Overall experimental aim 🧠🌾

Goal: Determine what the rice amino acid transporter OsAAP7 does—especially which amino acids it transports, where it acts in the cell, and how changing OsAAP7 affects tillering and yield, plus the downstream molecular pathways (N metabolism + hormones) that shift when OsAAP7 is altered.

Core strategy (big picture):

1. Use natural variation (haplotypes in 521 rice varieties) to link OsAAP7 expression with agronomic traits.
2. Create genetic perturbations:
   • Overexpression (OE) lines (too much OsAAP7)
   • CRISPR knockout (C) lines (OsAAP7 disabled)
3. Test mechanism:
   • Where the protein is (subcellular localization)
   • Where the gene is expressed (promoter-GUS)
   • What substrates it transports (yeast complementation + fluorescent amino acid uptake)
   • What changes in plant physiology (amino acid levels, hydroponics bud growth)
4. Use RNA-seq to connect the phenotype to gene-expression pathways.

Methods and protocols (with aim + concept + steps)

1) Promoter sequence variation + haplotype analysis (521 varieties) 🌍🧬

Aim

Identify promoter SNP patterns (haplotypes) of OsAAP7 and test whether different haplotypes correlate with OsAAP7 expression, tillering, and yield.

Concept/idea

Promoter variants can change transcription → change OsAAP7 expression → potentially change nitrogen/amino acid handling → affect branching/tillering and yield.

Steps (as described)

1. Extract SNP variants in the OsAAP7 promoter from 521 rice varieties using RiceVarMap 2.0.
2. Grow the 521 varieties in field conditions (Huaxi District, Guizhou; May–Oct).
3. Apply fertilizer: 270 kg·ha⁻¹, with N/P/K = 19%/7%/14%.
4. Identify 3 haplotypes (Hap1–Hap3) in the OsAAP7 promoter.
5. Measure OsAAP7 expression (Hap1 vs Hap2 emphasized) using qRT-PCR.
   • Primers designed using Primer Premier 5 (listed in Table S1).
6. Test associations between haplotypes and agronomic traits (tiller number, yield, etc.).

2) Building plant genetic constructs + making transgenic rice 🌱🧪

This includes overexpression, CRISPR knockout, and promoter-GUS.

Aim

Generate rice lines where OsAAP7 is:

• overexpressed (gain-of-function),
• knocked out (loss-of-function),
• or where its promoter drives GUS to visualize expression patterns.

Concept/idea

If OsAAP7 controls amino acid transport and branching, then changing its dosage should shift:

• amino acid accumulation/uptake
• bud outgrowth (tillering)
• yield traits

A) Overexpression vector (OsAAP7-OE)

Steps

1. Take OsAAP7 cDNA (1491 bp).
2. Clone into pCAMBIA1306 under the 35S promoter.
3. Use restriction enzymes BamHI and KpnI for cloning.
4. Transform into wild-type rice ZH11 via Agrobacterium workflow (details below).

B) CRISPR/Cas9 knockout vector (OsAAP7-C)

Key detail: because OsAAP7 had SNP mutation sites on exon 4, they used double targets aimed at exon 4 “to improve knockout efficiency.”

Steps

1. Design two CRISPR target sites targeting exon 4 of OsAAP7.
2. Construct CRISPR/Cas9 editing vector (paper describes the double-target strategy; primer design supported by SnapGene/Primer Premier 5; primer sequences in Table S1).
3. Transform into rice ZH11 via Agrobacterium workflow.

C) Promoter-GUS construct (pOsAAP7::GUS)

Steps

1. Amplify 2113 bp OsAAP7 promoter fragment upstream of coding region.
2. Insert into pCAMBIA1391Z upstream of GUS using BamHI and EcoRI.
3. Transform into rice via Agrobacterium workflow.

D) Agrobacterium transformation + transgenic line generation

Aim: deliver constructs into rice genome.

Steps

1. Transform each plasmid into Agrobacterium.
2. Infect calli from japonica rice ZH11 with Agrobacterium to generate transgenic plants.
3. Use the T2 generation for subsequent experiments.

3) Field growth + agronomic trait measurements 🌾📏

Aim

Quantify how OsAAP7 perturbation changes tillering and grain yield in real field conditions.

Concept/idea

Tiller number is a major yield component; field phenotyping tests whether OsAAP7 is agronomically relevant.

Steps

1. Plant ZH11, OE lines, CRISPR lines in two experimental fields (Huaxi and Lingshui, Guizhou University).
2. Apply fertilizer: 270 kg·ha⁻¹ with N/P/K = 19%/7%/14%.
3. At rice maturing stage, count 30 plants per line:
   • tiller number
   • grain yield per plant
4. Confirm genotypes:
   • OE lines: confirm higher OsAAP7 expression by qRT-PCR
   • CRISPR lines: confirm edits by sequencing

(In the Results section they also describe their selection workflow across T0→T1→T2, verifying Cas9 absence in T2 and selecting specific OE and C lines, but the “core method” is the field phenotyping + verification approach.)

4) Subcellular localization (OsAAP7-GFP in protoplasts) 🔬🟩🟥

Aim

Determine where OsAAP7 protein localizes inside the cell.

Concept/idea

Transporter location (plasma membrane vs ER membrane, etc.) affects how it controls amino acid distribution (e.g., uptake vs intracellular trafficking).

Steps

1. Amplify OsAAP7 cDNA.
2. Fuse OsAAP7 to GFP in an HBT plasmid (creates OsAAP7–GFP fusion).
3. Transiently transform construct into rice protoplasts.
4. Co-express AtWAK2-mCherry as an ER membrane marker.
5. Observe fluorescence using confocal microscopy (Nikon).
6. Primers designed with Primer Premier 5 (Table S1).

5) Promoter activity mapping: GUS staining + paraffin sectioning 🧫🔵✂️

Aim

Map where OsAAP7 is expressed across tissues and development stages, and visualize which cell types show promoter activity.

Concept/idea

Transporter function depends on expression location (roots vs shoots, vascular tissues vs parenchyma, etc.). Promoter-GUS gives spatial expression patterns.

Steps: GUS staining

1. Use pOsAAP7::GUS transgenic lines.
2. Perform staining on different tissues (vegetative + reproductive stages) using GUS Blue Kit (Huayueyang).
3. Follow a previously described histochemical staining method (they cite prior method).
4. Photograph tissues using a stereomicroscope (Olympus).

Steps: paraffin-slicing (histology)

1. Fix tissues using solution: 50% ethanol : 10% formaldehyde : 5% acetic acid.
2. Dehydrate by reducing water using ethanol gradient 50% → 100%.
3. Embed (“bury”) tissue in paraffin.
4. Section with a Leica slicing instrument.
5. Examine sections using a Zeiss microscope.

6) Yeast complementation assay (substrate transport test) 🍞🧬

Aim

Test whether OsAAP7 can functionally transport specific amino acids using a yeast mutant that lacks its own amino acid uptake capacity.

Concept/idea

If the yeast mutant can’t grow on a specific amino acid as the only nitrogen source, introducing a working transporter should “rescue” growth.

Steps (solid media spotting)

1. Transform into yeast mutant 22Δ10α:
   • empty vector control
   • pDR196-OsAAP7 construct
2. Use wild-type yeast strain 23344c as growth control.
3. Perform transformation using Yeast Transformation Kit (Coolaber).
4. Plate transformants on SD–Ura solid medium and incubate 30°C for 2–4 days.
5. Pick colonies and grow in YPDA liquid until OD600 ≈ 0.6–1.0.
6. Centrifuge 5000 rpm, 2 min (1.5 mL tube).
7. Discard supernatant; wash pellet with ddH2O 3–4 times.
8. Dilute yeast with ddH2O to defined absorbance values.
9. Spot 7 µL of each dilution onto uracil-free YNB solid medium (also lacking amino acids, NH4⁺, uracil).
10. Add one sole N source per condition:

• 3 mM (NH4)2SO4, or
• 3 mM of a single amino acid (various tested).

11. Use serial dilutions to OD600 = 0.1, 0.01, 0.001, 0.0001 (dilutions 1, 10, 100, 1000).
12. Invert plates; incubate at 30°C for 2–3 days.
13. Document growth with camera.

Steps (liquid growth curves)

1. Grow yeast expressing OsAAP7 vs empty vector in liquid medium where sole N source is:
   • 3 mM Phe, 3 mM Lys, 3 mM Leu, or 3 mM (NH4)2SO4.
2. Measure OD600 every 5 hours for 50 hours to compare growth rates.

7) Total nitrogen + nitrogen utilization efficiency + amino acid profiling 📊🧪

Aim

Quantify:

• Total nitrogen concentration
• NUtE (Nitrogen utilization efficiency)
• free amino acid concentrations in tissues

Concept/idea

If OsAAP7 moves amino acids, it should shift internal amino acid pools and potentially N economy, which relates to growth and yield.

A) Total N measurement

1. Measure total N using Total nitrogen analyzer (SKD-100, Peiou).

B) NUtE calculation

NUtE (%) = Grain yield (g) / (Grain N concentration (g) + Straw N concentration (g)) × 100

C) Free amino acid extraction + HPLC

1. Weigh 1 g sample.
2. Incubate in 10 mL 80% ethanol at 80°C for 20 min.
3. Transfer supernatant and repeat extraction steps (they indicate repeated steps).
4. Dry solution at 80°C to remove ethanol and water.
5. Add 1 mL of 0.5 mM NaOH.
6. Centrifuge 14,000 rpm for 20 min.
7. Filter supernatant through a 2 µm filter membrane.
8. Analyze filtrate by HPLC (Agilent 1260).

8) Fluorescent amino acid uptake assay 🌈➡️🌱

Aim

Directly test uptake/transport capacity for specific amino acids in seedlings.

Concept/idea

If OsAAP7 increases transport, OE seedlings should accumulate stronger fluorescence after exposure to fluorescently labeled amino acids; CRISPR lines should show weaker signals.

Steps

1. Use seedlings of ZH11, OsAAP7 OE, and OsAAP7 CRISPR lines.
2. Treat with 1 mM fluorescent amino acid:
   • Arg, Phe, or Lys
3. Incubate for 2 h, 6 h, or 10 h.
4. Image seedlings using Chemiluminescence Apparatus (Qinxiang).
5. Quantify fluorescence intensity using Bandscan.

9) Hydroponic culture + axillary bud measurements 💧🌱📏

Aim

Test how external amino acids affect axillary bud outgrowth in different genotypes (WT vs OE vs knockout).

Concept/idea

If OsAAP7 modulates amino acid internal levels/transport, the plant’s growth response to amino acids (especially in buds) should differ across genotypes.

Steps

1. Grow seedlings in basic rice nutrient solution for 1 week, containing:
   • 1 mM NH4NO3
   • 0.32 mM NaH2PO4
   • 0.51 mM K2SO4
   • 1.0 mM CaCl2
   • 1.65 mM MgSO4
   • 8.9 µM MnSO4
   • 0.5 µM Na2MoO4
   • 18.4 µM H3BO3
   • 0.14 µM ZnSO4
   • 0.16 µM CuSO4
   • 40.0 µM FeSO4
2. Transfer seedlings to solution containing:
   • 1.0 mM NH4NO3, plus
   • one amino acid: Arg or Phe or Lys (supplemented condition).
3. Measure lengths of first and second axillary buds using stereomicroscope (Olympus).
4. Growth setup:
   • Boxes: 525 × 360 × 230 mm
   • Phytotron conditions: 30°C for 14 h and 25°C for 10 h (day/night cycle).
5. Replace nutrient solution every 3 days.

10) RNA extraction + cDNA synthesis + qRT-PCR 🧫➡️🧬➡️📈

Aim

Quantify gene expression (e.g., OsAAP7 across tissues/lines) and validate expression patterns.

Concept/idea

qRT-PCR provides targeted quantification to confirm genotype effects (OE up, knockout altered) and tissue expression patterns.

Steps

1. Extract total RNA using TRIzol (Vazyme) following manufacturer instructions.
2. Use ~3 µg total RNA for first-strand cDNA synthesis with M-MLV reverse transcriptase (Vazyme).

qRT-PCR reaction setup (10 µL)

• 1 µL cDNA
• 1 µL primers
• 5 µL Mix (Vazyme)
• 3 µL ddH2O

qRT-PCR cycling

• 95°C 2 min (1 cycle)
• 35 cycles:
  • 95°C 30 s
  • 60°C 30 s
  • 72°C 30 s
• 72°C 1 min (1 cycle)

PCR amplification for OsAAP7 cDNA/promoter (20 µL)

• 1 µL DNA
• 10 µL Mix (Vazyme)
• 1.5 µL primers
• 6 µL ddH2O

PCR cycling

• 95°C 3 min (1 cycle)
• 30–35 cycles:
  • 95°C 30 s
  • 50–68°C 30 s
  • 72°C 2 min
• 72°C 10 min (1 cycle)

11) RNA-seq on axillary buds + differential expression analysis 🧬📚

Aim

Identify which pathways and genes change in axillary buds when OsAAP7 is overexpressed or knocked out.

Concept/idea

Transcriptome profiling links phenotype (bud growth/tillering) to underlying molecular pathways (N transport, hormone signaling, etc.).

Steps

1. Collect axillary buds from 60 seedlings per genotype:
   • ZH11
   • OsAAP7 OE1
   • OsAAP7 C1
2. Each replicate has 0.5 g tissue (replicate weight stated).
3. RNA-seq performed by Personalbio (China).
4. Data deposited to NCBI (accession details not in the methods text excerpt, but location stated).
5. Align clean reads to rice genome: Oryza sativa IRGSP-1.0.
6. Count reads using featureCounts.
7. Identify DEGs using DESeq with thresholds:
   • p < 0.05
   • fold-change > 1.4

12) Statistics + plotting 📉✅

Aim

Test whether observed differences are statistically significant and present results clearly.

Concept/idea

Use standard hypothesis tests (t-test, Tukey-Kramer) appropriate for comparing groups; visualize with established tools.

Steps/tools

1. Make statistical charts in GraphPad Prism 8.
2. Make heatmaps in TBtools.
3. Use SPSS (IBM) for:
   • t-test
   • Tukey-Kramer multiple range test
4. Significance notation:
   • • P<0.05
   • ** P<0.01
   • *** P<0.001
5. Data shown as mean ± standard deviation.

Quick “method map” (how the pieces fit) 🧩

• Natural promoter haplotypes → expression differences → trait associations
• Genetic manipulation (OE vs CRISPR) → field phenotypes (tillers/yield/NUtE)
• Mechanism tests
  • Where? (GFP localization)
  • Where expressed? (GUS + sections)
  • What transported? (yeast rescue + fluorescent uptake)
  • What accumulates? (HPLC amino acid profiling)
  • What happens to buds? (hydroponics + bud length)
• Why (pathways)? (RNA-seq + DEG enrichment + heatmaps)

Check your understanding (one question)

If you had to explain it in one sentence: what does the yeast complementation assay prove about OsAAP7?