The Plant Journal

Bauer, Claudia S.; Hoth, Stefan; Haga, Ken; Philippar, Katrin; Aoki, Naohiro; Hedrich, Rainer

doi: 10.1046/j.1365-313X.2000.00844.xpmid: 11069689

Recently, two K+ channel genes, ZMK1 and ZMK2, were isolated from maize coleoptiles. They are expressed in the cortex and vasculature, respectively. Expression in Xenopus oocytes characterized ZMK1 as an inwardly rectifying K+ channel activated by external acidification, while ZMK2 mediates voltage‐independent and proton‐inhibited K+ currents. In search of the related gene products in planta, we applied the patch–clamp technique to protoplasts isolated from the cortex and vasculature of Zea mays coleoptiles and mesocotyls. In the cortex, a 6–8 pS K+ channel gave rise to inwardly rectifying K+ currents. Like ZMK1, this channel was activated by apoplastic acidification. In contrast, protoplasts from vascular tissue expressing the sucrose transporter ZmSUT1 were dominated by largely voltage‐independent K+ currents with a single‐channel conductance of 22 pS. The pronounced sensitivity to the extracellular protons Ca2+, Cs+ and Ba2+ is reminiscent of ZMK2 properties in oocytes. Thus, the dominant K+ channels in cortex and vasculature most likely represent the gene products of ZMK1 and ZMK2. Our studies on the ZMK2‐like channels represent the first in planta analysis of a K+ channel that shares properties with the AKT3 K+ channel family.

Ohmiya, Yasunori; Samejima, Masahiro; Shiroishi, Masahiro; Amano, Yoshihiko; Kanda, Takahisa; Sakai, Fukumi; Hayashi, Takahisa

doi: 10.1046/j.1365-313x.2000.00860.xpmid: 11069690

Suspension‐cultured poplar (Populus alba) cells produce two distinct endo‐1,4‐β‐glucanases, one of which is released in the extracellular culture medium and the other localized in their walls. Two cDNA clones, PopCel1 and PopCel2, isolated from a poplar cDNA library, encode the extracellular and the wall‐bound endo‐1,4‐β‐glucanases, respectively, based upon deduced amino acid sequences. The products of these two genes contained domains conserved in endo‐1,4‐β‐glucanase (family 9) and showed 91.5% amino acid identity. The levels of both PopCel1 and PopCel2 mRNAs increased during the lag phase of growth and decreased rapidly during the linear phase. After the levels had decreased, they were again increased by addition of sucrose to the culture medium and further enhanced by the addition of 2,4‐dichlorophenoxyacetic acid (2,4‐D) in the presence of sucrose. The accumulation of the mRNAs was correlated with the solubilization of cello‐oligosaccharides. Cello‐oligosaccharides and xyloglucan were also solubilized from the wall preparations of poplar cells incubated with enzyme preparations from the extracellular culture medium and walls. An antibody against both PopCel proteins reduced the production of cello‐oligosaccharides by the extracellular enzyme by 90% and that by the wall‐bound enzyme by 55%, and also prevented xyloglucan solubilization. The results show that the accumulation of poplar endo‐1,4‐β‐glucanases is regulated indirectly by auxin in the presence of sucrose and can act on cellulose in suspension‐cultured poplar cells.

Chatfield, Steven P.; Stirnberg, Petra; Forde, Brian G.; Leyser, Ottoline

doi: 10.1046/j.1365-313x.2000.00862.xpmid: 11069691

Apically derived auxin has long been known to inhibit lateral bud growth, but since it appears not to enter the bud, it has been proposed that its inhibitory effect is mediated by a second messenger. Candidates include the plant hormones ethylene, cytokinin and abscisic acid. We have developed a new assay to study this phenomenon using the model plant Arabidopsis. The assay allows study of the effects of both apical and basal hormone applications on the growth of buds on excised nodal sections. We have shown that apical auxin can inhibit the growth of small buds, but larger buds were found to have lost competence to respond. We have used the assay with nodes from wild‐type and hormone‐signalling mutants to test the role of ethylene, cytokinin and abscisic acid in bud inhibition by apical auxin. Our data eliminate ethylene as a second messenger for auxin‐mediated bud inhibition. Similarly, abscisic acid signalling is not to be required for auxin action, although basally applied abscisic can enhance inhibition by apical auxin and apically applied abscisic acid can reduce it. By contrast, basally applied cytokinin was found to release lateral buds from inhibition by apical auxin, while apically applied cytokinin dramatically increased the duration of inhibition. These results are consistent with cytokinin acting independently to regulate bud growth, rather than as a second messenger for auxin. However, in the absence of cytokinin‐signalling mutants, a role for cytokinin as a second messenger for auxin cannot be ruled out.

Finnemann, Jørgen; Schjoerring, Jan K.

doi: 10.1046/j.1365-313x.2000.00863.xpmid: 11069692

Regulation of the cytosolic isozyme of glutamine synthetase (GS1; EC 6.3.1.2) was studied in leaves of Brassica napus L. Expression and immunodetection studies showed that GS1 was the only active GS isozyme in senescing leaves. By use of [γ‐32P]ATP followed by immunodetection, it was shown that GS1 is a phospho‐protein. GS1 is regulated post‐translationally by reversible phosphorylation catalysed by protein kinases and microcystin‐sensitive serine/threonine protein phosphatases. Dephosphorylated GS1 is much more susceptible to degradation than the phosphorylated form. The phosphorylation status of GS1 changes during light/dark transitions and depends in vitro on the ATP/AMP ratio. Phosphorylated GS1 interacts with 14‐3‐3 proteins as verified by two different methods: a His‐tag 14‐3‐3 protein column affinity method combined with immunodetection, and a far‐Western method with overlay of 14‐3‐3–GFP. The degree of interaction with 14‐3‐3‐proteins could be modified in vitro by decreasing or increasing the phosphorylation status of GS1. Thus, the results demonstrate that 14‐3‐3 protein is an activator molecule of cytosolic GS and provide the first evidence of a protein involved in the activation of plant cytosolic GS. The role of post‐translational regulation of cytosolic GS and interactions between phosphorylated cytosolic GS and 14‐3‐3 proteins in senescing leaves is discussed in relation to nitrogen remobilization.

Hanin, Moez; Mengiste, Tesfaye; Bogucki, Augustyn; Paszkowski, Jerzy

doi: 10.1046/j.1365-313x.2000.00867.xpmid: 11069693

The Arabidopsis MIM gene encodes a protein belonging to the SMC family (structure maintenance of chromosomes) which is required for intrachromosomal homologous recombination (ICR). Both ICR and MIM gene expression are enhanced by DNA‐damaging treatments, suggesting that MIM is a factor limiting DNA repair by homologous recombination (HR) under genotoxic stress. We tested this hypothesis by measuring the levels of recombination in the mim mutant under genotoxic stress, using methyl methanesulfonate. Although the mutant clearly showed diminished basal and induced levels of ICR, enhancement of ICR by DNA‐damaging treatments was similar to that observed in the wild type. This suggests that the MIM gene product is required for DNA repair by HR, but is not critical for HR induction. To determine whether enhanced availability of MIM would increase basal HR levels in Arabidopsis, we examined ICR frequencies in transgenic Arabidopsis strains overexpressing the MIM gene after ectopic insertion of additional MIM copies. Two independent lines showed a twofold increase in ICR frequency relative to the wild type. Thus MIM is required for efficient ICR in plants, and its manipulation can be used to change homologous recombination frequencies. Since MIM is one of the components responsible for chromatin dynamics, our results suggest that the chromatin environment determines the frequency of homologous recombination.

, ; Kazuoka, Toru; Torikai, Satomi; Kikuchi, Hiromi; Oeda, Kenji

doi: 10.1046/j.1365-313x.2000.00864.xpmid: 11069694

Arabidopsis thaliana plants showed an increased tolerance to high‐intensity light when pre‐exposed to medium‐intensity light. This response, known as light acclimatization, depended on the quantity of light, the period of irradiation, and the quality of light. Among characterized acclimatization‐induced cDNA clones, we identified a zinc finger protein rhl41 (responsive to high light) gene, that was rapidly up‐regulated in proportion to the time of irradiation and the light intensity. Transgenic Arabidopsis plants over‐expressing the rhl41 gene showed an increased tolerance to high‐intensity light, and also morphological changes of thicker and dark green leaves. Interestingly, the palisade parenchyma was highly developed in the leaves of the transgenic plants, which is one of the long‐term acclimatization responses in Arabidopsis plants. The anthocyanin content (a light protectant) as well as the chlorophyll content also increased. Antisense transgenic plants exhibited decreased tolerance to high irradiation. We propose that the RHL41 zinc finger protein has a key role in the acclimatization response to changes in light intensity.

Dewdney, Julia; Reuber, T. Lynne; Wildermuth, Mary C.; Devoto, Alessandra; Cui, Jianping; Stutius, Lisa M.; Drummond, Emma P.; Ausubel, Frederick M.

doi: 10.1046/j.1365-313x.2000.00870.xpmid: 11069695

To identify components of the defense response that limit growth of a biotrophic fungal pathogen, we isolated Arabidopsis mutants with enhanced disease susceptibility to Erysiphe orontii. Our initial characterization focused on three mutants, eds14, eds15, and eds16. None of these is considerably more susceptible to a virulent strain of the bacterial pathogen Pseudomonas syringae pv. maculicola (Psm). All three mutants develop a hypersensitive response when infiltrated with Psm expressing the avirulence gene avrRpt2, which activates resistance via the LZ‐NBS/LRR resistance protein encoded by RPS2. The growth of Psm(avrRpt2), while somewhat greater in the mutants than in the wild type, is less than growth of the isogenic virulent strain. These results indicate that resistance mediated via LZ‐NBS/LRR R genes is functional. Analysis of the growth of avirulent Peronospora parasitica strains showed that the resistance pathway utilized by TIR‐NBS/LRR R genes is also operative in all three mutants. Surprisingly, only eds14 and eds16 were more susceptible to Erysiphe cichoracearum. Analysis of the expression profiles of PR‐1, BGL2, PR‐5 and PDF1.2 in eds14, eds15, and eds16 revealed differences from the wild type for all the lines. In contrast, these mutants were not significantly different from wild type in the deposition of callose at sites of E. orontii penetration. All three mutants have reduced levels of salicylic acid after infection. eds16 was mapped to the lower arm of chromosome I and found by complementation tests to be allelic to the salicylic acid‐deficient mutant sid2.

Pham, Xuan Hoi; Reddy , Malireddy Kodandarami; Ehtesham, Nasreen Z.; Matta, Bharati; Tuteja, Narendra

doi: 10.1046/j.1365-313x.2000.00869.xpmid: 11069696

DNA helicases play an essential role in all aspects of nucleic acid metabolism, by providing a duplex‐unwinding function. This is the first report of the isolation of a cDNA (1.6 kb) clone encoding functional DNA helicase from a plant (pea, Pisum sativum). The deduced amino‐acid sequence has eight conserved helicase motifs of the DEAD‐box protein family. It is a unique member of this family, containing DESD and SRT motifs instead of DEAD/H and SAT. The encoded 45.5 kDa protein has been overexpressed in bacteria and purified to homogeneity. The purified protein contains ATP‐dependent DNA and RNA helicase, DNA‐dependent ATPase, and ATP‐binding activities. The protein sequence contains striking homology with eIF‐4A, which has not so far been reported as DNA helicase. The antibodies against pea helicase inhibit in vitro translation. The gene is expressed as 1.6 kb mRNA in different organs of pea. The enzyme is localized in the nucleus and cytosol, and unwinds DNA in the 3′ to 5′ direction. The pea helicase interacts with pea topoisomerase I protein and stimulates its activity. These results suggest that pea DNA helicase could be an important multifunctional protein involved in protein synthesis, maintaining the basic activities of the cell, and in upregulation of topoisomerase I activity. The discovery of such a protein with intrinsic multiple activity should make an important contribution to our better understanding of DNA and RNA transactions in plants.

Marin‐Olivier, Marianne; Chevalier, Tony; Fobis‐Loisy, Isabelle; Dumas, Christian; Gaude, Thierry

doi: 10.1046/j.1365-313x.2000.00874.xpmid: 11069697

The pollen grains of angiosperms are usually desiccated at maturity. Following pollination, pollen hydrates on the stigma surface before germination takes place. Rehydration is an essential step for the success of pollination and depends on the movement of water from the stigmatic cells. This water flow has been shown to be biologically regulated, and components of both pollen and stigma surfaces have been demonstrated to play a role in the control of pollen hydration. Regulation of water transport between animal or plant cells involves membrane proteins, designated aquaporins, which possess water‐channel activity. Such molecules may be candidates for controlling pollen hydration, and consequently we investigated whether aquaporins are present in the pollen and stigma cells in Brassica oleracea. Here, we report the identification of two new aquaporin genes, Bo‐PIP1b1 and Bo‐PIP1b2, which are highly homologous to PIP1b from Arabidopsis thaliana. Both Bo‐PIP1b1 and Bo‐PIP1b2 proteins are active water channels when expressed in Xenopus oocytes. Expression of Bo‐PIP1b1 and Bo‐PIP1b2 was observed in reproductive organs as well as in vegetative tissues. Interestingly, the use of a Bo‐PIP1b2 cDNA probe revealed that PIP1‐like transcripts were not present in the pollen grains or in the stigma papillae, but were present in the stigma cell layers underlying the papillar cells. This observation suggests that water flow between the pollen and stigma papillae may be dependent on aquaporins expressed in cells that are not directly in contact with the pollen grain.

Bouvier, Florence; Suire, Claude; D'Harlingue, Alain; Backhaus, Ralph A.; Camara, Bilal

doi: 10.1046/j.1365-313x.2000.00875.xpmid: 11069698

The nature of isoprenoids synthesized in plants is primarily determined by the specificity of prenyltransferases. Several of these enzymes have been characterized at the molecular level. The compartmentation and molecular regulation of geranyl diphosphate (GPP), the carbon skeleton that is the backbone of myriad monoterpene constituents involved in plant defence, allelopathic interactions and pollination, is poorly understood. We describe here the cloning and functional expression of a GPP synthase (GPPS) from Arabidopsis thaliana. Immunohistological analyses of diverse non‐secretory and secretory plant tissues reveal that GPPS and its congeners, monoterpene synthase, deoxy‐xylulose phosphate synthase and geranylgeranyl diphosphate synthase, are equally compartmentalized and distributed in non‐green plastids as well in chloroplasts of photosynthetic cells. This argues that monoterpene synthesis is not solely restricted to specialized secretory structures but can also occur in photosynthetic parenchyma. These data provide new information as to how monoterpene biosynthesis is compartmentalized and induced de novo in response to biotic and abiotic stress in diverse plants.