Natural variation in phosphorylation of photosystem II proteins in Arabidopsis thaliana: is it caused by genetic variation in the STN kinases?

Reversible phosphorylation of photosystem II (PSII) proteins is an important regulatory mechanism that can protect plants from changes in ambient light intensity and quality. We hypothesized that there is natural variation in this process in Arabidopsis (Arabidopsis thaliana), and that this results from genetic variation in the STN7 and STN8 kinase genes. To test this, Arabidopsis accessions of diverse geographical origins were exposed to two light regimes, and the levels of phospho-D1 and phospho-light harvesting complex II (LHCII) proteins were quantified by western blotting with anti-phosphothreonine antibodies. Accessions were classified as having high, moderate or low phosphorylation relative to Col-0. This variation could not be explained by the abundance of the substrates in thylakoid membranes. In genotypes with atrazine-resistant forms of the D1 protein, low D1 and LHCII protein phosphorylation was observed, which may be due to low PSII efficiency, resulting in reduced activation of the STN kinases. In the remaining genotypes, phospho-D1 levels correlated with STN8 protein abundance in high-light conditions. In growth light, D1 and LHCII phosphorylation correlated with longitude and in the case of LHCII phosphorylation also with temperature variability. This suggests a possible role of natural variation in PSII protein phosphorylation in the adaptation of Arabidopsis to diverse environments.     

A collection of INDEL markers for map-based cloning in seven Arabidopsis accessions

The availability of a comprehensive set of resources including an entire annotated reference genome, sequenced alternative accessions, and a multitude of marker systems makes Arabidopsis thaliana an ideal platform for genetic mapping. PCR markers based on INsertions/DELetions (INDELs) are currently the most frequently used polymorphisms. For the most commonly used mapping combination, Columbia×Landsberg erecta (Col-0×Ler-0), the Cereon polymorphism database is a valuable resource for the generation of polymorphic markers. However, because the number of markers available in public databases for accessions other than Col-0 and Ler-0 is extremely low, mapping using other accessions is far from straightforward. This issue arose while cloning mutations in the Wassilewskija (Ws-4) background. In this work, approaches are described for marker generation in Ws-4 x Col-0. Complementary strategies were employed to generate 229 INDEL markers. Firstly, existing Col-0/Ler-0 Cereon predicted polymorphisms were mined for transferability to Ws-4. Secondly, Ws-0 ecotype Illumina sequence data were analyzed to identify INDELs that could be used for the development of PCR-based markers for Col-0 and Ws-4. Finally, shotgun sequencing allowed the identification of INDELs directly between Col-0 and Ws-4. The polymorphism of the 229 markers was assessed in seven widely used Arabidopsis accessions, and PCR markers that allow a clear distinction between the diverged Ws-0 and Ws-4 accessions are detailed. The utility of the markers was demonstrated by mapping more than 35 mutations in a Col-0×Ws-4 combination, an example of which is presented here. The potential contribution of next generation sequencing technologies to more traditional map-based cloning is discussed.     

A Protein Phosphorylation Threshold for Functional Stacking of Plant Photosynthetic Membranes

Phosphorylation of photosystem II (PSII) proteins affects macroscopic structure of thylakoid photosynthetic membranes in chloroplasts of the model plant Arabidopsis. In this study, light-scattering spectroscopy revealed that stacking of thylakoids isolated from wild type Arabidopsis and the mutant lacking STN7 protein kinase was highly influenced by cation (Mg⁺⁺) concentrations. The stacking of thylakoids from the stn8 and stn7stn8 mutants, deficient in STN8 kinase and consequently in light-dependent phosphorylation of PSII, was increased even in the absence of Mg⁺⁺. Additional PSII protein phosphorylation in wild type plants exposed to high light enhanced Mg⁺⁺-dependence of thylakoid stacking. Protein phosphorylation in the plant leaves was analyzed during day, night and prolonged darkness using three independent techniques: immunoblotting with anti-phosphothreonine antibodies; Diamond ProQ phosphoprotein staining; and quantitative mass spectrometry of peptides released from the thylakoid membranes by trypsin. All assays revealed dark/night-induced increase in phosphorylation of the 43 kDa chlorophyll-binding protein CP43, which compensated for decrease in phosphorylation of the other PSII proteins in wild type and stn7, but not in the stn8 and stn7stn8 mutants. Quantitative mass spectrometry determined that every PSII in wild type and stn7 contained on average 2.5±0.1 or 1.4±0.1 phosphoryl groups during day or night, correspondingly, while less than every second PSII had a phosphoryl group in stn8 and stn7stn8. It is postulated that functional cation-dependent stacking of plant thylakoid membranes requires at least one phosphoryl group per PSII, and increased phosphorylation of PSII in plants exposed to high light enhances stacking dynamics of the photosynthetic membranes.     

Accession-dependent action potentials in Arabidopsis

Plant excitability, as measured by the appearance and circulation of action potentials (APs) after biotic and abiotic stress treatments, is a far lesser and more versatile phenomenon than in animals. To examine the genetic basis of plant excitability we used different Arabidopsis thaliana accessions. APs were induced by wounding (W) with a subsequent deposition (D) of 5 μL of 1 M KCl onto adult leaves. This treatment elicited transient voltage responses (APs) that were detected by 2 extracellular electrodes placed at a distance from the wounding location over an experimental time of 150 min. The first electrode (e1) was placed at the end of the petiole and the beginning of the leaf, and the second (e2) electrode was placed on the petiole near the center of the rosette. All accessions (Columbia (Col), Wassilewskija (Ws) and Landsberg erecta (Ler)) responded to the W & D treatment. After W & D treatment was performed on 100 plants for each accession, the number of APs ranged from 0 to 37 (median 8, total 940), 0 to 16 (median 5, total 528) and 0 to 18 (median 2, total 296) in Col, Ws and Ler, respectively. Responding plants (>0 APs) showed significantly different behaviors depending on their accessions of origin (i.e., Col 91, Ws 83 and Ler 76%). Some AP characteristics, such as amplitude and speed of propagation from e1 to e2 (1.28 mm s⁻¹), were the same for all accessions, whereas the average duration of APs was similar in Col and Ws, but different in Ler. Self-sustained oscillations were observed more frequently in Col than Ws and least often in Ler, and the mean oscillation frequency was more rapid in Col, followed by Ws, and was slowest in Ler. In general, Col was the most excitable accession, followed by Ws, and Ler was the least excitable; this corresponded well with voltage elicited action potentials. In conclusion, part of Arabidopsis excitability in AP responses is genetically pre-determined.     

Loss‐of‐function of OsSTN8 suppresses the photosystem II core protein phosphorylation and interferes with the photosystem II repair mechanism in rice (Oryza sativa)

STN8 kinase is involved in photosystem II (PSII) core protein phosphorylation (PCPP). To examine the role of PCPP in PSII repair during high light (HL) illumination, we characterized a T–DNA insertional knockout mutant of the rice (Oryza sativa) STN8 gene. In this osstn8 mutant, PCPP was significantly suppressed, and the grana were thin and elongated. Upon HL illumination, PSII was strongly inactivated in the mutants, but the D1 protein was degraded more slowly than in wild‐type, and mobilization of the PSII supercomplexes from the grana to the stromal lamellae for repair was also suppressed. In addition, higher accumulation of reactive oxygen species and preferential oxidation of PSII reaction center core proteins in thylakoid membranes were observed in the mutants during HL illumination. Taken together, our current data show that the absence of STN8 is sufficient to abolish PCPP in osstn8 mutants and to produce all of the phenotypes observed in the double mutant of Arabidopsis, indicating the essential role of STN8‐mediated PCPP in PSII repair.     

Genetic Architecture of Natural Variation of Telomere Length in Arabidopsis thaliana

Telomeres represent the repetitive sequences that cap chromosome ends and are essential for their protection. Telomere length is known to be highly heritable and is derived from a homeostatic balance between telomeric lengthening and shortening activities. Specific loci that form the genetic framework underlying telomere length homeostasis, however, are not well understood. To investigate the extent of natural variation of telomere length in Arabidopsis thaliana, we examined 229 worldwide accessions by terminal restriction fragment analysis. The results showed a wide range of telomere lengths that are specific to individual accessions. To identify loci that are responsible for this variation, we adopted a quantitative trait loci (QTL) mapping approach with multiple recombinant inbred line (RIL) populations. A doubled haploid RIL population was first produced using centromere-mediated genome elimination between accessions with long (Pro-0) and intermediate (Col-0) telomere lengths. Composite interval mapping analysis of this population along with two established RIL populations (Ler-2/Cvi-0 and Est-1/Col-0) revealed a number of shared and unique QTL. QTL detected in the Ler-2/Cvi-0 population were examined using near isogenic lines that confirmed causative regions on chromosomes 1 and 2. In conclusion, this work describes the extent of natural variation of telomere length in A. thaliana, identifies a network of QTL that influence telomere length homeostasis, examines telomere length dynamics in plants with hybrid backgrounds, and shows the effects of two identified regions on telomere length regulation.     

Phosphorylation of Photosystem II Controls Functional Macroscopic Folding of Photosynthetic Membranes in Arabidopsis

Photosynthetic thylakoid membranes in plants contain highly folded membrane layers enriched in photosystem II, which uses light energy to oxidize water and produce oxygen. The sunlight also causes quantitative phosphorylation of major photosystem II proteins. Analysis of the Arabidopsis thaliana stn7xstn8 double mutant deficient in thylakoid protein kinases STN7 and STN8 revealed light-independent phosphorylation of PsbH protein and greatly reduced N-terminal phosphorylation of D2 protein. The stn7xstn8 and stn8 mutants deficient in light-induced phosphorylation of photosystem II had increased thylakoid membrane folding compared with wild-type and stn7 plants. Significant enhancement in the size of stacked thylakoid membranes in stn7xstn8 and stn8 accelerated gravity-driven sedimentation of isolated thylakoids and was observed directly in plant leaves by transmission electron microscopy. Increased membrane folding, caused by the loss of light-induced protein phosphorylation, obstructed lateral migration of the photosystem II reaction center protein D1 and of processing protease FtsH between the stacked and unstacked membrane domains, suppressing turnover of damaged D1 in the leaves exposed to high light. These findings show that the high level of photosystem II phosphorylation in plants is required for adjustment of macroscopic folding of large photosynthetic membranes modulating lateral mobility of membrane proteins and sustained photosynthetic activity.The use of captured sunlight energy to split water and drive oxygenic photosynthesis by photosystem II (PSII) (Barber, 2006) inevitably generates reactive oxygen species and causes oxidative damage to the PSII protein pigment complex. The light-induced damage to PSII, in particular to the D1 reaction center protein, requires PSII repair to sustain its photosynthetic function (Takahashi and Murata, 2008). Impairment and degradation of D1 increase with rising light intensities, and this protein has the fastest turnover rate among the photosynthetic proteins of plants, algae, and cyanobacteria (Yokthongwattana and Melis, 2006). However, in plants, the PSII is segregated in highly stacked membrane layers of very large thylakoid membranes (Andersson and Anderson, 1980; Kirchhoff et al., 2008), which are densely folded to fit inside chloroplasts (Mullineaux, 2005; Shimoni et al., 2005). As a consequence, the PSII repair cycle in plants is slower than in cyanobacteria (Yokthongwattana and Melis, 2006), and it includes migration of the PSII complex from the stacked membrane domains (grana) to the unstacked membranes (stroma lamellae), where proteolysis and insertion of a newly synthesized D1 protein occurs (Baena-Gonzalez and Aro, 2002; Yokthongwattana and Melis, 2006). High light also causes quantitative phosphorylation of the membrane surface–exposed regions of D1, D2, CP43, and PsbH proteins of PSII in plants (Rintamäki et al., 1997; Vener et al., 2001), but the function of this phosphorylation is largely unknown and reports on its importance for the D1 protein turnover are conflicting (Bonardi et al., 2005; Tikkanen et al., 2008).Phosphorylation of the PSII proteins in Arabidopsis thaliana depends mostly on the light-activated protein kinase STN8 (Vainonen et al., 2005), while the STN7 kinase is essential for phosphorylation of the light-harvesting proteins of PSII (Bellafiore et al., 2005; Bonardi et al., 2005; Tikkanen et al., 2006). An earlier study on Arabidopsis mutants lacking both STN7 and STN8 (stn7xstn8), as well as only STN8, concluded that protein phosphorylation was not essential for PSII repair (Bonardi et al., 2005), while more recent work revealed a dramatic retardation in D1 degradation under high light in the stn8 and stn7xstn8 mutants (Tikkanen et al., 2008). Moreover, it was shown that the lack of PSII phosphorylation resulted in accumulation of photodamaged PSII complexes and in general oxidative damage of photosynthetic proteins in the thylakoid membranes under high light (Tikkanen et al., 2008). The other study revealed that the stn7xstn8 double mutant grown under natural field conditions produced 41% less seeds than wild-type plants (Frenkel et al., 2007), which also indicated physiological importance of thylakoid protein phosphorylation in maintenance of plant fitness.To uncover the function of light-dependent protein phosphorylation in plant photosynthetic membranes, we performed a detailed analysis of the Arabidopsis mutants deficient in the protein kinases STN7 and STN8. The earlier published results on protein phosphorylation analyses in the stn7xstn8 mutant of Arabidopsis were restricted to antiphosphothreonine antibody-based immunodetection and did not reveal any phosphorylation of PSII core proteins (Bonardi et al., 2005; Tikkanen et al., 2008). Using a mass spectrometry (MS) approach and immunoblot analyses with two complementary antiphosphothreonine antibodies, we find remaining light-independent phosphorylation of PsbH and D2 proteins of PSII in stn7xstn8. We demonstrate that degradation and aggregation patterns of the D1 protein in stn7xstn8 differ from those in wild-type, stn7, and stn8 plants. We also observe a reproducible delay in the degradation of D1 in high light–treated leaves of stn7xstn8 and stn8 compared with the wild-type and stn7 plants. Finally, we show that phosphorylation of PSII proteins modulates macroscopic rearrangements of the entire membrane network of plant thylakoids, which facilitates lateral mobility of membrane proteins, required for repair and sustained activity of PSII.     

Short-term down-regulation of zeaxanthin epoxidation in Arabidopsis thaliana in response to photo-oxidative stress conditions

The epoxidation of zeaxanthin (Zx) to violaxanthin after exposure to different light stress conditions has been studied in Arabidopsis (Arabidopsis thaliana). Formation of Zx was induced by illumination of intact leaves for up to 8 h at different light intensities and temperatures. The kinetics of epoxidation was found to be gradually retarded with increasing light stress during pre-illumination, indicating a gradual down-regulation of the Zx epoxidase activity. Retardation of the epoxidation rates by a factor of up to 10 was inducible either by increasing the light intensity or by extending the illumination time or by decreasing the temperature during pre-illumination. The retardation of the epoxidation kinetics was correlated with a decrease of the PSII quantum efficiency after the pre-illumination treatment. Experiments with the stn7/stn8 mutant of Arabidopsis indicated that the thylakoid protein kinases STN7 and STN8, which are required for the phosphorylation of PSII proteins, are not involved in the short-term down-regulation of Zx epoxidation. However, the retardation of Zx epoxidation was maintained in thylakoids isolated from pre-illuminated leaves, indicating that a direct modification of the Zx epoxidase is most likely involved in the light-induced down-regulation.     

The STN8 kinase‐PBCP phosphatase system is responsible for high‐light‐induced reversible phosphorylation of the PSII inner antenna subunit CP29 in rice

Reversible phosphorylation of thylakoid light‐harvesting proteins is a mechanism to compensate for unbalanced excitation of photosystem I (PSI) versus photosystem II (PSII) under limiting light. In monocots, an additional phosphorylation event on the PSII antenna CP29 occurs upon exposure to excess light, enhancing resistance to light stress. Different from the case of the major LHCII antenna complex, the STN7 kinase and its related PPH1 phosphatase were proven not to be involved in CP29 phosphorylation, indicating that a different set of enzymes act in the high‐light (HL) response. Here, we analyze a rice stn8 mutant in which both PSII core proteins and CP29 phosphorylation are suppressed in HL, implying that STN8 is the kinase catalyzing this reaction. In order to identify the phosphatase involved, we produced a recombinant enzyme encoded by the rice ortholog of AtPBCP, antagonist of AtSTN8, which catalyzes the dephosphorylation of PSII core proteins. The recombinant protein was active in dephosphorylating P‐CP29. Based on these data, we propose that the activities of the OsSTN8 kinase and the antagonistic OsPBCP phosphatase, in addition to being involved in the repair of photo‐damaged PSII, are also responsible for the HL‐dependent reversible phosphorylation of the inner antenna CP29.     

Cooperative ethylene and jasmonic acid signaling regulates selenite resistance in Arabidopsis

Selenium (Se) is an essential element for many organisms, but excess Se is toxic. To better understand plant Se toxicity and resistance mechanisms, we compared the physiological and molecular responses of two Arabidopsis (Arabidopsis thaliana) accessions, Columbia (Col)-0 and Wassilewskija (Ws)-2, to selenite treatment. Measurement of root length Se tolerance index demonstrated a clear difference between selenite-resistant Col-0 and selenite-sensitive Ws-2. Macroarray analysis showed more pronounced selenite-induced increases in mRNA levels of ethylene- or jasmonic acid (JA)-biosynthesis and -inducible genes in Col-0 than in Ws-2. Indeed, Col-0 exhibited higher levels of ethylene and JA. The selenite-sensitive phenotype of Ws-2 was attenuated by treatment with ethylene precursor or methyl jasmonate (MeJA). Conversely, the selenite resistance of Col-0 was reduced in mutants impaired in ethylene or JA biosynthesis or signaling. Genes encoding sulfur (S) transporters and S assimilation enzymes were up-regulated by selenite in Col-0 but not Ws-2. Accordingly, Col-0 contained higher levels of total S and Se and of nonprotein thiols than Ws-2. Glutathione redox status was reduced by selenite in Ws-2 but not in Col-0. Furthermore, the generation of reactive oxygen species by selenite was higher in Col-0 than in Ws-2. Together, these results indicate that JA and ethylene play important roles in Se resistance in Arabidopsis. Reactive oxygen species may also have a signaling role, and the resistance mechanism appears to involve enhanced S uptake and reduction.     

Quantitative trait loci involved in regulating seed oil composition in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and their evolutionary implications

Fatty acid composition is an important determinant of seed oil quality. Overall, 72 QTL for 12 fatty acid traits that control seed oil composition were identified in four recombinant inbred line (RIL) populations (Ler-0 × Sha, Ler-0 × Col-4, Ler-2 × Cvi, Ler-0 × No-0) of Arabidopsis thaliana. The identified QTL explained 3.2–79.8% of the phenotypic variance; 33 of the 59 QTL identified in the Ler-0 × Sha and the Ler-0 × Col RIL populations co-located with several a priori candidate genes for seed oil composition. QTL for fatty acids 18:1, 18:2, 22:1, and fatty acids synthesized in plastids was identified in both Ler-0 × Sha and Ler-0 × Col-4 RIL populations, and QTL for 16:0 was identified in the Ler-0 × Sha and Ler-0 × No-0 RIL populations providing strong support for the importance of these QTL in determining seed oil composition. We identified melting point QTL in three RIL populations, and fatty acid QTL collocated with two of them, suggesting that the loci could be under selection for altering the melting point of seed oils to enhance adaptation and could be useful for breeding purposes. Nuclear-cytoplasmic interactions and epistasis were rare. Analysis of the genetic correlations between these loci and other fatty acids indicated that these correlations would tend to strongly enhance selection for desirable fatty acids.     

Core protein phosphorylation facilitates the repair of photodamaged photosystem II at high light

Phosphorylation of photosystem II (PSII) reaction center protein D1 has been hypothesised to function as a signal for the migration of photodamaged PSII core complex from grana membranes to stroma lamellae for concerted degradation and replacement of the photodamaged D1 protein. Here, by using the mutants with impaired capacity (stn8) or complete lack (stn7 stn8) in phosphorylation of PSII core proteins, the role of phosphorylation in PSII photodamage and repair was investigated. We show that the lack of PSII core protein phosphorylation disturbs the disassembly of PSII supercomplexes at high light, which is a prerequisite for efficient migration of damaged PSII complexes from grana to stroma lamellae for repair. This results in accumulation of photodamaged PSII complexes, which in turn results, upon prolonged exposure to high light (HL), in general oxidative damage of photosynthetic proteins in the thylakoid membrane.     

Thylakoid protein phosphorylation in dynamic regulation of photosystem II in higher plants

In higher plants, the photosystem (PS) II core and its several light harvesting antenna (LHCII) proteins undergo reversible phosphorylation cycles according to the light intensity. High light intensity induces strong phosphorylation of the PSII core proteins and suppresses the phosphorylation level of the LHCII proteins. Decrease in light intensity, in turn, suppresses the phosphorylation of PSII core, but strongly induces the phosphorylation of LHCII. Reversible and differential phosphorylation of the PSII-LHCII proteins is dependent on the interplay between the STN7 and STN8 kinases, and the respective phosphatases. The STN7 kinase phosphorylates the LHCII proteins and to a lesser extent also the PSII core proteins D1, D2 and CP43. The STN8 kinase, on the contrary, is rather specific for the PSII core proteins. Mechanistically, the PSII-LHCII protein phosphorylation is required for optimal mobility of the PSII-LHCII protein complexes along the thylakoid membrane. Physiologically, the phosphorylation of LHCII is a prerequisite for sufficient excitation of PSI, enabling the excitation and redox balance between PSII and PSI under low irradiance, when excitation energy transfer from the LHCII antenna to the two photosystems is efficient and thermal dissipation of excitation energy (NPQ) is minimised. The importance of PSII core protein phosphorylation is manifested under highlight when the photodamage of PSII is rapid and phosphorylation is required to facilitate the migration of damaged PSII from grana stacks to stroma lamellae for repair. The importance of thylakoid protein phosphorylation is highlighted under fluctuating intensity of light where the STN7 kinase dependent balancing of electron transfer is a prerequisite for optimal growth and development of the plant. This article is part of a Special Issue entitled: Photosystem II.     

Thylakoid Protein Phosphorylation in Higher Plant Chloroplasts Optimizes Electron Transfer under Fluctuating Light

Several proteins of photosystem II (PSII) and its light-harvesting antenna (LHCII) are reversibly phosphorylated according to light quantity and quality. Nevertheless, the interdependence of protein phosphorylation, nonphotochemical quenching, and efficiency of electron transfer in the thylakoid membrane has remained elusive. These questions were addressed by investigating in parallel the wild type and the stn7, stn8, and stn7 stn8 kinase mutants of Arabidopsis (Arabidopsis thaliana), using the stn7 npq4, npq4, npq1, and pgr5 mutants as controls. Phosphorylation of PSII-LHCII proteins is strongly and dynamically regulated according to white light intensity. Yet, the changes in phosphorylation do not notably modify the relative excitation energy distribution between PSII and PSI, as typically occurs when phosphorylation is induced by “state 2” light that selectively excites PSII and induces the phosphorylation of both the PSII core and LHCII proteins. On the contrary, under low-light conditions, when excitation energy transfer from LHCII to reaction centers is efficient, the STN7-dependent LHCII protein phosphorylation guarantees a balanced distribution of excitation energy to both photosystems. The importance of this regulation diminishes at high light upon induction of thermal dissipation of excitation energy. Lack of the STN7 kinase, and thus the capacity for equal distribution of excitation energy to PSII and PSI, causes relative overexcitation of PSII under low light but not under high light, leading to disturbed maintenance of fluent electron flow under fluctuating light intensities. The physiological relevance of the STN7-dependent regulation is evidenced by severely stunted phenotypes of the stn7 and stn7 stn8 mutants under strongly fluctuating light conditions.Several proteins of PSII and its light-harvesting antenna (LHCII) are reversibly phosphorylated by the STN7 and STN8 kinase-dependent pathways according to the intensity and quality of light (Bellafiore et al., 2005; Bonardi et al., 2005). The best-known phosphorylation-dependent phenomenon in the thylakoid membrane is the state transition: a regulatory mechanism that modulates the light-harvesting capacity between PSII and PSI. According to the traditional view, “state 1” prevails when plants are exposed to far-red light (state 1 light), which selectively excites PSI. Alternatively, thylakoids are in “state 2” when plants are exposed to blue or red light (state 2 light), favoring PSII excitation. In state 1, the yield of fluorescence from PSII is higher in comparison with state 2 (for review, see Allen and Forsberg, 2001). State transitions are dependent on the phosphorylation of LHCII proteins (Bellafiore et al., 2005) and their association with PSI proteins, particularly PSI-H (Lunde et al., 2000). Under state 2 light, both the PSII core and LHCII proteins are strongly phosphorylated, whereas the state 1 light induces dephosphorylation of both the PSII core and LHCII phosphoproteins (Piippo et al., 2006; Tikkanen et al., 2006). In nature, however, such extreme changes in light quality rarely occur. The intensity of light, on the contrary, fluctuates frequently in all natural habitats occupied by photosynthetic organisms, thus constantly modulating the extent of thylakoid protein phosphorylation in a highly dynamic manner (Tikkanen et al., 2008a).The regulation of PSII-LHCII protein phosphorylation by the quantity of light is much more complex than the regulatory circuits induced by the state 1 and state 2 lights. Whereas changes in light quality induce a concurrent increase or decrease in the phosphorylation levels of both the PSII core (D1, D2, and CP43) and LHCII (Lhcb1 and Lhcb2) proteins, the changes in white light intensity may influence the kinetics of PSII core and LHCII protein phosphorylation in higher plant chloroplasts even in opposite directions (Tikkanen et al., 2008a). Indeed, it is well documented that low light (LL; i.e. lower than that generally experienced during growth) induces strong phosphorylation of LHCII but relatively weak phosphorylation of the PSII core proteins. Exposure of plants to high light (HL) intensities, on the contrary, promotes the phosphorylation of PSII core proteins but inhibits the activity of the LHCII kinase, leading to dephosphorylation of LHCII proteins (Rintamäki et al., 2000; Hou et al., 2003).Thylakoid protein phosphorylation induces dynamic migrations of PSII-LHCII proteins along the thylakoid membrane (Bassi et al., 1988; Iwai et al., 2008) and modulation of thylakoid ultrastructure (Chuartzman et al., 2008). According to the traditional state transition theory, the phosphorylation of LHCII proteins decreases the antenna size of PSII and increases that of PSI, which is reflected as a quenched fluorescence emission from PSII. Alternatively, subsequent dephosphorylation of LHCII increases the antenna size of PSII and decreases that of PSI, which in turn is seen as increased PSII fluorescence (Bennett et al., 1980; Allen et al., 1981; Allen and Forsberg, 2001). This view was recently challenged based on studies with thylakoid membrane fractions, revealing that modulations in the relative distribution of excitation energy between PSII and PSI by LHCII phosphorylation specifically occur in the areas of grana margins, where both PSII and PSI function under the same antenna system, and the energy distribution between the photosystems is regulated via a more subtle mechanism than just the robust migration of phosphorylated LHCII (Tikkanen et al., 2008b). It has also been reported that most of the PSI reaction centers are located in the grana margins in a close vicinity to PSII-LHCII-rich grana thylakoids (Kaftan et al., 2002), providing a perfect framework for the regulation of excitation energy distribution from LHCII to both PSII and PSI.When considering the natural light conditions, the HL intensities are the only known light conditions that in higher plant chloroplasts specifically dephosphorylate only the LHCII proteins but not the PSII core proteins. However, such light conditions do not lead to enhanced function of PSII. Instead, the HL conditions strongly down-regulate the function of PSII via nonphotochemical quenching of excitation energy (NPQ) and PSII photoinhibition (for review, see Niyogi, 1999). On the other hand, after dark acclimation of leaves and relaxation of NPQ, PSII functions much more efficiently when plants/leaves are transferred to LL despite strong phosphorylation of LHCII, as compared with the low phosphorylation state of LHCII upon transfer to HL conditions.The delicate regulation of thylakoid protein phosphorylation in higher plant chloroplasts according to prevailing light intensity is difficult to integrate with the traditional theory of state transitions (i.e. the regulation of the absorption cross-section of PSII and PSI by reversible phosphorylation of LHCII). Moreover, besides LHCII proteins, reversible phosphorylation of the PSII core proteins may also play a role in dynamic light acclimation of plants. Recently, we demonstrated that the PSII core protein phosphorylation is a prerequisite for controlled turnover of the PSII reaction center protein D1 upon photodamage (Tikkanen et al., 2008a). This, however, does not exclude the possibility that the strict regulation of PSII core protein phosphorylation is also connected to the regulation of light harvesting and photosynthetic electron transfer. Moreover, the interactions between PSII and LHCII protein phosphorylation, nonphotochemical quenching, and cyclic electron flow around PSI in the regulation of photosynthetic electron transfer reactions remain poorly understood. To gain a deeper insight into such regulatory networks, we explored the effect of strongly fluctuating white light on chlorophyll (chl) fluorescence in Arabidopsis (Arabidopsis thaliana) mutants differentially deficient in PSII-LHCII protein phosphorylation and/or the regulatory systems of NPQ.     

Natural variation of H3K27me3 distribution between two Arabidopsis accessions and its association with flanking transposable elements

Background

Histone H3 lysine 27 tri-methylation and lysine 9 di-methylation are independent repressive chromatin modifications in Arabidopsis thaliana. H3K27me3 is established and maintained by Polycomb repressive complexes whereas H3K9me2 is catalyzed by SUVH histone methyltransferases. Both modifications can spread to flanking regions after initialization and were shown to be mutually exclusive in Arabidopsis.

Results

We analyzed the extent of natural variation of H3K27me3 in the two accessions Landsberg erecta (Ler) and Columbia (Col) and their F1 hybrids. The majority of H3K27me3 target genes in Col were unchanged in Ler and F1 hybrids. A small number of Ler-specific targets were detected and confirmed. Consistent with a cis-regulatory mechanism for establishing H3K27me3, differential targets showed allele-specific H3K27me3 in hybrids. Five Ler-specific targets showed the active mark H3K4me3 in Col and for this group, differential H3K27me3 enrichment accorded to expression variation. On the other hand, the majority of Ler-specific targets were not expressed in Col, Ler or 17 other accessions. Instead of H3K27me3, the antagonistic mark H3K9me2 and other heterochromatic features were observed at these loci in Col. These loci were frequently flanked by transposable elements, which were often missing in the Ler genome assembly.

Conclusion

There is little variation in H3K27me3 occupancy within the species, although H3K27me3 targets were previously shown as overrepresented among differentially expressed genes. The existing variation in H3K27me3 seems mostly explained by flanking polymorphic transposable elements. These could nucleate heterochromatin, which then spreads into neighboring H3K27me3 genes, thus converting them to H3K9me2 targets.     

Effector-Triggered Immune Response in Arabidopsis thaliana Is a Quantitative Trait

We identified loci responsible for natural variation in Arabidopsis thaliana (Arabidopsis) responses to a bacterial pathogen virulence factor, HopAM1. HopAM1 is a type III effector protein secreted by the virulent Pseudomonas syringae strain Pto DC3000. Delivery of HopAM1 from disarmed Pseudomonas strains leads to local cell death, meristem chlorosis, or both, with varying intensities in different Arabidopsis accessions. These phenotypes are not associated with differences in bacterial growth restriction. We treated the two phenotypes as quantitative traits to identify host loci controlling responses to HopAM1. Genome-wide association (GWA) of 64 Arabidopsis accessions identified independent variants highly correlated with response to each phenotype. Quantitative trait locus (QTL) mapping in a recombinant inbred population between Bur-0 and Col-0 accessions revealed genetic linkage to regions distinct from the top GWA hits. Two major QTL associated with HopAM1-induced cell death were also associated with HopAM1-induced chlorosis. HopAM1-induced changes in Arabidopsis gene expression showed that rapid HopAM1-dependent cell death in Bur-0 is correlated with effector-triggered immune responses. Studies of the effect of mutations in known plant immune system genes showed, surprisingly, that both cell death and chlorosis phenotypes are enhanced by loss of EDS1, a regulatory hub in the plant immune-signaling network. Our results reveal complex genetic architecture for response to this particular type III virulence effector, in contrast to the typical monogenic control of cell death and disease resistance triggered by most type III effectors.     

Protein kinases and phosphatases involved in the acclimation of the photosynthetic apparatus to a changing light environment

Photosynthetic organisms are subjected to frequent changes in light quality and quantity and need to respond accordingly. These acclimatory processes are mediated to a large extent through thylakoid protein phosphorylation. Recently, two major thylakoid protein kinases have been identified and characterized. The Stt7/STN7 kinase is mainly involved in the phosphorylation of the LHCII antenna proteins and is required for state transitions. It is firmly associated with the cytochrome b₆f complex, and its activity is regulated by the redox state of the plastoquinone pool. The other kinase, Stl1/STN8, is responsible for the phosphorylation of the PSII core proteins. Using a reverse genetics approach, we have recently identified the chloroplast PPH1/TAP38 and PBPC protein phosphatases, which counteract the activity of STN7 and STN8 kinases, respectively. They belong to the PP2C-type phosphatase family and are conserved in land plants and algae. The picture that emerges from these studies is that of a complex regulatory network of chloroplast protein kinases and phosphatases that is involved in light acclimation, in maintenance of the plastoquinone redox poise under fluctuating light and in the adjustment to metabolic needs.     

HMA3 is a key factor for differences in Cd- and Zn-related phenotype between <Emphasis Type="Italic">Arabidopsis</Emphasis> Ws and Col-0 ecotypes

Columbia-0 (Col-0) appears to be less tolerant to cadmium (Cd) than the Wassilewskija (Ws) ecotype that exhibits the full Heavy Metal ATPase3 (HMA3) coding sequence. However, the physiological and molecular mechanisms of HMA3 encoded by point mutation genes in Col-0 remain unknown. In this study, we investigate whether the different metal-related phenotype observed in Col-0 (with HMA3 mutation) when compared to that of Ws (functional HMA3) is a result only of the HMA3 mutation. This investigation was carried out with a further study using plant materials as follows: Ws and Col-0 ecotypes, two HMA3 (Ws) overexpressing lines in Col-0, hma3 knock-out line in Ws. The results indicate that the Col-0 and hma3 mutant in Ws were less tolerant to Cd and Zn because HMA3 has lost the function of sequestration of Cd and Zn into the root vacuoles, thereby readily translocating Cd and Zn to the aerial parts. In addition, the root-to-shoot metal translocation rates of the Ws- and HMA3-overexpressing lines were lower than those of the Col-0 and hma3 mutants. These results indicate that HMA3 is important for the Cd and Zn detoxification in Arabidopsis.     

Phos-tag-based approach to study protein phosphorylation in the thylakoid membrane

Protein phosphorylation is a fundamental post-translational modification in all organisms. In photoautotrophic organisms, protein phosphorylation is essential for the fine-tuning of photosynthesis. The reversible phosphorylation of the photosystem II (PSII) core and the light-harvesting complex of PSII (LHCII) contribute to the regulation of photosynthetic activities. Besides the phosphorylation of these major proteins, recent phosphoproteomic analyses have revealed that several proteins are phosphorylated in the thylakoid membrane. In this study, we utilized the Phos-tag technology for a comprehensive assessment of protein phosphorylation in the thylakoid membrane of Arabidopsis. Phos-tag SDS-PAGE enables the mobility shift of phosphorylated proteins compared with their non-phosphorylated isoform, thus differentiating phosphorylated proteins from their non-phosphorylated isoforms. We extrapolated this technique to two-dimensional (2D) SDS-PAGE for detecting protein phosphorylation in the thylakoid membrane. Thylakoid proteins were separated in the first dimension by conventional SDS-PAGE and in the second dimension by Phos-tag SDS-PAGE. In addition to the isolation of major phosphorylated photosynthesis-related proteins, 2D Phos-tag SDS-PAGE enabled the detection of several minor phosphorylated proteins in the thylakoid membrane. The analysis of the thylakoid kinase mutants demonstrated that light-dependent protein phosphorylation was mainly restricted to the phosphorylation of the PSII core and LHCII proteins. Furthermore, we assessed the phosphorylation states of the structural domains of the thylakoid membrane, grana core, grana margin, and stroma lamella. Overall, these results demonstrated that Phos-tag SDS-PAGE is a useful biochemical tool for studying in vivo protein phosphorylation in the thylakoid membrane protein.