Plant Molecular Biology

Skinner, Jeffrey; Zitzewitz, Jarislav; Szűcs, Péter; Marquez-Cedillo, Luis; Filichkin, Tanya; Amundsen, Keenan; Stockinger, Eric; Thomashow, Michael; Chen, Tony; Hayes, Patrick

doi: 10.1007/s11103-005-2498-2pmid: 16244905

CBFs are key regulators in the Arabidopsis cold signaling pathway. We used Hordeum vulgare (barley), an important crop and a diploid Triticeae model, to characterize the CBF family from a low temperature tolerant cereal. We report that barley contains a large CBF family consisting of at least 20 genes (HvCBFs) comprising three multigene phylogenetic groupings designated the HvCBF1-, HvCBF3-, and HvCBF4-subgroups. For the HvCBF1- and HvCBF3-subgroups, there are comparable levels of phylogenetic diversity among rice, a cold-sensitive cereal, and the cold-hardy Triticeae. For the HvCBF4-subgroup, while similar diversity levels are observed in the Triticeae, only a single ancestral rice member was identified. The barley CBFs share many functional characteristics with dicot CBFs, including a general primary domain structure, transcript accumulation in response to cold, specific binding to the CRT motif, and the capacity to induce cor gene expression when ectopically expressed in Arabidopsis. Individual HvCBF genes differed in response to abiotic stress types and in the response time frame, suggesting different sets of HvCBF genes are employed relative to particular stresses. HvCBFs specifically bound monocot and dicot cor gene CRT elements in vitro under both warm and cold conditions; however, binding of HvCBF4-subgroup members was cold dependent. The temperature-independent HvCBFs activated cor gene expression at warm temperatures in transgenic Arabidopsis, while the cold-dependent HvCBF4-subgroup members of three Triticeae species did not. These results suggest that in the Triticeae – as in Arabidopsis – members of the CBF gene family function as fundamental components of the winter hardiness regulon.

Chai, Mao-Feng; Chen, Qi-Jun; An, Rui; Chen, Ye-Miao; Chen, Jia; Wang, Xue-Chen

doi: 10.1007/s11103-005-6802-ypmid: 16244906

As one of terminal electron acceptors in photosynthetic electron transport chain, NADP receives electron and H+ to synthesize NADPH, an important reducing energy in chlorophyll synthesis and Calvin cycle. NAD kinase (NADK), the catalyzing enzyme for the de novo synthesis of NADP from substrates NAD and ATP, may play an important role in the synthesis of NADPH. NADK activity has been observed in different sub-cellular fractions of mitochondria, chloroplast, and cytoplasm. Recently, two distinct NADK isoforms (NADK1 and NADK2) have been identified in Arabidopsis. However, the physiological roles of NADKs remain unclear. In present study, we investigated the physiological role of Arabidiposis NADK2. Sub-cellular localization of the NADK2–GFP fusion protein indicated that the NADK2 protein was localized in the chloroplast. The NADK2 knock out mutant (nadk2) showed obvious growth inhibition and smaller rosette leaves with a pale yellow color. Parallel to the reduced chlorophyll content, the expression levels of two POR genes, encoding key enzymes in chlorophyll synthesis, were down regulated in the nadk2 plants. The nadk2 plants also displayed hypersensitivity to environmental stresses provoking oxidative stress, such as UVB, drought, heat shock and salinity. These results suggest that NADK2 may be a chloroplast NAD kinase and play a vital role in chlorophyll synthesis and chloroplast protection against oxidative damage.

Valot, Benoît; Dieu, Marc; Recorbet, Ghislaine; Raes, Martine; Gianinazzi, Silvio; Dumas-Gaudot, Eliane

doi: 10.1007/s11103-005-8269-2pmid: 16244907

A sub-cellular proteomic approach was carried out to monitor membrane-associated protein modifications in response to the arbuscular mycorrhizal (AM) symbiosis. Membrane proteins were extracted from Medicago truncatula roots either inoculated or not with the AM fungus Glomus intraradices. Comparative two-dimensional electrophoresis revealed that 36 spots were differentially displayed in response to the fungal colonization including 15 proteins induced, 3 up-regulated and 18 down-regulated. Among them, seven proteins were found to be commonly down-regulated in AM-colonized and phosphate-fertilized roots. Twenty-five spots out of the 36 of interest could be identified by matrix assisted laser desorption/ionisation-time of flight and/or tandem mass spectrometry analyses. Excepting an acid phosphatase and a lectin, none of them was previously reported as being regulated during AM symbiosis. In addition, this proteomic approach allowed us for the first time to identify AM fungal proteins in planta.

Zhu, Huifen; Qian, Weiqiang; Lu, Xuzhong; Li, Dongping; Liu, Xin; Liu, Kunfan; Wang, Daowen

doi: 10.1007/s11103-005-0183-0pmid: 16244908

Purple acid phosphatases (PAPs) are metallo-phosphoesterases. Their expression and function have not been systematically investigated in higher plants. In this work, we compared the transcript levels of 28 Arabidopsis PAP (AtPAP) genes in five Arabidopsis organs. The 28 members, although differed in their expression patterns in vegetative organs, were all transcribed in flower. Furthermore, the transcription of seven members (AtPAPs 6, 11, 14, 19, 23, 24 and 25) occurred predominantly in the flower. To begin dissecting the role of AtPAP genes in flower development, further expression and functional analyses were conducted using AtPAP23. Histochemical staining of transgenic plants expressing AtPAP23 promoter-beta-glucuronidase (GUS) gene construct revealed that AtPAP23 transcription was strong in flower apical meristems, but became restricted to petals and anther filaments in fully developed flower. A GST (glutathione S-transferase) fusion protein of AtPAP23 (GST:AtPAP23) was expressed in bacterial cells, and was found to contain significant amounts of Fe and Mn (whereas the control GST protein contained none). In biochemical tests, GST:AtPAP23 showed typical acid phosphatase activities. The fusion protein was also highly active on phosphoserine, but not phosphotyrosine. Despite its highly specific expression pattern and the demonstrated biochemical function of its protein product, the RNAi (RNA interference), T-DNA knock-out and overexpression lines of AtPAP23 were indistinguishable from wild type plants in the development of flower (or other organs). Interestingly, the Fe and Mn contents were found significantly increased in AtPAP23 overexpression lines, which may offer a new direction for further functional studies of AtPAPs in Arabidopsis.

Sós-Hegedűs, Anita; Lovas, Ágnes; Kondrák, Mihály; Kovács, Gabriella; Bánfalvi, Zsófia

doi: 10.1007/s11103-005-0354-zpmid: 16244909

Previously, it was shown that low temperature (≤ 15 °C) inhibits RNA silencing-mediated defence by the control of siRNA generation. In contrast, we have found nine antisense potato lines out of 24 in which RNA silencing was not inhibited at low temperature. In these lines, the extent of endogenous repression varied in leaves and was found to be different in roots and tubers. In order to address the contribution of gene dosage and repetitive structure of the transgene loci to the temperature dependence/independence of antisense-mediated gene-silencing DNA gel blot analysis was performed. Interestingly, none of the studied features correlated with the observed silencing effect. In addition, the insertion of vector backbone sequences into the potato genome did not influence the temperature dependence. RNA-directed DNA methylation was detected in the majority of antisense lines, however, it was also independent of the type of RNA silencing. Thus, it is feasible that chromosomal flanking sequences or the chromatin structure surrounding the insertion determine which silencing pathway is activated.

Tang, Wei; Charles, Thomas; Newton, Ronald

doi: 10.1007/s11103-005-0451-zpmid: 16244910

Transcription factors play an important role in regulating gene expression in response to stress and pathogen tolerance. We describe here that overexpression of an ERF/AP2 pepper transcription factor (CaPF1) in transgenic Virginia pine (Pinus virginiana Mill.) confers tolerance to heavy metals Cadmium, Copper, and Zinc, to heat, and to pathogens Bacillus thuringiensis and Staphylococcus epidermidis, as by the survival rate of transgenic plants and the number of decreasing pathogen cells in transgenic tissues. Measurement of antioxidant enzymes ascorbate peroxidase (APOX), glutathione reductase (GR), and superoxide dismutase (SOD) activities demonstrated that the level of the enzyme activities was higher in transgenic Virginia pine plants overexpressing the CaPF1 gene, which may protect cells from the oxidative damage caused by stresses, compared to the controls. Constitutive overexpression of CaPF1 gene enhanced organ growth by increasing organ size and cell numbers in transgenic Virginia pine plants over those in control plants.

Lim, Jun; Jung, Jee; Lim, Chae; Lee, Mi-Hyun; Kim, Bong; Kim, Miran; Bruce, Wesley; Benfey, Philip

doi: 10.1007/s11103-005-0578-ypmid: 16244911

The SCARECROW (SCR) gene in Arabidopsis is required for asymmetric cell divisions responsible for ground tissue formation in the root and shoot. Previously, we reported that Zea mays SCARECROW (ZmSCR) is the likely maize ortholog of SCR. Here we describe conserved and divergent aspects of ZmSCR. Its ability to complement the Arabidopsis scr mutant phenotype suggests conservation of function, yet its expression pattern during embryogenesis and in the shoot system indicates divergence. ZmSCR expression was detected early during embryogenesis and localized to the endodermal lineage in the root, showing a gradual regionalization of expression. Expression of ZmSCR appeared to be analogous to that of SCR during leaf formation. However, its absence from the maize shoot meristem and its early expression pattern during embryogenesis suggest a diversification of ZmSCR in the patterning processes in maize. To further investigate the evolutionary relationship of SCR and ZmSCR, we performed a phylogenetic analysis using Arabidopsis, rice and maize SCARECROW-LIKE genes (SCLs). We found SCL23 to be the most closely related to SCR in both eudicots and monocots, suggesting that a gene duplication resulting in SCR and SCL23 predates the divergence of dicots and monocots.

Fusada, Naoki; Masuda, Tatsuru; Kuroda, Hirofumi; Shimada, Hiroshi; Ohta, Hiroyuki; Takamiya, Ken-ichiro

doi: 10.1007/s11103-005-0579-xpmid: 16244912

Cytokinins and light activate the transcription of the cucumber NADPH-protochlorophyllide reductase (POR) gene. We have previously reported that 2.3 kb of the 5′-region of this gene contains a cis-element that is responsive to cytokinin. In this study, to identify the cytokinin-responsive cis-element corresponding to chlorophyll biosynthesis and chloroplast development, we performed transient expression assays in etiolated cucumber cotyledons. A 5′-deletional analysis indicated that a 411-bp fragment (−451 to −40 bp) contained at least one of the cis-elements related to cytokinin-responsiveness. Gel mobility shift assays also detected cytokinin-enhanced binding in this region. DNase I footprinting analysis, using a 150-bp fragment (−490 to −340 bp) as the probe, identified the cytokinin-enhanced protected sequence as 5′-ATATTAGTGATAT-3′. More detailed gel mobility shift and competition analyses identified 5′-TATTAG-3′ as the sequence critical for cytokinin-enhanced binding. Mutations in the identified sequence in the transient expression assay caused a reduced but retained cytokinin-responsiveness, as well as low reporter activity of untreated control. These results suggest that the identified sequence is a novel cis-element exhibiting cytokinin-dependent protein binding in vitro, which may function effectively when interacting with other cytokinin-related elements. The effects of this element on the chloroplast development are discussed in relation to other cytokinin-related elements.

Kościańska, Edyta; Kalantidis, Kriton; Wypijewski, Krzysztof; Sadowski, Jan; Tabler, Martin

doi: 10.1007/s11103-005-0668-xpmid: 16244913

In this study we analyse several aspects of cytoplasmic RNA silencing by agroinfiltration of DNA constructs encoding single- and double-stranded RNAs derived from a GFP transgene and from the endogenous Virp1 gene. Both types of inductors resulted after 2–4 days in much higher concentration of siRNAs in the agroinfiltrated zone than normally seen during systemic silencing. More specifically, infiltration of two transgene hairpin constructs resulted in elevated levels of siRNAs. However, differences between the two constructs were observed: the antisense–sense arrangement was more effective than the sense–antisense order. For both double-stranded forms, we observed a relative increase of the 24-mer size class of siRNAs. When a comparable hairpin construct of the endogenous Virp1 gene was assayed, the portion of the 24-mer siRNA class remained low as observed for all kinds of single-stranded inducers. The lack of increase of Virp1-derived 24-mers was independent of the expression level, as demonstrated by agroinfiltration into a transgenic plant that overexpressed Virp1 and showed the same pattern. Using transducer constructs, we could detect within a week transitive silencing from GFP to GUS sequences in the infiltrated zone and in either direction 5′–3′ and 3′–5′. Conversely, for the endogenous Virp1 gene neither transitive silencing nor the induction of systemic silencing could be observed. These results are discussed in view of the current models of RNA silencing.

Ellerström, M.; Reidt, W.; Ivanov, R.; Tiedemann, J.; Melzer, M.; Tewes, A.; Moritz, T.; Mock, H.-P.; Sitbon, F.; Rask, L.; Bäumlein, H.

doi: 10.1007/s11103-005-0669-9pmid: 16244914