Arabidopsis seedlings were grown in short day conditions (10h L/14h D, 20°C). These conditions were applied in order to prevent the plant from entering the reproductive stage. Using micro scissors, plants were collected from 14 days old whose cotyledons and hypocotyls were removed.
Extracted molecule
total RNA
Extraction protocol
Total RNA (15 μg) was extracted with the RNeasy RNA isolation kit (Qiagen).
Label
biotin
Label protocol
Labeled cRNA was prepared and hybridized to Affymetrix ATH1 GeneChips, according to the manufacturer's guidelines (Affymetrix, Santa Clara, CA)
Hybridization protocol
Labeled cRNA was prepared and hybridized to Affymetrix ATH1 GeneChips, according to the manufacturer's guidelines (Affymetrix, Santa Clara, CA)
Scan protocol
Labeled cRNA was prepared and hybridized to Affymetrix ATH1 GeneChips, according to the manufacturer's guidelines (Affymetrix, Santa Clara, CA)
Description
In most angiosperms, polarity of leaves in the adaxial-abaxial sides could be associated with differences in morphology and anatomy that reflect the function of either side surfaces. Since dissecting these two parts is technically difficult, examples of metabolic pathways activated in one side of leaves and not in the other are scarce. In this study we used a developmental genetic approach to elucidate the molecular machinery that controls polarity in Arabidopsis leaves. One significant outcome of this strategy was the discovery that expression of three R2-R3 MYB factors, controlling a major pathway of secondary metabolism in Arabidopsis, was localized to the abaxial regions of young leaves. Herein we present in planta evidence that these three genes together with a second group of genes that includes the previously described ATR1 factor and its closest homolog, play a critical role in the transcriptional control of the biosynthetic pathways leading to the formation of the two most prominent classes of GSs in Arabidopsis. Silencing of either one of these two groups of genes using synthetic microRNAs resulted in reduced levels of GSs in leaves. Overexpression of the MYB28-like clade members (MYB28, MYB29 and MYB76) and the ATR1-like members [MYB51, MYB34 (ATR1)] in Arabidopsis led to induction of gene expression and metabolism associated with aliphatic glucosinolates (AGs) and indole glucosinolates (IGs), respectively. At the same time, overexpression of either clade members suppressed expression and metabolism associated with the other pathway, signifying a reciprocal, negative feedback control in between the two GSs pathways. Furthermore, we found that both members of the ATR1-like clade were not only key controllers of IGs homeostasis, but also effected metabolism of auxin and the phytoalexin camalexin. The availability of plants producing high levels of either IGs or AGs allowed us to compare between the capacities of these two types of GSs to deter herbivory. The results demonstrated that AGs exhibit more potent deterrent properties against the whitefly, Bemisia tabaci, a major agricultural pest of field and horticultural crops. Taken as a whole, this work provides a framework for dissecting the spatial regulation of metabolic pathways associated with secondary metabolism and its co-evolution with insect behavior.
