GDP-L-fucose is required for boundary definition in plants

Mutation of MURUS1 suppresses the highly serrated leaf phenotype that results from overexpression of CUP-SHAPED COTYLEDON 2 in Arabidopsis, revealing that GDP-L-fucose is involved in defining tissue boundaries during plant development.


Supplementary Protocol : Qpixies Macro For ImageJ
This ImageJ macro allows the semi-automatic identification of three parameters associated with a fluorescence signal: a zone with a signal level above the local background level; a zone with a user-defined area in which the signal is maximum; and a local background zone. By automatically defining a local background centred around the zone to be quantified, this macro minimizes the variability of the quantification that may result from user bias in the definition of the initial zone to be analysed. The input images can be single calibrated TIFF pictures or stacks of multiple calibrated TIFF slices corresponding to different channels and/or different exposure times.

Optional denoising and Manual Selection of the Zone Analysed
An optional image denoising step can be performed through the application of a Gaussian blur.
The user manually defines a large zone enclosing the region to be quantified.

Definition of the Object
The Object (here a part of leaf) is distinguished from the background by thresholding the image.

Definition of the BrightestArea
The BrightestArea is defined as a continuous zone in which the pixel intensity is above the mean intensity plus two times the standard deviation of the mean (mean+2SD) of the pixel intensity within the Object.

Definition of the BackGround
The BackGround is defined as the Object from which is excluded the Brightest Area.

Refining the SignalEstimation
In step 3, the mean intensity is calculated in the Object, which includes the zone with a strong signal and the background zone. This leads to an overestimation of the pixel intensity in the background and hence an underestimation of the signal. To overcome this, the SignalEstimation is defined here as a continuous zone in which the pixel intensity is above the mean+2SD of the pixel intensity within the Background.

Definition of the TargetArea
The background is calculated in a zone that depends on the Object defined by the user in step 1. Therefore, analysis of the same image with different Objects may lead to different final results. To limits this, we introduce this step that defines the TargetArea. The TargetArea is defined as the intersection of the Object with a Disk centred on the centre of the SignalEstimation and a radius equal to the radius of SignalEstimation multiplied by a userdefined constant (here 2).

Definition of the LocalBackground
The LocalBackground is defined as the TargetArea from which the SignalEstimation is excluded

Definition of the RawSignal
The RawSignal is calculated taking into account the local background: it is defined as a continuous zone in which the pixel intensity is above the mean+2SD of the pixel intensity within the LocalBackground.

Definition of the Signal
To refine the Signal zone, the steps 7 and 8 are iterated 10 times, or until the change in area of the RawSignal between two iterations is <1%.

Definition of the SignalMaximum
The SignalMaximum is defined as continuous zone with an area defined by the user and that corresponds to the intersection of the Signal and a disk centred on the centre of the Signal.
The output of the macro is the area, mean pixel intensity and SD of the three zones defined (Signal, SignalMaximum and LocalBackground). If the initial image contains multiples slices, these parameters will be measured for each zone in each slice.

Raw Signal
SignalMaximum