Systems Biology of Tomato Fruit Development: Combined Transcript, Protein, and Metabolite Analysis of Tomato Transcription Factor (nor,

rin) and Ethylene Receptor (Nr) Mutants Reveals Novel Regulatory Interactions
Osorio et al. Plant Physiology, September 2011, Vol. 157, pp. 405-425

BACKGROUND
Tomato (Solanum lycopersicum) is an established model to study eshy fruit development and ripening. small genome size (950 Mb) relatively short life cycle stable genetic transformation

BACKGROUND
Tomato ripening is regulated independently and cooperatively by ethylene and transcription factors: including nonripening (nor) and ripening-inhibitor (rin) Mutations of nor, rin, and the ethylene receptor Neverripe (Nr), which block ethylene perception and inhibit ripening, have proven to be great tools for advancing our understanding of the developmental programs regulating ripening. In this paper, the authors present systems analysis of nor, rin, and Nr at the transcriptomic, proteomic, and metabolomic levels during development and ripening.

RESULTS
Transcriptomic Proling Investigated differential transcript accumulation using two-color simultaneous hybridizations of the TOM1 array during tomato development for the wild type, Nr, nor and rin (10 time points) Expression analysis in a condensed PageMap display
light reaction photosystem:

RESULTS
Metabolic Proling Primary metabolite levels in the WT, nor, rin and Nr:

RESULTS
Proteomic Analysis Comparison of changes in mRNA and cognate protein abundance (mutant/wild type):

RESULTS
Network Analysis Visualization of transcript-protein-metabolite correlation:

RESULTS
Potential genetic regulatory network centered on ethylene governing tomato fruit development and ripening:

DISCUSSION
Ripening Mutations Provide Tools to Parse Transcription and Hormonal Control Exploitation of rin and nor make us able to define system under the influence of transcriptional
rin and nor genes operate upstream of crucial ripening activity nor is upstream of rin since ethylene/ ripening related gene expression appear to be more extensively inhibited in nor In rin, fruits remain responsive to ethylene in that ethyleneresponsive genes are induced by exogenous ethylene For nor, fruits remaining unripe in response to ethylene rin and nor fruits fail to complete normal ripening because the fruits fail to produce climecteric ethylene

Exploitation of Nr enable us to define the system under ethylene control

Nr gene interfere ethylene perception and significantly delay fruit ripening

DISCUSSION
Systems Analysis of Ripening Control Using Ripening Mutations Transcriptional analysis on the 3 ripening mutant revealed how ethylene, via nor, rin and Nr, influences the expression of hundred genes during development both prior to the onset of and during ripening These prove that ethylene plays in governing biochemical, physiochemical, and developmental processes throughout tomato development

DISCUSSION
Different Regulation of Ethylene-Related Genes in nor, rin, and Nr Normal development:
Ethylene synthesis-related genes - up-regulated in breaker stage fruits Ethylene signal transduction genes - up-regulated in late ripe stage

Mutants (nor, rin and Nr):

Ethylene signal transduction unaltered across development and ripening

There are genes in rin and Nr show up-regulation, but unaltered in nor Linear order of genes: nor-rin-Nr
nor higher order regulator than rin or Nr (ethylene) Ethylene synthesis gene promoters directly interact with rin

DISCUSSION
Characterization of the Fruit Ethyleneome Demonstrates Regulated Metabolomic Flux toward Ethylene Synthesis Ethylene synthesized from S-adenosylmethionine (SAM) SAM - potential to participate in polyamine or ethylene biosynthesis or both From transcriptomic and metabolomic data in this study, it show that ethylene biosynthesis may dominate the flux through SAM Polyamine accumulation in transgenic tomato lines deficient in the expression of enzymes of polyamine biosynthesis revealed a shift in SAM flux concomitant with an increase of ethylene and a decrease of aspartate level

DISCUSSION
nor, rin, and Nr Displayed Metabolic Shifts

Malate
decreased (during ripening) in wild type less decreased in rin & Nr unaltered in nor

Ethylene feedback regulates its own synthesis not only at the transcriptional level but also the metabolic level Sucrose degradation (late stages of ripening):
up-regulation: rin & Nr down-regulation: nor

Phenylalanine act as alternative respiratory substrates when carbohydrates are not abundant
Gradually increased during normal ripening also in the three mutants

DISCUSSION
Cell Wall Metabolism Is Affected in nor, rin, and Nr

Ripening related changes in tomato

progressive & extensive loss of firmness (part of cell wall disassembly)

Disassembly of hemicellulosic & pectin cell wall polysaccharides

reduced in rin fruit, diminished in nor

Cell wall-related metabolite (galacturonate, Gal, Ara, Xyl, Rib, Rha)

increased in wild type no changes in nor, rin & Nr

Polygalacturonase protein accumulation (ethylene & additional transcriptional effects)

strong reduction in nor and rin compared with Nr

DISCUSSION
Others Metabolic Changes Shown by nor, rin and Nr

Ascorbate metabolism (up-regulation of ascorbaterelated genes during ripening as well as ascorbate accumulation)
only in Nr neither nor nor rin showed changes in this pathway

Nr, nor & rin all showed some carotenoid production

normal food ripening (42 DAP) nor & rin (earlier stages, 39 & 41 DAP) Nr (later stage, 52 DAP)

Levels of organic acids correlate strongly with genes associated pathway

the metabolic intermediates in ripening is important

DISCUSSION
Protein Variations as Compared with Transcriptomic Data in nor, rin, and Nr A total of 158 proteins showed variations in abundance during ripening or equivalent stages of development in nor, rin & Nr than wild type The highest no of differently expressed proteins with respect to the wild type were at the ripe stage (52 DAP) among the three mutants. nor & rin shared a greater no of common protein (especially at 52 DAP)
nor & rin act together in the ethylene signalling cascade

32% (42 out of 133) of protein & transcript pairs decreased or increased in parallel during ripening
this correlation was particularly apparent at the late ripening stage (52 DAP) a high no of proteins that showed no correlations with the corresponding transcripts belonged to primary metabolism

CONCLUSION
This combination of transcriptome, proteome, and targeted metabolite analysis has helped to rene the ethylene-regulated transcriptome of tomato fruit and added to our knowledge the role of ethylene in both protein and metabolite regulation in tomato ripening. nor and rin act together in a cascade to control ripening (nor operates upstream of rin). Identify areas of metabolism that seem to be of high importance to the ripening process, such as hormones and cell wall metabolism in ethylene perception. These outputs will provide potential targets for the engineering of metabolism to facilitate the controlled modulation of ripening in tomato fruit.

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