Plant material and growth conditions
Cleome gynandra L. (Millenium Seedbank, Kew) plants were grown for 10 days in soil under long-day conditions with fluence rates of 150 µmol photon m−2 s−1 and a temperature of 23 °C.
Sorghum bicolor L. Moench inbred line BTx623 (USDA-ARS-SPA, Lubbock, TX, USA) was used as the genetic background for northern blot analyses. Sorghum plants were grown in soil in a greenhouse, with the natural diurnal light period in Oxford (UK), and were supplemented with 500 µmol photon m−2 s−1 when necessary, and up to 14 h in winter. The average daytime temperature was 28 °C and the average night temperature was 20 °C. Sorghum bicolor L. hybrid line Tx430 (Pioneer Hi-Bred, Plainview, TX, USA) was used as the genetic background for Illumina sequencing. Plants were grown in soil in a greenhouse, with the natural diurnal light period in Duesseldorf (Germany) and were supplemented with 300 µmol photon m−2 s−1 when necessary, and up to 14 h in winter. Average daytime temperature was 25 °C and average night temperature was 19 °C.
Oryza sativa var. japonica cv. Dongjin was used as the genetic background for all rice experiments. Rice plants were grown as described for the BTx623 sorghum line. Osglk1 and Osglk2 single mutants were grown and crossed in the glasshouse at the International Rice Research Institute (IRRI, Los Banos, Philippines). T1 seeds of the Osglk1-2 single mutant and T3 homozygous seeds of the Osglk2-2 mutant were incubated at 45 °C for 5 days to break seed dormancy, germinated on MS medium in petri dishes at 30 °C for 7 days, and then transplanted to pots containing soil. Plants were grown with a day/night temperature of 30/22 ± 3 °C and 65–85 % relative humidity. Osglk1-2 single mutants were PCR screened for the RNAi transgene and only PCR-positive plants were transplanted to pots. One-third of these plants should be homozygous for the transgene and two-thirds should be heterozygous.
To identify GLK genes, BLASTP was used to search all of the annotated land plant proteomes on Phytozome v8.0 (http://www.phytozome.net) plus the potato genome sequence (http://potatogenomics.plantbiology.msu.edu/), using the ZmGLK1 amino acid sequence as a query. Results for searches against each proteome were filtered manually to identify GLK genes (distinguished from other GARP family genes by an AREAEAA motif (consensus motif) at the C terminal of the DNA-binding domain). To ensure that all putative GLK genes were identified the amino acid sequences encoded by 5 GLK genes representing a wide range of angiosperm lineages (AtGLK1, GmGLKD, VvGLK, ZmGlk1, OsGLK2) were aligned using MAFFT (Katoh et al. 2005). This alignment was converted to a hidden Markov model and used to search Phytozome v8.0 plant and algal proteomes with an iterative HMMer search algorithm described previously (Eddy 1998; Kelly et al. 2011).
Phylogenetic trees of the identified GLK genes were inferred using both Bayesian and maximum likelihood methods. Protein sequences were aligned using MergeAlign (Collingridge and Kelly 2012). A 100 bootstrap maximum likelihood tree was inferred using RAxML (Stamatakis 2006) employing the LG model of sequence evolution (Le and Gascuel 2008) and CAT rate heterogeneity. A 50 % majority-rule consensus tree was calculated from the 100 bootstrap replicates using the python module dendropy (Sukumaran and Holder 2010). Bayesian phylogenetic trees were inferred using mrbayes v3.1.2 (Huelsenbeck and Ronquist 2001) with gamma-distributed substitution rate variation approximated by four discrete categories and shape parameter estimated from the data. The “covarion” model (Galtier 2001) was implemented and four chains were employed, each with a temperature of 0.2. Tree inference was made from a random start tree and allowed to run for 2,500,000 generations. The time taken to reach stationary phase was approximately 700,000 generations and thus the final 1,800,000 trees sampled every 200 generations were used to infer posterior probabilities on topology.
Identification of Osglk2 insertional mutants
Osglk2 T-DNA insertion lines (PFG-3A-13668.L) were ordered from RiceGE: Rice Functional Genomic Express Database http://signal.salk.edu/cgi-bin/RiceGE (An et al. 2003). 15 lines of T2 seeds were received (PFG-3A-13668-01 to PFG-3A-13668-15). DNA was extracted from five seedlings of each line, and PCR was performed using forward (5′-CAATTATGCGGTAGCAGCTG-3′) and reverse (5′-TCTCTGTCCAATAAAATCGAACTTC-3′) primers flanking the insertion, and a T-DNA right border primer (5′-AACGCTGATCAATTCCACAG-3′). The forward and reverse primers were used as a pair to generate a 1,072-bp fragment of the wild-type allele. The forward primer and T-DNA right border primer were used as a pair to generate a shorter fragment of the insertion allele. PCR conditions were 35 cycles of: 95 °C for 30 s, 53 °C for 30 s, 72 °C for 1.5 min. Lines containing the insertion allele were carried through to DNA gel blot analysis.
Generation of Osglk1 RNAi mutant lines
Osglk1 single mutant lines were generated by RNAi knock down of the OsGLK1 gene (Os06g24070) in O. sativa Dongjin. A 305-bp sequence of the OsGLK1 GCT-box (fragment 2 in Fig. 4a) was used as the target sequence. The sequence was first inserted downstream of the potato GA20 oxidase intron in the pUC-RNAi vector (Fang et al. 2008), as a BamHI/XbaI fragment in the sense orientation. The same sequence was then inserted in the antisense orientation into the BglII/SpeI sites of the pUC-RNAi construct that contained the sense fragment. To create the binary construct, the fragment comprising sense and antisense sequences of OsGLK1, separated by the potato GA20 oxidase intron, was excised from pUC-RNAi and inserted into the Pst1 site of pXQAct (Fang et al. 2008) between the rice actin1 promoter and Ocs terminator. Agrobacterium-mediated transformation into wild-type Dongjin callus was performed as described (Nishimura et al. 2006). After selection with G418 and PCR validation, seven regenerated plants were obtained that contained the RNAi construct.
Generation of Osglk1,glk2 double-mutant lines
To generate a double mutant, a 395-bp sequence between the OsGLK1 gene DNA-binding domain and GCT-box (fragment 1 in Fig. 4a) was used to create an RNAi construct as shown earlier. This construct was transformed into Osglk2-2 mutant callus. After selection with G418 and PCR validation, 20 regenerated plants were obtained that contained the RNAi construct. Unfortunately, none of the regenerated double mutants produced viable seed. An F2 population that segregated double mutants was therefore generated by crossing a homozygous Osglk2-2 single mutant line with a hemizygous Osglk1-2 knockdown line. The resultant F1 progeny were selfed to generate a segregating F2 population.
Isolation of BS and M cells
For northern blot analysis, BS and M cells were separated from fully expanded 3rd leaves of S. bicolor inbred line BTx623. M cells were separated enzymatically from leaf tissue essentially as described by Sheen and Bogorad (1985), but with vanadyl ribonucleoside complex omitted from the protoplast washing buffer. Bundle sheath strands were isolated mechanically using a household blender. Leaves were blended and filtered through 60 µM mesh using buffers described by Westhoff et al. (1991). Cell preparations were checked microscopically for purity and immediately frozen in liquid nitrogen before storage at −80 °C. For Illumina sequencing, M and BS cells were separated enzymatically as described previously (Wyrich et al. 1998).
C. gynandra BS and M cells were isolated by laser capture microdissection (LCM). Mature leaf tissue was harvested 4 h after dawn and immediately infiltrated with ethanol: acetic acid (3:1, v/v). The tissue was processed through a dehydration series of ethanol and Histoclear and then replaced by Paraplast Xtra. Leaf sections were floated in ethanol on MembraneSlide 1.0 PEN (Zeiss). LCM was performed using Arcturus XT (Life Technologies) and M and BS cells were captured using HS adhesive caps (Life Technologies) following the manufacturer’s instructions.
DNA and RNA analysis
Genomic DNA was isolated using a modified CTAB method (Murray and Thompson 1980). Total leaf RNA was isolated by guanidinium thiocyanate–phenol–chloroform extraction as described by Waters et al. (2008). RNA was extracted from separated sorghum BS and M cells as described by Sheen and Bogorad (1985) (for northern blot analysis) or by Wyrich et al. (1998) (for Illumina sequencing).
Total RNA from BS or M cells of C. gynandra harvested by LCM was extracted from three independent replicates using a Picopure RNA isolation kit (Life Technologies) and DNAse treatment. RNA integrity was assessed on a Bioanalyzer 2100 RNA picochip (Agilent). At least 5 ng of RNA for each sample was subsequently amplified through two rounds of amplification using the RiboAmp HS plus RNA amplification kit (Life Technologies).
For Illumina sequencing, RNA from five cell preparations of 10-day-old sorghum seedlings was pooled and the mRNA content was purified using the Oligotex mRNA Midi Kit (Qiagen). cDNA was produced using the SMARTer PCR cDNA Synthesis Kit (Clontech) and sent to GATC Biotech AG (Konstanz, Germany) for 40 bp Illumina sequencing using a standard library preparation protocol. Following standard GATC quality filtering, raw reads were mapped to sorghum Sbi1_4 gene models (http://genome.jgi-psf.org/Sorbi1/Sorbi1.info.html) using Bowtie 0.12.8 (Langmead et al. 2009) in the –v alignment mode with up to 3 mismatches and the –best option activated. Differentially expressed genes were calculated using a significance test (Audic and Claverie 1997) followed by a Bonferroni correction.
For real-time PCR, first-strand cDNA was synthesized from 5 ng amplified RNA using Superscript II (Invitrogen). Real-Time PCR was performed using SYBRgreen Jumpstart (Sigma) in a rotor-gene-Q system (Qiagen). Relative transcript levels were calculated based on Actin 7 levels. Primer sequences were as follows—CgGLK1: 5′-TCCGACTTGTGCACCGTATGATGT-3′ and 5′-ACCGAATGCCAAATGGAACGACAC-3′; CgGLK2: 5′-AAAGTTACGGGAGACGGTGGGAAA-3′ and 5′-CACGAATTTCCGGTGCAATTCCGA-3′; CgACT7: 5′-TCCGACCCGATGTGATGTTATGGT-3′ and 5′-CAATCACTTTCCGGCTGCAACCAA-3′.
DNA and RNA gel blots were prepared and hybridized in 0.45 M NaCl at 65 °C as described previously (Langdale et al. 1988a), using gene-specific probes as follows: SbGLK1 (transcript bases 1558–1864), SbGLK2 (transcript bases 2029–2346), ZmPEPC (pTN1, Langdale et al. 1988a), ZmRbcS (pJL10, Langdale et al. 1988a), OsGLK1 (transcript bases 1543–1856), OsGLK2 (transcript bases 2044–2325), NPTII, GUS (290 bp from the 5′ end of the cDNA amplified using primers 5′-ATGTTACGTCCTGTAG-3′ and 5′-ACTTTGCCGTAATGAGTGACC-3′). Blots were visualized and quantified using a Molecular FX phosphorimager (Bio-Rad, http://www.bio-rad.com/).
Light and transmission electron microscopy
For light microscopy, thick sections were prepared according to Yamada et al. (2009). One-month-old leaf blades were vacuum infiltrated for 10 min with fixation buffer [50 mM PIPES–NaOH, pH 6.9, 4 mM MgSO4, 10 mM EGTA, 0.1 % (w/v) Triton X-100, 200 µM phenylmethylsulfonyl fluoride, 5 % (v/v) formaldehyde and 1 % (v/v) glutaraldehyde] and then incubated at 4 °C overnight. The fixed segments were then embedded in 5 % (w/v) agar and sectioned at 70–80 µm with a Vibratome Series 1000 Sectioning System. Alternatively, leaf samples were fixed overnight in FAA (4 % formaldehyde, 5 % acetic acid, 50 % ethanol) and embedded in Paraplast Plus. Thin sections (8 µm) were cut using a rotary microtome and stained with Safranin/Fast Green as described previously (Langdale 1994). Sections were viewed and photographed with a Leica DMRB microscope.
For transmission electron microscopy, leaf samples were fixed in the dark by immersion in ice-cold fixative (4 % paraformaldehyde, 3 % glutaraldehyde in 0.05 M potassium phosphate buffer, pH 7) followed by vacuum infiltration. Subsequent steps were performed as described previously (Waters et al. 2008). Samples were stained sequentially with 2 % w/v OsO4 and 0.5 % w/v uranyl acetate and embedded in TAAB 812 resin (TAAB Laboratory Equipment, http://www.taab.co.uk). 0.1 µm sections were stained with 0.2 % w/v lead citrate, rinsed in deionized water, and then examined using a Zeiss (LEO) Omega 912 electron microscope. Digital images were captured using the SIS package (Soft Imaging Software GmbH, http://www.soft-imaging.net).
Chlorophyll was extracted from 2-month-old rice plants with replicates from four different plants assayed per line. Leaf tissues of the same fresh weight (200 mg) were ground in liquid nitrogen and resuspended in 80 % acetone. After incubation overnight in the dark at 4 °C, cell debris was pelleted by centrifugation for 1 min at 15,000g and the absorbance of the supernatant was measured at 663 and 645 nm on a Unicam UV4 UV/Vis Spectrometer. Total chlorophyll was calculated as (8.02 × A663 + 20.29 × A645) × V/1,000 × W, where V = volume of the extract (ml); W = weight of fresh leaves (g) (Arnon 1949).