Oxidative modification of LHC II associated with photosystem II and PS I-LHC I-LHC II membranes.

Under aerobic conditions the production of Reactive Oxygen Species (ROS) by electron transport chains is unavoidable, and occurs in both autotrophic and heterotrophic organisms. In photosynthetic organisms both Photosystem II (PS II) and Photosystem I (PS I), in addition to the cytochrome b6/f complex, are demonstrated sources of ROS. All of these membrane protein complexes exhibit oxidative damage when isolated from field-grown plant material. An additional possible source of ROS in PS I and PS II is the distal, chlorophyll-containing light-harvesting array LHC II, which is present in both photosystems. These serve as possible sources of 1O2 produced by the interaction of 3O2 with 3chl* produced by intersystem crossing. We have hypothesized that amino acid residues close to the sites of ROS generation will be more susceptible to oxidative modification than distant residues. In this study, we have identified oxidized amino acid residues in a subset of the spinach LHC II proteins (Lhcb1 and Lhcb2) that were associated with either PS II membranes (i.e. BBYs) or PS I-LHC I-LHC II membranes, both of which were isolated from field-grown spinach. We identified oxidatively modified residues by high-resolution tandem mass spectrometry. Interestingly, two different patterns of oxidative modification were evident for the Lhcb1 and Lhcb2 proteins from these different sources. In the LHC II associated with PS II membranes, oxidized residues were identified to be located on the stromal surface of Lhcb1 and, to a much lesser extent, Lhcb2. Relatively few oxidized residues were identified as buried in the hydrophobic core of these proteins. The LHC II associated with PS I-LHC I-LHC II membranes, however, exhibited fewer surface-oxidized residues but, rather a large number of oxidative modifications buried in the hydrophobic core regions of both Lhcb1 and Lhcb2, adjacent to the chlorophyll prosthetic groups. These results appear to indicate that ROS, specifically 1O2, can modify the Lhcb proteins associated with both photosystems and that the LHC II associated with PS II membranes represent a different population from the LHC II associated with PS I-LHC I-LHC II membranes.

The RNA-binding protein RBP45D of Arabidopsis promotes transgene silencing and flowering time.

RNA-directed DNA methylation (RdDM) helps to defend plants against invasive nucleic acids. In the canonical form of RdDM, 24-nt small interfering RNAs (siRNAs) are produced by DICER-LIKE 3 (DCL3). The siRNAs are loaded onto ARGONAUTE (AGO) proteins leading ultimately to de novo DNA methylation. Here, we introduce the Arabidopsis thaliana prors1 (LUC) transgenic system, in which 24-nt siRNAs are generated to silence the promoter-LUC construct. A forward genetic screen performed with this system identified, besides known components of RdDM (NRPD2A, RDR2, AGO4 and AGO6), the RNA-binding protein RBP45D. RBP45D is involved in CHH (where H is A, C or T) DNA methylation, and maintains siRNA production originating from the LUC transgene. RBP45D is localized to the nucleus, where it is associated with small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). RNA-Seq analysis showed that in CRISPR/Cas-mediated rbp-ko lines FLOWERING LOCUS C (FLC) mRNA levels are upregulated and several loci differentially spliced, among them FLM. In consequence, loss of RBP45D delays flowering, presumably mediated by the release of FLC levels and/or alternative splicing of FLM. Moreover, because levels and processing of transcripts of known RdDM genes are not altered in rbp-ko lines, RBP45D should have a more direct function in transgene silencing, probably independent of the canonical RdDM pathway. We suggest that RBP45D facilitates siRNA production by stabilizing either the precursor RNA or the slicer protein. Alternatively, RBP45D could be involved in chromatin modifications, participate in retention of Pol IV transcripts and/or in Pol V-dependent lncRNA retention in chromatin to enable their scaffold function.

Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation.

Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~ 1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25-30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.

Light acclimation of shade-tolerant and light-resistant Tradescantia species: induction of chlorophyll a fluorescence and P700 photooxidation, expression of PsbS and Lhcb1 proteins.

In this work, we have compared photosynthetic performance and expression of the PsbS and Lhcb1 proteins in two contrast ecotypes of Tradescantia species, T. fluminensis (shade-tolerant) and T. sillamontana (light-resistant), grown at two intensities of light: 50-125 µmol photons m-2 s-1 (low light, LL) and 875-1000 µmol photons m-2 s-1 (high light, HL). Using the EPR method for measuring the P700 content, we have found that LL-grown plants of both species have higher (by a factor of ≈1.7-1.8) contents of PSI per fresh weight unit as compared to HL-grown plants. Acclimation of plants to LL or HL irradiation also influences the Chl(a + b) level and expression of the PsbS and Lhcb1 proteins. Immunoblotting analysis showed that acclimation to HL stimulates (by a factor of ≈1.7-1.8) the level of PsbS related to the total number of P700 centers. In light-resistant species T. sillamontana, the ratio PsbS/P700 is about 2-times higher than in shade-tolerant species T. fluminensis grown under the same conditions. This should enhance the capacity of their leaves for protection against the light stress. In agreement with these observations, the capacity of leaves for NPQ induction was enhanced during plant acclimation to HL. Kinetic studies of P700 photooxidation and light-induced changes in the yield of Chl a fluorescence also revealed that the short-term regulation of electron transport processes in chloroplasts, which manifested themselves in the kinetics of [Formula: see text] induction and the rate of Chl a fluorescence quenching, occurred more rapidly in HL-grown plants than in LL-grown plants. Thus, both factors, enhanced expression of PsbS and more rapid response of the photosynthetic electron transport chain to dark-to-light transitions should increase the capacity of HL-grown plants for their resistance to rapid fluctuations of solar light.

The specific localizations of phosphorylated Lhcb1 and Lhcb2 isoforms reveal the role of Lhcb2 in the formation of the PSI-LHCII supercomplex in Arabidopsis during state transitions.

State transitions are an important photosynthetic short-term response that maintains the excitation balance between photosystems I (PSI) and II (PSII). In plants, when PSII is preferentially excited, LHCII, the main heterotrimeric light harvesting complex of PSII, is phosphorylated by the STN7 kinase, detaches from PSII and moves to PSI to equilibrate the relative absorption of the two photosystems (State II). When PSI is preferentially excited LHCII is dephosphorylated by the PPH1 (TAP38) phosphatase, and returns to PSII (State I). Phosphorylation of LHCII that remain bound to PSII has also been observed. Although the kinetics of LHCII phosphorylation are well known from a qualitative standpoint, the absolute phosphorylation levels of LHCII (and its isoforms) bound to PSI and PSII have been little studied. In this work we thoroughly investigated the phosphorylation level of the Lhcb1 and Lhcb2 isoforms that compose LHCII in PSI-LHCII and PSII-LHCII supercomplexes purified from WT and state transition mutants of Arabidopsis thaliana. We found that, at most, 40% of the monomers that make up PSI-bound LHCII trimers are phosphorylated. Phosphorylation was much lower in PSII-bound LHCII trimers reaching only 15-20%. Dephosphorylation assays using a recombinant PPH1 phosphatase allowed us to investigate the role of the two isoforms during state transitions. Our results strongly suggest that a single phosphorylated Lhcb2 is sufficient for the formation of the PSI-LHCII supercomplex. These results are a step towards a refined model of the state transition phenomenon and a better understanding of the short-term response to changes in light conditions in plants.

Ultraviolet-B responses of nuclear genes encoding light-harvesting complex II proteins in pea (Pisum sativum) are altered by norflurazon- and photobleaching-induced chloroplast changes.

The role of functional and intact chloroplasts in mediating the ultraviolet-B (UV-B, 290-320 nm) regulation of two nuclear genes encoding light-harvesting complex II proteins in pea (Pisum sativum L. cv. Extra Early Alaska) was studied. Plants with chloroplasts lacking or containing carotenoids and functional photosystem II were obtained by growth under dim red light (0.2 µmol m-2 s-1 ) in the presence or absence of norflurazon (NF), an inhibitor of carotenoid biosynthesis. The NF-treatment resulted in an increase in AB80 (lhcb1* 2) mRNA but no substantial change in Cab-8 (lhcb1* 4) mRNA, indicating that the mRNA accumulations for AB80 and Cab-8 were differently correlated with the presence and absence of carotenoids. The mRNA levels for both Cab-8 and AB80 in the NF-treated plants were reduced to the same extent by partially photobleaching the chloroplasts with 3 h of higher intensity white light (W, 110 µmol m-2 s-1 ), suggesting that chloroplast integrity was equally important for transcript accumulation of both genes. The mRNAs of both Cab-8 and AB80 in non-NF-treated control plants were decreased by UV-B irradiation, with the level of AB80 mRNA reduced to a greater extent. The UV-B-induced mRNA reduction of both genes was inhibited by NF. The difference between the UV-B responses of the two genes was unaffected by NF, but was abolished by photobleaching the NF-treated plants prior to the UV-B irradiation. Therefore, the presence of carotenoids enhanced rather than prevented the UV-B down-regulation, and the difference in UV-B responses of the two genes may be dependent on chloroplast integrity.