Acclimation to elevated CO2 affects the C/N balance by reducing de novo N-assimilation.

Plants exposed to elevated atmospheric CO2 concentrations show an increased photosynthetic activity. However, after prolonged exposure, the activity declines. This acclimation to elevated CO2 is accompanied by a rise in the carbon-to-nitrogen ratio of the biomass. Hence, increased sugar accumulation and sequential downregulation of photosynthetic genes, as well as nitrogen depletion and reduced protein content, have been hypothesized as the cause of low photosynthetic performance. However, the reason for reduced nitrogen content in plants at high CO2 is unclear. Here, we show that reduced photorespiration at increased CO2 -to-O2 ratio leads to reduced de novo assimilation of nitrate, thus shifting the C/N balance. Metabolic modeling of acclimated and non-acclimated plants revealed the photorespiratory pathway to function as a sink for already assimilated nitrogen during the light period, providing carbon skeletons for de novo assimilation. At high CO2 , low photorespiratory activity resulted in diminished nitrogen assimilation and eventually resulted in reduced carbon assimilation. For the hpr1-1 mutant, defective in reduction of hydroxy-pyruvate, metabolic simulations show that turnover of photorespiratory metabolites is expanded into the night. Comparison of simulations for hpr1-1 with those for the wild type allowed investigating the effect of a perturbed photorespiration on N-assimilation.

Phosphorylations of the Abutilon Mosaic Virus Movement Protein Affect Its Self-Interaction, Symptom Development, Viral DNA Accumulation, and Host Range.

The genome of bipartite geminiviruses in the genus Begomovirus comprises two circular DNAs: DNA-A and DNA-B. The DNA-B component encodes a nuclear shuttle protein (NSP) and a movement protein (MP), which cooperate for systemic spread of infectious nucleic acids within host plants and affect pathogenicity. MP mediates multiple functions during intra- and intercellular trafficking, such as binding of viral nucleoprotein complexes, targeting to and modification of plasmodesmata, and release of the cargo after cell-to-cell transfer. For Abutilon mosaic virus (AbMV), phosphorylation of MP expressed in bacteria, yeast, and Nicotiana benthamiana plants, respectively, has been demonstrated in previous studies. Three phosphorylation sites (T221, S223, and S250) were identified in its C-terminal oligomerization domain by mass spectrometry, suggesting a regulation of MP by posttranslational modification. To examine the influence of the three sites on the self-interaction in more detail, MP mutants were tested for their interaction in yeast by two-hybrid assays, or by Förster resonance energy transfer (FRET) techniques in planta. Expression constructs with point mutations leading to simultaneous (triple) exchange of T221, S223, and S250 to either uncharged alanine (MPAAA), or phosphorylation charge-mimicking aspartate residues (MPDDD) were compared. MPDDD interfered with MP-MP binding in contrast to MPAAA. The roles of the phosphorylation sites for the viral life cycle were studied further, using plant-infectious AbMV DNA-B variants with the same triple mutants each. When co-inoculated with wild-type DNA-A, both mutants infected N. benthamiana plants systemically, but were unable to do so for some other plant species of the families Solanaceae or Malvaceae. Systemically infected plants developed symptoms and viral DNA levels different from those of wild-type AbMV for most virus-plant combinations. The results indicate a regulation of diverse MP functions by posttranslational modifications and underscore their biological relevance for a complex host plant-geminivirus interaction.

Different forms of African cassava mosaic virus capsid protein within plants and virions.

One geminiviral gene encodes the capsid protein (CP), which can appear as several bands after electrophoresis depending on virus and plant. African cassava mosaic virus-Nigeria CP in Nicotiana benthamiana, however, yielded one band (~ 30 kDa) in total protein extracts and purified virions, although its expression in yeast yielded two bands (~ 30, 32 kDa). Mass spectrometry of the complete protein and its tryptic fragments from virions is consistent with a cleaved start M1, acetylated S2, and partial phosphorylation at T12, S25 and S62. Mutants for additional potentially modified sites (N223A; C235A) were fully infectious and formed geminiparticles. Separation in triton acetic acid urea gels confirmed charge changes of the CP between plants and yeast indicating differential phosphorylation. If the CP gene alone was expressed in plants, multiple bands were observed like in yeast. A high turnover rate indicates that post-translational modifications promote CP decay probably via the ubiquitin-triggered proteasomal pathway.

Differential methylation of the circular DNA in geminiviral minichromosomes.

Geminiviral minichromosomes were purified to explore epigenetic modifications. The levels of methylation in their covalently closed circular DNA were examined with the help of methylation-dependent restriction (MdR). DNA with 12 superhelical turns was preferentially modified, indicating minichromosomes with 12 nucleosomes leaving an open gap. MdR digestion yielded a specific product of genomic length, which was cloned and Sanger-sequenced, or amplified following ligation-mediated rolling circle amplification and deep-sequenced (circomics). The conventional approach revealed a single cleavage product indicating specific methylations at the borders of the common region. The circomics approach identified considerably more MdR sites in a preferential distance to each other of ~200 nts, which is the DNA length in a nucleosome. They accumulated in regions of nucleosome-free gaps, but scattered also along the genomic components. These results may hint at a function in specific gene regulation, as well as in virus resistance.

Properties of African Cassava Mosaic Virus Capsid Protein Expressed in Fission Yeast.

The capsid proteins (CPs) of geminiviruses combine multiple functions for packaging the single-stranded viral genome, insect transmission and shuttling between the nucleus and the cytoplasm. African cassava mosaic virus (ACMV) CP was expressed in fission yeast, and purified by SDS gel electrophoresis. After tryptic digestion of this protein, mass spectrometry covered 85% of the amino acid sequence and detected three N-terminal phosphorylation sites (threonine 12, serines 25 and 62). Differential centrifugation of cell extracts separated the CP into two fractions, the supernatant and pellet. Upon isopycnic centrifugation of the supernatant, most of the CP accumulated at densities typical for free proteins, whereas the CP in the pellet fraction showed a partial binding to nucleic acids. Size-exclusion chromatography of the supernatant CP indicated high order complexes. In DNA binding assays, supernatant CP accelerated the migration of ssDNA in agarose gels, which is a first hint for particle formation. Correspondingly, CP shifted ssDNA to the expected densities of virus particles upon isopycnic centrifugation. Nevertheless, electron microscopy did not reveal any twin particles, which are characteristic for geminiviruses.

Early function of the Abutilon mosaic virus AC2 gene as a replication brake.

UNLABELLED: The C2/AC2 genes of monopartite/bipartite geminiviruses of the genera Begomovirus and Curtovirus encode important pathogenicity factors with multiple functions described so far. A novel function of Abutilon mosaic virus (AbMV) AC2 as a replication brake is described, utilizing transgenic plants with dimeric inserts of DNA B or with a reporter construct to express green fluorescent protein (GFP). Their replicational release upon AbMV superinfection or the individual and combined expression of epitope-tagged AbMV AC1, AC2, and AC3 was studied. In addition, the effects were compared in the presence and in the absence of an unrelated tombusvirus suppressor of silencing (P19). The results show that AC2 suppresses replication reproducibly in all assays and that AC3 counteracts this effect. Examination of the topoisomer distribution of supercoiled DNA, which indicates changes in the viral minichromosome structure, did not support any influence of AC2 on transcriptional gene silencing and DNA methylation. The geminiviral AC2 protein has been detected here for the first time in plants. The experiments revealed an extremely low level of AC2, which was slightly increased if constructs with an intron and a hemagglutinin (HA) tag in addition to P19 expression were used. AbMV AC2 properties are discussed with reference to those of other geminiviruses with respect to charge, modification, and size in order to delimit possible reasons for the different behaviors. IMPORTANCE: The (A)C2 genes encode a key pathogenicity factor of begomoviruses and curtoviruses in the plant virus family Geminiviridae. This factor has been implicated in the resistance breaking observed in agricultural cotton production. AC2 is a multifunctional protein involved in transcriptional control, gene silencing, and regulation of basal biosynthesis. Here, a new function of Abutilon mosaic virus AC2 in replication control is added as a feature of this protein in viral multiplication, providing a novel finding on geminiviral molecular biology.

Three C-terminal phosphorylation sites in the Abutilon mosaic virus movement protein affect symptom development and viral DNA accumulation.

The Abutilon mosaic virus (AbMV, Geminiviridae) DNA B component encodes a movement protein (MP), which facilitates viral transport within plants and affects pathogenicity. The presence of phosphorylated serine and threonine residues was confirmed for MP expressed in yeast and Nicotiana benthamiana by comparative Western blot analysis using phospho-amino acid- and MP-specific immunodetection. Mass spectrometry of yeast-derived MP identified three phosphorylation sites located in the C-terminal domain (Thr-221, Ser-223 and Ser-250). To assess their functional relevance in plants, several point mutations were generated in the MP gene of DNA B, which replace Thr-221, Ser-223 and Ser-250, either singly or in combinations, with either an uncharged alanine or a phosphorylation-mimicking aspartate residue. When co-inoculated with DNA A, all mutants were infectious. In systemically infected plants the symptoms and/or viral DNA accumulation were significantly altered for several of the mutants.

Mitochondrial plasmids of sugar beet amplified via rolling circle method detected during curtovirus screening.

Crops of sugar beet have been considerably impaired by infection with Beet curly top virus (BCTV) during the past decades. Quick and reliable diagnostic techniques are therefore desirable to detect this circular single-stranded DNA-containing geminivirus. Techniques combining either tissue printing or blot hybridization, or rolling circle amplification (RCA) and restriction fragment length polymorphism (RFLP) were compared. Although they easily detected BCTV with certainty, both exhibited apparent false positive results which have been scrutinized in closer detail. Uninfected control plants revealed unspecific signals due to probe attachment on tissue blots, and dominant fragment patterns upon RCA/RFLP which did not hybridize with BCTV-specific probes. Cloning and sequencing of these DNA fragments showed that they were amplified from mitochondrial plasmids. Examination of their genome structure revealed no relationship with geminiviruses or their satellites.