Genetically engineered eucalyptus expressing pesticidal proteins from Bacillus thuringiensis for insect resistance: a risk assessment evaluation perspective.

Eucalyptus covers approximately 7.5 million hectares in Brazil and serves as the primary woody species cultivated for commercial purposes. However, native insects and invasive pests pose a significant threat to eucalyptus trees, resulting in substantial economic losses and reduced forest productivity. One of the primary lepidopteran pests affecting eucalyptus is Thyrinteina arnobia (Stoll, 1782) (Lepidoptera: Geometridae), commonly referred to as the brown looper caterpillar. To address this issue, FuturaGene, the biotech division of Suzano S.A., has developed an insect-resistant (IR) eucalyptus variety, which expresses Cry pesticidal proteins (Cry1Ab, Cry1Bb, and Cry2Aa), derived from Bacillus thuringiensis (Bt). Following extensive safety assessments, including field trials across various biomes in Brazil, the Brazilian National Technical Commission of Biosafety (CTNBio) recently approved the commercialization of IR eucalyptus. The biosafety assessments involved the analysis of molecular genomics, digestibility, thermostability, non-target organism exposure, degradability in the field, and effects on soil microbial communities and arthropod communities. In addition, in silico studies were conducted to evaluate allergenicity and toxicity. Results from both laboratory and field studies indicated that Bt eucalyptus is as safe as the conventional eucalyptus clone for humans, animals, and the environment, ensuring the secure use of this insect-resistant trait in wood production.

Safety Assessment of the CP4 EPSPS and NPTII Proteins in Eucalyptus.

Glyphosate herbicide treatment is essential to sustainable Eucalyptus plantation management in Brazil. Eucalyptus is highly sensitive to glyphosate, and Suzano/FuturaGene has genetically modified eucalyptus to tolerate glyphosate, with the aim of both protecting eucalyptus trees from glyphosate application damage and improving weed management. This study presents the biosafety results of the glyphosate-tolerant eucalyptus event 751K032, which expresses the selection marker neomycin phosphotransferase II (NPTII) enzyme and CP4-EPSPS, a glyphosate-tolerant variant of plant 5-enolpyruvyl-shikimate-3-phosphate synthase enzyme. The transgenic genetically modified (GM) event 751K032 behaved in the plantations like conventional non-transgenic eucalyptus clone, FGN-K, and had no effects on arthropods and soil microorganisms. The engineered NPTII and CP4 EPSPS proteins were heat-labile, readily digestible, and according to the bioinformatics analyses, unlikely to cause an allergenic or toxic reaction in humans or animals. This assessment of the biosafety of the glyphosate-tolerant eucalyptus event 751K032 concludes that it is safe to be used for wood production.

Multiple Gibberellin Receptors Contribute to Phenotypic Stability under Changing Environments.

The pleiotropic and complex gibberellin (GA) response relies on targeted proteolysis of DELLA proteins mediated by a GA-activated GIBBERELLIN-INSENSITIVE DWARF1 (GID1) receptor. The tomato (Solanum lycopersicum) genome encodes for a single DELLA protein, PROCERA (PRO), and three receptors, SlGID1a (GID1a), GID1b1, and GID1b2, that may guide specific GA responses. In this work, clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR associated protein 9-derived gid1 mutants were generated and their effect on GA responses was studied. The gid1 triple mutant was extremely dwarf and fully insensitive to GA. Under optimal growth conditions, the three receptors function redundantly and the single gid1 mutants exhibited very mild phenotypic changes. Among the three receptors, GID1a had the strongest effects on germination and growth. Yeast two-hybrid assays suggested that GID1a has the highest affinity to PRO. Analysis of lines with a single active receptor demonstrated a unique role for GID1a in protracted response to GA that was saturated only at high doses. When the gid1 mutants were grown in the field under ambient changing environments, they showed phenotypic instability, the high redundancy was lost, and gid1a exhibited dwarfism that was strongly exacerbated by the loss of another GID1b receptor gene. These results suggest that multiple GA receptors contribute to phenotypic stability under environmental extremes.

The Putative O-Linked N-Acetylglucosamine Transferase SPINDLY Inhibits Class I TCP Proteolysis to Promote Sensitivity to Cytokinin.

Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) is a putative serine and threonine O-linked N-acetylglucosamine transferase (OGT). While SPY has been shown to suppress gibberellin signaling and to promote cytokinin (CK) responses, its catalytic OGT activity was never demonstrated and its effect on protein fate is not known. We previously showed that SPY interacts physically and functionally with TCP14 and TCP15 to promote CK responses. Here, we aimed to identify how SPY regulates TCP14/15 activities and how these TCPs promote CK responses. We show that SPY activity is required for TCP14 stability. Mutation in the putative OGT domain of SPY (spy-3) stimulated TCP14 proteolysis by the 26S proteasome, which was reversed by mutation in CULLIN1 (CUL1), suggesting a role for SKP, CUL1, F-box E3 ubiquitin ligase in TCP14 proteolysis. TCP14 proteolysis in spy-3 suppressed all TCP14 misexpression phenotypes, including the enhanced CK responses. The increased CK activity in TCP14/15-overexpressing flowers resulted from increased sensitivity to the hormone and not from higher CK levels. TCP15 overexpression enhanced the response of the CK-induced synthetic promoter pTCS to CK, suggesting that TCP14/15 affect early steps in CK signaling. We propose that posttranslational modification of TCP14/15 by SPY inhibits their proteolysis and that the accumulated proteins promote the activity of the CK phosphorelay cascade in developing Arabidopsis leaves and flowers.

Uncovering DELLA-Independent Gibberellin Responses by Characterizing New Tomato procera Mutants.

Gibberellin (GA) regulates plant development primarily by triggering the degradation/deactivation of the DELLA proteins. However, it remains unclear whether all GA responses are regulated by DELLAs. Tomato (Solanum lycopersicum) has a single DELLA gene named PROCERA (PRO), and its recessive pro allele exhibits constitutive GA activity but retains responsiveness to external GA. In the loss-of-function mutant pro(ΔGRAS), all examined GA developmental responses were considerably enhanced relative to pro and a defect in seed desiccation tolerance was uncovered. As pro, but not pro(ΔGRAS), elongation was promoted by GA treatment, pro may retain residual DELLA activity. In agreement with homeostatic feedback regulation of the GA biosynthetic pathway, we found that GA20oxidase1 expression was suppressed in pro(ΔGRAS) and was not affected by exogenous GA3. In contrast, expression of GA2oxidase4 was not affected by the elevated GA signaling in pro(ΔGRAS) but was strongly induced by exogenous GA3. Since a similar response was found in Arabidopsis thaliana plants with impaired activity of all five DELLA genes, we suggest that homeostatic GA responses are regulated by both DELLA-dependent and -independent pathways. Transcriptome analysis of GA-treated pro(ΔGRAS) leaves suggests that 5% of all GA-regulated genes in tomato are DELLA independent.

Cytosolic activity of the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1A.

Arabidopsis has three gibberellin receptors, GIBBERELLIN INSENSITIVE DWARF1 A (GID1A), GID1B and GID1C. All GID1s are localized to the nucleus but have also been detected in the cytoplasm. Since other major gibberellin signaling components have been localized to the nucleus, the current model suggests that gibberellin perception and signaling is nuclear. In this work we investigated whether GID1A can perceive gibberellin and initiate a signaling cascade in the cytosol. We have generated transgenic plants expressing GID1A fused to green florescence protein (GFP) and either a nuclear export signal (NES) or a nuclear localization signal (NLS), in the background of the gid1a gid1c (gid1ac) double mutant. Analyses by confocal microscopy confirmed the localization of GFP-GID1A-NES only in the cytosol and of GFP-GID1A-NLS only in the nucleus. Surprisingly, both recombinant proteins were able to complement the gid1ac mutation and to restore growth and responsiveness to gibberellin. However, transgenic seeds and seedlings expressing GFP-GID1A-NLS were more sensitive to gibberellin than those expressing GFP-GID1A-NES. The results of this study suggest that GID1A can bind gibberellin in the cytosol to initiate gibberellin signaling and responses. It is possible that activated cytosolic GID1A interacts with DELLA proteins before they enter the nucleus and induces a conformational transition that leads to DELLA's deactivation or degradation in the nucleus.

Assembly of coordination nanostructures via ligand derivatization of oxide surfaces.

A scheme is presented for the construction of coordination nanostructures on oxide surfaces (glass, Si/SiO2, quartz), based on application of epoxy-terminated monolayers as anchors for covalent grafting of ligands. Two ligands bearing amine groups were reacted with epoxysilane monolayers on oxide surfaces, providing ligand-terminated substrates. The ligands employed were (i) a pyridine moiety, used for subsequent binding of cobalt tetraphenylporphine (CoTPP), and (ii) deferoxamine (DFX), which contains hydroxamic acid moieties, used for subsequent construction of various Zr4+-based coordination layers. The results suggest that a dense ligand layer was obtained in both cases, allowing the formation of coordination overlayers on the oxide surfaces. The growth of coordinated layers was similar to analogous overlayers assembled on Au substrates, indicating that high ligand coverage is achieved by the epoxy-amine surface reaction. Epoxy-based functionalization of oxide substrates is a mild and efficient method for preparing high-quality coordination overlayers. Moreover, the method makes use of commercially available silane and amine reactants, providing the basis for wide application.