The molecular mechanisms underlying the synthesis of large cell wall polysaccharides in plant cells are not fully understood. Here, we report that 2 atypical endo-β-1,4-mannanases (MANs), which are not secreted and do not degrade glucomannan in the cell wall, play a role in glucomannan synthesis. Among the 6 MANs in Arabidopsis (Arabidopsis thaliana), AtMAN2 and AtMAN5 contain a transmembrane domain at their N-terminal region instead of a signal peptide. Subcellular localization using MAN protein fused to a fluorescent protein demonstrated that AtMAN2 localizes to the endomembrane system, including the Golgi apparatus, in xylem and interfascicular fiber cells. An Arabidopsis man2 man5 double mutant lost 65% of glucomannan in the cell walls of the inflorescence stem. Immunostaining and immunoelectron microscopic observation also revealed that the man2 man5 double mutant loses glucomannan in the cell walls to about the same extent as the csla2 csla9 double mutant, which lacks major glucomannan synthases. Gene complementation experiments showed that the enzymatic activities of AtMAN2 and AtMAN5 are important for the synthesis of cell wall glucomannan. Arabidopsis possesses another atypical MAN, AtMAN6, with an HDEL retention signal at its C-terminus. However, mutation of AtMAN6 did not affect glucomannan content in the cell walls, suggesting distinct functions for these MANs. This study has identified AtMAN2 and AtMAN5 as factors necessary for normal glucomannan synthesis in Arabidopsis, along with GDP-mannose-generating enzymes and CslAs, and suggests that glucomannan hydrolysis by these MANs contributes to maintaining glucomannan synthesis.

Brassinosteroids (BRs) are important regulators of plant growth and stress responses. To identify novel transcription factors that regulate BR responses, we screened plants generated using chimeric repressor gene-silencing technology (CRES-T) in which transcription factors were converted into chimeric repressors by the fusion of the SRDX plant-specific repression domain with brassinazole (Brz), an inhibitor of BR biosynthesis. We found that a chimeric repressor of the homeobox transcription factor BRASSINOSTEROID-RELATED-HOMEOBOX-1 (BHB1-sx) induced a Brz-insensitive phenotype with a long hypocotyl, a slender dwarf growth habit, and epinastic leaves. Furthermore, the expression of BR-biosynthesis and BR-inducible genes was enhanced in BHB1-sx. In contrast, ectopic expression of BHB1 (BHB1-ox) resulted in a Brz-hypersensitive phenotype with a short hypocotyl, a dwarf habit, and hyponastic leaves, and the expression of BR-associated genes was repressed. Transcriptome analysis revealed that the expression of auxin-related genes was down-regulated in BHB1-ox plants, whereas that of stress-related genes was up-regulated, indicating that BHB1 negatively regulates BR responses. The expression of BHB1 was induced by BR and stress conditions, and the BHB1-ox plants showed higher tolerance to salt stress compared with the wild type, whereas the bhb1 mutant showed lower tolerance. Our results indicate that BHB1 might function as a hub factor mediating the crosstalk between salt stress and BR signaling.

Apomixis, defined as the transfer of maternal germplasm to offspring without fertilization, enables the fixation of F1-useful traits, providing advantages in crop breeding. However, most apomictic plants require pollination to produce endosperm. Endosperm is essential for embryogenesis and its development is suppressed until fertilization. We show that expression of a chimeric repressor of the Elongation of Siliques without Pollination 3 (ESP3) gene (Pro35S:ESP3-SRDX) induces ovule enlargement without fertilization in Arabidopsis thaliana. The ESP3 gene encodes a protein similar to the FWA homeodomain transcription factor containing a StAR-related lipid-transfer (START) domain. However, ESP3 lacks the homeobox-encoding region. Genes related to the cell cycle and sugar metabolism were upregulated in unfertilized Pro35S:ESP3-SRDX ovules similar as in fertilized seeds, while those related to autophagy were downregulated similar to fertilized seeds. Unfertilized Pro35S:ESP3-SRDX ovules partially nourished embryos when only the egg was fertilized, accumulating hexoses without central cell proliferation. ESP3 may regulate nutrient flow during seed development, and ESP3-SRDX could be a useful tool for complete apomixis that does not require pseudo-fertilization.