ORCID IDs: 0000-0003-1551-4699 (M.P.); 0000-0002-2315-6900 (M.B.).Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is the most abundant enzyme in plants and is responsible for CO 2 fixation during photosynthesis. This enzyme is assembled from eight large subunits (RbcL) encoded by a single chloroplast gene and eight small subunits (RbcS) encoded by a nuclear gene family. Rubisco is primarily found in the chloroplasts of mesophyll (C3 plants), bundlesheath (C4 plants), and guard cells. In certain species, photosynthesis also takes place in the secretory cells of glandular trichomes, which are epidermal outgrowths (hairs) involved in the secretion of specialized metabolites. However, photosynthesis and, in particular, Rubisco have not been characterized in trichomes. Here, we show that tobacco (Nicotiana tabacum) trichomes contain a specific Rubisco small subunit, NtRbcS-T, which belongs to an uncharacterized phylogenetic cluster (T). This cluster contains RbcS from at least 33 species, including monocots, many of which are known to possess glandular trichomes. Cluster T is distinct from the cluster M, which includes the abundant, functionally characterized RbcS isoforms expressed in mesophyll or bundle-sheath cells. Expression of NtRbcS-T in Chlamydomonas reinhardtii and purification of the full Rubisco complex showed that this isoform conferred higher V max and K m values as well as higher acidic pH-dependent activity than NtRbcS-M, an isoform expressed in the mesophyll. This observation was confirmed with trichome extracts. These data show that an ancient divergence allowed for the emergence of a so-far-uncharacterized RbcS cluster. We propose that secretory trichomes have a particular Rubisco uniquely adapted to secretory cells where CO 2 is released by the active specialized metabolism.

Summary Nicotiana tabacum suspension cells have been widely used to produce monoclonal antibodies, but the yield of secreted antibodies is usually low probably because of proteolytic degradation. Most IgGs that have been expressed in suspension cells have been of the human IgG1 isotype. In this study, we examined whether other isotypes displayed the same sensitivity to proteolytic degradation and whether the choice of plant host species mattered. Human serum IgG displayed different degradation profiles when incubated in spent culture medium from N. tabacum, Nicotiana benthamiana or Arabidopsis thaliana suspension cells. Zymography showed that the protease profile was host species dependent. Three human isotypes, IgG1, IgG2 and IgG4, and a mouse IgG2a were provided with the same heavy‐ and light‐chain variable regions from an anti‐human IgM antibody and expressed in N. tabacum cv. BY‐2 and A. thaliana cv. Col‐0 cells. Although all tested isotypes were detected in the extracellular medium using SDS‐PAGE and a functional ELISA, up to 10‐fold differences in the level of intact antibody were found according to the isotype expressed, to the host species and to the culture conditions. In the best combination (BY‐2 cells secreting human IgG1), we reported accumulation of more than 30 mg/L of intact antibody in culture medium. The possibility of using IgG constant regions as a scaffold to allow stable accumulation of antibodies with different variable regions was demonstrated for human IgG2 and mouse IgG2a.

No abstract

Key message3 6 pRbcS-T1 and pMALD1, two new trichome-specific promoters of Nicotiana tabacum, were 3 7 identified and their strength and specificity were compared to those of previously described 3 8 promoters in this species. Abstract 4 1 Nicotiana tabacum has emerged as a suitable host for metabolic engineering of terpenoids and 4 2 derivatives in tall glandular trichomes, which actively synthesize and secrete specialized 4 3 metabolites. However, implementation of an entire biosynthetic pathway in glandular 4 4 trichomes requires the identification of trichome-specific promoters to appropriately drive the 4 5 expression of the transgenes needed to set up the desired pathway. In this context, RT-qPCR 4 6 analysis was carried out on wild-type N. tabacum plants to compare the expression pattern 4 7 and gene expression level of NtRbS-T1 and NtMALD1, two newly identified genes expressed 4 8 in glandular trichomes, with those of NtCYP71D16, NtCBTS2α, NtCPS2, and NtLTP1, which 4 9 were reported in the literature to be specifically expressed in glandular trichomes. The latter 5 0 were previously investigated separately, preventing any accurate comparison of their 5 1 expression level. We show that NtRbcS-T1 and NtMALD1 are specifically expressed in 5 2 glandular trichomes like NtCYP71D16, NtCBTS2α, and NtCPS2, while NtLTP1 was also 5 3 expressed in other leaf tissues as well as in the stem. Transcriptional fusions of each of the six 5 4promoters to the GUS-VENUS reporter gene were introduced in N. tabacum by 5 5