Steven R. Rodermel

The immutans variegation locus of Arabidopsis defines a mitochondrial alternative oxidase homolog that functions during early chloroplast biogenesis.

Nuclear gene–induced variegation mutants provide a powerful system to dissect interactions between the genetic systems of the nucleus–cytoplasm, the chloroplast, and the mitochondrion. The immutans (im) variegation mutation of Arabidopsis is nuclear and recessive and results in the production of green- and white-sectored leaves. The green sectors contain cells with normal chloroplasts, whereas the white sectors are heteroplastidic and contain cells with abnormal, pigment-deficient plastids as well as some normal chloroplasts. White sector formation can be promoted by enhanced light intensities, but sectoring becomes irreversible early in leaf development. The white sectors accumulate the carotenoid precursor phytoene. We have positionally cloned IM and found that the gene encodes a 40.5-kD protein with sequence motifs characteristic of alternative oxidase, a mitochondrial protein that functions as a terminal oxidase in the respiratory chains of all plants. However, phylogenetic analyses revealed that the IM protein is only distantly related to these other alternative oxidases, suggesting that IM is a novel member of this protein class. We sequenced three alleles of im, and all are predicted to be null. Our data suggest a model of variegation in which the IM protein functions early in chloroplast biogenesis as a component of a redox chain responsible for phytoene desaturation but that a redundant electron transfer function is capable of compensating for IM activity in some plastids and cells.

Variegation mutants and mechanisms of chloroplast biogenesis

Variegated plants typically have green- and white-sectored leaves. Cells in the green sectors contain normal-appearing chloroplasts, whereas cells in the white sectors lack pigments and appear to be blocked at various stages of chloroplast biogenesis. Variegations can be caused by mutations in nuclear, chloroplast or mitochondrial genes. In some plants, the green and white sectors have different genotypes, but in others they have the same (mutant) genotype. One advantage of variegations is that they provide a means of studying genes for proteins that are important for chloroplast development, but for which mutant analysis is difficult, either because mutations in a gene of interest are lethal or because they do not show a readily distinguishable phenotype. This paper focuses on Arabidopsis variegations, for which the most information is available at the molecular level. Perhaps the most interesting of these are variegations caused by defective nuclear gene products in which the cells of the mutant have a uniform genotype. Two questions are of paramount interest: (1) What is the gene product and how does it function in chloroplast biogenesis? (2) What is the mechanism of variegation and why do green sectors arise in plants with a uniform (mutant) genotype? Two paradigms of variegation mechanism are described: immutans (im) and variegated2 (var2). Both mechanisms emphasize compensating activities and the notion of plastid autonomy, but redundant gene products are proposed to play a role in var2, but not in im. It is hypothesized that threshold levels of certain activities are necessary for normal chloroplast development.

Susceptibility to the Sugar Beet Cyst Nematode Is Modulated by Ethylene Signal Transduction in Arabidopsis thaliana

Previously, we identified Arabidopsis thaliana mutant rhd1-4 as hypersusceptible to the sugar beet cyst nematode Heterodera schachtii. We assessed rhd1-4 as well as two other rhd1 alleles and found that each exhibited, in addition to H. schachtii hypersusceptibility, decreased root length, increased root hair length and density, and deformation of the root epidermal cells compared with wild-type A. thaliana ecotype Columbia (Col-0). Treatment of rhd1-4 and Col-0 with the ethylene inhibitors 2-aminoethoxyvinylglycine and silver nitrate and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid suggests that the rhd1-4 hypersusceptibility and root morphology phenotypes are the result of an increased ethylene response. Assessment of known ethylene mutants further support the finding that ethylene plays a role in mediating A. thaliana susceptibility to H. schachtii because mutants that overproduce ethylene (eto1-1, eto2, and eto3) are hypersusceptible to H. schachtii and mutants that are ethylene-insensitive (etr1-1, ein2-1, ein3-1, eir1-1, and axr2) are less susceptible to H. schachtii. Because the ethylene mutants tested show altered susceptibility and altered root hair density and length, a discrimination between the effects of altered ethylene signal transduction and root hair density on susceptibility was accomplished by analyzing the ttg and gl2 mutants, which produce ectopic root hairs that result in greatly increased root hair densities while maintaining normal ethylene signal transduction. The observed normal susceptibilities to H. schachtii of ttg and g12 indicate that increased root hair density, per se, does not cause hypersusceptibility. Furthermore, the results of nematode attraction assays suggest that the hypersusceptibility of rhd1-4 and the ethylene-overproducing mutant eto3 may be the result of increased attraction of H. schachtii-infective juveniles to root exudates of these plants. Our findings indicate that rhd1 is altered in its ethylene response and that ethylene signal transduction positively influences plant susceptibility to cyst nematodes.

Photosynthesis, Rubisco activity and amount, and their regulation by transcription in senescing soybean leaves

Senescence is a phase of leaf ontogeny marked by declining photosynthetic activity that, in soybean (Glycine max [L.] Merr.), is paralleled by a decline in chloroplast function. Soybean leaves have different patterns of decline in photosynthetic capacity and chloroplast function associated with nodal position and sink activity. The objective of this work was to determine whether leaves from nodes 3 and 6 of soybean, which show these different patterns, are similarly regulated with respect to ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity and content and also to ascertain the degree of regulation of Rubisco content by transcription. Leaves from nodes 3 and 6 of field-grown soybean plants were sampled periodically from the time of their unfolding until near death. In situ CO2-exchange rate (CER) increased to a maximal level in both leaves and then declined slowly. For node 3 leaves the decline was progressive, but for node 6 leaves the decline was arrested at about 75% of maximum CER for a period of about 20 d, coincident with the onset of rapid seed growth, before a short period of very rapid decline immediately preceding leaf death. Rubisco activities and Rubisco content were directly correlated with CER in the leaves exhibiting the two different patterns. Rubisco activation ratio was similar for the two leaves and did not change throughout development. The primary regulator of photosynthesis at the physiological level, thus, was the amount of Rubisco protein. Decreases in Rubisco holoenzyme during senescence of both leaves were accompanied by coordinate decreases in the levels of mRNAs for the small and large subunits of Rubisco, suggesting that the decrease in Rubisco enzyme amounts during soybean leaf senescence is due to slower transcription rates and that levels of these mRNAs are coordinately controlled during senescence as they are during chloroplast development. However, plastid DNA template availability and posttranscriptional controls may also influence Rubisco content during senescence of these leaves. We conclude that soybean leaf photosynthesis likely unfolds according to a single developmental program but that modifications can be superimposed upon this program to maximize photosynthetic rates.

Elevated CO2 Effects during Leaf Ontogeny (A New Perspective on Acclimation).

For many plants growth in elevated CO2 leads to reduced rates of photosynthesis. To examine the role that leaf ontogeny plays in the acclimation response, we monitored photosynthesis and some related parameters at short intervals throughout the ontogenetic development of tobacco (Nicotiana tabacum L.) leaves under ambient (350 [mu]L L-1)- and high (950 [mu]L L-1)-CO2 conditions. The pattern of photosynthetic rate over time was similar between the two treatments and consistent with the expected pattern for a typical dicot leaf. However, the photosynthesis pattern in high-CO2-grown tobacco was shifted temporally to an earlier maximum and subsequent senescent decline. Ribulose-1,5-biphosphate carboxylase/oxygenase activity appeared to be the main factor regulating photosynthetic rates in both treatments. Therefore, we propose a new model for interpreting the acclimation response. Lowered photosynthetic rates observed during acclimation appear to be the result of a shift in the timing of the normal photosynthetic stages of leaf ontogeny to an earlier onset of the natural decline in photosynthetic rates associated with senescence.

Photosynthetic Redox Imbalance Governs Leaf Sectoring in the Arabidopsis thaliana Variegation Mutants immutans, spotty, var1, and var2

We hypothesized that chloroplast energy imbalance sensed through alterations in the redox state of the photosynthetic electron transport chain, measured as excitation pressure, governs the extent of variegation in the immutans mutant of Arabidopsis thaliana. To test this hypothesis, we developed a nondestructive imaging technique and used it to quantify the extent of variegation in vivo as a function of growth temperature and irradiance. The extent of variegation was positively correlated (R2 = 0.750) with an increase in excitation pressure irrespective of whether high light, low temperature, or continuous illumination was used to induce increased excitation pressure. Similar trends were observed with the variegated mutants spotty, var1, and var2. Measurements of greening of etiolated wild-type and immutans cotyledons indicated that the absence of IMMUTANS increased excitation pressure twofold during the first 6 to 12 h of greening, which led to impaired biogenesis of thylakoid membranes. In contrast with IMMUTANS, the expression of its mitochondrial analog, AOX1a, was transiently upregulated in the wild type but permanently upregulated in immutans, indicating that the effects of excitation pressure during greening were also detectable in mitochondria. We conclude that mutations involving components of the photosynthetic electron transport chain, such as those present in immutans, spotty, var1, and var2, predispose Arabidopsis chloroplasts to photooxidation under high excitation pressure, resulting in the variegated phenotype.

IMMUTANS Does Not Act as a Stress-Induced Safety Valve in the Protection of the Photosynthetic Apparatus of Arabidopsis during Steady-State Photosynthesis

IMMUTANS (IM) encodes a thylakoid membrane protein that has been hypothesized to act as a terminal oxidase that couples the reduction of O2 to the oxidation of the plastoquinone (PQ) pool of the photosynthetic electron transport chain. Because IM shares sequence similarity to the stress-induced mitochondrial alternative oxidase (AOX), it has been suggested that the protein encoded by IM acts as a safety valve during the generation of excess photosynthetically generated electrons. We combined in vivo chlorophyll fluorescence quenching analyses with measurements of the redox state of P700 to assess the capacity of IM to compete with photosystem I for intersystem electrons during steady-state photosynthesis in Arabidopsis (Arabidopsis thaliana). Comparisons were made between wild-type plants, im mutant plants, as well as transgenics in which IM protein levels had been overexpressed six (OE-6×) and 16 (OE-16×) times. Immunoblots indicated that IM abundance was the only major variant that we could detect between these genotypes. Overexpression of IM did not result in increased capacity to keep the PQ pool oxidized compared to either the wild type or im grown under control conditions (25°C and photosynthetic photon flux density of 150 μmol photons m−2 s−1). Similar results were observed either after 3-d cold stress at 5°C or after full-leaf expansion at 5°C and photosynthetic photon flux density of 150 μmol photons m−2 s−1. Furthermore, IM abundance did not enhance protection of either photosystem II or photosystem I from photoinhibition at either 25°C or 5°C. Our in vivo data indicate that modulation of IM expression and polypeptide accumulation does not alter the flux of intersystem electrons to P700+ during steady-state photosynthesis and does not provide any significant photoprotection. In contrast to AOX1a, meta-analyses of published Arabidopsis microarray data indicated that IM expression exhibited minimal modulation in response to myriad abiotic stresses, which is consistent with our functional data. However, IM exhibited significant modulation in response to development in concert with changes in AOX1a expression. Thus, neither our functional analyses of the IM knockout and overexpression lines nor meta-analyses of gene expression support the model that IM acts as a safety valve to regulate the redox state of the PQ pool during stress and acclimation. Rather, IM appears to be strongly regulated by developmental stage of Arabidopsis.

Flexibility in photosynthetic electron transport: The physiological role of plastoquinol terminal oxidase (PTOX)☆

Oxygenic photosynthesis depends on a highly conserved electron transport system, which must be particularly dynamic in its response to environmental and physiological changes, in order to avoid an excess of excitation energy and subsequent oxidative damage. Apart from cyclic electron flow around PSII and around PSI, several alternative electron transport pathways exist including a plastoquinol terminal oxidase (PTOX) that mediates electron flow from plastoquinol to O(2). The existence of PTOX was first hypothesized in 1982 and this was verified years later based on the discovery of a non-heme, di-iron carboxylate protein localized to thylakoid membranes that displayed sequence similarity to the mitochondrial alternative oxidase. The absence of this protein renders higher plants susceptible to excitation pressure dependant variegation combined with impaired carotenoid synthesis. Chloroplasts, as well as other plastids (i.e. etioplasts, amyloplasts and chromoplasts), fail to assemble organized internal membrane structures correctly, when exposed to high excitation pressure early in development. While the role of PTOX in plastid development is established, its physiological role under stress conditions remains equivocal and we postulate that it serves as an alternative electron sink under conditions where the acceptor side of PSI is limited. The aim of this review is to provide an overview of the past achievements in this field and to offer directions for future investigative efforts. Plastoquinol terminal oxidase (PTOX) is involved in an alternative electron transport pathway that mediates electron flow from plastoquinol to O(2). This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.

Functional Redundancy of AtFtsH Metalloproteases in Thylakoid Membrane Complexes

FtsH is an ATP-dependent metalloprotease found in bacteria, mitochondria, and plastids. Arabidopsis (Arabidopsis thaliana) contains 12 AtFtsH proteins, three in the mitochondrion and nine in the chloroplast. Four of the chloroplast FtsH proteins are encoded by paired members of closely related genes (AtFtsH1 and 5, and AtFtsH2 and 8). We have previously reported that AtFtsH2 and 8 are interchangeable components of AtFtsH complexes in the thylakoid membrane. In this article, we show that the var1 variegation mutant, which is defective in AtFtsH5, has a coordinate reduction in the AtFtsH2 and 8 pair, and that the levels of both pairs are restored to normal in var1 plants that overexpress AtFtsH1. Overexpression of AtFtsH1, but not AtFtsH2/VAR2, normalizes the pattern of var1 variegation, restoring a nonvariegated phenotype. We conclude that AtFtsH proteins within a pair, but not between pairs, are interchangeable and functionally redundant, at least in part. We further propose that the abundance of each pair is matched with that of the other pair, with excess subunits being turned over. The variegation phenotype of var1 (as well as var2, which is defective in AtFtsH2) suggests that a threshold concentration of subunits is required for normal chloroplast function. AtFtsH1, 2, 5, and 8 do not show evidence of tissue or developmental specific expression. Phylogenetic analyses revealed that rice (Oryza sativa) and Arabidopsis share a conserved core of seven FtsH subunit genes, including the AtFtsH1 and 5 and AtFtsH2 and 8 pairs, and that the structure of the present-day gene families can be explained by duplication events in each species following the monocot/dicot divergence.

Expression of an Arabidopsis phosphoglycerate mutase homologue is localized to apical meristems, regulated by hormones, and induced by sedentary plant-parasitic nematodes

We previously isolated a partial soybean cDNA clone whose transcript abundance is increased upon infection by the sedentary, endoparasitic soybean cyst nematode Heterodera glycines. We now isolated the corresponding full-length cDNA and determined that the predicted gene product was similar to the group of cofactor-dependent phosphoglycerate mutase/bisphosphoglycerate mutase enzymes (PGM/bPGM; EC 5.4.2.1/5.4.2.4). We designated the corresponding soybean gene GmPGM. PGM and bPGM are key catalysts of glycolysis that have been well characterized in animals but not plants. Using the GmPGM cDNA sequence, we identified a homologous Arabidopsis thaliana gene, which we designatedAtPGM. Histochemical GUS analyses of transgenic Arabidopsis plants containing theAtPGM promoter::GUS construct revealed that the AtPGM promoter directs GUS expression in uninfected plants only to the shoot and root apical meristems. In infected plants, GUS staining also is evident in the nematode feeding structures induced by the cyst nematode Heterodera schachtii and by the root-knot nematode Meloidogyne incognita. Furthermore, we discovered that the AtPGM promoter was down-regulated by abscisic acid and hydroxyurea, whereas it was induced by sucrose, oryzalin, and auxin, thereby revealing expression characteristics typical of genes with roles in meristematic cells. Assessment of the auxin-inducible AUX1 gene promoter (a gene coding for a polar auxin transport protein) similarly revealed feeding cell and meristem expression, suggesting that auxin may be responsible for the observed tissue specificity of the AtPGM promoter. These results provide first insight into the possible roles of PGM/bPGM in plant physiology and in plant-pathogen interactions.