High-resolution imaging as a tool for identifying quantitative trait loci that regulate photomorphogenesis in Arabidopsis thaliana.

A primary component of seedling establishment is the photomorphogenic response as seedlings emerge from the soil. This process is characterized by a reduced growth rate in the hypocotyl, increased root growth, opening of the apical hook and expansion of the cotyledons as photosynthetic organs. While fundamental to plant success, the photomorphogenic response can be highly variable. Additionally, studies of Arabidopsis thaliana are made difficult by subtle differences in growth rate between individuals. High-resolution imaging and computational processing have emerged as useful tools for quantification of such phenotypes. This study sought to: (i) develop an imaging methodology which could capture changes in growth rate as seedlings transition from darkness to blue light in real time, and (ii) apply this methodology to single-quantitative trait locus (QTL) analysis using the Cvi × Ler recombinant inbred line (RIL) mapping population. Significant differences in the photomorphogenic response were observed between the parent lines and analysis of 158 RILs revealed a wide range of growth rate phenotypes. Quantitative trait locus analysis detected significant loci associated with dark growth rate on chromosome 5 and significant loci associated with light growth rate on chromosome 2. Candidate genes associated with these loci, such as the previously characterized ER locus, highlight the application of this approach for QTL analysis. Genetic analysis of Landsberg lines without the erecta mutation also supports a role for ER in modulating the photomorphogenic response, consistent with previous QTL analyses of this population. Strengths and limitations of this methodology are presented, as well as means of improvement.

Electrophysiological study of Arabidopsis ABCB4 and PIN2 auxin transporters: Evidence of auxin activation and interaction enhancing auxin selectivity.

Polar auxin transport through plant tissue strictly requires polarly localized PIN proteins and uniformly distributed ABCB proteins. A functional synergy between the two types of membrane protein where their localizations overlap may create the degree of asymmetric auxin efflux required to produce polar auxin transport. We investigated this possibility by expressing ABCB4 and PIN2 in human embryonic kidney cells and measuring whole-cell ionic currents with the patch-clamp technique and CsCl-based electrolytes. ABCB4 activity was 1.81-fold more selective for Cl- over Cs+ and for PIN2 the value was 2.95. We imposed auxin gradients and determined that ABCB4 and PIN2 were 12-fold more permeable to the auxin anion (IAA-) than Cl-. This measure of the intrinsic selectivity of the transport pathway was 21-fold when ABCB4 and PIN2 were co-expressed. If this increase occurs in plants, it could explain why asymmetric PIN localization is not sufficient to create polar auxin flow. Some form of co-action or synergy between ABCB4 and PIN2 that increases IAA- selectivity at the cell face where both occur may be important. We also found that auxin stimulated ABCB4 activity, which may contribute to a self-reinforcement of auxin transport known as canalization.

Dominant gain-of-function mutations in transmembrane domain III of ERS1 and ETR1 suggest a novel role for this domain in regulating the magnitude of ethylene response in Arabidopsis.

Prior work resulted in identification of an Arabidopsis mutant, eer5-1, with extreme ethylene response in conjunction with failure to induce a subset of ethylene-responsive genes, including AtEBP. EER5, which is a TREX-2 homolog that is part of a nucleoporin complex, functions as part of a cryptic aspect of the ethylene signaling pathway that is required for regulating the magnitude of ethylene response. A suppressor mutagenesis screen was carried out to identify second site mutations that could restore the growth of ethylene-treated eer5-1 to wild-type levels. A dominant gain-of-function mutation in the ethylene receptor ETHYLENE RESPONSE SENSOR 1 (ERS1) was identified, with the ers1-4 mutation being located in transmembrane domain III at a point nearly equivalent to the previously described etr1-2 mutation in the other Arabidopsis subfamily I ethylene receptor, ETHYLENE RESPONSE 1 (ETR1). Although both ers1-4 and etr1-2 partially suppress the ethylene hypersensitivity of eer5-1 and are at least in part REVERSION TO ETHYLENE SENSITIVITY 1 (RTE1)-dependent, ers1-4 was additionally found to restore the expression of AtEBP in ers1-4;eer5-1 etiolated seedlings after ethylene treatment in an EIN3-dependent manner. Our work indicates that ERS1-regulated expression of a subset of ethylene-responsive genes is related to controlling the magnitude of ethylene response, with hyperinduction of these genes correlated with reduced ethylene-dependent growth inhibition.

A loss-of-function mutation in the nucleoporin AtNUP160 indicates that normal auxin signalling is required for a proper ethylene response in Arabidopsis.

As part of a continuing effort to elucidate mechanisms that regulate the magnitude of ethylene signalling, an Arabidopsis mutant with an enhanced ethylene response was identified. Subsequent characterization of this loss-of-function mutant revealed severe hypocotyl shortening in the presence of saturating ethylene along with increased expression in leaves of a subset of ethylene-responsive genes. It was subsequently determined by map-based cloning that the mutant (sar1-7) represents a loss-of-function mutation in the previously described nucleoporin AtNUP160 (At1g33410, SAR1). In support of previously reported results, the sar1-7 mutant partially restored auxin responsiveness to roots of an rce1 loss-of-function mutant, indicating that AtNUP160/SAR1 is required for proper expression of factors responsible for the repression of auxin signalling. Analysis of arf7-1/sar1-7 and arf19-1/sar1-7 double mutants revealed that mutations affecting either ARF7 or ARF19 function almost fully blocked manifestation of the sar1-7-dependent ethylene hypersensitivity phenotype, suggesting that ARF7- and ARF19-mediated auxin signalling is responsible for regulating the magnitude of and/or competence for the ethylene response in Arabidopsis etiolated hypocotyls. Consistent with this, addition of auxin to ethylene-treated seedlings resulted in severe hypocotyl shortening, reminiscent of that seen for other eer (enhanced ethylene response) mutants, suggesting that auxin functions in part synergistically with ethylene to control hypocotyl elongation and other ethylene-dependent phenomena.

FERONIA is a key modulator of brassinosteroid and ethylene responsiveness in Arabidopsis hypocotyls.

Ethylene signaling is a complex pathway that has been intensively analyzed partly due to its importance to the manifestation of horticultural phenomena, including fruit ripening and tissue senescence. In order to further our understanding of how this pathway is regulated, a screen for Arabidopsis mutants with increased ethylene response was conducted. From this, a mutant was identified as having a dark-grown hypocotyl that is indistinguishable from Col-0 wt in the presence of the ethylene perception inhibitor AgNO3, yet has extreme responsiveness to even low levels of ethylene. Map-based cloning of the mutation revealed a T-DNA insertion in the coding sequence of the receptor-like kinase FERONIA, which is required for normal pollen tube reception and cell elongation in a currently unknown capacity. In contrast to a previous report, analysis of our feronia knockout mutant shows it also has altered responsiveness to brassinosteroids, with etiolated fer-2 seedlings being partially brassinosteroid insensitive with regard to promotion of hypocotyl elongation. Our results indicate that FERONIA-dependent brassinosteroid response serves to antagonize the effect of ethylene on hypocotyl growth of etiolated seedlings, with loss of proper brassinosteroid signaling disrupting this balance and leading to a greater impact of ethylene on hypocotyl shortening.