The Plant Cell's struggle with qPCR

Earlier this year, I posted an article from The Plant Cell listing a series of recommendations for qPCR analysis [Eleven golden rules of quantitative RT-PCR].

The Plant Cell (and I can only guess because they are tired of receiving crappy, unreproducible or badly analyzed qPCR results or, even worse, "semiquantitative" RT-PCRs -see below- ) has recently published yet another article along the same line which "provides guidelines for the experimental design and statistical analysis of qRT-PCR data from the statistician's perspective"¹.

From The Plant Cell's Editor in Chief ²:

these are guidelines; any attempt to impose such analysis as standard while we are still struggling to persuade authors of the deficiencies of "semiquantitative" RT-PCR would be a difficult, if not impossible, task.

C'mon people.... nonquantitative semiquantitative RT-PCR? Really? And trying to get it into The Plant Cell (that by the way, has the highest impact factor of primary research journals in plant biology)?

She also commented (although on a previous Editorial)³:

Over the past 2 years, The Plant Cell has taken steps to remove "semiquantitative" RT-PCR from the pages of the journal

Good for The Plant Cell.

Please make the Editors's physical pain (which I assume they get when they see authors drawing quantitative conclusions in PCR analysis, from gels stained with EtBr) stop and follow their not-yet-imposed guidelines.


¹ Rieu I, Powers SJ. (2009) Real-Time Quantitative RT-PCR: Design, Calculations, and Statistics. The Plant Cell 21:1031-1033 (2009)
² Martin C (2009) Guidelines for Quantitative RT-PCR. The Plant Cell 21:1023
³ Martin C (2008) Refining Our Standards. The Plant Cell 20:1727

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Snapshots: Auxin

The plant hormone Auxin is a key player during pattern formation and organogenesis and is involved in virtually every aspect of plant growth and development.

Growing evidence suggests that its signaling pathway may be more complex than previously anticipated. During the past 15-20 years we have been able to identify important components of its signaling pathway and generate models to explain how auxin regulates different developmental processes ^1,2.

The latest "Snapshot" from Cell, depicts "Auxin Signaling and Transport".
For a full list of SnapShots, click here. I'm sure you'll find something interesting.

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¹ Lau S, Jürgens G, De Smet I (2008) The evolving complexity of the auxin pathway. Plant Cell. 2008 Jul;20(7):1738-46.
² Mockaitis K, Estelle M (2008) Auxin receptors and plant development: a new signaling paradigm. Annu Rev Cell Dev Biol. 2008;24:55-80.

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System-wide molecular evidence for phenotypic buffering in Arabidopsis

In this Brief Communication, in a very nice issue of Nature Genetics, the authors profiled several Arabidopsis lines for a system-wide genetical genomics¹ study of molecular variation, integrating transcript, protein and metabolite data from a population of recombinant inbred lines² (RILs) of this model plant.

It appears that most of the genome and transcript variation between the two parental accession used for generating the RILs (and of the RILs themselves), is 'buffered' and does not result on phenotypic variation.

The largest fraction of molecular variants is silent at the phenotypic level and only a few influential 'hot spots' regions cause a mejor phenotypic variation across a range of environmental conditions

Interestingly, and despite the generalized buffering, the authors described six QTL (quantitative trait loci) hotspots,

which seem to correspond to a few molecular 'breakpoints' of an otherwise robust regulatory system

This is somewhat related to an article I posted recently on phenotypic variation buffering in yeast. In general, the idea seems to be that most of the variation in a species remains silent phenotypically, but alterations in certain genes or sequences may unleash the accumulated genetic variation and it can then be manifested at the phenotypic level. In the case of the yeast article, the authors proposed these genes were network hubs.
Although the authors comment that most of the reported hotspots could be linked to well-studied genes, such as CRY2 or HUA2, it must me emphasized that due to the somewhat imperfect resolution of QTL mapping, maybe other genes or regions may be involved in the observed traits.
Buffering and release of variation may be widespread phenomenon; for example, in both Drosophila and Arabidopsis, altered Hsp90 function results in pleiotropic phenotypic effects dependent on the underlying genotype.

I leave you with the final sentence of this article

variation in a multitude of Arabidopsis complex traits can be explained to a considerable extent by only a few QTL hot spots

Here's the abstract and reference:

System-wide molecular evidence for phenotypic buffering in Arabidopsis

Fu J, Keurentjes JJ, Bouwmeester H, America T, Verstappen FW, Ward JL, Beale MH, de Vos RC, Dijkstra M, Scheltema RA, Johannes F, Koornneef M, Vreugdenhil D, Breitling R, Jansen RC.
[1] Groningen Bioinformatics Centre, University of Groningen, 9751NN Haren, The Netherlands. [2] Department of Genetics, University Medical Centre Groningen, University of Groningen, 9700RB Groningen, The Netherlands. [3] These authors contributed equally to this work.

We profiled 162 lines of Arabidopsis for variation in transcript, protein and metabolite abundance using mRNA microarrays, two-dimensional polyacrylamide gel electrophoresis, gas chromatography time-of-flight mass spectrometry, liquid chromatography quadrupole time-of-flight mass spectrometry, and proton nuclear magnetic resonance. We added all publicly available phenotypic data from the same lines and mapped quantitative trait loci (QTL) for 40,580 molecular and 139 phenotypic traits. We found six QTL hot spots with major, system-wide effects, suggesting there are six breakpoints in a system otherwise buffered against many of the 500,000 SNPs.

Jingyuan Fu, Joost J B Keurentjes, Harro Bouwmeester, Twan America, Francel W A Verstappen, Jane L Ward, Michael H Beale, Ric C H de Vos, Martijn Dijkstra, Richard A Scheltema, Frank Johannes, Maarten Koornneef, Dick Vreugdenhil, Rainer Breitling, Ritsert C Jansen (2009). System-wide molecular evidence for phenotypic buffering in Arabidopsis Nature Genetics, 41 (2), 166-167 DOI: 10.1038/ng.308

¹ I know this may be a new concept for many of you, and instead of explaining it myself, and give a possibly complex definition, I'd rather direct you to an article on Trends in Genetics (2001) 17:388-391.
²See Broman KW. Genetics. 2005 Feb;169(2):1133-46.

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Plant Biology Video Contest

Chlorofilms.org is organizing a contest for the best new videos illustrating different aspects of plant life (from the molecular and cellular aspects of plant biology to the whole plant and ecosystem levels). You can enter the contest simply by uploading your video to Youtube and filling an entry form at the organizer’s site. They must be, however, intended for the general public.

For those thinking ‘why in earth would I enter such a contest?” you may find it interesting to know that there are up to $8,000 US in prizes:
Grand Prize is $1,000; multiple categories will be eligible for 1st Prizes of $500; 2nd Prizes of $250; and Honorable Mentions.

Check the rules and more details at Chlorofilms’ website.

(banner from The Arabidopsis Information Resource (TAIR) website)

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Two novel GPCR-type G proteins are abscisic acid receptors in Arabidopsis

Pandey S, Nelson DC, Assmann SM.

Biology Department, 208 Mueller Laboratory, Penn State University, University Park, PA 16802, USA.

In plants, G proteins modulate signaling by the stress hormone, abscisic acid (ABA). We identify and characterize two novel Arabidopsis proteins that show homology to an orphan vertebrate GPCR (GPR89) and interact with the sole Arabidopsis G protein alpha subunit, GPA1, but also have intrinsic GTP-binding and GTPase activity. We have named these proteins GPCR-type G proteins (GTG1 and GTG2). Arabidopsis mutants lacking both GTG1 and GTG2 exhibit ABA hyposensitivity. GTG1 and GTG2 bind ABA specifically. The GDP-bound form of the GTGs exhibits greater ABA binding than the GTP-bound form, the GTPase activity of the GTGs is inhibited by GPA1, and gpa1 null mutants exhibit ABA-hypersensitive phenotypes. These results predict that, unusually, it is the GDP-bound, not the GTP-bound, form of the GTGs that actively relays the signal. We propose that GTG proteins function both as a new type of G protein and as a class of membrane-localized ABA receptors.

Cell. 2009 Jan 9;136(1):136-48.

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MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays

Popescu SC, Popescu GV, Bachan S, Zhang Z, Gerstein M, Snyder M, Dinesh-Kumar SP.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA.

Signaling through mitogen-activated protein kinases (MPKs) cascades is a complex and fundamental process in eukaryotes, requiring MPK-activating kinases (MKKs) and MKK-activating kinases (MKKKs). However, to date only a limited number of MKK-MPK interactions and MPK phosphorylation substrates have been revealed. We determined which Arabidopsis thaliana MKKs preferentially activate 10 different MPKs in vivo and used the activated MPKs to probe high-density protein microarrays to determine their phosphorylation targets. Our analyses revealed known and novel signaling modules encompassing 570 MPK phosphorylation substrates; these substrates were enriched in transcription factors involved in the regulation of development, defense, and stress responses. Selected MPK substrates were validated by in planta reconstitution experiments. A subset of activated and wild-type MKKs induced cell death, indicating a possible role for these MKKs in the regulation of cell death. Interestingly, MKK7- and MKK9-induced death requires Sgt1, a known regulator of cell death induced during plant innate immunity. Our predicted MKK-MPK phosphorylation network constitutes a valuable resource to understand the function and specificity of MPK signaling systems.

Genes Dev. 2008 Dec 18. [Epub ahead of print]

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Eleven golden rules of quantitative RT-PCR

Udvardi MK, Czechowski T, Scheible WR.

Plant Cell. 2008 Jul;20(7):1736-7. Epub 2008 Jul 29

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This is an important paper for plant biologists, considering that "the use of the term ‘semi-quantitative’ (and hence, semi-quantitative assays from which quantitative conclusions are to be made) are not acceptable in The Plant Cell; instead, assays must be shown to be sufficiently quantitative to support a conclusion of changes in levels" (thus, you should use qPCR)

In this sense, the following paper should also be taken into account: 

Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Czechowski T et al. Plant Physiol. 2005 Sep;139(1):5-17

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AGO1-miR173 complex initiates phased siRNA formation in plants

Montgomery TA, Yoo SJ, Fahlgren N, Gilbert SD, Howell MD, Sullivan CM, Alexander A, Nguyen G, Allen E, Ahn JH, Carrington JC.

Molecular and Cellular Biology Program, Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA.

MicroRNA (miRNA)-guided cleavage initiates entry of primary transcripts into the transacting siRNA (tasiRNA) biogenesis pathway involving RNA-DEPENDENT RNA POLYMERASE6, DICER-LIKE4, and SUPPRESSOR OF GENE SILENCING3. Arabidopsis thaliana TAS1 and TAS2 families yield tasiRNA that form through miR173-guided initiation-cleavage of primary transcripts and target several transcripts encoding pentatricopeptide repeat proteins and proteins of unknown function. Here, the TAS1c locus was modified to produce synthetic (syn) tasiRNA to target an endogenous transcript encoding PHYTOENE DESATURASE and used to analyze the role of miR173 in routing of transcripts through the tasiRNA pathway. miR173 was unique from other miRNAs in its ability to initiate TAS1c-based syn-tasiRNA formation. A single miR173 target site was sufficient to route non-TAS transcripts into the pathway to yield phased siRNA. We also show that miR173 functions in association with ARGONAUTE 1 (AGO1) during TAS1 and TAS2 tasiRNA formation, and we provide data indicating that the miR173-AGO1 complex possesses unique functionality that many other miRNA-AGO1 complexes lack.

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