Activation tagging identifies Arabidopsis transcription factor AtMYB68 for heat and drought tolerance at yield determining reproductive stages.

Heat stress occurring at reproductive stages can result in significant and permanent damage to crop yields. However, previous genetic studies in understanding heat stress response and signaling were performed mostly on seedling and plants at early vegetative stages. Here we identify, using a developmentally defined, gain-of-function genetic screen with approximately 18 000 Arabidopsis thaliana activation-tagged lines, a mutant that maintained productive seed set post-severe heat stress during flowering. Genome walking indicated this phenotype was caused by the insertion of 35S enhancers adjacent to a nuclear localized transcription factor AtMYB68. Subsequent overexpression analysis confirmed that AtMYB68 was responsible for the reproductive heat tolerance of the mutant. Furthermore, these transgenic Arabidopsis plants exhibited enhanced abscisic acid sensitivity at and post-germination, reduced transpirational water loss during a drought treatment, and enhanced seed yield under combined heat and drought stress during flowering. Ectopic expression of AtMYB68 in Brassica napus driven either by 35S or by heat-inducible promoter recapitulated the enhanced reproductive heat stress and drought tolerance phenotypes observed in the transgenic Arabidopsis. The improvement to heat stress is likely due to enhanced pollen viability observed in the transgenic plants. More importantly, the transgenic canola showed significant yield advantages over the non-transgenic controls in multiple locations, multiple season field trials under various drought and heat stress conditions. Together these results suggest that AtMYB68 regulate plant stress tolerance at the most important yield determining stage of plant development, and is an effective target for crop yield protection under current global climate volatility.

EDEM1 accelerates the trimming of alpha1,2-linked mannose on the C branch of N-glycans.

Glycoprotein folding and degradation in the endoplasmic reticulum (ER) is mediated by the ER quality control system. Mannose trimming plays an important role by forming specific N-glycans that permit the recognition and sorting of terminally misfolded conformers for ERAD (ER-associated degradation). The EDEM (ER degradation enhancing alpha-mannosidase-like protein) subgroup of proteins belonging to the Class I alpha1,2-mannosidase family (glycosylhydrolase family 47) has been shown to enhance ERAD. We recently reported that overexpression of EDEM3 enhances glycoprotein ERAD with a concomitant increase in mannose-trimming activity in vivo. Herein, we report that overexpression of EDEM1 produces Glc(1)Man(8)GlcNAc(2) isomer C on terminally misfolded null Hong Kong alpha1-antitrypsin (NHK) in vivo. Levels of this isomer increased throughout the chase period and comprised approximately 10% of the [(3)H]mannose-labeled N-glycans on NHK after a 3-h chase. Furthermore, overexpression of EDEM1 E220Q containing a mutation in a conserved catalytic residue essential for alpha1,2-mannosidase activity did not yield detectable levels of Glc(1)Man(8)GlcNAc(2) isomer C. Yet, the same extent of NHK ERAD-enhancement was observed in both EDEM1 and EDEM1 E220Q overexpressing cells. This can be attributed to both wild-type and mutant EDEM1 inhibiting aberrant NHK dimer formation. We further analyzed the N-glycan profile of total cellular glycoproteins from HepG2 cells stably overexpressing EDEM1 and found that the relative amount of Man(7)GlcNAc(2) isomer A, which lacks the terminal B and C branch mannoses, was increased compared to parental HepG2 cells. Based on this observation, we conclude that EDEM1 activity trims mannose from the C branch of N-glycans in vivo.

High root biomass production in anchored Arabidopsis plants grown in axenic sucrose supplemented liquid culture.

There are many benefits to growing Arabidopsis in solution-based media, especially when large amounts of root tissue are required for molecular and biochemical studies. Roots grown in soil are brittle and tend to break easily when removed from their substrate. We have developed an axenic liquid culture system that simplifies growing large amounts of roots from intact plants. This technique consists of germinating 15 seeds on 2.5 cm2 stainless steel screens placed on half-strength semisolid Murashige and Skoog medium containing 1% or 2% sucrose. The screens anchor and support the plantlets in an upright position while keeping the roots and shoots separate. The seedlings are transferred with forceps to 125-mL wide-mouth Erlenmeyer flasks containing 10 mL of half-strength Murashige and Skoog liquid medium and 1% sucrose. The flasks are placed onto a floor rotary shaker under fluorescent lights. After 3 days, the sucrose is increased to 3% and the volume to 15 mL for 7 days. During any further experimental manipulations, sucrose is not supplied. The media is changed every 3-4 days to replenish the nutrients. The presence of sucrose in the media dramatically increases the biomass, and large amounts of root tissue can easily be harvested.

Stimulation of ERAD of misfolded null Hong Kong alpha1-antitrypsin by Golgi alpha1,2-mannosidases.

Terminally misfolded or unassembled proteins are degraded by the cytoplasmic ubiquitin-proteasome pathway in a process known as ERAD (endoplasmic reticulum-associated protein degradation). Overexpression of ER alpha1,2-mannosidase I and EDEMs target misfolded glycoproteins for ERAD, most likely due to trimming of N-glycans. Here we demonstrate that overexpression of Golgi alpha1,2-mannosidase IA, IB, and IC also accelerates ERAD of terminally misfolded human alpha1-antitrypsin variant null (Hong Kong) (NHK), and mannose trimming from the N-glycans on NHK in 293 cells. Although transfected NHK is primarily localized in the ER, some NHK also co-localizes with Golgi markers, suggesting that mannose trimming by Golgi alpha1,2-mannosidases can also contribute to NHK degradation.

Respiratory distress and neonatal lethality in mice lacking Golgi alpha1,2-mannosidase IB involved in N-glycan maturation.

There are three mammalian Golgi alpha1,2-mannosidases, encoded by different genes, that form Man5GlcNAc2 from Man(8-9)GlcNAc2 for the biosynthesis of hybrid and complex N-glycans. Northern blot analysis and in situ hybridization indicate that the three paralogs display distinct developmental and tissue-specific expression. The physiological role of Golgi alpha1,2-mannosidase IB was investigated by targeted gene ablation. The null mice have normal gross appearance at birth, but they display respiratory distress and die within a few hours. Histology of fetal lungs the day before birth indicate some delay in development, whereas neonatal lungs show extensive pulmonary hemorrhage in the alveolar region. No significant histopathological changes occur in other tissues. No remarkable ultrastructural differences are detected between wild type and null lungs. The membranes of a subset of bronchiolar epithelial cells are stained with lectins from Phaseolus vulgaris (leukoagglutinin and erythroagglutinin) and Datura stramonium in wild type lungs, but this staining disappears in lungs from null mice. Mass spectrometry of N-glycans from different tissues shows no significant changes in global N-glycans of null mice. Therefore, only a few glycoproteins required for normal lung function depend on alpha1,2-mannosidase IB for maturation. There are no apparent differences in the expression of several lung epithelial cell and endothelial cell markers between null and wild type mice. The alpha1,2-mannosidase IB null phenotype differs from phenotypes caused by ablation of other enzymes in N-glycan biosynthesis and from other mouse gene disruptions that affect pulmonary development and function.

EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming.

Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM (ER degradation-enhancing alpha-mannosidase-like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I alpha-mannosidases (glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded alpha1-antitrypsin variant (null (Hong Kong)) and of TCRalpha. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded alpha1-AT null (Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent alpha1,2-mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of alpha1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has alpha1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM.

Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I.

Misfolded glycoproteins synthesized in the endoplasmic reticulum (ER) are degraded by cytoplasmic proteasomes, a mechanism known as ERAD (ER-associated degradation). In the present study, we demonstrate that ERAD of the misfolded genetic variant-null Hong Kong alpha1-antitrypsin is enhanced by overexpression of the ER processing alpha1,2-mannosidase (ER ManI) in HEK 293 cells, indicating the importance of ER ManI in glycoprotein quality control. We showed previously that EDEM, an enzymatically inactive mannosidase homolog, interacts with misfolded alpha1-antitrypsin and accelerates its degradation (Hosokawa, N., Wada, I., Hasegawa, K., Yorihuzi, T., Tremblay, L. O., Herscovics, A., and Nagata, K. (2001) EMBO Rep. 2, 415-422). Herein we demonstrate a combined effect of ER ManI and EDEM on ERAD of misfolded alpha1-antitrypsin. We also show that misfolded alpha1-antitrypsin NHK contains labeled Glc1Man9GlcNAc and Man5-9GlcNAc released by endo-beta-N-acetylglucosaminidase H in pulse-chase experiments with [2-3H]mannose. Overexpression of ER ManI greatly increases the formation of Man8GlcNAc, induces the formation of Glc1Man8GlcNAc and increases trimming to Man5-7GlcNAc. We propose a model whereby the misfolded glycoprotein interacts with ER ManI and with EDEM, before being recognized by downstream ERAD components. This detailed characterization of oligosaccharides associated with a misfolded glycoprotein raises the possibility that the carbohydrate recognition determinant triggering ERAD may not be restricted to Man8GlcNAc2 isomer B as previous studies have suggested.