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The C[O.sub.2]-concentrating mechanism (CCM) of Chlamydomonas reinhardtii and other microalgal species is essential for photosynthetic growth in most natural settings. A great deal has been learned regarding the CCM in cyanobacteria, including identification of inorganic carbon (Ci; C[O.sub.2] and HC[O.sup.-.sub.3]) transporters; however, specific knowledge of analogous transporters has remained elusive in eukaryotic microalgae such as C. reinhardtii. Here we investigated whether the limiting-C[O.sub.2]--inducible, putative ABC-type transporter HLA3 might function as a HC[O.sup.-.sub.3] transporter by evaluating the effect of pH on growth, photosynthetic Ci affinity, and [[sup.14]C]-Ci uptake in very low C[O.sub.2] conditions following RNA interference (RNAi) knockdown of HLA3 mRNA levels in wild-type and mutant cells. Although knockdown of HLA3 mRNA alone resulted in only modest but high-pH--dependent decreases in photosynthetic Ci affinity and Ci uptake, the combination of nearly complete knockdown of HLA3 mRNA with mutations in LCIB (which encodes limiting-Ci--inducible plastid-localized protein required for normal Ci uptake or accumulation in low-C[O.sub.2] conditions) and/or simultaneous, apparently off-target knockdown of LCIA mRNA (which encodes limiting-Ci--inducible plastid envelope protein reported to transport HC[O.sup.-.sub.3]) resulted in dramatic decreases in growth, Ci uptake, and photosynthetic Ci affinity, especially at pH 9, at which HC[O.sup.-.sub.3] is the predominant form of available Ci. Collectively, the data presented here provide compelling evidence that HLA3 is directly or indirectly involved in HC[O.sup.-.sub.3] transport, along with additional evidence supporting a role for LCIA in chloroplast envelope HC[O.sup.-.sub.3] transport and a role for LCIB in chloroplast Ci accumulation. ABC transporter | C[O.sub.2]-concentrating mechanism | microalgae | RNAi

Biosyntheses of chlorophyll and heme in oxygenic phototrophs share a common trunk pathway that diverges with insertion of magnesium or iron into the last common intermediate, protoporphyrin IX. Since both tetrapyrroles are pro-oxidants, it is essential that their metabolism is tightly regulated. Here, we establish that heme-derived linear tetrapyrroles (bilins) function to stimulate the enzymatic activity of magnesium chelatase (MgCh) via their interaction with GENOMES UNCOUPLED 4 (GUN4) in the model green alga Chlamydomonas reinhardtii. A key tetrapyrrole-binding component of MgCh found in all oxygenic photosynthetic species, CrGUN4, also stabilizes the bilin-dependent accumulation of protoporphyrin IX-binding CrCHLH1 subunit of MgCh in light-grown C. reinhardtii cells by preventing its photooxidative inactivation. Exogenous application of biliverdin IXa reverses the loss of CrCHLH1 in the bilin-deficient heme oxygenase (hmox1) mutant, but not in the gun4 mutant. We propose that these dual regulatory roles of GUN4: bilin complexes are responsible for the retention of bilin biosynthesis in all photosynthetic eukaryotes, which sustains chlorophyll biosynthesis in an illuminated oxic environment. [ABSTRACT FROM AUTHOR]

Phytochrome photosensors control a vast gene network in streptophyte plants, acting as master regulators of diverse growth and developmental processes throughout the life cycle. In contrast with their absence in known chlorophyte algal genomes and most sequenced prasinophyte algal genomes, a phytochrome is found in Micromonas pusilla, a widely distributed marine picoprasino-phyte (<2 urn cell diameter). Together with phytochromes identified from other prasinophyte lineages, we establish that prasinophyte and streptophyte phytochromes share core light-input and signaling-output domain architectures except for the loss of C-terminal response regulator receiver domains in the streptophyte phytochrome lineage. Phylogenetic reconstructions robustly support the presence of phytochrome in the common progenitor of green algae and land plants. These analyses reveal a monophy-letic clade containing streptophyte, prasinophyte, cryptophyte, and glaucophyte phytochromes implying an origin in the eukary-otic ancestor of the Archaeplastida. Transcriptomic measurements reveal diurnal regulation of phytochrome and bilin chromophore biosynthetic genes in Micromonas. Expression of these genes precedes both light-mediated phytochrome redistribution from the cytoplasm to the nucleus and increased expression of photosynthesis-associated genes. Prasinophyte phytochromes perceive wavelengths of light transmitted farther through seawater than the red/far-red light sensed by land plant phytochromes. Prasinophyte phytochromes also retain light-regulated histidine kinase activity lost in the streptophyte phytochrome lineage. Our studies demonstrate that light-mediated nuclear translocation of phytochrome predates the emergence of land plants and likely represents a widespread signaling mechanism in unicellular algae. [ABSTRACT FROM AUTHOR]

Plant phytochromes are photoswitchable red/far-red photoreceptors that allow competition with neighboring plants for photosynthetically active red light. In aquatic environments, red and far-red light are rapidly attenuated with depth; therefore, photosynthetic species must use shorter wavelengths of light. Nevertheless, phytochrome-related proteins are found in recently sequenced genomes of many eukaryotic algae from aquatic environments. We examined the photosensory properties of seven phytochromes from diverse algae: four prasinophyte (green algal) species, the heterokont (brown algal) Ectocarpus siliculosus, and two glaucophyte species. We demonstrate that algal phytochromes are not limited to red and far-red responses. Instead, different algal phytochromes can sense orange, green, and even blue light. Characterization of these previously undescribed photosensors using CD spectroscopy supports a structurally heterogeneous chromophore in the far-red- absorbing photostate. Our study thus demonstrates that extensive spectral tuning of phytochromes has evolved in phylogenetically distinct lineages of aquatic photosynthetic eukaryotes. [ABSTRACT FROM AUTHOR]

The maintenance of functional chloroplasts in photosynthetic eukaryotes requires real-time coordination of the nuclear and plastid genomes. Tetrapyrroles play a significant role in plastid-to-nucleus retrograde signaling in plants to ensure that nuclear gene expression is attuned to the needs of the chloroplast. Well-known sites of synthesis of chlorophyll for photosynthesis, plant chloroplasts also export heme and heme-derived linear tetrapyrroles (bilins), two critical metabolites respectively required for essential cellular activities and for light sensing by phytochromes. Here we establish that Chlamydomonas reinhardtii, one of many chlorophyte species that lack phytochromes, can synthesize bilins in both plastid and cytosol compartments. Genetic analyses show that both pathways contribute to iron acquisition from extracellular heme, whereas the plastid-localized pathway is essential for light-dependent greening and phototrophic growth. Our discovery of a bilin-dependent nuclear gene network implicates a widespread use of bilins as retrograde signals in oxygenic photosynthetic species. Our studies also suggest that bilins trigger critical metabolic pathways to detoxify molecular oxygen produced by photosynthesis, thereby permitting survival and phototrophic growth during the light period. [ABSTRACT FROM AUTHOR]

Many virus genomes encode proteases that facilitate infection. The molecular mechanism of plant recognition of viral proteases is largely unexplored. Using the system of Vigna unguiculata and cowpea mosaic virus (CPMV), we identified a cowpea lipid transfer protein (LTP1) which interacts with CPMV-encoded 24KPro, a cysteine protease, but not with the enzymatically inactive mutant 24KPro(C166A). Biochemical assays showed that LTP1 inhibited 24KPro proteolytic cleavage of the coat protein precursor large coat protein-small coat protein. Transient overexpression of LTP1 in cowpea reduced CPMV infection, whereas RNA interference-mediated LTP1 silencing increased CPMV accumulation in cowpea. LTP1 is mainly localized in the apoplast of uninfected plant cells, and after CPMV infection, most of the LTP1 is relocated to intracellular compartments, including chloroplast. Moreover, in stable LTP1 -transgenic Nicotiana benthamiana plants, LTP1 repressed soybean mosaic virus (SMV) nuclear inclusion a protease activity, and accumulation of SMV was significantly reduced. We propose that cowpea LTP1 suppresses CPMV and SMV accumulation by directly inhibiting viral cysteine protease activity., Competing Interests: Competing interests statement:The authors declare no competing interest.

Biosyntheses of chlorophyll and heme in oxygenic phototrophs share a common trunk pathway that diverges with insertion of magnesium or iron into the last common intermediate, protoporphyrin IX. Since both tetrapyrroles are pro-oxidants, it is essential that their metabolism is tightly regulated. Here, we establish that heme-derived linear tetrapyrroles (bilins) function to stimulate the enzymatic activity of magnesium chelatase (MgCh) via their interaction with GENOMES UNCOUPLED 4 (GUN4) in the model green alga Chlamydomonas reinhardtii A key tetrapyrrole-binding component of MgCh found in all oxygenic photosynthetic species, Cr GUN4, also stabilizes the bilin-dependent accumulation of protoporphyrin IX-binding Cr CHLH1 subunit of MgCh in light-grown C. reinhardtii cells by preventing its photooxidative inactivation. Exogenous application of biliverdin IXα reverses the loss of Cr CHLH1 in the bilin-deficient heme oxygenase ( hmox1 ) mutant, but not in the gun4 mutant. We propose that these dual regulatory roles of GUN4:bilin complexes are responsible for the retention of bilin biosynthesis in all photosynthetic eukaryotes, which sustains chlorophyll biosynthesis in an illuminated oxic environment., Competing Interests: The authors declare no competing interest.

The CO(2)-concentrating mechanism (CCM) of Chlamydomonas reinhardtii and other microalgal species is essential for photosynthetic growth in most natural settings. A great deal has been learned regarding the CCM in cyanobacteria, including identification of inorganic carbon (Ci; CO(2) and HCO(3)(-)) transporters; however, specific knowledge of analogous transporters has remained elusive in eukaryotic microalgae such as C. reinhardtii. Here we investigated whether the limiting-CO(2)-inducible, putative ABC-type transporter HLA3 might function as a HCO(3)(-) transporter by evaluating the effect of pH on growth, photosynthetic Ci affinity, and [(14)C]-Ci uptake in very low CO(2) conditions following RNA interference (RNAi) knockdown of HLA3 mRNA levels in wild-type and mutant cells. Although knockdown of HLA3 mRNA alone resulted in only modest but high-pH-dependent decreases in photosynthetic Ci affinity and Ci uptake, the combination of nearly complete knockdown of HLA3 mRNA with mutations in LCIB (which encodes limiting-Ci-inducible plastid-localized protein required for normal Ci uptake or accumulation in low-CO(2) conditions) and/or simultaneous, apparently off-target knockdown of LCIA mRNA (which encodes limiting-Ci-inducible plastid envelope protein reported to transport HCO(3)(-)) resulted in dramatic decreases in growth, Ci uptake, and photosynthetic Ci affinity, especially at pH 9, at which HCO(3)(-) is the predominant form of available Ci. Collectively, the data presented here provide compelling evidence that HLA3 is directly or indirectly involved in HCO(3)(-) transport, along with additional evidence supporting a role for LCIA in chloroplast envelope HCO(3)(-) transport and a role for LCIB in chloroplast Ci accumulation.