Maintenance of the cytosolic Na+/K+ ratio under saline conditions is crucial for plants. HKT-type Na+ transporters play a key role in keeping low cytosolic Na+ concentrations thus retaining a low Na+/K+ ratio, that reduces Na+ toxicity and causing high salinity stress tolerance. Two HKT-type transporters, AtHKT1 from Arabidopsis and TsHKT1;2 from Thellungiella salsuginea, that share high DNA and protein sequence identities, are distinguished by fundamentally different ion selection and salinity stress behavior. On the level of transcription, TsHKT1;2 is dramatically induced upon salt stress, whereas AtHKT1 is downregulated. TsHKT1;2-RNAi lines show severe potassium deficiency and are also sensitive to high [Na+]. We have validated the ability of the TsHKT1;2 protein to act as an efficient K+ transporter in the presence of high [Na+] by expression in yeast cells. K+ specificity is based on amino acid differences in the pore of the transporter protein relative to AtHKT1.
HKT1; halophyte; salt stress; Na+/K+ ratio; Thellungiella salsuginea
Auxin, a plant hormone, plays crucial roles in diverse aspects of plant growth and development reacting to and integrating environmental stimuli. Indole-3-acetic acid (IAA) is the major plant auxin that is synthesized by members of the YUCCA (YUC) family of flavin monooxygenases that catalyse a rate-limiting step. Although the paths to IAA biosynthesis are characterized in Arabidopsis, little is known about the corresponding components in potato. Recently, we isolated eight putative StYUC (Solanum tuberosum YUCCA) genes and five putative tryptophan aminotransferase genes in comparison to those found in Arabidopsis.1 The specific domains of YUC proteins were well conserved in all StYUC amino acid sequences. Transgenic potato (Solanum tuberosum cv. Jowon) overexpressing AtYUC6 showed high-auxin and enhanced drought tolerance phenotypes. The transgenic potatoes also exhibited reduced levels of ROS (reactive oxygen species) compared to control plants. We therefore propose that YUCCA and TAA families in potato would function in the auxin biosynthesis. The overexpression of AtYUC6 in potato establishes enhanced drought tolerance through regulated ROS homeostasis.
ArabidopsisYUCCA6; auxin; drought; potato; reactive oxygen species
In plants, microRNA399 (miR399) is a major regulator of phosphate (Pi) homeostasis by way of post-transcriptional mechanisms including transcript cleavage and transcriptional repression. Although miRNA genomic organization, biogenesis, and mode of action in plants are known, the regulatory mechanisms affecting miRNAs are poorly understood. We have shown that AtMYB2 functions as a transcriptional activator for miR399f expression in the context of phosphate homeostasis. AtMYB2 directly binds to a MYB-binding site in the promoter of the miR399f precursor and regulates miR399f expression. In addition, AtMYB2 transcripts are induced under Pi deficiency. The overexpression of AtMYB2 affects root system architecture (RSA), indicated by suppression of primary root growth and enhanced development of root hairs. AtMYB2 and miR399f are expressed and localized in the same tissues under Pi limitation. This study establishes that AtMYB2 regulates Pi-starvation responses (PSR) by activating of miR399f transcript, suggesting that an analysis of this miRNA promoter could reveal the existence and extent of crosstalk with other signaling mechanisms.
Arabidopsis MYB2; microRNA399; phosphate; phytohormones; signaling
The initiation of flowering in Arabidopsis is retarded or abolished by environmental stresses. Focusing on salt stress, we provide a molecular explanation for this well-known fact. A protein complex consisting of GI, a clock component important for flowering and SOS2, a kinase activating the [Na+] antiporter SOS1, exists under no stress conditions. GI prevents SOS2 from activating SOS1. In the presence of NaCl, the SOS2/GI complex disintegrates and GI is degraded. SO2, together with the Ca2+-activated sensor of sodium ions, SOS3, activates SOS1. In gi mutants, SOS1 is constitutively activated and gi plants are more highly salt tolerant than wild type Arabidopsis. The model shows GI as a transitory regulator of SOS pathway activity whose presence or amount connects flowering to environmental conditions.
GIGANTEA (GI); SOS1; SOS2; SOS3; high salinity; Arabidopsis
Reversible posttranslational modification of proteins by the action of small ubiquitin-like modifier (SUMO) peptide (sumoylation) has been known to participate in various biological processes in eukaryotes. However, much less is known about the role of sumoylation in plants. In our recent paper to which we write this Addendum, we show that loss of SIZ1, a SUMO E3 ligase, results in a highly increased SA-mediated defense signaling through a PAD4-dependent pathway. This signaling leads to constitutively expressed pathogen related (PR) genes and to increased disease resistance to a virulent bacterial pathogen. These findings significantly increase our understanding of the role of sumoylation in the plant defense system.
plant innate immunity; salicylic acid (SA); small ubiquitin-like modifer (SUMO); plant-pathogen interactions
Nitric oxide (NO) is known for its role in the activation of plant defense responses. To examine the involvement and mode of action of NO in plant defense responses, we introduced calmodulin-dependent mammalian neuronal nitric oxide synthase (nNOS), which controls the CaMV35S promoter, into wild-type and NahG tobacco plants. Constitutive expression of nNOS led to NO production and triggered spontaneous induction of leaf lesions. Transgenic plants accumulated high amounts of H2O2, with catalase activity lower than that in the wild type. nNOS transgenic plants contained high levels of salicylic acid (SA), and they induced an array of SA-, jasmonic acid (JA)-, and/or ethylene (ET)-related genes. Consequently, NahG co-expression blocked the induction of systemic acquired resistance (SAR)-associated genes in transgenic plants, implying SA is involved in NO-mediated induction of SAR genes. The transgenic plants exhibited enhanced resistance to a spectrum of pathogens, including bacteria, fungi, and viruses. Our results suggest a highly ranked regulatory role for NO in SA-, JA-, and/or ET-dependent pathways that lead to disease resistance.
nitric oxide (NO); nitric oxide synthase (NOS); plant defense signaling; reactive oxygen species; salicylic acid
Adiponectin is a mammalian hormone that exerts anti-diabetic, anti-cancer and cardioprotective effects through interaction with its major ubiquitously expressed plasma membrane localized receptors, AdipoR1 and AdipoR2. Here, we report a Saccharomyces cerevisiae based method for investigating agonist-AdipoR interactions that is amenable for high-throughput scale-up and can be used to study both AdipoRs separately. Agonist-AdipoR1 interactions are detected using a split firefly luciferase assay based on reconstitution of firefly luciferase (Luc) activity due to juxtaposition of its N- and C-terminal fragments, NLuc and CLuc, by ligand induced interaction of the chimeric proteins CLuc-AdipoR1 and APPL1-NLuc (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif 1-NLuc) in a S. cerevisiae strain lacking the yeast homolog of AdipoRs (Izh2p). The assay monitors the earliest known step in the adiponectin-AdipoR anti-diabetic signaling cascade. We demonstrate that reconstituted Luc activity can be detected in colonies or cells using a CCD camera and quantified in cell suspensions using a microplate reader. AdipoR1-APPL1 interaction occurs in absence of ligand but can be stimulated specifically by agonists such as adiponectin and the tobacco protein osmotin that was shown to have AdipoR-dependent adiponectin-like biological activity in mammalian cells. To further validate this assay, we have modeled the three dimensional structures of receptor-ligand complexes of membrane-embedded AdipoR1 with cyclic peptides derived from osmotin or osmotin-like plant proteins. We demonstrate that the calculated AdipoR1-peptide binding energies correlate with the peptides’ ability to behave as AdipoR1 agonists in the split luciferase assay. Further, we demonstrate agonist-AdipoR dependent activation of protein kinase A (PKA) signaling and AMP activated protein kinase (AMPK) phosphorylation in S. cerevisiae, which are homologous to important mammalian adiponectin-AdipoR1 signaling pathways. This system should facilitate the development of therapeutic inventions targeting adiponectin and/or AdipoR physiology.
Thellungiella parvula1 is related to Arabidopsis thaliana and is endemic to saline, resource-poor habitats2, making it a model for the evolution of plant adaptation to extreme environments. Here we present the draft genome for this extremophile species. Exclusively by next generation sequencing, we obtained the de novo assembled genome in 1,496 gap-free contigs, closely approximating the estimated genome size of 140 Mb. We anchored these contigs to seven pseudo chromosomes without the use of maps. We show that short reads can be assembled to a near-complete chromosome level for a eukaryotic species lacking prior genetic information. The sequence identifies a number of tandem duplications that, by the nature of the duplicated genes, suggest a possible basis for T. parvula’s extremophile lifestyle. Our results provide essential background for developing genomically influenced testable hypotheses for the evolution of environmental stress tolerance.
Peroxiredoxins (Prxs), which are classified into three isotypes in plants, play important roles in protection systems as peroxidases or molecular chaperones. The three Prx isotypes of Chinese cabbage, namely C1C-Prx, C2C-Prx, and C-PrxII, have recently been identified and characterized. The present study compares their molecular properties and biochemical functions to gain insights into their concerted roles in plants. The three Prx isotype genes were differentially expressed in tissue- and developmental stage-specific manners. The transcript level of the C1C-Prx gene was abundant at the seed stage, but rapidly decreased after imbibitions. In contrast, the C2C-Prx transcript was not detected in the seeds, but its expression level increased at germination and was maintained thereafter. The C-PrxII transcript level was mild at the seed stage, rapidly increased for 10 days after imbibitions, and gradually disappeared thereafter. In the localization analysis using GFP-fusion proteins, the three isotypes showed different cellular distributions. C1C-Prx was localized in the cytosol and nucleus, whereas C2C-Prx and C-Prx were found mainly in the chloroplast and cytosol, respectively. In vitro thiol-dependent antioxidant assays revealed that the relative peroxidase activities of the isotypes were C-PrxII > C2C-Prx > C1C-Prx. C1C-Prx and C2C-Prx, but not C-PrxII, prevented aggregation of malate dehydrogenase as a molecular chaperone. Taken together, these results suggest that the three isotypes of Prx play specific roles in the cells in timely and spatially different manners, but they also cooperate with each other to protect the plant.
chaperone; peroxidase; plant peroxiredoxin
The traditional focus on the central dogma of molecular biology, from gene through RNA to protein, has now been replaced by the recognition of an additional mechanism. The new regulatory mechanism, post-translational modifications to proteins, can actively alter protein function or activity introducing additional levels of functional complexity by altering cellular and sub-cellular location, protein interactions and the outcome of biochemical reaction chains. Modifications by ubiquitin (Ub) and ubiquitin-like modifiers systems are conserved in all eukaryotic organisms. One of them, small ubiquitin-like modifier (SUMO) is present in plants. The SUMO mechanism includes several isoforms of proteins that are involved in reactions of sumoylation and de-sumoylation. Sumoylation affects several important processes in plants. Outstanding among those are responses to environmental stresses. These may be abiotic stresses, such as phosphate deficiency, heat, low temperature, and drought, or biotic stressses, as well including defense reactions to pathogen infection. Also, the regulations of flowering time, cell growth and development, and nitrogen assimilation have recently been added to this list. Identification of SUMO targets is material to characterize the function of sumoylation or desumoylation. Affinity purification and mass spectrometric identification have been done lately in plants. Further SUMO noncovalent binding appears to have function in other model organisms and SUMO interacting proteins in plants will be of interest to plant biologists who dissect the dynamic function of SUMO. This review will discuss results of recent insights into the role of sumoylation in plants.
Arabidopsis; SUMO; sumoylation
Reversible conjugation of the small ubiquitin modifier (SUMO) peptide to proteins (SUMOylation) plays important roles in cellular processes in animals and yeasts. However, little is known about plant SUMO targets. To identify SUMO substrates in Arabidopsis and to probe for biological functions of SUMO proteins, we constructed 6xHis-3xFLAG fused AtSUMO1 (HFAtSUMO1) controlled by the CaMV35S promoter for transformation into Arabidopsis Col-0. After heat treatment, an increased sumoylation pattern was detected in the transgenic plants. SUMO1-modified proteins were selected after two-dimensional gel electrophoresis (2-DE) image analysis and identified using matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). We identified 27 proteins involved in a variety of processes such as nucleic acid metabolism, signaling, metabolism, and including proteins of unknown functions. Binding and sumoylation patterns were confirmed independently. Surprisingly, MCM3 (At5G46280), a DNA replication licensing factor, only interacted with and became sumoylated by AtSUMO1, but not by SUMO1ΔGG or AtSUMO3. The results suggest specific interactions between sumoylation targets and particular sumoylation enzymes.
Arabidopsis; mass spectrometry; proteomics; SUMO binding proteins; Sumoylation
Transcriptional repression of pathogen defense-related genes is essential for plant growth and development. Several proteins are known to be involved in the transcriptional regulation of plant defense responses. However, mechanisms by which expression of defense-related genes are regulated by repressor proteins are poorly characterized. Here, we describe the in planta function of CBNAC, a calmodulin-regulated NAC transcriptional repressor in Arabidopsis. A T-DNA insertional mutant (cbnac1) displayed enhanced resistance to a virulent strain of the bacterial pathogen Pseudomonas syringae DC3000 (PstDC3000), whereas resistance was reduced in transgenic CBNAC overexpression lines. The observed changes in disease resistance were correlated with alterations in pathogenesis-related protein 1 (PR1) gene expression. CBNAC bound directly to the PR1 promoter. SNI1 (suppressor of nonexpressor of PR genes1, inducible 1) was identified as a CBNAC-binding protein. Basal resistance to PstDC3000 and derepression of PR1 expression was greater in the cbnac1 sni1 double mutant than in either cbnac1 or sni1 mutants. SNI1 enhanced binding of CBNAC to its cognate PR1 promoter element. CBNAC and SNI1 are hypothesized to work as repressor proteins in the cooperative suppression of plant basal defense.
Salinity is an abiotic stress that limits both yield and the expansion of agricultural crops to new areas. In the last 20 years our basic understanding of the mechanisms underlying plant tolerance and adaptation to saline environments has greatly improved owing to active development of advanced tools in molecular, genomics, and bioinformatics analyses. However, the full potential of investigative power has not been fully exploited, because the use of halophytes as model systems in plant salt tolerance research is largely neglected. The recent introduction of halophytic Arabidopsis-Relative Model Species (ARMS) has begun to compare and relate several unique genetic resources to the well-developed Arabidopsis model. In a search for candidates to begin to understand, through genetic analyses, the biological bases of salt tolerance, 11 wild relatives of Arabidopsis thaliana were compared: Barbarea verna, Capsella bursa-pastoris, Hirschfeldia incana, Lepidium densiflorum, Malcolmia triloba, Lepidium virginicum, Descurainia pinnata, Sisymbrium officinale, Thellungiella parvula, Thellungiella salsuginea (previously T. halophila), and Thlaspi arvense. Among these species, highly salt-tolerant (L. densiflorum and L. virginicum) and moderately salt-tolerant (M. triloba and H. incana) species were identified. Only T. parvula revealed a true halophytic habitus, comparable to the better studied Thellungiella salsuginea. Major differences in growth, water transport properties, and ion accumulation are observed and discussed to describe the distinctive traits and physiological responses that can now be studied genetically in salt stress research.
Germination; halophytes; ion contents; root elongation; stomata; water relations
Much is already known about the function and functioning of the three genes that make up the SOS (Salt-Overly-Sensitive) pathway in plants, but recent studies indicate that the linkage between external increases in salinity and stress protection provided by genes SOS1, SOS2 and SOS3 is more complex than previously appreciated. It has recently been shown that the engineered reduced expression of the sodium/proton antiporter SOS1 affected several pathways indicating a role for SOS1 that exceeds its known function as an antiporter. Interference with expression of SOS1, characterized as a sodium/proton antiporter in the halophyte Thellungiella salsuginea converted Thellungiella into an essentially glycophytic species.
SOS1 function; Thellungiella salsuginea; halophytism
A mutation of AtSOS1 (Salt Overly Sensitive 1), a plasma membrane Na+/H+-antiporter in Arabidopsis thaliana, leads to a salt-sensitive phenotype accompanied by the death of root cells under salt stress. Intracellular events and changes in gene expression were compared during a non-lethal salt stress between the wild type and a representative SOS1 mutant, atsos1-1, by confocal microscopy using ion-specific fluorophores and by quantitative RT-PCR. In addition to the higher accumulation of sodium ions, atsos1-1 showed inhibition of endocytosis, abnormalities in vacuolar shape and function, and changes in intracellular pH compared to the wild type in root tip cells under stress. Quantitative RT-PCR revealed a dramatically faster and higher induction of root-specific Ca2+ transporters, including several CAXs and CNGCs, and the drastic down-regulation of genes involved in pH-homeostasis and membrane potential maintenance. Differential regulation of genes for functions in intracellular protein trafficking in atsos1-1 was also observed. The results suggested roles of the SOS1 protein, in addition to its function as a Na+/H+ antiporter, whose disruption affected membrane traffic and vacuolar functions possibly by controlling pH homeostasis in root cells.
Arabidopsis thaliana; endocytosis; salinity tolerance; SOS1
Sessile plants have developed a very delicate system to sense diverse kinds of endogenous developmental cues and exogenous environmental stimuli by using a simple Ca2+ ion. Calmodulin (CaM) is the predominant Ca2+ sensor and plays a crucial role in decoding the Ca2+ signatures into proper cellular responses in various cellular compartments in eukaryotes. A growing body of evidence points to the importance of Ca2+ and CaM in the regulation of the transcriptional process during plant responses to endogenous and exogenous stimuli. Here, we review recent progress in the identification of transcriptional regulators modulated by Ca2+ and CaM and in the assessment of their functional significance during plant signal transduction in response to biotic and abiotic stresses and developmental cues.
Abiotic/environmental stress; calcium signaling/transport; gene expression
Pn-AMP1, Pharbitis nil antimicrobial peptide 1, is a small cysteine-rich peptide implicated in host-plant defense. We show here that Pn-AMP1 causes depolarization of the actin cytoskeleton in Saccharomyces cerevisiae and Candida albicans. Pn-AMP1 induces rapid depolarization of actin cables and patches within 15 min. Increased osmolarity or temperature induces transient actin depolarization and results in increased sensitivity to Pn-AMP1, while cells conditioned to these stresses show less sensitivity. Mutations in components of a cell wall integrity pathway (Wsc1p, Rom2p, Bck1p and Mpk1p), which regulate actin repolarization, result in increased sensitivity to Pn-AMP1. A genetic screen reveals that mutations in components of the α-1,6-mannosyltransferase complex (Mnn10p, Mnn11p and Och1p), which regulate mannosylation of cell wall proteins, confer resistance to Pn-AMP1. FITC-conjugated Pn-AMP1 localizes to the outer surface of the cell with no significant staining observed in spheroplasts. Taken together, these results indicate that cell wall proteins are determinants of resistance to Pn-AMP1, and the ability of a plant defense protein to induce actin depolarization is important for its antifungal activity.
Actin cytoskeleton; Cell wall integrity pathway; Hevein-like peptide; Plant antifungal protein; Yeast
Loss-of-function siz1 mutations caused early flowering under short days. siz1 plants have elevated salicylic acid (SA) levels, which are restored to wild-type levels by expressing nahG, bacterial salicylate hydroxylase. The early flowering of siz1 was suppressed by expressing nahG, indicating that SIZ1 represses the transition to flowering mainly through suppressing SA-dependent floral promotion signaling under short days. Previous results have shown that exogenous SA treatment does not suppress late flowering of autonomous pathway mutants. However, the siz1 mutation accelerated flowering time of an autonomous pathway mutant, luminidependens, by reducing the expression of FLOWERING LOCUS C (FLC), a floral repressor. This result suggests that SIZ1 promotes FLC expression, possibly through an SA-independent pathway. Evidence indicates that SIZ1 is required for the full activation of FLC expression in the late-flowering FRIGIDA background. Interestingly, increased FLC expression and late flowering of an autonomous pathway mutant, flowering locus d (fld), was not suppressed by siz1, suggesting that SIZ1 promotes FLC expression by repressing FLD. Consistent with this, SIZ1 facilitates sumoylation of FLD that can be suppressed by mutations in three predicted sumoylation motifs in FLD (i.e. FLDK3R). Furthermore, expression of FLDK3R in fld protoplasts strongly reduced FLC transcription compared with expression of FLD, and this affect was linked to reduced acetylation of histone 4 in FLC chromatin. Taken together, the results suggest that SIZ1 is a floral repressor that not only represses the SA-dependent pathway, but also promotes FLC expression by repressing FLD activity through sumoylation, which is required for full FLC expression in a FRIGIDA background.
SIZ1; SA; flowering; SUMO; FLD; FLC
For microfabrications of biochips with micro fluidic channels, a large number of microfabrication techniques based on silicon or glass-based Micro-Electro-Mechanical System (MEMS) technologies were proposed in the last decade. In recent years, for low cost and mass production, polymer-based microfabrication techniques by microinjection molding and micro hot embossing have been proposed. These techniques, which require a proper photoresist, mask, UV light exposure, developing, and electroplating as a pre-process, are considered to have some problems. In this study, we propose a new microfabrication technology which consists of micro end-milling and powder blasting. This technique could be directly applied to fabricate the metal mold without any preprocesses. The metal mold with micro-channels is machined by micro end-milling, and then, burrs generated in the end-milling process are removed by powder blasting. From the experimental results, micro end-milling combined with powder blasting could be applied effectively for fabrication of the injection mold of biochips with micro fluidic channels.
Biochip; Micro Fluidic Channel; Powder Blasting; Micro End-milling; Deburring
The myristoylated calcium sensor SOS3 and its interacting protein kinase, SOS2, play critical regulatory roles in salt tolerance. Mutations in either of these proteins render Arabidopsis thaliana plants hypersensitive to salt stress. We report here the isolation and characterization of a mutant called enh1-1 that enhances the salt sensitivity of sos3-1 and also causes increased salt sensitivity by itself. ENH1 encodes a chloroplast-localized protein with a PDZ domain at the N-terminal region and a rubredoxin domain in the C-terminal part. Rubredoxins are known to be involved in the reduction of superoxide in some anaerobic bacteria. The enh1-1 mutation causes enhanced accumulation of reactive oxygen species (ROS), particularly under salt stress. ROS also accumulate to higher levels in sos2-1 but not in sos3-1 mutants. The enh1-1 mutation does not enhance sos2-1 phenotypes. Also, enh1-1 and sos2-1 mutants, but not sos3-1 mutants, show increased sensitivity to oxidative stress. These results indicate that ENH1 functions in the detoxification of reactive oxygen species resulting from salt stress by participating in a new salt tolerance pathway that may involve SOS2 but not SOS3.
Calmodulin (CaM) is involved in defense responses in plants. In soybean (Glycine max), transcription of calmodulin isoform 4 (GmCaM4) is rapidly induced within 30 min after pathogen stimulation, but regulation of the GmCaM4 gene in response to pathogen is poorly understood. Here, we used the yeast one-hybrid system to isolate two cDNA clones encoding proteins that bind to a 30-nt A/T-rich sequence in the GmCaM4 promoter, a region that contains two repeats of a conserved homeodomain binding site, ATTA. The two proteins, GmZF-HD1 and GmZF-HD2, belong to the zinc finger homeodomain (ZF-HD) transcription factor family. Domain deletion analysis showed that a homeodomain motif can bind to the 30-nt GmCaM4 promoter sequence, whereas the two zinc finger domains cannot. Critically, the formation of super-shifted complexes by an anti-GmZF-HD1 antibody incubated with nuclear extracts from pathogen-treated cells suggests that the interaction between GmZF-HD1 and two homeodomain binding site repeats is regulated by pathogen stimulation. Finally, a transient expression assay with Arabidopsis protoplasts confirmed that GmZF-HD1 can activate the expression of GmCaM4 by specifically interacting with the two repeats. These results suggest that the GmZF-HD1 and –2 proteins function as ZF-HD transcription factors to activate GmCaM4 gene expression in response to pathogen.
This study investigated the feasibility of the micro powder blasting technique for the micro fabrication of sensor structures using the Pyrex glass to replace the existing silicon-based acceleration sensor fabrication processes. As the preliminary experiments, the effects of the blasting pressure, the mass flow rate of abrasive and the number of nozzle scanning times on erosion depth of the Pyrex and the soda lime glasses were examined. From the experimental results, optimal blasting conditions were selected for the Pyrex glass machining. The dimensions of the designed glass sensor was 1.7×1.7×0.6mm for the vibrating mass, and 2.9×0.7×0.2mm for the cantilever beam. The machining results showed that the dimensional errors of the machined glass sensor ranged from 3 μm in minimum to 20 μm in maximum. These results imply that the micro powder blasting method can be applied for the micromachining of glass-based acceleration sensors to replace the exiting method.
Acceleration sensor; micro powder blasting; micromachining, masking, etching