Suppression of NDA-type alternative mitochondrial NAD(P)H dehydrogenases in arabidopsis thaliana modifies growth and metabolism, but not high light stimulation of mitochondrial electron transport.

The plant respiratory chain contains several pathways which bypass the energy-conserving electron transport complexes I, III and IV. These energy bypasses, including type II NAD(P)H dehydrogenases and the alternative oxidase (AOX), may have a role in redox stabilization and regulation, but current evidence is inconclusive. Using RNA interference, we generated Arabidopsis thaliana plants simultaneously suppressing the type II NAD(P)H dehydrogenase genes NDA1 and NDA2. Leaf mitochondria contained substantially reduced levels of both proteins. In sterile culture in the light, the transgenic lines displayed a slow growth phenotype, which was more severe when the complex I inhibitor rotenone was present. Slower growth was also observed in soil. In rosette leaves, a higher NAD(P)H/NAD(P)⁺ ratio and elevated levels of lactate relative to sugars and citric acid cycle metabolites were observed. However, photosynthetic performance was unaffected and microarray analyses indicated few transcriptional changes. A high light treatment increased AOX1a mRNA levels, in vivo AOX and cytochrome oxidase activities, and levels of citric acid cycle intermediates and hexoses in all genotypes. However, NDA-suppressing plants deviated from the wild type merely by having higher levels of several amino acids. These results suggest that NDA suppression restricts citric acid cycle reactions, inducing a shift towards increased levels of fermentation products, but do not support a direct association between photosynthesis and NDA proteins.

Trichoderma viride cellulase induces resistance to the antibiotic pore-forming peptide alamethicin associated with changes in the plasma membrane lipid composition of tobacco BY-2 cells.

BACKGROUND: Alamethicin is a membrane-active peptide isolated from the beneficial root-colonising fungus Trichoderma viride. This peptide can insert into membranes to form voltage-dependent pores. We have previously shown that alamethicin efficiently permeabilises the plasma membrane, mitochondria and plastids of cultured plant cells. In the present investigation, tobacco cells (Nicotiana tabacum L. cv Bright Yellow-2) were pre-treated with elicitors of defence responses to study whether this would affect permeabilisation. RESULTS: Oxygen consumption experiments showed that added cellulase, already upon a limited cell wall digestion, induced a cellular resistance to alamethicin permeabilisation. This effect could not be elicited by xylanase or bacterial elicitors such as flg22 or elf18. The induction of alamethicin resistance was independent of novel protein synthesis. Also, the permeabilisation was unaffected by the membrane-depolarising agent FCCP. As judged by lipid analyses, isolated plasma membranes from cellulase-pretreated tobacco cells contained less negatively charged phospholipids (PS and PI), yet higher ratios of membrane lipid fatty acid to sterol and to protein, as compared to control membranes. CONCLUSION: We suggest that altered membrane lipid composition as induced by cellulase activity may render the cells resistant to alamethicin. This induced resistance could reflect a natural process where the plant cells alter their sensitivity to membrane pore-forming agents secreted by Trichoderma spp. to attack other microorganisms, and thus adding to the beneficial effect that Trichoderma has for plant root growth. Furthermore, our data extends previous reports on artificial membranes on the importance of lipid packing and charge for alamethicin permeabilisation to in vivo conditions.

Regulation of callose synthase activity in situ in alamethicin-permeabilized Arabidopsis and tobacco suspension cells.

BACKGROUND: The cell wall component callose is mainly synthesized at certain developmental stages and after wounding or pathogen attack. Callose synthases are membrane-bound enzymes that have been relatively well characterized in vitro using isolated membrane fractions or purified enzyme. However, little is known about their functional properties in situ, under conditions when the cell wall is intact. To allow in situ investigations of the regulation of callose synthesis, cell suspensions of Arabidopsis thaliana (Col-0), and tobacco (BY-2), were permeabilized with the channel-forming peptide alamethicin. RESULTS: Nucleic acid-binding dyes and marker enzymes demonstrated alamethicin permeabilization of plasma membrane, mitochondria and plastids, also allowing callose synthase measurements. In the presence of alamethicin, Ca2+ addition was required for callose synthase activity, and the activity was further stimulated by Mg2+ Cells pretreated with oryzalin to destabilize the microtubules prior to alamethicin permeabilization showed significantly lower callose synthase activity as compared to non-treated cells. As judged by aniline blue staining, the callose formed was deposited both at the cell walls joining adjacent cells and at discrete punctate locations earlier described as half plasmodesmata on the outer walls. This pattern was unaffected by oryzalin pretreatment, showing a quantitative rather than a qualitative effect of polymerized tubulin on callose synthase activity. No callose was deposited unless alamethicin, Ca2+ and UDP-glucose were present. Tubulin and callose synthase were furthermore part of the same plasma membrane protein complex, as judged by two-dimensional blue native SDS-PAGE. CONCLUSION: Alamethicin permeabilization allowed determination of callose synthase regulation and tubulin interaction in the natural crowded cellular environment and under conditions where contacts between the cell wall, the plasma membrane and cytoskeletal macromolecules remained. The results also suggest that alamethicin permeabilization induces a defense response mimicking the natural physical separation of cells (for example when intercellulars are formed), during which plasmodesmata are transiently left open.

Suppression of the external mitochondrial NADPH dehydrogenase, NDB1, in Arabidopsis thaliana affects central metabolism and vegetative growth.

Ca(2+)-dependent oxidation of cytosolic NADPH is mediated by NDB1, which is an external type II NADPH dehydrogenase in the plant mitochondrial electron transport chain. Using RNA interference, the NDB1 transcript was suppressed by 80% in Arabidopsis thaliana plants, and external Ca(2+)-dependent NADPH dehydrogenase activity became undetectable in isolated mitochondria. This was linked to a decreased level of NADP(+) in rosettes of the transgenic lines. Sterile-grown transgenic seedlings displayed decreased growth specifically on glucose, and respiratory metabolism of (14)C-glucose was increased. On soil, NDB1-suppressing plants had a decreased vegetative biomass, but leaf maximum quantum efficiency of photosystem II and CO2 assimilation rates, as well as total respiration, were similar to the wild-type. The in vivo alternative oxidase activity and capacity were also similar in all genotypes. Metabolic profiling revealed decreased levels of sugars, citric acid cycle intermediates, and amino acids in the transgenic lines. The NDB1-suppression induced transcriptomic changes associated with protein synthesis and glucosinolate and jasmonate metabolism. The transcriptomic changes also overlapped with changes observed in a mutant lacking ABAINSENSITIVE4 and in A. thaliana overexpressing stress tolerance genes from rice. The results thus indicate that A. thaliana NDB1 modulates NADP(H) reduction levels, which in turn affect central metabolism and growth, and interact with defense signaling.

An alternatively spliced domain of the NDC1 NAD(P)H dehydrogenase gene strongly influences the expression of the ACTIN2 reference gene in Arabidopsis thaliana.

In plant respiratory chains, alternative pathways for NAD(P)H oxidation are mediated by type II NAD(P)H dehydrogenases belonging to the NDA, NDB, and NDC families. For the latter type, Arabidopsis thaliana contains a single gene, NDC1, whose functional role has not previously been analyzed in the plant. We found that A. thaliana NDC1 is alternatively spliced. Four base pairs at the 3' end of intron 5 are spliced out in NDC1-1, but retained in the NDC1-2 mRNA, which therefore contains a truncated reading frame. Both variants are conserved in dicotyledonous and monocotyledonous plants and their relative abundance varies between organs and in response to light. Three analyzed NDC1 T-DNA insertion lines all displayed an early bolting phenotype. A dramatic upregulation of ACTIN2 was characteristic of two lines containing T-DNA inserts upstream of intron 5, whereas a line with an insertion downstream of the NDC1-2 reading frame had an ACTIN2 expression level identical to the wildtype. Thus, the alternatively spliced 5' domain of NDC1 strongly influences the expression of the functionally unrelated ACTIN2, which is a common reference gene for quantitative RT-PCR. Also for other reference genes, strong expressional effects were observed when comparing various mutants and wildtypes in microarray databases.