SummaryArabidopsis seeds release large capsules of mucilaginous polysaccharides, which are shaped by an intricate network of cellulosic microfibrils. Cellulose synthase complexes is guided by the microtubule cytoskeleton, but it is unclear which proteins mediate this process in the seed coat epidermis (SCE).Using reverse genetics, we identified IQ67 DOMAIN 9 (IQD9) and KINESIN LIGHT CHAIN-RELATED 1 (KLCR1) as two highly expressed genes during seed development and comprehensively characterized their roles for cell wall polysaccharide biosynthesis and cortical microtubule (MT) organization.Mutations in IQD9 as well as in KLCR1 lead to compact mucilage capsules with aberrant cellulose distribution, which can be rescued by transgene complementation. Double mutant analyses revealed that their closest paralogs (IQD10 and KLCR2, respectively) are not required for mucilage biosynthesis. IQD9 physically interacts with KLCR1 and localizes to cortical MTs to maintain their organization in SCE cells. Similar to the previously identified TONNEAU1 (TON1) RECRUITING MOTIF 4 (TRM4) protein, IQD9 is required to maintain the velocity of cellulose synthases.Our results demonstrate that IQD9, KLCR1 and TRM4 are MT-associated proteins that are required for seed mucilage architecture. This study provides the first direct evidence that members of the IQD, KLCR and TRM families have overlapping roles in guiding the distribution of cell wall polysaccharides. Therefore, SCE cells provide an attractive system to further decipher the complex genetic regulation of polarized cellulose deposition.

Background The carbohydrate polymers that encapsulate plants cells have benefited humans for centuries and have valuable biotechnological uses. In the past five years, exciting possibilities have emerged in the engineering of polysaccharide-based biomaterials. Despite impressive advances on bacterial cellulose-based hydrogels, comparatively little is known about how plant hemicelluloses can be reconstituted and modulated in cells suitable for biotechnological purposes.Results Here, we assembled cellulose synthase-like A (CSLA) enzymes using an optimized Pichia pastoris platform to produce tunable heteromannan (HM) polysaccharides in yeast. By swapping the domains of plant mannan and glucomannan synthases, we engineered chimeric CSLA proteins that made β-1,4-linked mannan in quantities surpassing those of the native enzymes while minimizing the burden on yeast growth. Prolonged expression of a glucomannan synthase from Amorphophallus konjac was toxic to yeast cells: reducing biomass accumulation and ultimately leading to compromised cell viability. However, an engineered glucomannan synthase as well as CSLA pure mannan synthases and a CSLC glucan synthase did not inhibit growth. Interestingly, Pichia cell size could be increased or decreased depending on the composition of the CSLA protein sequence. HM yield and glucose incorporation could be further increased by co-expressing chimeric CSLA proteins with a MANNAN-SYNTHESIS-RELATED (MSR) co-factor from Arabidopsis thaliana.Conclusion The results provide novel routes for the engineering of polysaccharide-based biomaterials that are needed for a sustainable bioeconomy. The characterization of chimeric cellulose synthase-like enzymes in yeast offers an exciting avenue to produce plant polysaccharides in a tunable manner. Furthermore, cells modified with non-toxic plant polysaccharides such as β-mannan offer a modular chassis to produce and encapsulate sensitive cargo such as therapeutic proteins.

Water deficit impairs growth and survival of plants. Many water stress responses are under control of abscisic acid (ABA) but little is known about growth control under osmotic stress. Based on the previously described growth-promoting activity of the peptide hormone phytosulfokine (PSK), we hypothesized that it may contribute to growth regulation under water stress conditions. To test this hypothesis, we analyzed the Arabidopsis thaliana PSK receptor (PSKR) null mutant pskr1-3 pskr2-1 under mannitol and drought stress. In particular under mild water stress, fresht weight and photosynthetic efficiency were more reduced in pskr1-3 pskr2-1 than in wild type. Hydroponic and grafting experiments showed that PSKR signaling was not required for long-distance signaling from mannitol-stressed roots to shoot but rather for cell growth promotion in the shoot. Unlike wild type, pskr1-3 pskr2-1 shoots did not accumulate ABA in response to mannitol, showed misregulation of ABA synthesis genes and elevated expression of ABI1 and ABI2, repressors of ABA signaling whereas application of ABA partially reversed shoot growth inhibition by mannitol in pskr1-3 pskr2-1. In turn, mannitol and ABA induced expression of PSK3 and PSKR1, and ABA promoted expression of PSK2 and PSK4 revealing feedback regulatory loops between PSKR and osmotic stress signaling.HighlightPhytosulfokine receptor signaling regulates ABA synthesis and signaling genes and promotes ABA accumulation in the shoot of water-stressed plants and maintains leaf growth and photosynthetic efficiency which ensures plant health.

0

In plants, the cortical endoplasmic reticulum (ER) network is connected to the plasma membrane (PM) through the ER-PM contact sites (EPCSs), whose structures are maintained by EPCS resident proteins and the cytoskeleton.1-7 Strong co-alignment between EPCSs and the cytoskeleton is observed in plants,1,8 but little is known of how the cytoskeleton is maintained and regulated at the EPCS. Here, we have used a yeast-two-hybrid screen and subsequent in vivo interaction studies in plants by fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) analysis to identify two microtubule binding proteins, KLCR1 (kinesin-light-chain-related protein 1) and IQD2 (IQ67-domain 2), that interact with the actin binding protein NET3C and form a component of plant EPCS that mediates the link between the actin and microtubule networks. The NET3C-KLCR1-IQD2 module, acting as an actin-microtubule bridging complex, has a direct influence on ER morphology and EPCS structure. Their loss-of-function mutants, net3a/NET3C RNAi, klcr1, or iqd2, exhibit defects in pavement cell morphology, which we suggest is linked to the disorganization of both actin filaments and microtubules. In conclusion, our results reveal a novel cytoskeletal-associated complex, which is essential for the maintenance and organization of cytoskeletal structure and ER morphology at the EPCS and for normal plant cell morphogenesis.

Secretions from glandular trichomes potentially protect plants against a variety of aggressors. In the tomato clade of the Solanum genus, glandular trichomes of wild species produce a rich source of chemical diversity at the leaf surface. Previously, 7-epi-zingiberene produced in several accessions of Solanum habrochaites was found to confer resistance to whiteflies (Bemisia tabaci) and other insect pests. Here, we report the identification and characterisation of 9-hydroxy-zingiberene (9HZ) and 9-hydroxy-10,11-epoxyzingiberene (9H10epoZ), two derivatives of 7-epi-zingiberene produced in glandular trichomes of S. habrochaites LA2167. Using a combination of transcriptomics and genetics, we identified a gene coding for a cytochrome P450 oxygenase, ShCYP71D184, that is highly expressed in trichomes and co-segregates with the presence of the zingiberene derivatives. Transient expression assays in Nicotiana benthamiana showed that ShCYP71D184 carries out two successive oxidations to generate 9HZ and 9H10epoZ. Bioactivity assays showed that 9-hydroxy-10,11-epoxyzingiberene in particular exhibits substantial toxicity against B. tabaci and various microorganisms including Phytophthora infestans and Botrytis cinerea. Our work shows that trichome secretions from wild tomato species can provide protection against a wide variety of organisms. In addition, the availability of the genes encoding the enzymes for the pathway of 7-epi-zingiberene derivatives makes it possible to introduce this trait in cultivated tomato by precision breeding.

Tomato (Solanum lycopersicum L.) type VI glandular trichomes that occur on the surface of leaves, stems, young fruits and flowers produce and store a blend of volatile monoterpenes and sesquiterpenes. These compounds play important roles in the interaction with pathogens and herbivorous insects. Although the function of terpene synthases in the biosynthesis of volatile terpenes in tomato has been comprehensively investigated, the deciphering of their transcriptional regulation is only just emerging. We selected transcription factors that are over-expressed in trichomes based on existing transcriptome data and silenced them individually by virus-induced gene silencing. Of these, SlSCL3, a scarecrow-like (SCL) subfamily transcription factor, led to a significant decrease in volatile terpene content and expression of the corresponding terpene synthase genes when its transcription level was downregulated. Overexpression of SlSCL3 dramatically increased both the volatile terpene content and glandular trichome size, whereas its homozygous mutants showed reduced terpene biosynthesis. However, its heterozygous mutants also showed a significantly elevated volatile terpene content and enlarged glandular trichomes, similar to the overexpression plants. SlSCL3 modulates the expression of terpene biosynthetic pathway genes by transcriptional activation, but neither direct protein–DNA binding nor interaction with known regulators was observed. Moreover, transcript levels of the endogenous copy of SlSCL3 were decreased in the overexpression plants but increased in the heterozygous and homozygous mutants, suggesting feedback repression of its own promoter. Taken together, our results provide new insights into the role of SlSCL3 in the complex regulation of volatile terpene biosynthesis and glandular trichome development in tomato.

While Arabidopsis seed coat epidermal cells have become an excellent genetic system to study the biosynthesis and structural roles of various cell wall polymers, the physiological function of the secreted mucilaginous polysaccharides remains ambiguous. Seed mucilage is shaped by two distinct classes of highly substituted hemicelluloses along with cellulose and structural proteins, but their interplay has not been explored.We deciphered the functions of four distinct classes of cell wall polymers by generating a series of double mutants with defects in heteromannan, xylan, cellulose, or the arabinogalactan protein SALT-OVERLY SENSITIVE 5 (SOS5), and evaluating their impact on mucilage architecture and seed germination during salt stress.We discovered that muci10 seeds, lacking heteromannan branches, had elevated tolerance to salt stress, while heteromannan elongation mutants exhibited reduced germination in calcium chloride (CaCl2). By contrast, xylan made by MUCILAGE-RELATED21 (MUCI21) was found to be required for the adherence of mucilage pectin to microfibrils made by CELLULOSE SYNTHASE5 (CESA5) as well as to a SOS5-mediated network.Our results indicate that the substitution of xylan and glucomannan in seeds can fine-tune mucilage adherence and salt tolerance, respectively. The study of germinating seeds can thus provide insights into the synthesis, modification and function of complex glycans.

- The plant apoplast is a harsh environment in which hydrolytic enzymes, especially proteases, accumulate during pathogen infection. However, the defense functions of most apoplastic proteases remain largely elusive. - We show here that a newly identified small cysteine-rich secreted protein PC2 from the potato late blight pathogen Phytophthora infestans induces immunity in Solanum plants only after cleavage by plant apoplastic subtilisin-like proteases, such as tomato P69B.- A minimal 61 amino acid core peptide carrying two key cysteines, conserved widely in most oomycete species, is sufficient for PC2-induced cell death. Furthermore, we showed that Kazal-like protease inhibitors, such as EPI1, produced by P. infestans prevent PC2 cleavage and dampen PC2 elicited host immunity. - This study reveals that cleavage of pathogen proteins to release immunogenic peptides is an important function of plant apoplastic proteases.

Mannan is a class of cell wall polysaccharides widespread in the plant kingdom. Mannan structure and properties vary according to species and organ. The cell walls of cereal grains have been extensively studied due to their role in cereal processing and to their beneficial effect on human health as dietary fiber. Recently, we showed that mannan in wheat (Triticum aestivum) grain endosperm has a linear structure of β-1,4-linked mannose residues. The aim of this work was to study the biosynthesis and function of wheat grain mannan. We showed that mannan is deposited in the endosperm early during grain development, and we identified candidate mannan biosynthetic genes expressed in the endosperm. The functional study in wheat was unsuccessful therefore our best candidate genes were expressed in heterologous systems. The endosperm-specificTaCslA12 gene expressed in Pichia pastoris and in an Arabidopsis thaliana mutant depleted in glucomannan led to the production of wheat-like linear mannan lacking glucose residues and with moderate acetylation. Therefore, this gene encodes a mannan synthase and is likely responsible for the synthesis of wheat endosperm mannan.