Tomohiro Kiyosue

A nuclear gene encoding mitochondrial proline dehydrogenase, an enzyme involved in proline metabolism, is upregulated by proline but downregulated by dehydration in Arabidopsis.

Proline is one of the most common compatible osmolytes in water-stressed plants. The accumulation of proline in dehydrated plants is caused both by the activation of proline biosynthesis and by the inactivation of proline degradation; a decrease in the level of accumulated proline in rehydrated plants is caused both by the inhibition of proline biosynthesis and by the activation of proline degradation. The proline biosynthetic pathway has been well characterized, but the degradation of proline is poorly understood. Sequence analysis of an Arabidopsis cDNA clone, ERD5 (for early responsive to dehydration stress), isolated from plants dehydrated for 1 hr, revealed that it encodes a protein with identity to products of the yeast PUT1 (for proline utilization) gene (23.6% over 364 amino acids) and the Drosophila sluggish-A gene (34.5% over 255 amino acids). Their gene products are precursors of proline oxidases (dehydrogenase) (EC 1.5.99.8), which are the first enzymes involved in the conversion of proline to glutamic acid. Proline oxidase is localized in mitochondria. RNA gel blot analysis demonstrated that transcripts of the ERD5 gene were undetectable when plants had been dehydrated for 10 hr, but large amounts of the transcript accumulated when plants subsequently were rehydrated. Elevated levels of the transcript were also found in plants that had been incubated in a medium that contained proline. Immunologically, we showed that the product of ERD5 is localized in the mitochondrial fraction and accumulates in response to proline in cultured cells. Fusion genes for ERD5 and PUT1 complemented a put1 mutant of yeast, allowing put1 to grow with proline as the source of nitrogen. These results suggest that ERD5 encodes a precursor of proline dehydrogenase (oxidase), which is regulated at the level of mRNA accumulation in both dehydrated and rehydrated plants.

Chloroplast unusual positioning1 is essential for proper chloroplast positioning.

The intracellular distribution of organelles is a crucial aspect of effective cell function. Chloroplasts change their intracellular positions to optimize photosynthetic activity in response to ambient light conditions. Through screening of mutants of Arabidopsis defective in chloroplast photorelocation movement, we isolated six mutant clones in which chloroplasts gathered at the bottom of the cells and did not distribute throughout cells. These mutants, termed chloroplast unusual positioning (chup), were shown to belong to a single genetic locus by complementation tests. Observation of the positioning of other organelles, such as mitochondria, peroxisomes, and nuclei, revealed that chloroplast positioning and movement are impaired specifically in this mutant, although peroxisomes are distributed along with chloroplasts. The CHUP1 gene encodes a novel protein containing multiple domains, including a coiled-coil domain, an actin binding domain, a Pro-rich region, and two Leu zipper domains. The N-terminal hydrophobic segment of CHUP1 was expressed transiently in leaf cells of Arabidopsis as a fusion protein with the green fluorescent protein. The fusion protein was targeted to envelope membranes of chloroplasts in mesophyll cells, suggesting that CHUP1 may localize in chloroplasts. A glutathione S-transferase fusion protein containing the actin binding domain of CHUP1 was found to bind F-actin in vitro. CHUP1 is a unique gene identified that encodes a protein required for organellar positioning and movement in plant cells.

Cloning of cDNAs for genes that are early-responsive to dehydration stress (ERDs) inArabidopsis thaliana L.: identification of three ERDs as HSP cognate genes

InArabidopsis thaliana L., accumulation of abscisic acid (ABA) began to increase 2 h after plants had been subjected to dehydration stress and reached maximum levels after 10h. Differential hybridization was used to isolate 26Arabidopsis cDNAs with gene expression induced by a 1 h dehydration treatment. The cDNA clones were classified into 16 groups based on Southern blot hybridization, and named ERD (early-responsive todehydration) clones. Partial sequencing of the cDNA clones revealed that three ERDs were identical to those of HSP cognates (Athsp70-1, Athsp81-2, and ubiquitin extension protein). Dehydration stress strongly induced the expression of genes for the three ERDs, while application of ABA, which is known to act as a signal transmitter in dehydration-stressed plants, did not significantly affect the ERD gene expression. This result suggests that these HSP cognates are preferentially responsive to dehydration stress inA. thaliana, and that signaling pathways for the expression of these genes under conditions of dehydration stress are not mainly mediated by ABA. We also discuss the possible functions of these three ERD gene products against dehydration stress.

Responses of ferns to red light are mediated by an unconventional photoreceptor

Efficient photosynthesis is essential for plant survival. To optimize photosynthesis, plants have developed several photoresponses. Stems bend towards a light source (phototropism), chloroplasts move to a place of appropriate light intensity (chloroplast photorelocation) and stomata open to absorb carbon dioxide. These responses are mediated by the blue-light receptors phototropin 1 (phot1) and phototropin 2 (phot2) in Arabidopsis (refs 1–5). In some ferns, phototropism and chloroplast photorelocation are controlled by red light as well as blue light. However, until now, the photoreceptor mediating these red-light responses has not been identified. The fern Adiantum capillus-veneris has an unconventional photoreceptor, phytochrome 3 (phy3), which is a chimaera of the red/far-red light receptor phytochrome and phototropin. We identify here a function of phy3 for red-light-induced phototropism and for red-light-induced chloroplast photorelocation, by using mutational analysis and complementation. Because phy3 greatly enhances the sensitivity to white light in orienting leaves and chloroplasts, and PHY3 homologues exist among various fern species, this chimaeric photoreceptor may have had a central role in the divergence and proliferation of fern species under low-light canopy conditions.

AUREOCHROME, a photoreceptor required for photomorphogenesis in stramenopiles

A blue light (BL) receptor was discovered in stramenopile algae Vaucheria frigida (Xanthophyceae) and Fucus distichus (Phaeophyceae). Two homologs were identified in Vaucheria; each has one basic region/leucine zipper (bZIP) domain and one light–oxygen–voltage (LOV)-sensing domain. We named these chromoproteins AUREOCHROMEs (AUREO1 and AUREO2). AUREO1 binds flavin mononucleotide via its LOV domain and forms a 390-nm-absorbing form, indicative of formation of a cysteinyl adduct to the C(4a) carbon of the flavin mononucleotide upon BL irradiation. The adduct decays to the ground state in ≈5 min. Its bZIP domain binds the target sequence TGACGT. The AUREO1 target binding was strongly enhanced by BL treatment, implying that AUREO1 functions as a BL-regulated transcription factor. The function of AUREO1 as photoreceptor for BL-induced branching is elucidated through RNAi experiments. RNAi of AUREO2 unexpectedly induces sex organ primordia instead of branches, implicating AUREO2 as a subswitch to initiate development of a branch, but not a sex organ. AUREO sequences are also found in the genome of the marine diatom Thalassiosira pseudonana (Bacillariophyceae), but are not present in green plants. AUREOCHROME therefore represents a BL receptor in photosynthetic stramenopiles.

Characterization of two cDNAs (ERD11 and ERD13) for dehydration-inducible genes that encode putative glutathione S-transferases in Arabidopsis thaliana L

Two cDNA clones, designated ERD11 and ERD13, isolated from a cDNA library from Arabidopsis thaliana L. plants dehydrated for 1 h were sequenced and characterized. These clones encoded polypeptides that were homologous to glutathione S‐transferases of tobacco and maize. Genomic Southern hybridization suggested that there are a few additional genes showing high similarity to the ERD11 gene in the Arabidopsis genome. The expression of the genes for ERD11 and ERD13 was induced by dehydration, but was not affected by the application of four plant growth regulators, 2,4‐dichlorophenoxyacetic acid 6‐benzylaminopurine, abscisic acid, or gibberellic acid.

Phototropins Mediate Blue and Red Light-Induced Chloroplast Movements in Physcomitrella patens

Phototropin is the blue-light receptor that mediates phototropism, chloroplast movement, and stomatal opening in Arabidopsis. Blue and red light induce chloroplast movement in the moss Physcomitrella patens. To study the photoreceptors for chloroplast movement in P. patens, four phototropin genes (PHOTA1, PHOTA2, PHOTB1, and PHOTB2) were isolated by screening cDNA libraries. These genes were classified into two groups (PHOTA and PHOTB) on the basis of their deduced amino acid sequences. Then phototropin disruptants were generated by homologous recombination and used for analysis of chloroplast movement. Data revealed that blue light-induced chloroplast movement was mediated by phototropins in P. patens. Both photA and photB groups were able to mediate chloroplast avoidance, as has been reported for Arabidopsis phot2, although the photA group contributed more to the response. Red light-induced chloroplast movement was also significantly reduced in photA2photB1photB2 triple disruptants. Because the primary photoreceptor for red light-induced chloroplast movement in P. patens is phytochrome, phototropins may be downstream components of phytochromes in the signaling pathway. To our knowledge, this work is the first to show a function for the phototropin blue-light receptor in a response to wavelengths that it does not absorb.

Isolation and characterization of a cDNA that encodes ECP31, an embryogenic-cell protein from carrot.

A full-length cDNA for ECP31, an embryogenic cell protein from carrot (Daucus carota L.) with a Mr of 31000 (Kiyosue T, Satoh S, Kamada H, Harada H (1991) Plant Physiol 95: 1077–1083), was isolated from a cDNA library prepared from embryogenic cells using PCR-amplified DNA as a probe. The genomic Southern blot analysis revealed that there are two or three genes for ECP31 in the carrot genome. The transcripts of ECP31 accumulated in the peripheral regions of clusters of embryogenic cells and disappeared in the course of somatic embryogenesis that was induced by transfer of the embryogenic cells to auxin-free media. The cDNA encodes a polypeptide of 256 amino acids, and the calculated molecular weight of this polypeptide is 26 111. The deduced amino acid sequence shows a high degree (62.2%) of similarity to that of a protein that is abundant during late embryogenesis of cotton (LEA D34; Baker JC, Steele C, Dure III (1988) Plant Mol Biol 11: 227–291). The mRNAs for ECP31 started to accumulate in zygotic embryos at a late stage of embryogenesis but were undetectable in mature embryos within 24 h after imbibition of seeds. In dry fruits (seeds), the transcripts were detected only in zygotic embryos by in situ hybridization. The level of ECP31 transcripts increased after treatment with abscisic acid (ABA) in torpedo-shaped somatic embryos but not in seven-day-old seedlings. These results suggest that both embryo-specific factor(s) and ABA are involved in the expression of the gene for ECP31.

Stress induced somatic embryogenesis in carrot and its application to synthetic seed production

SummaryWhen apical meristems of carrot (Daucus carota L. cv. US-Harumakigosun) seedlings were cultured on hormone-free Murashige and Skoog’s (MS) medium with 0.7 M sucrose or 0.25–1 mM cadmium ion, then transferred to hormone-free MS medium with 0.1 M sucrose, somatic embryos were formed on the surface of the explants without visible callus formation. Somatic embryos were also formed on malformed seedlings, when carrot seeds were treated with hypochlorite solution at a high concentration and sown on hormone-free MS medium with 0.09 M sucrose. These somatic embryos were fractionated by passing through stainless steel sieves with different pore sizes, encapsulated in calcium alginate gel, and placed in plastic petri dishes under sterile conditions. These synthetic seeds germinated 1 to 2 weeks after the beginning of the culture. In the case of synthetic seeds containing a single embryo, the frequency of the seeds which developed both a radicle and a green bud was about 30–50% in large embryos induced by the treatment with sucrose, cadmium or sodium hypochlorite, and about 15% in 2,4-D induced embryos. When 2,4-D induced embryos were encapsulated and sown, numerous secondary and tertiary embryos were formed but they did not develop into normal seedlings.

ERD6, a cDNA clone for an early dehydration-induced gene of Arabidopsis, encodes a putative sugar transporter

Previously, we constructed a cDNA library from Arabidopsis plants that were exposed to dehydration stress for 1 h and obtained the ERD6 clone. Here we report that the ERD6 cDNA consists of 1741 bp and encodes a polypeptide of 496 amino acids having a predicted molecular weight of 54,354. The putative polypeptide of ERD6 is related to those of sugar transporters of bacteria, yeasts, plants and mammals. Hydropathy analysis revealed that ERD6 protein has 12 putative transmembrane domains and a central hydrophilic region. Sequences that are conserved at the ends of the 6th and 12th membrane-spanning domains of sugar transporters are also present in ERD6. These data suggest that ERD6 encodes a sugar transporter. Genomic Southern blots indicate that the ERD6 gene is a member of a multigene family in the Arabidopsis genome. The expression of the ERD6 gene was induced not only by dehydration but also by cold treatment.