Tsuneaki Takami

Efficient Operation of NAD(P)H Dehydrogenase Requires Supercomplex Formation with Photosystem I via Minor LHCI in Arabidopsis

In higher plants, the chloroplast NAD(P)H dehydrogenase (NDH) complex mediates photosystem I (PSI) cyclic and chlororespiratory electron transport. We reported previously that NDH interacts with the PSI complex to form a supercomplex (NDH-PSI). In this study, NDH18 and FKBP16-2 (FK506 Binding Protein 16-2), detected in the NDH-PSI supercomplex by mass spectrometry, were shown to be NDH subunits by the analysis of their knockdown lines. On the basis of extensive mutant characterization, we propose a structural model for chloroplast NDH, whereby NDH is divided into four subcomplexes. The subcomplex A and membrane subcomplex are conserved in cyanobacteria, but the subcomplex B and lumen subcomplex are specific to chloroplasts. Two minor light-harvesting complex I proteins, Lhca5 and Lhca6, were required for the full-size NDH-PSI supercomplex formation. Similar to crr pgr5 double mutants that completely lack cyclic electron flow activity around PSI, the lhca6 pgr5 double mutant exhibited a severe defect in growth. Consistent with the impaired NDH activity, photosynthesis was also severely affected in mature leaves of lhca6 pgr5. We conclude that chloroplast NDH became equipped with the novel subcomplexes and became associated with PSI during the evolution of land plants, and this process may have facilitated the efficient operation of NDH.

Role of the Low-Molecular-Weight Subunits PetL, PetG, and PetN in Assembly, Stability, and Dimerization of the Cytochrome b6f Complex in Tobacco

The cytochrome b6f (Cyt b6f) complex in flowering plants contains nine conserved subunits, of which three, PetG, PetL, and PetN, are bitopic plastid-encoded low-molecular-weight proteins of largely unknown function. Homoplastomic knockout lines of the three genes have been generated in tobacco (Nicotiana tabacum ‘Petit Havana’) to analyze and compare their roles in assembly and stability of the complex. Deletion of petG or petN caused a bleached phenotype and loss of photosynthetic electron transport and photoautotrophy. Levels of all subunits that constitute the Cyt b6f complex were faintly detectable, indicating that both proteins are essential for the stability of the membrane complex. In contrast, ΔpetL plants accumulate about 50% of other Cyt b6f subunits, appear green, and grow photoautotrophically. However, ΔpetL plants show increased light sensitivity as compared to wild type. Assembly studies revealed that PetL is primarily required for proper conformation of the Rieske protein, leading to stability and formation of dimeric Cyt b6f complexes. Unlike wild type, phosphorylation levels of the outer antenna of photosystem II (PSII) are significantly decreased under state II conditions, although the plastoquinone pool is largely reduced in ΔpetL, as revealed by measurements of PSI and PSII redox states. This confirms the sensory role of the Cyt b6f complex in activation of the corresponding kinase. The reduced light-harvesting complex II phosphorylation did not affect state transition and association of light-harvesting complex II to PSI under state II conditions. Ferredoxin-dependent plastoquinone reduction, which functions in cyclic electron transport around PSI in vivo, was not impaired in ΔpetL.

Guard Cell Chloroplasts Are Essential for Blue Light-Dependent Stomatal Opening in Arabidopsis

Blue light (BL) induces stomatal opening through the activation of H+-ATPases with subsequent ion accumulation in guard cells. In most plant species, red light (RL) enhances BL-dependent stomatal opening. This RL effect is attributable to the chloroplasts of guard cell, the only cells in the epidermis possessing this organelle. To clarify the role of chloroplasts in stomatal regulation, we investigated the effects of RL on BL-dependent stomatal opening in isolated epidermis, guard cell protoplasts, and intact leaves of Arabidopsis thaliana. In isolated epidermal tissues and intact leaves, weak BL superimposed on RL enhanced stomatal opening while BL alone was less effective. In guard cell protoplasts, RL enhanced BL-dependent H+-pumping and DCMU, a photosynthetic electron transport inhibitor, eliminated this effect. RL enhanced phosphorylation levels of the H+-ATPase in response to BL, but this RL effect was not suppressed by DCMU. Furthermore, DCMU inhibited both RL-induced and BL-dependent stomatal opening in intact leaves. The photosynthetic rate in leaves correlated positively with BL-dependent stomatal opening in the presence of DCMU. We conclude that guard cell chloroplasts provide ATP and/or reducing equivalents that fuel BL-dependent stomatal opening, and that they indirectly monitor photosynthetic CO2 fixation in mesophyll chloroplasts by absorbing PAR in the epidermis.

De Novo Biosynthesis of Fatty Acids Plays Critical Roles in the Response of the Photosynthetic Machinery to Low Temperature in Arabidopsis

The Arabidopsis thaliana kas3 mutant was isolated based on the hypersensitivity of PSII to low temperature using a Chl fluorescence imaging technique. Chl content was lower in kas3 seedlings cultured at 23 degrees C than in the wild type, but PSII activity was only mildly affected. However, after the chilling treatment at 4 degrees C for 7 d, PSII activity was severely impaired in kas3. PSII was more sensitive to light at 4 degrees C in the presence of lincomycin, suggesting that the kas3 mutation accelerates at least the PSII photodamage. The kas3 mutation causes an amino acid alteration in 3-ketoacyl-ACP synthase III (KasIII), leading to the partial loss of the de novo synthesis pathway for fatty acids in plastids. Consequently, the total fatty acid level was reduced to 75% of the wild-type level in kas3 at 23 degrees C and was further reduced to 60% at 4 degrees C. The composition of fatty acids was also slightly affected in kas3 at both 4 and 23 degrees C. Consistent with the results of the electron transport analysis, the chilling treatment also destabilized PsaA and cytochrome (Cyt) f and D1 in kas3. An analysis of double mutants with pgr1 conditionally defective in Cyt b(6)f activity and with var2 defective in FtsH protease suggested that the kas3 mutation has pleiotropic effects on chloroplast function, probably impacting both the Cyt b(6)f activity and translation in chloroplasts at 23 degrees C. The full activity of KasIII is required for the biogenesis of the intact electron transport machinery in thylakoid membranes and is especially important for the process of responding to low temperature.

Subfunctionalization of Sigma Factors during the Evolution of Land Plants Based on Mutant Analysis of Liverwort (Marchantia polymorpha L.) MpSIG1

Sigma factor is a subunit of plastid-encoded RNA polymerase that regulates the transcription of plastid-encoded genes by recognizing a set of promoters. Sigma factors have increased in copy number and have diversified during the evolution of land plants, but details of this process remain unknown. Liverworts represent the basal group of embryophytes and are expected to retain the ancestral features of land plants. In liverwort (Marchantia polymorpha L.), we isolated and characterized a T-DNA-tagged mutant (Mpsig1) of sigma factor 1 (MpSIG1). The mutant did not show any visible phenotypes, implying that MpSIG1 function is redundant with that of other sigma factors. However, quantitative reverse-transcription polymerase chain reaction and RNA gel blot analysis revealed that genes related to photosynthesis were downregulated, resulting in the minor reduction of some protein complexes. The transcript levels of genes clustered in the petL, psaA, psbB, psbK, and psbE operons of liverwort were lower than those in the wild type, a result similar to that in the SIG1 defective mutant in rice (Oryza sativa). Overexpression analysis revealed primitive functional divergence between the SIG1 and SIG2 proteins in bryophytes, whereas these proteins still retain functional redundancy. We also discovered that the predominant sigma factor for ndhF mRNA expression has been diversified in liverwort, Arabidopsis (Arabidopsis thaliana), and rice. Our study shows the ancestral function of SIG1 and the process of functional partitioning (subfunctionalization) of sigma factors during the evolution of land plants.

De Novo Biosynthesis of Fatty Acids is Important for Maintenance of Photochemical Activity under Low Temperature Environments in Arabidopsis

The Arabidopsis thaliana kas3 mutant was isolated based on its hypersensitivity of photosystem (PS) II to low temperature using a chlorophyll (Chl) fluorescence imaging system. Chl content was lower in kas3 seedlings cultured at 23 °C than in the wild type, but maximum PSII activity was only mildly affected. We also clarified that the activity and levels of photosynthetic electron transport machinery were reduced after chilling treatment. The kas3 mutation causes an amino acid alteration in 3-ketoacyl-ACP synthase III (KasIII) which catalyzes the first decarboxy condensation step in de novo fatty biosynthesis in plastids. The defect in KasIII led to the partial loss of the de novo synthesis pathway for fatty acids in plastids. Consequently, the total fatty acid level was reduced to 75% of the wild-type level in kas3 at 23 °C and was further reduced to 60% at 4 °C. The full activity of KasIII is required for the biogenesis of intact photosynthetic machinery in thylakoid membranes and is especially important for the process responding to low temperature.