Degradation of the Endoplasmic Reticulum by Autophagy during Endoplasmic Reticulum Stress in Arabidopsis

In this article, we show that the endoplasmic reticulum (ER) in Arabidopsis thaliana undergoes morphological changes in structure during ER stress that can be attributed to autophagy. ER stress agents trigger autophagy as demonstrated by increased production of autophagosomes. In response to ER stress, a soluble ER marker localizes to autophagosomes and accumulates in the vacuole upon inhibition of vacuolar proteases. Membrane lamellae decorated with ribosomes were observed inside autophagic bodies, demonstrating that portions of the ER are delivered to the vacuole by autophagy during ER stress. In addition, an ER stress sensor, INOSITOL-REQUIRING ENZYME-1b (IRE1b), was found to be required for ER stress–induced autophagy. However, the IRE1b splicing target, bZIP60, did not seem to be involved, suggesting the existence of an undiscovered signaling pathway to regulate ER stress–induced autophagy in plants. Together, these results suggest that autophagy serves as a pathway for the turnover of ER membrane and its contents in response to ER stress in plants.     

Increase in catalase-3 activity as a response to use of alternative catabolic substrates during sucrose starvation

Periods of carbohydrate deprivation are commonly encountered by plant cells. Plants respond to this nutrient stress by the mobilization of stored carbohydrates and the reallocation of other cellular macromolecules to degradative pathways. Previously we identified a number of metabolic genes that are upregulated in Arabidopsis thaliana cells during sucrose starvation. One of the genes identified encodes acyl-CoA oxidase-4 (ACX4, EC 1.3.3.6), a peroxisomal acyl-CoA oxidase that is unique to plants and involved in β-oxidation of short-chain fatty acids. Here we demonstrate that ACX4 activity increases during sucrose starvation, indicating a shift to a catabolic breakdown of fatty acids as a source of available carbon. This suggests a role for degradation of short-chain fatty acids in the response to sucrose starvation, leading in turn to the production of toxic H₂O₂. Catalase-3 (CAT3, EC 1.11.1.6) activity also increases during starvation as a direct response to the increase in oxidative stress caused by the rapid activation of alternative catabolic pathways, including a specific increase in ACX4 activity. Any disruption in ACX4 expression or in β-oxidation of fatty acids in general prevents this increase in catalase activity and expression. We hypothesize that CAT3 activity increases to remove the H₂O₂ produced by alternative catabolic processes induced during the carbohydrate shortages caused by extended periods of low-light conditions.     

Interactions between syntaxins identify at least five SNARE complexes within the Golgi/prevacuolar system of the Arabidopsis cell.

The syntaxin family of soluble N-ethyl maleimide sensitive factor adaptor protein receptors (SNAREs) is known to play an important role in the fusion of transport vesicles with specific organelles. Twenty-four syntaxins are encoded in the genome of the model plant Arabidopsis thaliana. These 24 genes are found in 10 gene families and have been reclassified as syntaxins of plants (SYPs). Some of these gene families have been previously characterized, with the SYP2-type syntaxins being found in the prevacuolar compartment (PVC) and the SYP4-type syntaxins on the trans-Golgi network (TGN). Here we report on two previously uncharacterized syntaxin groups. The SYP5 group is encoded by a two-member gene family, whereas SYP61 is a single gene. Both types of syntaxins are localized to multiple compartments of the endomembrane system, including the TGN and the PVC. These two groups of syntaxins form SNARE complexes with each other, and with other Arabidopsis SNAREs. On the TGN, SYP61 forms complexes with the SNARE VTI12 and either SYP41 or SYP42. SYP51 and SYP61 interact with each other and with VTI12, most likely also on the TGN. On the PVC, a SYP5-type syntaxin interacts specifically with a SYP2-type syntaxin, as well as the SNARE VTI11, forming a SNARE complex likely involved in TGN-to-PVC trafficking.     

Hydrogen formation in nearly stoichiometric amounts from glucose by a Rhodopseudomonas sphaeroides mutant.

Rhodopseudomonas sphaeroides produces molecular H2 and CO2 from reduced organic compounds which serve as electron sources and from light which provides energy in the form of adenosine 5'-triphosphate. This process is mediated by a nitrogenase enzyme. A mutant has been found that, unlike the wild type, will quantitatively convert glucose to H2 and CO2. Techniques for isolating other strains capable of utilizing other unusual electron sources are presented. Metabolism of glucose by the wild-type strain leads to an accumulation of gluconate. The isolated mutant strain does not appear to accumulate gluconate.     

Stimulation of dark CO2 fixation by ammonia in isolated mesophyll cells of Papaver somniferum L.

Addition of 2 mM ammonium ion to isolated mesophyll cells ofPapaver somniferum resulted in a 3-fold or greater increasein their rate of dark ¹⁴C fixation, while even 0.1 mM NH₄⁺ nearlydoubled that rate. The most rapid increase in the labeling ofa metabolite occurred in aspartate, and was accompanied by adecrease in the steady-state level of labeled phosphoenolpyruvate.No change in labeled pyruvate level occurred, and alanine labelingdeclined. Ammonium ion addition had no effect on the respiratoryrate of these cells in the dark. We conclude that NH₄⁺ stimulatesphosphoenolpyruvate carboxylation in this system, but has nodetectable effect on the pyruvate kinase reaction in the dark.Our results are compared with earlier findings and the possibleregulatory function of ammonia is discussed. (Received July 23, 1979; )     

Kinetics of light-dark CO2 fixation and glucose assimilation by Aphanocapsa 6714.

Cells of Aphanocapsa 6714 were subjected to alternating ligh-dark periods (flashing-light experiments). The corresponding activation (in the light) and inactivation (in the dark) of the reductive pentose cycle was measured, in vivo, from initial rates of 14CO2 incorporation and also by changes in the total concentration of 14C and 32P in soluble metabolites. Two principle sites of metabolic regulation were detected: (i) CO2 fixation was inactivated 15 to 20 s after removal of the light source, but reactivated rapidly on reentering the light; (ii) hydrolysis of fructose-1,6-diphosphate (FDP) and sedoheptulose-1,7-diphosphate (SDP) by their respective phosphatase(s) (FDP + SDPase) was rapidly inhibited in the dark but only slowly reactivated in the light. The time required for reactivation of FDP + SDPase, in the light, was on the order of 20 to 30 s. As a consequence of the timing of these inactivation-reactivation reactions, newly fixed CO2 accumulated in the FDP and SDP pools during the flashing-light experiments. Changes in the concentrations of the adenylate pools (mainly in the levels of adenosine 5'-triphosphate and adenosine diphosphate) were fast in comparison to the inactivation-reactivation reactions in the reductive pentose cycle. Thus, these regulatory effects may not be under the control of the adenylates in this organism. The activation of CO2 fixation in the light is at least in part due to activation of phosphoribulokinase, which is required for formation of ribulose-1,5-diphoshate, the carboxylation substrate. Phosphoribulokinase activity in crude extracts was found to be dependent on the presence of strong reducing agents such as dithiothreitol, but not significantly dependent on adenylate levels, although adenosine 5'-triphosphate is a substrate.     

Effects of glycine hydroxamate, carbon dioxide, and oxygen on photorespiratory carbon and nitrogen metabolism in spinach mesophyll cells

The effects of added glycine hydroxamate on the photosynthetic incorporation of ¹⁴CO₂ into metabolites by isolated mesophyll cells of spinach (Spinacia oleracea L.) was investigated under conditions favorable to photorespiratory (PR) metabolism (0.04% CO₂ and 20% O₂) and under conditions leading to nonphotorespiratory (NPR) metabolism (0.2% CO₂ and 2.7% O₂). Glycine hydroxamate (GH) is a competitive inhibitor of the photorespiratory conversion of glycine to serine, CO₂ and NH₄⁺. During PR fixation, addition of the inhibitor increased glycine and decreased glutamine labeling. In contrast, labeling of glycine decreased under NPR conditions. This suggests that when the rate of glycolate synthesis is slow, the primary route of glycine synthesis is through serine rather than from glycolate. GH addition increased serine labeling under PR conditions but not under NPR conditions. This increase in serine labeling at a time when glycine to serine conversion is partially blocked by the inhibitor may be due to serine accumulation via the “reverse” flow of photorespiration from 3-P-glycerate to hydroxypyruvate when glycine levels are high. GH increased glyoxylate and decreased glycolate labeling. These observations are discussed with respect to possible glyoxylate feedback inhibition of photorespiration.     

The pre-vacuolar t-SNARE AtPEP12p forms a 20S complex that dissociates in the presence of ATP

Many proteins are transported to the plant vacuole through the secretory pathway in small transport vesicles by a series of vesicle budding and fusion reactions. Vesicles carrying vacuolar cargo bud from the trans-Golgi network are thought to fuse with a pre-vacuolar compartment before being finally transported to the vacuole. In mammals and yeast, the fusion of a vesicle with its target organelle is mediated by a 20S protein complex containing membrane and soluble proteins that appear to be conserved between different species. A number of membrane proteins have been identified in plants that show sequence similarity with a family of integral membrane proteins (t-SNAREs) on target organelles that are required for the fusion of transport vesicles with that organelle. However, the biochemical function of these proteins has remained elusive. Here, we demonstrate for the first time the formation of a 20S complex in plants that has characteristics of complexes involved in vesicle fusion. This complex contains AtPEP12p, an Arabidopsis protein thought to be involved in protein transport to the prevacuolar compartment. In addition, we have shown that AtPEP12p can bind to alpha-SNAP, indicating that AtPEP12p does indeed function as a SNAP receptor or SNARE. These preliminary data suggest that AtPEP12p may function jointly with alpha-SNAP and NSF in the fusion of transport vesicles containing vacuolar cargo proteins with the pre-vacuolar compartment.     

AtVPS45 complex formation at the trans-Golgi network

The Sec1p family of proteins are thought to be involved in the regulation of vesicle fusion reactions through interaction with t-SNAREs (target soluble N-ethylmaleimide-sensitive factor attachment protein receptors) at the target membrane. AtVPS45 is a member of this family from Arabidopsis thaliana that we now demonstrate to be present on the trans-Golgi network (TGN), where it colocalizes with the vacuolar cargo receptor AtELP. Unlike yeast Vps45p, AtVPS45 does not interact with, or colocalize with, the prevacuolar t-SNARE AtPEP12. Instead, AtVPS45 interacts with two t-SNAREs, AtTLG2a and AtTLG2b, that show similarity to the yeast t-SNARE Tlg2p. AtTLG2a and -b each colocalize with AtVPS45 at the TGN; however, AtTLG2a is in a different region of the TGN than AtTLG2b by immunogold electron microscopy. Therefore, we propose that complexes containing AtVPS45 and either AtTLG2a or -b define functional subdomains of the TGN and may be required for different trafficking events. Among other Arabidopsis SNAREs, AtVPS45 antibodies preferentially coprecipitate AtVTI1b over the closely related isoform AtVTI1a, implying that AtVTI1a and AtVTI1b also have distinct functions within the cell. These data point to a functional complexity within the plant secretory pathway, where proteins encoded by gene families have specialized functions, rather than functional redundancy.     

YKT6 is a core constituent of membrane fusion machineries at the Arabidopsis trans-Golgi network

SNARE complex formation is essential for membrane fusion in exocytotic and vacuolar trafficking pathways. Vesicle-associated (v-) SNARE associates with a target membrane (t-) SNARE to form a SNARE complex bridging two membranes, which may facilitate membrane fusion. The Arabidopsis genome encodes a large number of predicted SNARE proteins that might function primarily as fusogens for vesicle transport in endomembrane systems. The SNAREs SYP41, SYP61 and VTI12 reside in the trans-Golgi network and have been proposed to function together in vesicle fusion with this organelle. Here, we use a liposome fusion assay to demonstrate that VTI12 and either SYP41 or SYP61, but not both, are required for membrane fusion. This indicates that SYP41 and SYP61 are likely to function in independent vesicle fusion reactions in Arabidopsis. In addition, we have identified two new functionally interchangeable components, YKT61 and YKT62, that show sequence similarity to the multifunctional yeast SNARE YKT6. Both YKT61 and YKT62 interact with SYP41 and are essential for membrane fusion mediated by either SYP41 or SYP61. These results therefore define the core constituents required for membrane fusion at the Arabidopsis trans-Golgi network.