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An antisense construct targeting the C4 isoform of NADP-malic enzyme (ME), the primary enzyme decarboxylating malate in bundle sheath cells to supply CO2 to Rubisco, was used to transform the dicot Flaveria bidentis. Transgenic plants (α-NADP-ME) exhibited a 34% to 75% reduction in NADP-ME activity relative to the wild type with no visible growth phenotype. We characterized the effect of reducing NADP-ME on photosynthesis by measuring in vitro photosynthetic enzyme activity, gas exchange, and real-time carbon isotope discrimination (Ɗ). In α-NADP-ME plants with less than 40% of wild-type NADP-ME activity, CO2 assimilation rates at high intercellular CO2 were significantly reduced, whereas the in vitro activities of both phosphoenolpyruvate carboxylase and Rubisco were increased. Ɗ measured concurrently with gas exchange in these plants showed a lower Ɗ and thus a lower calculated leakiness of CO2 (the ratio of CO2 leak rate from the bundle sheath to the rate of CO2 supply). Comparative measurements on antisense Rubisco small subunit F. bidentis plants showed the opposite effect of increased Ɗ and leakiness. We use these measurements to estimate the C4 cycle rate, bundle sheath leak rate, and bundle sheath CO2 concentration. The comparison of α-NADP-ME and antisense Rubisco small subunit demonstrates that the coordination of the C3 and C4 cycles that exist during environmental perturbations by light and CO2 can be disrupted through transgenic manipulations. Furthermore, our results suggest that the efficiency of the C4 pathway could potentially be improved through a reduction in C4 cycle activity or increased C3 cycle activity.

Plant viral vectors are valuable tools for heterologous gene expression, and because of virus-induced gene silencing (VIGS), they also have important applications as reverse genetics tools for gene function studies. Viral vectors are especially useful for plants such as soybean (Glycine max) that are recalcitrant to transformation. Previously, two generations of bean pod mottle virus (BPMV; genus Comovirus) vectors have been developed for overexpressing and silencing genes in soybean. However, the design of the previous vectors imposes constraints that limit their utility. For example, VIGS target sequences must be expressed as fusion proteins in the same reading frame as the viral polyprotein. This requirement limits the design of VIGS target sequences to open reading frames. Furthermore, expression of multiple genes or simultaneous silencing of one gene and expression of another was not possible. To overcome these and other issues, a new BPMV-based vector system was developed to facilitate a variety of applications for gene function studies in soybean as well as in common bean (Phaseolus vulgaris). These vectors are designed for simultaneous expression of multiple foreign genes, insertion of noncoding/antisense sequences, and simultaneous expression and silencing. The simultaneous expression of green fluorescent protein and silencing of phytoene desaturase shows that marker gene-assisted silencing is feasible. These results demonstrate the utility of this BPMV vector set for a wide range of applications in soybean and common bean, and they have implications for improvement of other plant virus-based vector systems.

Although flowering regulatory mechanisms have been extensively studied in Arabidopsis (Arabidopsis thaliana), those in other species have not been well elucidated. Here, we investigated the role of OsMADS51, a type I MADS-box gene in the short-day (SD) promotion pathway in rice (Oryza sativa). In SDs OsMADS51 null mutants flowered 2 weeks later than normal, whereas in long days loss of OsMADS51 had little effect on flowering. Transcript levels of three flowering regulators—Ehd1, OsMADS14, and Hd3a—were decreased in these mutants, whereas those of OsGI and Hd1 were unchanged. Ectopic expression of OsMADS51 caused flowering to occur about 7 d earlier only in SDs. In ectopic expression lines, transcript levels of Ehd1, OsMADS14, and Hd3a were increased, but those of OsGI and Hd1 remained the same. These results indicate that OsMADS51 is a flowering promoter, particularly in SDs, and that this gene functions upstream of Ehd1, OsMADS14, and Hd3a. To further investigate the relationship with other flowering promoters, we generated transgenic plants in which expression of Ehd1 or OsGI was suppressed. In Ehd1 RNA interference plants, OsMADS51 expression was not affected, supporting our conclusion that the MADS-box gene functions upstream of Ehd1. However, in OsGI antisense plants, the OsMADS51 transcript level was reduced. In addition, the circadian expression pattern for this MADS-box gene was similar to that for OsGI. These results demonstrate that OsMADS51 functions downstream of OsGI. In summary, OsMADS51 is a novel flowering promoter that transmits a SD promotion signal from OsGI to Ehd1.

Phosphoenolpyruvate carboxylase (PEPC; EC4.1.1.31) plays a key role during C(4) photosynthesis. The enzyme is activated by metabolites such as glucose-6-phosphate and inhibited by malate. This metabolite sensitivity is modulated by the reversible phosphorylation of a conserved serine residue near the N terminus in response to light. The phosphorylation of PEPC is modulated by a protein kinase specific to PEPC (PEPC-PK). To explore the role PEPC-PK plays in the regulation of C(4) photosynthetic CO(2) fixation, we have transformed Flaveria bidentis (a C(4) dicot) with antisense or RNA interference constructs targeted at the mRNA of this PEPC-PK. We generated several independent transgenic lines where PEPC is not phosphorylated in the light, demonstrating that this PEPC-PK is essential for the phosphorylation of PEPC in vivo. Malate sensitivity of PEPC extracted from these transgenic lines in the light was similar to the malate sensitivity of PEPC extracted from darkened wild-type leaves but greater than the malate sensitivity observed in PEPC extracted from wild-type leaves in the light, confirming the link between PEPC phosphorylation and the degree of malate inhibition. There were, however, no differences in the CO(2) and light response of CO(2) assimilation rates between wild-type plants and transgenic plants with low PEPC phosphorylation, showing that phosphorylation of PEPC in the light is not essential for efficient C(4) photosynthesis for plants grown under standard glasshouse conditions. This raises the intriguing question of what role this complexly regulated reversible phosphorylation of PEPC plays in C(4) photosynthesis.

Pectinesterase (PE; E.C. 3.1.1.11) is an enzyme responsible for the demethylation of galacturonyl residues in high-molecular-weight pectin and is believed to play an important role in cell wall metabolism. In this study, Pmeu1, a ubiquitously expressed PE gene, has been characterized by antisense suppression in tomato (Solanum lycopersicum). Transgenic tomato plants showed reduced PE activity levels in both green fruit and leaf tissue to around 65% and 25% of that found in wild-type plants, respectively. Pmeu1 was observed to encode a salt-dependent PE isoform that correlated with PE1 as previously described in fruit tissue. Silencing of Pmeu1 did not result in any detectable phenotype within the leaf tissue despite the gene product representing the major isoform in this tissue. In comparison, silencing in fruit resulted in an enhancement to the rate of softening during ripening. The role of PMEU1 in fruit ripening is discussed.

Plant leaves, harvesting light energy and fixing CO2, are a major source of foods on the earth. Leaves undergo developmental and physiological shifts during their lifespan, ending with senescence and death. We characterized the key regulatory features of the leaf transcriptome during aging by analyzing total- and small-RNA transcriptomes throughout the lifespan of Arabidopsis (Arabidopsis thaliana) leaves at multidimensions, including age, RNA-type, and organelle. Intriguingly, senescing leaves showed more coordinated temporal changes in transcriptomes than growing leaves, with sophisticated regulatory networks comprising transcription factors and diverse small regulatory RNAs. The chloroplast transcriptome, but not the mitochondrial transcriptome, showed major changes during leaf aging, with a strongly shared expression pattern of nuclear transcripts encoding chloroplast-targeted proteins. Thus, unlike animal aging, leaf senescence proceeds with tight temporal and distinct interorganellar coordination of various transcriptomes that would be critical for the highly regulated degeneration and nutrient recycling contributing to plant fitness and productivity.

The efficacy of using Serial Analysis of Gene Expression (SAGE) to analyze the transcriptome of the model dicotyledonous plant Arabidopsis was assessed. We describe an iterative tag-to-gene matching process that exploits the availability of the whole genome sequence of Arabidopsis. The expression patterns of 98% of the annotated Arabidopsis genes could theoretically be evaluated through SAGE and using an iterative matching process 79% could be identified by a tag found at a unique site in the genome. A total of 145,170 reliable experimental tags from two Arabidopsis leaf tissue SAGE libraries were analyzed, of which 29,632 were distinct. The majority (93%) of the 12,988 experimental tags observed greater than once could be matched within the Arabidopsis genome. However, only 78% were matched to a single locus within the genome, reflecting the complexities associated with working in a highly duplicated genome. In addition to a comprehensive assessment of gene expression in Arabidopsis leaf tissue, we describe evidence of transcription from pseudo-genes as well as evidence of alternative mRNA processing and anti-sense transcription. This collection of experimental SAGE tags could be exploited to assist in the on-going annotation of the Arabidopsis genome.

The coordinated induced expression of β-oxidation genes is essential to provide the energy supply for germination and postgerminative development. However, very little is known about other functions of β-oxidation in nonreserve organs. We have identified a gene-specific pattern of induced β-oxidation gene expression in wounded leaves of Arabidopsis. Mechanical damage triggered the local and systemic induction of only ACX1 among acyl-coenzyme A oxidase (ACX) genes, and KAT2/PED1 among 3-ketoacyl-coenzyme A thiolase (KAT) genes in Arabidopsis. In turn, wounding induced KAT5/PKT2 only systemically. Although most of the β-oxidation genes were activated by wound-related factors such as dehydration and abscisic acid, jasmonic acid (JA) induced only ACX1 and KAT5. Reduced expression of ACX1 or KAT2 genes, in transgenic plants expressing their corresponding mRNAs in antisense orientation, correlated with defective wound-activated synthesis of JA and with reduced expression of JA-responsive genes. Induced expression of JA-responsive genes by exogenous application of JA was unaffected in those transgenic plants, suggesting that ACX1 and KAT2 play a major role in driving wound-activated responses by participating in the biosynthesis of JA in wounded Arabidopsis plants.

Galactomannan biosynthesis in legume seed endosperms involves two Golgi membrane-bound glycosyltransferases, mannan synthase and galactomannan galactosyltransferase (GMGT). GMGT specificity is an important factor regulating the distribution and amount of (1→6)-α-galactose (Gal) substitution of the (1→4)-β-linked mannan backbone. The model legume Lotus japonicus is shown now to have endospermic seeds with endosperm cell walls that contain a high-Gal galactomannan (mannose [Man]/Gal = 1.2-1.3). Galactomannan biosynthesis in developing L. japonicus endosperms has been mapped, and a cDNA encoding a functional GMGT has been obtained from L. japonicus endosperms during galactomannan deposition. L. japonicus has been transformed with sense, antisense, and sense/antisense (“hairpin loop”) constructs of the GMGT cDNA. Some of the sense, antisense, and sense/antisense transgenic lines exhibited galactomannans with altered (higher) Man/Gal values in their (T1 generation) seeds, at frequencies that were consistent with posttranscriptional silencing of GMGT. For T1 generation individuals, transgene inheritance was correlated with galactomannan composition and amount in the endosperm. All the azygous individuals had unchanged galactomannans, whereas those that had inherited a GMGT transgene exhibited a range of Man/Gal values, up to about 6 in some lines. For Man/Gal values up to 4, the results were consistent with lowered Gal substitution of a constant amount of mannan backbone. Further lowering of Gal substitution was accompanied by a slight decrease in the amount of mannan backbone. Microsomal membranes prepared from the developing T2 generation endosperms of transgenic lines showed reduced GMGT activity relative to mannan synthase. The results demonstrate structural modification of a plant cell wall polysaccharide by designed regulation of a Golgi-bound glycosyltransferase.

Arabidopsis plants were transformed with acyl carrier protein (ACP)-4 in antisense conformation driven by the cauliflower mosaic virus 35S promoter. It was hypothesized that reduction of ACP4 in leaf tissue would result in a reduction in lipid biosynthesis and, in addition, affect fatty acid composition and leaf physiology. Several transgenic lines have been generated with reduced ACP4 protein in leaf tissue. Dramatic reductions in ACP4 resulted in a reduction of leaf lipid content (22%–60%) based on fresh leaf weight and a bleached appearance and reduced photosynthetic efficiency. In addition, a decrease in 16:3 as a percentage of the total fatty acid composition was noted. There were no changes in leaf lipid class distribution; however, there was a decrease in the relative amount of 16:3 in monogalactosyldiacylglycerol. These results suggest that ACP4 plays a major role in the biosynthesis of fatty acids for chloroplast membrane development. Alterations in the ACP isoform profile of Arabidopsis leaf also appear to alter the flow of fatty acids between the prokaryotic and eukaryotic pathways for assembly of galactolipids. However, it has not yet been determined if the changes in fatty acid composition are due to changes in the profile of ACP isoforms, or if they are actually a reaction to a reduction in fatty acid precursors.

Exposure of oilseed rape (Brassica napus) plants to increasing leaf temperatures between 15°C and 25°C increased photorespiratory NH4 + production from 0.7 to 3.5 μmol m−2 s−1. Despite the 5-fold increase in the rate of NH4 + production, the NH4 + concentration in root and leaf tissue water and xylem sap dropped significantly, whereas that in the leaf apoplastic fluid remained constant. The in vitro activity of glutamine synthetase (GS) in both leaves and roots also increased with temperature and in all cases substantially exceeded the observed rates of photorespiratory NH4 + production. The surplus of GS in oilseed rape plants was confirmed using GS2 antisense plants with 50% to 75% lower in vitro leaf GS activity than in the wild type. Despite the substantial reduction in GS activity, there was no tendency for antisense plants to have higher tissue NH4 + concentrations than wild-type plants and no overall correlation between GS activity and tissue NH4 + concentration was observed. Antisense plants exposed to leaf temperatures increasing from 14°C to 27°C or to a trifold increase in the O2 to CO2 ratio did not show any change in steady-state leaf tissue NH4 + concentration or in NH3emission to the atmosphere. The antisense plants also had similar leaf tissue concentrations of glutamine, glycine, and serine as the wild type, whereas glutamate increased by 38%. It is concluded that photorespiration does not control tissue or apoplastic levels of NH4 + in oilseed rape leaves and, as a consequence, that photorespiration does not exert a direct control on leaf atmosphere NH3 fluxes.

The plant mitochondrial electron transport chain is branched such that electrons at ubiquinol can be diverted to oxygen via the alternative oxidase (AOX). This pathway does not contribute to ATP synthesis but can dampen the mitochondrial generation of reactive oxygen species. Here, we establish that transgenic tobacco (Nicotiana tabacum L. cv Petit Havana SR1) cells lacking AOX (AS8 cells) show increased susceptibility to three different death-inducing compounds (H2O2, salicylic acid [SA], and the protein phosphatase inhibitor cantharidin) in comparison with wild-type cells. The timing and extent of AS8 cell death are very similar among the three treatments and, in each case, are accompanied by the accumulation of oligonucleosomal fragments of DNA, indicative of programmed cell death. Death induced by H2O2 or SA occurs by a mitochondria-dependent pathway characterized by cytochrome c release from the mitochondrion. Conversely, death induced by cantharidin occurs by a pathway without any obvious mitochondrial involvement. The ability of AOX to attenuate these death pathways may relate to its ability to maintain mitochondrial function after insult with a death-inducing compound or may relate to its ability to prevent chronic oxidative stress within the mitochondrion. In support of the latter, long-term treatment of AS8 cells with an antioxidant compound increased the resistance of AS8 cells to SA- or cantharidin-induced death. The results indicate that plants maintain both mitochondria-dependent and -independent pathways of programmed cell death and that AOX may act as an important mitochondrial “survival protein” against such death.

Tocopherols, synthesized by photosynthetic organisms, are micronutrients with antioxidant properties that play important roles in animal and human nutrition. Because of these health benefits, there is considerable interest in identifying the genes involved in tocopherol biosynthesis to allow transgenic alteration of both tocopherol levels and composition in agricultural crops. Tocopherols are generated from the condensation of phytyldiphosphate and homogentisic acid (HGA), followed by cyclization and methylation reactions. Homogentisate phytyltransferase (HPT) performs the first committed step in this pathway, the phytylation of HGA. In this study, bioinformatics techniques were used to identify candidate genes,slr1736 and HPT1, that encode HPT fromSynechocystis sp. PCC 6803 and Arabidopsis, respectively. These two genes encode putative membrane-bound proteins, and contain amino acid residues highly conserved with other prenyltransferases of the aromatic type. A Synechocystissp. PCC 6803 slr1736 null mutant obtained by insertional inactivation did not accumulate tocopherols, and was rescued by the Arabidopsis HPT1 ortholog. The membrane fraction of wild-type Synechocystis sp. PCC 6803 was capable of catalyzing the phytylation of HGA, whereas the membrane fraction from the slr1736 null mutant was not. The microsomal membrane fraction of baculovirus-infected insect cells expressing the Synechocystis sp. PCC 6803slr1736 were also able to perform the phytylation reaction, verifying HPT activity of the protein encoded by this gene. In addition, evidence that antisense expression of HPT1in Arabidopsis resulted in reduced seed tocopherol levels, whereas seed-specific sense expression resulted in increased seed tocopherol levels, is presented.

The specific functions of the light-harvesting proteins Lhca2 and Lhca3 were studied in Arabidopsis ecotype Colombia antisense plants in which the proteins were individually repressed. The antisense effect was specific in each plant, but levels of Lhca proteins other than the targeted products were also affected. The contents of Lhca1 and Lhca4 were unaffected, but Lhca3 (in Lhca2-repressed plants) was almost completely depleted, and Lhca2 decreased to about 30% of wild-type levels in Lhca3-repressed plants. This suggests that the Lhca2 and Lhca3 proteins are in physical contact with each other and that they require each other for stability. Photosystem I fluorescence at 730 nm is thought to emanate from pigments bound to Lhca1 and Lhca4. However, fluorescence emission and excitation spectra suggest that Lhca2 and Lhca3, which fluoresce in vitro at 680 nm, also could contribute to far-red fluorescence in vivo. Spectral forms with absorption maxima at 695 and 715 nm, apparently with emission maxima at 702 and 735 nm, respectively, might be associated with Lhca2 and Lhca3.

The root-hypocotyl of Arabidopsis produces a relatively large amount of secondary vascular tissue when senescence is delayed by the removal of inflorescences, and plants are grown at low population density. Peptidase zymograms prepared from isolated xylem and phloem revealed the existence of distinct proteolytic enzyme profiles within these tissues. cDNA libraries were constructed from isolated xylem and bark of the root-hypocotyl and screened for cDNAs coding for cysteine, serine, and aspartic peptidases. Three cDNAs, two putative papain-type cysteine peptidases (XCP1 and XCP2) and one putative subtilisin-type serine peptidase (XSP1), were identified from the xylem library for further analysis. Using RNA gel blots it was determined that these peptidases were expressed in the xylem and not in the bark. Quantitative reverse transcriptase-polymerase chain reaction confirmed the RNA gel-blot results and revealed high levels of XCP1 and XCP2 mRNA in stems and flowers of the infloresence. A poly-histidine-tagged version of XCP1 was purified from Escherichia coli by denaturing metal-chelate chromatography. Following renaturation, the 40-kD recombinant XCP1 was not proteolytically active. Activation was achieved by incubation of recombinant XCP1 at pH 5.5 and was dependent on proteolytic processing of the 40-kD inactive polypeptide to a 26-kD active peptidase.

Superoxide dismutases (SODs) are key components of the plant antioxidant defense system. While plastidic and cytosolic isoforms have been extensively studied, the importance of mitochondrial SOD at a cellular and whole-plant level has not been established. To address this, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated in which expression of AtMSD1, encoding the mitochondrial manganese (Mn)SOD, was suppressed by antisense. The strongest antisense line showed retarded root growth even under control growth conditions. There was evidence for a specific disturbance of mitochondrial redox homeostasis in seedlings grown in liquid culture: a mitochondrially targeted redox-sensitive green fluorescent protein was significantly more oxidized in the MnSOD-antisense background. In contrast, there was no substantial change in oxidation of cytosolically targeted redox-sensitive green fluorescent protein, nor changes in antioxidant defense components. The consequences of altered mitochondrial redox status of seedlings were subtle with no widespread increase of mitochondrial protein carbonyls or inhibition of mitochondrial respiratory complexes. However, there were specific inhibitions of tricarboxylic acid (TCA) cycle enzymes (aconitase and isocitrate dehydrogenase) and an inhibition of TCA cycle flux in isolated mitochondria. Nevertheless, total respiratory CO2 output of seedlings was not decreased, suggesting that the inhibited TCA cycle enzymes can be bypassed. In older, soil-grown plants, redox perturbation was more pronounced with changes in the amount and/or redox poise of ascorbate and glutathione. Overall, the results demonstrate that reduced MnSOD affects mitochondrial redox balance and plant growth. The data also highlight the flexibility of plant metabolism with TCA cycle inhibition having little effect on overall respiratory rates.

The maize (Zea mays) late pollen gene ZmMADS2 belongs to the MIKC type of MADS box transcription factor genes. Here, we report that ZmMADS2, which forms a homodimer in yeast (Saccharomyces cerevisiae), is required for anther dehiscence and pollen maturation. Development of anthers and pollen was arrested at 1 d before dehiscence in transgenic plants expressing the ZmMADS2-cDNA in antisense orientation. Temporal and spatial expression analyses showed high amounts of ZmMADS2 transcripts in endothecium and connective tissues of the anther at 1 d before dehiscence and in mature pollen after dehiscence. Transient transformation of maize and tobacco (Nicotiana tabacum) pollen with the luciferase reporter gene under the control of different ZmMADS2 promoter deletion constructs demonstrated the functionality and tissue specificity of the promoter. Transgenic maize plants expressing a ZmMADS2-green fluorescent protein fusion protein under control of the ZmMADS2 promoter were used to monitor protein localization during anther maturation and pollen tube growth. High amounts of the fusion protein accumulate in degenerating nuclei of endothecial and connective cells of the anther. A possible function of ZmMADS2 during anther dehiscence and pollen maturation and during pollen tube growth is discussed.

Transgenic tobacco (Nicotiana tabacum) with altered levels of mitochondrial alternative oxidase (AOX) were used to examine the potential role of this electron transport chain protein in resistance to tobacco mosaic virus. We examined the effect of AOX expression on the salicylic acid-induced resistance in susceptible plants and the resistance responses of plants harboring the N-gene. A lack of AOX did not compromise the ability of salicylic acid treatment to heighten the resistance of susceptible plants. In plants with the N-gene, a lack of AOX did not compromise the ability of the hypersensitive response to restrict the virus or the ability of the plant to develop systemic acquired resistance. Overexpression of AOX did not heighten the resistance of susceptible plants, but did result in smaller hypersensitive response lesions, suggesting a link between mitochondrial function and this programmed cell death event. We conclude that AOX is not a critical component of the previously characterized salicylhydroxamic acid-sensitive pathway important in viral resistance.

Phospholipase D (PLD), which hydrolyzes phospholipids into free head groups and phosphatidic acid (PA), may regulate cellular processes through the production of lipid and lipid-derived messengers. We have genetically abrogated PLDα, the most prevalent isoform of PLD in plants, and the depletion of PLDα in Arabidopsis decreased the levels of PA and superoxide production in Arabidopsis leaf extracts. Addition of PA promoted the synthesis of superoxide in the PLDα-depleted plants, as measured by chemiluminescence and superoxide dismutase-inhibitable, NADPH-dependent reduction of cytochrome c and nitroblue tetrazolium. The PA-enhanced generation of superoxide was associated mainly with microsomal membranes. Among various lipids tested, PA was the most effective stimulator with the optimal concentrations between 100 and 200 μm. The PA-promoted production of superoxide was observed also in leaves directly infiltrated with PA. The added PA was more effective in stimulating superoxide generation in the PLDα-depleted leaves than in the PLDα-containing, wild-type leaves, suggesting that PA produced in the cell was more effective than added PA in promoting superoxide production. These data indicate that PLD plays a role in mediating superoxide production in plants through the generation of PA as a lipid messenger.