Among many glycoproteins within the plant secretory system, KORRIGAN1 (KOR1), a membrane-anchored endo-β-1,4-glucanase involved in cellulose biosynthesis, provides a link between N-glycosylation, cell wall biosynthesis, and abiotic stress tolerance. After insertion into the endoplasmic reticulum, KOR1 cycles between the trans-Golgi network (TGN) and the plasma membrane (PM). From the TGN, the protein is targeted to growing cell plates during cell division. These processes are governed by multiple sequence motifs and also host genotypes. Here, we investigated the interaction and hierarchy of known and newly identified sorting signals in KOR1 and how they affect KOR1 transport at various stages in the secretory pathway. Conventional steady-state localization showed that structurally compromised KOR1 variants were directed to tonoplasts. In addition, a tandem fluorescent timer technology allowed for differential visualization of young versus aged KOR1 proteins, enabling the analysis of single-pass transport through the secretory pathway. Observations suggest the presence of multiple checkpoints/branches during KOR1 trafficking, where the destination is determined based on KOR1’s sequence motifs and folding status. Moreover, growth analyses of dominant PM-confined KOR1-L⁴⁸L⁴⁹→A⁴⁸A⁴⁹ variants revealed the importance of active removal of KOR1 from the PM during salt stress, which otherwise interfered with stress acclimation.

Pentyl leafy volatiles (PLV) are C₅ volatiles produced from polyunsaturated fatty acids by plant 13-lipoxygenases (13-LOX) in concert with other lipid metabolizing enzymes. Unlike related C₆ volatiles (GLV, green leafy volatiles), little is known about biosynthesis and the physiological function of PLV in plants. Zea mays LOX6 (ZmLOX6) is an unusual plant LOX that lacks lipid oxygenation activity but acts as a hydroperoxide lyase hypothesized to be specifically involved in PLV synthesis. We overexpressed ZmLOX6 in Arabidopsis thaliana and established that it indeed produces PLVs. Overexpression of ZmLOX6 caused mild chlorotic phenotype and induced a similar phenotype in untransformed Col-0 plants grown in close proximity, suggesting airborne signals, such as PLVs, are responsible for the phenotype. PLV production, dependency on the substrate from endogenous 13-LOX(s), and likely competition with endogenous 13-oxylipin pathway were consistent with the model that ZmLOX6 functions as a hydroperoxide lyase. The abundance of individual PLVs was differentially affected by ZmLOX6 overexpression, and the new profile indicated that ZmLOX6 had reaction products distinct from endogenous PLV-producing activities in the Arabidopsis host plants. ZmLOX6 overexpression also induced new hormonal status, which is likely responsible for increased attraction and propagation of aphids, nonetheless improving host plant tolerance to aphid infestation.

Cellular dynamics of KORRIGAN 1 (KOR1) is closely linked with cellulose biosynthesis and plant osmotic stress tolerance. Cycling of KOR1 between the plasma membrane (PM) and trans-Golgi Network (TGN) is maintained by sequence motifs and protein structures that are recognized by cellular transport and quality control mechanisms. Several mutations in KOR1, as well as in the host genetic background, promote the mistargeting of KOR1 and induce KOR1 accumulation in the tonoplast (TP). Yet, little is known about how retention and sorting of KOR1 are regulated in the PM-TGN cycle. Forward genetic screening for GFP-KOR1 mislocalizing phenotype resulted in several mutant lines with different localization patterns or signal intensity of GFP-KOR1. One of the identified mutants were disrupted at UDP-glucose:glycoprotein glucosyltransferase (UGGT) locus, which is essential for the protein quality control in the ER. Our finding suggests the mis/unfolded structure of KOR1 triggers the TP targeting.

Complex N-glycans are asparagine (N)-linked branched sugar chains attached to secretory proteins in eukaryotes. They are produced by modification of N-linked oligosaccharide structures in the endoplasmic reticulum and Golgi apparatus. Complex N-glycans formed in the Golgi apparatus are often assigned specific roles unique to the host organism, with their roles in plants remaining largely unknown. Using inhibitor (kifunensine, KIF) hypersensitivity as read out, we identified Arabidopsis mutants that require complex N-glycan modification. Among >100 KIF-sensitive mutants, one showing abnormal secretory organelles and a salt-sensitive phenotype contained a point mutation leading to amino acid replacement (G69R) in ARFA1E, a small Arf1-GTPase family protein presumably involved in vesicular transport. In vitro assays showed that the G69R exchange interferes with protein activation. In vivo, ARFA1EG69R caused dominant-negative effects, altering the morphology of the endoplasmic reticulum, Golgi apparatus, and trans-Golgi network (TGN). Post-Golgi transport (endocytosis/endocytic recycling) of the essential glycoprotein KORRIGAN1, one of the KIF sensitivity targets, is slowed down constitutively as well as under salt stress in the ARFA1EG69R mutant. Because regulated cycling of plasma membrane proteins is required for stress tolerance of the host plants, the ARFA1EG69R mutant established a link between KIF-targeted luminal glycoprotein functions/dynamics and cytosolic regulators of vesicle transport in endosome-/cell wall-associated tolerance mechanisms.