Rosanne Quinnell

The major light-harvesting pigment protein of Acaryochloris marina.

The major light‐harvesting protein complex containing chlorophyll (Chl) d was isolated from Acaryochloris marina thylakoid membranes. Isolation was achieved by detergent solubilisation followed by separation on 6–40% sucrose gradients using ultracentrifugation. The best Chl d yield (70%) used 0.3% dodecyl maltoside, 0.15% octyl glucoside, 0.05% zwittergent 3‐14 with the detergent:total Chl d ratio around 10:1 (w/w). Characterisation of the light‐harvesting pigment protein complex (lhc) involved non‐denaturing electrophoresis, SDS–PAGE, absorbance and fluorescence spectroscopy. The main polypeptide in the lhc was shown to be ca. 34 kDa and to contain Chl d and Chl a, indicating that the Acaryochloris lhc is similar to that of prochlorophytes. The Chl a level varied with the culture conditions, which is consistent with previous findings.

Malic enzyme activity in bacteroids from soybean nodules

SUMMARY: Soluble extracts of Bradyrhizobium japonicum bacteroids from soybean root nodules showed substantial rates of NAD+ and NADP+ reduction which were malate and MnCl2 dependent. Pyruvate was formed stoichiometrically and the NAD- and NADP-dependent rates were additive, indicating the presence of two malic enzymes. The NADP-dependent malic enzyme had a high affinity for malate (apparent K m = 0·1 mM) and was stimulated by ammonium. The NAD-dependent malic enzyme had a lower affinity for malate (apparent K m = 1·9 mM) and was stimulated by potassium and ammonium salts. The maximum velocities of the two enzymes were similar and of comparable magnitude to the activities of tricarboxylic acid cycle enzymes in the extracts. Possible roles of the malic enzymes in the metabolism of malate and succinate in bacteroids are discussed.

Formyl group modification of chlorophyll a: a major evolutionary mechanism in oxygenic photosynthesis

We discuss recent advances in chlorophyll research in the context of chlorophyll evolution and conclude that some derivations of the formyl side chain arrangement of the porphyrin ring from that of the Chl a macrocycle can extend the photosynthetic active radiation (PAR) of these molecules, for example, Chl d and Chl f absorb light in the near-infrared region, up to ∼750 nm. Derivations such as this confer a selective advantage in particular niches and may, therefore, be beneficial for photosynthetic organisms thriving in light environments with particular light signatures, such as red- and near-far-red light-enriched niches. Modelling of formyl side chain substitutions of Chl a revealed yet unidentified but theoretically possible Chls with a distinct shift of light absorption properties when compared to Chl a.

Isolation of Symbiosomes and The Symbiosome Membrane Complex from The Zoanthid Zoanthus Robustus

A. Kazandjian, V.A. Shepherd, M. Rodriguez-Lanetty, W. Nordemeier, A.W.D. Larkum and R.G. Quinnell. 2008. Isolation of symbiosomes and the symbiosome membrane complex from the zoanthid Zoanthus robustus. Phycologia 47: 294–306. DOI: 10.2216/07-23.1 The zoanthid Zoanthus robustus was used as a model organism to develop procedures for isolating pure symbiosomes and symbiosome membranes. The symbiosome is comprised of a zooxanthella (Symbiodinium sp.) cell that divides rarely and is separated from the host gastrodermal cytoplasm by a symbiosome multimembrane complex. Devising a method to isolate membranes at the interface between the symbiotic partners is a critical first step in characterising the molecular components involved in the metabolic trafficking necessary to sustain an effective symbiosis. After zoanthid gastrodermal cells were extracted, symbiosomes were released by mechanical disruption, recovered by centrifugation, and then purified using discontinuous sucrose gradient centrifugation. The material forming the membrane complex around symbiosomes proved highly resistant to disruption. Methods used to dissociate this interface from symbionts included (1) Triton X-100 detergent solubilisation, (2) osmotic shock with mechanical disruption, and (3) vigorous mechanical disruptions, where powerful shearing forces were used, combined with a series of sucrose density gradient centrifugation steps. The lipophilic styryl fluorochrome FM 1-43, at a concentration of 30 µM, selectively labelled the symbiosome membrane complex, both for isolated symbiosomes and those in hospite. Other cell membranes, including plasma membranes, endoplasmic reticulum, tonoplast, and organelle membranes, were not visibly labelled at this concentration. The selective labelling of the symbiosome membrane complex remained stable even after long exposure times (3 h). At 30 µM concentration, FM 1-43 also labelled symbiosome membrane fragments isolated using methods (1), (2) and (3). Method (3) proved to be the most effective in producing a fraction enriched in FM-143-labelled membrane material, which we call a symbiosome membrane complex. Transmission electron microscopy, together with confocal and conventional epifluorescence microscopy of the FM 1-43-stained preparations, was used to validate the purity of symbiosome preparations and to infer the complexity of the symbiosome membrane complex. This membrane complex has regions where the membranes contributed by the alga are appressed, and punctate regions whose function remains unclear.

Chlorophyll d as the major photopigment in Acaryochloris marina

Chlorophyll (Chl) d is the major pigment in the photosystems (PS) and light-harvesting complex(es) of Acaryochloris marina. Chl a is present in small and variable amounts in PSII and in the light-harvesting complex(es). Isolated PSII complex showed a major fluorescence emission peak at 725 nm and a smaller emission peak due to Chl d at 701 nm, while the PSI complex showed two pools of Chl d, one with emission at 730 nm and the other at 709 nm at 77 K. In PSI and PSII of classical cyanobacteria and of higher plants, where Chl a is the predominant pigment rather than Chl d, these differences are not as pronounced. Light energy absorbed by phycobiliproteins was also active in these Chl d emissions. The major light-harvesting pigment protein is similar to the prochlorophyte Chl-binding protein (pcb) and had a major emission peak at 711 nm. In Cyanobacteria an iron-stress induced Chl-binding protein (isiA) forms a polymeric ring around PSI, and so the effect(s) of iron stress on A. marina where investigated. No clear evidence could be deduced for the formation of an isiA protein under iron stress and no clear changes in the proportion of Chl d :Chl a could be discerned although phycobilins showed a decreased under iron-stress conditions. That Chl d replaces Chl a in all its functions in A. marina is clear; the advantage of this evolutionary development appears to be to enable A. marina to absorb far-red light which occurs in environments where red light is filtered out by other photosynthetic organisms.

Transport of symbiotic zooxanthellae in mesogleal canals of Zoanthus robustus

In symbiotic dinoflagellate–cnidarian associations, regulation of algal numbers is an essential feature during steady state conditions (Reimer 1971; Falkowski et al. 1993). During mass bleaching events there is massive loss of zooxanthellae due to processes that are poorly understood (Gates et al. 1992; Jones and Yellowlees 1997). Under normal environmental conditions, the symbiotic algal population is maintained by the lowlevel expulsion of algal cells from the host coelenteron (=gastrovascular cavity); this low-level expulsion is thought to balance increases in endosymbiont numbers that occur from algal division, thereby maintaining a constant algal density (Hoegh-Guldberg et al. 1987). Although symbiotic algae normally exist only in endodermal cells, here we describe the presence of zooxanthellae within canals which are distributed within the host’s mesoglea and which connect to mesenteries. This finding implies that some zooxanthellae discharged from host endodermal cells are transported through mesogleal ducts to the inner cavity of mesenteries, from where they are finally degraded and/or expelled into the coelenteron. Most zooxanthellae reside within the endodermal host cells but in certain cnidarian species zooxanthellae are also found within the ectodermal and mesogleal layers (Hyman 1940), which, always, at least in theory, are separated from the coelenteron. After zooxanthellae are released, either by exocytosis or through whole host cell detachment (Gates et al. 1992) they become submerged in a three-dimensional matrix of host tissue, which is relatively distant from the coelenteron, which is the site from where they will be ejected finally through the host pharynx and oral aperture. Some studies have suggested that zooxanthellae from scleractinian hosts are normally extruded from the gastrodermis (endodermal cells) and accumulated directly into the coelenteron (Titlyanov et al. 1996). However, this direct algal cell discharge from the coelenteron might happen only in endodermal cells lining the coelenteron, but not from those located in the inner cell layers of the host tissue (Fig. 1a). There is some other work (Yonge 1966; Reimer 1971; Trench 1974) that has shown that some released zooxanthellae are ejected into the coelenteron from mesenterial cavities, in which partial algal degradation seems to take place (Trench 1974; Titlyanov et al. 1996). Algae then amass on the lateral lobes of the mesenterial filaments, which are rolled and finally extruded from the body as a mass called a ‘‘zooxanthellae body or pellet’’ (Yonge 1966; Reimer 1971). However, how the zooxanthellae, that are released from the inner host tissue, reach the mesenteries was unclear.

Photosynthesis of an epiphytic resurrection fern Davallia angustata (Wall .ex Hook. & Grev.)

Davallia (Pachypleuria) angustata (Wall. ex Hook. & Grev.) is a common epiphytic fern that grows on tree trucks and palm trees in south-east Asia. The plant is a resurrection plant, capable of rapid recovery from desiccation, but is not a CAM plant like some other epiphytic ferns. Under well-watered conditions Davallia shows a diurnal cycle of photosynthesis with maxima in mid-morning ~0900 hours (solar time). Under optimum conditions, the optimum irradiance (Eopt) = 879.3 ± 65.31 μmol photons m–2 s–1 or ~45% of full sunlight qualifying it as a sun plant. The maximum photosynthetic electron transport rate (ETRmax) was 77.77 ± 3.423 μmol e– m–2 s–1 or, on a Chl a basis 350 ± 36.0 μmol g–1 (Chl a) s–1. The photosynthetic efficiency (α0) is α0 = 0.2404 ± 0.02076 e– photon–1 or 1.082 ± 0.137 e– photon m2 g–1 (Chl a). Eopt and maximum photosynthesis (ETRmax) are directly proportional to one another (y = mx, r = 0.8813, P < <0.001). The slope of the line is the average photosynthetic efficiency at optimum irradiance (ETRmax/Eopt or αEopt = 0.07505 ± 0.00262 e– photon–1), equivalent to a mean asymptotic photosynthetic efficiency (α0) of 0.2040 ± 0.00712 e– photon–1. This simple relationship between ETRmax and Eopt does not appear to have been noted before. There is some accumulation of titratable acid in the morning but no accumulation of organic acids at night. Davallia is not a CAM plant. A simple pulse amplitude modulation (PAM) protocol shows that Davallia is a homiochlorophyllous resurrection plant.

The Function of MgDVP in a Chlorophyll d -Containing Organism

The cyanobacterium Acaryochloris marina is an exceptional organism utilising chlorophyll d (Chl d) as its major photosynthetic pigment. Acaryochloris cells contain 90–99% Chl d with minor amounts of chlorophyll a and a chlorophyll c-like pigment. These unusual characteristics make it an excellent candidate to study various aspects of photosynthesis driven by Chl d. However, little is known about the pathway of Chl d biosynthesis. We specifically designed HPLC methods to analyse pigment compositions of Acaryochloris. This enabled us to detect intermediate products of the chlorophyll biosynthesis. We identified Mg-Protoporphyrin IX monomethyl ester (MgPMe) and Mg-2,4-divinyl pheoporphyrin (MgDVP) and the environmental factors influencing their concentration levels. HPLC-facilitated analysis of pigments from Acaryochloris cells cultured under various light quantities was performed; light stress conditions induced an increase in the ratio of MgDVP to Chl d. Pigment analysis of Acaryochloris cells grown under oxygen-stressed conditions demonstrated a decrease in MgDVP levels. We propose that the Chl d biosynthesis pathway favours an aerobic environment despite the fact that Acaryochloris cells can survive under anaerobic conditions.

Mobile Botany: Smart Phone Photography in Laboratory Classes Enhances Student Engagement

Abstract In our first-year university botany classes at Charles Sturt University, we noticed that in laboratory class, students were taking photographs of their specimens with the dissecting and compound microscopes using their mobile phones. Student-generated images as “learning objects” were used to enhance the engagement of all students, including Distance Education students who used images provided by the on-campus students. The Distance Education students did all the laboratory work at an intensive residential school, and they were encouraged to take images; these were shared with on-campus students, making them aware of the laboratory practical work they were yet to do. In other cases, images from students were incorporated into lectures and tutorials, preparing students for the lab exam. Botany students have shared their photomicrographs with their friends and family via social media. We saw interesting examples of students excitedly describing their images to non-science friends, teaching them what they were learning! In the second year, students were also encouraged to use their phones to capture their own images of plant specimens to help them master plant identification. Although we do not have any quantitative evidence of these activities enhancing student learning, it was evident that those students who took and shared their own images were more engaged in the learning process.