Evolution of GOLDEN2-LIKE gene function in C(3) and C (4) plants

Abstract

A pair of GOLDEN2-LIKE transcription factors is required for normal chloroplast development in land plant species that encompass the range from bryophytes to angiosperms. In the C(4) plant maize, compartmentalized function of the two GLK genes in bundle sheath and mesophyll cells regulates dimorphic chloroplast differentiation, whereas in the C(3) plants Physcomitrella patens and Arabidopsis thaliana the genes act redundantly in all photosynthetic cells. To assess whether the cell-specific function of GLK genes is unique to maize, we analyzed gene expression patterns in the C(4) monocot Sorghum bicolor and C(4) eudicot Cleome gynandra. Compartmentalized expression was observed in S. bicolor, consistent with the development of dimorphic chloroplasts in this species, but not in C. gynandra where bundle sheath and mesophyll chloroplasts are morphologically similar. The generation of single and double mutants demonstrated that GLK genes function redundantly in rice, as in other C(3) plants, despite the fact that GLK gene duplication in monocots preceded the speciation of rice, maize and sorghum. Together with phylogenetic analyses of GLK gene sequences, these data have allowed speculation on the evolutionary trajectory of GLK function. Based on current evidence, most species that retain single GLK genes belong to orders that contain only C(3) species. We therefore propose that the ancestral state is a single GLK gene, and hypothesize that GLK gene duplication enabled sub-functionalization, which in turn enabled cell-specific function in C(4) plants with dimorphic chloroplasts. In this scenario, GLK gene duplication preconditioned the evolution of C(4) physiology that is associated with chloroplast dimorphism.

Fig. 1

GLK gene phylogeny. Bayesian phylognetic tree of GLK genes. Posterior probabilities (B) and bootstrap support values (ML) where appropriate are shown at branch nodes. Sequences highlighted in green are non-angiosperm GLK genes, in purple are C₃ species with a single GLK gene, in blue are C₃ species with GLK duplicates and in red are C₄ species with GLK duplicates. Characterized GLK genes are annotated with numbers (i.e. GLK1, GLK2), those that have not been previously described are annotated with letters (i.e. GLKA-GLKD). GLK sequences correspond to the gene accessions shown in Supplemental Table S1. Asterisk indicates gene duplication in the Poales

Fig. 2

Analysis of GLK gene expression in leaves of Sorghum bicolor and Cleome gynandra. a Gel blot of 10 µg total RNA extracted from total leaf (T), mesophyll (M) or bundle sheath (BS) cells of sorghum. The blot was hybridized with SbGLK1 and SbGLK2, and with maize PEPC (M cell-specific) and RbcS (BS cell-specific) sequences to confirm the purity of the cell preparations. Ethidium bromide stained ribosomal RNA bands are shown as loading controls. b Transcript levels of SbGLK1 and SbGLK2 in BS and M cells of sorghum as determined by 40 bp Illumina RNA sequencing, and quantified as reads per million (RPM) to two decimal places. Sorghum PEPC (M cell-specific) and NADP-ME (BS cell-specific) transcript levels demonstrate the purity of the cell extracts. Significance of differential gene expression between M and BS samples was calculated as described in the “Results”. c Bootstrapped maximum likelihood phylogenetic tree of a subset of GLK genes from the Brassicales with the Aquilegia (Ranunculales) gene used as the outgroup. d qPCR of CgGLK1 and CgGLK2 with RNA extracted from C. gynandra M and BS cells separated by LCM. Values are shown relative to Actin7 transcript levels. Bars and error bars represent means and standard errors of three biological replicates, respectively. e Log2 of the ratio of BS/M transcript levels as determined by qPCR as in c. CgPPC2 (M cell-specific) and CgNADME2 (BS cell-specific) ratios confirm the purity of the cell preparations

Fig. 3

Identification of Osglk2 single mutants. a Schematic of T-DNA insertion in the OsGLK2 gene. XhoI restriction sites are indicated with arrows. Left and right T-DNA borders (LB, RB), the position of the B-glucuronidase (GUS) gene and of the OsGLK2 fragment used for hybridization in b are shown. b Gel blot analysis of XhoI digested DNA from wild-type (Dongjin) and from lines segregating a T-DNA insertion in the OsGLK2 gene. The position of the OsGLK2 fragment used for hybridization is shown in a. Asterisks indicate homozygous mutant lines. c The same blot as in b hybridized with a GUS gene fragment to determine transgene copy number. d RNA gel blot analysis of replicate wild-type (WT), and Osglk2 T₂ single mutant lines. Blots were hybridized with both OsGLK1 and OsGLK2. Ethidium bromide staining of 25S rRNA is shown as a loading control

Fig. 4

Generation of Osglk1 and double-mutant lines. a Alignment of OsGLK1 and OsGLK2 sequences showing the position of the two fragments used for RNAi knockdown of OsGLK1. Fragment 1 is a 395 bp sequence between the DNA-binding domain and GCT-box, and fragment 2 is a 305 bp sequence spanning the GCT-box. b Gel blot of HindIII digested DNA from wild-type (WT) and T₁ Osglk1 knockdown lines. Blots were hybridized with an NPTII fragment from the transformation vector so that the number of hybridizing fragments would reveal the transgene copy number in the genome. c RNA gel blot analysis of replicate wild-type (WT) and Osglk1 T₁ single mutant lines. Blots were hybridized with both OsGLK1 and OsGLK2. Ethidium bromide staining of 25S rRNA is shown as a loading control. d RNA gel blot analysis of replicate wild-type (WT) and T₀ Osglk1,glk2-2 double-mutant lines. Blots were hybridized with both OsGLK1 and OsGLK2. Ethidium bromide staining of 25S rRNA is shown as a loading control. e Phenotype of 2 week old regenerated Osglk1,glk2-2 double-mutant seedlings alongside wild-type (WT), Osglk1-2 and Osglk2-2 single mutant seedlings germinated from seeds

Fig. 5

Characterization of Osglk1-2, glk2-2 double mutants. a Gel blot analysis of HindIII digested DNA from wild-type plants and from F3 and F4 double-mutant plants. The blot was hybridized with an NPTII fragment to determine OsGLK1 RNAi transgene copy number. b, c RNA gel blot analysis of replicate wild-type and double-mutant plants. Blots were hybridized with OsGLK1 (b) and OsGLK2 (c) fragments. Ethidium bromide staining of 25S rRNA is shown as a loading control. d–f Phenotype of wild-type and double-mutant plants demonstrating pale green leaves (d, e) and pale green inflorescences (f) in the double mutant. g, h Representative phenotypes of single and double mutants (g) used for measurement of chlorophyll content (h). Values are the average ± SE of 4 measurements. FW fresh weight

Fig. 6

Leaf anatomy of Osglk single and double mutants. a–f Fresh (a, b) and safranin/fast green stained wax embedded (c–f) sections of wild-type (a, c), regenerated Osglk1,glk2 double (b) Osglk1-2,glk2-2 double (d), Osglk1-2 single (e) and Osglk2-2 single (f) mutants. Black arrowheads point to BS cell chloroplasts. Scale bar 50 µm. g–n Transmission electron micrographs of chloroplasts in M cells (g, i, k, m) and BS cells (h, j, l, n) of wild-type (g, h), Osglk1-2 single mutant (i, j), Osglk2-2 single mutant (k, l) and Osglk1-2,glk2-2 double mutant (m, n). Asterisks in n denotes an adjoining M cell. Black arrows point to granal lamellae; white arrow to disorganized lamellae and white arrowheads to vesicles. Scale bar 1 µm