LABORATORY PROCEDURES

2.2.1 RNA Extraction

RNA was extracted from ground frozen (-80ᵒC) leaf material using a modified version of the hot phenol extraction method of Verwoerd et al. (1989). A first extraction was performed with hot (80ᵒC) 1:1 phenol/extraction buffer (0.1M Tris/HCl, 0.1M LiCl, 1% SDS, 10mM EDTA, pH 8.0) and chloroform / isoamyl alcohol (IAA) (24:1). A second extraction of the aqueous phase was performed with chloroform/IAA. The aqueous phase was than mixed with 1 volume 4M LiCl to let the RNA precipitate overnight at 4ᵒC. The RNA was then collected

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by centrifugation and the pellet washed with 70% ethanol. After a DNase treatment with 5.0 units of RNase-free DNase (Promega), another chloroform/IAA extraction was performed. The RNA was then precipitated by adding 1/10 volume 3M sodium acetate and 2.75 volume ethanol to the aqueous phase and incubation at -20ᵒC. After collecting the RNAs by centrifugation the pellet was washed with 70% ethanol and solved in 50 µl DEPC-treated water. Quality and quantity of the RNAs were checked by measuring the concentration with a Nano Drop™ ND-1000 photo- spectrometer (Thermo Scientific, USA) and by TAE-agarose electrophoresis (1% gel).

2.2.2 cDNA Synthesis

cDNA was synthesised using Superscript III reverse transcriptase (Invitrogen). 2.0 µg RNA, 1.0 µl 10 mM dT-adapter primers and H2ODepc to a total volume of

13.0 µl were mixed and incubated at 70 ᵒC for 10 minutes. After chilling on ice 4.0 µL 5x buffer, 1.0 µl 0.1M DTT, 1.0 µl 10mM dNTPs and 1.0 µl Superscript III enzyme were added and the solution was first incubated for 5 minutes at room temperature, then 90 minutes at 50ᵒC, and finally 15 minutes at 70ᵒC. In some cases 5.0 µl H2ODEPC was then added to a total volume of 25.0 µl.

2.2.3 Semi-Quantitative PCR

1.0 µl of cDNA was used for semi-quantitative PCR (sq-PCR) in a reaction volume of 15.0 µl containing 0.3 µl of both sense and antisense primer, 7.5 µl RedTaq mix (Sigma) and 5.9 µl H2O. The PCR regime consisted of an initial 2

minutes at 94ᵒC, then an x number of cycles with 30 seconds at 94ᵒC, 20 seconds at 57 ᵒC (except for total NAC expression at 60ᵒC) and 40 seconds at 72 ᵒC, and finally 5 minutes at 72 ᵒC. The primers and the number of PCR cycles (during the linear phase of amplification) depended on the gene analysed (Table 2.6). All primers were designed to give PCR products of approximately 500bp.

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Table 2.6:Primers used for semi-quantitative PCR. * are TIGR plant transcript assemblies. † annealing temperature of 60ᵒC instead of 57ᵒC.

Gene Accession number Sense primer Antisense primer Number of cycles

Actin2 TC234027 CCTTCAATGTTCCAGCCATGTA ATAGTTGAGCCACCACTGAGCA 25

CCT TC266579 CGAGCTCTACAACCCCATGC CGGTAAGTCCAGCCAGGAATC 32 DNA-binding TC240748 GTTGAAGCGAGTCATCCACTTG CGAAGATCGTCCAGAGTATCAGC 32 F-box TC257395 GGTTCCGTTCGGTTGCTTATC GCACACGACGGAGGAAGTAAG 30 GLK1 TA110567_4565* GCATGCCGGTGTAAGTTCAGC GTGGTATCATCGGCGTGGATAA 38 MYBa TC275306 GCTCATACAGCTGCAACAGACA CTGTGTATTAAGGTGGTGGCTGA 33 MYBb TC263812 CTAACCTCACCATTTCAGGAAGC CATGGCTGAGAAGATAGGACGAG 23 MYC TA70705_4565* ACGCCATCTCCTACATCAACG CGGTGGTGAGTAATAGGATCACG 32 NAC

endogenous

TA67563_4565* CAAGGAAGACTGGGTGCTATGC GGAACAGACACTGCATCAACCA 33

NAC total† TA67563_4565* GGCAGGGAGTGGTACTTCTTCAG CACTTGCTCGATGGTGTTCATC 31 NAC

transgenic

TA67563_4565* CAAGGAAGACTGGGTGCTATGC (= same as endogenous NAC)

GAGAGAGACTGGTGATTTCAGCG (35S terminator primer)

40

PTF1 TC253044 CTAATGGAAACAGTGCCAGTGC GTGCCTTTCTGGTTGGATATGG 31

RBCS TC263601 AAGCCAGAGTGCCTCCTCCTA GTACGCGTCAGGGTACTCCTTC 17

RING TC268503 CAACATTTGGCTGAGAATGACC GATCAACTATGCCCTGCATTTG 33

SAG12 TC63758 AGAGAACGGCAAGGACTACTGG GTCGTGATGCAAATGTTTACGCG 27

WRKY total TC271537 BE406842 CTTCGTCCAAGAACAGCAAGAAC CTCATAAACCCATGTCCCCTTG 33 WRKY transgenic TC271537 BE406842 GACGTCGGCGCCCAGTCCATCATGAATA GAGAGAGACTGGTGATTTCAGCG (35S terminator primer) 40 ZFP-TF TC235744 CTTGGTGGTGACTACGCCAAC TGATGATGTCGACTGCRGGAC 33

All PCR samples were run on a 1.2% TAE-agarose gel with ethidium-bromide staining (3.0 µl per 100 ml gel), keeping replicates together in the cases all samples did not fit on one gel. An example gel is shown in Figure 2.6. In the case of the Hereward samples 11.0 µl of PCR product was run on a gel with medium-sized wells (comb of 20 on 100 ml gel). The band intensities were analysed using GeneSnap (6.00.21) and GeneTools (3.02.00) Analysis Software (Syngene, Cambridge). For the Avalon x Cadenza and the transgenic wheat samples, 5.0 µl of the PCR product was loaded into small wells (comb of 30 on 100 ml gel). The band intensities were analysed using GeneSnap (7.8.1.0) and GeneTools (4.1.2.0) Analysis Software (Syngene, Cambridge).

Expression of the actin2 reference gene was measured first for all samples to assess differences in cDNA concentrations between the samples. actin2

expression of all samples was related to the sample with the highest expression to get “conversion factors” for normalisation of other genes:

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( ⁄ ). These factors were multiplied with expression values of all other genes to normalise for differences in cDNA concentrations. For all experiments except the Hereward samples, an extra normalisation step was introduced: the relative expression of each sample was related to the 500bp band of 1.0 µl of the GeneRuler 100bp DNA ladder (Fermentas) to get comparable numbers for all the genes. However, since the number of PCR cycles differed between genes (Table 2.6), expression of different genes cannot be compared. Therefore the expression measured was relative rather than absolute, and only indicative of relative differences in expression of a single gene between different samples.

Figure 2.6: Example of a gel of semi-quantitative PCR. Expression of the

actin2 reference gene (top) was similar in all samples while expression of the

RBCS marker gene (bottom) in the same samples differed depending on senescence status of the leaves. The ladder is the GeneRuler 100bp DNA ladder (Fermentas).

2.2.4 Genomic DNA Extraction

Genomic DNA (gDNA) was extracted from ground frozen leaf material (1-2 cm section) according to a protocol modified from Lin et al. (2001). 600 µl DNA extraction buffer (0.1M Tris-HCl pH 8.0, 10mM EDTA pH 8.0, 1.0M KCl, 0.75% PVP-40, 3.6 g/L NaHSO4) was added and the samples were incubated at 65ᵒC

while shaking. 200 µl of 5M KAc (pH 5.2) was added and the samples were mixed and then centrifuged for 5 min at maximum speed. 0.6 volume

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isopropanol was added to the supernatant and the samples were precipitated for 10 minutes at -20ᵒC. After 10 minutes centrifugation the pellet was washed with 70% ethanol. The pellet was than dried and solved in 200 µl 10mM Tris-HCl buffer (pH 8.0) containing 200 µg RNase A.

2.2.5 PCR Screening for Transgenic Plants

The primers used for the screening of plants containing a NAC-overexpression construct were a primer on the NAC gene (NAC-CK208366for = 5’- CAA GGA AGA CTG GGT GCT ATG C- 3’) and a primer on the 35S-terminator (35S- TermRev = 5’- CCC GGG ATC TGG ATT TTA GTA CTG GAT -3’), so that the endogenous NAC would not be detected. For the WRKY-RNAi plants the same 35S reverse primer was used together with a primer based on the border of spacer and WRKY sequence (WRKY-RNAi-Rev = 5’- GAC GTC GGC GCC CAG TCC ATC ATG AAT A -3’). 0.2 µl of gDNA (or 1 µl 10x diluted gDNA) was used for PCR in a reaction volume of 15 µl (0.3 µl of both the sense and antisense primers, 7.5 µl RedTaq mix (Sigma), rest H2O). The PCR regime is an initial 2

minutes at 94ᵒC, then 40 cycles with 30 seconds at 94ᵒC, 20 seconds at 57 ᵒC and 40 seconds at 72 ᵒC, and finally 5 minutes at 72 ᵒC. The product was run on a 1%-TAE-gel with ethidium-bromide staining. The product sizes were 653 and 485 bp for NAC and WRKY respectively.

2.2.6 Nitrogen

The nitrogen concentration of dried plant material was analysed using either a CNS 2000 or a TruSpec Combustion Analyser (both from LECO Corporation, USA) via the Dumas combustion method (Dumas, 1831). The samples were first milled and then oven-dried overnight at 80ᵒC, after which either 300 mg (CNS 2000) or 150 mg (TruSpec) samples were weighed out for analysis. Total

nitrogen contents were calculated according to the formula:

. The nitrogen harvest index was calculated according to the formula:

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2.2.7 Thousand Grain Weight

Thousand grain weights (TGW) were determined by counting out two sets of 500 seeds, drying them overnight at 105ᵒC and then weighing them. Two sets of 500 were used instead of one of 1000 to be able to check whether there was variation within the sample (so if a representative subsample was taken).