NKS1/ELMO4 is an integral protein of a pectin synthesis protein complex and maintains Golgi morphology and cell adhesion in Arabidopsis.

Adjacent plant cells are connected by specialized cell wall regions, called middle lamellae, which influence critical agricultural characteristics, including fruit ripening and organ abscission. Middle lamellae are enriched in pectin polysaccharides, specifically homogalacturonan (HG). Here, we identify a plant-specific Arabidopsis DUF1068 protein, called NKS1/ELMO4, that is required for middle lamellae integrity and cell adhesion. NKS1 localizes to the Golgi apparatus and loss of NKS1 results in changes to Golgi structure and function. The nks1 mutants also display HG deficient phenotypes, including reduced seedling growth, changes to cell wall composition, and tissue integrity defects. These phenotypes are comparable to qua1 and qua2 mutants, which are defective in HG biosynthesis. Notably, genetic interactions indicate that NKS1 and the QUAs work in a common pathway. Protein interaction analyses and modeling corroborate that they work together in a stable protein complex with other pectin-related proteins. We propose that NKS1 is an integral part of a large pectin synthesis protein complex and that proper function of this complex is important to support Golgi structure and function.

Mechanochemical feedback mediates tissue bending required for seedling emergence.

Tissue bending is vital to plant development, as exemplified by apical hook formation during seedling emergence by bending of the hypocotyl. How tissue bending is coordinated during development remains poorly understood, especially in plants where cells are attached via rigid cell walls. Asymmetric distribution of the plant hormone auxin underlies differential cell elongation during apical hook formation. Yet the underlying mechanism remains unclear. Here, we demonstrate spatial correlation between asymmetric auxin distribution, methylesterified homogalacturonan (HG) pectin, and mechanical properties of the epidermal layer of the hypocotyl in Arabidopsis. Genetic and cell biological approaches show that this mechanochemical asymmetry is essential for differential cell elongation. We show that asymmetric auxin distribution underlies differential HG methylesterification, and conversely changes in HG methylesterification impact the auxin response domain. Our results suggest that a positive feedback loop between auxin distribution and HG methylesterification underpins asymmetric cell wall mechanochemical properties to promote tissue bending and seedling emergence.

Cell wall polysaccharides are mislocalized to the Vacuole in echidna mutants.

During cell wall biosynthesis, the Golgi apparatus is the platform for cell wall matrix biosynthesis and the site of packaging, of both matrix polysaccharides and proteins, into secretory vesicles with the correct targeting information. The objective of this study was to dissect the post-Golgi trafficking of cell wall polysaccharides using echidna as a vesicle traffic mutant of Arabidopsis thaliana and the pectin-secreting cells of the seed coat as a model system. ECHIDNA encodes a trans-Golgi network (TGN)-localized protein, which was previously shown to be required for proper structure and function of the secretory pathway. In echidna mutants, some cell wall matrix polysaccharides accumulate inside cells, rather than being secreted to the apoplast. In this study, live cell imaging of fluorescent protein markers as well as transmission electron microscopy (TEM)/immunoTEM of cryofixed seed coat cells were used to examine the consequences of TGN disorganization in echidna mutants under conditions of high polysaccharide production and secretion. While in wild-type seed coat cells, pectin is secreted to the apical surface, in echidna, polysaccharides accumulate in post-Golgi vesicles, the central lytic vacuole and endoplasmic reticulum-derived bodies. In contrast, proteins were partially mistargeted to internal multilamellar membranes in echidna. These results suggest that while secretion of both cell wall polysaccharides and proteins at the TGN requires ECHIDNA, different vesicle trafficking components may mediate downstream events in their secretion from the TGN.

Group III-A XTH genes of Arabidopsis encode predominant xyloglucan endohydrolases that are dispensable for normal growth.

The molecular basis of primary wall extension endures as one of the central enigmas in plant cell morphogenesis. Classical cell wall models suggest that xyloglucan endo-transglycosylase activity is the primary catalyst (together with expansins) of controlled cell wall loosening through the transient cleavage and religation of xyloglucan-cellulose cross links. The genome of Arabidopsis (Arabidopsis thaliana) contains 33 phylogenetically diverse XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/HYDROLASE (XTH) gene products, two of which were predicted to be predominant xyloglucan endohydrolases due to clustering into group III-A. Enzyme kinetic analysis of recombinant AtXTH31 confirmed this prediction and indicated that this enzyme had similar catalytic properties to the nasturtium (Tropaeolum majus) xyloglucanase1 responsible for storage xyloglucan hydrolysis during germination. Global analysis of Genevestigator data indicated that AtXTH31 and the paralogous AtXTH32 were abundantly expressed in expanding tissues. Microscopy analysis, utilizing the resorufin ß-glycoside of the xyloglucan oligosaccharide XXXG as an in situ probe, indicated significant xyloglucan endohydrolase activity in specific regions of both roots and hypocotyls, in good correlation with transcriptomic data. Moreover, this hydrolytic activity was essentially completely eliminated in AtXTH31/AtXTH32 double knockout lines. However, single and double knockout lines, as well as individual overexpressing lines, of AtXTH31 and AtXTH32 did not demonstrate significant growth or developmental phenotypes. These results suggest that although xyloglucan polysaccharide hydrolysis occurs in parallel with primary wall expansion, morphological effects are subtle or may be compensated by other mechanisms. We hypothesize that there is likely to be an interplay between these xyloglucan endohydrolases and recently discovered apoplastic exo-glycosidases in the hydrolytic modification of matrix xyloglucans.