—Invited Paper—

Measuring plant colors

Ichiro Kasajima . . . . . . . 63[PDF]

Plant colors such as ‘green leaf ’ and ‘red apple’ are often described based on human sense, even in scientific papers. On the other hand, colors are measured based on colorimetric principles in some papers, especially in the studies of horticultural plants. The science of color measurements (‘colorimetry’) is not included in any of the popular lectures in schools and universities, thus the principles of color measurements would not be understood by most researchers. The present review will overview the principles of colorimetry, and will introduce colorimetric methods which can be used for scientific measurement of plant colors. That is to say, the reflection spectrum of visible light (380–780 nm) is measured at 5-nm intervals on the surface of leaves or petals in ‘Spectrometric Color Measurement’ (SCM). The spectral data is multiplied with RGB or XYZ color matching functions and integrated to obtain RGB or XYZ intensities. Alternatively, approximate RGB values are directly obtained in ‘Photographic Color Measurement’ (PCM). RGB/XYZ intensities are further calculated to obtain ‘hue’, ‘saturation’, and ‘lightness’, the three factors of colors. Colorimetric insights into genetic regulations (such as MYB gene) and physiological regulations (such as alexandrite effect) of plant colors are also described.

Original Papers

Fractal based complexity analysis of wheat root system under different heavy metals

Tao Li, Uma Maheswari Rajagoplan, Hirofumi Kadono . . . . . . . 77[PDF]

In this study, fractal geometry was applied to characterize the complexity of the root system morphology of wheat plants under the exposure of heavy metals, namely cadmium (Cd), copper (Cu) and zinc (Zn). We proposed a measure called, relative complexity index (RCI), a ratio based on fractal dimension (FD) before and after exposure to heavy metals. FDs were calculated by box-counting method with digitized and skeletonized images of roots of wheat plants cultivated in hydroculture system. RCI, and relative weight were mesuared under different concentrations of Cd (0.001, 0.01 and 0.05 mM), Cu (0.016, 0.4 and 1.2 mM) and Zn (0.3 and 0.75 mM). Results showed significant reduction of RCI for Cd stress with 0.01 and 0.05, all Cu concentrations and promotion at all zinc concentrations. In comparison, no statistically significant changes were found in conventional relative weight measurement at low concentrations of Cu, Cd and Zn. RCI were more sensitive and were reliable in reflecting the influence of heavy metals than the conventional measure. These results imply that RCI can be an effective measure of the negative and positive effects of heavy metals on the development of complexity of root system under heavy metal exposures.

Aberrant endosperm formation caused by reduced production of major allergen proteins in a rice flo2 mutant that confers low-protein accumulation in grains

Hiroshi Teramura, Kyoko Kondo, Masato Suzuki, Hiroaki Kobayashi,Tsutomu Furukawa, Hiroaki Kusanoa, Hiroaki Shimada . . . . . . . 85[PDF] [Supplement]

Rice flo2 mutation produces grains showing a reduced amount of storage proteins. Using Nipponbare and the flo2 mutant, we created rice transformants that showed defective production of major allergen proteins RA14 and RA33 (14–16 kDa and 33 kDa allergen proteins, respectively) by RNAi introduction. The knock-down transformant generated using Nipponbare showed greatly reduced accumulation of both allergen proteins, normal growth, and production of a sufficient amount of normal-shaped seeds. F1 seeds were obtained by crossing between the transformants containing RNAi genes to RA14 and RA33, and showed decreased accumulation of both proteins. However, a peculiar phenotype was observed in the flo2 transformants that lacked accumulation of RA14 or RA33. They showed significantly reduced fertility. A wrinkled grain feature was found on the transformant lacking accumulation of RA14. F1 seeds obtained by crossing these transformants showed significantly lower fertility. F2 seeds showed decreases in both allergen proteins but morphological abnormality with small and severely wrinkled features. These results indicated that it is hard to obtain any transformant lacking accumulation of these allergen proteins using the flo2 mutant, whereas a knock-down transformant of both allergen protein genes was obtained when a wild-type Nipponbare was used as a host. These facts strongly suggest that RA14 and RA33 have some roles in rice seeds.

Micropropagation of Solanum quitoense var. quitoense by apical bud, petiole and hypocotyl culture

Bernardo Gutiérrez, María Mercedes Cobo, Miguel Orellana, Joely Vega, Venancio Arahana, Viviana Jaramillo, María de Lourdes Torres . . . . . . . 91[PDF] [Supplement]

The development of in vitro propagation methods can improve the current commercial use and conservation of plants like naranjilla (Solanum quitoense), a distinctive Andean crop and key emerging agricultural product. In the present study, we report in vitro culture protocols for naranjilla apical buds, hypocotyls and petioles. In apical bud culture, MS medium supplemented with 0.10 mg l−1 1-naphtaleneacetic acid (NAA) produced longer plantlets with greater number of leaves. Hypocotyl culture yielded higher number of shoots when using older explants in MS medium supplemented with different combinations of NAA, 6-benzylaminopurine (BAP) and gibberellic acid (GA3). Petiole culture produced a significantly higher number of shoots per explant, with more abundant and bigger leaves, when using MS medium supplemented with 0.02 mg l−1 NAA, 4.50 mg l−1 BAP and 1.00 mg l−1 GA3. A factorial analysis reveals that the interaction between GA3 and NAA/BAP plays an important role in shoot regeneration. These results provide new tools for the in vitro regeneration of naranjilla plants, improving on previously reported protocols for this species by using alternative explant types and regeneration protocols.

—Invited Paper—

Monitoring autophagy in rice tapetal cells during pollen maturation

Shigeru Hanamata, Jumpei Sawada, Bunki Toh, Seijiro Ono, Kazunori Ogawa, Togo Fukunaga, Ken-Ichi Nonomura, Takamitsu Kurusu, Kazuyuki Kuchitsu . . . . . . . 99[PDF] [Supplement1] [Supplement2]

We have previously shown that autophagy is required for post meiotic anther development including programmed cell death-mediated degradation of the tapetum and pollen maturation in rice. However, the spatiotemporal dynamics of autophagy in the tapetum remain poorly understood. We here established an in vivo imaging technique to analyze the dynamics of autophagy in rice tapetum cells by expressing green fluorescent protein-tagged AtATG8, a marker for autophagosomes. 3D-imaging analysis revealed that the number of autophagosomes/autophagy-related structures is extremely low at the tetrad stage (stage 8), and autophagy is dramatically induced at the uninucleate stages (stage 9–10) throughout the tapetal cells during anther development. The present monitoring system for autophagy offers a powerful tool to analyze the regulation of autophagy in rice tapetal cells during pollen maturation.

Short Communications
—Invited Paper—

Involvement of the membrane-localized ubiquitin ligase ATL8 in sugar starvation response in Arabidopsis

Yongming Luo, Shoki Aoyama, Yoichiro Fukao, Yukako Chiba, Takeo Sato, Junji Yamaguchi . . . . . . . 107[PDF] [Supplement]

As major components of the ubiquitin system, ubiquitin ligases mediate the transfer of ubiquitin to specific target substrates, thereby playing important roles in regulating a wide range of cellular processes. The Arabidopsis Tóxicos en Levadura (ATL) family is a group of plant-specific RING-type ubiquitin ligases with N-terminal transmembrane-like domains. To date, 91 ATL isoforms have been identified in the Arabidopsis genome, with some reported to regulate plant responses to environmental stresses. However, the functions of most ATLs remain unclear. This study showed that ATL8 isa sugar starvation response gene and that ATL8 expression was significantly increased by sugar starvation conditions butrepressed by exogenous sugar supply. The ATL8 protein was found to possess ubiquitin ligase activity in vitro and to localize to membrane-bound compartments in plant cells. In addition, Starch Synthase 4 was identified as a putative interactor with ATL8, suggesting that ATL8 may be involved in modulating starch accumulation in response to sugar availability. These findings suggest that ATL8 functions as a membrane-localized ubiquitin ligase likely to be involved in the adaptation of Arabidopsis plants to sugar starvation stress.


De novo transcriptome analysis of needles of Thujopsis dolabrata var. hondae

Shiro Suzuki, Hideyuki Suzuki, Koji Tanaka, Masaomi Yamamura, Daisuke Shibata, Toshiaki Umezawa . . . . . . . 113[PDF] [Supplement1] [Supplement2] [Supplement3] [Supplement4] [Supplement5]

Podophyllotoxin is a starting material of the semisynthetic anticancer medicines etoposide, teniposide, and etopophos. The major plant source of podophyllotoxin is rhizomes of Podophyllum hexandrum, which is a Himalayan endangered species; therefore, alternative sources of podophyllotoxin or bioproduction systems have been pursued to avoid exploiting this limited natural resource. In this paper, we report de novo transcriptome analysis of Thujopsis dolablata var. hondae, which accumulates the podophyllotoxin derivatives (deoxypodophyllotoxin and β-peltatin A methyl ether) in its needles. We analyzed transcriptomes of the T. dolablata var. hondae young needles to obtain the sequences that putatively encode O-methyltransferases, cytochrome P450s, and a 2-oxoglutarate dependent dioxygenase because these protein families are responsible for podophyllotoxin-related compound formation in P. hexandrum. The resulting transcriptomes contained considerable numbers of coding sequences classified into the three protein families. Our results are a genetic basis for identifying genes involved in the biosynthesis of podophyllotoxin and related compounds and also for future metabolic engineering of podophyllotoxin in heterologous hosts.

Agroinfiltration-based efficient transient protein expression in leguminous plants

Takuya Suzaki, Mai Tsuda, Hiroshi Ezura, Brad Day, Kenji Miura . . . . . . . 119[PDF] [Supplement]

Transient protein expression is an effective tool to rapidly unravel novel gene functions, such as transcriptional activity of promoters and sub-cellular localization of proteins. However, transient expression is not applicable to some species and varieties because of insufficient expression levels. We recently developed one of the strongest agroinfiltrationbased transient protein expression systems for plant cells, termed ‘Tsukuba system.’ About 4 mg/g fresh weight of protein expression in Nicotiana benthamiana was obtained using this system. The vector pBYR2HS, which contains a geminiviral replication system and a double terminator, can be used in various plant species and varieties, including lettuces, eggplants, tomatoes, hot peppers, and orchids. In this study, we assessed the applicability of the Tsukuba system to several species of legumes, including Lotus japonicus, soybean Glycine max, and common bean Phaseolus vulgaris. The GFP protein was transiently expressed in the seedpods of all examined legume species, however, protein expression in leaves was observed only in P. vulgaris. Taken together, our system is an effective tool to examine gene function rapidly in several legume species based on transient protein expression.