Long-term abscisic acid promotes golden2-like1 degradation through constitutive photomorphogenic 1 in a light intensity-dependent manner to suppress chloroplast development.

Abiotic stress, a serious threat to plants, occurs for extended periods in nature. Abscisic acid (ABA) plays a critical role in abiotic stress responses in plants. Therefore, stress responses mediated by ABA have been studied extensively, especially in short-term responses. However, long-term stress responses mediated by ABA remain largely unknown. To elucidate the mechanism by which plants respond to prolonged abiotic stress, we used long-term ABA treatment that activates the signalling against abiotic stress such as dehydration and investigated mechanisms underlying the responses. Long-term ABA treatment activates constitutive photomorphogenic 1 (COP1). Active COP1 mediates the ubiquitination of golden2-like1 (GLK1) for degradation, contributing to lowering expression of photosynthesis-associated genes such as glutamyl-tRNA reductase (HEMA1) and protochlorophyllide oxidoreductase A (PORA), resulting in the suppression of chloroplast development. Moreover, COP1 activation and GLK1 degradation upon long-term ABA treatment depend on light intensity. Additionally, plants with COP1 mutation or exposed to higher light intensity were more sensitive to salt stress. Collectively, our results demonstrate that long-term treatment of ABA leads to activation of COP1 in a light intensity-dependent manner for GLK1 degradation to suppress chloroplast development, which we propose to constitute a mechanism of balancing normal growth and stress responses upon the long-term abiotic stress.

Production of active human iduronate-2-sulfatase (IDS) enzyme in Nicotiana benthamiana.

Many strategies have been developed to produce high levels of biologically active recombinant proteins in plants for biopharmaceutical purposes. However, the production of an active form of human iduronate-2-sulfatase (hIDS) for the treatment of Hunter syndrome by enzyme replacement therapy (ERT) is challenging due to the requirement for cotranslational modification by a formylglycine-producing enzyme encoded by sulfatase modifying factor 1 (hSUMF1) at the Cys84 residue, which converts it to C(alpha)-formylglycine. In this study, we have shown that hIDS can be highly expressed in N. benthamiana by using different constructs. Among them, BiP-GB1-L-dCBD1-2L-8xHis-L-6xHis-3L-EK-hIDS-HDEL (GB1-CBD1-hIDS) showed a high expression level when transiently co-expressed with the turnip crinkle virus gene silencing suppressor P38 and GB1-fused human calreticulin (GB1-CRT1) as a folding enhancer. The hSUMF1 was co-expressed with hIDS for cotranslational modification. The full-length recombinant proteins were purified using Ni2+-NTA affinity resin followed by enterokinase treatment to obtain tag-free hIDS. The N-terminal fragment was removed using microcrystalline cellulose (MCC) beads. The purified active form of hIDS can successfully cleave the sulfate group from an artificial substrate, 4-nitrocatechol sulfate, at a similar level to commercial hIDS expressed in animal cells. These results suggest that plants could be a promising platform for the production of recombinant hIDS.

Design of an artificial transcriptional system for production of high levels of recombinant proteins in tobacco (Nicotiana benthamiana).

Plants have recently received much attention as a means of producing recombinant proteins because they are easy to grow at a low cost and at a large scale. Although many plant protein expression systems have been developed, there remains a need for improved systems that deliver high yields of recombinant proteins. Transcription of the recombinant gene is a key step in increasing the yield of recombinant proteins. However, revealed strong promoters, terminators, and transcription factors that have been identified do not necessarily lead to high level production of recombinant proteins. Thus, in this study, a robust expression system was designed to produce high levels of recombinant protein consisting of a novel hybrid promoter, FM'M-UD, coupled with an artificial terminator, 3PRt. FM'M-UD contained fragments from three viral promoters (the promoters of Mirabilis mosaic caulimovirus (MMV) full-length transcript, the MMV subgenomic transcript, and figwort mosaic virus subgenomic transcript) and two types of cis-acting elements (four GAL4 binding sites and two zinc finger binding sites). The artificial terminator, 3PRt, consisted of the PINII and 35S terminators plus RB7, a matrix attachment region. The FM'M-UD promoter increased protein levels of reporters GFP, RBD : SD1 (part of S protein from SARS-CoV-2), and human interleukin-6 (hIL6) by 4-6-fold, 2-fold, and 6-fold, respectively, relative to those of the same reporters driven by the CaMV 35S promoter. Furthermore, when the FM'M-UD/3PRt expression cassette was expressed together with GAL4/TAC3d2, an artificial transcription factor that bound the GAL4 binding sites in FM'M-UD, levels of hIL6 increased by 10.7-fold, relative to those obtained from the CaMV 35S promoter plus the RD29B terminator. Thus, this novel expression system led to the production of a large amount of recombinant protein in plants.

The B1 Domain of Streptococcal Protein G Serves as a Multi-Functional Tag for Recombinant Protein Production in Plants.

Plants have long been considered a cost-effective platform for recombinant production. A recently recognized additional advantage includes the low risk of contamination of human pathogens, such as viruses and bacterial endotoxins. Indeed, a great advance has been made in developing plants as a "factory" to produce recombinant proteins to use for biopharmaceutical purposes. However, there is still a need to develop new tools for recombinant protein production in plants. In this study, we provide data showing that the B1 domain of Streptococcal protein G (GB1) can be a multi-functional domain of recombinant proteins in plants. N-terminal fusion of the GB1 domain increased the expression level of various target proteins ranging from 1.3- to 3.1-fold at the protein level depending on the target proteins. GB1 fusion led to the stabilization of the fusion proteins. Furthermore, the direct detection of GB1-fusion proteins by the secondary anti-IgG antibody eliminated the use of the primary antibody for western blot analysis. Based on these data, we propose that the small GB1 domain can be used as a versatile tag for recombinant protein production in plants.