Crystallization of the Photosystem II core complex and its chlorophyll binding subunit CP43 from transplastomic plants of Nicotianatabacum

Photosystem II from transplastomic plants of Nicotiana tabacum with a hexahistidine tag at the N-terminal end of the PsbE subunit (α-chain of the cytochrome b559) was purified according to the protocol of Fey et al. (BBA 12:1501–1509, 2008). The protein sample was then subjected to two additional gel filtration runs in order to increase its homogeneity and to standardize the amount of detergent. Large three dimensional crystals of the core complex were obtained. Crystals of one of its chlorophyll binding subunits (CP43) in isolation grew in very similar conditions that differed only in the concentration of the detergent. Diffraction of Photosystem II and CP43 crystals at various synchrotron beamlines was limited to a resolution of 7 and 14 Å, respectively. In both cases the diffraction quality was insufficient for an unambiguous assignment of the crystallographic lattice or space group

A codon-optimized luciferase from Gaussia princeps facilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii has emerged as a superb model species in plant biology. Although the alga is easily transformable, the low efficiency of transgene expression from the Chlamydomonas nuclear genome has severely hampered functional genomics research. For example, poor transgene expression is held responsible for the lack of sensitive reporter genes to monitor gene expression in vivo, analyze subcellular protein localization or study protein–protein interactions. Here, we have tested the luciferase from the marine copepod Gaussia princeps (G-Luc) for its suitability as a sensitive bioluminescent reporter of gene expression in Chlamydomonas. We show that a Gaussia luciferase gene variant, engineered to match the codon usage in the Chlamydomonas nuclear genome, serves as a highly sensitive reporter of gene expression from both constitutive and inducible algal promoters. Its bioluminescence signal intensity greatly surpasses previously developed reporters for Chlamydomonas nuclear gene expression and reaches values high enough for utilizing the reporter as a tool to monitor responses to environmental stresses in vivo and to conduct high-throughput screenings for signaling mutants in Chlamydomonas

Artemisinin Inhibits Chloroplast Electron Transport Activity: Mode of Action

Artemisinin, a secondary metabolite produced in Artemisia plant species, besides having antimalarial properties is also phytotoxic. Although, the phytotoxic activity of the compound has been long recognized, no information is available on the mechanism of action of the compound on photosynthetic activity of the plant. In this report, we have evaluated the effect of artemisinin on photoelectron transport activity of chloroplast thylakoid membrane. The inhibitory effect of the compound, under in vitro condition, was pronounced in loosely and fully coupled thylakoids; being strong in the former. The extent of inhibition was drastically reduced in the presence of uncouplers like ammonium chloride or gramicidin; a characteristic feature described for energy transfer inhibitors. The compound, on the other hand, when applied to plants (in vivo), behaved as a potent inhibitor of photosynthetic electron transport. The major site of its action was identified to be the QB; the secondary quinone moiety of photosystemII complex. Analysis of photoreduction kinetics of para-benzoquinone and duroquinone suggest that the inhibition leads to formation of low pool of plastoquinol, which becomes limiting for electron flow through photosystemI. Further it was ascertained that the in vivo inhibitory effect appeared as a consequence of the formation of an unidentified artemisinin-metabolite rather than by the interaction of the compound per se. The putative metabolite of artemisinin is highly reactive in instituting the inhibition of photosynthetic electron flow eventually reducing the plant growth

Graft-Transmitted siRNA Signal from the Root Induces Visual Manifestation of Endogenous Post-Transcriptional Gene Silencing in the Scion

In plants, post-transcriptional gene silencing (PTGS) spreads systemically, being transmitted from the silenced stock to the scion expressing the corresponding transgene. It has been reported that a graft-transmitted siRNA signal can also induce PTGS of an endogenous gene, but this was done by top-grafting using silenced stock. In the present study involving grafting of Nicotiana benthamiana, we found that PTGS of an endogenous gene, glutamate-1-semialdehyde aminotransferase (GSA), which acts as a visible marker of RNAi via inhibition of chlorophyll synthesis, was manifested along the veins of newly developed leaves in the wild-type scion by the siRNA signal synthesized only in companion cells of the rootstock

Surplus Photosynthetic Antennae Complexes Underlie Diagnostics of Iron Limitation in a Cyanobacterium

Chlorophyll fluorescence from phytoplankton provides a tool to assess iron limitation in the oceans, but the physiological mechanism underlying the fluorescence response is not understood. We examined fluorescence properties of the model cyanobacterium Synechocystis PCC6803 and a ΔisiA knock-out mutant of the same species grown under three culture conditions which simulate nutrient conditions found in the open ocean: (1) nitrate and iron replete, (2) limiting-iron and high-nitrate, representative of natural high-nitrate, low-chlorophyll regions, and (3) iron and nitrogen co-limiting. We show that low variable fluorescence, a key diagnostic of iron limitation, results from synthesis of antennae complexes far in excess of what can be accommodated by the iron-restricted pool of photosynthetic reaction centers. Under iron and nitrogen co-limiting conditions, there are no excess antennae complexes and variable fluorescence is high. These results help to explain the well-established fluorescence characteristics of phytoplankton in high-nutrient, low-chlorophyll ocean regions, while also accounting for the lack of these properties in low-iron, low-nitrogen regions. Importantly, our results complete the link between unique molecular consequences of iron stress in phytoplankton and global detection of iron stress in natural populations from space

Metabolic Engineering of Potato Carotenoid Content through Tuber-Specific Overexpression of a Bacterial Mini-Pathway

BACKGROUND: Since the creation of “Golden Rice”, biofortification of plant-derived foods is a promising strategy for the alleviation of nutritional deficiencies. Potato is the most important staple food for mankind after the cereals rice, wheat and maize, and is extremely poor in provitamin A carotenoids. METHODOLOGY: We transformed potato with a mini-pathway of bacterial origin, driving the synthesis of beta-carotene (Provitamin A) from geranylgeranyl diphosphate. Three genes, encoding phytoene synthase (CrtB), phytoene desaturase (CrtI) and lycopene beta-cyclase (CrtY) from Erwinia, under tuber-specific or constitutive promoter control, were used. 86 independent transgenic lines, containing six different promoter/gene combinations, were produced and analyzed. Extensive regulatory effects on the expression of endogenous genes for carotenoid biosynthesis are observed in transgenic lines. Constitutive expression of the CrtY and/or CrtI genes interferes with the establishment of transgenosis and with the accumulation of leaf carotenoids. Expression of all three genes, under tuber-specific promoter control, results in tubers with a deep yellow (“golden”) phenotype without any adverse leaf phenotypes. In these tubers, carotenoids increase approx. 20-fold, to 114 mcg/g dry weight and beta-carotene 3600-fold, to 47 mcg/g dry weight. CONCLUSIONS: This is the highest carotenoid and beta-carotene content reported for biofortified potato as well as for any of the four major staple foods (the next best event being “Golden Rice 2”, with 31 mcg/g dry weight beta-carotene). Assuming a beta-carotene to retinol conversion of 6∶1, this is sufficient to provide 50% of the Recommended Daily Allowance of Vitamin A with 250 gms (fresh weight) of “golden” potatoes

Competing charge transfer pathways at the photosystem II-electrode interface.

The integration of the water-oxidation enzyme photosystem II (PSII) into electrodes allows the electrons extracted from water oxidation to be harnessed for enzyme characterization and to drive novel endergonic reactions. However, PSII continues to underperform in integrated photoelectrochemical systems despite extensive optimization efforts. Here we carried out protein-film photoelectrochemistry using spinach and Thermosynechococcus elongatus PSII, and we identified a competing charge transfer pathway at the enzyme-electrode interface that short-circuits the known water-oxidation pathway. This undesirable pathway occurs as a result of photo-induced O2 reduction occurring at the chlorophyll pigments and is promoted by the embedment of PSII in an electron-conducting fullerene matrix, a common strategy for enzyme immobilization. Anaerobicity helps to recover the PSII photoresponse and unmasks the onset potentials relating to the QA/QB charge transfer process. These findings impart a fuller understanding of the charge transfer pathways within PSII and at photosystem-electrode interfaces, which will lead to more rational design of pigment-containing photoelectrodes in general.This work was supported by the U.K. Engineering and Physical Sciences Research Council (EP/H00338X/2 to E. Reisner), the U.K. Biology and Biotechnological Sciences Research Council (BB/K010220/1 to E. Reisner), a Marie Curie International Incoming Fellowship (PIIF-GA-2012-328085 RPSII to J.J.Z.). N.P. was supported by the Winton Fund for the Physics of Sustainability. E. Romero. and R.v.G. were supported by the VU University Amsterdam, the Laserlab-Europe Consortium, the TOP grant (700.58.305) from the Foundation of Chemical Sciences part of NWO, the Advanced Investigator grant (267333, PHOTPROT) from the European Research Council, and the EU FP7 project PAPETS (GA 323901). R.v.G. gratefully acknowledges his `Academy Professor' grant from the Royal Netherlands Academy of Arts and Sciences (KNAW). We would also like to thank Miss Katharina Brinkert and Prof A. William Rutherford for a sample of T. elongatus PSII, and H. v. Roon for preparation of the spinach PSII samples

Role of the Arabidopsis PIN6 auxin transporter in auxin homeostasis and auxin-mediated development

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.Christopher I. Cazzonelli, Marleen Vanstraelen, Sibu Simon, Kuide Yin, Ashley Carron-Arthur, Nazia Nisar, Gauri Tarle, Abby J. Cuttriss¤, Iain R. Searle, Eva Benkova, Ulrike Mathesius, Josette Masle, Jiří Friml, Barry J. Pogso

Overexpression of the Rieske FeS protein of the Cytochrome b 6 f complex increases C4 photosynthesis in Setaria viridis.

C4 photosynthesis is characterised by a CO2 concentrating mechanism that operates between mesophyll and bundle sheath cells increasing CO2 partial pressure at the site of Rubisco and photosynthetic efficiency. Electron transport chains in both cell types supply ATP and NADPH for C4 photosynthesis. Cytochrome b 6 f is a key control point of electron transport in C3 plants. To study whether C4 photosynthesis is limited by electron transport we constitutively overexpressed the Rieske FeS subunit in Setaria viridis. This resulted in a higher Cytochrome b 6 f content in mesophyll and bundle sheath cells without marked changes in the abundances of other photosynthetic proteins. Rieske overexpression plants showed better light conversion efficiency in both Photosystems and could generate higher proton-motive force across the thylakoid membrane underpinning an increase in CO2 assimilation rate at ambient and saturating CO2 and high light. Our results demonstrate that removing electron transport limitations can increase C4 photosynthesis

Cryo-EM structure of the spinach cytochrome b6 f complex at 3.6 Å resolution.

The cytochrome b6 f (cytb6 f ) complex has a central role in oxygenic photosynthesis, linking electron transfer between photosystems I and II and converting solar energy into a transmembrane proton gradient for ATP synthesis1-3. Electron transfer within cytb6 f occurs via the quinol (Q) cycle, which catalyses the oxidation of plastoquinol (PQH2) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation2. In higher plants, cytb6 f also acts as a redox-sensing hub, pivotal to the regulation of light harvesting and cyclic electron transfer that protect against metabolic and environmental stresses3. Here we present a 3.6 Å resolution cryo-electron microscopy (cryo-EM) structure of the dimeric cytb6 f complex from spinach, which reveals the structural basis for operation of the Q cycle and its redox-sensing function. The complex contains up to three natively bound PQ molecules. The first, PQ1, is located in one cytb6 f monomer near the PQ oxidation site (Qp) adjacent to haem bp and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Qp site and another that permits it, supporting a gating function for the chlorophyll a involved in redox sensing. PQ2 straddles the intermonomer cavity, partially obstructing the PQ reduction site (Qn) on the PQ1 side and committing the electron transfer network to turnover at the occupied Qn site in the neighbouring monomer. A conformational switch involving the haem cn propionate promotes two-electron, two-proton reduction at the Qn site and avoids formation of the reactive intermediate semiquinone. The location of a tentatively assigned third PQ molecule is consistent with a transition between the Qp and Qn sites in opposite monomers during the Q cycle. The spinach cytb6 f structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis