Agnieszka Zygadlo

Sulfur starvation in rice: the effect on photosynthesis, carbohydrate metabolism, and oxidative stress protective pathways

Sulfur-deficient plants generate a lower yield and have a reduced nutritional value. The process of sulfur acquisition and assimilation play an integral role in plant metabolism, and response to sulfur deficiency involves a large number of plant constituents. Rice (Oryza sativa) is the second most consumed cereal grain, and the effects of sulfur deprivation in rice were analyzed by measuring changes in photosynthesis, carbohydrate metabolism, and antioxidants. The photosynthetic apparatus was severely affected under sulfur deficiency. The Chl content was reduced by 49% because of a general reduction of PSII and PSI and the associated light-harvesting antenna. The PSII efficiency was 31% lower at growth light, and the ability of PSI to photoreduce NADP+ was decreased by 61%. The Rubisco content was also significantly reduced in the sulfur-deprived plants. The imbalances between PSII and PSI, and between photosynthesis and carbon fixation led to a general over-reduction of the photosynthetic electron carriers (higher 1-q(P)). Chromatographic analysis showed that the level of monosaccharides was lower and starch content higher in the sulfur-deprived plants. In contrast, no changes in metabolite levels were found in the tricarboxylic acid or Calvin cycle. The level of the thiol-containing antioxidant, GSH, was 70% lower and the redox state was significantly more oxidized. These changes in GSH status led to an upregulation of the cytosolic isoforms of GSH reductase and monodehydroascorbate reductase. In addition, alternative antioxidants like flavonoids and anthocyanins were increased in the sulfur-deprived plants.

Intrinsically unstructured phosphoprotein TSP9 regulates light harvesting in Arabidopsis thaliana.

Thylakoid-soluble phosphoprotein of 9 kDa, TSP9, is an intrinsically unstructured plant-specific protein [Song, J., et al. (2006) Biochemistry 45, 15633-15643] with unknown function but established associations with light-harvesting proteins and peripheries of both photosystems [Hansson, M., et al. (2007) J. Biol. Chem. 282, 16214-16222]. To investigate the function of this protein, we used a combination of reverse genetics and biochemical and fluorescence measurement methods in Arabidopsis thaliana. Differential gene expression analysis of plants with a T-DNA insertion in the TSP9 gene using an array of 24000 Arabidopsis genes revealed disappearance of high light-dependent induction of a specific set of mostly signaling and unknown proteins. TSP9-deficient plants had reduced levels of in vivo phosphorylation of light-harvesting complex II polypeptides. Recombinant TSP9 was phosphorylated in light by thylakoid membranes isolated from the wild-type and mutant plants lacking STN8 protein kinase but not by the thylakoids deficient in STN7 kinase, essential for photosynthetic state transitions. TSP9-lacking mutant and RNAi plants with downregulation of TSP9 showed reduced ability to perform state transitions. The nonphotochemical quenching of chlorophyll fluorescence at high light intensities was also less efficient in the mutant compared to wild-type plants. Blue native electrophoresis of thylakoid membrane protein complexes revealed that TSP9 deficiency increased relative stability of photosystem II dimers and supercomplexes. It is concluded that TSP9 regulates plant light harvesting acting as a membrane-binding protein facilitating dissociation of light-harvesting proteins from photosystem II.

Knock-out of the chloroplast-encoded PSI-J subunit of photosystem I in Nicotiana tabacum.

The plastid‐encoded psaJ gene encodes a hydrophobic low‐molecular‐mass subunit of photosystem I (PSI) containing one transmembrane helix. Homoplastomic transformants with an inactivated psaJ gene were devoid of PSI‐J protein. The mutant plants were slightly smaller and paler than wild‐type because of a 13% reduction in chlorophyll content per leaf area caused by an ≈ 20% reduction in PSI. The amount of the peripheral antenna proteins, Lhca2 and Lhca3, was decreased to the same level as the core subunits, but Lhca1 and Lhca4 were present in relative excess. The functional size of the PSI antenna was not affected, suggesting that PSI‐J is not involved in binding of light‐harvesting complex I. The specific PSI activity, measured as NADP+ photoreduction in vitro, revealed a 55% reduction in electron transport through PSI in the mutant. No significant difference in the second‐order rate constant for electron transfer from reduced plastocyanin to oxidized P700 was observed in the absence of PSI‐J. Instead, a large fraction of PSI was found to be inactive. Immunoblotting analysis revealed a secondary loss of the luminal PSI‐N subunit in PSI particles devoid of PSI‐J. Presumably PSI‐J affects the conformation of PSI‐F, which in turn affects the binding of PSI‐N. This together renders a fraction of the PSI particles inactive. Thus, PSI‐J is an important subunit that, together with PSI‐F and PSI‐N, is required for formation of the plastocyanin‐binding domain of PSI. PSI‐J is furthermore important for stability or assembly of the PSI complex.

The Properties of the Positively Charged Loop Region in PSI-G Are Essential for Its “Spontaneous” Insertion into Thylakoids and Rapid Assembly into the Photosystem I Complex

The PSI-G subunit of photosystem I (PSI) is an 11-kDa membrane protein that plays an important role in electron transport between plastocyanin and PSI and is involved in the stability of the PSI complex. Within the complex, the PSI-G subunit is bound to PSI-B and is in contact with Lhca1. PSI-G has two transmembrane spans connected by a positively charged stromal loop. The loop is inaccessible to proteases, indicating a tightly bound location within the PSI complex. Here, we have studied the insertion mechanism and assembly of PSI-G. We show that the protein inserts into thylakoids by a direct or “spontaneous” pathway that does not involve the activities of any known chloroplast protein-targeting machinery. Surprisingly, the positively charged stromal loop region plays a major role in this process. Mutagenesis or deletions within this region almost invariably lead to a marked lowering of insertion efficiency, strongly indicating a critical role for the loop in the organization of the transmembrane regions prior to or during membrane insertion. Finally, we have examined the assembly of newly inserted PSI-G into the PSI complex, since very little is known of the assembly pathway for this large multimeric complex. Interestingly, we find that inserted PSI-G can be found within the full PSI complex within the import assay time frame after insertion into thylakoids, strongly suggesting that PSI-G normally associates at the end of the assembly process. This is consistent with its location on the periphery of the complex.

Insertion of the plant photosystem I subunit G into the thylakoid membrane.

Subunit G of photosystem I is a nuclear‐encoded protein, predicted to form two transmembrane α‐helices separated by a loop region. We use in vitro import assays to show that the positively charged loop domain faces the stroma, whilst the N‐ and C‐termini most likely face the lumen. PSI‐G constructs in which a His‐ or Strep‐tag is placed at the C‐terminus or in the loop region insert with the same topology as wild‐type photosystem I subunit G (PSI‐G). However, the presence of the tags in the loop make the membrane‐inserted protein significantly more sensitive to trypsin, apparently by disrupting the interaction between the loop and the PSI core. Knock‐out plants lacking PSI‐G were transformed with constructs encoding the C‐terminal and loop‐tagged PSI‐G proteins. Experiments on thylakoids from the transgenic lines show that the C‐terminally tagged versions of PSI‐G adopt the same topology as wild‐type PSI‐G, whereas the loop‐tagged versions affect the sensitivity of the loop region to trypsin, thus confirming the in vitro observations. Furthermore, purification of PSI complexes from transgenic plants revealed that all the tagged versions of PSI‐G are incorporated and retained in the PSI complex, although the C‐terminally tagged variants of PSI‐G were preferentially retained. This suggests that the loop region of PSI‐G is important for proper integration into the PSI core. Our experiments demonstrate that it is possible to produce His‐ and Strep‐tagged PSI in plants, and provide further evidence that the topology of membrane proteins is dictated by the distribution of positive charges, which resist translocation across membranes.

Involvement of TSP9 Phosphoprotein in Balancing the Photosynthetic Light Harvesting Process in Arabidopsis thaliana

TSP9 is the thylakoid soluble phosphoprotein of 9 kDa, an intrinsically unstructured plant specific protein that has earlier been found interacting with light harvesting proteins and both photosystems in photosynthetic thylakoid membranes from spinach. We investigated the function of this protein in Arabidopsis thaliana using reverse genetics, biochemical and fluorometric methods.\ Recombinant TSP9 was used as a substrate for light-induced phosphorylation by thylakoid membranes isolated from wild type and two mutant lines lacking either STN7 or STN8 protein kinases. TSP9 was found phosphorylated by STN7 kinase. Phosphorylation of light-harvesting complex II was found reduced in knockout plants incapable of expressing TSP9. Chlorophyll fluorescence studies showed reduced ability of the TSP9 lacking mutant to perform the photosynthetic state transitions, as well as decreased non-photochemical quenching as compared with the wild type plants. We propose a\ role for this small soluble protein in the regulation of light harvesting in Arabidopsis.