BRANCHLESS TRICHOMES links cell shape and cell cycle control in Arabidopsis trichomes

Endoreplication, also called endoreduplication, is a modified cell cycle in which DNA is repeatedly replicated without subsequent cell division. Endoreplication is often associated with increased cell size and specialized cell shapes, but the mechanism coordinating DNA content with shape and size remains obscure. Here we identify the product of the BRANCHLESS TRICHOMES (BLT) gene, a protein of hitherto unknown function that has been conserved throughout angiosperm evolution, as a link in coordinating cell shape and nuclear DNA content in endoreplicated Arabidopsis trichomes. Loss-of-function mutations in BLT were found to enhance the multicellular trichome phenotype of mutants in the SIAMESE (SIM) gene, which encodes a repressor of endoreplication. Epistasis and overexpression experiments revealed that BLT encodes a key regulator of trichome branching. Additional experiments showed that BLT interacts both genetically and physically with STICHEL, another key regulator of trichome branching. Although blt mutants have normal trichome DNA content, overexpression of BLT results in an additional round of endoreplication, and blt mutants uncouple DNA content from morphogenesis in mutants with increased trichome branching, further emphasizing its role in linking cell shape and endoreplication.     

Trichome cell growth in Arabidopsis thaliana can be derepressed by mutations in at least five genes

Leaf trichomes in Arabidopsis are unicellular epidermal hairs with a branched morphology. They undergo successive endoreduplication rounds early during cell morphogenesis. Mutations affecting trichome nuclear DNA content, such as triptychon or glabra3, alter trichome branching. We isolated new mutants with supernumerary trichome branches, which fall into three unlinked complementation groups: KAKTUS and the novel loci, POLYCHOME and RASTAFARI. They map to chromosomes IV, II, and V, respectively. The trichomes of these mutants presented an increased DNA content, although to a variable extent. The spindly-5 mutant, which displays a constitutive gibberellin response, also produces overbranched trichomes containing more nuclear DNA. We analyzed genetic interactions using double mutants and propose that two independent pathways, defined by SPINDLY and TRIPTYCHON, act to limit trichome growth. KAKTUS and POLYCHOME might have redundant actions mediating gibberellin control via SPINDLY. The overall leaf polysomaty was not notably affected by these mutations, suggesting that they affect the control of DNA synthesis in a tissue- or cell type-specific manner. Wild-type tetraploids also produce overbranched trichomes; they displayed a shifted polysomaty in trichomes and in the whole leaf, suggesting a developmental program controlling DNA increases via the counting of endoreduplication rounds.     

How plants split hairs

The unicellular three-branched trichomes, or 'hairs', of Arabidopsis provide a model system for studying cell morphogenesis in plants. Recent results, including the characterization of a newly identified mutant with multicellular trichomes, have led to a new view of how trichome morphogenesis might be controlled.     

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.     

Control of endoreduplication of trichome by RPT2a, a subunit of the 19S proteasome in Arabidopsis

The ubiquitin/26S proteasome pathway plays a central role in the degradation of short-lived regulatory proteins to control many cellular events. The Arabidopsis knockout mutant rpt2a, which contains a defect in the AtRPT2a subunit of the 26S proteasome regulatory particle, showed enlarged leaves caused by increased cell size that correlated with increased ploidy caused by extended endoreduplication. To clarify the role of RPT2a in endoreduplication control, trichome development was genetically examined in further detail. RHL1 and GL3 encode proteins that have a role in the positive regulation of endocycle progression in trichomes. The rhl1 mutants are stalled at 8C and have trichomes with only a single branch. The rpt2a mutation did not alter the rhl1 mutant phenotype, and trichomes of double rpt2a rhl1 mutants resembled that of single rhl1 mutants. On the other hand, the rpt2a mutation suppressed the gl3 phenotype (stalled at 16C, two trichome branches), and trichomes of the double rpt2a gl3 mutant resembled those of the wild type (WT) plants. Together, these data suggest that RPT2a functions to negatively regulate endocycle progression following completion of the third endoreduplication step mediated by RHL1 (8C–16C).     

Leaf hairs influence phytopathogenic fungus infection and confer an increased resistance when expressing a Trichoderma alpha-1,3-glucanase

The leaf surface of a very large number of plant species are covered by trichomes. Non-glandular trichomes are specialized unicellular or multicellular structures that occur in many different plant species and function in xenobiotic detoxification and protecting the plant against pest attack. By analysing the susceptibility of trichome mutants, evidence is provided that indicates the influence of leaf trichomes on foliar fungal infections in Arabidopsis thaliana, probably by facilitating the adhesion of the fungal spores/hyphae to the leaf surface. A decreased trichome number in the hairless Arabidopsis mutant gl1 enhances tolerance against the necrotrophic fungus Botrytis cinerea. By contrast, the try mutant shows an increased susceptibility to both fungal infection and accumulation. Trichome density does not influence infection by the soil-borne pathogen Rhizoctonia solani. In addition, the influence of trichomes on foliar infection is supported by targeting the high-level expression of the Trichoderma harzianum alpha-1,3-glucanase protein to the specialized cell structures. Trichome expression of this anti-fungal hydrolase shows a significant resistance to infection by the foliar pathogen Botrytis cinerea. Resistance to this fungus is not dependent on the constitutive induction of the salicylic or jasmonic defence signalling pathways, but the presence of the alpha-1,3-glucanase protein in trichomes.     

Arabidopsis nucleoporin CPR5 controls trichome cell death through the core cell cycle regulator CKI

• The Arabidopsis trichome is a polyploid epidermal cell resulting from multiple rounds of endocycles. The CYCLIN‐DEPENDENT KINASE INHIBITOR (CKI) family proteins are core cell cycle regulators that promote the endocycle. CONSTITUTIVE EXPRESSION OF PR GENES 5 (CPR5) is a plant‐specific nucleoporin. It has been found that two Arabidopsis CKI, SIAMESE (SIM) and SIAMESE‐RELATED 1 (SMR1), function downstream of CPR5 to activate plant effector‐triggered cell death. The sim smr1 double mutants form multicellular and clustered trichomes, while the cpr5 mutants produce dead and branchless trichomes. This study explored roles of the CPR5‐CKI signalling pathway in trichome cell cycle transition.
• To examine the underlying mechanism of how cell cycle transition is regulated in plant trichomes, Trypan blue staining, flow cytometry, scanning electron microscopy (SEM) and nuclear DNA measurement were conducted.
• The native promoter‐driven CKI and GUS fusion reporter showed that both SIM and SMR1 proteins were preferentially expressed in trichomes. The cpr5‐induced dead and branchless trichomes were fully suppressed by the sim smr1 double mutant, suggesting that SIM and SMR1 function downstream of CPR5 in trichome development. Flow cytometry analysis showed that as compared to the number of 2C (C = DNA content in a haploid nucleus) cells, the number of 4C cells significantly increased, whereas that of polyploidy cells (8C and 16C) dramatically decreased in the cpr5 mutant. The elevated 4C/2C ratio in the cpr5 mutant is consistent with de‐repression of pro‐endocycle regulators SIM and SMR1. The polyploidy cells (8C and 16C) may be selectively targeted to cell death, which is therefore attributed to the branchless trichomes in the cpr5 mutant. Nuclear DNA content analysis demonstrated that the nuclear DNA content of trichomes in the cpr5 sim mutant was significantly higher than in the sim mutant, indicating that CPR5 is a negative endocycle regulator in trichomes.
• This study reveals that the CPR5‐CKI signalling pathway controls trichome cell cycle transition and excessive endocycles are required for cell death in plant trichomes.

     

A contradictory GLABRA3 allele helps define gene interactions controlling trichome development in Arabidopsis

Previously characterized Arabidopsis gl3 mutants have trichomes that are smaller, less branched and undergo fewer rounds of endoreplication than wild-type trichomes. A new gl3 mutant, called gl3-sst, has oddly shaped trichomes that over expand during early development, undergo more endoreduplication and that have a striking nuclear morphology. The mutant nuclei consist of many interconnected lobes; however, only a single set of polytene-like chromosomes reside in the mutant nuclei. The predicted gl3-sst polypeptide has a Leu to Phe substitution (codon 78) within a region responsible for protein-protein interaction. Yeast interaction assays comparing GL3 with gl3-sst proteins show that the mutant protein interaction with GL1 and TTG1 is decreased by 75% and 50%, respectively, but there is no difference in its interaction with TRY. Furthermore, TRY has the ability to prevent the GL1 GL3 interaction and the GL1 gl3-sst interaction is even more sensitive to TRY. Analysis of plants expressing functional GFP-tagged versions of GL1, GL3 and TRY show that the proteins are localized in trichome nuclei. These results have been used to model trichome initiation in terms of protein interactions and threshold levels of activator complex.     

Chromatin assembly factor CAF-1 is required for cellular differentiation during plant development

Chromatin assembly factor CAF-1 facilitates the formation of nucleosomes on newly replicated DNA in vitro. However, the role of CAF-1 in development is poorly understood because mutants are not available in most multicellular model organisms. Biochemical evidence suggests that FASCIATA1, FASCIATA2 and MSI1 form CAF-1 in Arabidopsis thaliana. Because fasciata mutants are viable, CAF-1 is not essential for cell division in plants. Arabidopsis CAF-1 mutants have defects in shoot apical meristems; in addition, CAF-1 is required to establish seedling architecture, leaf size and trichome differentiation. CAF-1 is needed to restrict branching of trichomes on rosette leaves. Increased trichome branching in CAF-1 mutants is not strictly correlated with increased nuclear DNA content. In addition, fas2 glabra3 double mutants show an additive genetic interaction, demonstrating that CAF-1 acts genetically parallel to the GLABRA3-containing, endoreduplication-coupled trichome branching pathway. However, CAF-1 is often needed to restrict endoreduplication, because seedlings of most CAF-1 mutants have increased ploidy. Notably, in the Landsberg erecta background, loss of CAF-1 does not affect ploidy, demonstrating that loss of CAF-1 can be compensated in some Arabidopsis accessions. These results reveal that the functions of FAS1, FAS2 and MSI1 are not restricted to meristems, but are also needed to control genome replication at multiple steps of development.     

The Arabidopsis STICHEL gene is a regulator of trichome branch number and encodes a novel protein

Here, we analyze the STICHEL (STI) gene, which plays an important role in the regulation of branch number of the unicellular trichomes in Arabidopsis. We have isolated the STI locus by positional cloning and confirmed the identity by sequencing seven independent sti alleles. The STI gene encodes a protein of 1,218 amino acid residues containing a domain with sequence similarity to the ATP-binding eubacterial DNA-polymerase III gamma-subunits. Because endoreduplication was found to be normal in sti mutants the molecular function of STI in cell morphogenesis is not linked to DNA replication and, therefore, postulated to represent a novel pathway. Northern-blot analysis shows that STI is expressed in all organs suggesting that STI function is not trichome specific. The analysis of sti alleles and transgenic lines overexpressing STI suggests that STI regulates branching in a dosage-dependent manner.     

SIAMESE, a plant-specific cell cycle regulator, controls endoreplication onset in Arabidopsis thaliana

Recessive mutations in the SIAMESE (SIM) gene of Arabidopsis thaliana result in multicellular trichomes harboring individual nuclei with a low ploidy level, a phenotype strikingly different from that of wild-type trichomes, which are single cells with a nuclear DNA content of approximately 16C to 32C. These observations suggested that SIM is required to suppress mitosis as part of the switch to endoreplication in trichomes. Here, we demonstrate that SIM encodes a nuclear-localized 14-kD protein containing a cyclin binding motif and a motif found in ICK/KRP (for Interactors of Cdc2 kinase/Kip-related protein) cell cycle inhibitor proteins. Accordingly, SIM was found to associate with D-type cyclins and CDKA;1. Homologs of SIM were detected in other dicots and in monocots but not in mammals or fungi. SIM proteins are expressed throughout the shoot apical meristem, in leaf primordia, and in the elongation zone of the root and are localized to the nucleus. Plants overexpressing SIM are slow-growing and have narrow leaves and enlarged epidermal cells with an increased DNA content resulting from additional endocycles. We hypothesize that SIM encodes a plant-specific CDK inhibitor with a key function in the mitosis-to-endoreplication transition.     

Differential regulation of trichome formation on the adaxial and abaxial leaf surfaces by gibberellins and photoperiod in Arabidopsis thaliana (L.) Heynh.

In wild-type (WT) Columbia and Landsberg erecta ecotypes of Arabidopsis thaliana (L.) Heynh., trichomes are present on the adaxial surfaces of all rosette leaves but are absent from the abaxial surfaces of the first-formed leaves. We have determined that both long-day (LD) photoperiod and gibberellin (GA) stimulate trichome formation. WT plants grown in LD conditions produce the first abaxial trichome on earlier leaves than plants grown in short-day (SD) conditions. Photoperiod sensitivity of abaxial trichome formation on WT plants develops gradually over time, reaching the maximum sensitivity about 24 d after germination. Application of gibberellic acid to WT plants growing in SD conditions accelerates the onset of abaxial trichomes. Conversely, application of 20 to 80 mg L-1 paclobutrazol, a GA biosynthesis inhibitor, to wild-type plants suppresses trichome initiation on the abaxial epidermis. The GA-deficient mutants ga1-5 and ga4-1 and the GA-insensitive mutant gai-1 exhibit delayed onset of abaxial trichomes when grown in LD conditions. The null mutant ga1-3 produces completely glabrous leaves when grown in SD conditions. Application of gibberellic acid to glabrous ga1-3 plants consistently induces earlier formation of trichomes on the adaxial epidermis than on the abaxial epidermis, demonstrating a difference between the adaxial and abaxial surfaces in their response to GA with regard to trichome formation.     

The actin cytoskeleton is required to elaborate and maintain spatial patterning during trichome cell morphogenesis in Arabidopsis thaliana

Arabidopsis thaliana trichomes provide an attractive model system to dissect molecular processes involved in the generation of shape and form in single cell morphogenesis in plants. We have used transgenic Arabidopsis plants carrying a GFP-talin chimeric gene to analyze the role of the actin cytoskeleton in trichome cell morphogenesis. We found that during trichome cell development the actin microfilaments assumed an increasing degree of complexity from fine filaments to thick, longitudinally stretched cables. Disruption of the F-actin cytoskeleton by actin antagonists produced distorted but branched trichomes which phenocopied trichomes of mutants belonging to the 'distorted' class. Subsequent analysis of the actin cytoskeleton in trichomes of the distorted mutants, alien, crooked, distorted1, gnarled, klunker and wurm uncovered actin organization defects in each case. Treatments of wild-type seedlings with microtubule-interacting drugs elicited a radically different trichome phenotype characterized by isotropic growth and a severe inhibition of branch formation; these trichomes did not show defects in actin cytoskeleton organization. A normal actin cytoskeleton was also observed in trichomes of the zwichel mutant which have reduced branching. ZWICHEL, which was previously shown to encode a kinesin-like protein is thought to be involved in microtubule-linked processes. Based on our results we propose that microtubules establish the spatial patterning of trichome branches whilst actin microfilaments elaborate and maintain the overall trichome pattern during development.     

植物不同类型表皮毛调控模型研究进展

表皮毛是植物地上部分表皮细胞向外突出延伸的特化毛状结构,不仅可以保护植物免受病虫的危害,还具有一定的经济和药用价值,对其调控的分子机制的阐明有利于植物的分子设计育种和遗传改良。近年来,模式植物拟南芥表皮毛形成的调控模式基本被阐明,其他植物表皮毛的调控机制也取得很大进展。鉴于此,文中综述了拟南芥和棉花(单细胞表皮毛)及番茄和青蒿(多细胞表皮毛)在基因和激素水平上对表皮毛的发育调控,同时简要介绍了其他典型单、双子叶植物表皮毛相关的研究进展,最后,展望了植物表皮毛的研究方向和应用前景。     

CPR5 is involved in cell proliferation and cell death control and encodes a novel transmembrane protein.

Plants often respond to pathogens by sacrificing cells at the infection site. This type of programmed cell death is mimicked by the constitutive pathogene response5 (cpr5) mutant in Arabidopsis in the absence of pathogens, suggesting a role for CPR5 in programmed cell death control. The analysis of the cellular phenotypes of two T-DNA-tagged cpr5 alleles revealed an additional role for CPR5 in the regulation of endoreduplication and cell division. In cpr5 mutant trichomes, endoreduplication cycles stop after two rounds instead of four, and trichome cells have fewer branches than normal. Eventually, cpr5 trichomes die, the nucleus disintegrates, and the cell collapses. Similarly, leaf growth stops earlier than in wild-type, and, frequently, regions displaying spontaneous cell death are observed. The cloning of the CPR5 gene revealed a novel putative transmembrane protein with a cytosolic domain containing a nuclear-targeting sequence. The dual role of CPR5 in cell proliferation and cell death control suggests a regulatory link between these two processes.     

ULTRASTRUCTURE OF GLANDULAR TRICHOMES OF LEAVES OF NICOTIANA TABACUM L., cv XANTHI

Electron microscopy confirms previous light microscope observations that tobacco leaf trichomes are glandular and that there are two different types. Both the tall trichome (multicellular stalk, unicellular or multicellular head) and the short trichome (unicellular stalk; multicellular head) exhibit characteristics common to gland cells—a dense cytoplasm, numerous mitochondria, and little vacuolation. The tall trichome contains structurally well developed chloroplasts and an elaborate network of endoplasmic reticulum. The short trichome contains undifferentiated plastids and endoplasmic reticulum which parallels the nucleus and plasmalemma. Few dictyosomes are seen either in the short trichome or in the tall trichome. The short trichome appears to undergo structural changes concurrently with the appearance of secretory product within the cells. The most noticeable change is the formation of the extraplasmic space between the cell wall and the plasmalemma. Electron dense secretory product is observed between the plasmalemma and the cell wall and within the intercellular spaces.