Recently, plastidial carbonic anhydrase (CA, EC 4.2.1.1) cDNA clones encoding functional CA enzymes were isolated from a nonphotosynthetic cotton tissue. The role of CA in photosynthetic tissues have been well characterized, however there is almost no information for the role of CA in nonphotosynthetic tissues. A survey of relative CA transcript abundance and enzyme activity in different cotton organs revealed that there was substantial CA expression in cotyledons of seedlings and embryos, both nonphotosynthetic tissues. To gain insight into the role(s) of CA, I examined CA expression in cotyledons of seedlings during post-germinative growth at different environmental conditions. CA expression in cotyledons of seedlings increased from 18 h to 72 h after germination in the dark. Seedlings exposed to light had about a 2-fold increase in CA activities when compared with seedlings kept in the dark, whereas relative CA transcript levels were essentially the same. Manipulation of external CO2 environments [zero, ambient (350 ppm), or high (1000 ppm)] modulated coordinately the relative transcript abundance of CA (and rbcS) in cotyledons, but did not affect enzyme activities. On the other hand, regardless of the external CO2 conditions seedlings exposed to light exhibited increase CA activity, concomitant with Rubisco activity and increased chlorophyll content. Our data revealed that steady-state levels of CA and rbcS transcripts are regulated at the transcriptional level in response to external CO2 conditions, while CA and Rubisco activities are modulated at the post-transcriptional level by light. Thus CA expression in cotyledons during post-germinative growth may be to “prime” cotyledons for the transition at the subcellular level for the transition from plastids to chloroplasts, where it provides CO2 for Rubisco during photosynthesis. Furthermore, CA expression increased during embryo maturation similar to oil accumulation. Specific sulfonamide inhibitors of CA activity significantly reduced the rate of [14C]-acetate incorporation into total lipids in cotton embryos and tobacco leaves and cell suspensions in vivo and in vitro. Similar results were obtained in chloroplasts isolated from leaves of transgenic CA antisense-suppressed tobacco plants (5% of wildtype activity). Collectively, these results support the notion that CA plays several physiological roles in nonphotosynthetic tissues.

Plastid genomes of photosynthetic flowering plants are usually highly conserved in both structure and gene content. However, the plastomes of parasitic and mycoheterotrophic plants may be released from selective constraint due to the reduction or loss of photosynthetic ability. Here we present the greatly reduced and highly divergent, yet functional, plastome of the nonphotosynthetic holoparasite Hydnora visseri (Hydnoraceae, Piperales). The plastome is 27 kb in length, with 24 genes encoding ribosomal proteins, ribosomal RNAs, tRNAs and a few non-bioenergetic genes, but no genes related to photosynthesis. The inverted repeat and the small single copy region are only ~1.5 kb, and intergenic regions have been drastically reduced. Despite extreme reduction, gene order and orientation are highly similar to the plastome of Piper cenocladum, a related photosynthetic plant in Piperales. Gene sequences in Hydnora are highly divergent and several complementary approaches using the highest possible sensitivity were required for identification and annotation of this plastome. Active transcription is detected for all of the protein coding genes in the plastid genome, and one of two introns is appropriately spliced out of rps12 transcripts. The whole genome shotgun read depth is 1,400X coverage for the plastome, while the mitochondrial genome is covered at 40X and the nuclear genome at 2X. Despite the extreme reduction of the genome and high sequence divergence, the presence of syntenic, long transcriptionally-active open reading frames with distant similarity to other plastid genomes and a high plastome stoichiometry relative to the mitochondrial and nuclear genomes suggests that the plastome remains functional in Hydnora visseri. A four stage model of gene reduction, including the potential for complete plastome loss, is proposed to account for the range of plastid genomes in nonphotosynthetic plants.

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Plastid genomes of photosynthetic flowering plants are usually highly conserved in both structure and gene content. However, the plastomes of parasitic and mycoheterotrophic plants may be released from selective constraint due to the reduction or loss of photosynthetic ability. Here we present the greatly reduced and highly divergent, yet functional, plastome of the nonphotosynthetic holoparasite Hydnora visseri (Hydnoraceae, Piperales). The plastome is 27 kb in length, with 24 genes encoding ribosomal proteins, ribosomal RNAs, tRNAs and a few non-bioenergetic genes, but no genes related to photosynthesis. The inverted repeat and the small single copy region are only ~1.5 kb, and intergenic regions have been drastically reduced. Despite extreme reduction, gene order and orientation are highly similar to the plastome of Piper cenocladum, a related photosynthetic plant in Piperales. Gene sequences in Hydnora are highly divergent and several complementary approaches using the highest possible sensitivity were required for identification and annotation of this plastome. Active transcription is detected for all of the protein coding genes in the plastid genome, and one of two introns is appropriately spliced out of rps12 transcripts. The whole genome shotgun read depth is 1,400X coverage for the plastome, while the mitochondrial genome is covered at 40X and the nuclear genome at 2X. Despite the extreme reduction of the genome and high sequence divergence, the presence of syntenic, long transcriptionally-active open reading frames with distant similarity to other plastid genomes and a high plastome stoichiometry relative to the mitochondrial and nuclear genomes suggests that the plastome remains functional in Hydnora visseri. A four stage model of gene reduction, including the potential for complete plastome loss, is proposed to account for the range of plastid genomes in nonphotosynthetic plants.