Light-independent Chlorophyll Biosynthesis Research Articles

Plastome Reduction in the Only Parasitic Gymnosperm Parasitaxus Is Due to Losses of Photosynthesis but Not Housekeeping Genes and Apparently Involves the Secondary Gain of a Large Inverted Repeat.

Plastid genomes (plastomes) of parasitic plants undergo dramatic reductions as the need for photosynthesis relaxes. Here, we report the plastome of the only known heterotrophic gymnosperm Parasitaxus usta (Podocarpaceae). With 68 unique genes, of which 33 encode proteins, 31 tRNAs, and four rRNAs in a plastome of 85.3-kb length, Parasitaxus has both the smallest and the functionally least capable plastid genome of gymnosperms. Although the heterotroph retains chlorophyll, all genes for photosynthesis are physically or functionally lost, making photosynthetic energy gain impossible. The pseudogenization of the three plastome-encoded light-independent chlorophyll biosynthesis genes chlB, chlL, and chlN implies that Parasitaxus relies on either only the light-dependent chlorophyll biosynthesis pathway or another regulation system. Nesting within a group of gymnosperms known for the absence of the large inverted repeat regions (IRs), another unusual feature of the Parasitaxus plastome is the existence of a 9,256-bp long IR. Its short length and a gene composition that completely differs from those of IR-containing gymnosperms together suggest a regain of this critical, plastome structure-stabilizing feature. In sum, our findings highlight the particular path of lifestyle-associated reductive plastome evolution, where structural features might provide additional cues of a continued selection for plastome maintenance.

Presence and absence of light-independent chlorophyll biosynthesis among Chlamydomonas green algae in an ice-covered Antarctic lake

ABSTRACTThe cold, permanently ice-covered waters of Lake Bonney, Antarctica, may seem like an uninviting place for an alga, but they are home to a diversity of photosynthetic life, including Chlamydomonas sp. UWO241, a psychrophile residing in the deep photic zone. Recently, we found that UWO241 has lost the genes responsible for light-independent chlorophyll biosynthesis, which is surprising given that this green alga comes from a light-limited environment and experiences extended periods of darkness during the Antarctic winter. Why discard such a process? We argued that it might be linked to the very high dissolved oxygen concentration of Lake Bonney at the depth at which UWO241 is found. Oxygen is the Achilles’ heel of the key enzyme involved in light-independent chlorophyll biosynthesis: DPOR. If this hypothesis is true, then other algae in Lake Bonney should also be susceptible to losing DPOR, such as Chlamydomonas sp. ICE-MDV, which predominantly resides in the chemocline, a depth with an even higher oxygen concentration than that where UWO241 exists. Here, we report that, contrary to our earlier prediction, ICE-MDV has maintained the genes encoding DPOR. We briefly discuss the implications of this finding in relation to the loss of light-independent chlorophyll synthesis in UWO241.

LTS3 gene controls light-independent chlorophyll biosynthesis in green algae Chlamydomonas reinhardtii

The genetic control of light-independent chlorophyll biosynthesis in plant cells has been investigated using Chlamydomonas reinhardtii Lts3-mutants defective in dark chlorophyll biosynthesis on the stage before protochlorophyllide to chlorophyllide conversion. In heterotrophic conditions the mutants are unable to synthesize chlorophyll and accumulate protoporphyrins, after illumination they are greening. The mutants were tested for pigment contents, activity of enzymes and expression of the genes, encoding these enzymes. The LTS3 gene has been identified by positional cloning, and the predicted LTS3 protein appeared to be a GATA transcription factor, which activate the expression of genes encoded chlorophyll biosynthesis enzymes: Mg-chelatase and glutamate 1-semialdehyde aminotransferase in the dark, and possibly, important for adaptation of plant cells for autotrophic conditions.

A novel insight into the regulation of light-independent chlorophyll biosynthesis in Larix decidua and Picea abies seedlings

Light-independent chlorophyll (Chl) biosynthesis is a prerequisite for the assembly of photosynthetic pigment-protein complexes in the dark. Dark-grown Larix decidua Mill. seedlings synthesize Chl only in the early developmental stages and their Chl level rapidly declines during the subsequent development. Our analysis of the key regulatory steps in Chl biosynthesis revealed that etiolation of initially green dark-grown larch cotyledons is connected with decreasing content of glutamyl-tRNA reductase and reduced 5-aminolevulinic acid synthesizing capacity. The level of the Chl precursor protochlorophyllide also declined in the developing larch cotyledons. Although the genes chlL, chlN and chlB encoding subunits of the light-independent protochlorophyllide oxidoreductase were constitutively expressed in the larch seedlings, the accumulation of the ChlB subunit was developmentally regulated and ChlB content decreased in the fully developed cotyledons. The efficiency of chlB RNA-editing was also reduced in the mature dark-grown larch seedlings. In contrast to larch, dark-grown seedlings of Picea abies (L.) Karst. accumulate Chl throughout their whole development and show a different control of ChlB expression. Analysis of the plastid ultrastructure, photosynthetic proteins by Western blotting and photosynthetic parameters by gas exchange and Chl fluorescence measurements provide additional experimental proofs for differences between dark and light Chl biosynthesis in spruce and larch seedlings.

Evolutionary Dynamics of Light-Independent Protochlorophyllide Oxidoreductase Genes in the Secondary Plastids of Cryptophyte Algae

Plastid genes encoding light-independent protochlorophyllide oxidoreductase (LIPOR) subunits were isolated from cryptophyte algae, the first example of such genes in plastids of secondary endosymbiotic origin. The presence of functional and nonfunctional copies of LIPOR genes in cryptophytes suggests that light-independent chlorophyll biosynthesis is a nonessential pathway in these organisms.

Experimental approach to elucidating the mechanism of light-independent chlorophyll biosynthesis in greening barley.

Etiolated seedlings of angiosperms illuminated for a few hours and then returned to darkness continue to accumulate chlorophyll (Chl) a ([Popov and Dilova, 1969][1]; [Adamson et al., 1997][2], and refs. therein). In leaves of barley ( Hordeum vulgare ) seedlings and leaves of Tradescancia albiflora

Partial recovery of light-independent chlorophyll biosynthesis in the chlL-deletion mutant of Synechocystis sp. PCC 6803.

A chlL-deletion mutant of Synechocystis sp. PCC 6803 designated as chlL- was unable to make significant amounts of chlorophyll in darkness. However, an apparent pseudorevertant has been generated spontaneously that can synthesize an increased amount of chlorophyll under light-activated heterotrophic growth conditions. Under these conditions, the chlorophyll content in this pseudorevertant was about 20% of that in the wild-type strain and about 4 times more than that in the original and in the recently recreated chlL-deletion mutant. This is paralleled by increased performance of dark-grown cells in terms of chlorophyll fluorescence induction and oxygen evolution rates in the pseudorevertant versus in the original mutant. PCR analysis confirmed that the chlL- -pseudorevertant mutant still lacked the chlL gene. These results imply that the light-independent chlorophyll biosynthesis pathway was partly recovered.

Chlorophyll a availability affects psbA translation and D1 precursor processing in vivo in Synechocystis sp. PCC 6803.

Transcript accumulation and translation of psbA as well as processing of the D1 precursor protein were investigated in relation to chlorophyll availability in vivo in cyanobacterial strains lacking photosystem I (PS I). The psbA transcript level was almost independent of chlorophyll availability and was approximately 3-fold lower in darkness than in continuous light (5 microE m-2 s-1). Upon illumination, it reached a steady-state level within several hours. Upon growth under light-activated heterotrophic growth conditions (LAHG) in the PS I-less strain, D1 synthesis occurred immediately upon illumination. However, in PS I-less/chlL- cells, which lacked the light-independent chlorophyll biosynthesis pathway and had very low chlorophyll levels after LAHG growth, very little D1 synthesis occurred upon illumination, and the synthesis rate increased with time. This result suggests a translational control of D1 biosynthesis related to chlorophyll availability. Upon illumination, initially a high level of the nonprocessed D1 precursor was observed by pulse labeling and immunodetection in LAHG-grown PS I-less/chlL- cells but not in PS I-less cells. A significant amount of the D1 precursor eventually was processed to mature D1, and the half-life of the D1 precursor decreased as the chlorophyll content of the cells increased. The D1 processing enzyme CtpA was found to be present at similar levels regardless illumination or chlorophyll levels. We conclude that, directly or indirectly, chlorophyll availability is needed for D1 translation as well as for efficient processing of the D1 precursor.

Influence of polyamine inhibitors on light-independent and light-dependent chlorophyll biosynthesis and on the photosynthetic rate

The mutant C-2A′ of the unicellular green alga Scenedesmus obliquus behaves at 32°C like higher plants in that it accumulates protochlorophyllide in darkness and converts it to chlorophyll when exposed to light. By lowering the growth temperature from 32 to 22°C, protochlorophyllide can be reduced in darkness. Thus this organism allowed us to study the effects of the inhibition of several enzymes of polyamine biosynthesis on light-dependent and light-independent chlorophyll biosynthesis. Reduction of the intracellular level of putrescine by the use of 1,4-diamino-2-butanone inhibitor blocked the light-independent chlorophyll biosynthesis (dramatically increasing the photochlorophyllide levels with parallel reduction of chlorophyll). The influence of the inhibitor in question is even more dramatic in light-dependent chlorophyll biosynthesis, where small concentrations of 1,4-diamino-2-butanone resulted in considerable reduction of chlorophyll during illumination. The above reduction of chlorophyll was accompanied by an increase in photosynthetic activity and respiration rate, which suggests the formation of a photosynthetic apparatus that behaves similarly to one adapted to high light intensities. Decreases in the intracellular levels of spermidine and spermine by the inhibitors cyclohexylamine and 1,3-diaminopropane respectively, although not showing changes in light-independent chlorophyll biosynthesis, do exhibit a regular reduction of light-dependent chlorophyll biosynthesis and a decrease in the photosynthetic activity as well as in the respiration rate. These characteristics are typical for a photosynthetic apparatus adapted to low light intensities with a higher antenna composition per reaction centre. The possible effects of the reduction of putrescine, spermidine and spermine levels on the stabilization-destabilization of photosynthetic subcomplexes are discussed.

Light-independent chlorophyll biosynthesis: involvement of the chloroplast gene chlL (frxC).

The Chlamydomonas reinhardtii chloroplast gene chlL (frxC) is shown to be involved in the light-independent conversion of protochlorophyllide to chlorophyllide. The polypeptide encoded by chlL contains a striking 53% amino acid sequence identity with the bacteriochlorophyll (bch) biosynthesis bchL gene product in the photosynthetic bacterium Rhodobacter capsulatus. In a previous analysis, we demonstrated that bchL was involved in light-independent protochlorophyllide reduction, thereby implicating chlL in light-independent protochlorophyllide reduction in photosynthetic eukaryotes. To perform a functional/mutational analysis of chlL, we utilized particle gun-mediated transformation to disrupt the structural sequence of chlL at its endogenous locus in the chloroplast genome of Chlamydomonas. Transformants for which the multicopy chloroplast genome was homoplasmic for the disrupted chlL allele exhibit a "yellow-in-the-dark" phenotype that we demonstrated to be a result of the dark accumulation of protochlorophyllide. The presence of a chlL homolog in distantly related bacteria and nonflowering land plants, which are thought to be capable of synthesizing chlorophyll in the dark, was also demonstrated by cross-hybridization analysis. In contrast, we observed no cross-hybridization of a probe of chlL to DNA samples from representative angiosperms that require light for chlorophyll synthesis, in support of our conclusion that chlL is involved in light-independent chlorophyll biosynthesis. The role of chlL in protochlorophyllide reduction as well as recent evidence that both light-independent and light-dependent protochlorophyllide reductases may be of bacterial origin are discussed.