Leaf Plasmodesmata Respond Differently to TMV, ToBRFV and TYLCV Infection.

Macromolecule and cytosolic signal distribution throughout the plant employs a unique cellular and intracellular mechanism called plasmodesmata (PD). Plant viruses spread throughout plants via PD using their movement proteins (MPs). Viral MPs induce changes in plasmodesmata's structure and alter their ability to move macromolecule and cytosolic signals. The developmental distribution of a family member of proteins termed plasmodesmata located proteins number 5 (PDLP5) conjugated to GFP (PDLP5-GFP) is described here. The GFP enables the visual localization of PDLP5 in the cell via confocal microscopy. We observed that PDLP5-GFP protein is present in seed protein bodies and immediately after seed imbibition in the plasma membrane. The effect of three different plant viruses, the tobacco mosaic virus (TMV), tomato brown rugose fruit virus (ToBRFV, tobamoviruses), and tomato yellow leaf curl virus (TYLCV, begomoviruses), on PDLP5-GFP accumulation at the plasmodesmata was tested. In tobacco leaf, TMV and ToBRFV increased PDLP5-GFP amount at the plasmodesmata of cell types compared to control. However, there was no statistically significant difference in tomato leaf. On the other hand, TYLCV decreased PDLP5-GFP quantity in plasmodesmata in all tomato leaf cells compared to control, without any significant effect on plasmodesmata in tobacco leaf cells.

Functional analysis of MYB alleles from Solanum chilense and Solanum lycopersicum in controlling anthocyanin levels in heterologous tobacco plants.

Flavonoids are natural pigments occurring in plants and are present in fruits, leaves, stems, roots, and flowers. Tobacco plants transformed with an MYB regulatory gene from either Solanum chilense (Sc) or S. lycopersicum (Sl) demonstrate that ScANT1 induces a higher level of anthocyanin accumulation in comparison to SlANT1 and that this gene is sufficient to promote increased anthocyanin levels. We compared the aptitude of ScANT1 protein to induce anthocyanin accumulation to that of SlANT1 protein in tobacco plants. We also tested the effect of amino acid substitutions in ScANT1 and SlANT1. We examined these synthetic alleles' effect following the over-expression of additional anthocyanin synthesis regulators, such as the tomato bHLH (SlJAF13) protein. Our results show that the amino acid changes that differentiate ScANT1 from SlANT1 are the main contributors to the advantage that ScANT1 has over SlANT1 in anthocyanin accumulation per transcript unit. We further demonstrated that altering the amino acid composition of SlANT1 could increase anthocyanin accumulation, while reciprocally modifying ScANT1 lowers the anthocyanin level. These results confirm the increased anthocyanin level in tobacco is attributed to the amino acid differences between ScANT1 and SlANT1. We also show that the co-expression of SlJAF13 with SlANT1 in tobacco plants represses the anthocyanin production.

Shoot Regeneration Is Not a Single Cell Event.

Shoot regeneration is a key tool of modern plant biotechnology. While many researchers use this process empirically, very little is known about the early molecular genetic factors and signaling events that lead to shoot regeneration. Using tobacco as a model system, we found that the inductive events required for shoot regeneration occur in the first 4-5 days following incubation on regeneration medium. Leaf segments placed on regeneration medium did not produce shoots if removed from the medium before four days indicating this time frame is crucial for the induction of shoot regeneration. Leaf segments placed on regeneration medium for longer than five days maintain the capacity to produce shoots when removed from the regeneration medium. Analysis of gene expression during the early days of incubation on regeneration medium revealed many changes occurring with no single expression pattern evident among major gene families previously implicated in developmental processes. For example, expression of Knotted gene family members increased during the induction period, whereas transcription factors from the Wuschel gene family were unaltered during shoot induction. Expression levels of genes involved in cell cycle regulation increased steadily on regeneration medium while expression of NAC genes varied. No obvious possible candidate genes or developmental processes could be identified as a target for the early events (first few days) in the induction of shoot regeneration. On the other hand, observations during the early stages of regeneration pointed out that regeneration does not occur from a single cell but a group of cells. We observed that while cell division starts just as leaf segments are placed on regeneration medium, only a group of cells could become shoot primordia. Still, these primordia are not identifiable during the first days.

Expression of flowering locus T2 transgene from Pyrus communis L. delays dormancy and leaf senescence in Malus×domestica Borkh, and causes early flowering in tobacco.

Annual and perennial plants represent two different evolutionary strategies based on differential synchronization of their reproductive development. The mobile signal protein FLOWERING LOCUS T (FT) plays a central role in mediating the onset of reproduction in both plant types. Two novel FT-like genes from pear (Pyrus communis)-PcFT1 and PcFT2-were isolated, and their expression profiles were determined for one annual cycle. The effects of PcFT2 on flowering were investigated in annual (tobacco) and perennial (apple) plants by means of grafting and generating transgenic plants. Long-distance graft transmission of PcFT2 in both annual and perennial plants was confirmed using a 35S::PcFT2-YFP construct. Ectopic overexpression of PcFT2 caused early flowering in tobacco but not in apple. Transgenic apples were less sensitive to short-day-induced dormancy, and this phenotype was also observed in wild-type apples grafted onto the transgenic plants. Comparison of PcFT2 protein structure to the paralogous FT proteins from apple and pear showed alterations that could influence protein structure and thus the florigen-activation complex. PcFT2 protein seems to function by promoting flowering as all other FT proteins in the annual plant tobacco while in the perennial plant apple PcFT2 does not promote flowering but delays senescence. This observation may hint to a modified function of FT2 in perennial plants.

A Novel Route Controlling Begomovirus Resistance by the Messenger RNA Surveillance Factor Pelota.

Tomato yellow leaf curl virus (TYLCV) is a devastating disease of tomato (Solanum lycopersicum) that can be effectively controlled by the deployment of resistant cultivars. The TYLCV-resistant line TY172 carries a major recessive locus for TYLCV resistance, designated ty-5, on chromosome 4. In this study, the association between 27 polymorphic DNA markers, spanning the ty-5 locus, and the resistance characteristics of individual plants inoculated with TYLCV in 51 segregating recombinant populations were analyzed. These analyses localized ty-5 into a 425 bp region containing two transversions: one in the first exon of a gene encoding the tomato homolog of the messenger RNA surveillance factor Pelota (Pelo), and a second in its proximal promoter. Analyses of susceptible and resistant lines revealed that the relative transcript level of the gene remained unchanged, regardless of whether the plants were infected with TYLCV or not. This suggests that the polymorphism discovered in the coding region of the gene controls the resistance. Silencing of Pelo in a susceptible line rendered the transgenic plants highly resistant, while in the resistant line TY172 had no effect on symptom development. In addition, over-expression of the susceptible allele of the gene in the resistant TY172 line rendered it susceptible, while over-expression of the resistant allele in susceptible plants had no effect. These results confirm that Pelo is the gene controlling resistance at the ty-5 locus. Pelo, implicated in the ribosome recycling-phase of protein synthesis, offers an alternative route to promote resistance to TYLCV and other viruses.

Expression of mitochondrial gene fragments within the tapetum induce male sterility by limiting the biogenesis of the respiratory machinery in transgenic tobacco.

Plant mitochondrial genomes (mtDNAs) are large and undergo frequent recombination events. A common phenotype that emerges as a consequence of altered mtDNA structure is cytoplasmic-male sterility (CMS). The molecular basis for CMS remains unclear, but it seems logical that altered respiration activities would result in reduced pollen production. Analysis of tobacco (Nicotiana tabacum) mtDNAs indicated that CMS-associated loci often contain fragments of known organellar genes. These may assemble with organellar complexes and thereby interfere with normal respiratory functions. Here, we analyzed whether the expression of truncated fragments of mitochondrial genes (i.e. atp4, cox1 and rps3) may induce male sterility by limiting the biogenesis of the respiratory machinery. cDNA fragments corresponding to atp4f, cox1f and rps3f were cloned in-frame to a mitochondrial localization signal and a C-termini HA-tag under a tapetum-specific promoter and introduced to tobacco plants by Agrobacterium-mediated transformation. The constructs were then analyzed for their effect on mitochondrial activity and pollen fertility. Atp4f, Cox1f and Rps3f plants demonstrated male sterility phenotypes, which were tightly correlated with the expression of the recombinant fragments in the floral meristem. Fractionation of native organellar extracts showed that the recombinant ATP4f-HA, COX1f-HA and RPS3f-HA proteins are found in large membrane-associated particles. Analysis of the respiratory activities and protein profiles indicated that organellar complex I was altered in Atp4f, Cox1f and Rps3f plants.

ANTHOCYANIN1 from Solanum chilense is more efficient in accumulating anthocyanin metabolites than its Solanum lycopersicum counterpart in association with the ANTHOCYANIN FRUIT phenotype of tomato.

Anthocyanins are flavonoid metabolites contributing attractive colors and antioxidant qualities to the human diet. Accordingly, there is a growing interest in developing crops enriched with these compounds. Fruits of the cultivated tomato, Solanum (S.) lycopersicum, do not normally produce high levels of anthocyanins. However, several wild tomato species yield anthocyanin-pigmented fruits, and this trait has been introgressed into the cultivated tomato. Two genes encoding homologous R2R3 MYB transcription factors, termed ANT1 and AN2, were previously genetically implicated in anthocyanin accumulation in tomato fruit peels of the ANTHOCYANIN FRUIT (AFT) genotype originating from S. chilense. Here we compared transgenic tomato plants constitutively over-expressing the S. lycopersicum (35S::ANT1 ( L ) ) or the S. chilense (35S::ANT1 ( C )) allele, and show that each displayed variable levels of purple pigmentation in vegetative as well as reproductive tissues. However, 35S::ANT1 ( C ) was significantly more efficient in producing anthocyanin pigments, attributed to its gene coding-sequence rather than to its transcript levels. These results expand the potential of enhancing anthocyanin levels through engineering coding-sequence polymorphisms in addition to the transcriptional alterations commonly used. In addition, a segregating population obtained from a recombinant genotype revealed that the native ANT1, and not AN2, is fully associated with the AFT phenotype and that ANT1 alone can generate the characteristic phenotype of anthocyanin accumulation in AFT fruits. Our results therefore provide further support to the hypothesis that ANT1 is the gene responsible for anthocyanin accumulation in fruits of the AFT genotype.

Overexpression of UV-DAMAGED DNA BINDING PROTEIN 1 links plant development and phytonutrient accumulation in high pigment-1 tomato.

Fruits of tomato plants carrying the high pigment-1 mutations hp-1 and hp-1(w) are characterized by an increased number of plastids coupled with enhanced levels of functional metabolites. Unfortunately, hp-1 mutant plants are also typified by light-dependent retardation in seedling and whole-plant growth and development, which limits their cultivation. These mutations were mapped to the gene encoding UV-DAMAGED DNA BINDING PROTEIN 1 (DDB1) and, recently, fruit-specific RNA interference studies have demonstrated an increased number of plastids and enhanced carotenoid accumulation in the transgenic tomato fruits. However, whole-plant overexpression of DDB1, required to substantiate its effects on seedling and plant development and to couple them with fruit phenotypes, has heretofore been unsuccessful. In this study, five transgenic lines constitutively overexpressing normal DDB1 in hp-1 mutant plants were analysed. Eleven-day-old seedlings, representing these lines, displayed up to approximately 73- and approximately 221-fold overexpression of the gene in hypocotyls and cotyledons, respectively. This overexpression resulted in statistically significant reversion to the non-mutant developmental phenotypes, including more than a full quantitative reversion. This reversion of phenotypes was generally accompanied by correlated responses in chlorophyll accumulation and altered expression of selected light signalling genes: PHYTOCHROME A, CRYPTOCHROME 1, ELONGATED HYPOCOTYL 5, and the gene encoding CHLOROPHYLL A/B-BINDING PROTEIN 4. Cumulatively, these results provide the missing link between DDB1 and its effects on tomato plant development.

Light signaling genes and their manipulation towards modulation of phytonutrient content in tomato fruits.

Due to its economic importance, ease of genetic manipulation, cultivation and processing, the tomato plant has been a target for increasing and diversifying content of fruit phytonutrients by transgenic and non-transgenic approaches. The tomato high pigment (hp) mutations exemplify the latter alternative and due to their positive effect on fruit lycopene content, they were introgressed into elite tomato germplasm for cost effective extraction of this important carotenoid. Interestingly, hp mutant fruits are also characterized by higher fruit levels of other functional metabolites, phenotypes caused by mutations in central genes regulating light signal-transduction. This gene identification suggests that modulation of light signaling machinery in plants may be highly effective towards manipulation of fruit phytonutrients but has never been thoroughly reviewed. This review therefore summarizes the progress which has been made on this valuable approach, emphasizing the consequences of transgenic modulation of light signaling components on the functional properties of the tomato fruit.

Molecular aspects of Anthocyanin fruit tomato in relation to high pigment-1.

The tomato Anthocyanin fruit (Aft) genotype is characterized by purple color in skin and outer pericarp of its fruits due to higher levels of anthocyanins-flavonoid metabolites. Our objectives were to carry out metabolic and molecular characterization of this genotype, emphasizing its interaction with the high pigment-1 (hp-1) mutation, known to increase flavonoids in tomato fruits. These objectives fit the growing interest in developing tomato fruits with higher levels of functional metabolites. Our results show that 1) Aft fruits are also characterized by significantly higher levels of the flavonols quercetin and kaempferol, thus enhancing their functional value; 2) the tomato Anthocyanin1 (Ant1) gene, encoding a Myb transcription factor, displayed nucleotide and amino acid polymorphisms between the Aft genotype and cultivated genotypes; 3) a DNA marker based on Ant1 showed that the Aft trait is encoded by a single locus on chromosome 10 fully associated with Ant1; and 4) double homozygotes Aft/Aft hp-1/hp-1 plants displayed a more-than-additive effect on the production of fruit anthocyanidins and flavonols. This effect was manifested by approximately 5-, 19-, and 33-fold increase of petunidin, malvidin, and delphinidin, respectively, in the double mutants compared with the cumulative levels of their parental lines.