A chimeric Lhcb::Nia gene: an inducible counter selection system for mutants in the phytochrome signal transduction pathway

Abstract

One approach towards understanding the transduction pathways of phytochromes is the selection of mutants impaired in various steps. We report here the construction of an inducible counter-selection system for such mutants employing the enzyme nitrate reductase. This enzyme can convert the benign substrate analogue chlorate to the toxic product chlorite, resulting in severe growth inhibition. An Arabidopsis thaliana nitrate reductase gene (Nial*2) was placed under the regulation of an Arabidopsis thaliana light-harvesting chlorophyll a/b protein (Lhcb1*3) promoter that is phytochrome-responsive. The chimeric Lhcb::Nia gene was transformed into A. thaliana. Homozygous transformant lines were selected and grown in the absence of nitrate and the presence of L-glutamine, conditions that substantially inhibited the expression of the endogenous nitrate reductase genes. In darkness seedlings of the transformed lines were resistant to chlorate; however, when seedlings were grown with intermittent red light, increased sensitivity to chlorate was observed. This sensitivity was correlated with an increase in both Nia1*2 RNA levels and nitrate reductase activity. The resistant seedlings were clearly distinguishable from the sensitive ones based on hypocotyl length, with no overlap in this parameter between the two populations. Thus, this system should allow for the selection of mutants that are impaired in phytochrome regulation of the transcription of Lhcb genes.

Nitrate assimilation in plant shoots depends on photorespiration

“…We used three genotypes of Arabidopsis thaliana cv. Columbia: (i) the wild type, (ii) a transgenic line harboring the chimeric gene Lhch1*3::Nia1*2 that overexpresses one form of NO 3 Ϫ reductase (18), and (iii) a genotype with mutations in both structural genes for NO 3 Ϫ reductase, nia1 nia2 (19). Seeds were germinated on plates filled with a dilute Murashige-Skoog medium (2.3 g⅐liter Ϫ1 ) in 0.75% Phytagar (GIBCO͞BRL).…”

Responses of Arabidopsis and Wheat to Rising CO2 Depend on Nitrogen Source and Nighttime CO2 Levels

“…For the gas exchange experiments, we also used two Arabidopsis 'Columbia' genotypes that exhibited different levels of NR activity (Rachmilevitch et al, 2004): a transgenic line harboring the chimeric gene Lhch1*3::Nia1*2 (the Arabidopsis nitrate reductase gene under the regulation of the light-harvesting chlorophyll a/b protein promoter) that had about twice the NR activity of the wild type (Heimer et al, 1995) and a genotype with mutations in both structural genes for NR, nia1 nia2, which had little detectable NR activity (Wilkinson and Crawford, 1993).…”

shygrl1Is a Mutant Affected in Multiple Aspects of Photomorphogenesis

“…Lhcb1*3::Nia1*2 Expression Seeds of the transgenic line A4 (Heimer et al, 1995), which were homozygous for the Lhcb1*3::Nia1*2 chimeric gene construct depicted in Figure 1 were mutagenized. Plants grown from these seeds were selfed, and the progeny of these plants (the M2 generation) were used to select potential mutants of interest.…”

“…Part of the Lhcb1*3 (1.3 kb) promoter, which extends through ϩ14 of transcription start, was fused to 3.5 kb of Nia1*2 genomic DNA containing its own transcription termination sequences. Construction of the transformation vector and selection of the stably transformed A4 line was described in detail in Heimer et al (1995).…”

“…Nitrate reductase (NR), an enzyme that reduces nitrate to nitrite, can also convert the nontoxic compound chlorate to chlorite, which is toxic to plants. We had previously found that the growth of the transgenic line was severely inhibited when grown on chlorate under conditions in which the phytochrome system was activated by intermittent red (IR) light (Heimer et al, 1995). This strategy has led to the identification of a locus that we have designated shygrl1 (shg1) because when mutated, it causes short stature, yellow-green leaves, and reduced accumulation of Lhcb genes.…”