ordinated during leaf development. This co-ordination between processes has long been explained by dependence between both processes. Most studies on dicotyledonous leaf development indicate that leaf expansion rate depends on the number of cells in the leaf. We tested this hypothesis with a large range of environmental conditions and different plant species. Accordingly, we found a strong correlation between both absolute leaf expansion rate and leaf cell number. However, we showed that this relationship is not necessarily causal because it can be simulated by the hypothesis of independence between cell division and tissue expansion according to Green's theory of growth (1976). Received 23 February 2000; accepted 3 March 2000     

Tryptophan deficiency affects organ growth by retarding cell expansion in Arabidopsis

Tryptophan (Trp) is an essential amino acid required not only for protein synthesis but also for the production of many plant metabolites, including the hormone auxin. Mutations that disrupt Trp biosynthesis result in various developmental defects in plant organs, but how Trp affects organ growth and development remains unclear. Here, we identify an Arabidopsis mutant, small organ1 ( smo1/trp2-301 ), which exhibits a reduction in the size of its aerial organs as a result of the retardation of growth by cell expansion, rather than by the retardation of growth by cell proliferation. smo1/trp2-301 contains a lesion in TSB1 that encodes a predominantly expressed Trp synthase β-subunit, and is allelic with trp2 mutants. Further analyses show that in trp2 leaf cells, the nuclear endoreduplication is impaired and chloroplast development is delayed. Furthermore, cell expansion and leaf growth in trp2 can be restored by the exogenous application of Trp, but not by auxin, and the general protein synthesis is not apparently affected in trp2 mutants. Our findings suggest that the deficiency in Trp or its derivatives is a growth-limiting factor for cell expansion during plant organogenesis.     

Gibberellin indirectly promotes chloroplast biogenesis as a means to maintain the chloroplast population of expanded cells

Chloroplast biogenesis needs to be well coordinated with cell division and cell expansion during plant growth and development to achieve optimal photosynthesis rates. Previous studies showed that gibberellins (GAs) regulate many important plant developmental processes, including cell division and cell expansion. However, the relationship between chloroplast biogenesis with cell division and cell expansion, and how GA coordinately regulates these processes, remains poorly understood. In this study, we showed that chloroplast division was significantly reduced in the GA‐deficient mutants of Arabidopsis (ga1‐3) and Oryza sativa (d18‐AD), accompanied by the reduced expression of several chloroplast division‐related genes. However, the chloroplasts of both mutants exhibited increased grana stacking compared with their respective wild‐type plants, suggesting that there might be a compensation mechanism linking chloroplast division and grana stacking. A time‐course analysis showed that cell expansion‐related genes tended to be upregulated earlier and more significantly than the genes related to chloroplast division and cell division in GA‐treated ga1‐3 leaves, suggesting the possibility that GA may promote chloroplast division indirectly through impacting leaf mesophyll cell expansion. Furthermore, our cellular and molecular analysis of the GA‐response signaling mutants suggest that RGA and GAI are the major repressors regulating GA‐induced chloroplast division, but other DELLA proteins (RGL1, RGL2 and RGL3) also play a role in repressing chloroplast division in Arabidopsis. Taken together, our data show that GA plays a critical role in controlling and coordinating cell division, cell expansion and chloroplast biogenesis through influencing the DELLA protein family in both dicot and monocot plant species.     

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.     

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.     

Shade avoidance responses are mediated by the ATHB-2 HD-zip protein, a negative regulator of gene expression.

The ATHB-2 gene encoding an homeodomain-leucine zipper protein is rapidly and strongly induced by changes in the ratio of red to far-red light which naturally occur during the daytime under the canopy and induce in many plants the shade avoidance response. Here, we show that elevated ATHB-2 levels inhibit cotyledon expansion by restricting cell elongation in the cotyledon-length and -width direction. We also show that elevated ATHB-2 levels enhance longitudinal cell expansion in the hypocotyl. Interestingly, we found that ATHB-2-induced, as well as shade-induced, elongation of the hypocotyl is dependent on the auxin transport system. In the root and hypocotyl, elevated ATHB-2 levels also inhibit specific cell proliferation such as secondary growth of the vascular system and lateral root formation. Consistent with the key role of auxin in these processes, we found that auxin is able to rescue the ATHB-2 lateral root phenotype. We also show that reduced levels of ATHB-2 result in reciprocal phenotypes. Moreover, we demonstrate that ATHB-2 functions as a negative regulator of gene expression in a transient assay. Remarkably, the expression in transgenic plants of a derivative of ATHB-2 with the same DNA binding specificity but opposite regulatory properties results in a shift in the orientation of hypocotyl cell expansion toward radial expansion, and in an increase in hypocotyl secondary cell proliferation. A model of ATHB-2 function in the regulation of shade-induced growth responses is proposed.     

Arabidopsis thaliana organellar DNA polymerase IB mutants exhibit reduced mtDNA levels with a decrease in mitochondrial area density

Plant organelle genomes are complex and the mechanisms for their replication and maintenance remain unclear. Arabidopsis thaliana has two DNA polymerase genes, DNA polymerase IA (polIA) and polIB, that are dual targeted to mitochondria and chloroplasts and are differentially expressed in primary plant tissues. PolIB gene expression occurs at higher levels in tissues not primary for photosynthesis. Arabidopsis T‐DNA polIB mutants have a 30% reduction in relative mitochondrial DNA (mtDNA) levels, but also exhibit a 70% increase in polIA gene expression. The polIB mutant shows an increase in mitochondrial numbers but a significant decrease in mitochondrial area density within the hypocotyl epidermis, shoot apex and root tips. Chloroplast numbers are not significantly different in mesophyll protoplasts. These mutants do not have a significant difference in total dark mitorespiration levels but exhibit a difference in light respiration levels and photosynthesis capacity. Organelle‐encoded genes for components of respiration and photosynthesis are upregulated in polIB mutants. The mutants exhibited slow growth in conjunction with a decreased rate of cell expansion and other secondary phenotypic effects. Evidence suggests that early plastid development and DNA levels are directly affected by a polIB mutation but are resolved to wild‐type levels over time. However, mitochondria numbers and DNA levels never reach wild‐type levels in the polIB mutant. We propose that both polIA and polIB are required for mtDNA replication. The results suggest that polIB mutants undergo an adjustment in cell homeostasis, enabling them to maintain functional mitochondria at the cost of normal cell expansion and plant growth.     

Genome-wide Expression Profiling in Seedlings of the Arabidopsis Mutant uro that is Defective in the Secondary Cell Wall Formation

Plant secondary growth is of tremendous importance, not only for plant growth and development but also for economic usefulness. Secondary tissues such as xylem and phloem are the conducting tissues in plant vascular systems, essentially for water and nutrient transport, respectively. On the other hand, products of plant secondary growth are important raw materials and renewable sources of energy. Although advances have been recently made towards describing molecular mechanisms that regulate secondary growth, the genetic control for this process is not yet fully understood. Secondary cell wall formation in plants shares some common mechanisms with other plant secondary growth processes. Thus, studies on the secondary cell wall formation using Arabidopsis may help to understand the regulatory mechanisms for plant secondary growth. We previously reported phenotypic characterizations of an Arabidopsis semi-dominant mutant, upright rosette （uro）, which is defective in secondary cell wall growth and has an unusually soft stem. Here, we show that lignification in the secondary cell wall in uro is aberrant by analyzing hypocotyl and stem. We also show genome-wide expression profiles of uro seedlings, using the Affymetrix GeneChip that contains approximately 24 000 Arabidopsis genes. Genes identified with altered expression levels include those that function in plant hormone biosynthesis and signaling, cell division and plant secondary tissue growth. These results provide useful information for further characterizations of the regulatory network in plant secondary cell wall formation.     

Exit from proliferation during leaf development in Arabidopsis thaliana: a not-so-gradual process

Early leaf growth is sustained by cell proliferation and subsequent cell expansion that initiates at the leaf tip and proceeds in a basipetal direction. Using detailed kinematic and gene expression studies to map these stages during early development of the third leaf of Arabidopsis thaliana, we showed that the cell-cycle arrest front did not progress gradually down the leaf, but rather was established and abolished abruptly. Interestingly, leaf greening and stomatal patterning followed a similar basipetal pattern, but proliferative pavement cell and formative meristemoid divisions were uncoordinated in respect to onset and persistence. Genes differentially expressed during the transition from cell proliferation to expansion were enriched in genes involved in cell cycle, photosynthesis, and chloroplast retrograde signaling. Proliferating primordia treated with norflurazon, a chemical inhibitor of retrograde signaling, showed inhibited onset of cell expansion. Hence, differentiation of the photosynthetic machinery is important for regulating the exit from proliferation.     

Recent advances in the genetic regulation of the shape of simple leaves

Leaves are major photosynthetic organs, and their diverse shapes and sizes allow adaptation to the natural environment. The early control of leaf shape and size depends on the control of the rate and plane of cell division at the shoot apical meristem and the polarity-dependent cell differentiation in the leaf primordium. In this review, we first summarize knowledge regarding several genes that control the initial stages of leaf formation and leaf polarity (e.g. adaxial–abaxial polarity, symmetry, and flat morphology). Formation of the lateral leaf morphology involves co-ordination of the rates of division and enlargement of leaf cells. Thus, we also summarize information on a number of genes that control these stages of two-dimensional lateral leaf growth (e.g. polarized cell expansion, specific control of cell proliferation, and integration of cell proliferation and expansion). In addition, we discuss several recently identified microRNAs, which are important factors affecting the development of leaf shape via control of spatial and temporal expression of target gene families. We focus on the genetic regulation of leaf shape in the model plant Arabidopsis thaliana from the perspective of spatial and temporal balance among cell proliferation, enlargement, and differentiation, with special emphasis on the results of our own studies.     

控制植物器官大小的分子机理

植物器官大小是植物形态的一个重要特征并受严格的遗传调控。器官大小与两个不同的过程有关：细胞扩张和细胞分裂。分子遗传分析已经鉴定了许多基因,这些基因通过作用于其中一个或两个过程来影响器官的最终大小。某种植物个体间器官大小的差异是由控制该器官特征的基因表达水平变化引起的,通过拟南芥的遗传分析显示这些基因是如何受控制或被修饰的。以上这些资料阐明了植物如何确定继续或停止生长,同时也提供了改变植物积累生物量的方法。     

Sterols regulate development and gene expression in Arabidopsis

Sterols are important not only for structural components of eukaryotic cell membranes but also for biosynthetic precursors of steroid hormones. In plants, the diverse functions of sterol-derived brassinosteroids (BRs) in growth and development have been investigated rigorously, yet little is known about the regulatory roles of other phytosterols. Recent analysis of Arabidopsis fackel (fk) mutants and cloning of the FK gene that encodes a sterol C-14 reductase have indicated that sterols play a crucial role in plant cell division, embryogenesis, and development. Nevertheless, the molecular mechanism underlying the regulatory role of sterols in plant development has not been revealed. In this report, we demonstrate that both sterols and BR are active regulators of plant development and gene expression. Similar to BR, both typical (sitosterol and stigmasterol) and atypical (8, 14-diene sterols accumulated in fk mutants) sterols affect the expression of genes involved in cell expansion and cell division. The regulatory function of sterols in plant development is further supported by a phenocopy of the fk mutant using a sterol C-14 reductase inhibitor, fenpropimorph. Although fenpropimorph impairs cell expansion and affects gene expression in a dose-dependent manner, neither effect can be corrected by applying exogenous BR. These results provide strong evidence that sterols are essential for normal plant growth and development and that there is likely a BR-independent sterol response pathway in plants. On the basis of the expression of endogenous FK and a reporter gene FK::beta-glucuronidase, we have found that FK is up-regulated by several growth-promoting hormones including brassinolide and auxin, implicating a possible hormone crosstalk between sterol and other hormone-signaling pathways.     

Characterization of the AE7 gene in Arabidopsis suggests that normal cell proliferation is essential for leaf polarity establishment

The formation of leaf polarity is critical for leaf morphogenesis. In this study, we characterized and cloned an Arabidopsis gene, AS1/2 ENHANCER7 (AE7), which is required for both leaf adaxial-abaxial polarity formation and normal cell proliferation. The ae7 mutant exhibited leaf adaxial-abaxial polarity defects and double mutants combining ae7 with the leaf polarity mutants as1 (asymmetric leaves1), as2, rdr6 (RNA-dependent RNA polymerase6) or ago7/zip (argonaute7/zippy) all resulted in plants with an apparently enhanced loss of adaxial leaf identity. In addition, ae7 also showed decreased cell proliferation in both leaves and roots, compensated by increased cell sizes in leaves. AE7 encodes a protein conserved in many eukaryotic organisms, ranging from unicellular yeasts to humans; however, the functions of AE7 family members from other species have not been reported. In situ hybridization revealed that AE7 is expressed in a spotted pattern in plant tissues, similar to cell-cycle marker genes such as HISTONE4. Moreover, the ae7 endoploidy and expression analysis of several cell-cycle marker genes in ae7 suggest that the AE7 gene is required for cell cycle progression. As the previously characterized 26S proteasome and ribosome mutants also affect both leaf adaxial-abaxial polarity and cell proliferation, similar to the defects in ae7, we propose that normal cell proliferation may be essential for leaf polarity establishment. Possible models for how cell proliferation influences leaf adaxial-abaxial polarity establishment are discussed.     

Regulation of cell expansion by the <Emphasis Type="Italic"> DISTORTED </Emphasis>genes in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>: actin controls the spatial organization of microtubules

The control of the directionality of cell expansion was investigated using a class of eight genes, the so-called DISTORTED (DIS) genes, that are required for proper expansion of leaf trichomes in Arabidopsis thaliana. By tracing the separation of latex beads placed on the trichome surface, we demonstrate that trichomes grow by diffuse rather than tip growth, and that in dis mutants deviations from the normal orientation of growth can occur in all possible directions. We could not detect any differences in intracellular organization between wild-type and dis-group mutants by electron microscopy. The analysis of double mutants showed that although the expression of the dis phenotype is generally independent of branching and endoreduplication, dis mutations act synthetically in combination lesions in the ZWI gene, which encodes a kinesin motor protein. Using a MAP4:GFP marker line, we show that the organization of cortical microtubules is affected in dis-group mutants. The finding that most dis-group mutants have actin defects suggested to us that actin is involved in organizing the orientation of microtubules. By analyzing the microtubule organization in plants treated with drugs that bind to actin, we verified that actin is involved in the positioning of cortical microtubules and thereby in plant cell expansion.