REGULATOR OF BULB BIOGENESIS1 (RBB1) Is Involved in Vacuole Bulb Formation in Arabidopsis

<div><p>Vacuoles are dynamic compartments with constant fluctuations and transient structures such as trans-vacuolar strands and bulbs. Bulbs are highly dynamic spherical structures inside vacuoles that are formed by multiple layers of membranes and are continuous with the main tonoplast. We recently carried out a screen for mutants with abnormal trafficking to the vacuole or aberrant vacuole morphology. We characterized <i>regulator of bulb biogenesis1-1</i> (<i>rbb1-1</i>), a mutant in Arabidopsis that contains increased numbers of bulbs when compared to the parental control. <i>rbb1-1</i> mutants also contain fewer transvacuolar strands than the parental control, and we propose the hypothesis that the formation of transvacuolar strands and bulbs is functionally related. We propose that the bulbs may function transiently to accommodate membranes and proteins when transvacuolar strands fail to elongate. We show that RBB1 corresponds to a very large protein of unknown function that is specific to plants, is present in the cytosol, and may associate with cellular membranes. RBB1 is involved in the regulation of vacuole morphology and may be involved in the establishment or stability of trans-vacuolar strands and bulbs.</p></div

<i>regulator of bulb biogenesis1</i> contains many bulbs in many cell types.

<p>(a-h) <i>rbb1-1</i> mutants have more bulbs than the parental line. GFP-TIP2;1 localization in the parental line (WT, a-d) or <i>rbb1-1</i> (e-h). Four-day-old seedlings were imaged by confocal microscopy to visualize morphology of the vacuole in the epidermis of hypocotyl (a, e) and cotyledon (b, f), and in root epidermis and cortex (c, g). The bulb phenotype was also observed in rosette leaves of 6-week-old plants (d, h). Scale bar = 20 μm. (i-n) GFP-TIP2;1 and RFP-TIP1;1 co-localize in <i>rbb1-1</i> bulbs. The hypocotyls of 4-day-old dark-grown seedlings expressing GFP-TIP2;1 (green, i, l) and RFP-TIP1;1 (magenta, j, m) in the parental line (i-k) and <i>rbb1-1</i> mutants (l-n) are shown. Merged images (k, n) are also shown. Scale bar: 20 μm. (o) <i>rbb1-1</i> mutants do not accumulate higher levels of GFP-TIP2;1 in seedlings. Immunoblot of GFP-TIP2;1 accumulation in the parental line and <i>rbb1-1</i> using antibodies against GFP (α-GFP) and Calreticulin (α-CRT) as loading control.</p

GFP-TIP2;1 trafficking to the vacuole is similar between <i>rbb1-1</i> and the parental line.

<p>(a-f) BFA inhibits trafficking of TIP1;1YFP but not GFP-TIP2;1 in the parental line or <i>rbb1-1</i>. Four-day-old seedlings from the parental (GFP-TIP2;1, a, d), <i>rbb1-1</i> (b, e) or TIP1;1-YFP (c, f) were exposed to 0.1% DMSO (control, a, b, c) or 75 μM BFA (d, e, f) for 3 hours and hypocotyl cells were imaged. BFA compartments are indicated with arrows. Scale: 20 μm. (g-r) C834 inhibits GFP-TIP2;1 trafficking in the parental line and <i>rbb1-1</i>. Three-day-old seedlings from the parental line and <i>rbb1-1</i> were exposed to 0.5% DMSO (g-i, m-o) or 55 μM C834 (j-l, p-r) for 48 h, and root cells were imaged in the microscope. Signal from GFP-TIP2;1 (green, g, j, m, p), the ER marker mCherry-HDEL (magenta, h, k, n, p) or the merged image is shown (i, l, o, r). Co-localization of GFP with mCherry-HDEL at the ER is indicated with arrowheads. Scale bar: 20 μm.</p

The <i>RBB1</i> locus corresponds to <i>At5g40450</i>.

<p>(a) Schematic structure of the <i>RBB1</i> locus indicating the positions of <i>rbb1</i> alleles. Exons are indicated as black boxes and introns are indicated as white boxes. 5' and 3' UTRs are indicated in gray. Triangles indicate T-DNA insertions of SALK lines. The position of three diagnostic RT-PCR amplicons at the 5’ end, middle and 3’ end are shown. (b) Accumulation of <i>RBB1</i> transcripts as determined by RT-PCR with gene specific primers in seedlings from Col-0 (WT), <i>rbb1-2</i>, the GFP-TIP2;1 parental line and <i>rbb1-1</i>. Three pairs of gene specific primers were used to detect the transcript at the 5' end, middle, and 3' end of the <i>RBB1</i> gene as indicated in (a). Numbers represent amplicon size. (c-e) Lack of genetic complementation between <i>rbb1-1</i> and <i>rbb1-2</i>. Hypocotyls of four-day-old dark-grown seedlings from the parental control (WT, c), <i>rbb1-1</i> (d) and the F₁ progeny from a cross between <i>rbb1-1</i> and <i>rbb1-2</i> (<i>rbb1-1/rbb1-2</i> double heterozygotes, e) were imaged by confocal microscopy. Scale bar: 20μm. (f-g) <i>rbb1-2</i> contains bulbs in a similar manner as <i>rbb1-1</i>. Four-day-old seedlings were stained with 2 μM Lysotracker Red for 2 hours to label the vacuole in Col-0 (WT) and <i>rbb1-2</i>. Bulbs are indicated with arrows. Scale: 20 μm. (h) <i>RBB1</i> is expressed in many developmental stages. The accumulation of <i>RBB1</i> transcripts was determined by RT-PCR with gene specific primers in various tissues of 4 and 7-day old Col-0 (WT) plants. Two pairs of gene specific primers detected the 5' end and 3' end of <i>RBB1</i> gene as indicated in (a).</p

3xYpet-RBB1 is a cytoplasmic protein that associates with the tonoplast.

<p>(a-c) A 3xYpet-RBB1 fusion complements the bulb phenotype of <i>rbb1-2</i>. Four-day-old seedlings from <i>rbb1-2</i> (a) or <i>rbb1-2</i> 3xYpet-RBB1 (b, c) were stained with Lysotracker Red (magenta, a, b) to label the vacuole. The signal from 3xYpet-RBB1 (c) for the complemented plant is shown. (d-f) 3xYpet-RBB1 does not co-localize with FM4-64. Images show the cotyledon of 7-d-old seedlings expressing 3xYpet-RBB1 (d) stained with 5 μM FM4-64 (e) and the merged image (f). (g-i) 3xYpet-RBB1 and RFP-SYP22 do not co-localize. Cotyledons of 7-d-old seedlings expressing 3xYpet-RBB1 (g) and RFP-SYP22 (h) were imaged. Merged image (i) is shown. (j-l) 3xYpet-RBB1 and Lysotracker Red do not co-localize in rosette leaves. Seedlings from the 3xYpet-RBB1 line were stained with Lysotracker Red. Signal from 3xYpet-RBB1 (j), Lysotracker Red (k) and the merged image (l) are shown. Arrowheads indicate the localization of 3xYpet-RBB1 at the tip of elongating trans-vacuolar strands. (m-o) GFP molecules can also label the tips of transvacuolar strands. Seedlings from a 35::GFP marker line were stained with Lysotracker Red. The GFP signal (m), Lysotracker Red (n) and the merged image (o) are shown. Arrowheads indicate the tip of TVS. All scale bars = 20 μm. (p) 3xYpet-RBB1 accumulates in the soluble (S100) and membrane pellet (P100) fractions from whole seedlings. Immunoblot of 3xYpet-RBB1 soluble (S100) and membrane (P100) fractions from Col-0 and 3xYpet-RBB1 transgenic plants using antibodies against GFP (α-GFP), cFBPase (α-cFBPase, control for soluble fraction), and Plasma Membrane H+ATPase (α-H+ATPase, control for membrane fraction).</p

Developmental progression of the <i>rbb1-1</i> phenotype and effects from light.

<p>(a-j) The bulb phenotype is apparent in hypocotyls after 3 d of germination. Germinating seedlings from the parental line (GFP-TIP2;1, a-e) and <i>rbb1-1</i> mutants (f-j) were imaged after 0–4 days of incubation. (k-n) The bulb phenotype of <i>rbb1-1</i> is present in light- and dark-grown seedlings. GFP-TIP2;1 localization in hypocotyls was visualized in 4-d-old seedlings from the parental line (WT, k, m) or <i>rbb1-1</i> (l, n) when grown under light (k, l) or dark (m, n) conditions. n = 36 cells from 6 seedlings. All scale bars = 20 μm. (o) Dark treatment does not alter the <i>rbb1-1</i> phenotype. The number of bulbs was counted in hypocotyls from parental line and <i>rbb1-1</i> seedlings that were grown in the light or in the dark as in k-n. n = 9–15 seedlings. Bars represent standard error. (p) <i>rbb1-1</i> hypocotyls have fewer TVS after 3 d of germination. Seedlings were germinated in the light and TVS were counted after 2 days of incubation. n = 50 cells from 5 seedlings. Bars represent standard error. * Significantly different to the parental line in a t-test (P ≤ 0.01).</p

Molecular mechanisms of endomembrane trafficking in plants

Endomembrane trafficking is essential for all eukaryotic cells. The best-characterized membrane trafficking organelles include the endoplasmic reticulum (ER), Golgi apparatus, early and recycling endosomes, multivesicular body, or late endosome, lysosome/vacuole, and plasma membrane. Although historically plants have given rise to cell biology, our understanding of membrane trafficking has mainly been shaped by the much more studied mammalian and yeast models. Whereas organelles and major protein families that regulate endomembrane trafficking are largely conserved across all eukaryotes, exciting variations are emerging from advances in plant cell biology research. In this review, we summarize the current state of knowledge on plant endomembrane trafficking, with a focus on four distinct trafficking pathways: ER-to-Golgi transport, endocytosis, trans-Golgi network-to-vacuole transport, and autophagy. We acknowledge the conservation and commonalities in the trafficking machinery across species, with emphasis on diversity and plant-specific features. Understanding the function of organelles and the trafficking machinery currently nonexistent in well-known model organisms will provide great opportunities to acquire new insights into the fundamental cellular process of membrane trafficking

A small molecule inhibitor partitions two distinct pathways for trafficking of tonoplast intrinsic proteins in Arabidopsis.

Tonoplast intrinsic proteins (TIPs) facilitate the membrane transport of water and other small molecules across the plant vacuolar membrane, and members of this family are expressed in specific developmental stages and tissue types. Delivery of TIP proteins to the tonoplast is thought to occur by vesicle-mediated traffic from the endoplasmic reticulum to the vacuole, and at least two pathways have been proposed, one that is Golgi-dependent and another that is Golgi-independent. However, the mechanisms for trafficking of vacuolar membrane proteins to the tonoplast remain poorly understood. Here we describe a chemical genetic approach to unravel the mechanisms of TIP protein targeting to the vacuole in Arabidopsis seedlings. We show that members of the TIP family are targeted to the vacuole via at least two distinct pathways, and we characterize the bioactivity of a novel inhibitor that can differentiate between them. We demonstrate that, unlike for TIP1;1, trafficking of markers for TIP3;1 and TIP2;1 is insensitive to Brefeldin A in Arabidopsis hypocotyls. Using a chemical inhibitor that may target this BFA-insensitive pathway for membrane proteins, we show that inhibition of this pathway results in impaired root hair growth and enhanced vacuolar targeting of the auxin efflux carrier PIN2 in the dark. Our results indicate that the vacuolar targeting of PIN2 and the BFA-insensitive pathway for tonoplast proteins may be mediated in part by common mechanisms

Molecular mechanisms of endomembrane trafficking in plants

Endomembrane trafficking is essential for all eukaryotic cells. The best-characterized membrane trafficking organelles include the endoplasmic reticulum (ER), Golgi apparatus, early and recycling endosomes, multivesicular body, or late endosome, lysosome/vacuole, and plasma membrane. Although historically plants have given rise to cell biology, our understanding of membrane trafficking has mainly been shaped by the much more studied mammalian and yeast models. Whereas organelles and major protein families that regulate endomembrane trafficking are largely conserved across all eukaryotes, exciting variations are emerging from advances in plant cell biology research. In this review, we summarize the current state of knowledge on plant endomembrane trafficking, with a focus on four distinct trafficking pathways: ER-to-Golgi transport, endocytosis, trans-Golgi network-to-vacuole transport, and autophagy. We acknowledge the conservation and commonalities in the trafficking machinery across species, with emphasis on diversity and plant-specific features. Understanding the function of organelles and the trafficking machinery currently nonexistent in well-known model organisms will provide great opportunities to acquire new insights into the fundamental cellular process of membrane trafficking