Non-photosynthetic Plants Research Articles

SCALE DEPENDENCE OF ABSORPTION OF PHOTOSYNTHETICALLY ACTIVE RADIATION IN TERRESTRIAL ECOSYSTEMS

The fraction of photosynthetically active radiation absorbed by plant canopies (fAPAR) is a critical biophysical variable for extrapolating ecophysiological measurements from the leaf to landscape scale. Quantification of fAPAR determinants at the landscape level is needed to improve the interpretation of remote sensing data, to facilitate its use in constraining ecosystem process models, and to improve synoptic-scale links between carbon and nutrient cycles. Most canopy radiation budget studies have focused on light attenuation in plant canopies, with little regard for the importance of the scale-dependent biophysical and structural factors (e.g., leaf and stem optical properties, leaf and stem area, and extent of vegetation structural types) that ultimately determine fAPAR at canopy and landscape scales. Most studies have also assumed that nonphotosynthetic vegetation (litter and stems) contributes little to fAPAR. Using a combined field measurement and radiative transfer modeling approach, we quantified (a) the relative role of the leaf-, canopy-, and landscape-level factors that determine fAPAR in terrestrial ecosystems and (b) the magnitude of PAR absorption by grass litter and woody plant stems. Variability in full spectral-range (400–2500 nm) reflectance/transmittance and PAR (400–700 nm) absorption at the level of individual leaf, stem, and litter samples was quantified for a wide array of broadleaf arborescent and grass species along a 900-km north–south Texas savanna transect. Among woody growth forms, leaf reflectance and transmittance spectra were statistically comparable between populations, species within a genus, and functional types (deciduous vs. evergreen, legume vs. nonlegume). Within the grass life-form, spectral properties were statistically comparable between species and C3/C4 physiologies. We found that tissue-level PAR absorption among species, genera, functional groups, and growth forms and between climatologically diverse regions was statistically similar, and for fresh leaves, it represented the most spectrally similar region of the shortwave spectrum. Subsequent modeling analyses indicated that the measured range of leaf, woody stem, and litter optical properties explained only a small proportion of the variance in tree and grass canopy fAPAR. However, the presence of nonphotosynthetic vegetation (e.g., stem and litter) had a significant effect on canopy fAPAR. In trees with a leaf area index (LAI) <3.0, stem surfaces increased canopy fAPAR by 10–40%. Standing grass litter canopies absorbed almost as much PAR as green grass canopies. Modeling the radiation regime in plant canopies should therefore account for the absorption of PAR by nonphotosynthetic plant components. Failure to do so may lead to overestimates of primary production, especially in woodlands, savannas, and shrublands dominated by species with optically thin canopies and in grasslands that accumulate senescent material. Further sensitivity analyses revealed that the extent and LAI of vegetation structural types (trees and grasses) were the dominant controls on savanna landscape-level fAPAR, accounting for 60–80% of the total variation. Variation in leaf-level and all other canopy-level factors contributed individually to explain only a small proportion (<11%) of the variance in landscape fAPAR; however, when considered as a group, they accounted for 20–40% of the variation in landscape fAPAR. These results emphasize the need for more mechanistic analyses of canopy-level radiative transfer, and subsequent carbon flux and trace gas processes, in plant canopies and across landscapes comprising heterogeneous mixtures of plant growth forms and life-forms.

Alternate paths of evolution for the photosynthetic gene rbcL in four nonphotosynthetic species of Orobanche.

We have determined the nucleotide sequence for the Rubisco large subunit from four holoparasitic species of Orobanche. Intact open reading frames are present in two species (O. corymbosa and O. fasciculata), whereas the remaining species (O. cernua and O. ramosa) have rbcL pseudogenes. Sequences for rbcL 5'-UTRs from species of Orobanche have few changes in the promoter and ribosome binding sites compared to photosynthetic higher plants. Comparison of rbcL 3'-UTR sequences for Nicotiana, Ipomoea, Cuscuta, and Orobanche reveal that nucleotide sequences from parasitic plants have regions capable of forming stem-loop structures, but 56-69 nt are deleted upstream of the stem-loop in the parasitic plants compared to their photosynthetic relatives. Although rbcL pseudogenes of O. cernua and O. ramosa have many large and small deletions, few indels are shared in common, implying that their common ancestor probably had an intact rbcL reading frame. Intact rbcL reading frames in O. corymbosa and O. fasciculata retain a bias of synonymous over nonsynonymous substitutions and deduced protein sequences are consistent with potentially functional Rubisco large subunit proteins. A conservative model of random substitution processes in pseudogene sequences estimates that the probability is low (P < 0.028) that these sequences would retain an open reading frame by chance. Species of Orobanche have either had recent photosynthetic ancestors, implying multiple independent losses of photosynthesis in this genus, or the rbcL gene may serve an unknown function in some nonphotosynthetic plants.

Complete Gene Map of the Plastid-like DNA of the Malaria Parasite Plasmodium falciparum

Malaria parasites, and other parasitic protists of the Phylum Apicomplexa, carry a plastid-like genome with greatly reduced sequence complexity. This 35 kb DNA circle resembles the plastid DNA of non-photosynthetic plants, encoding almost exclusively components involved in gene expression. The complete gene map described here includes genes for duplicated large and small subunit rRNAs, 25 species of tRNA, three subunits of a eubacterial RNA polymerase, 17 ribosomal proteins, and a translation elongation factor. In addition, it codes for an unusual member of the Clp family of chaperones, as well as an open reading frame of unknown function found in red algal plastids. Transcription is polycistronic. This plastid-like DNA molecule is conserved in several genera of apicomplexans and is conjectured to have been acquired by an early progenitor of the Phylum by secondary endosymbiosis. The function of the organelle (plastid) carrying this DNA remains obscure, but appears to be specified by genes transferred to the nucleus.

Free radical formation in non-photosynthetic plant tissues — an overview

Free radical formation in non-photosynthetic plant tissues — an overview

Transcription, splicing and editing of plastid RNAs in the nonphotosynthetic plant Epifagus virginiana.

Expression of the vestigial plastid genome of the nonphotosynthetic, parasitic flowering plant Epifagus virginiana was examined by northern analysis and by characterization of cDNAs. Probes for each of 12 plastid genes tested hybridized to all lanes of northern blots containing total RNA prepared from stems and fruits of Epifagus and from leaves of tobacco. Certain transcript patterns in Epifagus plastids are highly complex and similar to those of tobacco operons. In contrast, genes such as rps2, which have become orphaned in Epifagus as a result of evolutionary loss of formerly cotranscribed genes, show simpler transcript patterns in Epifagus than in tobacco. Sizing and sequencing of cDNAs generated by reverse transcriptase-PCR for three genes, rps12, rpl2, and clpP, show that their transcripts are properly cis- and/or trans-spliced at the same five group II intron insertion sites used in photosynthetic plants. A single, conventional C-->U edit in rps12 was found among the total of 1401 nucleotides of cDNA sequence that was determined for the three genes. An octanucleotide sequence identical to a putative guide RNA of plant organelles and perfectly complementary to the rps12 edit site itself was identified just 200 bp upstream of the edit site. These data, together with previous results from the complete sequencing of the Epifagus plastid genome, provide compelling evidence that this degenerate genome is nonetheless expressed and functional. Analysis of the putative maturase MatK, encoded by the group II intron of trnK in photosynthetic land plants but by a freestanding gene in Epifagus, leads us to hypothesize that it acts 'in trans' to assist the splicing of group II introns other than the one in which it is normally encoded.

Differentiation of Six Distinct Thioredoxins in Seeds of the Soybean

The thioredoxin profile of non-photosynthetic plant tissues, in particular of seeds, is much less known than that of green leaves. We have systematically fractionated and assayed the low molecular weight, heat-stable proteins present in extracts from soybean seeds by a variety of chromatographic and enzymatic methods. A multiplicity of six thioredoxins has been established, closely comparable to the pattern previously observed in soybean leaves (Häberlein,1991). Purification to homogeneity of the six proteins was achieved by FPLC. Thioredoxin activities in vitro towards soybean chloroplast NADP-MDH, fructosebisphosphatase, NADPH thioredoxin reductase, and E. coli ribonucleotide reductase have been measured. Some of the seed thioredoxins exhibit higher molecular weights, stronger binding to anion exchange columns, and much lower specificity in enzyme assays than their counterparts isolated from leaves, suggesting the presence of unspecialized storage forms in the dry seed.

Effect of Paraquat on Dark-GrownPhaseolus vulgarisCells

Paraquat is known to affect all green plants and other eukaryotic organisms including mammalian cells. The aim of this study was to improve the understanding of paraquat toxicity in nonphotosynthetic plant cells using dark-grown kidney bean cells in tissue culture. It is shown that uptake of paraquat is an active process, and that paraquat inhibits cell growth, reduces DNA synthesis, and inhibits the activity of hydroxypyruvate reductase while enhancing the activity of glutathione reductase which is involved in cellular defense against oxidant stress. Additionally, it is demonstrated that iron ions are involved in paraquat toxicity. We conclude that uptake of paraquat into cells is via polyamine channels and that the deleterious effects of paraquat on these nonphotosynthetic cells are mediated by iron.

Identification of a Major Soluble Protein in Mitochondria from Nonphotosynthetic Tissues as NAD-Dependent Formate Dehydrogenase

In many plant species, one of the most abundant soluble proteins (as judged by two-dimensional polyacrylamide gel electrophoresis) in mitochondria from nongreen tissues is a 40-kD polypeptide that is relatively scarce in mitochondria from photosynthetic tissues. cDNA sequences encoding this polypeptide were isolated from a lambda gt11 cDNA expression library from potato (Solanum tuberosum L.) by screening with a specific antibody raised against the 40-kD polypeptide. The cDNA sequence contains an open reading frame of 1137 nucleotides whose predicted amino acid sequence shows strong homology to an NAD-dependent formate dehydrogenase (EC 1.2.1.2) from Pseudomonas sp. 101. Comparison of the cDNA sequence with the N-terminal amino acid sequence of the mature 40-kD polypeptide suggests that the polypeptide is made as a precursor with a 23-amino acid presequence that shows characteristics typical of mitochondrial targeting signals. The identity of the polypeptide was confirmed by assaying the formate dehydrogenase activity in plant mitochondria from various tissues and by activity staining of mitochondrial proteins run on native gels combined with antibody recognition. The abundance and distribution of this protein suggest that higher plant mitochondria from various nonphotosynthetic plant tissues (tubers, storage roots, seeds, dark-grown shoots, cauliflower heads, and tissues grown in vitro) might contain a formate-producing fermentation pathway similar to those described in bacteria and algae.

Structure and expression of the nuclear gene coding for the chloroplast ribosomal protein L21: developmental regulation of a housekeeping gene by alternative promoters.

We have cloned and sequenced the nuclear gene of the chloroplast ribosomal protein L21 (rpl21) of Spinacia oleracea. The gene consists of five exons and four introns. All introns are located in the sequence which corresponds to the Escherichia coli-like central core of the protein. L21 mRNA is present in photosynthetic (leaves) and nonphotosynthetic (roots and seeds) plant organs, although large quantitative differences exist. Primer extension and S1 nuclease mapping experiments revealed the existence of two types of transcripts in leaves. The two corresponding start sites were defined as P1 and P2. In roots and seeds, we found only the shorter of the two transcripts (initiated at P2). The nucleotide sequence surrounding P2 resembles promoters for housekeeping and vertebrate r-protein genes. Analysis of several promoter constructions by transient expression confirmed that both transcripts originate from transcription initiation. Results are interpreted to mean that the expression of the rpl21 gene is regulated by alternative promoters. One of the promoters (P2) is constitutive, and the other one (P1) is specifically induced in leaves, i.e., its activation should be related to the transformation of amyloplasts or proplastids to chloroplasts. The gene thus represents the first example of a housekeeping gene which is regulated by the organ-specific usage of alternative promoters. Primer extension analysis and S1 nuclease mapping of another nucleus-encoded chloroplast ribosomal protein gene (rps1) give evidence that the same type of regulation by two-promoter usage might be a more general phenomenon of plant chloroplast-related ribosomal protein genes. Preliminary results indicate that presence of conserved sequences within the rpl21 and rps1 promoter regions which compete for the same DNA binding activities.

Rapid evolution of the plastid translational apparatus in a nonphotosynthetic plant: Loss or accelerated sequence evolution of tRNA and ribosomal protein genes

The vestigial plastid genome of Epifagus virginiana (beechdrops), a nonphotosynthetic parasitic flowering plant, is functional but lacks six ribosomal protein and 13 tRNA genes found in the chloroplast DNAs of photosynthetic flowering plants. Import of nuclear gene products is hypothesized to compensate for many of these losses. Codon usage and amino acid usage patterns in Epifagus plastic genes have not been affected by the tRNA gene losses, though a small shift in the base composition of the whole genome (toward A+T-richness) is apparent. The ribosomal protein and tRNA genes that remain have had a high rate of molecular evolution, perhaps due to relaxation of constraints on the translational apparatus. Despite the compactness and extensive gene loss, one translational gene (infA, encoding initiation factor 1) that is a pseudogene in tobacco has been maintained intact in Epifagus.

Lack of a functional plastid tRNACys gene is associated with loss of photosynthesis in a lineage of parasitic plants

We recently reported that the gene for chloroplast tRNA(Cys)(GCA) is a pseudogene in the plastid DNA of Epifagus virginiana, a non-photosynthetic parasitic flowering plant in the family Orobanchaceae. Since this is the only tRNA(Cys) gene in the plastid genome, and since Epifagus appears to possess a functional plastid translational apparatus, it seems probable that nuclear-encoded tRNAs are imported into plastids to effect translation. In this study we have surveyed species closely related to Epifagus to establish how widespread the loss of this tRNA gene has been. We find that Conopholis americana, another non-photosynthetic parasite, lacks the gene altogether, but that seven closely-related photosynthetic plants (both parasitic and free-living) maintain an intact chloroplast tRNA(Cys) gene. Thus, the tRNA(Cys) gene appears to have become non-functional at the same time that photosynthetic ability was lost. This may be because the levels of putatively imported tRNAs are sufficient to meet the demands of plastid gene expression under nonphotosynthetic conditions only.

Plastid translation and transcription genes in a non-photosynthetic plant: intact, missing and pseudo genes.

The non-photosynthetic, parasitic flowering plant Epifagus virginiana has recently been shown to contain a grossly reduced plastid genome that has lost many photosynthetic and chloro-respiratory genes. We have cloned and sequenced a 3.9 kb domain of plastid DNA from Epifagus to investigate the patterns of evolutionary change in such a reduced genome and to determine which genes are still present and likely to be functional. This 3.9 kb domain is colinear with a 35.4 kb region of tobacco chloroplast DNA, differing from it by a minimum of 11 large deletions varying in length from 354 bp to 11.5 kb, as well as by a number of small deletions and insertions. The nine genes retained in Epifagus encode seven tRNAs and two ribosomal proteins and are coextensive and highly conserved in sequence with homologs in photosynthetic plants. This suggests that these genes are functional in Epifagus and, together with evidence that the Epifagus plastid genome is transcribed, implies that plastid gene products play a role in processes other than photosynthesis and gene expression. Genes that are completely absent include not only photosynthetic genes, but surprisingly, genes encoding three subunits of RNA polymerase, four tRNAs and one ribosomal protein. In addition, only pseudogenes are found for two other tRNAs. Despite these defunct tRNA genes, codon and amino acid usage in Epifagus protein genes is normal. We therefore hypothesize that the expression of plastid genes in Epifagus relies on the import of nuclear encoded tRNAs and RNA polymerase from the cytoplasm.

Characterization of a ferredoxin:NADP + oxidoreductase from a nonphotosynthetic plant tissue

A flavoprotein with properties similar to those of ferredoxin:NADP + oxidoreductases found in the leaves of higher plants has been purified to apparent homogeneity from bean sprouts, a nonphotosynthetic plant tissue. The absorbance and circular dichroism spectra of the bean sprout protein are similar to those of spinach leaf ferredoxin:NADP + oxidoreductase and an antibody raised against the spinach enzyme recognized the bean sprout enzyme. The bean sprout enzyme catalyzed ferredoxin-dependent electron transfer from NADPH to equine cytochrome c at a high rate but, unlike the spinach enzyme, exhibited little NADPH to 2,6-dichlorophenol indophenol diaphorase activity. The bean sprout enzyme forms a 1:1 electrostatically stabilized complex with ferredoxins isolated from either bean sprouts or spinach leaves.

Transcriptional regulation and DNA methylation of nuclear genes for photosynthesis in nongreen plant cells.

The transcripts of nuclear genes for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS), chlorophyll a/b-binding protein (cab), and extrinsic 33-kDa protein involved in photosystem II water oxidation (woxA) were not detectable in the white wild cultured cells of sycamore (Acer pseudoplatanus), in contrast to their high levels in the sibling green mutant cells and the constitutive expression of actin genes (act) in both cell types. We have examined the template activities of nuclear DNAs using the HeLa cell in vitro transcription system. All of the three photosynthesis genes from the green cell line and act from both cell types were well transcribed in vitro, but these photosynthesis genes from the white cell line were not, indicating that the transcriptional regulation is ascribable to DNA templates. Digestion of nuclear DNA with methyl-sensitive and -insensitive isoschizomeric endonucleases and the subsequent Southern hybridization showed that each gene has the identical recognition sites of restriction enzymes in the green and white cell lines, but some of the sites were methylated only in the photosynthesis genes in the white cells. There was observed a clear inverse relationship between the level of expressed transcripts and the extent of DNA methylation. Thus, it is inferred that the selective methylation of DNA is a likely mechanism for suppressing transcription of nuclear genes for photosynthesis in the nonphotosynthetic plant cells.

Cellular energy levels and their effect on male cell abortion in cytoplasmically male sterile lines of Petunia hybrida

Using an HPLC method it has proved possible to follow the levels of adenine and pyridine nucleotides in the anthers of normal and cytoplasmically male sterile (CMS) lines of Petunia hybrida. Well before the appearance of any structural differences, anthers of CMS plants begin to show lower ATP/ADP ratios. In anthers, as in other non-photosynthetic plant tissues, there is a strong correlation between the ATP/ADP ratio and levels of NADPH and, when NADPH was assayed in fertile and CMS anthers, very dramatic differences were discovered. In male fertile plants, the NADPH/NADP ratio differs strikingly between somatic and reproductive tissues and reaches a peak in anthers at the early prophase of meiosis. The ratio in male sterile anthers remains largely unchanged from that normally seen in somatic tissues over this period. A cytochemical localisation technique revealed that, at the stage of development in question, the major reserves of anther NADPH are held within the tapetal cells and that levels within CMS tapeta are strikingly lower than those of normal plants. These findings are discussed in the perspective of theories proposed to explain the operation of CMS, and also in terms of the now conclusive evidence that the genetical basis of CMS lies in changes in the organisation of the mitochondrial genome.

Constitutive transcription and regulation of gene expression in non-photosynthetic plastids of higher plants

The plastid genome in higher plants contains >50 genes for rRNAs, tRNAs and proteins for transcriptional and translational functions, besides the genes encoding photosynthetic proteins. Considering the totipotency of most higher plant cells and the differentiation capacity of plastids, it can be inferred that at least the genes for genetic functions must be constitutively expressed in all plant organs, including non-photosynthetic roots, to maintain a basal level of transcriptional and translational activities. To test this hypothesis, transcription, RNA accumulation and polysome formation were analyzed in root amyloplasts, and in plastids from hypocotyls and cotyledons of dark-grown spinach seedlings. The results for 10 representative genes show that they are constitutively transcribed at relative rates which are similar in root amyloplasts and leaf chloroplasts. The differential accumulation of their mRNAs in roots and other non-photosynthetic plant organs is controlled at the post-transcriptional level by a developmental program. Although mRNAs for photosynthetic proteins are detectable in root amyloplasts, some of them are specifically depleted from polysomes relative to mRNAs for ribosomal proteins. This translational discrimination does not result from modifications in splicing or 5'- and 3' -end processing of mRNAs for photosynthetic proteins, since processing is identical in root amyloplasts and leaf chloroplasts. The results support the model of constitutive transcription of the plastid genome, and indicate that the expression of most plastid genes in spinach plants is controlled primarily by post-transcriptional and translational mechanisms.

Evidence for the presence of a [2Fe-2S] ferredoxin in bean sprouts

An iron-sulfur protein with properties similar to those of ferredoxins found in the leaves of higher plants has been isolated from bean sprouts - a non-photosynthetic plant tissue. The bean sprout protein has a molecular mass of 12.5 kDa and appears to contain a single [2Fe-2S] cluster. The absorbance and circular dichroism spectra of the bean sprout protein resemble those of spinach leaf ferredoxin and the bean sprout protein can replace spinach ferredoxin as an electron donor for NADP + reduction, nitrite reduction and thioredoxin reduction by spinach leaf enzymes. Although the reduced bean sprout protein ( E m = −440 mV) is a slightly stronger reductant than spinach ferredoxin and appears to be less acidic than spinach ferredoxin, the two proteins are similar enough so that the bean sprout protein is recognized by an antibody raised against spinach ferredoxin.

Green Algae (Scenedesmus obliquus) Contain Three Thioredoxins of Regular Size

Abstract A comprehensive thioredoxin profile of Scenedesmus obliquus has been established by chromatography of heat-stable protein extracts on five different ion exchange, gel permeation, and affinity chromatography columns and using three different assay systems including homolo­gous S. obliquus ribonucleotide reductase, chloroplast fructose-bis-phosphatase, and NADP malate dehydrogenase. Four different thioredoxins were purified to homogeneity. Besides the large chloroplast thioredoxin f described previously, the algae contain three proteins of molecular weight 12,000 designated thioredoxin I, II, and III. They bind specifically to antibodies against E. coli thioredoxin. Chloroplast-free mutant algae (strain C-2A′) lack thioredoxin f but contain all three regular thioredoxins. Species I and II have very similar amino acid composition and enzyme-stimulating activities. They are considered cytoplasmic thioredoxins which serve as hydrogen donors in algal deoxyribonucleotide biosynthesis. Thioredoxin III is of low activity towards all the presently tested enzymes and its physiological role remains unknown; its role as a glutaredoxin could be excluded. All non-photosynthetic plant cells analyzed so far (mutant algae, seeds, and roots) contain a set of three regular-size thioredoxins.

Effect of 3-amino-1,2,4-triazole, a catalase inhibitor, on peroxide content of suspension-cultured pear fruit cells

Catalase activity in suspension-cultured pear ( Pyrus communis L. cv. Passe Crassane) fruit cells was substantially reduced by 3-amino-1,2,4-triazole (amitrole). Maximal reduction occurred within a few hours and no effects on cell growth or respiration were found over 48 h. There was little increase in content of H 2O 2 concomitant with catalase deactivation. This was also the case even when the free radical generators methyl viologen (paraquat) and ozone were given to the cells in conjunction with amitrole. Superoxide dismutase (SOD) activity remained constant with amitrole treatment. It is proposed that, at least in non-photosynthetic plant cells, reduction in catalase activity does not necessarily imply a rise in H 2O 2 content to levels toxic to the cell.

Proline Accumulation in Tubers of Jerusalem Artichoke

When separated whole tubers of Helianthus tuberosus L. were dried, and when tuber slices were plasmolysed in mannitol solutions, free proline accumulated in the tuber tissue. The amount of proline accumulated in tuber slices increased with the concentration of mannitol in the plasmolysing solution. A lag of approximately 24 h preceded proline accumulation in plasmolysed slices. During the lag period, the content of free arginine, a major component of the free amino acid pool, decreased by at least 50%. L-Indospicine, a competitive inhibitor of arginase (L-arginine amidinohydrolase EC 3.5.3.1) inhibited arginine catabolism in slices and also inhibited proline accumulation. On the other hand, cycloheximide, an inhibitor of protein synthesis, did not inhibit arginine loss or proline accumulation. An increase in the concentration of mannitol required for plasmolysis accompanied an increase in free proline in whole, dormant tubers stored at 4°C. These tubers are an example of a non-photosynthetic higher plant tissue in which free proline accumulates when the water potential decreases. The relationship between arginine catabolism and proline accumulation is discussed.