A receptor-channel trio conducts Ca2+ signalling for pollen tube reception.

Precise signalling between pollen tubes and synergid cells in the ovule initiates fertilization in flowering plants1. Contact of the pollen tube with the ovule triggers calcium spiking in the synergids2,3 that induces pollen tube rupture and sperm release. This process, termed pollen tube reception, entails the action of three synergid-expressed proteins in Arabidopsis: FERONIA (FER), a receptor-like kinase; LORELEI (LRE), a glycosylphosphatidylinositol-anchored protein; and NORTIA (NTA), a transmembrane protein of unknown function4-6. Genetic analyses have placed these three proteins in the same pathway; however, it remains unknown how they work together to enable synergid-pollen tube communication. Here we identify two pollen-tube-derived small peptides7 that belong to the rapid alkalinization factor (RALF) family8 as ligands for the FER-LRE co-receptor, which in turn recruits NTA to the plasma membrane. NTA functions as a calmodulin-gated calcium channel required for calcium spiking in the synergid. We also reconstitute the biochemical pathway in which FER-LRE perceives pollen-tube-derived peptides to activate the NTA calcium channel and initiate calcium spiking, a second messenger for pollen tube reception. The FER-LRE-NTA trio therefore forms a previously unanticipated receptor-channel complex in the female cell to recognize male signals and trigger the fertilization process.

Membrane receptor-mediated mechano-transduction maintains cell integrity during pollen tube growth within the pistil.

Invasive or penetrative growth is critical for developmental and reproductive processes (e.g., pollen tube penetration of pistils) and disease progression (e.g., cancer metastasis and fungal hyphae invasion). The invading or penetrating cells experience drastic changes in mechanical pressure from the surroundings and must balance growth with cell integrity. Here, we show that Arabidopsis pollen tubes sense and/or respond to mechanical changes via a cell-surface receptor kinase Buddha's Paper Seal 1 (BUPS1) while emerging from compressing female tissues. BUPS1-defective pollen tubes fail to maintain cell integrity after emergence from these tissues. The mechano-transduction function of BUPS1 is established by using a microfluidic channel device mimicking the mechanical features of the in vivo growth path. BUPS1-based mechano-transduction activates Rho-like GTPase from Plant 1 (ROP1) GTPase to promote exocytosis that facilitates secretion of BUPS1's ligands for mechanical signal amplification and cell wall rigidification in pollen tubes. These findings uncover a membrane receptor-based mechano-transduction system for cells to cope with the physical challenges during invasive or penetrative growth.

SMALL ORGAN4 Is a Ribosome Biogenesis Factor Involved in 5.8S Ribosomal RNA Maturation.

Ribosome biogenesis is crucial for cellular metabolism and has important implications for disease and aging. Human (Homo sapiens) glioma tumor-suppressor candidate region gene2 (GLTSCR2) and yeast (Saccharomyces cerevisiae) Nucleolar protein53 (Nop53) are orthologous proteins with demonstrated roles as ribosome biogenesis factors; knockdown of GLTSCR2 impairs maturation of 18S and 5.8S ribosomal RNAs (rRNAs), and Nop53 is required for maturation of 5.8S and 25S rRNAs. Here, we characterized SMALL ORGAN4 (SMO4), the most likely ortholog of human GLTSCR2 and yeast Nop53 in Arabidopsis (Arabidopsis thaliana). Loss of function of SMO4 results in a mild morphological phenotype; however, we found that smo4 mutants exhibit strong cytological and molecular phenotypes: nucleolar hypertrophy and disorganization, overaccumulation of 5.8S and 18S rRNA precursors, and an imbalanced 40S:60S ribosome subunit ratio. Like yeast Nop53 and human GLTSCR2, Arabidopsis SMO4 participates in 5.8S rRNA maturation. In yeast, Nop53 cooperates with mRNA transport4 (Mtr4) for 5.8S rRNA maturation. In Arabidopsis, we found that SMO4 plays similar roles in the 5.8S rRNA maturation pathway than those described for MTR4. However, SMO4 seems not to participate in the degradation of by-products derived from the 5'-external transcribed spacer (ETS) of 45S pre-rRNA, as MTR4 does.

Cyanide Boosting Copper Catalysis: A Mild Approach to Fluorescent Benzazole Derivatives from Nonemissive Schiff Bases in Biological Media.

An application of nucleophilic cyclization and oxidation of nonemissive Schiff bases via cyanide boosting copper catalysis to synthesize fluorescent benzazole derivatives in high conversion yield is disclosed. This approach is highlighted by broad substrate scope, fast reaction time, and mild conditions and can efficiently proceed in living cells or Arabidopsis root tissues. Furthermore, this methodology can be applied for selective detection of Cu2+ and CN-.

GABA operates upstream of H+-ATPase and improves salinity tolerance in Arabidopsis by enabling cytosolic K+ retention and Na+ exclusion.

The non-protein amino acid γ-aminobutyric acid (GABA) rapidly accumulates in plant tissues in response to salinity. However, the physiological rationale for this elevation remains elusive. This study compared electrophysiological and whole-plant responses of salt-treated Arabidopsis mutants pop2-5 and gad1,2, which have different abilities to accumulate GABA. The pop2-5 mutant, which was able to overaccumulate GABA in its roots, showed a salt-tolerant phenotype. On the contrary, the gad1,2 mutant, lacking the ability to convert glutamate to GABA, showed oversensitivity to salinity. The greater salinity tolerance of the pop2-5 line was explained by: (i) the role of GABA in stress-induced activation of H+-ATPase, thus leading to better membrane potential maintenance and reduced stress-induced K+ leak from roots; (ii) reduced rates of net Na+ uptake; (iii) higher expression of SOS1 and NHX1 genes in the leaves, which contributed to reducing Na+ concentration in the cytoplasm by excluding Na+ to apoplast and sequestering Na+ in the vacuoles; (iv) a lower rate of H2O2 production and reduced reactive oxygen species-inducible K+ efflux from root epidermis; and (v) better K+ retention in the shoot associated with the lower expression level of GORK channels in plant leaves.

Unmethyl-esterified homogalacturonan and extensins seal Arabidopsis graft union.

BACKGROUND: Grafting is a technique widely used in horticulture. The processes involved in grafting are diverse, and the technique is commonly employed in studies focusing on the mechanisms that regulate cell differentiation or response of plants to abiotic stress. Information on the changes in the composition of the cell wall that occur during the grafting process is scarce. Therefore, this study was carried out for analyzing the composition of the cell wall using Arabidopsis hypocotyls as an example. During the study, the formation of a layer that covers the surface of the graft union was observed. So, this study also aimed to describe the histological and cellular changes that accompany autografting of Arabidopsis hypocotyls and to perform preliminary chemical and structural analyses of extracellular material that seals the graft union. RESULTS: During grafting, polyphenolic and lipid compounds were detected, along with extracellular deposition of carbohydrate/protein material. The spatiotemporal changes observed in the structure of the extracellular material included the formation of a fibrillar network, polymerization of the fibrillar network into a membranous layer, and the presence of bead-like structures on the surface of cells in established graft union. These bead-like structures appeared either "closed" or "open". Only three cell wall epitopes, namely: LM19 (un/low-methyl-esterified homogalacturonan), JIM11, and JIM20 (extensins), were detected abundantly on the cut surfaces that made the adhesion plane, as well as in the structure that covered the graft union and in the bead-like structures, during the subsequent stages of regeneration. CONCLUSIONS: To the best of our knowledge, this is the first report on the composition and structure of the extracellular material that gets deposited on the surface of graft union during Arabidopsis grafting. The results showed that unmethyl-esterified homogalacturonan and extensins are together involved in the adhesion of scion and stock, as well as taking part in sealing the graft union. The extracellular material is of importance not only due to the potential pectin-extensin interaction but also due to its origin. The findings presented here implicate a need for studies with biochemical approach for a detailed analysis of the composition and structure of the extracellular material.

Ectopic expression of a pea apyrase enhances root system architecture and drought survival in Arabidopsis and soybean.

Ectoapyrases (ecto-NTPDases) function to decrease levels of extracellular ATP and ADP in animals and plants. Prior studies showed that ectopic expression of a pea ectoapyrase, psNTP9, enhanced growth in Arabidopsis seedlings and that the overexpression of the two Arabidopsis apyrases most closely related to psNTP9 enhanced auxin transport and growth in Arabidopsis. These results predicted that ectopic expression of psNTP9 could promote a more extensive root system architecture (RSA) in Arabidopsis. We confirmed that transgenic Arabidopsis seedlings had longer primary roots, more lateral roots, and more and longer root hairs than wild-type plants. Because RSA influences water uptake, we tested whether the transgenic plants could tolerate osmotic stress and water deprivation better than wild-type plants, and we confirmed these properties. Transcriptomic analyses revealed gene expression changes in the transgenic plants that helped account for their enhanced RSA and improved drought tolerance. The effects of psNTP9 were not restricted to Arabidopsis, because its expression in soybeans improved the RSA, growth, and seed yield of this crop and supported higher survival in response to drought. Our results indicate that in both Arabidopsis and soybeans, the constitutive expression of psNTP9 results in a more extensive RSA and improved survival in drought stress conditions.

Alterations of plant architecture and phase transition by the phytoplasma virulence factor SAP11.

As key mediators linking developmental processes with plant immunity, TCP (TEOSINTE-BRANCHED, CYCLOIDEA, PROLIFERATION FACTOR 1 and 2) transcription factors have been increasingly shown to be targets of pathogenic effectors. We report here that TB/CYC (TEOSINTE-BRANCHED/CYCLOIDEA)-TCPs are destabilized by phytoplasma SAP11 effectors, leading to the proliferation of axillary meristems. Although a high degree of sequence diversity was observed among putative SAP11 effectors identified from evolutionarily distinct clusters of phytoplasmas, these effectors acquired fundamental activity in destabilizing TB/CYC-TCPs. In addition, we demonstrate that miR156/SPLs and miR172/AP2 modules, which represent key regulatory hubs involved in plant phase transition, were modulated by Aster Yellows phytoplasma strain Witches' Broom (AY-WB) protein SAP11. A late-flowering phenotype with significant changes in the expression of flowering-related genes was observed in transgenic Arabidopsis plants expressing SAP11AYWB. These morphological and molecular alterations were correlated with the ability of SAP11 effectors to destabilize CIN (CINCINNATA)-TCPs. Although not all putative SAP11 effectors display broad-spectrum activities in modulating morphological and physiological changes in host plants, they serve as core virulence factors responsible for the witches' broom symptom caused by phytoplasmas.

Iris lactea var. chinensis (Fisch.) cysteine-rich gene llCDT1 enhances cadmium tolerance in yeast cells and Arabidopsis thaliana.

IlCDT1, a cysteine-rich protein, was isolated from Iris lactea var. chinensis (Fisch.) (I. lactea var. chinensis). Its transcription was up-regulated by the exogenous application of Cd. The truncated IlCDT1 (25-54) containing 14 Cys residues confers Cd tolerance to yeast as the intact IlCDT1, indicating that Cys residues are required for Cd tolerance presumably by chelating Cd. When the gene was constitutively expressed in A. thaliana, root length of transgenic lines was longer than that of wild-type under 100 µM or 200 µM Cd stress. However, Cd absorption in wild-type was more than in two trangenic lines under 100 µM Cd exposure. IlCDT1 may directly bind Cd, through chelating Cd and avoiding the Cd uptake into the cells. Together, IlCDT1 may be a promising gene for the Cd tolerance improvement. SUMMARY: Cysteine-rich gene llCDT1 enhances cadmium tolerance in yeast cells and Arabidopsis thaliana.

Changes in iron availability in Arabidopsis are rapidly sensed in the leaf vasculature and impaired sensing leads to opposite transcriptional programs in leaves and roots.

The OLIGOPEPTIDE TRANSPORTER 3 (OPT3) has recently been identified as a component of the systemic network mediating iron (Fe) deficiency responses in Arabidopsis. Reduced expression of OPT3 induces an over accumulation of Fe in roots and leaves, due in part by an elevated expression of the IRON-REGULATED TRANSPORTER 1. Here we show however, that opt3 leaves display a transcriptional program consistent with an Fe overload, suggesting that Fe excess is properly sensed in opt3 leaves and that the OPT3-mediated shoot-to-root signaling is critical to prevent a systemic Fe overload. We also took advantage of the tissue-specific localization of OPT3, together with other Fe-responsive genes, to determine the timing and location of early transcriptional events during Fe limitation and resupply. Our results show that the leaf vasculature responds more rapidly than roots to both Fe deprivation and resupply, suggesting that the leaf vasculature is within the first tissues that sense and respond to changes in Fe availability. Our data highlight the importance of the leaf vasculature in Fe homeostasis by sensing changes in apoplastic levels of Fe coming through the xylem and relaying this information back to roots via the phloem to regulate Fe uptake at the root level.

Plant architecture, auxin homeostasis and phenol content in Arabidopsis thaliana grown in cadmium- and zinc-enriched media.

A screening strategy using micropropagation glass tubes with a gradient of distances between germinating seeds and a metal-contaminated medium was used for studying alterations in root architecture and morphology of Arabidopsis thaliana treated with cadmium (Cd) and zinc (Zn) at the concentration of 10-20µM and 100-200µM, respectively. Metal concentrations in plant shoots and roots were measured by quadrupole inductively coupled plasma mass spectrometry. After 21days from germination, all plants in the tubes were scanned at high resolution and the root systems analyzed. The localization of indole-3-acetic acid (IAA) in the primary root and lateral root apices was monitored using DR5:GUS, LAX3:GUS and AUX1:GUS Arabidopsis transgenic lines. Total phenol content in leaves was measured spectrophotometrically. Shoot and root dry weight and leaf area did not change in Zn-exposed plants and significantly decreased in Cd-exposed plants, compared to control plants. Cadmium induced a reduction of root length, of mean number of roots and of total root surface. Both Cd- and Zn-exposed plants showed a reduced specific root length. This morphological behavior, together with an observed increase in root diameter in metal-exposed plants could be interpreted as compensatory growth, and the observed thicker roots could act as a barrier to protect root from the metals. In comparison with the apical localization of the IAA signal in the control plants, Zn generally reinforced the intensity of IAA signal, without affecting its localization. In Cd-exposed plants, IAA localization remained apical but weaker compared to control plants. Total phenols decreased in plants exposed to Zn and Cd. Therefore, we propose that the remodelling of the root architecture and the production of some secondary metabolites, such as IAA and phenols could be two responses of plants subjected to metal stress. This knowledge can open the way to future phytoremediation strategies of contaminated sites.

AtGRP3 Is Implicated in Root Size and Aluminum Response Pathways in Arabidopsis.

AtGRP3 is a glycine-rich protein (GRP) from Arabidopsis thaliana shown to interact with the receptor-like kinase AtWAK1 in yeast, in vitro and in planta. In this work, phenotypic analyses using transgenic plants were performed in order to better characterize this GRP. Plants of two independent knockout alleles of AtGRP3 develop longer roots suggesting its involvement in root size determination. Confocal microscopy analysis showed an abnormal cell division and elongation in grp3-1 knockout mutants. Moreover, we also show that grp3-1 exhibits an enhanced Aluminum (Al) tolerance, a feature also described in AtWAK1 overexpressing plants. Together, these results implicate AtGRP3 function root size determination during development and in Al stress.

Disaggregating polyploidy, parental genome dosage and hybridity contributions to heterosis in Arabidopsis thaliana.

Heterosis is the phenomenon whereby hybrid offspring of genetically divergent parents display superior characteristics compared with their parents. Although hybridity and polyploidy can influence heterosis in hybrid plants, the differential contributions of hybridity vs polyploidy to heterosis effects remain unknown. To address this question, we investigated heterosis effects on rosette size and growth rate of 88 distinct F1 lines of Arabidopsis thaliana consisting of diploids, reciprocal triploids and tetraploids in isogenic and hybrid genetic contexts. 'Heterosis without hybridity' effects on plant size can be generated in genetically isogenic F1 triploid plants. Paternal genome excess F1 triploids display positive heterosis, whereas maternal genome excess F1 s display negative heterosis effects. Paternal genome dosage increases plant size in F1 hybrid triploid plants by, on average, 57% (in contrast with 35% increase displayed by F1 diploid hybrids). Such effects probably derive from differential seed size, as the growth rate of triploids was similar to diploids. Tetraploid plants display a lower growth rate compared with other ploidies, whereas hybrids display increased early stage growth rate. By disaggregating heterosis effects caused by hybridity vs genome dosage, we advance our understanding of heterosis in plants and facilitate novel paternal genome dosage-based strategies to enhance heterosis effects in crop plants.

Induction of a dwarf phenotype with IBH1 may enable increased production of plant-made pharmaceuticals in plant factory conditions.

Year-round production in a contained, environmentally controlled 'plant factory' may provide a cost-effective method to produce pharmaceuticals and other high-value products. However, cost-effective production may require substantial modification of the host plant phenotype; for example, using dwarf plants can enable the growth of more plants in a given volume by allowing more plants per shelf and enabling more shelves to be stacked vertically. We show here that the expression of the chimeric repressor for Arabidopsis AtIBH1 (P35S:AtIBH1SRDX) in transgenic tobacco plants (Nicotiana tabacum) induces a dwarf phenotype, with reduced cell size. We estimate that, in a given volume of cultivation space, we can grow five times more AtIBH1SRDX plants than wild-type plants. Although, the AtIBH1SRDX plants also showed reduced biomass compared with wild-type plants, they produced about four times more biomass per unit of cultivation volume. To test whether the dwarf phenotype affects the production of recombinant proteins, we expressed the genes for anti-hepatitis B virus antibodies (anti-HBs) in tobacco plants and found that the production of anti-HBs per unit fresh weight did not significantly differ between wild-type and AtIBH1SRDX plants. These data indicate that P35S:AtIBH1SRDX plants produced about fourfold more antibody per unit of cultivation volume, compared with wild type. Our results indicate that AtIBH1SRDX provides a useful tool for the modification of plant phenotype for cost-effective production of high-value products by stably transformed plants in plant factory conditions.

PEA-CLARITY: 3D molecular imaging of whole plant organs.

Here we report the adaptation of the CLARITY technique to plant tissues with addition of enzymatic degradation to improve optical clearing and facilitate antibody probe penetration. Plant-Enzyme-Assisted (PEA)-CLARITY, has allowed deep optical visualisation of stains, expressed fluorescent proteins and IgG-antibodies in Tobacco and Arabidopsis leaves. Enzyme treatment enabled penetration of antibodies into whole tissues without the need for any sectioning of the material, thus facilitating protein localisation of intact tissue in 3D whilst retaining cellular structure.

Live cell imaging of FM4-64, a tool for tracing the endocytic pathways in Arabidopsis root cells.

Confocal live imaging of the amphiphilic styryl dye FM4-64 is a valuable technique to monitor organelle dynamics and in particular endocytic pathways. After application in plants, FM4-64 immediately stains the plasma membrane and is then integrated on vesicles following endomembrane system-dependent internalization processes. Over time, FM4-64 becomes distributed throughout the full vesicular network from the plasma membrane to the vacuole, including the components of the secretory pathways. Here we provide succinct examples of the many important developmental processes in plants that rely on endocytosis and describe two suitable methods to trace the endocytic pathways in Arabidopsis thaliana root cells based on the uptake of FM4-64.

The plasma membrane H(+) -ATPase AHA2 contributes to the root architecture in response to different nitrogen supply.

In this study the role of the plasma membrane (PM) H(+) -ATPase for growth and development of roots as response to nitrogen starvation is studied. It is known that root development differs dependent on the availability of different mineral nutrients. It includes processes such as initiation of lateral root primordia, root elongation and increase of the root biomass. However, the signal transduction mechanisms, which enable roots to sense changes in different mineral environments and match their growth and development patterns to actual conditions in the soil, are still unknown. Most recent comments have focused on one of the essential macroelements, namely nitrogen, and its role in the modification of the root architecture of Arabidopsis thaliana. As yet, not all elements of the signal transduction pathway leading to the perception of the nitrate stimulus, and hence to anatomical changes of the root, which allow for adaptation to variable ion concentrations in the soil, are known. Our data demonstrate that primary and lateral root length were shorter and lower in aha2 mutant lines compared with wild-type plants in response to a variable nitrogen source. This suggests that the PM proton pump AHA2 (Arabidopsis plasma membrane H(+) -ATPase isoform 2) is important for root growth and development during different nitrogen regimes. This is possible by controlling the pH homeostasis in the root during growth and development as shown by pH biosensors.

Physiological and morphological responses induced by α-particle radiation on Arabidopsis thaliana embryos.

Alpha (α)-particle radiation has been thoroughly studied in the occupational and residential environments, but biological mechanisms induced by α-particle radiation on plants are not clearly understood. In this study, radiation effects were examined using different total doses (1, 10, 100 Gy, respectively) of 241Am, α-particle on Arabidopsis embryos. No significant difference in the germination percentage was observed between the 3 levels of doses and the control. Germination speed and root length were increased by treatment with the 1-Gy dose of a-particles, and decreased by treatment with 10- and 100-Gy doses. Moreover, the bending degree of roots increased with radiation dose, and the roots showed an "S" shape when treated with the 100-Gy dose. Root bending under the 100-Gy dose was inhibited by scavengers of reactive oxygen species (ROS). Root gravitropism and root length may respond to the consistency of ROS induced by irradiation. Further analysis of the physiological effects revealed that an increase in a-particle radiation intensity enhanced the activity of catalase and the content of malondialdehyde, but superoxide dismutase activity was reduced by treatment with 100-Gy radiation of a-particles, suggesting that the high linear energy transfer of a-particles may cause a relatively high level of membrane lipid preoxidation and high accumulation of ROS. ROS showed both physiological and morphological responses following exposure to α-particle radiation in Arabidopsis embryos.

Tandem high-pressure freezing and quick freeze substitution of plant tissues for transmission electron microscopy.

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Ethylene Response Factor070 regulates root development and phosphate starvation-mediated responses.

Inorganic phosphate (Pi) availability is a major factor determining growth and consequently the productivity of crops. However, it is one of the least available macronutrients due to its high fixation in the rhizospheres. To overcome this constraint, plants have developed adaptive responses to better acquire, utilize, and recycle Pi. Molecular determinants of these adaptive mechanisms include transcription factors (TFs) that play a major role in transcriptional control, thereby regulating genome-scale networks. In this study, we have characterized the biological role of Arabidopsis thaliana Ethylene Response Factor070 (AtERF070), a Pi starvation-induced TF belonging to the Apetala2/Ethylene Response Factor family of TFs in Arabidopsis (Arabidopsis thaliana). It is localized to the nucleus and induced specifically in Pi-deprived roots and shoots. RNA interference-mediated suppression of AtERF070 led to augmented lateral root development resulting in higher Pi accumulation, whereas there were reductions in both primary root length and lateral root number in 12-d-old transgenic seedlings overexpressing AtERF070. When the overexpressing lines were grown to maturity under greenhouse conditions, they revealed a stunted bushy appearance that could be rescued by gibberellic acid application. Furthermore, a number of Pi starvation-responsive genes were modulated in AtERF070-overexpressing and RNA interference lines, thereby suggesting a potential role for this TF in maintaining Pi homeostasis.