Biology subjects

Staub, J. M.

Publications and source records attributed to Staub, J. M..

3 recordsLinked to original sources

bioRxiv2026

Chloroplast expression of Chlamydomonas glycolate dehydrogenase en route to an improved photorespiratory bypass

Successes with photorespiratory bypass pathway engineering have relied on nuclear transformation, requiring subcellular targeting and protein localization in the target organelles for photorespiration. In the current study, mitochondrial Chlamydomonas glycolate dehydrogenase (CrGDH) was directly expressed in chloroplasts of Chlamydomonas and tobacco, as the first enzymatic step for a photorespiratory bypass. Proof-of-concept experiments in Chlamydomonas were followed by transgenic tobacco lines that confirmed the chloroplast accumulation of active CrGDH protein. Photosynthetic rates and biomass were comparable or less than those of wild type plants under the tested conditions, indicating that chloroplast expression of CrGDH alone is insufficient to improve plant performance. These results suggest that additional downstream enzymes within a complete photorespiratory bypass are required to metabolize glyoxylate derived from CrGDH activity and thereby benefit growth. One of the chloroplast transformed lines (APP2882) accumulated CrGDH while maintaining wild type levels of photosynthesis and biomass, providing a best candidate chassis for engineering full photorespiratory bypass pathways.

plant biology↗

bioRxiv2025

Functional relocation of the maize chloroplast atpB gene to the nucleus restores photosynthetic competence to a gene-edited non-photosynthetic mutant

We present a novel approach to photosynthetic gene engineering in maize using a nuclear-encoded, chloroplast-targeted TALE-cytidine deaminase enzyme to create non-photosynthetic knockout mutants of the chloroplast rbcL gene. An off-target mutation in the adjacent atpB gene, encoding the {beta} subunit of ATP synthase, was consistently found in all edited lines, identified as pigment-deficient in tissue culture. These double mutants, carrying mutations in both genes, were purified to homoplasmy using unique leaf-base regeneration techniques. To test mutation complementation and identify the causal gene, nuclear transgenic lines expressing chloroplast-targeted RbcL and AtpB proteins were generated. The results show that nuclear expression of AtpB restores chlorophyll accumulation and supports wild-type growth in tissue culture.

plant biology↗

bioRxiv2022

Plastid double-strand RNA transgenes trigger small RNA-based gene silencing of nuclear-encoded genes

Plastid transformation technology has been widely used to express traits of potential commercial importance, though the technology has been limited to traits that function while sequestered in the organelle. Prior research indicates that plastid contents can escape from the organelle, suggesting a possible mechanism for engineering plastid transgenes to function in other cellular locations. To test this hypothesis, we created tobacco plastid transformants that express a fragment of the nuclear-encoded Phytoene desaturase (PDS) gene capable of catalyzing post-transcriptional gene silencing if RNA escape to the cytoplasm occurs. We found multiple lines of direct evidence that plastid-encoded PDS transgenes affect nuclear PDS gene silencing: knockdown of the nuclear-encoded PDS mRNA and/or its apparent translational inhibition, biogenesis of 21-nucleotide (nt) phased small interfering RNAs (phasiRNAs), and pigment deficient plants. Furthermore, plastid-expressed double-stranded RNA (dsRNA) with no cognate nuclear-encoded pairing partner also produced abundant 21-nt phasiRNAs in the cytoplasm, demonstrating that a nuclear-encoded template is not required for siRNA biogenesis. Our results indicate that RNA escape from plastids to the cytoplasm occurs broadly, with functional consequences that include entry into the gene silencing pathway. Furthermore, we uncover a method to produce plastid-encoded traits with functions outside of the organelle and open new fields of study in plastid development, compartmentalization and small RNA biogenesis.

plant biology↗