Invited Review

Recent advances in forest tree biotechnology

Shiro Suzuki, Hideyuki Suzuki. . . . . . . 1[PDF]

Forest trees produce an important feedstock, wood. Forest tree breeding programs have been traditionally carried out by selecting elite trees to enhance productivity and processability. Recently, however, a biotechnological approach has attracted much attention because it enables efficient and versatile improvement of forest trees. In the last decade, forest tree biotechnology has considerably progressed: genomic sequences of several forest tree species have been decoded, efficient Agrobacterim-mediated genetic transformation and regeneration systems have been established in a number of forest tree species, and many reports have been published on the metabolic engineering of a major wood component, lignin, in forest trees. However, in contrast to the metabolic engineering of lignin, the metabolic engineering of cellulose and hemicelluloses in forest trees awaits further development. The detrimental effects on tree growth are often concomitant with the metabolic engineering of wood components. To mitigate such effects, fine-tuned regulation of transgene expression, and the production of value-added products may be targeted in future forest tree biotechnology.

Original Papers

Overexpression of Arabidopsis FT gene in apple leads to perpetual owering

Norimitsu Tanaka, Ayano Ureshino, Narumi Shigeta, Naozumi Mimida, Sadao Komori, Sae Takahashi, Yuki Tanaka-Moriya, Masato Wada. . . . . . . 11[PDF]

Transgenic apple plants that overexpressed Arabidopsis FLOWERING LOCUS T (FT) under the control of the rolC promoter showed early flowering, while the introduction of FT driven by the 35S promoter induced flower development directly from transgenic apple callus in vitro, but vegetative growth was not maintained and the explant died. GFP–FT fusion proteins were detectable in transgenic apple tissues but never caused early flowering in the transformants. Under the control of the rolC promoter, the fused protein was localized in vascular tissues and fluorescence was detectable in companion cells in the stem and petiole. The transgenic apple lines that expressed AtFT driven by the rolC promoter showed differences in inflorescence architecture and floral organ number from those typical of nontransformed apple. Flowers of transgenic apple lines often contained more numerous petals, fewer stamens, and no pistils, and the pollen grains were incapable of germinating. The transgenic apple lines showed perpetual flowering without the requirement for low temperature and obvious photoperiodism. The expression patterns of six floral meristem identity genes and floral organ genes in flowers of transgenic apple lines were investigated. Among the floral meristem identity genes, expression of MdFT2 was suppressed and that of AFL2 was dramatically enhanced in the transformants. Of the floral organ genes, expression of MdMADS13, which functions as a class B gene, was unchanged from that of the flower of wild type one, whereas expression of MdMADS-NB, a possible class C gene, was suppressed.

Molecular cloning and characterization of coumarin glucosyltransferase in hairy roots of Pharbitis nil(Ipomoea nil)

Hideaki Kanoh, Moriyuki Kawauchi, Masanori Kuroyanagi, Toshihide Arima. . . . . . . 21[PDF]

The hairy roots induced from Pharbitis nil (Ipomoea nil) by infection of the plant pathogen Agrobacterium rhizogenes produced and released the phytoalexin of umbelliferone into the culture medium by treatment with copper sulfate. However, this umbelliferone was immediately recovered in the hairy roots as its glucoside, skimmin, after a few hours. To analyze the details of these phenomena, we searched for glucosyltransferase (GTase) genes related to skimmin production and isolated two full-length cDNAs, named PNgt1 and vPNgt2. They contain the plant secondary product glucosyltransferase (PSPG) box that is a common signature of plant GTases. Phylogenetic analyses of PNgt1 and PNgt2 indicated that they encode a broad substrate-specific GTase and an unclassified GTase, respectively. An enzyme assay of the recombinant PNGT1 protein showed a high glucosylation rate against coumarin and benzaldehyde derivatives, especially umbelliferone. The expression level of PNgt1 after treatment with umbelliferone showed a correlation with skimmin production. Moreover, we found the expression levels of PNgt1 were altered by the plant hormones salicylic acid and methyl jasmonate. These results suggest the importance of plant GTases in phytoalexin production and plant defense mechanisms.

Late flowering phenotype under ultra-short photoperiod (USP) in Arabidopsis thaliana

Haruna Sugiyama, Yu Natsui, Miyuki Hara, Kana Miyata, Rim Nessi, Tsuyoshi Mizoguchi. . . . . . . 29[PDF]

Many environmental and endogenous factors affect flowering time of plants. In Arabidopsis, there are three major pathways for the control of flowering time; the photoperiod or long-day (LD) pathway, autonomous/vernalization pathway and gibberellic acid (GA) pathway. The flowering regulation under most common photoperiods in Arabidopsis involves some floral activators such as CONSTANS (CO) and GIGANTEA (GI) and a circadian clock protein, EARLY FLOWERING 3 (ELF3). In this work, we examined the effect of ultra-short photoperiod (USP) on flowering time of three accessions (Ler, Col and Ws). All the wild-type plants tested showed remarkable delay of flowering under 3 h or less photoperiods, but different sensitivity to the USP was found in these accessions. Late flowering phenotype of plants with mutations in genes involved in the three major pathways was enhanced under the USP. Expression of FLOWERING LOCUS T (FT) encoding a florigen decreased under the USP. A mutation in the ELF3 gene in Ler largely suppressed the delay of flowering under the USP. These results suggest that floral regulation pathway under the USP may be independent of the three well-characterized pathways. ELF3 may play a key role in the USP pathway.

CO-EXPRESSED WITH CLOCK GENES LHY AND CCA1 1 (CEC1) is regulated by LHY and CCA1 and plays a key role in phase setting of GI in Arabidopsis thaliana

Miyuki Hara, Hiroshi Kamada, Tsuyoshi Mizoguchi. . . . . . . 35[PDF]

Many biological processes, including the control of flowering time, are regulated by the circadian clock. Although a number of clock-associated genes have been characterized in Arabidopsis thaliana (Arabidopsis), the complete molecular mechanisms of the circadian clock remain unclear. Here, we report that CO-EXPRESSED WITH CLOCK GENES LHY AND CCA1 1 (CEC1) plays an important role in circadian clock function in Arabidopsis. Three genes, CEC1, CEC2, and CEC3, are co-expressed with the clock genes LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1). The mutants, cec1 and cec2, exhibited an early flowering phenotype under long-day (LD) and continuous-light (LL) conditions, possibly through an increase in FLOWERING LOCUS T (FT) mRNA. In addition, rhythmic peaks of GIGANTEA (GI) expression were delayed in the cec1 mutant plants, but the period length and amplitude of GI expression were not affected under LD and LL. These results suggest that CEC1 might contribute to the modulation of circadian phases.

Short Communication

Increase in vascular pattern complexity caused by mutations in LHY and CCA1 in Arabidopsis thaliana under continuous light

Kohei Aihara, Satoshi Naramoto, Miyuki Hara, Tsuyoshi Mizoguchi. . . . . . . 43[PDF]

Circadian rhythms in Arabidopsis thaliana (Arabidopsis) are controlled by clock components such as LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1). Plants with mutations in both LHY and CCA1 (lhy;cca1) show a wavy leaf phenotype under continuous light (LL). The circadian clock regulates both the biosynthesis and signaling of plant hormones, including auxin. Auxin plays a key role in vascular pattern formation in leaves. For example, plants with mutations in either VASCULAR NETWORK DEFECTIVE 3 (VAN3) or FORKED 1 (FKD1) exhibit reduced complexity in their leaf vascular patterns. However, the molecular mechanism underlying the decrease in flatness of lhy;cca1 leaves under LL has not been elucidated. To address this question, the leaf vascular patterns of lhy;cca1 were compared with those of wild-type, van3, and fkd1 plants under LL. As reported previously, the numbers of areoles and branch points in van3 and fkd1 plants grown for 14 days under LL were much lower than those of wild-type plants. In contrast, the numbers of free ends, areoles, and branch points increased in lhy;cca1. This is the first demonstration of Arabidopsis mutants with increased vascular pattern complexity. Our results suggest that the circadian clock plays a key role in controlling the vascular pattern of leaves.

ARF6 and ARF8 contribute to tissue reunion in incised Arabidopsis inorescence stems

Weerasak Pitaksaringkarn, Sumie Ishiguro, Masashi Asahina, Shinobu Satoh. . . . . . . 49[PDF]

Previously, we found that auxin and wound-inducible hormones contributed to the control of tissue reunion in both the upper and lower parts of incised Arabidopsis stems by inducing the expression of ANAC071 and RAP2.6L, respectively. Here, we reveal how the expression of ANAC071 and RAP2.6L is controlled by auxin. An arf6 arf8 double mutant exhibited inhibition of cell division in pith tissue 1 week after incision; this was not true of either the single mutants or mutants in other ARF genes. ANAC071 and RAP2.6L expression was suppressed in incised stems of the arf6 arf8 mutant, but RAP2.6L expression was enhanced in non-incised stems. In addition, the expression of DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1), which encodes a jasmonic acid biosynthetic enzyme, was induced by the incision in both wild-type and arf6 arf8 stems, but overall expression level was suppressed in the arf6 arf8 mutant. We thus propose that auxin accumulation promotes ANAC071 expression via ARF6 and ARF8 activity in the upper part of incised stems, and that a reduced auxin level induces RAP2.6L expression in the lower part of the incision as a result of its release from the suppression caused by the action of ARF6 and ARF8-mediated auxin in non-incised stems. Moreover, auxin signaling via ARF6 and ARF8 is essential for jasmonic acid production, via the induction of DAD1, to increase RAP2.6L expression during tissue reunion.

A visible assay for meiotic homologous recombination in pollens of rice

Yoshinori Shingu, Yasuko Kanno, Takeshi Tokai, Takehiko Shibata, Kyo Wakasa. . . . . . . 55[PDF]

Meiotic homologous recombination generates genetic diversity for adaptations to environmental changes, and is the underlying principle of breeding by genetic crossing. Meiotic recombination in plants is solely manifested by the phenotypes of the progeny after a series of genetic crosses, and thus its assessment is laborious and time-consuming. A facile assay for meiotic recombination in higher plants, especially in crops, will innovate molecular breeding technology. In this study, we developed a fluorescence-based quantitative assay for meiotic gene conversion in rice pollens, using a pair of homologous genes: an active gene encoding cyan-fluorescent protein (CFP) as the recipient gene, and a yellowfluorescent protein (YFP) gene without a promoter as the donor gene for the conversion. Pollens emitting YFP fluorescence, representing meiotic gene conversion products, were detected at frequencies of 2.7×10−6 to 4.9×10−5 in five transgenic lines, whereas the mitotic gene conversion rate between the two genes was less than 10−6 in calli derived from one of these lines. Southern hybridization analyses of the transgenic lines revealed that the CFP and YFP genes were integrated at various independent loci in each line. This method provides a rapid technique to assess meiotic gene conversion.


Expression of genes encoding transporters and enzyme proteins in response to low-pH and high-aluminum treatments in Acacia mangium, a stress-tolerant leguminous tree

Syuhei Mizuno, Shin-ichi Ayabe, Hiroshi Uchiyama. . . . . . . 61[PDF]

Several genes of model and crop species that function in plant adaptation to acid soils have recently been characterized, but little is known about the molecular basis of the stress tolerance of woody plants. In the present study, using cell suspension cultures of Acacia mangium, a leguminous tree habituated to tropical acid soils, genes up-regulated in response to lowering the medium pH and addition of AlCl3 were screened by successive differential display and semiquantitative RT-PCRs, followed by full-length determination by RACE (rapid amplification of cDNA ends)-PCR. A total of 57 genes were shown to be induced by low-pH and/or aluminum stresses, and 44 full-length sequences were identified and cloned. They included genes of a multidrug and toxic compound extrusion transporter that secretes citrate ions to chelate aluminum, ATP-binding cassette transporters, a plasma membrane H+-ATPase, and a CYP94A, known in other plants to be responsive to low-pH and/or aluminum stresses. Genes that have not been recognized to be stress-responsive were also up-regulated by low-pH/aluminum treatments. Many of these genes were induced in the stressed A. mangium seedling roots. The cloned genes should provide the resources for the identification of the factors that play roles in the adaptation of A. mangium to acid soils.

Localization of transgene-derived friabilins in rice endosperm cells

Misa Fujiwara, Go Suzuki, Daiki Kudo, Haruna Oba, Yukio Wada, Hideo Wada, Naoki Wada, Sadequr Rahman, Kiichi Fukui, Yasuhiko Mukai. . . . . . . 67[PDF]

Puroindoline-a (Pina), puroindoline-b (Pinb) genes in the wheat hardness-locus region encode 15-kDa friabilin proteins, whose accumulation in the endosperm leads to grain softness texture. In wheat, the PINA and PINB friabilins are associated with starch granules in the endosperm cell, while there is no friabilin in rice. The rice endosperm structure consisting of compound starch granules is fundamentally different from that in wheat. We previously produced two different lines of transgenic rice plants with the large genomic fragment including Pina or Pinb of Aegilops tauschii. However, localization of exogenous friabilins in the rice endosperm cell still remains to be determined. In the present study, we stacked the two different transgenic rice lines. The F4 seeds of the stacked line, in which the homozygosity of the Pina and Pinb transgenes was checked by FISH analysis, were used for histochemical analysis of the endosperm cell. Immunodetection of PINA and PINB proteins using the Durotest antibody showed that wheat-derived friabilins were localized between compound starch granules as well as between starch granules in the rice endosperm cell. This suggests that such localization of the friabilins might prevent tight interaction between the compound starch granules and between the starch granules in the rice endosperm, leading to its soft texture.

Effects of silver nitrate on shoot regeneration of Artemisia annua L.

Caiyan Lei, Hong Wang, Benye Liu, Hechun Ye. . . . . . . 71[PDF]

In order to further increase the shoot regeneration frequency of Artemisia annua L., the effects of silver nitrate on this process was investigated in this study. Different concentration of silver nitrate was added to the shoot induction medium, which was MS basic medium containing 1.0 mgl−1 6-benzyladenine (6-BA) and 0.05 mg l−1 α-napthaleneacetic acid (NAA). When 2 mg l−1 silver nitrate was added to the shoot induction medium, the shoot induction frequency and shoot number per explants was significantly higher than that of the control (without silver nitrate). In addition, silver nitrate at all the tested concentrations could significantly reduce callus formation of the explants. Silver nitrate had also positive influence on shoot elongation in the first 20 days. Furthermore, silver nitrate did not affect the sensitivity of A. annua shoots to Kanamycin (KM); therefore, silver nitrate could be used to improve shoot regeneration capacity and frequency in A. annua genetic transformation.

In vitro evaluation of dehydration tolerance in AtDREB1A transgenic potatoes

Huu Duc Huynh, Takayoshi Shimazaki, Mie Kasuga, Kazuko Yamaguchi-Shinozaki, Akira Kikuchi, Kazuo N. Watanabe. . . . . . . 77[PDF]

Abiotic stresses have negative effects on potato growth and production. To enhance the abiotic stress tolerance of the commercial potato cultivar, Desiree, rd29A::AtDREB1A transgenic lines have been developed. The salinity and freeze tolerance of these transgenic lines has previously been demonstrated; however, their dehydration tolerance remains to be elucidated. First of all, we have tackled the reproducible tolerance evaluation methodology, which have been the hurdle for selecting the stable dehydration tolerant phenotypes in potato. A novel in vitro method was developed using the rotary liquid culture combined with PEG. This method enhanced oxygen diffusion into the medium and diminished root damage/injury during plant transfer, thereby reducing the side effects caused by hypoxia and penetration of osmotica. In the present study, we evaluated the dehydration tolerance of twelve transgenic potato lines, and seven of the transgenic lines showed enhanced dehydration tolerance in comparison with the non-transgenic line. However, we observed growth retardation in some dehydration tolerant lines. Therefore, balance between the dehydration tolerance and growth retardation of the transgenic plants should be considered. Four of seven of the transgenic lines displayed enhanced dehydration tolerance without growth retardation, and may represent good candidates for practical application.

Sequence variations in the flavonoid 3,5-hydroxylase gene associated with reddish ower phenotypes in three delphinium varieties

Natsuki Miyagawa, Yuzo Nishizaki, Taira Miyahara, Mitsutoshi Okamoto, Yukio Hirose, Yoshihiro Ozeki, Nobuhiro Sasaki. . . . . . . 83[PDF]

Although delphiniums are famed for their blue flowers, a few varieties display reddish flowers, such the pale-pink garden varieties ‘Ehime Kou 9 (Kou 9)’ and ‘F1 Super Happy Pink (SHP),’ and the orange-red flowered species Delphinium nudicaule (NDC). Blue delphinium flowers contain a delphinidin-derived anthocyanin, whereas the three varieties mentioned above have anthocyanins derived from the aglycone pelargonidin in their sepals. As flavonoid 3′,5′-hydroxylase (F3′5′H) is known to be the key enzyme in biosynthesis of delphinidin, we compared the structure and function of the F3′5′H gene in three varieties of delphiniums with reddish flowers to one that has blue flowers. We found that the F3′5′H gene of ‘Kou 9’ had a point mutation that generated a stop codon in the first exon. Genomic PCR analysis indicated that the ‘SHP’ variety lacked F3′5′H. Although the nucleotide sequence of the F3′5′H open reading frame was identical in ‘NDC’ to that of the wild type, it lacked an intron and no F3′5′H transcripts could be detected in this variety. We conclude that the red flower phenotypes of these delphiniums derive from independent mutations of the F3′5′H gene. This is the first report on the delphinium F3′5′H gene. At a practical level, these mutations should be of value for breeding new pink and red flower varieties.