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Cotyledon cells of dark-germinated angiosperms develop etioplasts that are plastids containing unique internal membranes called prolamellar bodies (PLBs). Protochlorophyllide (Pchlide), a precursor of chlorophyll, accumulates in PLBs and forms a ternary complex with NADPH and light-dependent NADPH:protochlorophyllide oxidoreductase (LPOR), which allows for the rapid formation of chlorophyll after illumination while avoiding photodamage. PLBs are 3D lattice structures formed by the lipid bilayer rich in monogalactosyldiacylglycerol (MGDG). Although MGDG was found to be required for the formation and function of the thylakoid membrane in chloroplasts in various plants, the roles of MGDG in PLB formation and etioplast development are largely unknown. To analyze the roles of MGDG in etioplast development, we suppressed MGD1 encoding the major isoform of MGDG synthase by using a dexamethasone-inducible artificial microRNA in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. Strong MGD1 suppression caused a 36% loss of MGDG in etiolated seedlings, together with a 41% decrease in total Pchlide content. The loss of MGDG perturbed etioplast membrane structures and impaired the formation of the photoactive Pchlide-LPOR-NADPH complex and its oligomerization, without affecting LPOR accumulation. The MGD1 suppression also impaired the formation of Pchlide from protoporphyrin IX via multiple enzymatic reactions in etioplast membranes, which suggests that MGDG is required for the membrane-associated processes in the Pchlide biosynthesis pathway. Suppressing MGD1 at several germination stages revealed that MGDG biosynthesis at an early germination stage is particularly important for Pchlide accumulation. MGDG biosynthesis may provide a lipid matrix for Pchlide biosynthesis and the formation of Pchlide-LPOR complexes as an initial step of etioplast development.

The development of plant chloroplasts is regulated by various developmental, environmental, and hormonal cues. In Arabidopsis (Arabidopsis thaliana), chloroplast development is repressed in roots via auxin signaling. However, roots develop chloroplasts when they are detached from the shoot. In contrast to auxin, cytokinin positively affects chloroplast development in roots, but the role and signaling pathway of cytokinin in the root greening response remain unclear. To understand the regulatory pathways of chloroplast development in the plant stress response, we examined the mechanisms underlying the conditional greening of detached roots. In wild-type Arabidopsis roots, shoot removal activates type B ARABIDOPSIS RESPONSE REGULATOR (ARR)-mediated cytokinin signaling and induces chlorophyll accumulation and photosynthetic remodeling. ARR1 and ARR12 are essential for up-regulating nucleus- and plastid-encoded genes associated with chloroplast development in detached roots. In this process, WOUND INDUCED DEDIFFERENTIATION1 and class B GATA transcription factors (B-GATAs) act upstream and downstream of ARRs, respectively. Overexpression of B-GATAs promotes root greening, as does shoot removal, dependent on a light signaling transcription factor, LONG HYPOCOTYL5. Auxin represses the root greening response independent of ARR signaling. GNC-LIKE (GNL), a B-GATA, is strongly up-regulated in detached roots via ARR1 and ARR12 but is repressed by auxin, so GNL may function at the point of convergence of cytokinin and auxin signaling in the root greening response.

Jasmonic acid (JA) and methyl jasmonate (MeJA), collectively known as JAs, regulate diverse physiological processes in plants, including the response to wounding. Recent reports suggest that a cyclopentenone precursor of JA, 12-oxo-phytodienoic acid (OPDA), can also induce gene expression. However, little is known about the physiological significance of OPDA-dependent gene expression. We used microarray analysis of approximately 21,500 Arabidopsis (Arabidopsis thaliana) genes to compare responses to JA, MeJA, and OPDA treatment. Although many genes responded identically to both OPDA and JAs, we identified a set of genes (OPDA-specific response genes [ORGs]) that specifically responded to OPDA but not to JAs. ORGs primarily encoded signaling components, transcription factors, and stress response-related genes. One-half of the ORGs were induced by wounding. Analysis using mutants deficient in the biosynthesis of JAs revealed that OPDA functions as a signaling molecule in the wounding response. Unlike signaling via JAs, OPDA signaling was CORONATINE INSENSITIVE 1 independent. These results indicate that an OPDA signaling pathway functions independently of JA/MeJA signaling and is required for the wounding response in Arabidopsis.

Tetrapyrrole compounds, such as chlorophylls, hemes, and phycobilins, are synthesized in many enzymatic steps. For regulation of the tetrapyrrole metabolic pathway, it is generally considered that several specific isoforms catalyzing particular enzymatic steps control the flow of tetrapyrrole intermediates by differential regulation of gene expression depending on environmental and developmental factors. However, the coordination of such regulatory steps and orchestration of the overall tetrapyrrole metabolic pathway are still poorly understood. In this study, we developed an original mini-array system, which enables the expression profiling of each gene involved in tetrapyrrole biosynthesis simultaneously with high sensitivity. With this system, we performed a transcriptome analysis of Arabidopsis seedlings in terms of the onset of greening, endogenous rhythm, and developmental control. Data presented here clearly showed that based on their expression profiles at the onset of greening, genes involved in tetrapyrrole biosynthesis can be classified into four categories, in which genes are coordinately regulated to control the biosynthesis. Moreover, genes in the same group were similarly controlled in an endogenous rhythmic manner but also by a developmental program. The physiological significance of these gene clusters is discussed.

To elucidate the mechanism of an irradiance-dependent adjustment in the chlorophyll (Chl) antenna size of Dunaliella salina, we investigated the regulation of expression of the Chl a oxygenase (CAO) and light-harvesting complex b (Lhcb) genes as a function of Chl availability in the photosynthetic apparatus. After a high-light to low-light shift of the cultures, levels of both CAO and Lhcb transcripts were rapidly induced by about 6-fold and reached a high steady-state level within 1.5 h of the shift. This was accompanied by repair of photodamaged photosystem II (PSII) reaction centers, accumulation of Chl a and Chl b (4:1 ratio), photosystem I (PSI), light-harvesting complex, and by enlargement of the Chl antenna size of both photosystems. In gabaculine-treated cells, induction of CAO and Lhcb transcripts was not affected despite substantial inhibition in de novo Chl biosynthesis. However, cells were able to synthesize and accumulate some Chl a and Chl b (1:1 ratio), resulting in a marked lowering of the Chl a to Chl b ratio in the presence of this inhibitor. Assembly incorporation of light-harvesting complex and a corresponding Chl antenna size increase, mostly for the existing photosystems, was noted in the presence of gabaculine. Repair of photodamaged PSII was not affected by gabaculine. However, assembly accumulation of new PSI was limited under such conditions. These results suggest a coordinate regulation of CAO and Lhcb gene transcription by irradiance, independent of Chl availability. The results are discussed in terms of different signal transduction pathways for the regulation of the photosynthetic apparatus organization by irradiance.

The first committed step of porphyrin synthesis in higher plants is the reduction of glutamyl-tRNA to glutamate 1-semialdehyde. This reaction is catalyzed by glutamyl-tRNA reductase, which is encoded by hemA genes. Two hemA cDNA clones (hemA1 and hemA2) were obtained from cucumber (Cucumis sativus) cotyledons by the PCR and cDNA library screening. They showed significant homology with published hemA sequences. Southern blot analysis of cucumber genomic DNA revealed that these genes are located at different loci and that there is another gene similar to the hemA genes. Accumulation of hemA1 mRNA was detected primarily in cotyledons and hypocotyls of greening cucumber seedlings, whereas that of hemA2 mRNA was detected in all tissues examined. Illumination of cucumber seedlings increased markedly the accumulation of hemA1 mRNA, but it did not induce remarkable changes in that of hemA2 mRNA. These findings suggest that hemA1 mRNA was accumulated in response to the demand of Chl synthesis in photosynthesizing tissues, whereas hemA2 mRNA was expressed in response to the demand of the synthesis of porphyrins other than chlorophylls.

Ferrochelatase is the enzyme that catalyzes the last step in the heme biosynthetic pathway. It catalyzes the insertion of iron (Fez+) into the tetrapyrrole ring of protoporphyrin IX to generate protoheme. Recently, we isolated mutants of Escherichia coli that were sensitive to visible light (Miyamoto et al., 1991; Nakahigashi et al., 1991), and these mutants [designated visA (=hemH) mutants] were shown to be the result of a mutation in the visA (=hemH) gene, which encodes ferrochelatase. The nucleotide sequences of genes that encode ferrochelatases from six different sources are available (E. coli, Miyamoto et al., 1991; Bacillus subtilis, Hansson and Hederstedt, 1992; Bradyrhizobium japonicum, Frustaci and O’Brian, 1992; yeast, Labbe-Bois, 1990; mouse, Taketani et al., 1990; human, Nakahashi et al., 1990), but none have been reported from higher plants. Here we report the isolation and the sequences of cDNAs that encode a ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) from barley (Hordeum vulgare L. cv Svalof‘s Bonus) and a ferrochelatase from cucumber (Cucumis sativus L. cv Aonagajibai). Both cDNAs were isolated from libraries in phage vector Xgtll. To screen the clones, we used a deletion mutant of E. coli (strain VSZOO), in which part of the gene for ferrochelatase had been deleted. Because VS200 mutants produce no heme, they cannot grow well even on complete medium. Therefore, we isolated large colonies of VS200 cells that formed among the tiny colonies after infection by the clones in the cDNA libraries of barley and cucumber. Phages obtained from the large colonies were tested for their ability to improve the poor growth of VS200 bacteria. To determine the nucleotide sequence of the cDNA insert in each selected phage clone, an EcoRI fragment was recloned into plasmid vector pUCl18 and serial-deletion clones were constructed by the step-wise method of Yanisch-Perron et al. (1985). The insert of a phage clone from the barley cDNA library was 1728 bp long and included a single open reading frame that encoded a polypeptide of 484 amino acids (Table I). Coincidentally, the insert from the cucumber cDNA library