Tomato transcriptome

As most children, growing up I showed little appreciation for what I had, coveting instead what I did not. Now I realize how fortunate I have been to have grown up half the time in a metropolis and the other half at the countryside. At the farm. A subsistence farm, although I truly loathe the term because we were not just subsisting but thriving off the land, as we planted and harvested a bit of everything and we had a specimen or four of almost all the farm animals, from bipeds to quadrupeds.

I got on this memory lane after reading the paper of Shinozaki et al. (2018) on tomatoes. It was a difficult read for me as it was punctured by many term definition lookups since botany evolved quite steeply since the last time I checked, about 25 years or so.

Briefly, the scientists grew tomato plants in a greenhouse at Cornell, NY. They harvested the fruit from 60 plants about 5 to 50 days after the flower was at its peak (DPA, days post anthesis) following this chart:

• Expanding [fruit] stage (harvested at 5, 10, 20, or 30 DPA)
• Mature Green stage (full-size green fruit, ≈ 39 DPA),
• Breaker stage (definite break in color from green to tannish-yellow with less than 10% of the surface, ≈ 42 DPA),
• Pink stage (50% pink or red color, ≈ 44 DPA),
• Light red stage (100% light red, ≈ 46 DPA),
• Red ripe stage (full red for 8 days, ≈ 50 DPA).

(simplified from the Methods section, p. 10, see pic)

Fig. 1 (partial from Shinozaki et al., 2018). A tissue/cell-based transcript profiling of developing tomato fruit. a Traced image of six targeted fruit tissues. Shaded areas of the total pericarp and the placenta were not harvested. b Traced image of five pericarp cells. c Representative pictures of harvested fruit spanning ten developmental stages. d Representative pictures of the stylar end of MG and Br stage fruit. DPA, days post anthesis; MG, mature green; Br, breaker; Pk, pink; LR, light red; RR, red ripe. Credit: DOI: 10.1038/s41467-017-02782-9. License: CC BY 4.0 IL.

Immediately after harvesting, the tomato was scanned with a micro-computed tomograph (micro-CT) to generate a 3D image of the fruit, including its internal structures. Then, the fruit was dissected by hand or laser, depending of its size, divided into various tissue types and then preserved either via snap-freezing in liquid nitrogen or standard tissue fixation for light or transmission electron microscopy. Finally, the researchers used kits to extract and analyze the RNA from their samples. And, last but not least, a lot of math & stats.

This is what I got out of it:

1. A total of 24,660 genes were uniquely expressed in various tomato cell types and at various stages of development.
2. The tomato ripens from within, meaning from the interior to the exterior and not the other way around.
3. The ripening seems to be a continuous process, starting before the ‘Breaker’ stage.
4. The ripening signals originate in the locular tissues (the goo around the seeds; it’s possible that the seeds themselves send the signals to the locular tissue to start the ripening process).
5. The flesh of the fruit is only one part of the tomato and the most investigated, but the other types of tissue are also important. For example, some genes responsible for aroma and flavor (CTOMT1, TOMLOXC) are predominantly or even exclusively expressed in the flesh, but some genes that improve the nutritional value (SlGAD3) are expressed mostly in the placenta.
6. The fruit can do photosynthesis, probably for the benefit of its seeds.
7. Each developmental stage is characterized by a distinct transcriptome profile (by inference, also a distinct proteomic profile, although not necessarily in exact correspondence)
8. Botany, like any serious science, is complicated.

Ah, I have been vindicated. By science, nonetheless! You see, in my pursuit to recapture the tomato taste of my childhood I sample various homegrown exemplars of Solanum lycopersicum derived both from more or less failed personal attempts with pots on the balcony and from various farmer’s market vendors. While I can understand – though not approve of – the industrial scale agro-growers’ practice to pick the tomatoes green, unripe and then artificially injecting them with ethylene to prolong shelf life, I completely fail to understand the picking them up when green by the sellers in the farmer’s markets. I had many surreal conversations with such vendors (I cannot call them farmers for the life of me) who more than once attempted to reassure me that 1) Everybody’s picking tomatoes green off the vine because that’s how it’s done and 2) Ripening happens on the window sill. In vain have I tried to explain the difference between ripen and rotten; in vain have I pointed out that color is only one indicator of ripening; in vain did I explain that during ripening on the vine the plant delivers certain substances to the fruit that lead to changes in the flesh composition to make it more nutritious for the future seedling, process that the aforesaid window sill does nor partake in. Alas, ultimately, my arguments (and my family’s last 400 years of farming experience) hit the wall of “I am growing tomatoes for three years now and I know what I’m doing. Are you buying or not?” As you might imagine, I end up going home frustrated and yet staring at some exorbitantly expensive and looking as sad as I feel greenish tomatoes.

For me, this is what Shinozaki et al. (2018) validated: Ripening is a complex process that involves a lot of physiological changes in the fruit, not merely some extra production of ethylene that can be conveniently supplied externally by a syringe or rotting on the window sill. Of course, there is nowhere in the paper that Shinozaki et al. (2018) say that. What they do say is this: “The ripening program is revealed as comprising gradients of gene expression, initiating in internal tissues then radiating outward, and basipetally along a latitudinal axis. We also identify spatial variations in the patterns of epigenetic control superimposed on ripening gradients” (Abstract). Tomayto, tomahto…

Now we know that… simply put, I’m right. Sometimes is good to be right. I am old enough to prefer happiness and tranquility over rightness & righteousness, but still young enough that sometimes, just sometimes, it feels good to be right. Yes, the Shinozaki et al. (2018) paper exists only for my vindication in my farmer’s market squabbles and not for providing a huge comprehensive atlas on the tomato transcriptome, along with an awesome spatiotemporal map showing the place and time of the expression of genes responsible for fruit ripening, quality traits and so on.

Good job, Shinozaki et al. (2018)!

REFERENCE: Shinozaki Y, Nicolas P, Fernandez-Pozo N, Ma Q, Evanich DJ, Shi Y, Xu Y, Zheng Y, Snyder SI, Martin LBB, Ruiz-May E, Thannhauser TW, Chen K, Domozych DS, Catalá C, Fei Z, Mueller LA, Giovannoni JJ, & Rose JKC (25 Jan 2018). High-resolution spatiotemporal transcriptome mapping of tomato fruit development and ripening. Nature Communications, 9(1):364. PMID: 29371663, PMCID: PMC5785480, DOI: 10.1038/s41467-017-02782-9. ARTICLE | FREE FULLTEXT PDF | The Tomato Expression Atlas database

By Neuronicus, 7 February 2018

Midichlorians, midichloria, and mitochondria

Nathan Lo is an evolutionary biologist interested in creepy crawlies, i.e. arthropods. Well, he’s Australian, so I guess that comes with the territory (see what I did there?). While postdoc’ing, he and his colleagues published a paper (Sassera et al., 2006) that would seem boring for anybody without an interest in taxonomy, a truly under-appreciated field.

The paper describes a bacterium that is a parasite for the mitochondria of a tick species called Ixodes ricinus, the nasty bugger responsible for Lyme disease. The authors obtained a female tick from Berlin, Germany and let it feed on a hamster until it laid eggs. By using genetic sequencing (you can use kits these days to extract the DNA, do PCR, gels and cloning, pretty much everything), electron microscopy (real powerful microscopes) and phylogenetic analysis (using computer softwares to see how closely related some species are) the authors came to the conclusion that this parasite they were working on is a new species. So they named it. And below is the full account of the naming, from the horse’s mouth, as it were:

“In accordance with the guidelines of the International Committee of Systematic Bacteriology, unculturable bacteria should be classified as Candidatus (Murray & Stackebrandt, 1995). Thus we propose the name ‘Candidatus Midichloria mitochondrii’ for the novel bacterium. The genus name Midichloria (mi.di.chlo′ria. N.L. fem. n.) is derived from the midichlorians, organisms within the fictional Star Wars universe. Midichlorians are microscopic symbionts that reside within the cells of living things and ‘‘communicate with the Force’’. Star Wars creator George Lucas stated that the idea of the midichlorians is based on endosymbiotic theory. The word ‘midichlorian’ appears to be a blend of the words mitochondrion and chloroplast. The specific epithet, mitochondrii (mi.to′chon.drii. N.L. n. mitochondrium -i a mitochondrion; N.L. gen. n. mitochondrii of a mitochondrion), refers to the unique intramitochondrial lifestyle of this bacterium. ‘Candidatus M. mitochondrii’ belongs to the phylum Proteobacteria, to the class Alphaproteobacteria and to the order Rickettsiales. ‘Candidatus M. mitochondrii’ is assigned on the basis of the 16S rRNA (AJ566640) and gyrB gene sequences (AM159536)” (p. 2539).

George Lucas gave his blessing to the Christening (of course he did).

Acknowledgements: Thanks go to Ms. BBD who prevented me from making a fool of myself (this time!) on the social media by pointing out to me that midichloria are real and that they are a mitochondrial parasite.

REFERENCE: Sassera D, Beninati T, Bandi C, Bouman EA, Sacchi L, Fabbi M, Lo N. (Nov. 2006). ‘Candidatus Midichloria mitochondrii’, an endosymbiont of the tick Ixodes ricinus with a unique intramitochondrial lifestyle. International Journal of Systematic and Evolutionary Microbiology, 56(Pt 11): 2535-2540. PMID: 17082386, DOI: 10.1099/ijs.0.64386-0. ABSTRACT | FREE FULLTEXT PDF 

By Neuronicus, 29 July 2017