From 77f2404e39b277af7e54be7413f44689d84255e7 Mon Sep 17 00:00:00 2001
From: Viktoria Petrova <vipet103@hhu.de>
Date: Tue, 4 Feb 2025 14:22:46 +0100
Subject: [PATCH 1/6] add titratable acidity assay and protocol

---
 assays/CMML_18-0008_HPLC-DAD/isa.assay.xlsx   | Bin 6907 -> 6907 bytes
 .../isa.assay.xlsx                            | Bin 7024 -> 7024 bytes
 assays/CMML_18-0008_LCqTOF/isa.assay.xlsx     | Bin 18499 -> 15579 bytes
 .../Maleckova2018_aba_mRNASeq/isa.assay.xlsx  | Bin 17188 -> 14477 bytes
 assays/TitratableAcidity/README.md            |   0
 assays/TitratableAcidity/dataset/.gitkeep     |   0
 assays/TitratableAcidity/isa.assay.xlsx       | Bin 0 -> 6898 bytes
 assays/TitratableAcidity/protocols/.gitkeep   |   0
 .../protocols/TitratableAcidityProtocol.md    |   3 +++
 studies/Maleckova2018_aba/isa.study.xlsx      | Bin 17183 -> 14597 bytes
 10 files changed, 3 insertions(+)
 create mode 100644 assays/TitratableAcidity/README.md
 create mode 100644 assays/TitratableAcidity/dataset/.gitkeep
 create mode 100644 assays/TitratableAcidity/isa.assay.xlsx
 create mode 100644 assays/TitratableAcidity/protocols/.gitkeep
 create mode 100644 assays/TitratableAcidity/protocols/TitratableAcidityProtocol.md

diff --git a/assays/CMML_18-0008_HPLC-DAD/isa.assay.xlsx b/assays/CMML_18-0008_HPLC-DAD/isa.assay.xlsx
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diff --git a/assays/CMML_18-0008_LCqTOF-MasshunterQuant/isa.assay.xlsx b/assays/CMML_18-0008_LCqTOF-MasshunterQuant/isa.assay.xlsx
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diff --git a/assays/TitratableAcidity/protocols/.gitkeep b/assays/TitratableAcidity/protocols/.gitkeep
new file mode 100644
index 00000000..e69de29b
diff --git a/assays/TitratableAcidity/protocols/TitratableAcidityProtocol.md b/assays/TitratableAcidity/protocols/TitratableAcidityProtocol.md
new file mode 100644
index 00000000..15a5cbc8
--- /dev/null
+++ b/assays/TitratableAcidity/protocols/TitratableAcidityProtocol.md
@@ -0,0 +1,3 @@
+## Titratable acidity
+
+Approximately 30 mg of ground leaf material was incubated in 500 µl 50% (v/v) methanol at 90 °C while shaking. After centrifugation at 13,000 g for 5 min at room temperature, 450 µl of the supernatant was collected and the remaining pellet was extracted once more. Both fractions were pooled and stored at –20 °C until use. Titratable acidity was measured using bromothymol blue (Carl Roth) as a pH indicator: 60 µl of extract was mixed with 40 µl distilled H2O and 4 µl bromothymol blue (1 mg/ml stock). Using the microinjector of a Synergy H1 (BioTek) plate reader, samples were titrated with 1 mM KOH in 5 µl steps. After each step, absorbance at 445 nm and 615 nm was recorded, and the ratio of the absorbances at 615/445 nm was calculated. Based on the 615/445 ratio of buffer standards (pH 4.6–7.5), the pH of analysed extracts was determined. From the volume of KOH required to titrate extracts to pH 7.0, the titratable acidity was calculated. Two extractions were performed from each sample, and each extract was titrated in three replicates.
\ No newline at end of file
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GitLab


From c40dab6807dc0f3bcdafee989ae046718e660a09 Mon Sep 17 00:00:00 2001
From: Viktoria Petrova <vipet103@hhu.de>
Date: Thu, 6 Feb 2025 14:55:09 +0100
Subject: [PATCH 2/6] fill out titratable acidity isa assay

---
 assays/CMML_18-0008_GC-MS/isa.assay.xlsx  | Bin 32937 -> 28843 bytes
 assays/CMML_18-0008_LCqTOF/isa.assay.xlsx | Bin 15579 -> 15580 bytes
 assays/TitratableAcidity/isa.assay.xlsx   | Bin 6898 -> 17253 bytes
 studies/Maleckova2018_aba/isa.study.xlsx  | Bin 14597 -> 14598 bytes
 4 files changed, 0 insertions(+), 0 deletions(-)

diff --git a/assays/CMML_18-0008_GC-MS/isa.assay.xlsx b/assays/CMML_18-0008_GC-MS/isa.assay.xlsx
index ddb29d440999a2b2c1a628e4adbe169d027a6df5..6283174d59ae9942458e3e9039f03670e9f2180d 100644
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F{|kl*9F710

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-- 
GitLab


From 9d2fae8d902cf3608424a9ec02e966e680b9d79b Mon Sep 17 00:00:00 2001
From: Viktoria Petrova <vipet103@hhu.de>
Date: Thu, 6 Feb 2025 15:01:11 +0100
Subject: [PATCH 3/6] add protein and starch measurement assay and protocol

---
 assays/ProteinAndStarchMeasurements/README.md |   0
 .../dataset/.gitkeep                          |   0
 .../isa.assay.xlsx                            | Bin 0 -> 6903 bytes
 .../protocols/.gitkeep                        |   0
 .../ProteinAndStarchMeasurementsProtocol.md   |   5 +++++
 assays/TitratableAcidity/isa.assay.xlsx       | Bin 17253 -> 17253 bytes
 6 files changed, 5 insertions(+)
 create mode 100644 assays/ProteinAndStarchMeasurements/README.md
 create mode 100644 assays/ProteinAndStarchMeasurements/dataset/.gitkeep
 create mode 100644 assays/ProteinAndStarchMeasurements/isa.assay.xlsx
 create mode 100644 assays/ProteinAndStarchMeasurements/protocols/.gitkeep
 create mode 100644 assays/ProteinAndStarchMeasurements/protocols/ProteinAndStarchMeasurementsProtocol.md

diff --git a/assays/ProteinAndStarchMeasurements/README.md b/assays/ProteinAndStarchMeasurements/README.md
new file mode 100644
index 00000000..e69de29b
diff --git a/assays/ProteinAndStarchMeasurements/dataset/.gitkeep b/assays/ProteinAndStarchMeasurements/dataset/.gitkeep
new file mode 100644
index 00000000..e69de29b
diff --git a/assays/ProteinAndStarchMeasurements/isa.assay.xlsx b/assays/ProteinAndStarchMeasurements/isa.assay.xlsx
new file mode 100644
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diff --git a/assays/ProteinAndStarchMeasurements/protocols/.gitkeep b/assays/ProteinAndStarchMeasurements/protocols/.gitkeep
new file mode 100644
index 00000000..e69de29b
diff --git a/assays/ProteinAndStarchMeasurements/protocols/ProteinAndStarchMeasurementsProtocol.md b/assays/ProteinAndStarchMeasurements/protocols/ProteinAndStarchMeasurementsProtocol.md
new file mode 100644
index 00000000..5890e9ef
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+++ b/assays/ProteinAndStarchMeasurements/protocols/ProteinAndStarchMeasurementsProtocol.md
@@ -0,0 +1,5 @@
+## Protein and Starch Measurements
+
+Approximately 25 mg of ground leaf material was used for extraction in 80% (v/v) ethanol with 10 mM 2-(N-morpholino)ethanesulfonic acid (pH 6). Extraction was performed three times with 500 µl extraction buffer while shaking for 1 hour at 90 °C. After each extraction step, samples were centrifuged at 12,000 g for 10 min and the supernatant was collected and pooled with previous fractions. From each sample, two extracts were prepared and stored at –20 °C until analysed. The protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).
+
+Pellets obtained during protein extraction were gelatinized with 300 µl 0.2 M NaOH at 90 °C for 40 minutes. After adjustment to pH 5–6, 200 µl hydrolysis buffer was added [20 mM acetate buffer, pH 4.8; 0.5 U α-amylase (10102814001, Roche), and 4 U amyloglucosidase (A7095, Roche)] and the mixture was incubated overnight at 37 °C. Glucose resulting from starch degradation was measured enzymatically: 10 µl of digest was added to 100 mM HEPES-NaOH (pH 7.5) with 10 mM MgCl2, 1 mM NADP+, 2 mM ATP, 0.1 U glucose-6-phosphate dehydrogenase (G6PDHII-RO, Roche), and 4 U hexokinase (11426362001, Roche). The final reaction volume was 200 µl and the increase of absorbance at 340 nm was monitored.
\ No newline at end of file
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GitLab


From 2dc785cf31b0ee4fe390294fda70c00cfa24ae22 Mon Sep 17 00:00:00 2001
From: Viktoria Petrova <vipet103@hhu.de>
Date: Thu, 6 Feb 2025 16:12:34 +0100
Subject: [PATCH 4/6] fill out protein and starch measurement isa assay

---
 .../isa.assay.xlsx                            | Bin 6903 -> 28205 bytes
 assays/TitratableAcidity/isa.assay.xlsx       | Bin 17253 -> 17252 bytes
 2 files changed, 0 insertions(+), 0 deletions(-)

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-- 
GitLab


From c63e2184e73addfd54c67f42fc82e37e58ff7cc7 Mon Sep 17 00:00:00 2001
From: Viktoria Petrova <vipet103@hhu.de>
Date: Thu, 6 Feb 2025 16:15:18 +0100
Subject: [PATCH 5/6] add protocol ref to rna seq isa assay

---
 assays/CMML_18-0008_LCqTOF/isa.assay.xlsx     | Bin 15580 -> 15580 bytes
 .../Maleckova2018_aba_mRNASeq/isa.assay.xlsx  | Bin 14477 -> 14677 bytes
 2 files changed, 0 insertions(+), 0 deletions(-)

diff --git a/assays/CMML_18-0008_LCqTOF/isa.assay.xlsx b/assays/CMML_18-0008_LCqTOF/isa.assay.xlsx
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-- 
GitLab


From b9af7455aba5d8de706a72bbc3cfafe041d3be15 Mon Sep 17 00:00:00 2001
From: Viktoria Petrova <vipet103@hhu.de>
Date: Thu, 20 Feb 2025 18:34:12 +0100
Subject: [PATCH 6/6] add gc-ms protocol and reference in isa assay

---
 assays/CMML_18-0008_GC-MS/isa.assay.xlsx     | Bin 28843 -> 29486 bytes
 assays/CMML_18-0008_GC-MS/protocols/GC-MS.md |   3 +++
 assays/TitratableAcidity/isa.assay.xlsx      | Bin 17252 -> 17253 bytes
 3 files changed, 3 insertions(+)
 create mode 100644 assays/CMML_18-0008_GC-MS/protocols/GC-MS.md

diff --git a/assays/CMML_18-0008_GC-MS/isa.assay.xlsx b/assays/CMML_18-0008_GC-MS/isa.assay.xlsx
index 6283174d59ae9942458e3e9039f03670e9f2180d..aef434cea1039931d53f11c4a3dec99b8df8644f 100644
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diff --git a/assays/CMML_18-0008_GC-MS/protocols/GC-MS.md b/assays/CMML_18-0008_GC-MS/protocols/GC-MS.md
new file mode 100644
index 00000000..6f481cbd
--- /dev/null
+++ b/assays/CMML_18-0008_GC-MS/protocols/GC-MS.md
@@ -0,0 +1,3 @@
+## Metabolite profiling
+
+Approximately 50 mg of ground leaf material was used to obtain extracts for metabolite profiling by gas chromatography-mass spectrometry (GC-MS) as described by Fiehn et al. (2000). Data analysis was performed using MassHunter Software, version B.07.00 (Agilent). For relative quantification, all metabolite peak areas were normalized to the peak area of the internal standard ribitol added prior to extraction and the amount of leaf material. From each sample, two extracts were prepared and analysed.
\ No newline at end of file
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-- 
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