Stress- and phospholipid signalling responses in Arabidopsis PLC4-KO and -overexpression lines under salt- and osmotic stress.

Several drought and salt tolerant phenotypes have been reported when overexpressing (OE) phospholipase C (PLC) genes across plant species. In contrast, a negative role for Arabidopsis PLC4 in salinity stress was recently proposed, showing that roots of PLC4-OE seedlings were more sensitive to NaCl while plc4 knock-out (KO) mutants were more tolerant. To investigate this apparent contradiction, and to analyse the phospholipid signalling responses associated with salinity stress, we performed root growth- and phospholipid analyses on plc4-KO and PLC4-OE seedlings subjected to salinity (NaCl) or osmotic (sorbitol) stress and compared these with wild type (WT). Only very minor differences between PLC4 mutants and WT were observed, which even disappeared after normalization of the data, while in soil, PLC4-OE plants were clearly more drought tolerant than WT plants, as was found earlier when overexpressing Arabidopsis PLC2, -3, -5, -7 or -9. We conclude that PLC4 plays no opposite role in salt-or osmotic stress and rather behaves like the other Arabidopsis PLCs.

The dynamics of touch-responsive gene expression in cereals.

Wind, rain, herbivores, obstacles, neighbouring plants, etc. provide important mechanical cues to steer plant growth and survival. Mechanostimulation to stimulate yield and stress resistance of crops is of significant research interest, yet a molecular understanding of transcriptional responses to touch is largely absent in cereals. To address this, we performed whole-genome transcriptomics following mechanostimulation of wheat, barley, and the recent genome-sequenced oat. The largest transcriptome changes occurred ±25 min after touching, with most of the genes being upregulated. While most genes returned to basal expression level by 1-2 h in oat, many genes retained high expression even 4 h post-treatment in barley and wheat. Functional categories such as transcription factors, kinases, phytohormones, and Ca2+ regulation were affected. In addition, cell wall-related genes involved in (hemi)cellulose, lignin, suberin, and callose biosynthesis were touch-responsive, providing molecular insight into mechanically induced changes in cell wall composition. Furthermore, several cereal-specific transcriptomic footprints were identified that were not observed in Arabidopsis. In oat and barley, we found evidence for systemic spreading of touch-induced signalling. Finally, we provide evidence that both the jasmonic acid-dependent and the jasmonic acid-independent pathways underlie touch-signalling in cereals, providing a detailed framework and marker genes for further study of (a)biotic stress responses in cereals.

Characterization of Histone H3 Gene Family Reveals That GmHH3-3 is Associated With Higher Seed Weight in Glycine max.

The main function of histone protein is to provide support to the structure of chromosomes. It helps in binding a long thread of DNA into a more condensed shape to fit into the nucleus. From histone variants, histone H3 (HH3) plays a crucial role in plant growth and development. Characterization of histones has not been reported in Glycine max till now. The objective of this study was to characterize the HH3 gene family for molecular breeding of G. max. In this study, 17 HH3 members in G. max were identified by performing local BLASTp using HH3 members from Arabidopsis as a query. Phylogenetic analysis classified HH3 genes in seven clades. Sequence logo analysis among Arabidopsis thaliana, Oryza sativa, and Glycine max showed a higher level of similarity in amino acids. Furthermore, conserveness of G. max HH3 genes was also confirmed by Gene Structure Display. Ten paralogous gene pairs were identified in GmHH3 genes in the Glycine max genome by conducting collinearity analysis. G. max HH3 genes have experienced strong purifying selection pressure, with limited functional divergence originating from the segmental and whole-genome duplication, as evidenced by the Ka/Ks ratio. The KASP marker was developed for GmHH3-3 gene. Genotyping was performed on 46 G. max genotypes. This differentiation was based upon the presence of either GmHH3-3-C or GmHH3-3-T allele in the CDS region. The results showed that G. max accessions containing the GmHH3-3-T allele at respective locus showed higher thousand seed weight than that of those accessions that contain the GmHH3-3-C allele. This research provides the basic information to further decipher the function of HH3 in soybean.

MCU proteins dominate in vivo mitochondrial Ca2+ uptake in Arabidopsis roots.

Ca2+ signaling is central to plant development and acclimation. While Ca2+-responsive proteins have been investigated intensely in plants, only a few Ca2+-permeable channels have been identified, and our understanding of how intracellular Ca2+ fluxes is facilitated remains limited. Arabidopsis thaliana homologs of the mammalian channel-forming mitochondrial calcium uniporter (MCU) protein showed Ca2+ transport activity in vitro. Yet, the evolutionary complexity of MCU proteins, as well as reports about alternative systems and unperturbed mitochondrial Ca2+ uptake in knockout lines of MCU genes, leave critical questions about the in vivo functions of the MCU protein family in plants unanswered. Here, we demonstrate that MCU proteins mediate mitochondrial Ca2+ transport in planta and that this mechanism is the major route for fast Ca2+ uptake. Guided by the subcellular localization, expression, and conservation of MCU proteins, we generated an mcu triple knockout line. Using Ca2+ imaging in living root tips and the stimulation of Ca2+ transients of different amplitudes, we demonstrated that mitochondrial Ca2+ uptake became limiting in the triple mutant. The drastic cell physiological phenotype of impaired subcellular Ca2+ transport coincided with deregulated jasmonic acid-related signaling and thigmomorphogenesis. Our findings establish MCUs as a major mitochondrial Ca2+ entry route in planta and link mitochondrial Ca2+ transport with phytohormone signaling.

Visual Outcomes of Microkeratome-Assisted Anterior Lamellar Keratoplasty in Keratoconus: 5-Year Results.

Purpose: To report 5-year results of microkeratome-assisted anterior lamellar keratoplasty (MK-ALK) in cases of keratoconus. Methods: Patients with advanced keratoconus and the thinnest corneal location 300 µ or more were recruited. A Carriazo-Barraquer microkeratome was used to remove a 200-µ cap from the recipient cornea, and to prepare a 300-µ anterior stromal graft from a donor cornea. A full-thickness crescentic incision was made in the posterior stromal recipient bed using a 6.5-mm suction trephine. The donor was sutured to the recipient bed using interrupted nylon sutures. The minimum follow-up was five years. Results: Twelve eyes of 12 patients were included. The mean age was 26 ± 8 years. None of the patients required conversion to penetrating keratoplasty. Mean logMAR uncorrected and best spectacle-corrected visual acuity, respectively, improved from 1.56 ± 0.24 and 1.18 ± 0.32 preoperatively, to 0.63 ± 0.38 and 0.18 ± 0.12, five years after surgery (P=0.001for both). There was also a statistically significant reduction of mean manifest spherical equivalent, refractive cylinder, and mean keratometry readings. Posterior stromal striations occurred in all patients immediately after surgery but resolved after a maximum of 3 months. At five-years, anterior segment optical coherence tomography revealed a clear interface in all cases and a mean graft thickness of 328 ± 27 µ. Conclusion: MK-ALK is a safe and effective procedure for advanced keratoconus. Where feasible, it may be the best choice for patients at high risk of poor outcomes with penetrating keratoplasty, such as young patients with atopic keratoconjunctivitis or Down's syndrome.

Touch signaling and thigmomorphogenesis are regulated by complementary CAMTA3- and JA-dependent pathways.

Plants respond to mechanical stimuli to direct their growth and counteract environmental threats. Mechanical stimulation triggers rapid gene expression changes and affects plant appearance (thigmomorphogenesis) and flowering. Previous studies reported the importance of jasmonic acid (JA) in touch signaling. Here, we used reverse genetics to further characterize the molecular mechanisms underlying touch signaling. We show that Piezo mechanosensitive ion channels have no major role in touch-induced gene expression and thigmomorphogenesis. In contrast, the receptor-like kinase Feronia acts as a strong negative regulator of the JA-dependent branch of touch signaling. Last, we show that calmodulin-binding transcriptional activators CAMTA1/2/3 are key regulators of JA-independent touch signaling. CAMTA1/2/3 cooperate to directly bind the promoters and activate gene expression of JA-independent touch marker genes like TCH2 and TCH4. In agreement, camta3 mutants show a near complete loss of thigmomorphogenesis and touch-induced delay of flowering. In conclusion, we have now identified key regulators of two independent touch-signaling pathways.

Phenotyping Tomato Root Developmental Plasticity in Response to Salinity in Soil Rhizotrons.

Plants have developed multiple strategies to respond to salt stress. In order to identify new traits related to salt tolerance, with potential breeding application, the research focus has recently been shifted to include root system architecture (RSA) and root plasticity. Using a simple but effective root phenotyping system containing soil (rhizotrons), RSA of several tomato cultivars and their response to salinity was investigated. We observed a high level of root plasticity of tomato seedlings under salt stress. The general root architecture was substantially modified in response to salt, especially with respect to position of the lateral roots in the soil. At the soil surface, where salt accumulates, lateral root emergence was most strongly inhibited. Within the set of tomato cultivars, H1015 was the most tolerant to salinity in both developmental stages studied. A significant correlation between several root traits and aboveground growth parameters was observed, highlighting a possible role for regulation of both ion content and root architecture in salt stress resilience.

Efficacy of addition of nepafenac 0.1% to steroid eye drops in prevention of post-phaco macular edema in high-risk eyes.

PURPOSE: To compare the efficacy of addition of nonsteroidal anti-inflammatory eye drops to steroidal eye drops with that of using postoperative steroidal anti-inflammatory eye drops alone in prevention of macular edema in high-risk patients. SETTING: Cairo University Hospital. DESIGN: This study was comparative prospective interventional randomized study. METHODS: This study included 100 cataractous eyes divided into five subgroups: 20 eyes of diabetic patients, 20 uveitic eyes, 20 traumatic cataracts, 20 glaucomatous eyes on topical prostaglandin analogs, and 20 eyes with posterior capsular rupture during phacoemulsification. Each subgroup of 20 was randomized between two groups of 10 eyes, group A received postoperative topical steroids alone and group B received both steroidal and nonsteroidal anti-inflammatory eye drops. RESULTS: There was significant increase in postoperative central foveal thickness as compared to preoperative values in both groups (60.9 ± 87.95 µ in group A and 25.52 ± 57.26 µ in group B) that was significantly more in group A (P value 0.016). There was significant difference in postoperative macular thickness between both groups (280.1 ± 86.0 µ and 246.80 ± 57.73 µ, respectively, in groups A and B) (P value = 0.012). There was no statistically significant difference between both groups in preoperative and postoperative corrected distance visual acuity and intraocular pressure. CONCLUSION: Addition of topical nepafenac eye drops to topical steroid drops significantly reduced the amount of pseudophakic macular edema after cataract surgery in high-risk eyes.

Halotropism is a response of plant roots to avoid a saline environment.

Tropisms represent fascinating examples of how plants respond to environmental signals by adapting their growth and development. Here, a novel tropism is reported, halotropism, allowing plant seedlings to reduce their exposure to salinity by circumventing a saline environment. In response to a salt gradient, Arabidopsis, tomato, and sorghum roots were found to actively prioritize growth away from salinity above following the gravity axis. Directionality of this response is established by an active redistribution of the plant hormone auxin in the root tip, which is mediated by the PIN-FORMED 2 (PIN2) auxin efflux carrier. We show that salt-induced phospholipase D activity stimulates clathrin-mediated endocytosis of PIN2 at the side of the root facing the higher salt concentration. The intracellular relocalization of PIN2 allows for auxin redistribution and for the directional bending of the root away from the higher salt concentration. Our results thus identify a cellular pathway essential for the integration of environmental cues with auxin-regulated root growth that likely plays a key role in plant adaptative responses to salt stress.

Effect of anodal versus cathodal transcranial direct current stimulation on stroke rehabilitation: a pilot randomized controlled trial.

OBJECTIVE: We compared the long-term effect of anodal versus cathodal transcranial direct current stimulation (tDCS) on motor recovery in patients after subacute stroke. METHODS: Forty patients with ischemic stroke undergoing rehabilitation were randomly assigned to 1 of 3 groups: Anodal, Cathodal (over-affected and unaffected hemisphere, respectively), and Sham. Each group received tDCS at an intensity of 2 mA for 25 minutes daily for 6 consecutive days over of the motor cortex hand area. Patients were assessed with the National Institutes of Health Stroke Scale (NIHSS), Orgogozo's MCA scale (OMCASS), the Barthel index (BI), and the Medical Research Council (MRC) muscle strength scale at baseline, after the sixth tDCS session and then 1, 2, and 3 months later. Motor cortical excitability was measured with transcranial magnetic stimulation (TMS) at baseline and after the sixth session. RESULTS: By the 3-month follow-up, all groups had improved on all scales with P values ranging from .01 to .0001. Improvement was equal in the Anodal and Cathodal groups. When these treated groups were combined and compared with Sham, significant interactions were seen for the OMCASS and BI scales of functional ability (P = .002 for each). There was increased cortical excitability of the affected hemisphere in all groups with the changes being greater in the real versus sham groups. There were borderline significant improvements in muscle strength. CONCLUSION: A brief course of 2 types of tDCS stimulation is superior to sham stimulation in enhancing the effect of rehabilitation training to improve motor recovery after stroke.

Heat stress activates phospholipase D and triggers PIP accumulation at the plasma membrane and nucleus.

Heat stress induces an array of physiological adjustments that facilitate continued homeostasis and survival during periods of elevated temperatures. Here, we report that within minutes of a sudden temperature increase, plants deploy specific phospholipids to specific intracellular locations: phospholipase D (PLD) and a phosphatidylinositolphosphate kinase (PIPK) are activated, and phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PIP(2)) rapidly accumulate, with the heat-induced PIP(2) localized to the plasma membrane, nuclear envelope, nucleolus and punctate cytoplasmic structures. Increases in the steady-state levels of PA and PIP(2) occur within several minutes of temperature increases from ambient levels of 20-25 degrees C to 35 degrees C and above. Similar patterns were observed in heat-stressed Arabidopsis seedlings and rice leaves. The PA that accumulates in response to temperature increases results in large part from the activation of PLD rather than the sequential action of phospholipase C and diacylglycerol kinase, the alternative pathway used to produce this lipid. Pulse-labelling analysis revealed that the PIP(2) response is due to the activation of a PIPK rather than inhibition of a lipase or a PIP(2) phosphatase. Inhibitor experiments suggest that the PIP(2) response requires signalling through a G-protein, as aluminium fluoride blocks heat-induced PIP(2) increases. These results are discussed in the context of the diverse cellular roles played by PIP(2) and PA, including regulation of ion channels and the cytoskeleton.

Phospholipid signaling responses in salt-stressed rice leaves.

Salinity is one of the major environmental factors limiting growth and productivity of rice plants. In this study, the effect of salt stress on phospholipid signaling responses in rice leaves was investigated. Leaf cuts were radiolabeled with 32P-orthophosphate and the lipids extracted and analyzed by thin-layer chromatography, autoradiography and phosphoimaging. Phospholipids were identified by co-migration of known standards. Results showed that 32P(i) was rapidly incorporated into the minor lipids, phosphatidylinositol bisphosphate (PIP2) and phosphatidic acid (PA) and, interestingly, also into the structural lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), which normally label relatively slowly, like phosphatidylcholine (PC) and phosphatidylinositol (PI). Only very small amounts of PIP2 were found. However, in response to salt stress (NaCl), PIP2 levels rapidly (<30 min) increased up to 4-fold, in a time- and dose-dependent manner. PA and its phosphorylated product, diacylglycerolpyrophosphate (DGPP), also increased upon NaCl stress, while cardiolipin (CL) levels decreased. All other phospholipid levels remained unchanged. PA signaling can be generated via the combined action of phospholipase C (PLC) and diacylglycerol kinase (DGK) or directly via phospholipase D (PLD). The latter can be measured in vivo, using a transphosphatidylation assay. Interestingly, these measurements revealed that salt stress inhibited PLD activity, indicating that the salt stress-induced PA response was not due to PLD activity. Comparison of the 32P-lipid responses in salt-tolerant and salt-sensitive cultivars revealed no significant differences. Together these results show that salt stress rapidly activates several lipid responses in rice leaves but that these responses do not explain the difference in salt tolerance between sensitive and tolerant cultivars.

Structure and sensory equipment of the ovipositor of Habrobracon hebetor (Say) (Hymenoptera: Braconidae).

The microsculpture of various structures of the ovipositor of Habrobracon hebetor (Say) (Hymenoptera: Braconidae) is described from scanning and transmission electron microscopy. These include: the ovipositor egg canal, valvillus, seal of the first valvulae, interlocking mechanism (olistheter) connecting the first and second valvulae, an olistheter-like interlocking mechanism connecting the two pieces of the first valvulae, annulation, microtrichia of the third valvulae, and the ovipositor sensory equipment. Better understanding of the microsculpture of these components may make their roles in stinging, oviposition, and the host selection process more clear.

Pathogenicity of two entomopathogenic hyphomycetes, Beauveria bassiana and Metarhizium anisopliae, to the housefly Musca domestica L.

The pathogenicity of three isolates of Metarhizium anisopliae and two isolates Beauveria bassiana originating from different insect species and localities was tested against housefly larvae. Only one isolate of M. anisopliae (Ma23) and the two isolates of B. bassiana (Bb2, Bb108) significantly (P<0.05) reduced the percentage of emerging adults. At the highest tested concentration 10(7) spores/ml. the reduction in the percentage of adult emergence was 93.8%. 61.3% and 49% for Bb2. Ma23 and Bb108. respectively. Based on the IC50 values Bb2 was the most virulent isolate followed by Ma23 and Bb108, (1.13. 1.7 and 4 x 10(6). respectively). The ability of adult flies to pick up conidiospores from contaminated media as well as the possibility of isolate recycling through cadavers of fungus-killed adults were also investigated. Adults were able to pick up conidiospores from the contaminated media. All contaminated adult cadavers subsequently sporulated when placed under high relative humidity conditions. When adult flies were exposed to mycotised cadavers 12. 75 and 76% of the adults exposed to Bb2, Ma23 and Bb108, respectively sporulated under high relative humidity conditions.