MPK12 is the gene responsible for "cool" leaf surface temperature130

alignment analysis of amino acid between Col-0 and Cvi-0 and the analysis of guard cell gene expression in Columbia. The nucleotide sequences of the candidate genes were downloaded from http://signal.salk.edu/atg1001/3.0 /gebrowser.php and were translated into amino acid sequences using an online tool, EMBOSS Transeq (http://www.ebi.ac.uk/Tools/st/emboss_transeq/). Subsequently, alignments of amino acid sequences in Col-0 and Cvi-0 were performed for each candidate gene (data not shown). Genes showing an amino acid difference between Col and Cvi had priority to be tested. In addition, guard cells gene expression data for each candidate gene of Col-0 was downloaded from http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi.

Genes with high expression levels in guard cells were tested firstly. As a result, genes such as AT2G45910, AT2G46070, AT2G46080, AT2G46090, AT2G46140, AT2G46150, AT2G46250, AT2G46280, and AT2G46310 acquired priority to be tested by both Cvi transformants with wild type Col alleles, and by Col knockout mutants.

3.3.2.7 MPK12 is the gene responsible for "cool" leaf surface temperature

Cvi transformants with wild type Col alleles were generated by first cloning nine Col-0 genes with their endogenous promoters into pBI121 vector, and then transforming these constructs into wild type Cvi-0 plants through floral dip, and selecting for

AT2G46240 BAG6 A member of Arabidopsis BAG (Bcl-2-associated athanogene) proteins.

AT2G46250 Myosin Myosin heavy chain-related.

AT2G46255 MIR159C Encodes a microRNA that targets several MYB family members.

AT2G46260 - BTB/POZ/Kelch-associated protein.

AT2G46270 GBF3 encodes a bZIP G-box binding protein.

AT2G46280 TRIP-1 Encodes a homolog of mammalian TGF-beta receptor interacting protein.

AT2G46290 WD40 Transducin/WD40 repeat-like superfamily protein.

AT2G46300 LEA3 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family.

AT2G46308 - Unknown protein.

AT2G46310 CRF5 CRF5 encodes one of the six cytokinin response factors.

AT2G46320 - Mitochondrial substrate carrier family protein.

AT2G46330 - AGP16:Encodes arabinogalactan protein (AGP16).

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Figure 3-39 Increased leaf surface temperature of Cvi transformants homozygous for Col MPK12 allele. Enhanced leaf surface temperatures in Cvi transformants homozygous for Col MPK12 allele A), and B) thermogram and leaf surface temperatures of Cvi transformants. Plants were 20 days old and grown under short day conditions (8 hours light / 16 hours dark photoperiod) at photon flux density of 150 μmol m^-2 s^-1, and 22°C and 50% relative humidity in the daytime and 17°C and 60% relative humidity at night. The scale bar donates 2 cm. C) Electrophoresis result indicates two successful transformation events. Water (-) and Cvi served as negative controls, PSK :: MPK12 plasmid DNA (+) served as positive control. A-B) n=3 biological replicates, mean ± SEM, **P<0.001 compared with corresponding wild types.

homozygous lines as carried out for RCAR1 (Ma et al., 2009). Successful transformants were identified by selecting stay-green seedlings grown on 1/2 sugar MS medium containing antibiotics kanamycin. The T1 transformants were then grown in soil for three months to amplify T2 seeds. Subsequently, leaf temperatures of transgenic lines harboring three genes (AT2G46070, AT2G46080, and AT2G46280 respectively) were analyzed (marked in Table 3-3). During this period, the MPK12 gene (AT2G46070), one of the candidate genes, was identified by another group to be responsible for the WUE of plants through an analysis of carbon isotope compositions of an RILs population derived from Ler-0 and Cvi-0. The leaf surface temperatures of our transgenic lines were analyzed and confirmed the results of MPK12 (At2G46070) being Cool1. Among these candidates, the Cvi line homozygous for the Col MPK12 allele (CviMPK12) showed a Col-like phenotype, with a leaf surface temperature of more than 1 °C higher than Cvi -0 wild type, while transgenic lines homozygous for other Col alleles did not show any leaf temperature differences (Fig.

3-39A and B). Electrophoresis data of the transgenic line CviMPK12 confirmed that

132 target constructs were transformed into Cvi-0 wild type plants (Fig. 3-39C).

Figure 3-40 Reduced leaf surface temperature of knockout MPK12 mutant mpk12-3 in Columbia background. A) Available T-DNA insertion lines and their insertion positions. B) Thermogram and C) leaf surface temperatures of mpk12-3 mutants. Plants were 28 days old and grown under well-watered conditions. Growth conditions as described in Fig. 3-38. The scale bar donates 2 cm. B-C) n=5 biological replicates, mean ± SEM, **P<0.001 compared with wild type Col.

In addition, available knockout lines from the five candidate genes (AT2G46070, AT2G46080, AT2G46090, AT2G46140 and AT2G46250) in accession Col-0 background were analyzed and the results of the MPK12 knockout line (mpk12-3) is shown. The MPK12 gene consists of six exons and five introns. Three knockout lines are available in the NASC stock center (Fig. 3-40A). The insertion sites of SALK074849 and SALK075365 are located in the first exon and that of GK103D08 in the fourth exon.

SALK074849 and SALK075365 have been reported either not to be homozygous or no T-DNA insertion event occurred (Lee, J. S. et al., 2009), whereas the insertion of GK103D08 was proven to be a homozygous mpk12 knockout. Analysis of thermal imaging showed a Cvi-like phenotype, 0.9 °C lower leaf surface temperature in mpk12-3 plants compared with wild type Col -0 (Fig. 3-40B and C).

Taken together, these results demonstrate that MPK12 is the gene contributing to the variation in leaf surface temperatures between Col-0 and Cvi-0.

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3.3.2.8 Amino acid alignments of MPK12

Figure 3-41 Alignment of deduced amino acid sequence of MPK12. A) Alignment of deduced amino acid sequence of MPK12 between Col-0 and Cvi-0. The black frame highlights the substitution from glycine of Col to arginine of Cvi. B) Alignment of deduced amino acid sequence of MPK12 compared to all other MPKs presented in Columbia. The black frame at position 53 highlights the conserved glycine A) and B) Texts on the left side indicate either names of accessions or names of MPKs, and

"Con." means consensus sequence. Numbers in the picture are the position of amino acid residues. Red represents a high consensus value (90%) while blue (between 50%

and 90%) and black (lower than 50%) indicate lower consensus.

In order to obtain a first hint for the variation in leaf temperature, the alignment of the amino acid sequence of MPK12 in Col-0 and Cvi-0 was analyzed. The result indicates a single substitution from glycine to arginine at residue 53, which falls within the kinase domain of the MPK12 protein (Fig. 3-41A). Further alignments of all MPKs from Arabidopsis Col-0 (Fig. 3-41B) and other available organisms (data not shown) showed that the substitution with arginine is unique for Cvi-0, while all other species showed strong conservation of a glycine residue at site 53.