Expansin genes expression in growing ovaries and grains of sunflower are tissue-specific and associate with final grain weight.

BACKGROUND: Grain weight (GW) is a key component of sunflower yield and quality, but may be limited by maternal tissues. Cell growth is influenced by expansin proteins that loosen the plant cell wall. This study aimed to identify spatio-temporal expression of EXPN genes in sunflower reproductive organ tissues (ovary, pericarp, and embryo) and evaluate correlations between reproductive organ growth and expansin genes expression. Evaluations involved eight different developmental stages, two genotypes, two source-sink treatments and two experiments. The genotypes evaluated are contrasting in GW (Alybro and confection variety RHA280) under two source-sink treatments (control and shaded) to study the interactions between grain growth and expansin genes expression. RESULTS: Ovaries and grains were sampled at pre- and post-anthesis, respectively. Final GW differed between genotypes and shading treatments. Shading treatment decreased final GW by 16.4 and 19.5% in RHA280 and Alybro, respectively. Relative expression of eight expansin genes were evaluated in grain tissues. EXPN4 was the most abundant expansin in the ovary tissue, while EXPN10 and EXPN7 act predominantly in ovary and pericarp tissues, and EXPN1 and EXPN15 in the embryo tissues. CONCLUSIONS: Specific expansin genes were expressed in ovary, pericarp and embryo in a tissue-specific manner. Differential expression among grain tissues was consistent between genotypes, source-sink treatments and experiments. The correlation analysis suggests that EXPN genes could be specifically involved in grain tissue extension, and their expression could be linked to grain size in sunflower.

Effect of tacrolimus on activity and expression of P-glycoprotein and ATP-binding cassette transporter A5 (ABCA5) proteins in hematoencephalic barrier cells.

Tacrolimus is an agent used in clinical immunosuppressive drug therapies. A wide spectrum of adverse effects has been reported in association with this immunosuppressor, including neurotoxic effect. The upper limit of therapeutic blood concentrations of tacrolimus has been described as 30 ng/ml in immunosuppressed patients. We investigated the effect of this therapeutic dose of tacrolimus on the expression and activity of the multidrug resistance protein 1 (MDR1 or Pgp, P-glycoprotein) and ATP-binding cassette transporters A5 (ABCA5) in human brain microvascular endothelial cells (HBMEC), derived from Blood-Brain Barrier (BBB) endothelium, these being the most predominantly expressed transcripts in these cells. The expression and activity of MDR1 transporter decreased with 30 ng/ml tacrolimus. The cell viability was not changed with the therapeutic dose used. By contrast, ABCA5 transcripts, of unknown role as yet, increased their expression at this concentration. We propose that the secondary cytotoxic effects of this immunosuppressor on CSN, besides the functional blockade related to multidrug resistance proteins, such as MDR1, and probably ABCA5, could be linked to variations in the expression levels of these proteins at the BBB.

Distinct functions for Arf guanine nucleotide exchange factors at the Golgi complex: GBF1 and BIGs are required for assembly and maintenance of the Golgi stack and trans-Golgi network, respectively.

We examined the relative function of the two classes of guanine nucleotide exchange factors (GEFs) for ADP-ribosylation factors that regulate recruitment of coat proteins on the Golgi complex. Complementary overexpression and RNA-based knockdown approaches established that GBF1 regulates COPI recruitment on cis-Golgi compartments, whereas BIGs appear specialized for adaptor proteins on the trans-Golgi. Knockdown of GBF1 and/or COPI did not prevent export of VSVGtsO45 from the endoplasmic reticulum (ER), but caused its accumulation into peripheral vesiculotubular clusters. In contrast, knockdown of BIG1 and BIG2 caused loss of clathrin adaptor proteins and redistribution of several TGN markers, but had no impact on COPI and several Golgi markers. Surprisingly, brefeldin A-inhibited guanine nucleotide exchange factors (BIGs) knockdown prevented neither traffic of VSVGtsO45 to the plasma membrane nor assembly of a polarized Golgi stack. Our observations indicate that COPII is the only coat required for sorting and export from the ER exit sites, whereas GBF1 but not BIGs, is required for COPI recruitment, Golgi subcompartmentalization, and cargo progression to the cell surface.

GBF1, a cis-Golgi and VTCs-localized ARF-GEF, is implicated in ER-to-Golgi protein traffic.

The formation and maturation of membrane carriers that transport cargo from the ER to the Golgi complex involves the sequential action of the coat protein complexes COPII and COPI. Recruitment of COPI to nascent carriers requires activation of ADP-ribosylation factors by a BrefeldinA-sensitive guanine nucleotide exchange factor. Using new antisera and a GFP-tagged protein, we demonstrate that the exchange factor GBF1 localized to both Golgi membranes and peripheral puncta, near but separate from ER exit sites. Live cell imaging revealed that GFP-GBF1 associates dynamically with both membranes through rapid exchange with a large cytosolic pool. Treatment with BrefeldinA dramatically altered this rapid exchange, causing accumulation of GBF1 on both Golgi and peripheral puncta before eventual redistribution to the ER in a microtubule-dependent manner. Measurement of diffusion coefficients and subcellular fractionation confirmed this shift in GBF1 from cytosolic to membrane bound. BrefeldinA-induced accumulation of GBF1 coincided with loss of COPI from peripheral puncta. Furthermore, recruitment of GBF1 to cargo-containing peripheral puncta coincided with recruitment of COPI, but not COPII. Strikingly, microinjection of anti-GBF1 antibodies specifically caused dissociation of COPI from membranes. These observations strongly suggest that GBF1 regulates COPI membrane recruitment in the early secretory pathway.

Characterization of alternatively spliced and truncated forms of the Arf guanine nucleotide exchange factor GBF1 defines regions important for activity.

Analysis of multiple transcripts for the Arf-specific guanine nucleotide exchange factor GBF1 identified three positions displaying small in-frame deletions and insertions. Sequencing of genomic DNA for CHO GBF1 and analysis of the human gene established that those variations were consistent with alternate splicing events. RT-PCR analysis of CHO mRNA confirmed that these small in-frame deletions occurred at significant and similar frequencies in both WT and BFA resistant CHO cells. These splice variants behaved like GBF1 in biological assays based on the observation that GBF1 is cytotoxic at high levels but will confer resistance to BFA when moderately overexpressed. Comparison of variants with larger deletions defined regions of 75 (exons 5-7) and 412 (exons 31-39) amino acid residues that were required for cell killing but were dispensable for promoting BFA resistance.