Plant Biotechnology Vol.34 No.2



Ubiquitin related enzymes and plant-specific ubiquitin ligase ATL family in tomato plants

Xingwen Li, Yoko Hasegawa, Yu Lu, Takeo Sato. . . . . . . 71 [PDF]

Ubiquitination is one of the fundamental post-translational modifications of proteins with ubiquitin, a conserved 76-amino acid protein present in eukaryotes, which is catalyzed by ubiquitin ligase. Compared with humans, the number of ubiquitin ligase genes is nearly double in plant species such as Arabidopsis and rice, suggesting that this enzyme plays critical roles in many aspects of plant growth, including development and abiotic and biotic environmental stress responses. In addition to its fundamental activities in eukaryotic cells, ubiquitin signaling mediates plant specific cellular functions, including phytohormone response, seed and fruit development, and biotic and abiotic stress responses. The ATL family is a RING-H2 type ubiquitin ligase widely conserved in plant species. We previously showed that the plant specific ubiquitin ligase ATL31 regulates the carbon/nitrogen-nutrient response and pathogen resistance in Arabidopsis, and we identified and characterized the basic biochemical function of an ATL31 homologue in tomato plants (Solanum lycopersicum L.). This protein, called SlATL31, may act as a ubiquitin ligase in tomato fruit. The tomato is a major crop plant and a model system for fleshy fruit development. This review provides an overview of the ubiquitin ligases and related enzymes, and highlights the ubiquitin ligase ATL family in tomato plants.

Original Papers

Tumorigenesis inheritance from the putative progenitor species of Nicotiana rustica

Haruka Takanashi, Wataru Marubashi. . . . . . . 79 [PDF]

Hybrid seedlings from crosses of Nicotiana rustica×N. langsdorffii and N. rustica×N. alata show tumors including teratomas and vitrification. In the present study, we attempted to elucidate the genetic background leading to tumorigenesis and vitrification from the viewpoint of the amphidiploidy of N. rustica. The species N. undulata, N. paniculata, and N. knightiana have been suggested to be the progenitors of N. rustica or closely related to its progenitors. We tested tumorigenesis in interspecific hybrids between these putative progenitors of N. rustica and N. langsdorffii or N. alata, which are the species in section Alatae. The hybrid seedlings were cultured in test tubes and their morphological characteristics were observed. According to previous reports, most of the hybrid seedlings from the crosses N. rustica×N. langsdorffii and N. rustica×N. alata formed tumors and showed vitrification. In crosses with every putative progenitor of N. rustica, a portion of hybrid seedlings formed tumors and showed vitrification. These observations suggested that N. rustica inherited the factors leading to expression of abnormal symptoms from its putative progenitors.
We also observed the influence of high temperature on the expression of abnormal symptoms of hybrid seedlings from the cross N. rustica×N. alata. While these hybrids developed teratomas and other tumors at 28°C, when cultured at 34°C, they did not show any abnormalities. This is the first report to show that phenotypic abnormalities in hybrid seedlings of N. rustica×N. alata are temperature sensitive.

Constitutive activation of plant-specific RAB5 GTPase confers increased resistance against adapted powdery mildew fungus

Noriko Inada, Kazuo Ebine, Emi Ito, Akihiko Nakano, Takashi Ueda. . . . . . . 89 [PDF]

The obligate biotrophic fungal pathogens that cause powdery mildew disease establish infection in living host cells by modifying host cellular functions, including membrane trafficking. Previously, we reported that two Arabidopsis thaliana RAB5 GTPases, plant-specific ARA6/RABF1 and canonical ARA7/RABF2b, accumulate at the extrahaustorial membrane (EHM), which surrounds the specialized infection hypha called the haustorium. In this study, we examined the role of ARA6 and ARA7, which regulate distinctive endosomal trafficking pathways, in plant–powdery mildew fungus interactions. Although ARA6- and ARA7-related mutants did not exhibit altered susceptibility to the A. thaliana–adapted powdery mildew fungus Golovinomyces orontii, overexpression of constitutively active ARA6, but not constitutively active ARA7, repressed proliferation of G. orontii. The repression of fungal proliferation was associated with accelerated formation of the callosic encasement around the haustorium. Furthermore, microscopic observation revealed an accumulation of the constitutively active form of ARA6, but not active ARA7, at the EHM. These results indicate that plant-specific ARA6 has a specific role in plant–powdery mildew fungus interaction, and manipulation of ARA6 activity could be a novel tool to overcome this plant disease.

Rapid identification of apple (Malus×domestica Borkh.) S alleles using sequencing-based DNA marker APPLid

Ichiro Kasajima, Teppei Kikuchi, Nobuyuki Yoshikawa. . . . . . . 97 [PDF]

All apple cultivars harbor the trait called self-incompatibility. Self-incompatibility represents that the pistils of the flowers are not successfully fertilized with own, the same cultivar’s pollens. Compatibility or incompatibility of apple flowers are determined by S alleles. For example, the most popular apple cultivar ‘Fuji’ possesses the S1 and S9 alleles (S1S9). Thus, ‘Fuji’ is incompatible with S1S9 cultivars, but is compatible with the cultivars possessing different combinations of S alleles such as S2S7 and S1S7. Apple S alleles have been identified by performing a series of allele-specific PCR amplifications, to detect more than ten different S alleles separately. Here, we developed a new type of sequencing-based DNA marker of the apple S-RNase gene, which identifies S alleles. This DNA marker was named APPLid (apple S-allele identifier). A 53-base region in the first coding sequence of S-RNase is the target of APPLid sequencing. Variation in nucleotide sequences in this APPLid sequence enables allele identifications. This region is amplified from apple genomic DNA by using a pair of degenerate primers. The forward primer is attached with ‘DS5 adaptor.’ After PCR amplification, electrophoresis and gel extraction of 177-bp DNA fragments, APPLid sequence is determined by direct sequencing with a sequencing primer. The APPLid sequences of 20 apple cultivars completely matched their S alleles, which include triploid cultivars. In conclusion, APPLid identifies apple S alleles (S1, S2, S3, S4, S5, S7, S9, S10, S20, S24, S25, S26, S27 and S28, so far) just by a single sequencing analysis.

Short Communication

Phosphorylation of rice sucrose synthase isoforms promotes the activity of sucrose degradation

Haruka Takeda, Muneyuki Niikura, Azusa Narumi, Hiromi Aoki, Tadamasa Sasaki, Hiroaki Shimada. . . . . . . 107 [PDF]

Sucrose is utilized as an initial material for production of the storage substances. Sucrose synthase reversibly catalyzes reactions of the sucrose degradation and its synthesis between sucrose with UDP and UDP-glucose with fructose. They also had the activity of the reactions for sucrose degradation of sucrose with ADP, and sucrose synthesis from ADP-glucose and fructose. Rice has three representative isoforms of sucrose synthase, Rsus1, Rsus2, and Rsus3, in which Rsus1 and Rsus3 are highly expressed in developing seeds. These three isoforms were phosphorylated by SPK, a calcium-dependent protein kinase. By phosphorylation, they showed increase of their reactivity for sucrose degradation on both reactions using UDP and ADP. In contrast, the synthetic activity of these isoforms was not altered by phosphorylation in any cases of the reactions with UDP-glucose and ADP-glucose. These results indicated that phosphorylation of sucrose synthase isoforms selectively led to enhance the reactivity for sucrose degradation.


Expression and protein localization analyses of Arabidopsis GLABRA3 (GL3) in tomato (Solanum lycopersicum) root epidermis

Rumi Tominaga-Wada, Kyosuke Ota, Naoto Hayashi, Koh Yamada, Ryosuke Sano, Takuji Wada. . . . . . . 115 [PDF]

The arrangement of root hair and non-hair cells in the root epidermis provides a useful model for understanding the cell fate determination system in plants. A network of related transcription factors, including GLABRA3 (GL3), influences the patterning of cell types in Arabidopsis. GL3 is expressed primarily in root hair cells and encodes a bHLH transcription factor, which inhibits root hair differentiation in Arabidopsis root epidermis. By transforming the GL3 promoter::GFP into tomato, we demonstrated that the Arabidopsis GL3 promoter can function in tomato root epidermis. GFP fluorescence was observed in almost all root epidermal cells in the GL3::GFP transgenic tomato plants, indicating that all root epidermal cells of tomato possess root hair cell identity similar to that of Arabidopsis root hair cells. This is consistent with the phenotype of the tomato root, in which all epidermal cells produce root hairs. Moreover, we observed the localization of a GL3:GFP fusion protein in GL3::GL3:GFP transgenic tomato; although GL3 is known to exclusively localize in non-hair cell nuclei in Arabidopsis root epidermis, GL3:GFP fluorescence was detected not in the nuclei but in the cytoplasm of transgenic tomato epidermal cells. These results suggest that the nuclear localization mechanism differs between tomato and Arabidopsis.

Isolation of GFP-tagged plasma membrane protein from Arabidopsis egg cells

Tomoko Igawa, Lixy Yamada, Hitoshi Sawada, Toshiyuki Mori. . . . . . . 119 [PDF]

Angiosperms possess a double fertilization system for sexual reproduction. Double fertilization is regulated by interactions among proteins localized in the plasma membrane of each sex gamete. A few plasma membrane resident proteins regulating double fertilization have been identified in male gametes. In contrast, no fertilization regulators in female gamete plasma membrane have been identified, largely due to difficulties in the isolation and collection of female gametes. We had produced Arabidopsis transgenic plant pDD45::GFP-AtPIP2;1 where the egg cell plasma membrane was specifically labeled with GFP (Igawa et al. 2013). The protein extract derived from approximately 200 pistils, which contained unfertilized and mature egg cells, was subjected to immunoprecipitation using anti-GFP antibody. As a result, both GFP and AtPIP2;1 were specifically detected in immunoprecipitated proteins from pistil tissues of pDD45::GFP-AtPIP2;1 transgenic plant, but not in those of wild type pistils. It was revealed that specific proteins expressed in the egg cells were successfully isolated from pistil cell population. The method described here showed the feasibility of isolating specific egg cell plasma membrane protein without gamete isolation and collection procedures.

In vitro regeneration and Agrobacterium-mediated transformation of male- sterile marigold (Tagetes erecta L.)

Nuoendagula, Mana Narushima, Mikiko Uesugi, Yasushi Murai, Yoshihiro Katayama, Yosuke Iimura, Shinya Kajita. . . . . . . 125 [PDF]

In this study, we devised a method for the in vitro regeneration and subsequent genetic transformation of male sterile marigold. To our knowledge, this is the first report of generation of transgenic plants with a single genotype of marigold via Agrobacterium-mediated transformation. We obtained four transgenic lines from two independent experiments with 496 leaf explants, which were inoculated by an Agrobacterium strain LBA4404 harboring the plasmid, pIG121-Hm. Although the efficiency of the transformation in our system was low, stable expression of uidA gene in adventitious shoots and compound leaves could be detected in β-glucuronidase histochemical analysis. This protocol contributes to the progress of genetic studies and molecular breeding of this species.