A novel RNA binding protein affects rbcL gene expression and is specific to bundle sheath chloroplasts in C4plants

Abstract

BackgroundPlants that utilize the highly efficient C4 pathway of photosynthesis typically possess kranz-type leaf anatomy that consists of two morphologically and functionally distinct photosynthetic cell types, the bundle sheath (BS) and mesophyll (M) cells. These two cell types differentially express many genes that are required for C4 capability and function. In mature C4 leaves, the plastidic rbcL gene, encoding the large subunit of the primary CO2 fixation enzyme Rubisco, is expressed specifically within BS cells. Numerous studies have demonstrated that BS-specific rbcL gene expression is regulated predominantly at post-transcriptional levels, through the control of translation and mRNA stability. The identification of regulatory factors associated with C4 patterns of rbcL gene expression has been an elusive goal for many years.ResultsRLSB, encoded by the nuclear RLSB gene, is an S1-domain RNA binding protein purified from C4 chloroplasts based on its specific binding to plastid-encoded rbcL mRNA in vitro. Co-localized with LSU to chloroplasts, RLSB is highly conserved across many plant species. Most significantly, RLSB localizes specifically to leaf bundle sheath (BS) cells in C4 plants. Comparative analysis using maize (C4) and Arabidopsis (C3) reveals its tight association with rbcL gene expression in both plants. Reduced RLSB expression (through insertion mutation or RNA silencing, respectively) led to reductions in rbcL mRNA accumulation and LSU production. Additional developmental effects, such as virescent/yellow leaves, were likely associated with decreased photosynthetic function and disruption of associated signaling networks.ConclusionsReductions in RLSB expression, due to insertion mutation or gene silencing, are strictly correlated with reductions in rbcL gene expression in both maize and Arabidopsis. In both plants, accumulation of rbcL mRNA as well as synthesis of LSU protein were affected. These findings suggest that specific accumulation and binding of the RLSB binding protein to rbcL mRNA within BS chloroplasts may be one determinant leading to the characteristic cell type-specific localization of Rubisco in C4 plants. Evolutionary modification of RLSB expression, from a C3 “default” state to BS cell-specificity, could represent one mechanism by which rbcL expression has become restricted to only one cell type in C4 plants.

Engineering chloroplasts to improve Rubisco catalysis: prospects for translating improvements into food and fiber crops

“…Unfortunately, generating site-directed mutants has Deletion of MRL1 has no impact of synthesis of Rubisco L-subunits (Johnson et al, 2010). However, the RLSB (RBCL RNA S1-binding domain) protein has been shown to regulate rbcL transcript abundance in C 3 (Yerramsetty et al, 2016) and C 4 plant chloroplasts (Bowman et al, 2013). (b) Representation of the cm TrL masterline plastome and the general rbcL transforming plasmid pLev4 (Whitney et al, 2011b), which is equipped with restriction sites Nhe1 at the 5 0 end and Sal1 or Xba1 at the 3 0 end for inserting foreign rbcL genes.…”

Regulation of Rubisco gene expression in C4 plants

“…Note the specific localization of the Rubisco protein and transcript to leaf BSC, with no accumulation in the MC. For the top panel, Rubisco immunolocalization was performed using antisera specific for the Rubisco LSU, and a secondary antibody conjugated with the fluorescent dye Alexafluor 546 as described [25 ]. Laser scanning confocal imaging reveals Rubisco localization in this Kranz-type C 4 leaf section as yellow colored fluorescence only within the chloroplasts of BSC, and not in the chloroplasts of MC (observed as blue autofluorescence).…”

Vaccination via Chloroplast Genetics: Affordable Protein Drugs for the Prevention and Treatment of Inherited or Infectious Human Diseases

“…Because plastid gene expression is primarily regulated at the post-transcriptional level (6), selection of 5′ and 3′ ntranslated regions (UTRs) (Figure 1 a ) is also crucial for promoting maximal gene expression. In particular, the stem-loop secondary structure of the 5′ UTR stabilizes mRNAs and promotes efficient translation, likely through specific interactions with RNA-binding proteins (RBPs) (8, 52, 107, 150). The 3′ UTR facilitates mRNA stability of plastid-expressed genes (128).…”