Cllmodulin in tip-growing plant cells,visualized by fluorescing calmodulin-binding phenothiazines

Calmodulin (CaM) was visualized light-microscopically by the fluorescent CaM inhibitors fluphenazine and chlorpromazine, both phenothiazines, during polar tip growth of pollen tubes of Lilium longiflorum, root hairs of Lepidium sativum, moss caulonema of Funaria hygrometrica, fungal hyphae of Achlya spec. and in the alga Acetabularia mediterranea, as well as during multipolar tip growth in Micrasterias denticulata. Young pollen tubes and root hairs showed tip fluorescence; at later stages and in the growing parts of the other subjects the fluorescence was almost uniform. After treatment with cytochalasin B, punctuate fluorescence occurred in the clear zone adjacent to the tip of pollen tubes. The observations indicate that there is CaM in all our tested systems detectable with this method. It may play a key role in starting polar growth. As in pollen tubes, CaM might be in part associated with the microfilament network at the tip, and thus regulate vesicle transport and cytoplasmic streaming.Abbreviations CaM calmodulin - CB cytochalasin B - CTC chlorotetracycline     

Tip growth: signaling in the apical dome

Signaling molecules, such as ROP/RAC GTPases and their regulators, reactive oxygen species (ROS) and phospholipids, play pivotal roles in the control of tip growth in pollen tubes and root hairs. They are often localized to the apical growing region of these cells, where their functions are tightly interconnected with cytoskeletal rearrangement and polar vesicle trafficking, which participate in tip growth as well as affect the generation and maintenance of the apical growing region. Recent advances in our understanding of the interface between these cellular activities and signaling in tip growth will be discussed.     

Pollen tube NAD(P)H oxidases act as a speed control to dampen growth rate oscillations during polarized cell growth

Reactive oxygen species (ROS) produced by NAD(P)H oxidases play a central role in plant stress responses and development. To better understand the function of NAD(P)H oxidases in plant development, we characterized the Arabidopsis thaliana NAD(P)H oxidases RBOHH and RBOHJ. Both proteins were specifically expressed in pollen and dynamically targeted to distinct and overlapping plasma membrane domains at the pollen tube tip. Functional loss of RBOHH and RBOHJ in homozygous double mutants resulted in reduced fertility. Analyses of pollen tube growth revealed remarkable differences in growth dynamics between Col–0 and rbohh–1 rbohj–2 pollen tubes. Growth rate oscillations of rbohh–1 rbohj–2 pollen tubes showed strong fluctuations in amplitude and frequency, ultimately leading to pollen tube collapse. Prior to disintegration, rbohh–1 rbohj–2 pollen tubes exhibit high‐frequency growth oscillations, with significantly elevated growth rates, suggesting that an increase in the rate of cell‐wall exocytosis precedes pollen tube collapse. Time‐lapse imaging of exocytic dynamics revealed that NAD(P)H oxidases slow down pollen tube growth to coordinate the rate of cell expansion with the rate of exocytosis, thereby dampening the amplitude of intrinsic growth oscillations. Using the Ca²⁺ reporter Yellow Cameleon 3.6, we demonstrate that high‐amplitude growth rate oscillations in rbohh–1 rbohj–2 pollen tubes are correlated with growth‐dependent Ca²⁺ bursts. Electrophysiological experiments involving double mutant pollen tubes and pharmacological treatments also showed that ROS influence K⁺ homeostasis. Our results indicate that, by limiting pollen tube growth, ROS produced by NAD(P)H oxidases modulate the amplitude and frequency of pollen tube growth rate oscillations.     

Calcium gradients in tip growing plant cells visualized by chlorotetracycline fluorescence

With chlorotetracycline (CTC)-fluorescence a tip-to-base Ca²⁺ gradient is visualized in all tested, tip-growing plant cells: pollen tubes of Lilium longiflorum, root hairs of Lepidium sativum, moss caulonema of Funaria hygrometrica, fungal hyphae of Achlya and in the alga Acetabularia mediterranea. The fluorescence gradients in the different species vary in intensity and extension. Sometimes a punctate mobile CTC-fluorescence, in the size range of mitochondria, is observed. Bursting cells lose their fluorescence rapidly, indicating a cytoplasmic localization of the gradient. Only in Acetabularia is the wall also fluorescent with CTC. The results are interpreted as evidence for a general role of a calcium gradient in tip growth.Abbreviation CTC chlorotetracycline     

Conserved function of Rho-related Rop/RAC GTPase signaling in regulation of cell polarity in Physcomitrella patens

Cell polarity is fundamentally important to growth and development in higher plants, from pollen tubes to root hairs. Basal land plants (mosses and ferns) also have cell polarity, developing protonemal apical cells that show polar tip growth. Flowering plants have a distinct group of Rho GTPases that regulate polarity in polarized cell growth. Rop/RAC signaling module components have been identified in non-flowering plants, but their roles remain unclear. To understand the importance and evolution of Rop/RAC signaling in polarity regulation in land plants, we examined the functions of PpRop and PpRopGEF in protonemal apical cells of the moss Physcomitrella patens. Inducible overexpression of PpRop2 or PpRopGEF3 caused depolarized growth of tip-growing apical cells. PpRop2 overexpression also caused aberrant cross wall formation. Fluorescent protein-tagged PpRop2 localized to the plasma membrane, including the cross wall membrane, and fluorescent-tagged PpRopGEF3 showed polarized localization to the tip region in apical cells. Thus, our results suggest common functions of PpRop and PpRopGEF in the tip-growing apical cells and the importance of a conserved Rop/RAC signaling module in the control of cell polarity in land plants.     

Male gametophyte defective 4 encodes a rhamnogalacturonan II xylosyltransferase and is important for growth of pollen tubes and roots in Arabidopsis

In flowering plants, the growth of pollen tubes is essential for the delivery of sperm to the egg cells. Although many factors (including cell‐wall properties) are involved in this process, little is known about the underlying molecular mechanisms that regulate the growth of pollen tubes. We report here the characterization of an Arabidopsis mutant male gametophyte defective 4 (mgp4) that is severely defective in pollen tube growth. The mgp4 mutation also impairs root growth of pollen‐rescued mgp4 mutant plants generated by expressing MGP4 cDNA under the control of a pollen grain/tube‐specific promoter. The MGP4 gene encodes a putative xylosyltransferase and is expressed in many organs/tissues, including pollen tubes and roots. MGP4 protein expressed in Pichia pastoris exhibited xylosyltransferase activity and transferred d ‐xylose onto l ‐fucose. The pectic polysaccharide rhamnogalacturonan II (RG‐II), isolated from 7‐day‐old pollen‐rescued mutant seedlings, exhibited a 30% reduction in 2‐O‐methyl d ‐xylose residues. Furthermore, an exogenous supply of boric acid enhanced RG‐II dimer formation and partially restored the root growth of the pollen‐rescued mutant seedlings. Taken together, these results suggest that MGP4 plays important roles in pollen tube and root growth by acting as a xylosyltransferase involved in the biosynthesis of pectic RG‐II.     

Roothairless5, which functions in maize (Zea mays L.) root hair initiation and elongation encodes a monocot‐specific NADPH oxidase

Root hairs are instrumental for nutrient uptake in monocot cereals. The maize (Zea mays L.) roothairless5 (rth5) mutant displays defects in root hair initiation and elongation manifested by a reduced density and length of root hairs. Map‐based cloning revealed that the rth5 gene encodes a monocot‐specific NADPH oxidase. RNA‐Seq, in situ hybridization and qRT‐PCR experiments demonstrated that the rth5 gene displays preferential expression in root hairs but also accumulates to low levels in other tissues. Immunolocalization detected RTH5 proteins in the epidermis of the elongation and differentiation zone of primary roots. Because superoxide and hydrogen peroxide levels are reduced in the tips of growing rth5 mutant root hairs as compared with wild‐type, and Reactive oxygen species (ROS) is known to be involved in tip growth, we hypothesize that the RTH5 protein is responsible for establishing the high levels of ROS in the tips of growing root hairs required for elongation. Consistent with this hypothesis, a comparative RNA‐Seq analysis of 6‐day‐old rth5 versus wild‐type primary roots revealed significant over‐representation of only two gene ontology (GO) classes related to the biological functions (i.e. oxidation/reduction and carbohydrate metabolism) among 893 differentially expressed genes (FDR <5%). Within these two classes the subgroups ‘response to oxidative stress’ and ‘cellulose biosynthesis’ were most prominently represented.     

Root hair growth in Arabidopsis thaliana is directed by calcium and an endogenous polarity

Tip growth of plant cells has been suggested to be regulated by a tip-focused gradient in cytosolic calcium concentration ([Ca²⁺]_c). However, whether this gradient orients apical growth or follows the driving force for this process remains unknown. Using localized photoactivation of the caged calcium ionophore Br-A23187 we have been able to artificially generate an asymmetrical calcium influx across the root hair tip. This led to a change in the direction of tip growth towards the high point of the new [Ca²⁺]_c gradient. Such reorientation of growth was transient and there was a return to the original direction within 15 min. Root hairs forced to change the direction of their growth by placing a mechanical obstacle in their path stopped, reoriented growth to the side, and grew past the mechanical blockage. However, as soon as the growing tip had cleared the obstacle, growth returned to the original direction. Confocal ratio imaging revealed that a tip-focused [Ca²⁺]_c gradient was always centered at the site of active growth. When the root hair changed direction the gradient also reoriented, and when growth returned to the original direction, so did the [Ca²⁺]_c gradient. This normal direction of apical growth of Arabidopsis thaliana (L.) Heynh. root hairs was found to be at a fixed angle from the root of 85 ± 6.7 degrees. In contrast, Tradescantia virginiana (L.) pollen tubes that were induced to reorient by touch or localized activation of the caged ionophore, did not return to the original growth direction, but continued to elongate in their new orientation. These results suggest that the tip-focused [Ca²⁺]_c gradient is an important factor in localizing growth of the elongating root hair and pollen tube to the apex. However, it is not the primary determinant of the direction of elongation in root hairs, suggesting that other information from the root is acting to continuously reset the growth direction away from the root surface. Received: 22 April 1997 / Accepted: 14 May 1997     

Multiple cyclic nucleotide‐gated channels coordinate calcium oscillations and polar growth of root hairs

Calcium gradients underlie polarization in eukaryotic cells. In plants, a tip‐focused Ca²⁺‐gradient is fundamental for rapid and unidirectional cell expansion during epidermal root hair development. Here we report that three members of the cyclic nucleotide‐gated channel family are required to maintain cytosolic Ca²⁺ oscillations and the normal growth of root hairs. CNGC6, CNGC9 and CNGC14 were expressed in root hairs, with CNGC9 displaying the highest root hair specificity. In individual channel mutants, morphological defects including root hair swelling and branching, as well as bursting, were observed. The developmental phenotypes were amplified in the three cngc double mutant combinations. Finally, cngc6/9/14 triple mutants only developed bulging trichoblasts and could not form normal root hair protrusions because they burst after the transition to the rapid growth phase. Prior to developmental defects, single and double mutants showed increasingly disturbed patterns of Ca²⁺ oscillations. We conclude that CNGC6, CNGC9 and CNGC14 fulfill partially but not fully redundant functions in generating and maintaining tip‐focused Ca²⁺ oscillations, which are fundamental for proper root hair growth and polarity. Furthermore, the results suggest that these calmodulin‐binding and Ca²⁺‐permeable channels organize a robust tip‐focused oscillatory calcium gradient, which is not essential for root hair initiation but is required to control the integrity of the root hair after the transition to the rapid growth phase. Our findings also show that root hairs possess a large ability to compensate calcium‐signaling defects, and add new players to the regulatory network, which coordinates cell wall properties and cell expansion during polar root hair growth.     

Vesicular trafficking, cytoskeleton and signalling in root hairs and pollen tubes

Root hairs and pollen tubes show strictly polar cell expansion called tip growth. Recent studies of tip growth in root hairs and pollen tubes have revealed that small GTPases of the Rab, Arf and Rho/Rac families, along with their regulatory proteins, are essential for spatio-temporal regulation of vesicular trafficking, cytoskeleton organization and signalling. ROP/RAC GTPases are involved in a multiplicity of functions including the regulation of cytoskeleton organization, calcium signalling and endocytosis in pollen tubes and root hairs. One of the most exciting recent discoveries is the preferential localization of vesicles of the trans-Golgi network (TGN), defined by specific RAB GTPases, in the apical "clear zone" and the definition of TGN as a bona fide organelle involved in both polarized secretion and endocytosis. The TGN is thought to serve the function of an early endosome in plants because it is involved in early endocytosis and rapid vesicular recycling of the plasma membrane in root epidermal cells.     

KINKY POLLEN encodes a SABRE-like protein required for tip growth in Arabidopsis and conserved among eukaryotes

In higher plants, pollen tubes and root hairs share an ancient growth process named tip growth. We have isolated three allelic Arabidopsis mutant lines showing kinky-shaped pollen tubes and, when homozygous, showing shorter and thicker root hairs. The ultrastructure of pollen tubes in these kinky pollen (kip) mutants is similar to that of the wild type; however, time-lapse studies suggest that aberrant pollen tube shape is caused by periodic growth arrests alternated with phases of tube axis reorientation. The KIP gene encodes a protein of 2587 amino acids that is predicted to be targeted to the secretory pathway. KIP mRNA was detected in all organs investigated but was most abundant in pollen and roots. KIP has putative homologues in many eukaryotes, including mammals and yeast, and is similar to the Arabidopsis SABRE gene, whose mutation causes a dwarf phenotype. The phenotype of the kip/sab double mutant suggests related functions for both genes, however, the KIP protein is mostly required for tip-growth.     

The plant beneficial rhizobacterium Achromobacter sp. 5B1 influences root development through auxin signaling and redistribution

Roots provide physical and nutritional support to plant organs that are above ground and play critical roles for adaptation via intricate movements and growth patterns. Through screening the effects of bacterial isolates from roots of halophyte Mesquite (Prosopis sp.) on Arabidopsis thaliana, we identified Achromobacter sp. 5B1 as a probiotic bacterium that influences plant functional traits. Detailed genetic and architectural analyses in Arabidopsis grown in vitro and in soil, cell division measurements, auxin transport and response gene expression and brefeldin A treatments demonstrated that root colonization with Achromobacter sp. 5B1 changes the growth and branching patterns of roots, which were related to auxin perception and redistribution. Expression analysis of auxin transport and signaling revealed a redistribution of auxin within the primary root tip of wild‐type seedlings by Achromobacter sp. 5B1 that is disrupted by brefeldin A and correlates with repression of auxin transporters PIN1 and PIN7 in root provasculature, and PIN2 in the epidermis and cortex of the root tip, whereas expression of PIN3 was enhanced in the columella. In seedlings harboring AUX1, EIR1, AXR1, ARF7ARF19, TIR1AFB2AFB3 single, double or triple loss‐of‐function mutations, or in a dominant (gain‐of‐function) mutant of SLR1, the bacterium caused primary roots to form supercoils that are devoid of lateral roots. The changes in growth and root architecture elicited by the bacterium helped Arabidopsis seedlings to resist salt stress better. Thus, Achromobacter sp. 5B1 fine tunes both root movements and the auxin response, which may be important for plant growth and environmental adaptation.     

Polarized cell growth in Arabidopsis requires endosomal recycling mediated by GBF1-related ARF exchange factors

Polarized tip growth is a fundamental cellular process in many eukaryotic organisms, mediating growth of neuronal axons and dendrites or fungal hyphae. In plants, pollen and root hairs are cellular model systems for analysing tip growth. Cell growth depends on membrane traffic. The regulation of this membrane traffic is largely unknown for tip-growing cells, in contrast to cells exhibiting intercalary growth. Here we show that in Arabidopsis, GBF1-related exchange factors for the ARF GTPases (ARF GEFs) GNOM and GNL2 play essential roles in polar tip growth of root hairs and pollen, respectively. When expressed from the same promoter, GNL2 (in contrast to the early-secretory ARF GEF GNL1) is able to replace GNOM in polar recycling of the auxin efflux regulator PIN1 from endosomes to the basal plasma membrane in non-tip growing cells. Thus, polar recycling facilitates polar tip growth, and GNL2 seems to have evolved to meet the specific requirement of fast-growing pollen in higher plants.     

The role of +TIPs in directional tip expansion

Aspergillus nidulans is an ideal model to study nuclear migration and intracellular transport by dynein and kinesin owing to its long neuron‐like hyphae, conserved transport mechanisms, and powerful genetics. In this organism, as in other filamentous fungi, microtubules have been implicated in patterning cell shape through polarized tip growth – the hallmark mode of growth that generates the elongated hyphae. Exactly how microtubules regulate tip growth is incompletely understood and remains a fascinating question for various cell types, such as pollen tubes and root hairs. Zeng et al. (2014) describe important new findings in A. nidulans regarding the role of EBA, the master regulator of microtubule plus end‐tracking proteins, in specifying microtubule dynamics required for directional tip growth at the hyphal tip.     

Signaling tip growth in plants

Tip growth is an extreme form of polar growth modulated by both intrinsic and extrinsic spatial cues. Pollen tubes and root hairs have been used as model systems to investigate tip growth signaling in higher plants. Recent studies have focused on tip-localized Ca2+ gradients and Rho GTPases in pollen tubes and a series of mutants affecting root hair tip growth. These molecular and genetic markers will serve as stepping stones towards uncovering tip growth pathways in plants.     

Boron deficiency alters cytosolic Ca2+ concentration and affects the cell wall components of pollen tubes in Malus domestica

• Boron (B) is essential for normal plant growth, including pollen tube growth. B deficiency influences various physiological and metabolic processes in plants. However, the underlying mechanism of B deficiency in pollen tube growth is not sufficiently understood. In the present research, the influence of B deficiency on apple (Malus domestica) pollen tube growth was studied and the possible regulatory mechanism evaluated.
• Apple pollen grains were cultured under different concentrations of B. Scanning ion‐selective electrode technique, fluorescence labelling and Fourier‐transform infrared (FTIR) analysis were used to detect calcium ion flux, cytosolic Ca²⁺ concentration ([Ca²⁺]cyt), actin filaments and cell wall components of pollen tubes.
• B deficiency inhibited apple pollen germination and induced retardation of tube growth. B deficiency increased extracellular Ca²⁺ influx and thus led to increased [Ca²⁺]cyt in the pollen tube tip. In addition, B deficiency modified actin filament arrangement at the pollen tube apex. B deficiency also altered the deposition of pollen tube wall components. Clear differences were not observed in the distribution patterns of cellulose and callose between control and B deficiency treated pollen tubes. However, B deficiency affected distribution patterns of pectin and arabinogalactan proteins (AGP). Clear ring‐like signals of pectins and AGP on control pollen tubes varied according to B deficiency. B deficiency further decreased acid pectins, esterified pectins and AGP content at the tip of the pollen tube, which were supported by changes in chemical composition of the tube walls.
• B appears to have an active role in pollen tube growth by affecting [Ca²⁺]cyt, actin filament assembly and pectin and AGP deposition in the pollen tube. These findings provide valuable information that enhances our current understanding of the mechanism regulating pollen tube growth.

     

Arabidopsis PROTEIN S‐ACYL TRANSFERASE4 mediates root hair growth

Polar growth of root hairs is critical for plant survival and requires fine‐tuned Rho of plants (ROP) signaling. Multiple ROP regulators participate in root hair growth. However, protein S‐acyl transferases (PATs), mediating the S‐acylation and membrane partitioning of ROPs, are yet to be found. Using a reverse genetic approach, combining fluorescence probes, pharmacological drugs, site‐directed mutagenesis and genetic analysis with related root‐hair mutants, we have identified and characterized an Arabidopsis PAT, which may be responsible for ROP2 S‐acylation in root hairs. Specifically, functional loss of PAT4 resulted in reduced root hair elongation, which was rescued by a wild‐type but not an enzyme‐inactive PAT4. Membrane‐associated ROP2 was significantly reduced in pat4, similar to S‐acylation‐deficient ROP2 in the wild type. We further showed that PAT4 and SCN1, a ROP regulator, additively mediate the stability and targeting of ROP2. The results presented here indicate that PAT4‐mediated S‐acylation mediates the membrane association of ROP2 at the root hair apex and provide novel insights into dynamic ROP signaling during plant tip growth.     

NtGNL1基因通过调控囊泡运输影响烟草根毛的极性生长

极性生长是植物生长发育中的常见现象,但囊泡运输与极性生长的关系还未完全明确。花粉管和根毛是植物细胞极性生长的典型模式。早期研究显示NtGNL1(Nicotiana tabacum GNOM-LIKE 1)通过调节囊泡的后高尔基体转运来影响烟草的花粉管生长。本文以NtGNL1 RNAi转基因植株为材料,研究NtGNL1基因在根毛生长中的作用。结果表明,NtGNL1 RNAi转基因植株的根毛生长明显滞后于野生型,且其根毛出现膨大、弯折、扭曲等形态,与NtGNL1 RNAi转基因植株的花粉管异常形态类似。q RT-PCR检测RNAi转基因株系根毛中PIN1、PIN2、GL2、ROP6、RHD6基因的m RNA表达量,显示PIN2和GL2的表达量显著下调,PIN1、ROP6和RHD6的表达量变化不明显。FM4-64染色表明烟草根表皮细胞和根毛的囊泡分布都受到影响,即NtGNL1基因也影响根毛中的囊泡运输。BFA处理加剧了囊泡的聚集程度,提示根毛尖端还存在其它对BFA敏感并调控囊泡运输的基因。以上证据显示,NtGNL1基因通过囊泡运输途径影响烟草根毛的极性生长,NtGNL1基因的表达下调也影响了PIN2和GL2的表达,从而间接影响根毛的极性生长。