Youn-Il Park

The Senescence-Induced Staygreen Protein Regulates Chlorophyll Degradation

Loss of green color in leaves results from chlorophyll (Chl) degradation in chloroplasts, but little is known about how Chl catabolism is regulated throughout leaf development. Using the staygreen (sgr) mutant in rice (Oryza sativa), which maintains greenness during leaf senescence, we identified Sgr, a senescence-associated gene encoding a novel chloroplast protein. Transgenic rice overexpressing Sgr produces yellowish-brown leaves, and Arabidopsis thaliana pheophorbide a oxygenase–impaired mutants exhibiting a stay-green phenotype during dark-induced senescence have reduced expression of Sgr homologs, indicating that Sgr regulates Chl degradation at the transcriptional level. We show that the leaf stay-greenness of the sgr mutant is associated with a failure in the destabilization of the light-harvesting chlorophyll binding protein (LHCP) complexes of the thylakoid membranes, which is a prerequisite event for the degradation of Chls and LHCPs during senescence. Transient overexpression of Sgr in Nicotiana benthamiana and an in vivo pull-down assay show that Sgr interacts with LHCPII, indicating that the Sgr-LHCPII complexes are formed in the thylakoid membranes. Thus, we propose that in senescing leaves, Sgr regulates Chl degradation by inducing LHCPII disassembly through direct interaction, leading to the degradation of Chls and Chl-free LHCPII by catabolic enzymes and proteases, respectively.

A novel α-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2

A 29.5 kDa intracellular α‐type carbonic anhydrase, designated Cah3, from the unicellular green alga Chlamydomonas reinhardtii is the first of this type discovered inside a photosynthetic eukaryote cell. We describe the cloning of a cDNA which encodes the protein. Immunoblot studies with specific antibodies raised against Cah3 demonstrate that the polypeptide is associated exclusively with the thylakoid membrane. The putative transit peptide suggests that Cah3 is directed to the thylakoid lumen, which is confirmed further by the presence of mature sized Cah3 after thermolysin treatment of intact thylakoids. Complementation of the high inorganic carbon concentration‐requiring mutant, cia‐3, with a subcloned cosmid containing the cah3 gene yielded transformants that grew on atmospheric levels of CO2 (0.035%) and contained an active 29.5 kDa α‐type carbonic anhydrase. Although, cia‐3 has reduced internal carbonic anhydrase activity, unexpectedly the level of Cah3 was similar to that of the wild‐type, suggesting that the mutant accumulates an inactive Cah3 polypeptide. Genomic sequence analysis of the mutant revealed two amino acid changes in the transit peptide. Results from photosynthesis and chlorophyll a fluorescence parameter measurements show that the cia‐3 mutant is photosynthetically impaired. Our results indicate that the carbonic anhydrase, extrinsically located within the chloroplast thylakoid lumen, is essential for growth of C.reinhardtii at ambient levels of CO2, and that at these CO2 concentrations the enzyme is required for optimal photosystem II photochemistry.

UNIFYING MODEL FOR THE PHOTOINACTIVATION OF PHOTOSYSTEM II IN VIVO UNDER STEADY-STATE PHOTOSYNTHESIS

We present a unifying mechanism for photoinhibition based on current obsevations from in vivo studies rather than from in vitro studies with isolated thylakoids or PS II membranes. In vitro studies have limited relevance for in vivo photoinhibition because very high light is used with photon exposures rarely encountered in nature, and most of the multiple, interacting, protective strategies of PS II regulation in living cells are not functional. It is now established that the photoinactivation of Photosystem II in vivo is a probability and light-dosage event which depends on the photons absorbed and not the irradiance per se. As the reciprocity law is obeyed and target theory analysis strongly suggests that only one photon is required, we propose that a single dominant molecular mechanism occurs in vivo with one photon inactivating PS II under limiting, saturating or sustained high light. Two mechanisms have been proposed for photoinhibition under high light, acceptor-side and donor-side photoinhibition [see Aro et al. (1994) Biochim Biophys Acta 1143: 113–134], and another mechanism for very low light, the low-light syndrome [Keren et al. (1995) J Biol Chem 270: 806–814]. Based on the exciton-radical pair equilibrium model of exciton dynamics, we propose a unifying mechanism for the photoinactivation of PS II in vivo under steady-state photosynthesis that depends on the generation and maintenance of increased concentrations of the primary radical pair, P680+Pheo−, and the different ways charge recombination is regulated under varying environmental conditions [Anderson et al. (1997) Physiol Plant 100: 214–223]. We suggest that the primary cause of damage to D1 protein is P680+, rather than singlet O2 formed from triplet P680, or other reactive oxygen species.

Functional Conservation of a Root Hair Cell-Specific cis-Element in Angiosperms with Different Root Hair Distribution Patterns

Vascular plants develop distinctive root hair distribution patterns in the root epidermis, depending on the taxon. The three patterns, random (Type 1), asymmetrical cell division (Type 2), and positionally cued (Type 3), are controlled by different upstream fate-determining factors that mediate expression of root hair cell-specific genes for hair morphogenesis. Here, we address whether these root hair genes possess a common transcriptional regulatory module (cis-element) determining cell-type specificity despite differences in the final root hair pattern. We identified Arabidopsis thaliana expansinA7 (At EXPA7) orthologous (and paralogous) genes from diverse angiosperm species with different hair distribution patterns. The promoters of these genes contain conserved root hair–specific cis-elements (RHEs) that were functionally verified in the Type-3 Arabidopsis root. The promoter of At EXPA7 (Type-3 pattern) also showed hair cell–specific expression in the Type 2 rice (Oryza sativa) root. Root hair–specific genes other than EXPAs also carry functionally homologous RHEs in their promoters. The RHE core consensus was established by a multiple alignment of functionally characterized RHEs from different species and by high-resolution analysis of At EXPA7 RHE1. Our results suggest that this regulatory module of root hair–specific genes has been conserved across angiosperms despite the divergence of upstream fate-determining machinery.

Expression of the isiA gene is essential for the survival of the cyanobacterium Synechococcus sp. PCC 7942 by protecting photosystem II from excess light under iron limitation

Iron deficiency is known to suppress primary productivity in both marine and freshwater ecosystems. In response to iron deficiency, certain cyanobacteria induce a chlorophyll (Chl)–protein complex, CP43′, which is encoded by the isiA gene. The deduced amino‐acid sequence of CP43′ predicts some structural similarity to the CP43 polypeptide of photosystem II, but the function of CP43′ remains uncertain. In order to assess its physiological role, the isiA gene of a cyanobacterium, Synechococcus sp. PCC7942, was inactivated by insertion mutagenesis (giving isiA− cells). Compared with isiA− cells, under iron deprivation, wild‐type cells showed both lower rates of photosystem II‐mediated O2 evolution at limiting light irradiances and decreased yields of room temperature Chl fluorescence at various irradiances. These observations strongly suggest that the decreased photosystem II activity in wild‐type cells with CP43′ is attributable to increased non‐radiative dissipation of light energy. In agreement with this hypothesis, isiA− cells were more susceptible to photoinhibition of photosynthesis than wild‐type cells, resulting in much slower growth rates under iron limitation. Based on these results, we suggest that CP43′ functions as a non‐radiative dissipator of light energy, thus protecting photosystem II from excessive excitation under iron‐deficient conditions.

Role of the Rice Hexokinases OsHXK5 and OsHXK6 as Glucose Sensors

The Arabidopsis (Arabidopsis thaliana) hexokinase 1 (AtHXK1) is recognized as an important glucose (Glc) sensor. However, the function of hexokinases as Glc sensors has not been clearly demonstrated in other plant species, including rice (Oryza sativa). To investigate the functions of rice hexokinase isoforms, we characterized OsHXK5 and OsHXK6, which are evolutionarily related to AtHXK1. Transient expression analyses using GFP fusion constructs revealed that OsHXK5 and OsHXK6 are associated with mitochondria. Interestingly, the OsHXK5ΔmTP-GFP and OsHXK6ΔmTP-GFP fusion proteins, which lack N-terminal mitochondrial targeting peptides, were present mainly in the nucleus with a small amount of the proteins seen in the cytosol. In addition, the OsHXK5NLS-GFP and OsHXK6NLS-GFP fusion proteins harboring nuclear localization signals were targeted predominantly in the nucleus, suggesting that these OsHXKs retain a dual-targeting ability to mitochondria and nuclei. In transient expression assays using promoter∷luciferase fusion constructs, these two OsHXKs and their catalytically inactive alleles dramatically enhanced the Glc-dependent repression of the maize (Zea mays) Rubisco small subunit (RbcS) and rice α-amylase genes in mesophyll protoplasts of maize and rice. Notably, the expression of OsHXK5, OsHXK6, or their mutant alleles complemented the Arabidopsis glucose insensitive2-1 mutant, thereby resulting in wild-type characteristics in seedling development, Glc-dependent gene expression, and plant growth. Furthermore, transgenic rice plants overexpressing OsHXK5 or OsHXK6 exhibited hypersensitive plant growth retardation and enhanced repression of the photosynthetic gene RbcS in response to Glc treatment. These results provide evidence that rice OsHXK5 and OsHXK6 can function as Glc sensors.

Near-UV cyanobacteriochrome signaling system elicits negative phototaxis in the cyanobacterium Synechocystis sp. PCC 6803

Positive phototaxis systems have been well studied in bacteria; however, the photoreceptor(s) and their downstream signaling components that are responsible for negative phototaxis are poorly understood. Negative phototaxis sensory systems are important for cyanobacteria, oxygenic photosynthetic organisms that must contend with reactive oxygen species generated by an abundance of pigment photosensitizers. The unicellular cyanobacterium Synechocystis sp. PCC6803 exhibits type IV pilus-dependent negative phototaxis in response to unidirectional UV-A illumination. Using a reverse genetic approach, together with biochemical, molecular genetic, and RNA expression profiling analyses, we show that the cyanobacteriochrome locus (slr1212/uirS) of Synechocystis and two adjacent response regulator loci (slr1213/uirR and the PatA-type regulator slr1214/lsiR) encode a UV-A–activated signaling system that is required for negative phototaxis. We propose that UirS, which is membrane-associated via its ETR1 domain, functions as a UV-A photosensor directing expression of lsiR via release of bound UirR, which targets the lsiR promoter. Constitutive expression of LsiR induces negative phototaxis under conditions that normally promote positive phototaxis. Also induced by other stresses, LsiR thus integrates light inputs from multiple photosensors to determine the direction of movement.

CP43′, the isiA Gene Product, Functions as an Excitation Energy Dissipator in the Cyanobacterium Synechococcus sp. PCC 7942¶

Abstract Under conditions of iron deficiency certain cyanobacteria induce a chlorophyll (Chl)-binding protein, CP43′, which is encoded by the isiA gene. We have previously suggested that CP43′ functions as a nonradiative dissipator of light energy. To further substantiate its functional role an isiA overexpression construct was introduced into the genome of a cyanobacterium Synechococcus sp. PCC 7942 (giving isiAoe cells). The presence of functional CP43′ in isiAoe cells was confirmed by Western blot as well as by the presence of a characteristic blueshift of the red Chl a absorption peak and a notable increase in the 77 K fluorescence peak at 685 nm. Compared to wild-type cells isiAoe cells, with induced CP43′, had both smaller functional antenna size and decreased yields of room temperature Chl fluorescence at various light irradiances. These observations strongly suggest that isiAoe cells, with induced CP43′, have an increased capacity for dissipating light energy as heat. In agreement with this hypothesis isiAoe cells were also more resistant to photoinhibition of photosynthesis than wild-type cells. Based on these results we have further strengthened the hypothesis that CP43′ functions as a nonradiative dissipator of light energy, thus protecting photosystem II from excessive excitation under iron-deficient conditions.

HY5 regulates anthocyanin biosynthesis by inducing the transcriptional activation of the MYB75/PAP1 transcription factor in Arabidopsis

Several positive transcription factors regulate Arabidopsis anthocyanin biosynthesis. HY5, a component of light‐signaling pathways, and PAP1, an R2R3‐MYB transcription factor, share common regulatory targets on anthocyanin biosynthesis genes. The epistatic interactions between the two transcription factors are currently unknown. To address this problem, we analyzed crosses between hy5 and pap1 mutants (hy5pap1) or pap1D overexpressors (hy5pap1D), performed chromatin immunoprecipitation‐qPCR, and determined the PAP1 promoter region through deletion analysis. The results show that HY5 regulates PAP1 expression via direct binding to G‐ and ACE‐boxes in the promoter region, which suggests bifurcate regulation of anthocyanin biosynthesis by HY5 via transcriptional activation of PAP1.

Role of a novel photosystem II‐associated carbonic anhydrase in photosynthetic carbon assimilation in Chlamydomonas reinhardtii

Intracellular carbonic anhydrases (CA) in aquatic photosynthetic organisms are involved in the CO2‐concentrating mechanism (CCM), which helps to overcome CO2 limitation in the environment. In the green alga Chlamydomonas reinhardtii, this CCM is initiated and maintained by the pH gradient created across the chloroplast thylakoid membranes by photosystem (PS) II‐mediated electron transport. We show here that photosynthesis is stimulated by a novel, intracellular α‐CA bound to the chloroplast thylakoids. It is associated with PSII on the lumenal side of the thylakoid membranes. We demonstrate that PSII in association with this lumenal CA operates to provide an ample flux of CO2 for carboxylation.