Expression, purification and analysis of an Arabidopsis recombinant CBL-interacting protein kinase3 (CIPK3) and its constitutively active form.

CIPK3 is a member of CBL (calcineurin B-like)-interacting serine-threonine protein kinases which play an important role in many developmental and adaptation processes in Arabidopsis. Studies conducted on members of this family such as SOS2, PKS8 and PKS11 have provided insight into how these kinases interact with their target substrates in the signal-response process. Since SOS2, PKS8 and PKS11 have low enzymatic activities in vitro and their amino acid sequences are homologous to that of CIPK3, it was assumed that CIPK3 would have a low enzymatic activity. To enhance CIPK3 enzyme activity, a constitutively active form, CIPK3T183D, was generated by a Thr(183) to Asp(183) substitution in the activation loop. To obtain proteins for analysis, glutathione S-transferase (GST) fusion protein system was used. Although both CIPK3 and CIPK3T183D were successfully expressed, they were found in inclusion bodies with three truncated proteins. Since the truncated proteins had a similar affinity to the GST-Bind Resin as the target protein, the one-step affinity purification could no longer be used. As an alternative, His fusion protein expression system was employed for protein production. Although both His-CIPK3 and His-CIPK3T183D also accumulated in inclusion bodies, they were expressed as a single protein species. A method involving Sarkosyl was developed for isolating and purifying the His fusion proteins. His-CIPK3 and His-CIPK3T183D produced were highly purified and enzymatically active. In addition, a 9-fold increase in kinase activity in His-CIPK3T183D was observed, indicating that Thr(183) to Asp(183) substitution in the activation loop of CIPK3 had succeeded in enhancing the kinase activity.

Transcriptome profiling and methyl homeostasis of an Arabidopsis mutant deficient in S-adenosylhomocysteine hydrolase1 (SAHH1).

Transcriptome profiling was conducted to detect genes whose expression is significantly changed in an Arabidopsis mutant deficient in S-adenosylhomocysteine hydrolase1 (SAHH1) during early seedling development when mutant phenotypes could be clearly observed. A total of 2,040 differentially expressed genes were identified, representing approximately 6.7% of the 30,385 DNA oligonucleotide targets on the microarray. Among these differential expressed genes, many were mapped to pathways essential to plant growth and development including those of primary, secondary and hormone metabolisms. A significant proportion of up-regulated genes encoded transposable elements which were mapped to the centromeric and pericentromeric regions of the Arabidopsis chromosomes that were analyzed. A number of down-regulated genes were found to be involved in root hair formation, which might have contributed to the root hair defective phenotype of the mutant. Analysis of genes encoding transposable elements and those associating with root hair development indicated that these genes were highly co-expressed during seedling development. Despite SAHH1 deficiency, the expression of genes encoding methyltransferase remained largely unchanged in the sahh1 mutant. Bisulfite sequencing analysis of the transposable elements and the FWA gene revealed that their sequences in the mutant were deficient of 5-methylcytosines. Analysis of mutant genomic DNA using restriction endonucleases that were unable to cut methylated DNA suggested a genome-wide hypomethylation had occurred in the mutant. These results indicated that SAHH1 plays a critical role in methyl homeostasis, and its deficiency is a major contributing factor to the change of global gene expression, metabolic pathways and activation of transposable elements in the sahh1 mutant.

Chlorophyll reduction in the seed of Brassica napus with a glutamate 1-semialdehyde aminotransferase antisense gene.

Chlorophyll reduction in the seed of Brassica can be achieved by downregulating its synthesis. To reduce chlorophyll synthesis, we have used a cDNA clone of Brassica napus encoding glutamate 1-semialdehyde aminotransferase (GSA-AT) to make an antisense construct for gene manipulation. Antisense glutamate 1-semialdehyde aminotransferase gene (Gsa) expression, directed by a Brassica napin promoter, was targeted specifically to the embryo of the developing seed. Transformants expressing antisense Gsa showed varying degrees of inhibition resulting in a range of chlorophyll reduction in the seeds. Seed growth and development were not affected by reduction of chlorophyll. Seeds from selfed transgenic plants germinated with high efficiency and growth of seedlings was vigorous. Seedlings from T2 transgenic lines segregated into three distinctive phenotypes: dark green, light green and yellow, indicating the dominant inheritance of Gsa antisense gene. These transgenic lines have provided useful materials for the development of a low chlorophyll seed variety of B. napus.