The new kid on the block: a dominant-negative mutation of phototropin1 enhances carotenoid content in tomato fruits.

Phototropins, the UVA-blue light photoreceptors, endow plants to detect the direction of light and optimize photosynthesis by regulating positioning of chloroplasts and stomatal gas exchange. Little is known about their functions in other developmental responses. A tomato Non-phototropic seedling1 (Nps1) mutant, bearing an Arg495His substitution in the vicinity of LOV2 domain in phototropin1, dominant-negatively blocks phototropin1 responses. The fruits of Nps1 mutant were enriched in carotenoids, particularly lycopene, compared with its parent, Ailsa Craig. On the contrary, CRISPR/CAS9-edited loss of function phototropin1 mutants displayed subdued carotenoids compared with the parent. The enrichment of carotenoids in Nps1 fruits is genetically linked with the mutation and exerted in a dominant-negative fashion. Nps1 also altered volatile profiles with high levels of lycopene-derived 6-methyl 5-hepten2-one. The transcript levels of several MEP and carotenogenesis pathway genes were upregulated in Nps1. Nps1 fruits showed altered hormonal profiles with subdued ethylene emission and reduced respiration. Proteome profiles showed a causal link between higher carotenogenesis and increased levels of protein protection machinery, which may stabilize proteins contributing to MEP and carotenogenesis pathways. The enhancement of carotenoid content by Nps1 in a dominant-negative fashion offers a potential tool for high lycopene-bearing hybrid tomatoes.

A dominant mutation in the light-oxygen and voltage2 domain vicinity impairs phototropin1 signaling in tomato.

In higher plants, blue light (BL) phototropism is primarily controlled by the phototropins, which are also involved in stomatal movement and chloroplast relocation. These photoresponses are mediated by two phototropins, phot1 and phot2. Phot1 mediates responses with higher sensitivity than phot2, and phot2 specifically mediates chloroplast avoidance and dark positioning responses. Here, we report the isolation and characterization of a Nonphototropic seedling1 (Nps1) mutant of tomato (Solanum lycopersicum). The mutant is impaired in low-fluence BL responses, including chloroplast accumulation and stomatal opening. Genetic analyses show that the mutant locus is dominant negative in nature. In dark-grown seedlings of the Nps1 mutant, phot1 protein accumulates at a highly reduced level relative to the wild type and lacks BL-induced autophosphorylation. The mutant harbors a single glycine-1484-to-alanine transition in the Hinge1 region of a phot1 homolog, resulting in an arginine-to-histidine substitution (R495H) in a highly conserved A'α helix proximal to the light-oxygen and voltage2 domain of the translated gene product. Significantly, the R495H substitution occurring in the Hinge1 region of PHOT1 abolishes its regulatory activity in Nps1 seedlings, thereby highlighting the functional significance of the A'α helix region in phototropic signaling of tomato.

Complex and shifting interactions of phytochromes regulate fruit development in tomato.

Tomato fruit ripening is a complex metabolic process regulated by a genetical hierarchy. A subset of this process is also modulated by light signalling, as mutants encoding negative regulators of phytochrome signal transduction show higher accumulation of carotenoids. In tomato, phytochromes are encoded by a multi-gene family, namely PHYA, PHYB1, PHYB2, PHYE and PHYF; however, their contribution to fruit development and ripening has not been examined. Using single phytochrome mutants phyA, phyB1 and phyB2 and multiple mutants phyAB1, phyB1B2 and phyAB1B2, we compared the on-vine transitory phases of ripening until fruit abscission. The phyAB1B2 mutant showed accelerated transitions during ripening, with shortest time to fruit abscission. Comparison of transition intervals in mutants indicated a phase-specific influence of different phytochrome species either singly or in combination on the ripening process. Examination of off-vine ripened fruits indicated that ripening-specific carotenoid accumulation was not obligatorily dependent upon light and even dark-incubated fruits accumulated carotenoids. The accumulation of transcripts and carotenoids in off-vine and on-vine ripened mutant fruits indicated a complex and shifting phase-dependent modulation by phytochromes. Our results indicate that, in addition to regulating carotenoid levels in tomato fruits, phytochromes also regulate the time required for phase transitions during ripening.

NEATTILL: A simplified procedure for nucleic acid extraction from arrayed tissue for TILLING and other high-throughput reverse genetic applications.

BACKGROUND: TILLING (Targeting Induced Local Lesions in Genomes) is a reverse genetics procedure for identifying point mutations in selected gene(s) amplified from a mutagenized population using high-throughput detection platforms such as slab gel electrophoresis, capillary electrophoresis or dHPLC. One essential pre-requisite for TILLING is genomic DNA isolation from a large population for PCR amplification of selected target genes. It also requires multiplexing of genomic DNA isolated from different individuals (pooling) in typically 8-fold pools, for mutation scanning, and to minimize the number of PCR amplifications, which is a strenuous and long-drawn-out work. We describe here a simplified procedure of multiplexing, NEATTILL (Nucleic acid Extraction from Arrayed Tissue for TILLING), which is rapid and equally efficient in assisting mutation detection. RESULTS: The NEATTILL procedure was evaluated for the tomato TILLING platform and was found to be simpler and more efficient than previously available methods. The procedure consisted of pooling tissue samples, instead of nucleic acid, from individual plants in 96-well plates, followed by DNA isolation from the arrayed samples by a novel protocol. The three variants of the NEATTILL procedure (vast, in-depth and intermediate) can be applied across various genomes depending upon the population size of the TILLING platform. The 2-D pooling ensures the precise confirmation of the coordinates of the positive mutant line while scanning complementary plates. Choice of tissue for arraying and nucleic acid isolation is discussed in detail with reference to tomato. CONCLUSION: NEATTILL is a convenient procedure that can be applied to all organisms, the genomes of which have been mutagenized and are being scanned for multiple alleles of various genes by TILLING for understanding gene-to-phenotype relationships. It is a time-saving, less labour intensive and reasonably cost-effective method. Tissue arraying can cut costs by up to 90% and minimizes the risk of exposing the DNA to nucleases. Before arraying, different tissues should be evaluated for DNA quality, as the case study in tomato showed that cotyledons rather than leaves are better suited for DNA isolation. The protocol described here for nucleic acid isolation can be generally adapted for large-scale projects such as insertional mutagenesis, transgenic confirmation, mapping and fingerprinting which require isolation of DNA from large populations.

Structural and functional analysis of rice genome.

Rice is an excellent system for plant genomics as it represents a modest size genome of 430 Mb. It feeds more than half the population of the world. Draft sequences of the rice genome, derived by whole-genome shotgun approach at relatively low coverage (4-6 X), were published and the International Rice Genome Sequencing Project (IRGSP) declared high quality (>10 X), genetically anchored, phase 2 level sequence in 2002. In addition, phase 3 level finished sequence of chromosomes 1, 4 and 10 (out of 12 chromosomes of rice) has already been reported by scientists from IRGSP consortium. Various estimates of genes in rice place the number at >50,000. Already, over 28,000 full-length cDNAs have been sequenced, most of which map to genetically anchored genome sequence. Such information is very useful in revealing novel features of macro- and micro-level synteny of rice genome with other cereals. Microarray analysis is unraveling the identity of rice genes expressing in temporal and spatial manner and should help target candidate genes useful for improving traits of agronomic importance. Simultaneously, functional analysis of rice genome has been initiated by marker-based characterization of useful genes and employing functional knock-outs created by mutation or gene tagging. Integration of this enormous information is expected to catalyze tremendous activity on basic and applied aspects of rice genomics.

Sequence analysis of the long arm of rice chromosome 11 for rice-wheat synteny.

The DNA sequence of 106 BAC/PAC clones in the minimum tiling path (MTP) of the long arm of rice chromosome 11, between map positions 57.3 and 116.2 cM, has been assembled to phase 2 or PLN level. This region has been sequenced to 10x redundancy by the Indian Initiative for Rice Genome Sequencing (IIRGS) and is now publicly available in GenBank. The region, excluding overlaps, has been predicted to contain 2,932 genes using different software. A gene-by-gene BLASTN search of the NCBI wheat EST database of over 420,000 cDNA sequences revealed that 1,143 of the predicted rice genes (38.9%) have significant homology to wheat ESTs (bit score >/= 100). Further BLASTN search of these 1,143 rice genes with the GrainGenes database of sequence contigs containing bin-mapped wheat ESTs allowed 113 of the genes to be placed in bins located on wheat chromosomes of different homoeologous groups. The largest number of genes, about one-third, mapped to the homoeologous group 4 chromosomes of wheat, suggesting a common evolutionary origin. The remaining genes were located on wheat chromosomes of different groups with significantly higher numbers for groups 3 and 5. Location of bin-mapped wheat contigs to chromosomes of all the seven homoeologous groups can be ascribed to movement of genes (transpositions) or chromosome segments (translocations) within rice or the hexaploid wheat genomes. Alternatively, it could be due to ancient duplications in the common ancestral genome of wheat and rice followed by selective elimination of genes in the wheat and rice genomes. While there exists definite conservation of gene sequences and the ancestral chromosomal identity between rice and wheat, there is no obvious conservation of the gene order at this level of resolution. Lack of extensive colinearity between rice and wheat genomes suggests that there have been many insertions, deletions, duplications and translocations that make the synteny comparisons much more complicated than earlier thought. However, enhanced resolution of comparative sequence analysis may reveal smaller conserved regions of colinearity, which will facilitate selection of markers for saturation mapping and sequencing of the gene-rich regions of the wheat genome.