An Arabidopsis protoplast isolation method reduces cytosolic acidification and activation of the chloroplast stress sensor SENSITIVE TO FREEZING 2

Chloroplasts adapt to freezing and other abiotic stresses in part by modifying their membranes. One key-remodeling enzyme is SENSITIVE TO FREEZING2 (SFR2). SFR2 is unusual because it does not respond to initial cold stress or cold acclimation, instead it responds during freezing conditions in Arabidopsis. This response has been shown to be sensitive to cytosolic acidification. The unique lipid products of SFR2 have also been detected in response to non-freezing stresses, but what causes SFR2 to respond in these stresses is unknown. Here, we investigate protoplast isolation as a representative of wounding stress. We show that SFR2 oligogalactolipid products accumulate during protoplast isolation. Notably, we show that protoplast cytosol is acidified during isolation. Modification of the buffers reduces oligogalactolipid accumulation, while prolonged incubation in the isolated state increases it. We conclude that SFR2 activation during protoplast isolation correlates with cytosolic acidification, implying that all SFR2 activation may be dependent on cytosolic acidification. We also conclude that protoplasts can be more gently isolated, reducing their stress

Phylloquinone (vitamin K 1 ) biosynthesis in plants: two peroxisomal thioesterases of lactobacillales origin hydrolyze 1,4‐dihydroxy‐2‐naphthoyl‐coa

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/1/TPJ_4972_sm_FigS3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/2/TPJ_4972_sm_TableS1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/3/TPJ_4972_sm_FigS2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/4/TPJ_4972_sm_TableS4.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/5/TPJ_4972_sm_FigS6.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/6/j.1365-313X.2012.04972.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/7/TPJ_4972_sm_FigS1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/8/TPJ_4972_sm_TableS3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/9/TPJ_4972_sm_FigS5.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/10/TPJ_4972_sm_TableS2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/92396/11/TPJ_4972_sm_FigS4.pd

An Arabidopsis protoplast isolation method reduces cytosolic acidification and activation of the chloroplast stress sensor SENSITIVE TO FREEZING 2

Chloroplasts adapt to freezing and other abiotic stresses in part by modifying their membranes. One key-remodeling enzyme is SENSITIVE TO FREEZING2 (SFR2). SFR2 is unusual because it does not respond to initial cold stress or cold acclimation, instead it responds during freezing conditions in Arabidopsis. This response has been shown to be sensitive to cytosolic acidification. The unique lipid products of SFR2 have also been detected in response to non-freezing stresses, but what causes SFR2 to respond in these stresses is unknown. Here, we investigate protoplast isolation as a representative of wounding stress. We show that SFR2 oligogalactolipid products accumulate during protoplast isolation. Notably, we show that protoplast cytosol is acidified during isolation. Modification of the buffers reduces oligogalactolipid accumulation, while prolonged incubation in the isolated state increases it. We conclude that SFR2 activation during protoplast isolation correlates with cytosolic acidification, implying that all SFR2 activation may be dependent on cytosolic acidification. We also conclude that protoplasts can be more gently isolated, reducing their stress

A morphological analysis of a hybrid swarm of native Ulmus rubra Muhl. and introduced U. pumila L. (Ulmaceae) in southeastern Nebraska

Volume: 2013-44Start Page: 1End Page: 2

Dual-Domain, Dual-Targeting Organellar Protein Presequences in Arabidopsis Can Use Non-AUG Start Codons

The processes accompanying endosymbiosis have led to a complex network of interorganellar protein traffic that originates from nuclear genes encoding mitochondrial and plastid proteins. A significant proportion of nucleus-encoded organellar proteins are dual targeted, and the process by which a protein acquires the capacity for both mitochondrial and plastid targeting may involve intergenic DNA exchange coupled with the incorporation of sequences residing upstream of the gene. We evaluated targeting and sequence alignment features of two organellar DNA polymerase genes from Arabidopsis thaliana. Within one of these two loci, protein targeting appeared to be plastidic when the 5′ untranslated leader region (UTR) was deleted and translation could only initiate at the annotated ATG start codon but dual targeted when the 5′ UTR was included. Introduction of stop codons at various sites within the putative UTR demonstrated that this region is translated and influences protein targeting capacity. However, no ATG start codon was found within this upstream, translated region, suggesting that translation initiates at a non-ATG start. We identified a CTG codon that likely accounts for much of this initiation. Investigation of the 5′ region of other nucleus-encoded organellar genes suggests that several genes may incorporate upstream sequences to influence targeting capacity. We postulate that a combination of intergenic recombination and some relaxation of constraints on translation initiation has acted in the evolution of protein targeting specificity for those proteins capable of functioning in both plastids and mitochondria

Combined whole-organ imaging at single-cell resolution and immunohistochemical analysis of prostate cancer and its liver and brain metastases.

Early steps of cancer initiation and metastasis, while critical for understanding disease mechanisms, are difficult to visualize and study. Here, we describe an approach to study the processes of initiation, progression, and metastasis of prostate cancer (PC) in a genetically engineered RapidCaP mouse model, which combines whole-organ imaging by serial two-photon tomography (STPT) and post hoc thick-section immunofluorescent (IF) analysis. STPT enables the detection of single tumor-initiating cells within the entire prostate, and consequent IF analysis reveals a transition from normal to transformed epithelial tissue and cell escape from the tumor focus. STPT imaging of the liver and brain reveal the distribution of multiple metastatic foci in the liver and an early-stage metastatic cell invasion in the brain. This imaging and data analysis pipeline can be readily applied to other mouse models of cancer, offering a highly versatile whole-organ platform to study in situ mechanisms of cancer initiation and progression