SummaryReverse transcription-polymerase chain reaction (RT-PCR) approaches have been used in a large proportion of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably

The development of shoot-borne roots, or adventitious roots, is indispensable for mass propagation of elite genotypes. It is a complex genetic trait with a high phenotypic plasticity due to multiple endogenous and environmental regulatory factors. We demonstrate here that a subtle balance of activator and repressor AUXIN RESPONSE FACTOR (ARF) transcripts controls adventitious root initiation. Moreover, microRNA activity appears to be required for fine-tuning of this process. Thus, ARF17, a target of miR160, is a negative regulator, and ARF6 and ARF8, targets of miR167, are positive regulators of adventitious rooting. The three ARFs display overlapping expression domains, interact genetically, and regulate each other's expression at both transcriptional and posttranscriptional levels by modulating miR160 and miR167 availability. This complex regulatory network includes an unexpected feedback regulation of microRNA homeostasis by direct and nondirect target transcription factors. These results provide evidence of microRNA control of phenotypic variability and are a significant step forward in understanding the molecular mechanisms regulating adventitious rooting.

Vegetative shoot-based propagation of plants, including mass propagation of elite genotypes, is dependent on the development of shoot-borne roots, which are also called adventitious roots. Multiple endogenous and environmental factors control the complex process of adventitious rooting. In the past few years, we have shown that the auxin response factors ARF6 and ARF8, targets of the microRNA miR167, are positive regulators of adventitious rooting, whereas ARF17, a target of miR160, is a negative regulator. We showed that these genes have overlapping expression profiles during adventitious rooting and that they regulate each other's expression at the transcriptional and posttranscriptional levels by modulating the homeostasis of miR160 and miR167. We demonstrate here that this complex network of transcription factors regulates the expression of three auxininducible Gretchen Hagen3 (GH3) genes, GH3.3, GH3.5, and GH3.6, encoding acyl-acid-amido synthetases. We show that these three GH3 genes are required for fine-tuning adventitious root initiation in the Arabidopsis thaliana hypocotyl, and we demonstrate that they act by modulating jasmonic acid homeostasis. We propose a model in which adventitious rooting is an adaptive developmental response involving crosstalk between the auxin and jasmonate regulatory pathways.

Quantitative RT-PCR (reverse transcription polymerase chain reaction, also known as qRT-PCR or real-time RT-PCR) has been used in large proportions of transcriptome analyses published to date. The accuracy of the results obtained by this method strongly depends on accurate transcript normalization using stably expressed genes, known as references. Statistical algorithms have been developed recently to help validate reference genes but, surprisingly, this robust approach is under-utilized in plants. Instead, putative 'housekeeping' genes tend to be used as references without any proper validation. The concept of normalization in transcript quantification is introduced here and the factors affecting its reliability in qRT-PCR are discussed in an attempt to convince molecular biologists, and non-specialists, that systematic validation of reference genes is essential for producing accurate, reliable data in qRT-PCR analyses, and thus should be an integral component of them.

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Despite an ever-increasing interest for the use of pectin-derived oligogalacturonides (OGs) as biological control agents in agriculture, very little information exists—mainly for technical reasons—on the nature and activity of the OGs that accumulate during pathogen infection. Here we developed a sensitive OG profiling method, which revealed unsuspected features of the OGs generated during infection of Arabidopsis thaliana with the fungus Botrytis cinerea. Indeed, in contrast to previous reports, most OGs were acetyl- and methylesterified, and 80% of them were produced by fungal pectin lyases, not by polygalacturonases. Polygalacturonase products did not accumulate as larger size OGs but were converted into oxidized GalA dimers. Finally, the comparison of the OGs and transcriptomes of leaves infected with B. cinerea mutants with reduced pectinolytic activity but with decreased or increased virulence, respectively, identified candidate OG elicitors. In conclusion, OG analysis provides insights into the enzymatic arms race between plant and pathogen and facilitates the identification of defense elicitors.

Pectin methylesterases (PME, EC. 3.1.1.11) are enzymes that demethylesterify plant cell wall pectins in muro. In Arabidopsis thaliana, putative PME proteins are thought to be encoded by a 66-member gene family. This study used real-time RT-PCR to gain an overview of the expression of the entire family at eight silique developmental stages, in flower buds and in vegetative tissue in the Arabidopsis. Only 15% of the PMEs were not expressed at any of the developmental stages studied. Among expressed PMEs, expression data could be clustered into five distinct groups: 19 PMEs highly or uniquely expressed in floral buds, 4 PMEs uniquely expressed at mid-silique developmental stages, 16 PMEs highly or uniquely expressed in silique at late developmental stages, 16 PMEs mostly ubiquitously expressed, and 1 PME with a specific expression pattern, i.e. not expressed during early silique development. Comparison of expression and phylogenetic profiles showed that, within phylogenetic group 2, all but one PME belong to the floral bud expression group. Similar results were shown for a subset of one of the phylogenetic group, which differed from others by containing most of the PMEs that do not possess any PRO part next to their catalytic part. Expression data were confirmed by two promoter:GUS transgenic plant analysis revealing a PME expressed in pollen and one in young seeds. Our results highlight the high diversity of PME expression profiles. They are discussed with regard to the role of PMEs in fruit development and cell growth.