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Xyloglucan (XyG) is an abundant component of the primary cell walls of most plants. While the structure of XyG has been well studied, much remains to be learned about its biosynthesis. Here we employed reverse genetics to investigate the role of Arabidopsis cellulose synthase like-C (CSLC) proteins in XyG biosynthesis. We found that single mutants containing a T-DNA in each of the five Arabidopsis CSLC genes had normal levels of XyG. However, higher-order cslc mutants had significantly reduced XyG levels, and a mutant with disruptions in all five CSLC genes had no detectable XyG. The higher-order mutants grew with mild tissue-specific phenotypes. Despite the apparent lack of XyG, the cslc quintuple mutant did not display significant alteration of gene expression at the whole-genome level, excluding transcriptional compensation. The quintuple mutant could be complemented by each of the five CSLC genes, supporting the conclusion that each of them encodes a XyG glucan synthase. Phylogenetic analyses indicated that the CSLC genes are widespread in the plant kingdom and evolved from an ancient family. These results establish the role of the CSLC genes in XyG biosynthesis, and the mutants described here provide valuable tools with which to study both the molecular details of XyG biosynthesis and the role of XyG in plant cell wall structure and function., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The unfolded protein response (UPR) is an ancient signaling pathway designed to protect cells from the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER). Because misregulation of the UPR is potentially lethal, a stringent surveillance signaling system must be in place to modulate the UPR. The major signaling arms of the plant UPR have been discovered and rely on the transcriptional activity of the transcription factors bZIP60 and bZIP28 and on the kinase and ribonuclease activity of IRE1, which splices mRNA to activate bZIP60. Both bZIP28 and bZIP60 modulate UPR gene expression to overcome ER stress. In this study, we demonstrate at a genetic level that the transcriptional role of bZIP28 and bZIP60 in ER-stress responses is antagonized by nonexpressor of PR1 genes 1 (NPR1), a critical redox-regulated master regulator of salicylic acid (SA)-dependent responses to pathogens, independently of its role in SA defense. We also establish that the function of NPR1 in the UPR is concomitant with ER stress-induced reduction of the cytosol and translocation of NPR1 to the nucleus where it interacts with bZIP28 and bZIP60. Our results support a cellular role for NPR1 as well as a model for plant UPR regulation whereby SA-independent ER stress-induced redox activation of NPR1 suppresses the transcriptional role of bZIP28 and bZIP60 in the UPR., Competing Interests: The authors declare no conflict of interest.

Eukaryotic cells require mechanisms to establish the proportion of cellular volume devoted to particular organelles. These mechanisms are poorly understood. From a screen for plastid-to-nucleus signaling mutants in Arabidopsis thaliana, we cloned a mutant allele of a gene that encodes a protein of unknown function that is homologous to two other Arabidopsis genes of unknown function and to FRIENDLY, which was previously shown to promote the normal distribution of mitochondria in Arabidopsis. In contrast to FRIENDLY, these three homologs of FRIENDLY are found only in photosynthetic organisms. Based on these data, we proposed that FRIENDLY expanded into a small gene family to help regulate the energy metabolism of cells that contain both mitochondria and chloroplasts. Indeed, we found that knocking out these genes caused a number of chloroplast phenotypes, including a reduction in the proportion of cellular volume devoted to chloroplasts to 50% of wild type. Thus, we refer to these genes as REDUCED CHLOROPLAST COVERAGE (REC). The size of the chloroplast compartment was reduced most in rec1 mutants. The REC1 protein accumulated in the cytosol and the nucleus. REC1 was excluded from the nucleus when plants were treated with amitrole, which inhibits cell expansion and chloroplast function. We conclude that REC1 is an extraplastidic protein that helps to establish the size of the chloroplast compartment, and that signals derived from cell expansion or chloroplasts may regulate REC1.

Tocopherols are nonpolar compounds synthesized and localized in plastids but whose genetic elimination specifically impacts fatty acid desaturation in the endoplasmic reticulum (ER), suggesting a direct interaction with ER-resident enzymes. To functionally probe for such interactions, we developed transorganellar complementation, where mutated pathway activities in one organelle are experimentally tested for substrate accessibility and complementation by active enzymes retargeted to a companion organelle. Mutations disrupting three plastid-resident activities in tocopherol and carotenoid synthesis were complemented from the ER in this fashion, demonstrating transorganellar access to at least seven nonpolar, plastid envelope-localized substrates from the lumen of the ER, likely through plastid:ER membrane interaction domains. The ability of enzymes in either organelle to access shared, nonpolar plastid metabolite pools redefines our understanding of the biochemical continuity of the ER and chloroplast with profound implications for the integration and regulation of organelle-spanning pathways that synthesize nonpolar metabolites in plants.

In plants, heat stress responses are controlled by heat stress transcription factors that are conserved among all eukaryotes and can be constitutively expressed or induced by heat. Heat-inducible transcription factors that are distinct from the "classical" heat stress transcription factors have also been reported to contribute to heat tolerance. Here, we show that bZIP28, a gene encoding a putative membrane-tethered transcription factor, is up-regulated in response to heat and that a bZIP28 null mutant has a striking heat-sensitive phenotype. The heat-inducible expression of genes that encode BiP2, an endoplasmic reticulum (ER) chaperone, and HSP26.5-P, a small heat shock protein, is attenuated in the bZIP28 null mutant. An estradiol-inducible bZIP28 transgene induces a variety of heat and ER stress-inducible genes. Moreover, heat stress appears to induce the proteolytic release of the predicted transcription factor domain of bZIP28 from the ER membrane, thereby causing its redistribution to the nucleus. These findings indicate that bZIP28 is an essential component of a membrane-tethered transcription factor-based signaling pathway that contributes to heat tolerance.

A challenging task in the study of the secretory pathway is the identification and localization of new proteins to increase our understanding of the functions of different organelles. Previous proteomic studies of the endomembrane system have been hindered by contaminating proteins, making it impossible to assign proteins to organelles. Here we have used the localization of organelle proteins by the isotope tagging technique in conjunction with isotope tags for relative and absolute quantitation and 2D liquid chromatography for the simultaneous assignment of proteins to multiple subcellular compartments. With this approach, the density gradient distributions of 689 proteins from Arabidopsis thaliana were determined, enabling confident and simultaneous localization of 527 proteins to the endoplasmic reticulum, Golgi apparatus, vacuolar membrane, plasma membrane, or mitochondria and plastids. This parallel analysis of endomembrane components has enabled protein steady-state distributions to be determined. Consequently, genuine organelle residents have been distinguished from contaminating proteins and proteins in transit through the secretory pathway.