The maize (Zea mays L.) roothairless 3 gene encodes a putative GPI-anchored, monocot-specific, COBRA-like protein that significantly affects grain yield

The rth3 ( roothairless 3 ) mutant is specifically affected in root hair elongation. We report here the cloning of the rth3 gene via a PCR-based strategy (amplification of insertion mutagenized sites) and demonstrate that it encodes a COBRA-like protein that displays all the structural features of a glycosylphosphatidylinositol anchor. Genes of the COBRA family are involved in various types of cell expansion and cell wall biosynthesis. The rth3 gene belongs to a monocot-specific clade of the COBRA gene family comprising two maize and two rice genes. While the rice ( Oryza sativa ) gene OsBC1L1 appears to be orthologous to rth3 based on sequence similarity (86% identity at the protein level) and maize/rice synteny, the maize ( Zea mays L.) rth3-like gene does not appear to be a functional homolog of rth3 based on their distinct expression profiles. Massively parallel signature sequencing analysis detected rth3 expression in all analyzed tissues, but at relatively low levels, with the most abundant expression in primary roots where the root hair phenotype is manifested. In situ hybridization experiments confine rth3 expression to root hair-forming epidermal cells and lateral root primordia. Remarkably, in replicated field trials involving near-isogenic lines, the rth3 mutant conferred significant losses in grain yield.     

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.     

Analyses of mutants of three genes that influence root hair development in Zea mays (Gramineae) suggest that root hairs are dispensable

Root hairs are specialized epidermal cells that are thought to play an important role in plant nutrition by facilitating the absorption of water and nutrients. Three maize mutants with abnormal root hair morphologies (rthl, rth2, and rth3) have been isolated from Mutator transposon stocks. All three root hair mutant phenotypes are controlled by single recessive alleles. The rthl mutant initiates normal-looking root hair primordia that fail to elongate. The normal-looking root hair primordia of the rth2 mutant elongate to only approximately one-fifth to one-fourth the length of wild type root hairs. Like rth1 primordia, rth3 primordia undergo little elongation. However, unlike the relatively normal-looking rth1 primordia, rth3 primordia are distinctly abnormal when viewed through a scanning electron microscope. The rth1 mutant exhibits pleiotropic nutrient deficiencies, while the rth2 and rth3 mutants grow vigorously. This finding suggests that under some environmental conditions, root hairs are less important to plant growth than has been previously thought. The rthl, rth2, and rth3 genes have been mapped to chromosomes 1L, 5L, and 1S, respectively, via crosses with BA translocation stocks. The rth2 allele exhibits reduced transmission through the male gametophyte, but a normal rate of transmission through female gametophytes; rth1 and rth3 are transmitted at normal rates.     

Microtubules guide root hair tip growth

The ability to establish cell polarity is crucial to form and function of an individual cell. Polarity underlies critical processes during cell development, such as cell growth, cell division, cell differentiation and cell signalling. Interphase cytoplasmic microtubules in tip-growing fission yeast cells have been shown to play a particularly important role in regulating cell polarity. By placing proteins that serve as spatial cues in the cell cortex of the expanding tip, microtubules determine the site where exocytosis, and therefore growth, takes place. Transport and the targeting of exocytotic vesicles to the very tip depend on the actin cytoskeleton. Recently, endoplasmic microtubules have been identified in tip-growing root hairs, which are an experimental system for plant cell growth. Here, we review the data that demonstrate involvement of microtubules in hair elongation and polarity of the model plants Medicago truncatula and Arabidopsis thaliana. Differences and similarities between the microtubule organization and function in these two species are discussed and we compare the observations in root hairs with the microtubule-based polarity mechanism in fission yeast.     

AtCYT-INV1, a neutral invertase,is involved in osmotic stress-induced inhibition on lateral root growth in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Neutral/Alkaline invertases are unique to plant and photosynthetic bacteria. The function of Neutral/Alkaline invertases in plant development is not clear so far. In this study, we isolated an Arabidopsis (Col-0) mutant insensitive to osmotic stress-induced inhibition on lateral root growth. Map-based cloning reveals that a neutral invertase gene (AtCYT-INV1) was point-mutated. The mutant Atcyt-inv1 showed short primary root, smaller size of leaves and siliques, and promotion of the reproductive compared to the wild type (WT). Carbohydrate measurement showed that sucrose is accumulated and glucose is reduced in the mutant Atcyt-inv1 under normal and 3% mannitol treatments. Taken together, AtCYT-INV1 plays multiple roles in plant development and is involved in osmotic stress-induced inhibition on lateral root growth by controlling the concentration of hexose in cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Arbuscular mycorrhizal colonization outcompetes root hairs in maize under low phosphorus availability

Background and AimsAn increase in root hair length and density and the development of arbuscular mycorrhiza symbiosis are two alternative strategies of most plants to increase the root–soil surface area under phosphorus (P) deficiency. Across many plant species, root hair length and mycorrhization density are inversely correlated. Root architecture, rooting density and physiology also differ between species. This study aims to understand the relationship among root hairs, arbuscular mycorrhizal fungi (AMF) colonization, plant growth, P acquisition and mycorrhizal-specific P_i transporter gene expression in maize.MethodsUsing nearly isogenic maize lines, the B73 wild type and the rth3 root hairless mutant, we quantified the effect of root hairs and AMF infection in a calcareous soil under P deficiency through a combined analysis of morphological, physiological and molecular factors.Key ResultsWild-type root hairs extended the rhizosphere for acid phosphatase activity by 0.5 mm compared with the rth3 hairless mutant, as measured by in situ zymography. Total root length of the wild type was longer than that of rth3 under P deficiency. Higher AMF colonization and mycorrhiza-induced phosphate transporter gene expression were identified in the mutant under P deficiency, but plant growth and P acquisition were similar between mutant and the wild type. The mycorrhizal dependency of maize was 33 % higher than the root hair dependency.ConclusionsThe results identified larger mycorrhizal dependency than root hair dependency under P deficiency in maize. Root hairs and AMF inoculation are two alternative ways to increase P_i acquisition under P deficiency, but these two strategies compete with each other.     

Glia re-sealed particles freshly prepared from adult rat brain are competent for exocytotic release of glutamate

Glial subcellular re-sealed particles (referred to as gliosomes here) were purified from rat cerebral cortex and investigated for their ability to release glutamate. Confocal microscopy showed that the glia-specific proteins glial fibrillary acidic protein (GFAP) and S-100, but not the neuronal proteins 95-kDa postsynaptic density protein (PSD-95), microtubule-associated protein 2 (MAP-2) and beta-tubulin III, were enriched in purified gliosomes. Furthermore, gliosomes exhibited labelling neither for integrin-alphaM nor for myelin basic protein, which are specific for microglia and oligodendrocytes respectively. The Ca2+ ionophore ionomycin (0.1-5 microm) efficiently stimulated the release of tritium from gliosomes pre-labelled with [3H]d-aspartate and of endogenous glutamate in a Ca(2+)-dependent and bafilomycin A1-sensitive manner, suggesting the involvement of an exocytotic process. Accordingly, ionomycin was found to induce a Ca(2+)-dependent increase in the vesicular fusion rate, when exocytosis was monitored with acridine orange. ATP stimulated [3H]d-aspartate release in a concentration- (0.1-3 mm) and Ca(2+)-dependent manner. The gliosomal fraction contained proteins of the exocytotic machinery [syntaxin-1, vesicular-associated membrane protein type 2 (VAMP-2), 23-kDa synaptosome-associated protein (SNAP-23) and 25-kDa synaptosome-associated protein (SNAP-25)] co-existing with GFAP immunoreactivity. Moreover, GFAP or VAMP-2 co-expressed with the vesicular glutamate transporter type 1. Consistent with ultrastructural analysis, several approximately 30-nm non-clustered vesicles were present in the gliosome cytoplasm. It is concluded that gliosomes purified from adult brain contain glutamate-accumulating vesicles and can release the amino acid by a process resembling neuronal exocytosis.     

Rab6, Rab8, and MICAL3 cooperate in controlling docking and fusion of exocytotic carriers

Rab6 is a conserved small GTPase that localizes to the Golgi apparatus and cytoplasmic vesicles and controls transport and fusion of secretory carriers [1]. Another Rab implicated in trafficking from the trans-Golgi to the plasma membrane is Rab8 [2-5]. Here we show that Rab8A stably associates with exocytotic vesicles in a Rab6-dependent manner. Rab8A function is not needed for budding or motility of exocytotic carriers but is required for their docking and fusion. These processes also depend on the Rab6-interacting cortical factor ELKS [1], suggesting that Rab8A and ELKS act in the same pathway. We show that Rab8A and ELKS can be linked by MICAL3, a member of the MICAL family of flavoprotein monooxygenases [6]. Expression of a MICAL3 mutant with an inactive monooxygenase domain resulted in a strong accumulation of secretory vesicles that were docked at the cell cortex but failed to fuse with the plasma membrane, an effect that correlated with the strongly reduced mobility of MICAL3. We propose that the monooxygenase activity of MICAL3 is required to regulate its own turnover and the concomitant remodeling of vesicle-docking protein complexes in which it is engaged. Taken together, the results of our study illustrate cooperation of two Rab proteins in constitutive exocytosis and implicates a redox enzyme in this process.     

Regulation of exocytosis in adrenal chromaffin cells: focus on ARF and Rho GTPases

Neurons and neuroendocrine cells release transmitters and hormones by exocytosis, a highly regulated process in which secretory vesicles or granules fuse with the plasma membrane to release their contents in response to a calcium trigger. Several stages have been recognized in exocytosis. After recruitment and docking at the plasma membrane, vesicles/granules enter a priming step, which is then followed by the fusion process. Cortical actin remodelling accompanies the exocytotic reaction, but the links between actin dynamics and trafficking events remain poorly understood. Here, we review the action of Rho and ADP-ribosylation factor (ARF) GTPases within the exocytotic pathway in adrenal chromaffin cells. Rho proteins are well known for their pivotal role in regulating the actin cytoskeleton. ARFs were originally identified as regulators of vesicle transport within cells. The possible interplay between these two families of GTPases and their downstream effectors provides novel insights into the mechanisms that govern exocytosis.     

Calmodulin is essential for assembling links necessary for exocytotic membrane fusion in Paramecium.

Calmodulin has long been suspected to be involved in calcium-regulated exocytosis but its precise site(s) of action has not yet been identified. In Paramecium, a genetic approach to the problem is possible as in vivo-selected mutations in the calmodulin gene that prevent the activation of some channels have been characterized. Three of these calmodulin mutants were examined for exocytotic capacity and the mutant cam1 was found to be defective for exocytosis at 35 degrees C. The loss of exocytotic capacity in cam1 cells can be restored by transformation with the wild-type calmodulin gene, demonstrating that its exocytotic lesion is indeed due to the mutation in the calmodulin gene. The cam1 mutant displays abnormal exocytotic sites at the non-permissive temperature: it lacks the links ('rosettes' of intramembranous particles in the plasma membrane and the fibrous 'connecting material') which normally connect plasma and trichocyst membranes. Upon shift of cam1 cells from the permissive to a non-permissive temperature, performed sites remain functional. These results demonstrate that calmodulin is necessary for the assembly of these links at the exocytotic site. These results do not, however, exclude the possibility of calmodulin also being involved in Ca(2+)-dependent steps of the stimulus-exocytosis coupling.     

拟南芥表皮毛发育突变体abt3-1的基因定位及表型分析

在发掘和鉴定调控植物表皮毛发育的新因子过程中,获得了一个表皮毛发育异常的拟南芥隐性突变体abt3-1(aberrantly branched trichome 3-1)。与野生型拟南芥(Col-0)相比,其表皮毛分支数目明显增加。另外,abt3-1还表现出植株小、叶形宽、叶色发灰、主根短等发育缺陷。利用图位克隆技术将该突变基因ABT3定位在1号染色体上,分子标记在F28G11#3与F4N21#1之间,物理距离为134kb。该研究将为进一步克隆ABT3基因及研究其在调控植物生长发育过程中的作用奠定基础。     

Rab6 regulates transport and targeting of exocytotic carriers

Constitutive exocytosis delivers newly synthesized proteins, lipids, and other molecules from the Golgi apparatus to the cell surface. This process is mediated by vesicles, which bud off the trans-Golgi network, move along cytoskeletal filaments, and fuse with the plasma membrane. Here, we show that the small GTPase Rab6 marks exocytotic vesicles and, together with the microtubule plus-end-directed motor kinesin-1, stimulates their processive microtubule-based transport to the cell periphery. Furthermore, Rab6 directs targeting of secretory vesicles to plasma-membrane sites enriched in the cortical protein ELKS, a known Rab6 binding partner. Our data demonstrate that although Rab6 is not essential for secretion, it controls the organization of exocytosis within the cellular space.     

The measurement of exocytosis in plant cells

Exocytosis is of vital importance to the growth and developmentof plant cells. It is a dynamic process in which vesicles bearingpolysaccharide precursors and proteins fuse with the plasmamembrane and release their contents. Equally important, newplasma membrane is delivered by exocytosis as secretory vesiclemembrane becomes incorporated. The requirements for polysaccharides,proteins and plasma membrane are very different in differentcell types, so there must be sophisticated mechanisms for ensuringdelivery of these materials to the correct cellular locationsat the appropriate time and, particularly in the case of membrane,their recovery and recycling. Currently, little is known ofthese mechanisms in plants, but new methods for measuring exocytosisare under development, and existing techniques have alreadycontributed data of considerable relevance. Here the methodsfor measuring exocytosis are described and evaluated, with emphasison the electrophysiological measurement of capacitance as arelatively non-invasive method, and on cell-free assays becauseof their potential importance in the identification of proteinsand other factors that control exocytosis in plant cells. Key words: Exocytosis, vesicle traffic, vesicle fusion, polysaccharides, cell wall, cell plate, root cap, secretion, patch-clamping     

Synthesis of vesicle cargo determines amplitude of Ca(2+)-sensitive exocytosis

Here we examine the potential coupling between the synthesis of secretory proteins and the sensitivity of exocytosis to the concentration of free Ca(2+) in the cytosol ([Ca(2+)](i)) in plant cell. We therefore monitor in tobacco protoplasts the excursion of the membrane capacitance in response to an elevation of [Ca(2+)](i) as a measure for exocytotic activity. The data show that a ramp like elevation of [Ca(2+)](i) generates in protoplasts from wild type plants and from transgenic plants, which overexpress the secreted α-amylase, an exocytotic burst with an initial steep and a subsequent slow phase. The largest capacitive burst is obtained in α-amylase producing plants and the amplitude of the [Ca(2+)](i) evoked C(m) excursion is a function of the amylase synthesis of the plants. The data support a model according to which plant cells have at least two serial [Ca(2+)](i) sensitive processes in the final steps of their exocytotic pathway. The overproduction of a secreted cargo does not affect the kinetics of this process but the number of vesicles in pools upstream of the [Ca(2+)](i) sensitive steps.     

v-SNAREs control exocytosis of vesicles from priming to fusion

SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca(2+)-dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.     

A rapid exocytosis mode in chromaffin cells with a neuronal phenotype

We have used astrocyte-conditioned medium (ACM) to promote the transdifferentiation of bovine chromaffin cells and study modifications in the exocytotic process when these cells acquire a neuronal phenotype. In the ACM-promoted neuronal phenotype, secretory vesicles and intracellular Ca2+ rise were preferentially distributed in the neurite terminals. Using amperometry, we observed that the exocytotic events also occurred mainly in the neurite terminals, wherein the individual exocytotic events had smaller quantal size than in undifferentiated cells. Additionally, duration of pre-spike current was significantly shorter, suggesting that ACM also modifies the fusion pore stability. After long exposure (7-9 days) to ACM, the kinetics of catecholamine release from individual vesicles was markedly accelerated. The morphometric analysis of vesicle diameters suggests that the rapid exocytotic events observed in neurites of ACM-treated cells correspond to the exocytosis of large dense-core vesicles (LDCV). On the other hand, experiments performed in EGTA-loaded cells suggest that ACM treatment promotes a better coupling between voltage-gated calcium channels (VGCC) and LDCV. Thus, our findings reveal that ACM promotes a neuronal phenotype in chromaffin cells, wherein the exocytotic kinetics is accelerated. Such rapid exocytosis mode could be caused at least in part by a better coupling between secretory vesicles and VGCC.     

Exocytosis in neuroendocrine cells: new tasks for actin

Most secretory cells undergoing calcium-regulated exocytosis in response to cell surface receptor stimulation display a dense subplasmalemmal actin network, which is remodeled during the exocytotic process. This review summarizes new insights into the role of the cortical actin cytoskeleton in exocytosis. Many earlier findings support the actin-physical-barrier model whereby transient depolymerization of cortical actin filaments permits vesicles to gain access to their appropriate docking and fusion sites at the plasma membrane. On the other hand, data from our laboratory and others now indicate that actin polymerization also plays a positive role in the exocytotic process. Here, we discuss the potential functions attributed to the actin cytoskeleton at each major step of the exocytotic process, including recruitment, docking and fusion of secretory granules with the plasma membrane. Moreover, we present actin-binding proteins, which are likely to link actin organization to calcium signals along the exocytotic pathway. The results cited in this review are derived primarily from investigations of the adrenal medullary chromaffin cell, a cell model that is since many years a source of information concerning the molecular machinery underlying exocytosis.