Trichome Growth Research Articles

SDG26 Is Involved in Trichome Control in Arabidopsis thaliana: Affecting Phytohormones and Adjusting Accumulation of H3K27me3 on Genes Related to Trichome Growth and Development.

Plant trichomes formed by specialized epidermal cells play a role in protecting plants from biotic and abiotic stresses and can also influence the economic and ornamental value of plant products. Therefore, further studies on the molecular mechanisms of plant trichome growth and development are important for understanding trichome formation and agricultural production. SET Domain Group 26 (SDG26) is a histone lysine methyltransferase. Currently, the molecular mechanism by which SDG26 regulates the growth and development of Arabidopsis leaf trichomes is still unclear. We found that the mutant of Arabidopsis (sdg26) possessed more trichomes on its rosette leaves compared to the wild type (Col-0), and the trichome density per unit area of sdg26 is significantly higher than that of Col-0. The content of cytokinins and jasmonic acid was higher in sdg26 than in Col-0, while the content of salicylic acid was lower in sdg26 than in Col-0, which is conducive to trichome growth. By measuring the expression levels of trichome-related genes, we found that the expression of genes that positively regulate trichome growth and development were up-regulated, while the negatively regulated genes were down-regulated in sdg26. Through chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we found that SDG26 can directly regulate the expression of genes related to trichome growth and development such as ZFP1, ZFP5, ZFP6, GL3, MYB23, MYC1, TT8, GL1, GIS2, IPT1, IPT3, and IPT5 by increasing the accumulation of H3K27me3 on these genes, which further affects the growth and development of trichomes. This study reveals the mechanism by which SDG26 affects the growth and development of trichomes through histone methylation. The current study provides a theoretical basis for studying the molecular mechanism of histone methylation in regulating leaf trichome growth and development and perhaps guiding the development of new crop varieties.

Comparative transcriptome analysis reveals key pathways and genes involved in trichome development in tea plant (Camellia sinensis).

Trichomes, which develop from epidermal cells, are considered one of the important characteristics of the tea plant [Camellia sinensis (L.) O. Kuntze]. Many nutritional and metabolomic studies have indicated the important contributions of trichomes to tea products quality. However, understanding the regulation of trichome formation at the molecular level remains elusive in tea plants. Herein, we present a genome-wide comparative transcriptome analysis between the hairless Chuyeqi (CYQ) with fewer trichomes and the hairy Budiaomao (BDM) with more trichomes tea plant genotypes, toward the identification of biological processes and functional gene activities that occur during trichome development. In the present study, trichomes in both cultivars CYQ and BDM were unicellular, unbranched, straight, and soft-structured. The density of trichomes was the highest in the bud and tender leaf periods. Further, using the high-throughput sequencing method, we identified 48,856 unigenes, of which 31,574 were differentially expressed. In an analysis of 208 differentially expressed genes (DEGs) encoding transcription factors (TFs), five may involve in trichome development. In addition, on the basis of the Gene Ontology (GO) annotation and the weighted gene co-expression network analysis (WGCNA) results, we screened several DEGs that may contribute to trichome growth, including 66 DEGs related to plant resistance genes (PRGs), 172 DEGs related to cell wall biosynthesis pathway, 29 DEGs related to cell cycle pathway, and 45 DEGs related to cytoskeleton biosynthesis. Collectively, this study provided high-quality RNA-seq information to improve our understanding of the molecular regulatory mechanism of trichome development and lay a foundation for additional trichome studies in tea plants.

The Plasmodesmata-Located β-1,3-Glucanase Enzyme PdBG4 Regulates Trichomes Growth in Arabidopsis thaliana.

Intercellular material transport and information transmission in plants are carried out through the plasmodesmata (PD). The amount of callose around the PD controls channel permeability. In plants, β-1,3-glucanase can degrade callose and affect plant growth and development. In this study, the gene producing PD-localized β-1,3-glucanase and regulating the leaf trichomes is identified and named PdBG4. Based on functional analysis through a series of genetic manipulation assays, we found that the high expression of PdBG4 was associated with strong PD permeability and short Arabidopsis thaliana leaf trichomes. Conversely, the low expression of PdBG4 correlated with weak PD permeability and long Arabidopsis thaliana leaf trichomes. This study revealed that the PdBG4 gene negatively modulates leaf trichome growth and development by regulating PD permeability.

A DNA-Free Editing Platform for Genetic Screens in Soybean via CRISPR/Cas9 Ribonucleoprotein Delivery.

CRISPR/Cas9-based ribonucleoprotein (RNP)-mediated system has the property of minimizing the effects related to the unwanted introduction of vector DNA and random integration of recombinant DNA. Here, we describe a platform based on the direct delivery of Cas9 RNPs to soybean protoplasts for genetic screens in knockout gene-edited soybean lines without the transfection of DNA vectors. The platform is based on the isolation of soybean protoplasts and delivery of Cas RNP complex. To empirically test our platform, we have chosen a model gene from the soybean genetic toolbox. We have used five different guide RNA (gRNA) sequences that targeted the constitutive pathogen response 5 (CPR5) gene associated with the growth of trichomes in soybean. In addition, efficient protoplast transformation, concentration, and ratio of Cas9 and gRNAs were optimized for soybean for the first time. Targeted mutagenesis insertion and deletion frequency and sequences were analyzed using both Sanger and targeted deep sequencing strategies. We were able to identify different mutation patterns within insertions and deletions (InDels) between + 5 nt and –30 bp and mutation frequency ranging from 4.2 to 18.1% in the GmCPR5 locus. Our results showed that DNA-free delivery of Cas9 complexes to protoplasts is a useful approach to perform early-stage genetic screens and anticipated analysis of Cas9 activity in soybeans.

The role of the cytoskeletal proteins MreB and FtsZ in multicellular cyanobacteria.

Multiseriate and true-branching cyanobacteria are at the peak of prokaryotic morphological complexity. However, little is known about the mechanisms governing multiplanar cell division and morphogenesis. Here, we study the function of the prokaryotic cytoskeletal proteins, MreB and FtsZ in Fischerella muscicola PCC 7414 and Chlorogloeopsis fritschii PCC 6912. Vancomycin and HADA labeling revealed a mixed apical, septal, and lateral trichome growth mode in F.muscicola, whereas C.fritschii exhibits septal growth. In all morphotypes from both species, MreB forms either linear filaments or filamentous strings and can interact with FtsZ. Furthermore, multiplanar cell division in F.muscicola likely depends on FtsZ dosage. Our results lay the groundwork for future studies on cytoskeletal proteins in morphologically complex cyanobacteria.

Detection of the Lychee Erinose Mite, Aceria litchii (Keifer) (Acari: Eriophyidae) in Florida, USA: A Comparison with Other Alien Populations.

The lychee erinose mite (LEM), Aceria litchii (Keifer) is a serious pest of lychee (Litchi chinensis Sonn.). LEM causes a type of gall called ‘erineum’ (abnormal felty growth of trichomes from the epidermis), where it feeds, reproduces and protects itself from biotic and abiotic adversities. In February of 2018, LEM was found in a commercial lychee orchard on Pine Island, Florida. Infestations were recorded on young leaves, stems, and inflorescences of approximately 30 young trees (1.5–3.0 yrs.) of three lychee varieties presenting abundant new growth. Although LEM is present in Hawaii, this mite is a prioritized quarantine pest in the continental USA and its territories. Florida LEM specimens showed small morphological differences from the original taxonomic descriptions of Keifer (1943) and Huang (2008). The observed differences are probably an artifact of the drawings in the original descriptions. Molecular comparisons were conducted on the DNA of LEM specimens from India, Hawaii, Brazil, Taiwan, Australia and Florida. The amplified COI fragment showed very low nucleotide variation among the locations and thus, could be used for accurate LEM identification. The ITS1 sequences and partial 5.8S fragments displayed no nucleotide differences for specimens from any of the locations except Australia. Consistent differences were observed in the ITS2 and 28S fragments. The ITS1-ITS2 concatenated phylogeny yielded two lineages, with Australia in one group and Hawaii, India, Brazil, Florida and Taiwan in another. Specimens from Taiwan and Florida present identical ITS and rDNA segments, suggesting a common origin; however, analysis of additional sequences is needed to confirm the origin of the Florida population.

Functional analysis and development of a CRISPR/Cas9 allelic series for a CPR5 ortholog necessary for proper growth of soybean trichomes

Developments in genomic and genome editing technologies have facilitated the mapping, cloning, and validation of genetic variants underlying trait variation. This study combined bulked-segregant analysis, array comparative genomic hybridization, and CRISPR/Cas9 methodologies to identify a CPR5 ortholog essential for proper trichome growth in soybean (Glycine max). A fast neutron mutant line exhibited short trichomes with smaller trichome nuclei compared to its parent line. A fast neutron-induced deletion was identified within an interval on chromosome 6 that co-segregated with the trichome phenotype. The deletion encompassed six gene models including an ortholog of Arabidopsis thaliana CPR5. CRISPR/Cas9 was used to mutate the CPR5 ortholog, resulting in five plants harboring a total of four different putative knockout alleles and two in-frame alleles. Phenotypic analysis of the mutants validated the candidate gene, and included intermediate phenotypes that co-segregated with the in-frame alleles. These findings demonstrate that the CPR5 ortholog is essential for proper growth and development of soybean trichomes, similar to observations in A. thaliana. Furthermore, this work demonstrates the value of using CRISPR/Cas9 to generate an allelic series and intermediate phenotypes for functional analysis of candidate genes and/or the development of novel traits.

Constitutive expression of NtabSPL6-1 in tobacco and Arabidopsis could change the structure of leaves and promote the development of trichomes

The coding sequence of NtabSPL6-1 was cloned by high-fidelity PCR with specific primers and was used in construction of a binary vector for overexpression. Wild-type Col-0 Arabidopsis plants and Qinyan95 tobacco leaves were transformed using floral dip and leaf disc methods, respectively. Phenotypic observation showed that constitutive expression of NtabSPL6-1 in Arabidopsis could promote the development of trichomes on leaf epidermis and influence the growth pattern of cauline leaves. In tobacco, ectopic expression of NtabSPL6-1 led to dwarfism of the plants and alteration of the leaf structure, accompanied by changes of the glandular trichomes in development. At the same time, the self-regulation capability of NtabSPL6-1 was determined by yeast two-hybrid system. The results indicated that SBP-C terminal domain and C terminal domain of NtabSPL6-1 possessed strong transcriptional activation ability; the intact protein, N terminal domain, and the first peptide fragment in N terminal domain possessed weak transcriptional activation ability; and the second and the third peptide fragments in N terminal domain had no transcriptional activation ability, suggesting the N terminal domain of NtabSPL6-1 could block the activity of the C terminal domain. NtabSPL6-1 may affect the resistance of plants to biotic stress factors indirectly by regulation of the trichome growth.

The Trihelix transcription factor GT2-like 1 (GTL1) promotes salicylic acid metabolism, and regulates bacterial-triggered immunity.

The Trihelix Transcription factor GT2-like 1 (GTL1) was previously shown to be a key regulator of ploidy-dependent trichome growth and drought tolerance. Here, we report that GTL1 plays an important role in coordinating plant immunity. We show that gtl1 mutants are compromised in the regulation of basal immunity, microbial pattern-triggered immunity (PTI) and effector-triggered RIN4-mediated immunity. Transcriptome analysis revealed that GTL1 positively regulates defense genes and inhibits factors that mediate growth and development. By performing hormonal measurements and chromatin-immunoprecipitation studies, we found GTL1 to coordinate genes involved in salicylic acid metabolism, transport and response. Interaction studies and comparative transcriptomics to known data sets revealed that GTL1 is part of the MPK4 pathway and regulates oppositely the expression of differentially expressed genes in mpk4 plants. We introduced the gtl1 mutation in the mpk4 mutant and thereby partially suppressed its dwarfism and the high resistance against a bacterial invader. Our data show that GTL1 is part of the MPK4 pathway and acts as a positive regulator of bacterial-triggered immunity and SA homeostasis.

Functional Analysis and RNA Sequencing Indicate the Regulatory Role of Argonaute1 in Tomato Compound Leaf Development.

Regardless of whether a leaf is simple or compound, the mechanism underlying its development will give rise to a full comprehension of plant morphogenesis. The role of Argonaute1 (AGO1) in the development of simple leaves has been established, but its role in the development of compound leaves remains to be characterized. In this paper, a virus-induced gene silencing (VIGS) strategy was used to dramatically down-regulate the expression of AGO1 ortholog in tomatoes, a model plant for research into compound leaves. AGO1-silenced tomato compound leaves exhibited morphological defects of leaf adaxial-abaxial and trichome development. Analysis of global gene expression profiles indicated that the silencing of AGO1 in tomato compound leaf caused significant changes in the expression of several critical genes, including Auxin Response Factor 4 (ARF4) and Non-expressor of PR5 (NPR5), which were involved in adaxial-abaxial formation and IAA15 that was found to contribute to growth of trichomes as well as Gibberellic Acid Insensitive (GAI) which participated in hormone regulation. Collectively, these results shed light on the complicated mechanism by which AGO1 regulates compound leaf development.

Ecology and phylogenetic affinity of the early Cambrian tubular microfossilMegathrix longus

AbstractAbundant, exquisitely preserved specimens of the enigmatic tubular microfossilMegathrix longusare reported from the early Cambrian Yanjiahe Formation in the Yangtze Gorges area. Studies of their morphology and taphonomy reveal that the inner cross‐wall ofMegathrix longusis topographically flat, the trichome terminus is blunt and closed, and incomplete cross‐walls are regularly intercalated between complete ones. The deformation of trichomes, as well as the corrugation of their cross‐walls, are postmortem features caused by compaction, rather than biological features as previously interpreted. Statistical analysis indicates that chamber division is accomplished by the closure of incomplete cross‐walls. This process, which contributed to trichome growth, is extremely similar to that of modern Oscillatoriaceae. Trichome fragmentation was also observed in a series of specimens, which closely resembles that of cyanobacteria, particularly the Oscillatoriaceae. The termini ofMegathrix longussuggest that it was planktonic. In general,Megathrix longusbears a close affinity to the extant Oscillatoriaceae (Cyanophyta).

Molecular characterization of a transcriptionally active Ty1/copia-like retrotransposon in Gossypium.

A transcriptionally active Ty1/copia -like retrotransposon was identified in the genome of Gossypium barbadense. The different heat activation of this element was observed in two tetraploid cotton species. Most retrotransposons from plants are transcriptionally silent, or activated under certain conditions. Only a small portion of elements are transcriptionally active under regular condition. A long terminal repeat (LTR) retrotransposon was isolated from the cultivated Sea Island cotton (H7124) genome during the investigation of the function of a homeodomain leucine zipper gene (HD1) in trichome growth. Insertion of this element in HD1 gene of At sub-genome was related to the trichomeless stem in Gossypium barbadense. The element, named as GBRE-1, had all features of a typical Ty1/copia retrotransposon and possessed high similarity to the members of ONSEN retrotransposon family. It was 4997 bp long, comprising a single 4110 bp open reading frame, which encoded 1369 amino acids including the conserved domains of gag and pol. The expression of GBRE-1 was detected under regular condition in G. barbadense and G. hirsutum, and its expression level was increased under heat-stress condition in G. hirsutum. Besides, its expression pattern was similar to that of the ONSEN retrotransposon. Abundant cis-regulatory motifs related to stress-response and transcriptional regulation were found in the LTR sequence. These results suggested that GBRE-1 was a transcriptionally active retrotransposon in Gossypium. To our knowledge, this is the first report of the isolation of a complete Ty1/copia-type retrotransposon with present-day transcriptional activity in cotton.

GTL1 keeps cell growth and nuclear ploidy under control

EMBO J (2012) 31 24, 4488–4501 doi:10.1038/emboj.2012.294; published online November092012 The endoreplication programme consists of multiple rounds of full genome replication in the absence of any intervening mitosis. This leads to an increase in nuclear ploidy that has been linked to the initiation of cell differentiation and growth. Recent work (Breuer et al, 2012) has identified the GTL1 transcription factor as a master regulator of cell growth through two different mechanisms: one, endoreplication dependent where GTL1 acts as a repressor of the anaphase-promoting complex (APC), and another as a regulator of endoreplication-independent cell growth.

Effects of UV-B Radiation and Periodic Desiccation on the Morphogenesis of the Edible Terrestrial Cyanobacterium Nostoc flagelliforme

The terrestrial cyanobacterium Nostoc flagelliforme Berk. et M. A. Curtis has been a popular food and herbal ingredient for hundreds of years. To meet great market demand and protect the local ecosystem, for decades researchers have tried to cultivate N. flagelliforme but have failed to get macroscopic filamentous thalli. In this study, single trichomes with 50 to 200 vegetative cells were induced from free-living cells by low light and used to investigate the morphogenesis of N. flagelliforme under low UV-B radiation and periodic desiccation. Low-fluence-rate UV-B (0.1 W m(-2)) did not inhibit trichome growth; however, it significantly increased the synthesis of extracellular polysaccharides and mycosporine-like amino acids and promoted sheath formation outside the trichomes. Under low UV-B radiation, single trichomes developed into filamentous thalli more than 1 cm long after 28 days of cultivation, most of which grew separately in liquid BG11 medium. With periodic desiccation treatment, the single trichomes formed flat or banded thalli that grew up to 2 cm long after 3 months on solid BG11 medium. When trichomes were cultivated on solid BG11 medium with alternate treatments of low UV-B and periodic desiccation, dark and scraggly filamentous thalli that grew up to about 3 cm in length after 40 days were obtained. In addition, the cultivation of trichomes on nitrogen-deficient solid BG11 medium (BG11(0)) suggested that nitrogen availability could affect the color and lubricity of newly developed thalli. This study provides promising techniques for artificial cultivation of N. flagelliforme in the future.

A WD40-repeat gene from Malus × domestica is a functional homologue of Arabidopsis thaliana TRANSPARENT TESTA GLABRA1

The WD40 repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) is involved in a multitude of developmental and biochemical reactions in Arabidopsis thaliana such as the production of seed coat colour and mucilage, pigmentation by anthocyanins as well as the formation of trichomes and root hairs. In this study, a putative TTG1 homologue was isolated from apple (Malus x domestica Borkh.) showing 80.2% identity to A. thaliana TTG1 on nucleotide and 90.7% similarity on amino acid level. The MdTTG1 candidate was able to activate the AtBAN promoter in cooperation with the A. thaliana transcription factors TT2 and TT8 in A. thaliana protoplasts. This indicates that the encoded protein can be integrated into the complex that activates BAN in A. thaliana, and that a similar complex might also be present in apple. When transformed into ttg1 mutants of A. thaliana, the apple sequence was able to restore trichome growth, anthocyanin production in young seedlings as well as proanthocyanidin production in seeds. Additionally, roots of complemented mutant plants showed root hair formation resembling wild type. These results show that the studied apple WD40 gene is a functional homologue of AtTTG1 and we refer to this gene as MdTTG1.

Quantitative analysis of heterogeneous spatial distribution of Arabidopsis leaf trichomes using micro X‐ray computed tomography

Quantitative morphological traits may be defined based on the 3D anatomy reconstructed from micro X-ray computed tomography (microCT) images. In this study, the heterogeneous spatial distribution of trichomes (hairs) on the adaxial leaf blade surface in Arabidopsis was evaluated in terms of 3D quantitative traits, including trichome number, average nearest-neighbour distance between trichomes, and proportion of large trichomes. The data reflect spatial heterogeneity in the radial direction, in that a greater number of trichomes were observed on the leaf blade margins relative to the non-margins, a distribution effect caused by the CAPRICE (CPC) and GLABRA3 (GL3) genes, which have previously been shown to affect trichome density. We further determined that the proportion of large trichomes on the blade mid-rib increases from the proximal end to the distal leaf tip in both wild-type plants and GL3 mutants. Our results indicate that the CPC [corrected] gene affects trichome distribution, rather than trichome growth, causing trichome initiation at the proximal base rather than the distal tip. On the other hand, CPC does affect trichome growth and developmental progression. Hence, quantitative phenotyping based on microCT enables precise phenotypic description for elucidation of gene control in morphological mutants.

A Genetic Regulatory Network in the Development of Trichomes and Root Hairs

Trichomes and root hairs differentiate from epidermal cells in the aerial tissues and roots, respectively. Because trichomes and root hairs are easily accessible, particularly in the model plant Arabidopsis, their development has become a well-studied model of cell differentiation and growth. Molecular genetic analyses using Arabidopsis mutants have demonstrated that the differentiation of trichomes and root hair/hairless cells is regulated by similar molecular mechanisms. Transcriptional complexes regulate differentiation into trichome cells and root hairless cells, and formation of the transcriptional complexes is inhibited in neighboring cells. Control of cell growth after fate determination has also been analyzed using Arabidopsis mutants. The progression of endoreduplication cycles, reorientation of microtubules, and organization of the actin cytoskeleton play important roles in trichome growth. Various cellular components such as ion channels, the actin cytoskeleton, microtubules and cell wall materials, and intracellular signal transduction act to establish and maintain root hair tip growth.

Early seedling growth and morphogenesis in the xantha1 mutant of sunflower with alteration of chloroplast biogenesis

In the xantha1 (xan1) mutant of sunflower (Helianthus annuus L.), the effects on organ anatomy and seedling growth did correlate to the alteration of chloroplast biogenesis. The xan1 seedlings grown under 165 µmol(photon) m−2 s−1 revealed a severely altered chloroplast ultrastructure in cotyledons and leaves. Cross-sections or clarified tissues of the xan1 cotyledons did not show evident alterations with respect to normal cotyledons suggesting that the impairment of chloroplast biogenesis has negligible consequences on embryonic leaves. By contrast, the analysis of xan1 leaves showed that the defects in chloroplast biogenesis were correlated to a drastic reduction of organ size and to a clear enhancement of the trichome growth. The differentiation of palisade and spongy parenchyma in cotyledons and leaves of the xan1 mutant was normal but both organs displayed a drastic reduction in the plastid number with respect to wild type. In addition, xan1 hypocotyls showed a reduced development of the main vascular bundles in comparison with normal seedlings and an undersized central cylinder of the primary root. The exogenous supply of sucrose was not sufficient to revert in vitro the deficit of xan1 growth and the constraints in morphogenetic processes.

The YORE-YORE gene regulates multiple aspects of epidermal cell differentiation in Arabidopsis.

We have identified a new Arabidopsis mutant, yore-yore (yre), which has small trichomes and glossy stems. Adhesion between epidermal cells was observed in the organs of the yre shoot. The cloned YRE had high homology to plant genes involved in epicuticular wax synthesis, such as ECERIFERUM1 (CER1) and maize GLOSSY1. The phenotype of transgenic plants harboring double-stranded RNA interference (dsRNAi) YRE was quite similar to that of the yre mutant. The amount of epicuticular wax extracted from leaves and stems of yre-1 was approximately one-sixth of that from the wild type. YRE promoter::GUS and in situ hybridization revealed that YRE was specifically expressed in cells of the L1 layer of the shoot apical meristem and young leaves, stems, siliques, and lateral root primordia. Strong expression was detected in developing trichomes. The trichome structure of cer1 was normal, whereas that of the yre cer1 double mutant was heavily deformed, indicating that epicuticular wax is required for normal growth of trichomes. Double mutants of yre and trichome-morphology mutants, glabra2 (gl2) and transparent testa glabra1 (ttg1), showed that the phenotype of the trichome structure was additive, suggesting that the wax-requiring pathway is distinct from the trichome development pathway controlled by GL2 and TTG1.

Requirements for Arabidopsis ATARP2 and ATARP3 during Epidermal Development

Plant cells employ the actin cytoskeleton to stably position organelles, as tracks for long distance transport, and to reorganize the cytoplasm in response to developmental and environmental cues [1]. While diverse classes of actin binding proteins have been implicated in growth control, the mechanisms of cytoskeletal reorganization and the cellular functions of specific actin filament arrays are unclear. Arabidopsis trichome morphogenesis includes distinct requirements for the microtubule and actin filament cytoskeletons. It also is a genetically tractable process that is providing new knowledge about cytoskeleton function in plants [2]. The “distorted group” of mutants defines a class of at least eight genes that are required during the actin-dependent phase of trichome growth. Using map-based cloning and a candidate gene approach, we identified mutations in ARP3 (ATARP3) and ARP2 (ATARP2) genes as the cause of the distorted1 (dis1) and wurm (wrm) phenotypes, respectively. ARP2 and ARP3 are components of the evolutionarily conserved ARP2/3 complex that nucleates actin filament polymerization [3]. Mutations in DIS1 and WRM caused severe trichome growth defects but had relatively mild effects on shoot development. DIS1 rescued the phenotype of Δarp3 when overexpressed in S. cerevisiae. Developing dis1 trichomes had defects in cytoplasmic actin bundle organization and reduced relative amounts of cytoplasmic actin filaments in developing branches.