Nuclear Mutation Inhibits Expression of the Chloroplast Gene That Encodes the Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

Abstract

Chlamydomonas reinhardtii mutant 76-SEN was recovered as a light-sensitive, acetate-requiring strain that failed to complement a chloroplast structural gene mutant of ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39). Further genetic analysis revealed that the new mutation was inherited in a mendelian pattern, indicating that it resides within the nucleus. The 76-5EN mutant lacks Rubisco holoenzyme but has wild-type levels of whole-chain electron transport activity and chlorophyll. During a 1-min pulse labeling with 35S0,2-, little or no Rubisco largesubunit synthesis occurred in the mutant. Nuclear-encoded small subunits were synthesized to a normal level and were subsequently degraded. When analyzed by northern hybridization, the 76-SEN mutant was found to have a decreased level of large-subunit mRNA. large-subunit mRNA synthesis also appeared to be reduced during a 10-min pulse labeling with [32P]orthophosphate, but the labeled mRNA was stable during a 1-h chase. These results indicate that a nuclear gene mutation specifically disrupts the accumulation of large-subunit mRNA within the chloroplast. A deeper understanding of the nature of the 76-SEN gene may be useful for manipulating the expression of the agronomically important Rubisco enzyme.Rubisco (EC 4.1.1.39) plays a pivotal role in photosynthesis because it catalyzes both the carboxylation and oxygenation of RuBP. The amount of carboxylation and the ratio of carboxylation to oxygenation ultimately determine net photosynthetic COP fixation (reviewed by Spreitzer, 1993). The Rubisco holoenzyme exists within the chloroplasts of plants and green algae as a hexadecamer composed of equal numbers of 55-kD large subunits and 15-kD small subunits. The chloroplast rbcL gene encodes the catalytic large subunits, whereas a family of nuclear RbcS genes encodes the small subunits (reviewed by Spreitzer, 1993).As a means for defining the structure/function relationships of chloroplast Rubisco, mutants of the green alga Chlamydomonas reinhardtii have been recovered by screening for defects in Rubisco holoenzyme stability or catalysis (Spreitzer et al., 1992 Thow et al., 1994).Only several Rubisco mutants have been recovered that arise from mutations in nuclear genes (Spreitzer et al., 1988(Spreitzer et al., , 1992. Two of these nuclear mutants have been analyzed in detail. The 552-28 mutation suppresses the temperatureconditional phenotype and altered enzyme properties of the 68-4"rbcL mutant (Chen et al., 1990(Chen et al., , 1993. The 68-4PP mutation causes a LeuZ9'-to-Phe substitution in the Rubisco large subunit, which, in the absence of the S52-2B suppressor mutation, decreases the carboxylase/oxygenase ratio and thermal stability of Rubisco (Chen et al., 1988(Chen et al., , 1993. A second nuclear mutant, named 68-1 lAR, was recovered as a temperature-conditional, acetate-requiring strain (Spreitzer et al., 1988). The 68-1 1AR mutation decreases the carboxylase/ oxygenase ratio and thermal stability of Rubisco (Gotor et al., 1994). Each of th...

bundle sheath defective2, a Mutation That Disrupts the Coordinated Development of Bundle Sheath and Mesophyll Cells in the Maize Leaf

“…It has been proposed that reduced chloroplast gene expression during leaf senescence results from decreased chloroplast function followed by increased chloroplast breakdown (Camp et al, 1982;Mae et al, 1984;Wardley et al, 1984;Ford and Shibles, 1988;Kura-Hotta et al, 1990). In RbcS antisense-inhibited transgenic tobacco, the developmental reduction of rbcL transcripts observed in older leaves has been correlated with a decrease in plastid DNA template availability (Jiang and Rodermel, 1995). In a similar fashion, the reduction in rbcL mRNA levels at the ' tips of bsd2-ml light-grown leaves could be a consequence of chloroplast breakdown.…”

“…For example, a point mutation in the tobacco rbcL gene that prevents holoenzyme assembly results in a 98% reduction in steady state levels of both large and small subunit proteins (Avni et al, 1989). Similarly, antisense inhibition of RbcS gene expression in transgenic tobacco prevents large subunit protein accumulation, although rbcL transcripts accumulate to normal levels and are translated (Rodermel et al, 1988;Jiang and Rodermel, 1995). ln bsd2-ml leaves, RbcS transcript accumulation profiles were essentially normal; however, the spatial and temporal regulation of rbcL transcripts was disrupted.…”

Elimination of the Chlamydomonas gene family that encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase

“…When the acetaterequiring insertional mutants were screened by SDS͞PAGE and Western blot analysis, one of the light-sensitive strains, named T60-3, was found to lack Rubisco large and small subunits. This is a common attribute of a number of Rubisco mutants that have primary defects in large-subunit synthesis or assembly (5,6,31,36). However, 35 SO 4 2Ϫ -labeling experiments indicated that little or no synthesis of either subunit occurred during a 1-min pulse (Fig.…”

“…Because the DNA integrates into the nuclear genome at apparently random loci, the transforming DNA can act as an insertional mutagen to disrupt functional genes (37,38). We initially decided to use this method to physically mark alleles of mutant nuclear genes that affect Rubisco expression, stability, and catalysis (28,36,39). A dark-grown cw d arg-7-8 mt Ϫ strain, which lacks a cell wall and requires arginine for growth (26), was transformed with plasmid pARG7.8, which contains the wild-type argininosuccinate lyase gene (26).…”

“…Filters were washed in 0.1ϫ SSPE͞0.1% SDS at 65ЊC and subjected to autoradiography at Ϫ70ЊC. Total RNA was isolated, resolved in agaroseformaldehyde gel, and blotted to nylon membrane (36). A 32 P-labeled PCR fragment of rbcL [bases 295-1245 relative to the 1428-bp coding region (35)] was used as a probe.…”