Emerging Research in Plant Cell Biology

Genomics-based breeding of economically important crops such as banana, coffee, cotton, potato, tobacco and wheat is often hampered by genome size, polyploidy and high repeat content. We adapted the sequence-based Whole Genome Profiling (WGP™) technology to gain insight into the polyploidy of the model plant Nicotiana tabacum (tobacco). N. tabacum is assumed to originate from a hybridization event between Nicotiana sylvestris and Nicotiana tomentosiformis about 200,000 years ago. This resulted in tobacco having a haploid genome size of 4,500 million base pairs, about four times larger than the related tomato and potato genomes. In this study a physical map containing 9,750 contigs of bacterial artificial chromosomes (BACs) was constructed. The average contig size was 462 kbp, and the calculated genome coverage equaled the estimated tobacco genome size. We used a method for determination of the ancestral origin of the genome by annotation of WGP sequence tags. This assignment agreed with the ancestral annotation available from the tobacco genetic map and may be used to investigate the evolution of homoeologous genome segments after polyploidization. The map generated will be an essential scaffold for the tobacco genome. We propose the combination of WGP physical mapping technology with tag profiling of ancestral lines as a generally applicable method to elucidate the ancestral origin of genome segments of polyploid species. The physical mapping of genes and their origins will enable application of biotechnology to polyploid plants aimed at accelerating and increasing the precision of breeding for abiotic and biotic stress resistance.

The tomato MADS-box transcription factor RIN acts as a master regulator of fruit ripening. Here, we identified MADS-box proteins that interact with RIN; we also provide evidence that these proteins act in the regulation of fruit ripening. We conducted a yeast two-hybrid screen of a cDNA library from ripening fruit, for genes encoding proteins that bind to RIN. The screen identified two MADS-box genes, FUL1 and FUL2 (previously called TDR4 and SlMBP7), both of which have high sequence similarity to Arabidopsis FRUITFULL. Expression analyses revealed that the FUL1 mRNA and FUL1 protein accumulate in a ripening-specific manner in tomato fruits and FUL2 mRNA and protein accumulate at the pre-ripening stage and throughout ripening. Biochemical analyses confirmed that FUL1 and FUL2 form heterodimers with RIN; this interaction required the FUL1 and FUL2 C-terminal domains. Also, the heterodimers bind to a typical target DNA motif for MADS-box proteins. Chromatin immunoprecipitation assays revealed that FUL1 and FUL2 bind to genomic sites that were previously identified as RIN-target sites, such as the promoter regions of ACS2, ACS4 and RIN. These findings suggest that RIN forms complexes with FUL1 and FUL2 and these complexes regulate expression of ripening-related genes. In addition to the functional redundancy between FUL1 and FUL2, we also found they have potentially divergent roles in transcriptional regulation, including a difference in genomic target sites.

We present the coupled use of specifically localized fluorescent gene markers and image processing for automated quantitative analysis of cell growth and genetic activity across living plant tissues. We used fluorescent protein markers to identify cells, create seeds and boundaries for the automatic segmentation of cell geometries and ratiometrically measure gene expression cell by cell in Arabidopsis thaliana.

Dehydration leads to different physiological and biochemical responses in plants. We analyzed the lipid composition and the expression of genes involved in lipid biosynthesis in the desiccation-tolerant plant Craterostigma plantagineum. A comparative approach was carried out with Lindernia brevidens (desiccation tolerant), and two desiccation-sensitive species, L. subracemosa and A. thaliana. In C. plantagineum the total lipid content remained constant while the lipid composition underwent major changes during desiccation. The most prominent change was the removal of monogalactosyldiacylglycerol (MGDG) from the thylakoids. Analysis of molecular species composition revealed that around 50% of 36:x (number of carbons in the acyl chains: number of double bonds) MGDG was hydrolyzed and diacylglycerol (DAG) used for phospholipid synthesis, while another MGDG fraction was converted into digalactosyldiacylglycerol via the DGD1/DGD2 pathway and subsequently into oligogalactolipids by SFR2. 36:x-DAG was also employed for the synthesis of triacylglycerol. Phosphatidic acid (PA) increased in C. plantagineum, L. brevidens, and L. subracemosa, in agreement with a role of PA as an intermediate of lipid turnover and of phospholipase D in signalling during desiccation. 34:x-DAG, presumably derived from de novo assembly, was converted into phosphatidylinositol (PI) in C. plantagineum and L. brevidens, but not in desiccation-sensitive plants, suggesting that PI is involved in acquisition of desiccation tolerance. The accumulation of oligogalactolipids and PI in the chloroplast and extraplastidial membranes, respectively, increases the concentration of hydroxyl groups and enhances the ratio of bilayer to nonbilayer forming lipids, thus contributing to protein and membrane stabilization.

The RNA-directed DNA methylation (RdDM) pathway is of central importance to the initiation and maintenance of transcriptional gene silencing in plants. DNA methylation is directed to target sequences in a mechanism that involves the production of small RNAs by RNA Polymerase IV and by long non-coding RNAs by RNA Polymerase V. DNA methylation then leads to the recruitment of histone modifying enzymes followed by the establishment of a silenced chromatin state. Recently MORC6, a member of the Microrchidia (MORC) family of adenosine triphosphatases (ATPases), has been shown to be involved in transcriptional gene silencing. However reports differed regarding whether MORC6 was involved in RdDM itself or acting downstream of DNA methylation to enable the formation of higher order chromatin structure. In this work we demonstrate that MORC6 is required for efficient RdDM at some target loci and using a GFP reporter system we observe that morc6 mutants give rise to a stochastic silencing phenotype. By using cell sorting to separate silenced and unsilenced cells, we show that release of silencing at this locus does not occur in the absence of a loss of DNA methylation. Thus our data supports a view that MORC6 can influence RdDM and that it is not only acting downstream of DNA methylation. Therefore for some loci, efficient initiation or maintenance of DNA methylation may depend on the ability to form higher order chromatin structure.

SummaryWhile the existence of environmental reservoirs of human pathogens is well established, less is known about the role of nonagricultural environments in emergence, evolution, and spread of crop pathogens.Here, we analyzed phylogeny, virulence genes, host range, and aggressiveness of Pseudomonas syringae strains closely related to the tomato pathogen P. syringae pv. tomato (Pto), including strains isolated from snowpack and streams.The population of Pto relatives in nonagricultural environments was estimated to be large and its diversity to be higher than that of the population of Pto and its relatives on crops. Ancestors of environmental strains, Pto, and other genetically monomorphic crop pathogens were inferred to have frequently recombined, suggesting an epidemic population structure for P. syringae. Some environmental strains have repertoires of type III-secreted effectors very similar to Pto, are almost as aggressive on tomato as Pto, but have a wider host range than typical Pto strains.We conclude that crop pathogens may have evolved through a small number of evolutionary events from a population of less aggressive ancestors with a wider host range present in nonagricultural environments.

peptides are interchangeable between precursor proteins. However, emerging evidence shows that different transit peptides contain different motifs specifying their preference for certain plastid types or ages. In this opinion article, we propose a ‘multi-selection and multi-order’ (M&M) model for transit peptide design, describing each transit peptide as an assembly of motifs for interacting with selected translocon components. These interactions determine the preference of the precursor for a particular plastid type or age. Furthermore, the order of the motifs varies among transit peptides, explaining why no consensus sequences have been identified through linear sequence comparison of all transit peptides as one group.