Reconstructing the chemical and structural characteristics of the plant cell wall represents a promising solution to overcoming lignocellulosic biomass recalcitrance to biochemical deconstruction. This study aims to leverage hydroxyproline (Hyp)‐
The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐
- PAR ID:
- 10081546
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Plant Biotechnology Journal
- Volume:
- 17
- Issue:
- 6
- ISSN:
- 1467-7644
- Page Range / eLocation ID:
- p. 1130-1141
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
Abstract O ‐glycosylation, a process unique to plant cell wall glycoproteins, as an innovative technology for de novo design and engineering in planta of Hyp‐O ‐glycosylated biopolymers (HypGP) that facilitate plant cell wall reconstruction. HypGP consisting of 18 tandem repeats of “Ser–Hyp–Hyp–Hyp–Hyp” motif or (SP4)18was designed and engineered into tobacco plants as a fusion peptide with either a reporter protein enhanced green fluorescence protein or the catalytic domain of a thermophilic E1 endoglucanase (E1cd) fromAcidothermus cellulolyticus . The engineered (SP4)18module was extensively Hyp‐O ‐glycosylated with arabino‐oligosaccharides, which facilitated the deposition of the fused protein/enzyme in the cell wall matrix and improved the accumulation of the protein/enzyme in planta by 1.5–11‐fold. The enzyme activity of the recombinant E1cd was not affected by the fused (SP4)18module, showing an optimal temperature of 80°C and optimal pH between 5 and 8. The plant biomass engineered with the (SP4)18‐tagged protein/enzyme increased the biomass saccharification efficiency by up to 3.5‐fold without having adverse impact on the plant growth. -
Summary Cytosolic calcium concentration ([Ca2+]cyt) and heterotrimeric G‐proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Cao) is as strong a stimulus for stomatal closure as the phytohormone abscisic acid (
ABA ), but underlying mechanisms remain elusive. Here, we report that the sole Arabidopsis heterotrimeric Gβ subunit,AGB 1, is required for four guard cell Caoresponses: induction of stomatal closure; inhibition of stomatal opening; [Ca2+]cytoscillation; and inositol 1,4,5‐trisphosphate (InsP3) production. Stomata in wild‐type Arabidopsis (Col) and in mutants of the canonical Gα subunit, , showed inhibition of stomatal opening and promotion of stomatal closure by Cao. By contrast, stomatal movements ofGPA 1agb1 mutants andagb1 /gpa1 double‐mutants, as well as those of theagg1agg2 Gγ double‐mutant, were insensitive to Cao. These behaviors contrast withABA ‐regulated stomatal movements, which involveGPA 1 andAGB 1/AGG 3 dimers, illustrating differential partitioning of G‐protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus‐specific encoding. knockouts retained reactive oxygen species andAGB 1NO production, but lostYC 3.6‐detected [Ca2+]cytoscillations in response to Cao, initiating only a single [Ca2+]cytspike. Experimentally imposed [Ca2+]cytoscillations restored stomatal closure inagb1 . Yeast two‐hybrid and bimolecular complementation fluorescence experiments revealed thatAGB 1 interacts with phospholipase Cs (PLCs), and Caoinduced InsP3 production in Col but not inagb1 . In sum, G‐protein signaling viaAGB 1/AGG 1/AGG 2 is essential for Cao‐regulation of stomatal apertures, and stomatal movements in response to Caoapparently require Ca2+‐induced Ca2+release that is likely dependent on Gβγ interaction withPLC s leading to InsP3 production. -
Summary The oligosaccharyltransferase (
OT ) complex catalyzesN ‐glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plantOT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified ArabidopsisOT complexes using the tandem affinity‐taggedOT subunitSTAUROSPORINE AND TEMPERATURE SENSITIVE 3a (STT 3a) expressed by an Arabidopsis protein super‐expression platform. Mass‐spectrometry analysis of the purified complexes identified three essentialOT subunits,OLIGOSACCHARYLTRANSFERASE 1 (OST 1),HAPLESS 6 (HAP 6),DEFECTIVE GLYCOSYLATION 1 (DGL 1), and a number of ribosomal subunits. Transmission‐electron microscopy showed thatSTT 3a becomes incorporated intoOT –ribosome super‐complexes formedin vivo , demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwisein planta interaction analyses of individualOT subunits demonstrated that all subunits identified in animalOT complexes are conserved in Arabidopsis and physically interact withSTT 3a. Genetic analysis of newly establishedOT subunit mutants for andOST 1 (DEFENDER AGAINST APOTOTIC DEATH ) family genes revealed thatDAD OST 1 andDAD 1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations in andDGL 1,OST 3/6 .STT 3a -
Summary The collaborative non‐self‐recognition model for S‐
RN ase‐based self‐incompatibility predicts that multiple S‐locus F‐box proteins (SLF s) produced by pollen of a givenS ‐haplotype collectively mediate ubiquitination and degradation of all non‐self S‐RN ases, but not self S‐RN ases, in the pollen tube, thereby resulting in cross‐compatible pollination but self‐incompatible pollination. We had previously used pollen extracts containingGFP ‐fused S2‐SLF 1 (SLF 1 with anS 2‐haplotype) ofPetunia inflata for co‐immunoprecipitation (Co‐IP ) and mass spectrometry (MS ), and identified PiCUL 1‐P (a pollen‐specific Cullin1), PiSSK 1 (a pollen‐specific Skp1‐like protein) and PiRBX 1 (a conventional Rbx1) as components of theSCFS 2–SLF 1complex. Using pollen extracts containing PiSSK 1:FLAG :GFP for Co‐IP /MS , we identified two additionalSLF s (SLF 4 andSLF 13) that were assembled intoSCFSLF complexes. As 17 genes (SLF toSLF 1 ) have been identified inSLF 17S 2andS 3pollen, here we examined whether all 17SLF s are assembled into similar complexes and, if so, whether these complexes are unique toSLF s. We modified the previous Co‐IP /MS procedure, including the addition of style extracts from four differentS ‐genotypes to pollen extracts containing PiSSK 1:FLAG :GFP , to perform four separate experiments. The results taken together show that all 17SLF s and anSLF ‐like protein,SLFL ike1 (encoded by anS ‐locus‐linked gene), co‐immunoprecipitated with PiSSK 1:FLAG :GFP . Moreover, of the 179 other F‐box proteins predicted byS 2andS 3pollen transcriptomes, only a pair with 94.9% identity and another pair with 99.7% identity co‐immunoprecipitated with PiSSK 1:FLAG :GFP . These results suggest thatSCFSLF complexes have evolved specifically to function in self‐incompatibility. -
Summary Respiration in leaves and the continued elevation in the atmospheric
CO 2concentration causeCO 2‐mediated reduction in stomatal pore apertures. Several mutants have been isolated for which stomatal responses to both abscisic acid (ABA ) andCO 2are simultaneously defective. However, there are only few mutations that impair the stomatal response to elevatedCO 2, but not toABA . Such mutants are invaluable in unraveling the molecular mechanisms of earlyCO 2signal transduction in guard cells. Recently, mutations in the mitogen‐activated protein (MAP ) kinase, , have been shown to partially impairMPK 12CO 2‐induced stomatal closure. Here, we show thatmpk12 plants, in which is stably silenced specifically in guard cells (MPK 4mpk12 mpk4 homozygous double‐mutants), completely lackGC CO 2‐induced stomatal responses and have impaired activation of guard cell S‐type anion channels in response to elevatedCO 2/bicarbonate. However,ABA ‐induced stomatal closure, S‐type anion channel activation andABA ‐induced marker gene expression remain intact in thempk12 mpk4 double‐mutants. These findings suggest thatGC MPK 12 andMPK 4 act very early inCO 2signaling, upstream of, or parallel to the convergence ofCO 2andABA signal transduction. The activities ofMPK 4 andMPK 12 protein kinases were not directly modulated byCO 2/bicarbonatein vitro , suggesting that they are not directCO 2/bicarbonate sensors. Further data indicate thatMPK 4 andMPK 12 have distinguishable roles in Arabidopsis and that the previously suggested role ofRHC 1 in stomatalCO 2signaling is minor, whereasMPK 4 andMPK 12 act as key components of early stomatalCO 2signal transduction.