Construction and analysis of transgenic tobacco expressing variant forms of calmodulin

Calcium has been implicated as an important signal transducer in a number of plant growth and developmental processes. An important modulator of calcium action is the multifunctional calcium-binding protein calmodulin. Previous work has shown that calmodulin posttranslational methylation of lysine-115 results in an attenuation of NAD kinase, a calmodulin-dependent enzyme.

The advent of transgenic plant technologies allows us to examine the importance in vivo of calmodulin and calmodulin methylation on growth and development. To study the role of posttranslational methylation in calmodulin function, a site-specific mutant of calmodulin, VU-3 (lys > arg 115) was utilized. This mutation blocks methylation of calmodulin at position 115. Transgenic tobacco (Nicotiana tabacum cv. Wisconsin 38) were generated which express either wild type (VU-1) or mutant (VU-3) calmodulin from the cauliflower mosaic 355 promoter. Hygromycin resistant plants were regenerated from tissue culture and self-pollinated to produce seed (F₁). This seed was tested for resistance to hygromycin and in the VU-1 lines the expected 3:1 ratio was observed indicating Mendelian inheritance of a single dominant gene. However, VU-3 seed segregated in an apparent 2:1 fashion and when VU-3 transgenics (F₀) were outcrossed to wild type tobacco abnormal ratios and seed viability were iii observed suggesting that simple Mendelian genetics do not apply. VU-3 lines have a high incidence of empty, presumably non-viable pollen (30-60%) and seed number per pod is ten- fold less than VU-1 or control plants. Besides the reproductive abnormalities observed in all VU-3 plants, these plants were also true dwarfs. Southern analysis of chromosomal DNA from transgenic plants revealed hybridizing bands on different restriction fragments indicating independent transgenic lines. Since, the phenotypes were observed with all VU-3 lines, it appears as if they are due to the expression of VU-3 calmodulin. These studies indicate that calmodulin methylation is important for normal growth and development.