NICHE ADAPTATION OF PROCHLOROCOCCUS ECOTYPES: INSIGHTS THROUGH COMPARATIVE GENOMICS

Hess, W. R.¹*, Rocap, G.², Steglich, C.¹, Post, A.², Ting, C. S.³ & Chisholm, S. W.^{2,3 1}Humboldt-University Berlin, Germany; Institute of Biology, Chausseestr. 117, D-10115 Berlin, Germany; ²Massachusetts Institute of Technology, Department of Civil and Environmental Engineering and ³Department of Biology, Massachusetts Institute of Technology, 15 Vassar Street, 48-425 MIT, Cambridge MA 02139, USA Prochlorococcus is an extremely small, chlorophyll b-containing oceanic cyanobacterium. Specific ecotypes of Prochlorococcus have adapted to different ecological conditions with regard to factors as light or the available nutrients. Such differentially adapted ecotypes are less than 3% divergent in their 16S rRNA sequences, which invites speculation as to how their specific gene content has diverged to reflect the particular niche of each strain. Complete genome sequences have been determined of two Prochlorococcus strains, MED4 and MIT9313. These two strains are representatives of high and low light-adapted ecotypes. Intriguing similarities between both genomes include their small size and compact organization (MED4: 1.7 Mbp and MIT9313: 2.3 Mbp), a gene cluster for RubisCo and carboxysomal proteins that is of obviously non-cyanobacterial origin, or genes for two different lycopene cyclases explaining how Prochlorococcus synthesizes alpha-carotene, a carotenoid that is not common to cyanobacteria. Several genes and operons which in cyanobacteria are involved in light harvesting, nitrate utilization or in the generation of circadian rhythms have been reduced to a different degree in the two compared genomes. MED4 has many more genes encoding high light inducible proteins and photolyases. In contrast, MIT9313 possesses more genes to build up more complex light harvesting structures, including a gene cluster to produce chromophorylated phycoerythrin. The latter represents an intermediate between the phycobiliproteins of non-chlorophyll b containing cyanobacteria and a degenerated phycoerythrin present in MED4. Screening of natural samples from the Red Sea suggests that a highly similar phycoerythrin form is wide-spread among high light-adapted ecotypes.     

Expression of PTHrP and its cognate receptor in the rheumatoid synovial microcirculation

Genome sequence analyses revealed the occurrence of two paralogous ppa genes potentially encoding distinct Family I inorganic pyrophosphatases (sPPases, EC3.6.1.1) in the marine unicellular cyanobacteria Prochlorococcus marinus strains MED4 and MIT9313 and Synechococcus sp. WH8102. Protein sequence alignment and phylogenetic analysis indicated that the ppa gene proper of cyanobacteria (ppa1) encodes a presumably inactive mutant enzyme whereas the second gene (ppa2) might encode an active sPPase closely related to those of some proteobacteria. Heterologous expression of the two cloned P. marinus MED4 ppa genes in Escherichia coli confirmed this proposal, only the inactive ppa1 product being immunodetected by anti-cyanobacterial sPPase antibodies. A possible scenario of ppa gene inactivation and replacement in the context of the postulated rapid diversification of marine unicellular cyanobacteria, the most abundant photosynthetic prokaryotes in the oceans, is discussed.     

Growth responses and photosynthetic characteristics of wild and phycoerythrin-deficient strains of <Emphasis Type="Italic">Hypnea musciformis</Emphasis> (Rhodophyta)

The phycoerythrin-deficient strain (green phenotype) of Hypnea musciformis (Rhodophyta) originated from a green branch, which had arisen as a spontaneous mutation in a wild plant (brown phenotype) collected from the Brazilian coast. The present study describes the growth responses to irradiance, photoperiod and temperature variations, pigment contents, and photosynthetic characteristics of the brown and green strains of H. musciformis. The results showed that growth rates increased as a function of irradiance (up to 40 μmol photons m⁻² s⁻¹) but, with further increase in irradiance (from 40 to 120 μmol photons m⁻² s⁻¹), became light-saturated and remained almost unchanged. The highest growth rates of the brown and green strains were observed in temperatures of 20–25°C under long (14:10 h LD) and short (10:14 h LD) photoperiods. The brown strain had higher growth rates than the green strain in the short photoperiod, which could be related to the high concentrations of phycobiliproteins. Phycoerythrin was not detected in the green strain. The brown strain had higher concentrations of allophycocyanin and phycoerythrin in the short photoperiod while the green strain had higher concentrations of phycocyanin. The brown strain presented higher photosynthetic efficiency (α), and lower saturation parameter (I_k) and compensation irradiance (I_c) than the green strain. The brown strain exhibited the characteristics of shade-adapted plants, and its higher value of photosynthetic efficiency could be attributed to the higher phycoerythrin concentrations. Results of the present study indicate that both colour strains of H. musciformis could be selected for aquaculture, since growth rates were similar (although in different optimal light conditions), as the green strain seems to be adapted to higher light levels than the brown strain. Furthermore, these colour strains could be a useful experimental system to understand the regulation of biochemical processes of photosynthesis and metabolism of light-harvesting pigments in red algae.     

Comparative molecular population genetics of phycoerythrin locus in <Emphasis Type="Italic">Prochlorococcus</Emphasis>

As the only remainder type of phycobiliproteins in Prochlorococcus, the actual role of phycoerythrin still remains unknown. Previous studies revealed that two different forms of phycoerythrin gene were found in two ecotypes of Prochlorococcus that are specifically adapted to either high light (HL) or low light (LL) conditions. Here we analyze patterns of phycoerythrin nucleotide variation in the HL- and LL-Prochlorococcus populations. Our analyses reveal a significantly greater number of non-synonymous fixed substitutions in peB and peA than expected based on interspecific comparisons. This pattern of excess non-synonymous fixed substitutions is not seen in other five phycoerythrin-related genes (peZ/V/Y/T/S). Several neutrality statistical tests indicate an excess of rare frequency polymorphisms in the LL-Prochlorococcus data, but an excess of intermediate frequency polymorphisms in the HL-Prochlorococcus data. Distributions of the positively selected sites identified using the likelihood ratio test, when mapped onto the phycoerythrin tertiary structure, reveal that HL- and LL-phycoerythrin should be under different selective patterns. These findings may provide insights into the likely role of selection at the phycoerythrin locus and motivate further research to unveil the function of phycoerythrin in Prochlorococcus.     

UV-induced phycobilisome dismantling in the marine picocyanobacterium <Emphasis Type="Italic">Synechococcus</Emphasis> sp. WH8102

The marine picocyanobacterium Synechococcus sp. WH8102 was submitted to ultraviolet (UV-A and B) radiations and the effects of this stress on reaction center II and phycobilisome integrity were studied using a combination of biochemical, biophysical and molecular biology techniques. Under the UV conditions that were applied (4.3 W m⁻² UV-A and 0.86 W m⁻² UV-B), no significant cell mortality and little chlorophyll degradation occurred during the 5 h time course experiment. However, pulse amplitude modulated (PAM) fluorimetry analyses revealed a rapid photoinactivation of reaction centers II. Indeed, a dramatic decrease of the D1 protein amount was observed, despite a large and rapid increase in the expression level of the psbA gene pool. Our results suggest that D1 protein degradation was accompanied (or followed) by the disruption of the N-terminal domain of the anchor linker polypeptide L_CM, which in turn led to the disconnection of the phycobilisome complex from the thylakoid membrane. Furthermore, time course analyses of in vivo fluorescence emission spectra suggested a partial dismantling of phycobilisome rods. This was confirmed by characterization of isolated antenna complexes by SDS-PAGE and immunoblotting analyses which allowed us to locate the disruption site of the rods near the phycoerythrin I—phycoerythrin II junction. In addition, genes encoding phycobilisome components, including α-subunits of all phycobiliproteins and phycoerythrin linker polypeptides were all down regulated in response to UV stress. Phycobilisome alteration could be the consequence of direct UV-induced photodamages and/or the result of a protease-mediated process.     

Chlorophyll fluorescence imaging of photosynthetic activity in sun and shade leaves of trees

The differences in pigment levels, photosynthetic activity and the chlorophyll fluorescence decrease ratio R _Fd (as indicator of photosynthetic rates) of green sun and shade leaves of three broadleaf trees (Platanus acerifolia Willd., Populus alba L., Tilia cordata Mill.) were compared. Sun leaves were characterized by higher levels of total chlorophylls a + b and total carotenoids x + c as well as higher values for the weight ratio chlorophyll (Chl) a/b (sun leaves 3.23–3.45; shade leaves: 2.74–2.81), and lower values for the ratio chlorophylls to carotenoids (a + b)/(x + c) (with 4.44–4.70 in sun leaves and 5.04–5.72 in shade leaves). Sun leaves exhibited higher photosynthetic rates P _N on a leaf area basis (mean of 9.1–10.1 μmol CO₂ m⁻² s⁻¹) and Chl basis, which correlated well with the higher values of stomatal conductance G _s (range 105–180 mmol m⁻² s⁻¹), as compared to shade leaves (G _s range 25–77 mmol m⁻² s⁻¹; P _N: 3.2–3.7 μmol CO₂ m⁻² s⁻¹). The higher photosynthetic rates could also be detected via imaging the Chl fluorescence decrease ratio R _Fd, which possessed higher values in sun leaves (2.8–3.0) as compared to shade leaves (1.4–1.8). In addition, via R _Fd images it was shown that the photosynthetic activity of the leaves of all trees exhibits a large heterogeneity across the leaf area, and in general to a higher extent in sun leaves than in shade leaves.     

Excess irradiance causes early symptoms of senescence during leaf expansion in photoautotrophically <Emphasis Type="Italic">in vitro</Emphasis> grown tobacco plants

Photosynthetic parameters, growth, and pigment contents were determined during expansion of the fourth leaf of in vitro photoautotrophically cultured Nicotiana tabacum L. plants at three irradiances [photosynthetically active radiation (400–700 nm): low, LI 60 μmol m⁻² s⁻¹; middle, MI 180 μmol m⁻² s⁻¹; and high, HI 270 μmol m⁻² s⁻¹]. During leaf expansion, several symptoms usually accompanying leaf senescence appeared very early in HI and then in MI plants. Symptoms of senescence in developing leaves were: decreasing chlorophyll (Chl) a+b content and Chl a/b ratio, decreasing both maximum (F_V/F_M) and actual (Φ_PS2) photochemical efficiency of photosystem 2, and increasing non-photochemical quenching. Nevertheless, net photosynthetic oxygen evolution rate (P _N) did not decrease consistently with decrease in Chl content, but exhibited a typical ontogenetic course with gradual increase. P _N reached its maximum before full leaf expansion and then tended to decline. Thus excess irradiance during in vitro cultivation did not cause early start of leaf senescence, but impaired photosynthetic performance and Chl content in leaves and changed their typical ontogenetic course.     

How Wind Affects the Photosynthetic Performance of Trees: Quantified with Chlorophyll a Fluorescence and Open-Top Chambers

Meteorological parameters inside and outside an open-top chamber (OTC) fumigation facility were recorded and the primary photosynthetic response of four tree species measured with chlorophyll (Chl) a fluorescence emission. Parameters extracted from the Chl a fluorescence transient were used to calculate photosynthetic activity of the leaves using a performance index. Measurements were made during the night throughout a single growing season. The seasonal primary photosynthetic performance in all species was significantly altered by growth in the OTCs, and the degree of response was dependent upon the species. Wind was an important effectual component of the altered environment. The average temperature was consistently 1.94±0.70 °C higher within the OTCs, whereas wind speed fluctuated substantially more between inside and outside the OTCs (0 to 8 m ⁻¹). There was a correlation between the photosynthetic performance index and wind speed in Fagus sylvatica, Fraxinus excelsior, and Prunus serotina. The response to wind was also particular to each species; the photosynthetic performance of F. sylvatica increased with wind speed (1 to 7 m s⁻¹), decreased with F. excelsior (0 to 6.5 m s⁻¹) and P. serotina (0 to 5.5 m s⁻¹). Abies alba, in contrast, was almost insensitive to wind. A model was proposed and tested for the conversion of the photosynthetic performance values collected in OTCs to predict the photosynthetic performance outside OTCs. The wide variety of responses to wind and temperature of the four species conformed to linear functions that describe the relationship of the wind speed and temperature responses with the difference in photosynthetic performance between the OTC and open environments. Specific coefficients for wind and temperature were proposed. The photosynthetic response to wind of each species depends on its ecophysiological specialisation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of light and temperature on the cyanobacterium <Emphasis Type="Italic">Arthronema africanum</Emphasis> - a prospective phycobiliprotein-producing strain

The effect of light intensity (50–300 μmol photons m⁻² s⁻¹) and temperature (15–50°C) on chlorophyll a, carotenoid and phycobiliprotein content in Arthronema africanum biomass was studied. Maximum growth rate was measured at 300 μmol photons m⁻² s⁻¹ and 36°C after 96 h of cultivation. The chlorophyll a content increased along with the increase in light intensity and temperature and reached 2.4% of dry weight at 150 μmol photons m⁻² s⁻¹ and 36°C, but it decreased at higher temperatures. The level of carotenoids did not change significantly under temperature changes at illumination of 50 and 100 μmol photons m⁻² s⁻¹. Carotenoids were about 1% of the dry weight at higher light intensities: 150 and 300 μmol photons m⁻² s⁻¹. Arthronema africanum contained C-phycocyanin and allophycocyanin but no phycoerythrin. The total phycobiliprotein content was extremely high, more than 30% of the dry algal biomass, thus the cyanobacterium could be deemed an alternative producer of C-phycocyanin. A highest total of phycobiliproteins was reached at light intensity of 150 μmol photons m⁻² s⁻¹ and temperature of 36°C, C-phycocyanin and allophycocyanin amounting, respectively, to 23% and 12% of the dry algal biomass. Extremely low (<15°C) and high temperatures (>47°C) decreased phycobiliprotein content regardless of light intensity.     

Enhancement of susceptivity to photoinhibition and photooxidation in rice chlorophyll <Emphasis Type="Italic">b</Emphasis>-less mutants

Two rice chlorophyll (Chl) b-less mutants (VG28-1, VG30-5) and the respective wild type (WT) plant (cv. Zhonghua No. 11) were analyzed for the changes in Chl fluorescence parameters, xanthophyll cycle pool, and its de-epoxidation state under exposure to strong irradiance, SI (1 700 μmol m⁻² s⁻¹). We also examined alterations in the chloroplast ultrastructure of the mutants induced by methyl viologen (MV) photooxidation. During HI (0–3.5 h), the photoinactivation of photosystem 2 (PS2) appeared earlier and more severely in Chl b-less mutants than in the WT. The decreases in maximal photochemical efficiency of PS2 in the dark (F_v/F_m), quantum efficiency of PS2 electron transport (Φ_PS2), photochemical quenching (q_P), as well as rate of photochemistry (P_rate), and the increases in de-epoxidation state (DES) and rate of thermal dissipation of excitation energy (D_rate) were significantly greater in Chl b-mutants compared with the WT plant. A relatively larger xanthophyll pool and 78–83 % conversion of violaxanthin into antheraxanthin and zeaxanthin in the mutants after 3.5 h of HI was accompanied with a high ratio of inactive/total PS2 (0.55–0.73) and high 1–q_P (0.57–0.68) which showed that the activities of the xanthophyll cycle were probably insufficient to protect the photosynthetic apparatus against photoinhibition. No apparent difference of chloroplast ultrastructure was found between Chl b-less mutants and WT plants grown under low, LI (180 μmol m⁻² s⁻¹) and high, HI (700 μmol m⁻² s⁻¹) irradiance. However, swollen chloroplasts and slight dilation of thylakoids occurred in both mutants and the WT grown under LI followed by MV treatment. These typical symptoms of photooxidative damage were aggravated as plants were exposed to HI. Distorted and loose scattered thylakoids were observed in particular in the Chl b-less mutants. A greater extent of photoinhibition and photooxidation in these mutants indicated that the susceptibility to HI and oxidative stresses was enhanced in the photosynthetic apparatus without Chl b most likely as a consequence of a smaller antenna size.     

Rapid evolutionary divergence of Photosystem I core subunits PsaA and PsaB in the marine prokaryote Prochlorococcus

The nucleotide sequences of the genes coding for the subunits of the Photosystem I (PS I) core, PsaA and PsaB were determined for the marine prokaryotic oxyphototrophs Prochlorococcus sp. MED4 (CCMP1378), P. marinus SS120 (CCMP1375) and Synechococcus sp. WH7803. Divergence of these sequences from those of both freshwater cyanobacteria and higher plants was remarkably high, given the conserved nature of PsaA and PsaB proteins. In particular, the PsaA of marine prokaryotes showed several specific insertions and deletions with regard to known PsaA sequences. Even in between the two Prochlorococcus strains, which correspond to two genetically different ecotypes with shifted growth irradiance optima, the sequence identity was only 80.2% for PsaA and 88.9% for PsaB. Possible causes and implications of the fast evolution rates of these two PS I core subunits are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Response of Photosynthetic Apparatus of Spring Barley (Hordeum vulgare L.) to Combined Effect of Elevated CO2 Concentration and Different Growth Irradiance

The response of barley (Hordeum vulgare L. cv. Akcent) to various photosynthetic photon flux densities (PPFDs) and elevated [CO₂] [700 μmol (CO₂) mol⁻¹; EC] was studied by gas exchange, chlorophyll (Chl) a fluorescence, and pigment analysis. In comparison with barley grown under ambient [CO₂] [350 μmol (CO₂) mol⁻¹; AC] the EC acclimation resulted in a decrease in photosynthetic capacity, reduced stomatal conductance, and decreased total Chl content. The extent of acclimation depression of photosynthesis, the most pronounced for the plants grown at 730 μmol m⁻² s⁻¹ (PPFD₇₃₀), may be related to the degree of sink-limitation. The increased non-radiative dissipation of absorbed photon energy for all EC plants corresponded to the higher de-epoxidation state of xanthophylls only for PPFD₇₃₀ barley. Further, a pronounced decrease in photosystem 2 (PS2) photochemical efficiency (given as F_V/F_M) for EC plants grown at 730 and 1 200 μmol m⁻² s⁻¹ in comparison with AC barley was related to the reduced epoxidation of antheraxanthin and zeaxanthin back to violaxanthin in darkness. Thus the EC conditions sensitise the photosynthetic apparatus of high-irradiance acclimated barley plants (particularly PPFD₇₃₀) to the photoinactivation of PS2. This revised version was published online in August 2006 with corrections to the Cover Date.     

The development of antenna complexes of barley (Hordeum vulgare cv. Akcent) under different light conditions as judged from the analysis of 77 K chlorophyll a fluorescence spectra

Using 77 K chlorophyll a (Chl a) fluorescence spectra in vivo, the development was studied of Photosystems II (PS II) and I (PS I) during greening of barley under intermittent light followed by continuous light at low (LI, 50 μmol m⁻² s⁻¹) and high (HI, 1000 μmol m⁻² s⁻¹) irradiances. The greening at HI intermittent light was accompanied with significantly reduced fluorescence intensity from Chl b excitation for both PS II (F685) and PS I (F743), in comparison with LI plants, indicating that assembly of light-harvesting complexes (LHC) of both photosystems was affected to a similar degree. During greening at continuous HI, a slower increase of emission from Chl b excitation in PS II as compared with PS I was observed, indicating a preferred reduction in the accumulation of LHC II. The following characteristics of 77 K Chl a fluorescence spectra documented the photoprotective function of an elevated content of carotenoids in HI leaves: (1) a pronounced suppression of Soret region of excitation spectra (410–450 nm) in comparison with the red region (670–690 nm) during the early stage of greening indicated a strongly reduced excitation energy transfer from carotenoids to the Chl a fluorescing forms within PS I and PS II; (2) changes in the shape of the excitation band of Chl b and carotenoids (460–490 nm) during greening under continuous light confirmed that the energy transfer from carotenoids to Chl a within PS II remained lower as compared with the LI plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Desulfurization of dibenzothiophene by Bacillus subtilis recombinants carrying dszABC and dszD genes

The desulfurization (dsz) genes from Rhodococcus erythropolis DS-3 were successfully integrated into the chromosomes of Bacillus subtilis ATCC 21332 and UV1 using an integration vector pDGSDN, yielding two recombinant strains, B. subtilis M29 and M28 in which the integrated dsz genes were expressed efficiently under the promoter, Pspac. The dibenzothiophene (DBT) desulfurization efficiency of M29 was 16.2 mg DBT l⁻¹ h⁻¹ at 36 h, significantly higher than that of R. erythropolis DS−3 and B. subtilis M28 and also showed no product inhibition. The interfacial tension of the supernatant fermented by M29 varied from 48 mN m⁻¹ to 4.2 mN m⁻¹, lower than that of the recombinant strain, M28, reveals that the biosurfactant secreted from M29 may have an important function in the DBT desulfurization process.     

Partial sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase and the phylogeny of Prochloron and Prochlorococcus (Prochlorales)

The prochlorophytes, oxygenic photosynthetic prokaryotes having no phycobiliprotein but possessing chlorophylls a and b, have been proposed to have a common ancestry with green chloroplasts, yet this is still controversal. We report here that partial sequence comparisons of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, including sequence data from two prochlorophytes, Prochlorococcus and Prochloron, indicate that Prochlorococcus is more closely related to a photosynthetic bacterium, Chromatium vinosum (-purple bacteria), than to cyanobacteria, while Prochloron is closely related to the prochlorophyte Prochlorothrix and to cyanobacteria. The molecular phylogenetic tree indicates that a common ancestor of Prochlorococcus and -purple bacteria branched off from the land plant lineage earlier than Prochloron, Prochlorothrix, and cyanobacteria.Correspondence to: A. Shimada     

The effect of elevated CO2 concentration on leaf chlorophyll and nitrogen contents in rice during post-flowering phases

The effect of elevated CO₂ concentration (CE) on leaf chlorophyll (Chl) and nitrogen (N) contents and photosynthetic rate (P_N) was evaluated during the post-flowering stages of rice grown at CE (570 ± 50 μmol mol⁻¹) in open top chamber (OTC), at ambient CO₂ concentration (∼ 365 μmol mol⁻¹) in OTC and at open field. Thirty-five day old seedlings were transplanted in OTCs or in field and allowed to grow till maturity. Chl and N contents were highest at the time of flowering and thereafter it started to decline. The rate of decline in Chl and N contents was faster in plants grown under CE mostly in later part of growth. Irrespective of treatment difference, flag leaf contained the highest amount of Chl and N than penultimate and third leaf. The higher P_N was observed in leaves under CE than in the leaves in other two growing conditions. Considering growth stage, P_N was the highest at flowering which reduced at the later part of growth due to degradation of Chl and N content of the leaf. Under CE it was 40.02 μmol m⁻² s⁻¹ at flowering and it reduced to only 14.77 μmol m⁻² s⁻¹ at maturity stage. The beneficial effect of CE in increasing leaf P_N may be maintained by applying extra dose of nitrogen at the later stages of plant growth.