Conditional repression of AUXIN BINDING PROTEIN1 reveals that it coordinates cell division and cell expansion during postembryonic shoot development in Arabidopsis and tobacco

AUXIN BINDING PROTEIN1 (ABP1) has long been characterized as a potentially important mediator of auxin action in plants. Analysis of the functional requirement for ABP1 during development was hampered because of embryo lethality of the null mutant in Arabidopsis thaliana. Here, we used conditional repression of ABP1 to investigate its function during vegetative shoot development. Using an inducible cellular immunization approach and an inducible antisense construct, we showed that decreased ABP1 activity leads to a severe retardation of leaf growth involving an alteration in cell division frequency, an altered pattern of endocycle induction, a decrease in cell expansion, and a change in expression of early auxin responsive genes. In addition, local repression of ABP1 activity in the shoot apical meristem revealed an additional role for ABP1 in cell plate formation and cell shape. Moreover, cells at the site of presumptive leaf initiation were more sensitive to ABP1 repression than other regions of the meristem. This spatial context-dependent response of the meristem to ABP1 inactivation and the other data presented here are consistent with a model in which ABP1 acts as a coordinator of cell division and expansion, with local auxin levels influencing ABP1 effectiveness.     

The auxin-binding protein 1 is essential for the control of cell cycle

The phytohormone auxin has been known for >50 years to be required for entry into the cell cycle. Despite the critical effects exerted by auxin on the control of cell division, the molecular mechanism by which auxin controls this pathway is poorly understood, and how auxin is perceived upstream of any change in the cell cycle is unknown. Auxin Binding Protein 1 (ABP1) is considered to be a candidate auxin receptor, triggering early modification of ion fluxes across the plasma membrane in response to auxin. ABP1 has also been proposed to mediate auxin-dependent cell expansion, and is essential for early embryonic development. We investigated whether ABP1 has a role in the cell cycle. Functional inactivation of ABP1 in the model plant cell system BY2 was achieved through cellular immunization via the conditional expression of a single-chain fragment variable (scFv). This scFv was derived from a well characterized anti-ABP1 monoclonal antibody previously shown to block the activity of the protein. We demonstrate that functional inactivation of ABP1 results in cell-cycle arrest, and provide evidence that ABP1 plays a critical role in regulation of the cell cycle by acting at both the G1/S and G2/M checkpoints. We conclude that ABP1 is essential for the auxin control of cell division and is likely to constitute the first step of the auxin-signalling pathway mediating auxin effects on the cell cycle.     

Overexpression of the Auxin Binding PROTEIN1 Modulates PIN-Dependent Auxin Transport in Tobacco Cells

Background

Auxin binding protein 1 (ABP1) is a putative auxin receptor and its function is indispensable for plant growth and development. ABP1 has been shown to be involved in auxin-dependent regulation of cell division and expansion, in plasma-membrane-related processes such as changes in transmembrane potential, and in the regulation of clathrin-dependent endocytosis. However, the ABP1-regulated downstream pathway remains elusive.

Methodology/Principal Findings

Using auxin transport assays and quantitative analysis of cellular morphology we show that ABP1 regulates auxin efflux from tobacco BY-2 cells. The overexpression of ABP1can counterbalance increased auxin efflux and auxin starvation phenotypes caused by the overexpression of PIN auxin efflux carrier. Relevant mechanism involves the ABP1-controlled vesicle trafficking processes, including positive regulation of endocytosis of PIN auxin efflux carriers, as indicated by fluorescence recovery after photobleaching (FRAP) and pharmacological manipulations.

Conclusions/Significance

The findings indicate the involvement of ABP1 in control of rate of auxin transport across plasma membrane emphasizing the role of ABP1 in regulation of PIN activity at the plasma membrane, and highlighting the relevance of ABP1 for the formation of developmentally important, PIN-dependent auxin gradients.     

Altered Expression of Auxin-binding Protein 1 Affects Cell Expansion and Auxin Pool Size in Tobacco Cells

Auxin-binding protein 1 (ABP1) has an essential role in auxin-dependent cell expansion, but its mechanisms of action remain unknown. Our previous study showed that ABP1-mediated cell expansion is auxin concentration dependent. However, auxin distribution in plant tissue is heterogeneous, complicating the interpretation of ABP1 function. In this study, we used cells in culture that have altered expression of ABP1 to address the mechanism of ABP1 action at the cellular level, because cells in culture have homogeneous cell types and could potentially circumvent the heterogeneous auxin-distributions inherent in plant tissues. We found that cells overexpressing ABP1 had altered sensitivity to auxin and were larger, with nuclei that have undergone endoreduplication, a finding consistent with other data that support an auxin extracellular receptor role for ABP1. These cells also had a higher free auxin pool size, which cannot be explained by altered auxin transport. In cells lacking detectable ABP1, a higher rate of auxin metabolism was observed. The results suggest that ABP1 has, beyond its proposed role as an auxin extracellular receptor, a role in mediating auxin availability.     

Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth

The distribution and biosynthesis of indole-3-acetic acid (IAA) was investigated during early plant development in Arabidopsis. The youngest leaves analysed, less than 0.5 mm in length, contained 250 pg mg(-1) of IAA and also exhibited the highest relative capacity to synthesize this hormone. A decrease of nearly one hundred-fold in IAA content occurred as the young leaves expanded to their full size, and this was accompanied by a clear shift in both pool size and IAA synthesis capacity. The correlation between high IAA content and intense cell division was further verified in tobacco leaves, where a detailed analysis revealed that dividing mesophyll tissue contained ten-fold higher IAA levels than tissue growing solely by elongation. We demonstrated that all parts of the young Arabidopsis plant can potentially contribute to the auxin needed for growth and development, as not only young leaves, but also all other parts of the plant such as cotyledons, expanding leaves and root tissues have the capacity to synthesize IAA de novo. We also observed that naphthylphthalamic acid (NPA) treatment induced tissue-dependent feedback inhibition of IAA biosynthesis in expanding leaves and cotyledons, but intriguingly not in young leaves or in the root system. This observation supports the hypothesis that there is a sophisticated tissue-specific regulatory mechanism for auxin biosynthesis. Finally, a strict requirement for maintaining the pool sizes of IAA was revealed as reductions in leaf expansion followed both decreases and increases in the IAA levels in developing leaves. This indicates that leaves are not only important sources for IAA synthesis, but that normal leaf expansion depends on rigorous control of IAA homeostasis.     

TONSOKU is expressed in S phase of the cell cycle and its defect delays cell cycle progression in Arabidopsis

TONSOKU(TSK)/MGOUN3/BRUSHY1 of Arabidopsis thaliana encodes a nuclear leucine-glycine-aspargine (LGN) domain protein implicated to be involved in genome maintenance, and mutants with defects in TSK show a fasciated stem with disorganized meristem structures. We identified a homolog of TSK from tobacco BY-2 cells (NtTSK), which showed high sequence conservation both in the LGN domain and in leucine-rich repeats with AtTSK. The NtTSK gene was expressed during S phase of the cell cycle in tobacco BY-2 cells highly synchronized for cell division. The tsk mutants of Arabidopsis contained an increased proportion of cells with 4C nuclei and cells expressing cyclin B1 compared with the wild type. These results suggest that TSK is required during the cell cycle and defects of TSK cause the arrest of cell cycle progression at G2/M phase.     

生长素结合蛋白cDNA的克隆及其在烟草中的表达

基于拟南芥内质网生长素结合蛋白基因的cDNA序列,设计合成了Ap5和Ap3两个引物,应用RT－PCR技术扩增了拟南芥的ABP基因。将该基因克隆在植物表达载体p35SSIN的35S启动子和Nos3’端之间,得到植物表达载体p35SE。通过农杆菌个导的方法对烟草SR1进行了转化,由分子杂交等检测证明,生长素结合蛋白基因已在烟草中表达,同时转基因烟草后代对生长素的敏感性明显增加。     

Heteroblasty in the moss,Aphanoregma patens (Physcomitrella patens), results from progressive modulation of a single fundamental leaf developmental programme

Abstract

Leaves at the apex of a mature Aphanoregma patens (Hedw.) Lindb. (Physcomitrella patens (Hedw.) Bruch Schimp. in B.S.G.) gametophore differ markedly in size and form from those at its base. To determine how these differences are produced during development, we first examined qualitative and quantitative differences between successive leaves along the stem and among leaves at different developmental stages. Differences between successive leaves were slight and cumulative. Local changes in cell number and size combined to produce a regularly shaped and approximately bilaterally symmetrical leaf suggesting that cell division and cell expansion are regionally regulated and coordinated at the organ level. The midrib and marginal teeth are discrete characters, which were prefigured by changes in cell shape in leaves that lacked these characters. In leaf primordia, cell proliferation was responsible for most of the changes in leaf form and size early in development and may have continued as cell expansion took over as the primary contributor to leaf growth and morphogenesis. Thus, leaf heteroblasty in Physcomitrella probably results from modulation of a single developmental programme by external and/or internal forces, which alter progressively in intensity as a gametophore grows. We applied exogenous cytokinin and auxin separately to growing cultures to explore their effects on leaf growth. Cytokinin and auxin stimulated leaf cell division and leaf cell elongation, respectively. Also, young upper leaves of gametophores exposed to exogenous auxin closely resembled basal leaves of untreated plants. Therefore, endogenous cytokinins and auxins may be among the modulating internal forces involved in leaf morphogenesis and the establishment of leaf heteroblasty.     

Expression of genomic AtCYCD2;1 in Arabidopsis induces cell division at smaller cell sizes: implications for the control of plant growth

The Arabidopsis (Arabidopsis thaliana) CYCD2;1 gene introduced in genomic form increased cell formation in the Arabidopsis root apex and leaf, while generating full-length mRNA, raised CDK/CYCLIN enzyme activity, reduced G1-phase duration, and reduced size of cells at S phase and division. Other cell cycle genes, CDKA;1, CYCLIN B;1, and the cDNA form of CYCD2;1 that produced an aberrantly spliced mRNA, produced smaller or zero increases in CDK/CYCLIN activity and did not increase the number of cells formed. Plants with a homozygous single insert of genomic CYCD2;1 grew with normal morphology and without accelerated growth of root or shoot, not providing evidence that cell formation or CYCLIN D2 controls growth of postembryonic vegetative tissues. At the root apex, cells progressed normally from meristem to elongation, but their smaller size enclosed less growth and a 40% reduction in final size of epidermal and cortical cells was seen. Smaller elongated cell size inhibited endoreduplication, indicating a cell size requirement. Leaf cells were also smaller and more numerous during proliferation and epidermal pavement and palisade cells attained 59% and 69% of controls, whereas laminas reached normal size. Autonomous control of expansion was therefore not evident in abundant cell types that formed tissues of root or leaf. Cell size was reduced by a greater number formed in a tissue prior to cell and tissue expansion. Initiation and termination of expansion did not correlate with cell dimension or number and may be determined by tissue-wide signals acting across cellular boundaries.     

Effect of auxin on the mitotic cell cycle in cultured leaf segments at different stages of development in wheat

Young leaves of Triticum timopheevi Zukh. show a defined gradient of development. One-mm-long sections from such leaves were cultured in vitro. At a low concentration of exogenous auxin, cells in the most basal, highly meristematic explants divided readily in culture, but in the absence of auxin they soon ceased dividing and were arrested in G1 and G2 of the mitotic cell cycle. In the region adjoining the meristem, where most cells were arrested in G1, very high concentrations of auxin had to be applied to reinitiate cell division, i.e. stimulate transitions from G1 to S-phase and from G2 to mitosis. Above this potentially auxin-responsive region, which represented less than 50% of the total leaf length, there followed tissue, which, when excised, showed nuclear DNA replication in a number of cells in the absence of auxin. However, the cells did not complete the mitotic cycle, either in the absence or presence of exogenous auxin. We suggest this loss of responsiveness is correlated with an uncoupling of auxin from the control of the cell cycle.     

转异源abp基因烟草的叶外植体不同培养中的形态发生

以转正义和反义abp基因[生长素结合蛋白（ABP）的基因]烟草及对照的叶外植体为材料,在附加2mg／L或1mg／L的6－BA和不同浓度的NAA的MS培养基上进行不定芽分化试验。在较低NAA浓度条件下,转正义abp基因烟草（SE2）的叶外植体分化的不定芽数高于对照（SR1）和转反abp基因烟草（AS3）,说明SE2对生长素的敏感性比SR1和AS3高;而在较高NAA浓度条件下,AS3分化的不定芽数大于SE2和SR1,说明AS3对生长素的敏感性比SE2和SR1低。在培养基中加入生长素极性运输抑制剂HFCA后,不定芽分化受到抑制,分化不定芽中部分叶的形态由两侧对称性生长转变为形成不对称的喇叭状叶。在HFCA浓度为0－7．5mg／L条件下,SE2的喇叭叶发生频率明显高于SR1和AS3,其中SE2和SR1的喇叭叶发生频率分别在HFCA浓度为6．5和7．5mg／L时达到最高以后保持平稳,而AS3的喇叭叶发生频率在HFCA处理浓度直到15mg／L时仍保持上升趋势。这些结果说明ABP具有结合外源生长素,从而促进芽器官分化的功能,同时可能作为生长素受体参与了生长素的极性运输。     

Model-based analysis of Arabidopsis leaf epidermal cells reveals distinct division and expansion patterns for pavement and guard cells

To efficiently capture sunlight for photosynthesis, leaves typically develop into a flat and thin structure. This development is driven by cell division and expansion, but the individual contribution of these processes is currently unknown, mainly because of the experimental difficulties to disentangle them in a developing organ, due to their tight interconnection. To circumvent this problem, we built a mathematic model that describes the possible division patterns and expansion rates for individual epidermal cells. This model was used to fit experimental data on cell numbers and sizes obtained over time intervals of 1 d throughout the development of the first leaf pair of Arabidopsis (Arabidopsis thaliana). The parameters were obtained by a derivative-free optimization method that minimizes the differences between the predicted and experimentally observed cell size distributions. The model allowed us to calculate probabilities for a cell to divide into guard or pavement cells, the maximum size at which it can divide, and its average cell division and expansion rates at each point during the leaf developmental process. Surprisingly, average cell cycle duration remained constant throughout leaf development, whereas no evidence for a maximum cell size threshold for cell division of pavement cells was found. Furthermore, the model predicted that neighboring cells of different sizes within the epidermis expand at distinctly different relative rates, which could be verified by direct observations. We conclude that cell division seems to occur independently from the status of cell expansion, whereas the cell cycle might act as a timer rather than as a size-regulated machinery.     

Long-term inhibition by auxin of leaf blade expansion in bean and Arabidopsis

The role of auxin in controlling leaf expansion remains unclear. Experimental increases to normal auxin levels in expanding leaves have shown conflicting results, with both increases and decreases in leaf growth having been measured. Therefore, the effects of both auxin application and adjustment of endogenous leaf auxin levels on midrib elongation and final leaf size (fresh weight and area) were examined in attached primary monofoliate leaves of the common bean (Phaseolus vulgaris) and in early Arabidopsis rosette leaves. Aqueous auxin application inhibited long-term leaf blade elongation. Bean leaves, initially 40 to 50 mm in length, treated once with alpha-naphthalene acetic acid (1.0 mm), were, after 6 d, approximately 80% the length and weight of controls. When applied at 1.0 and 0.1 mm, alpha-naphthalene acetic acid significantly inhibited long-term leaf growth. The weak auxin, beta-naphthalene acetic acid, was effective at 1.0 mm; and a weak acid control, benzoic acid, was ineffective. Indole-3-acetic acid (1 microm, 10 microm, 0.1 mm, and 1 mm) required daily application to be effective at any concentration. Application of the auxin transport inhibitor, 1-N-naphthylphthalamic acid (1% [w/w] in lanolin), to petioles also inhibited long-term leaf growth. This treatment also was found to lead to a sustained elevation of leaf free indole-3-acetic acid content relative to untreated control leaves. Auxin-induced inhibition of leaf growth appeared not to be mediated by auxin-induced ethylene synthesis because growth inhibition was not rescued by inhibition of ethylene synthesis. Also, petiole treatment of Arabidopsis with 1-N-naphthylphthalamic acid similarly inhibited leaf growth of both wild-type plants and ethylene-insensitive ein4 mutants.     

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.     

Quantitative analysis of cell division in leaves: methods,developmental patterns and effects of environmental conditions

In planta quantitative studies of cell cycle are necessary for examining the role of cell division in the response of plants to environmental conditions and to analyse the behaviour of transformed plants in this context. We present and discuss non-intrusive kinematic methods which allow estimating the duration of cell cycle with a high spatial resolution in the leaf. Different methods are proposed and discussed for monocotyledons and dicotyledons, and compared with methods involving the use of chemicals. In monocotyledon leaves, cell division is restricted to a limited zone near the leaf insertion point, twice as long in the mesophyll as in the epidermis. In dicotyledons, cell division occurs in the whole leaf with a uniform and constant cell cycle duration for a determinate number of cell cycles, representing about half of leaf development. Over several experiments, this number is well conserved in a given leaf zone in the absence of stresses, but larger near the leaf base than near the leaf tip. After that, cell cycle duration increases because cells are progressively blocked in G1 while the durations of S-G2-M phases do not change with time. Leaf temperature affects neither the distribution of nuclei in each phase of the cycle nor the number of cell cycles in a leaf. Water or light deficits both cause a partial blockage of nuclei in G1 during the stress only, thereby increasing cell cycle duration and decreasing final cell number. These results suggest that a strong developmental programme drives cell division in leaves, so a simple framework allows analysis of temporal patterns, of spatial gradients and of the effect of environmental conditions.     

Kinematic evidence that atmospheric nitrogen dioxide increases the rates of cell proliferation and enlargement to stimulate leaf expansion in Arabidopsis

To elucidate the stimulation of leaf growth by atmospheric nitrogen dioxide (NO₂), we performed a kinematic analysis of the eighth leaves of Arabidopsis thaliana (accession C24) plants grown for 17–35 days after sowing in the presence or absence of 50 ppb NO₂ (designated +NO₂ plants and –NO₂ plants, respectively). We found that the peak and mean values of the relative rates of leaf expansion, cell division and cell expansion were always greater in +NO₂ plants than in –NO₂ plants. No evidence for prolonged duration was obtained. Thus, NO₂ treatment increased the rates of both cell proliferation and enlargement to increase leaf size. Furthermore, a fold-change analysis showed that cell proliferation and enlargement differentially regulated NO₂-induced leaf expansion.     

Cell surface- and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis

Auxin is a multifunctional hormone essential for plant development and pattern formation. A nuclear auxin-signaling system controlling auxin-induced gene expression is well established, but cytoplasmic auxin signaling, as in its coordination of cell polarization, is unexplored. We found a cytoplasmic auxin-signaling mechanism that modulates the interdigitated growth of Arabidopsis leaf epidermal pavement cells (PCs), which develop interdigitated lobes and indentations to form a puzzle-piece shape in a two-dimensional plane. PC interdigitation is compromised in leaves deficient in either auxin biosynthesis or its export mediated by PINFORMED 1 localized at the lobe tip. Auxin coordinately activates two Rho GTPases, ROP2 and ROP6, which promote the formation of complementary lobes and indentations, respectively. Activation of these ROPs by auxin occurs within 30 s and depends on AUXIN-BINDING PROTEIN 1. These findings reveal Rho GTPase-based auxin-signaling mechanisms, which modulate the spatial coordination of cell expansion across a field of cells.     

Mechanisms of leaf tooth formation in Arabidopsis

Serration found along leaf margins shows species‐specific characters. Whereas compound leaf development is well studied, the process of serration formation is largely unknown. To understand mechanisms of serration development, we investigated distinctive features of cells that could give rise to tooth protrusion in the simple‐leaf plant Arabidopsis. After the emergence of a tooth, marginal cells, except for cells at the sinuses and tips, started to elongate rapidly. Localized cell division seemed to keep cells at the sinus smaller, rather than halt cell elongation. As leaves matured, the marginal cell number between teeth became similar in any given tooth. These results suggest that teeth are formed by repetition of an unknown mechanism that spatially monitors cell number and regulates cell division. We then examined the role of CUP‐SHAPED COTYLEDON 2 (CUC2) in serration development. cuc2‐3 forms fewer hydathodes and auxin maxima, visualized by DR5rev::GFP, at the leaf margin, suggesting that CUC2 patterns serration through the regulation of auxin. In contrast to a previous interpretation, comparison of leaf outlines revealed that CUC2 promotes outgrowth of teeth rather than suppression of growth at the sinuses. We found that mutants with increased CUC2 expression form ectopic tissues and mis‐express SHOOT MERISTEMLESS (STM) at the sinus between the enhanced teeth. Similar but infrequent STM expression was found in the wild type, indicating STM involvement in the serration of simple leaves. Our study provides insights into the morphological and molecular mechanisms for leaf development and tooth formation, and highlights similarities between serration and compound leaf development.