Plant Biotechnology Vol.33 No.4

Contents

Preface

Environmental responses of plants: Biological interactions in the homogenous population or community (mixed populations)

Tsuyoshi Mizoguchi, Masatoshi Funabashi. . . . . . . 211 [PDF]

Review

Synecological farming: Theoretical foundation on biodiversity responses of plant communities

Masatoshi Funabashi. . . . . . . 213 [PDF]

A novel farming method, namely synecological farming (synecoculture in short), based on theory and observation of synecology has been proposed as total optimization of productivity, product quality, environmental load and adaptation capacity to climate change. Synecoculture is designed on a variety of environmental responses within ecological optimum in high-density mixed polyculture where various edible species were intentionally introduced. The whole methodology can be considered as anthropogenic augmentation of ecosystem functioning that promotes dynamic biodiversity–productivity relationship prevalent in natural ecosystems.
 In this review we summarize the theoretical foundation to provide a systematic definition of synecoculture and clarify the relationship with existing farming methods. We also collate previously reported analyses of organic and mineral components in farm products, and outline their physiological characteristics and functions in response to culture environments.

Original Papers

The ERF transcription factor EPI1 is a negative regulator of dark-induced and jasmonate-stimulated senescence in Arabidopsis

KwiMi Chung, Toshitsugu Nakano, Sumire Fujiwara, Nobutaka Mitsuda, Namie Otsuki, Yayoi Tsujimoto-Inui, Yuki Naito, Masaru Ohme-Takagi, Kaoru Suzuki. . . . . . . 235 [PDF]

Identification of the factors involved in the regulation of senescence and the analysis of their function are important for both a biological understanding of the senescence mechanism and the improvement of agricultural productivity. In this study, we identified an ERF gene termed “ERF gene conferring Postharvest longevity Improvement 1” (EPI1) as a possible regulator of senescence in Arabidopsis. We found that EPI1 possesses transcriptional repression activity and that the transgenic plants overexpressing EPI1 and expressing its chimeric repressor, EPI1-SRDX, commonly suppressed the darkness-induced senescence in their excised aerial parts. These transgenic plants additionally maintained a high level of chlorophyll, even after the methyl jasmonate (MeJA) treatment, which stimulated senescence in the dark. In addition, we found that senescence-induced and -reduced genes are down- and upregulated, respectively, in the MeJA-treated transgenic plants under darkness. Our results suggest that EPI1 functions as a negative regulator of the dark-induced and JA-stimulated senescence.

WUSCHEL-RELATED HOMEOBOX 2 is a transcriptional repressor involved in lateral organ formation and separation in Arabidopsis

KwiMi Chung, Shingo Sakamoto, Nobutaka Mitsuda, Kaoru Suzuki, Masaru Ohme-Takagi, Sumire Fujiwara. . . . . . . 245 [PDF]

In this study, we characterized the function of WUSCHEL-RELATED HOMEOBOX 2 (WOX2) using overexpression, CRES-T, and VP16 fusion techniques. Although the function of WOX2 has been described mainly in embryogenesis, it was unclear whether it also plays a role in the post-embryogenic developmental stage. We found that WOX2 has transcriptional repression activity and that either overexpression of WOX2 or expression of its chimeric repressor causes severe growth defects and other morphological phenotypes by impairing plant organ formation and separation. By contrast, VP16-fused WOX2-expressing plants did not display such severe phenotypic defects. In addition, some of them displayed phenotypic defects such as fusion of organs and induction of undifferentiated cells in the boundary regions of organs where GUS staining was clearly observed in the proWOX2:GUS transgenic plants. We suggest that WOX2 is involved in regulation of lateral organ formation and separation during the post-embryogenic development processes.

ANAC075, a putative regulator of VASCULAR-RELATED NAC-DOMAIN7, is a repressor of flowering

Sumire Fujiwara, Nobutaka Mitsuda. . . . . . . 255 [PDF]

Fine-tuning of flowering timing is crucial for plants to survive and leave offspring and depends on various endogenous and environmental factors. Here we report the identification of a vascular transcription factor, ANAC075, a putative regulator of VASCULAR-RELATED NAC-DOMAIN7 (VND7), as a negative regulator of flowering in Arabidopsis. Loss of function of ANAC075 causes the upregulation of floral integrator genes and early flowering under both long- and short-day conditions. ANAC075 promoter activity was detected in vascular tissues, including phloem. Previous reports suggested that ANAC075 is a transcriptional activator involved in the secondary cell wall formation, implying that the promotion of flowering time in anac075 mutants is caused by the disruption of flowering-time gene regulation in phloem and/or vascular tissue formation.

Effects of varying light quality from single-peak blue and red light-emitting diodes during nursery period on flowering, photosynthesis, growth, and fruit yield of everbearing strawberry

Hideo Yoshida, Daiki Mizuta, Naoya Fukuda, Shoko Hikosaka, Eiji Goto. . . . . . . 267 [PDF]

We studied the effects of varying light quality on the flowering, photosynthetic rate and fruit yield of everbearing strawberry plants (Fragaria×ananassa Duch. ‘HS138’), which are long-day plants, to increase the efficiency of fruit production in plant factories. The plants were grown under continuous lighting using three types of blue and red LEDs (blue light peak wavelength: 405, 450, and 470 nm; red light peak wavelength: 630, 660, and 685 nm) during the nursery period. All blue light from the various peak LED types promoted more flowering compared with red light (630 and 660 nm except for 685 nm). The longer wavelength among the red light range positively correlated with earlier flowering, whereas the number of days to anthesis did not significantly differ among blue LED treatment wavelengths, irrespective of peak wavelength. The result of a similar experiment using the perpetual flowering Fragaria vesca accession Hawaii-4 representing a model strawberry species showed almost the same pattern of flowering response to light quality. These results suggest that long-day strawberry plants show similar flowering response to light quality. The photosynthetic rate under red light (660 nm) was higher than that under blue light (450 nm). However, the plants grown under red light showed lower photosynthetic capacity than those grown under blue light. Although the light color used to grow the seedlings showed no difference in the daily fruit production, blue light irradiation during the nursery period hastened harvesting because of the advance in flowering.

Naturally grown rucola, Eruca sativa, contains more α-linolenic acid than conventionally grown rucola

Kousaku Ohta, Tsuyoshi Takeshita, Masatoshi Funabashi, Shoji Oda. . . . . . . 277 [PDF]

The ratio of dietary n-6/n-3 polyunsaturated fatty acids is higher in the modern Western meal when compared with that of the Paleolithic era. Evidences have been accumulating that the extremely high ratio of dietary n-6/n-3 polyunsaturated fatty acids increases the risk of disease and the deterioration of physical conditions among humans. In this study, the ratio of linoleic acid (C18:2, n-6) and α-linolenic acid (C18:3, n-3) of rucola, Eruca sativa, was compared between naturally grown samples and conventional farming products (n=3 for each). We found that the naturally grown rucola contained significantly higher amount of α-linolenic acid (p=0.026), resulting in the lower ratio of linoleic acid and α-linolenic acid than the conventional (p=0.016). This finding suggests that vegetables cultured in conventional farming method could decrease the health promotion effects that the vegetables originally possess in natural environment.

Distribution of RAB5-positive multivesicular endosomes and the trans-Golgi network in root meristematic cells of Arabidopsis thaliana

Emi Ito, Tomohiro Uemura, Takashi Ueda, Akihiko Nakano. . . . . . . 281 [PDF]

In plant cells, the trans-Golgi network (TGN) is known to act as the early endocytic compartment, whereas RAB5-localizing multivesicular endosomes (MVEs) act as the later compartment. Land plants and certain green algal species possess plant-unique RAB5 homologs (ARA6/RABF1 in Arabidopsis thaliana) in addition to the orthologs of animal RAB5 (RHA1/RABF2a and ARA7/RABF2b in A. thaliana), and these two RAB5 members reside in substantially overlapping but different subpopulations of MVEs. Several studies indicate that the TGN and MVEs are closely related; however, the distribution of the two RAB5 groups in relation to the TGN remains elusive. Here, we quantitatively showed that ARA6 and ARA7 are closely associated with the TGN, and the subpopulation of ARA6 and ARA7 overlaps with the TGN in the root epidermal cells of A. thaliana.

Identification of barley landrace genotypes with contrasting salinity tolerance at vegetative growth stage

Mariem Ben Chikha, Kamel Hessini, Rim Nefissi Ourteni, Abdelwahed Ghorbel, Nejia Zoghlami. . . . . . . 287 [PDF]

In the present study, we evaluated the behavior of 21 Tunisian barley landraces under salt stress. The evaluation was performed using 14 morphological and physiological traits at vegetative growth stage under severe salt stress (200 and 250 mM). A multivariate analysis was used in order to select the genotypes with contrasting behavior towards salinity and to identify the major traits conferring salinity tolerance. According to the PCA analyses the genotypes exhibited diverse behavior with the salt stress concentration, indeed 3 different clustering profiles were obtained. Eleven quantitative characters were considered the most pertinent for the ranking of genotypes for salt tolerance. Among them the total fresh weight and the net CO2 assimilation rate were the most discriminating descriptors at 250 mM NaCl. These parameters allowed as the identification of the contrasting pair genotypes toward salinity. “Testour“ was classified as the most sensitive and “Enfidha” the most tolerant toward salinity stress. These findings would be of great relevance in breeding programs.

The FT-like gene PehFT in petunia responds to photoperiod and light quality but is not the main gene promoting light quality-associated flowering

Atsuko Tsukamoto, Tadayoshi Hirai, Dong Poh Chin, Masahiro Mii, Tsuyoshi Mizoguchi, Daiki Mizuta, Hideo Yoshida, Jorunn E. Olsen, Hiroshi Ezura, Naoya Fukuda. . . . . . . 297 [PDF]

In Arabidopsis, flowering is delayed under red light and induced under far red light and blue light. Studies suggest that the florigen, FLOWERING LOCUS T, is involved in the control of light quality-associated flowering in Arabidopsis. In petunia, similar to Arabidopsis, flowering is delayed under red light and induced under blue light, however its mechanism still remains unknown. Here we isolated a gene which has 75% amino acid sequence similarity with Arabidopsis FT (AtFT), named PehFT. By overexpressing PehFT in Arbidopsis and petunia, we tested its ability to induce flowering. Also, by conducting expression analyses of PehFT under different light quality treatments, we tested its response to light quality. We concluded that PehFT, like AtFT, is a gene which responds to photoperiod and light quality, but unlike AtFT, is not the main gene controlling the light quality-associated flowering.

Short Communication

Cellular and developmental function of ACAP type ARF-GAP proteins are diverged in plant cells

Satoshi Naramoto, Tomoko Dainobu, Hiroki Tokunaga, Junko Kyozuka, Hiroo Fukuda. . . . . . . 309 [PDF]

Vesicle transport is crucial for various cellular functions and development of multicellular organisms. ARF-GAP is one of the key regulators of vesicle transport and is diverse family of proteins. Arabidopsis has 15 ARF-GAP proteins and four members are classified as ACAP type ARF-GAP proteins. Our previous study identified that VASCULAR NETWORK DEFECTIVE3 (VAN3), an ACAP ARF-GAP, played crucial roles in leaf vascular formation. However, it remains question how other members of plant ACAP ARF-GAPs function in cellular and developmental processes. To characterize these, we analyzed spatial expression pattern and subcellular localization of VAN3 and three other ACAPs, so called VAN3-like proteins (VALs). Expression pattern analysis revealed that they were expressed in distinctive developmental processes. Subcellular localization analysis in protoplast cells indicated that in contrast to VAN3, which localizes on trans-Golgi networks/early endosomes (TGNs/EEs), VAL1 and VAL2 were localized on ARA6-labelled endosomes, and VAL3 resided mainly in the cytoplasm. These results indicated that VAN3 and VALs are differently expressed in a tissue level and function in different intracellular compartments, in spite of their significant sequence similarities. These findings suggested functional divergence among plant ACAPs. Cellular localizations of all members of animal ACAP proteins are identical. Therefore our findings also suggested that plant evolved ACAP proteins in plant specific manner.

Notes

A loss-of-function mutation in the DWARF4/PETANKO5 gene enhances the late-flowering and semi-dwarf phenotypes of the Arabidopsis clock mutant lhy-12;cca1-101 under continuous light without affecting FLC expression

Syunji Suzuki, Kana Miyata, Miyuki Hara, Kanae Niinuma, Hirokazu Tsukaya, Masahide Takase, Ryosuke Hayama, Tsuyoshi Mizoguchi. . . . . . . 315 [PDF]

The circadian clock plays important roles in the control of photoperiodic flowering in Arabidopsis. Mutations in the LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) genes (lhy;cca1) accelerate flowering under short days, whereas lhy;cca1 delays flowering under continuous light (LL). The lhy;cca1 mutant also exhibits short hypocotyls and petioles under LL. However, the molecular mechanisms underlying the regulation of both flowering time and organ lengths in the LHY/CCA1-dependent pathway are not fully understood. To address these questions, we performed EMS mutagenesis of the lhy-12;cca1-101 line and screened for mutations that enhance the lhy;cca1 phenotypes under LL. In this screen, we identified a novel allele of dwarf4 (dwf4) and named it petanko 5 (pta5). A similar level of enhancement of the delay in flowering was observed in these two dwf4 mutants when combined with the lhy;cca1 mutations. The lhy;cca1 and dwf4 mutations did not significantly affect the expression level of the floral repressor gene FLC under LL. Our results suggest that a defect in brassinosteroid (BR) signaling delayed flowering independent of the FLC expression level, at least in plants with the lhy;cca1 mutation grown under LL. The dwf4/pta5 mutation did not enhance the late-flowering phenotype of plants overexpressing SVP under LL, suggesting that SVP and BR function in a common pathway that controls flowering time. Our results suggest that the lhy;cca1 mutant exhibits delayed flowering due to both the BR signaling-dependent and -independent pathways under LL.

Density effects on late flowering mutants of Arabidopsis thaliana under continuous light

Kiwako Yamamoto, Kei Takahashi, Miyuki Hara, Kana Miyata, Ryosuke Hayama, Tsuyoshi Mizoguchi. . . . . . . 323 [PDF]

In general, plant growth is inhibited under high-density conditions, while it is promoted under low-density conditions. This is known as the “density effect”. Growing plants at high densities is often associated with an accelerated flowering time. Three major pathways [the long day (LD), gibberellic acid (GA), and autonomous/vernalization pathways] are known to play important roles in the control of flowering time. Circadian clock genes, namely, LHY, CCA1, GI, and ELF3, regulate the LD pathway. GAI and FCA control flowering via GA and autonomous pathways, respectively. The density effect on plant size is caused by specific factors such as the amount of nutrition obtained from the soil and touch frequency among plants. However, the molecular mechanism underlying the acceleration of flowering time due to density effects remains unclear. Here, we show the density effects on three Brassicaceae plants, namely, Brassica rapa var. nipposinica, Brassica napus, and Brassica chinensis f. honsaitai. They showed shorter stems and leaves when grown at high densities on soil under continuous light (LL). Shorter stems and leaves, as well as accelerated flowering times, were observed when a model plant, Arabidopsis thaliana, was grown under the same conditions. Unexpectedly, ethylene insensitive 2 (ein2) showed no differences in density effects in our experiments. The acceleration of flowering at higher densities was largely suppressed by gai, but not by gi, lhy;cca1, or fca. These results suggest that the promotion of flowering (as a density effect) is likely dependent on the GA pathway, but not the LD or autonomous pathways.

Density effects on semi-dwarf and early flowering mutants of Arabidopsis thaliana under continuous light

Kei Takahashi, Miyuki Hara, Kana Miyata, Ryosuke Hayama, Tsuyoshi Mizoguchi. . . . . . . 333 [PDF]

Plant growth promotion and inhibition under low- and high-density conditions (referred to as the density effect) has been studied extensively. Here, we show that such density effects were unaffected by the position of wild-type (WT) and gibberellic acid insensitive (gai) strains of Arabidopsis thaliana (Arabidopsis) within pots. Additionally, petanko 1 (pta1) and pta5 were newly discovered alleles of the ROTUNDIFOLIA 3 (ROT3) and DWARF 4 (DWF4) genes that are involved in brassinosteroid biosynthesis. Unlike gai, the semi-dwarf mutants of pta1 and pta5 exhibited normal flowering times and a shortening of rosette leaves at high densities. Moreover, the pta1 and pta5 variants suppressed flowering stem shortening at high densities. pta5, but not pta1 suppressed the reduction in silique number at intermediate densities. SPINDLY (SPY) is a negative regulator of GA signaling, while PHYTOCHROME B (PHYB) is a red-light photoreceptor. High-density growth did not reduce the flowering time of phyB mutants, but did affect that of spy mutants. Neither spy nor phyB suppressed the shortening of rosette leaves at high densities; however, spy suppressed flowering stem shortening. Moreover, spy suppressed the reduction of silique number at high densities, while and phyB promoted the decrease. These data suggest that GA, BR, and light signaling pathways play important roles in the density effect.