Microinjection of heme oxygenase genes rescues phytochrome-chromophore-deficient mutants of the moss Ceratodon purpureus

Abstract

In protonemal tip cells of the moss Ceratodon purpureus (Hedw.) Brid., phototropism and chlorophyll accumulation are regulated by the photoreceptor phytochrome. The mutant ptr116 lacks both responses as a result of a defect in the biosynthesis of phytochromobilin, the chromophore of phytochrome, at the point of biliverdin formation. The rescue of the phototropic response and of chlorophyll synthesis were tested by injecting different substances into tip cells of ptr116. Microinjection was first optimised with the use of fluorescent dyes and an expression plasmid containing a green fluorescent protein (GFP) gene. Injected phycocyanobilin, which substitutes for phytochromobilin, rescued both the phototropic response and light-induced chlorophyll accumulation in ptr116. The same results were obtained when expression plasmids with heme oxygenase genes of rat (HO-1) and Arabidopsis thaliana (L.) Heynh. (HY1) were injected. Heme oxygenase catalyses the conversion of heme into biliverdin. Whereas HY1 has a plastid target sequence and is presumably transferred to plastids, HO-1 is proposed to be cytosolic. The data show that ptr116 lacks heme oxygenase enzyme activity and indicate that heme oxygenases of various origin are active in Ceratodon bilin synthesis. In addition, it can be inferred from the data that the intracellular localisation of the expressed heme oxygenase is not important since the plastid enzyme can be replaced by a cytosolic one.

Photoreceptor Gene Families in Lower Plants

“…These phenotypes were rescued by feeding the tetrapyrrole biliverdin or phycocyanobilin but not protoporphylin or heme, suggesting that class 1 ptr mutants were impaired in the biosynthesis of the phytochrome chromophore, probably in the conversion of heme into biliverdin (Lamparter et al, , 1997Esch and Lamparter, 1998). Confirming these results, microinjection of heme oxygenase genes of rat or A. thaliana rescued class 1 ptr mutants (Brücker et al, 2000). Class 2 ptr mutants are aphototropic but do not show the defect in chlorophyll accumulation and have spectrally active phytochrome at a level similar to that of wild type Esch et al, 1999).…”

The moss Physcomitrella patens: methods and tools from cultivation to targeted analysis of gene function

“…The microinjection needles also have to be introduced into the cells at a precise region basal to the nucleus, because all attempts to inject the cells at the tip region resulted in the bursting of the cells. Microinjection of a green fluorescent protein (GFP) expression construct into P. patens protonema tip cells resulted in a transformation rate of approximately 50% (Brücker et al, 2000). As only the transient GFP expression had been analysed, whether this technique can be used to generate stable transgenic lines and targeted knockout mutants has not been assessed.…”

Photomorphogenesis of Mosses

“…The filaments formed during the days subsequent to the insertion grew towards unilateral red light and produced wild-type levels of chlorophyll. Microinjection is a powerful tool for the analysis of functional aspects of gene and metabolite activity at the cellular level, in the present case it allowed for a rapid assessment of the effect of heme oxygenase expression on phytochrome mutant phenotypes (Brücker et al, 2000).…”

“…For this purpose, the respective genes were cloned into an expression vector and placed directly into mutant tip cells by means of microinjection (Brücker et al, 2000). This method had been previously established for the microinjection of low and high molecular dye components, phycocyanobilin, and GFP expression constructs into mutant and wild type cells.…”

“…Protoplasts are directly accessible for PEG-mediated DNA transfer (Schaefer et al, 1991), which has become a routine technique for moss transformation. Other transformation techniques such as particle gun bombardment (Sawahel et al, 1992) or microinjection (Brücker et al, 2000) have also been established. The significance of mosses for plant science has further increased since Schaefer and Zryd found that in Physcomitrella patens the rate of homologous recombination is very high (Schaefer and Zryd, 1997).…”