Pollen tube cells elongate based on actin- dependent targeted secretion at the tip. Rho family small GTPases have been implicated in the regulation of related processes in animal and yeast cells. We have functionally characterized Rac type Rho family proteins that are expressed in growing pollen tubes. Expression of dominant negative Rac inhibited pollen tube elongation, whereas expression of constitutive active Rac induced depolarized growth. Pollen tube Rac was found to accumulate at the tip plasma membrane and to physically associate with a phosphatidylinositol monophosphate kinase (PtdIns P-K) activity. Phosphatidylinositol 4, 5-bisphosphate (PtdIns 4, 5-P2), the product of PtdIns P-Ks, showed a similar intracellular localization as Rac. Expression of the pleckstrin homology (PH)-domain of phospholipase C (PLC)-δ1, which binds specifically to PtdIns 4, 5-P2, inhibited pollen tube elongation. These results indicate that Rac and PtdIns 4, 5-P2 act in a common pathway to control polar pollen tube growth and provide direct evidence for a function of PtdIns 4, 5-P2 compartmentalization in the regulation of this process.

/pdf/rac-homologues-and-compartmentalized-phosphatidylinositol-4-32jfeqx6dr.pdf

Rac Homologues and Compartmentalized Phosphatidylinositol 4, 5-Bisphosphate Act in a Common Pathway to Regulate Polar Pollen Tube Growth

Triacylglycerol (TAG) biosynthesis is a principal metabolic pathway in most organisms, and TAG is the major form of carbon storage in many plant seeds. Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is the only acyltransferase enzyme that has been confirmed to contribute to TAG biosynthesis in Arabidopsis thaliana seeds. However, dgat1 null mutants display only a 20 to 40% decrease in seed oil content. To determine whether other enzymes contribute to TAG synthesis, candidate genes were expressed in TAG-deficient yeast, candidate mutants were crossed with the dgat1-1 mutant, and target genes were suppressed by RNA interference (RNAi). An in vivo role for phospholipid:diacylglycerol acyltransferase 1 (PDAT1; At5g13640) in TAG synthesis was revealed in this study. After failing to obtain double homozygous plants from crossing dgat1-1 and pdat1-2, further investigation showed that the dgat1-1 pdat1-2 double mutation resulted in sterile pollen that lacked visible oil bodies. RNAi silencing of PDAT1 in a dgat1-1 background or DGAT1 in pdat1-1 background resulted in 70 to 80% decreases in oil content per seed and in disruptions of embryo development. These results establish in vivo involvement of PDAT1 in TAG biosynthesis, rule out major contributions by other candidate enzymes, and indicate that PDAT1 and DGAT1 have overlapping functions that are essential for normal pollen and seed development of Arabidopsis.

/pdf/dgat1-and-pdat1-acyltransferases-have-overlapping-functions-ib4iebm2g6.pdf

DGAT1 and PDAT1 Acyltransferases Have Overlapping Functions in Arabidopsis Triacylglycerol Biosynthesis and Are Essential for Normal Pollen and Seed Development

Genome editing using the CRISPR/Cas9 system can be used to modify plant genomes, however, improvements in specificity and applicability are still needed in order for the editing technique to be useful in various plant species. Here, using genome editing mediated by a truncated gRNA (tru-gRNA)/Cas9 combination, we generated new alleles for OST2, a proton pump in Arabidopsis, with no off-target effects. By following expression of Cas9 and the tru-gRNAs, newly generated mutations in CRIPSR/Cas9 transgenic plants were detected with high average mutation rates of up to 32.8% and no off-target effects using constitutive promoter. Reducing nuclear localization signals in Cas9 decreased the mutation rate. In contrast, tru-gRNA Cas9 cassettes driven by meristematic- and reproductive-tissue-specific promoters increased the heritable mutation rate in Arabidopsis, showing that high expression in the germ line can produce bi-allelic mutations. Finally, the new mutant alleles obtained for OST2 exhibited altered stomatal closing in response to environmental conditions. These results suggest further applications in molecular breeding to improve plant function using optimized plant CRISPR/Cas9 systems.

/pdf/optimization-of-crispr-cas9-genome-editing-to-modify-abiotic-55j32rp0lz.pdf

Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants.

Benzophenones (BPs) are widely used in UV filters, fragrance enhancers, and plastic additives. In this study, the genotoxicity of 14 BPs was tested using the SOS/umu assay, and the related substituent contribution was disclosed. The results of this study revealed that the major contributor to the genotoxicity of the BPs was the ortho,para-di-substitution, and the increasing hydroxy substitution on the benzene ring. In addition, the higher the dispersion of the substituent species on the two benzene rings, the lower the genotoxicity exhibited by the compound. Furthermore, 2 dimensional and 3 dimenional quantitative structure-activity relationships (2D- and 3D-QSAR) studies indicated that hydrogen-bond interactions and electrostatic effects were determinants for the genotoxicity of the BPs. The current results provide useful information for the assessment of the potential ecological risk and health effects of BP-type UV filters.

Ecotoxicology and Environmental Safety

The Ecological Importance of Honey Bees and their Relevance to Ecotoxicology. Volatile and Semi-volatile Organic Compounds in Beehive Atmospheres. Risk Assessment of Plant Protection Products on Honey Bees: Regulatory Aspects. Acute Toxicity of Pesticides to Honey Bees. The Proboscis Extension Response: Assessing the Sublethal Effects of Pesticides on the Honey Bee. Effects of Imidacloprid on the Neural Processes of Memory in Honey Bees. Impact of Agrochemicals on Non-Apis Bees. Honey Bees as Indicators of Radionuclide Contamination: A Truly Useful Biomonitor. Cesium-134 and Cesium-137 in Fresh Honeys Collected after the Chernobyl Accident. The Role of Honey Bees in Environmental Monitoring in Croatia. Use of Honey Bees as Bioindicators of Environmental Pollution in Italy. Typology of French Acacia Honeys Based on their Concentrations in Metallic and Nonmetallic Elements. The Role of Insect-Resistant Transgenic Crops in Agriculture. Using Proteins to Assess the Potential Impacts of Genetically Modified Plants on Honey Bees. Direct and Indirect Effects of Genetically Modified Plants on the Honey Bee.

Honey Bees: Estimating the Environmental Impact of Chemicals

The expression of foreign genes in pollen may pose potential problems in the field release of transgenic plants, since pollen represents a route whereby foreign genes and their products may escape into the wider environment. The possible risks posed by crosshybridization with wild relatives have been extensively explored, but problems that may arise due to the expression of foreign gene products in pollen have not been so widely studied. The activities of the CaMV 35S and nos promoters in pollen in populations of stably transformed plants and in transient expression analysis are described. These promoters are commonly used in all areas of plant molecular biology research and their expression patterns will be of interest to those involved in field release studies. The results show that both promoters had no detectable pollen activity in Arabidopsis, but both showed activity in tobacco pollen. The CaMV 35S-gus gene fusion showed heritable expression levels in tobacco pollen of up to a maximum of 64.6 pmol 4-ML) min"1 mg ~1 total protein, nos promoter activity in transgenic tobacco pollen was highly variable, with GUS activities ranging from undetectable levels up to 2561 pmol 4-MU min"1 mg~1 total protein within the transgenic population. Histochemical staining of anther sections from 10-12 mm buds revealed that the CaMV 35S promoter had some activity in the vascular bundle, stomium and tapetum, while GUS expression from the nos promoter in sporophytic tissues was confined entirely to the stomium.

/pdf/activities-of-camv-35s-and-nos-promoters-in-pollen-3vdmmrlanr.pdf

Activities of CaMV 35S and nos promoters in pollen: implications for field release of transgenic plants

Methanol does not specifically inhibit endogenous β-glucuronidase (GUS) activity

Use of the GUS Reporter Gene

A potential problem in the field release of transgenic plants is the spread of foreign gene products via pollen. Therefore, the use of the tomato pollen-specific lat52 gene promoter was investigated as a means of targeting antisense RNA to pollen without affecting transgene expression elsewhere in the plant. A transgenic tobacco line, T115, which showed GUS expression in pollen, leaves and roots was retransformed with a construct containing the pollen-specific lat52 promoter driving the GUS encoding uid A gene in antisense orientation. From 24 independent transformants obtained, 19 showed a significant reduction in pollen GUS activity. Of these lines, four showed a reproducible antisense effect in pollen in the next generation, while it was shown in one line that GUS activity in leaves and roots was also unaffected. To ascertain the effectiveness of the antisense strategy to down-regulate very high levels of pollen expression, a lat52-gus antisense construct was introduced into tobacco lines containing lat52-gus, which had pollen GUS activity of up to 250 times greater than in line T115. Results showed that 30 out of 34 independent lines exhibited a significant antisense effect in pollen, confirming the effectiveness of pollen-targeted antisense startegy to reduce undesirable expression in pollen independent of expression level in pollen.

Antisense-mediated suppression of transgene expression targeted specifically to pollen

We have evaluated a novel system which allows the facile isolation of proteins and mRNA found specifically in wound site cells (Harikrishna et al. [1991] J.Ex. Bot. 42: 791–799) and has enabled the construction of cDNA libraries enriched for genes expressed at the wound surface. Standard differential and “cold plaque” screening indicated that over 40% of the clones in our cDNA libraries are differentially expressed in seedlings and wounded tissue. Sequence analysis of a number of wound-inducible clones has revealed that a diversity of novel genes are upregulated at wound sites, including a cytosolic Zn/Cu superoxide dismutase, a glycine-rich protein, S-Adenosyl-homocysteine hydrolase and a family of genes (AoPRl-3) related to the ‘intracellular’ class of PR proteins. These latter three gene families have been found by other laboratories to be upregulated in fungal elicitor-treated suspension cultured cells (Somssich et al. [1986], PNAS 83:2427–2430; Walter et al. [1990] MGG 222:353–360) and the data suggests that genes activated transiently at the wound surface (local induction) may be regulated by similar signal transduction pathways responsible for upregulating genes specifically induced around sites of attempted pathogen invasion in plants. The AoPRl gene family (Warner et al. [1992] PMB 19: 555–561) is massively and rapidly induced by wounding, its expression persisting for at least one week. A 1.1 kb upstream region of the AoPRl gene was fused to the GUS coding region and transferred to tobacco and potato. Histochemical analysis of transgenic plants (Warner et al [1992] The Plant J. In Press) has indicated that GUS activity is rapidly induced in the locality of the wound surface and around hypersensitive and necrotic lesions caused by fungal (Botrytis cinerea) and viral attack (PVY and TNV).

Gene Expression at Sites of Wounding, Pathogen Invasion & Phenylpropanoid Synthesis in Monocots and Dicots

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