Exam Article

Applied Molecular Cellular Biology

🌾 Big Picture: What Is This Paper About?

This study investigates OsAAP7, an amino acid transporter in rice, and shows that:

👉 High OsAAP7 activity suppresses tillering and yield 👉 Blocking OsAAP7 (via CRISPR/Cas9) increases tillers, nitrogen-use efficiency, and grain yield

In short: 🧬 Less OsAAP7 = more tillers = higher yield

The mechanism involves amino acid transport (Phe, Lys, Arg, Leu) and downstream effects on nitrogen signaling and plant hormones (auxin & cytokinin).

🌱 Background & Biological Context

🌾 Why Tillering Matters

Rice yield depends heavily on tiller number
Each tiller → one panicle → more grains
Tillering is tightly regulated by nitrogen availability

🧪 Nitrogen in Plants

Plants take up:

Inorganic N: nitrate (NO₃⁻), ammonium (NH₄⁺)
Organic N: amino acids

🔑 Amino acids are the main transport form of nitrogen inside rice

🚚 Amino Acid Transporters (AATs)

Plants use two major transporter families:

1️⃣ APC superfamily

CATs (cationic AA)
ACTs
Polyamine transporters

2️⃣ AAAP superfamily

AAPs (Amino Acid Permeases)
LHTs, ProTs, GATs, AUXs, ANTs

📌 OsAAP7 belongs to the AAP subfamily, which can transport multiple amino acids.

🧬 What Was Known Before?

Some rice AAPs:

Increase tillering (OsAAP1, OsAAP4, OsAAP14, OsAAP15)
Decrease tillering (OsAAP3, OsAAP5)

❓ OsAAP7 was largely uncharacterized

This paper fills that gap.

🧠 Study Goals

The authors aimed to:

Characterize natural variation of OsAAP7
Identify where and when OsAAP7 is expressed
Determine which amino acids OsAAP7 transports
Test how OsAAP7 affects:
- Tillering
- Yield
- Nitrogen use efficiency
Reveal molecular pathways involved (RNA-seq)

🧬 Natural Variation: Indica vs Japonica

🔍 Promoter Haplotype Analysis (521 varieties)

Hap1 → mostly indica
Hap2 → mostly japonica

📊 Key Findings

Hap1 → lower OsAAP7 expression
Hap1 → more tillers & higher yield
Hap2 → higher OsAAP7 expression
Hap2 → fewer tillers

💡 OsAAP7 expression negatively correlates with yield

🧫 Expression Pattern of OsAAP7

🧪 GUS Reporter Analysis

Vegetative stage

High expression: lateral roots, leaf sheaths, leaf blades
Low expression: root tips, stems

Reproductive stage

High expression: stems, leaf sheaths
Low expression: roots, panicles, leaf blades

📍 Expression is strongest in vascular and parenchyma tissues

🧬 Subcellular Localization

Using GFP fusion:

OsAAP7 localizes to the endoplasmic reticulum (ER) 🧱
This is different from many AAPs that sit on the plasma membrane

👉 Suggests intracellular amino acid regulation, not just uptake from soil

🧪 What Does OsAAP7 Transport?

🧫 Yeast Complementation Assays

OsAAP7 rescues yeast growth when:

Phenylalanine (Phe)
Lysine (Lys)
Leucine (Leu)
Arginine (Arg)

📌 Confirms transport capability

🌈 Fluorescent Amino Acid Uptake

Overexpression lines: strong fluorescence
Knockout lines: weak fluorescence

✔ OsAAP7 actively transports Phe, Lys, Arg (and Leu)

✂️ Genetic Engineering Approaches

🔽 Knockout Lines (CRISPR/Cas9)

Targeted deletion in exon 4
Cas9 fully segregated out by T2 generation

🌾 Field Phenotypes

🌱 Tillering

OE lines → fewer tillers
Knockouts → more tillers

🌾 Grain Yield

OE lines → lower yield
Knockouts → significantly higher yield

⚡ Nitrogen Use Efficiency (NUtE)

Increased in knockouts
Decreased in OE lines

📌 OsAAP7 is a negative regulator of yield

🧪 Amino Acid Accumulation (HPLC)

📈 Overexpression Lines

High accumulation of:
- Phenylalanine
- Lysine
- Arginine

📉 Knockout Lines

Reduced levels of these amino acids

🔎 Suggests that excess basic & neutral amino acids suppress bud growth

🌿 Hydroponic Experiments: Axillary Bud Growth

🧫 Low amino acid concentrations

OE lines: enhanced second bud growth

🧪 High amino acid concentrations

OE lines: strong inhibition of bud elongation
Knockouts: enhanced growth

💡 Amino acid concentration matters, not just presence

🧠 RNA-seq: Molecular Mechanism

🔬 Differential Expression

Hundreds of DEGs in axillary buds
Clear separation of OE, WT, and knockout lines (PCA)

🧬 Affected Pathways

Nitrogen metabolism

Ammonium & nitrate transporters (AMTs, NPFs)

Hormones

Auxin signaling ↑ in OE lines
Cytokinin biosynthesis ↑ in knockout lines

📌 OsAAP7 coordinates:

N signaling
Auxin (inhibits buds)
Cytokinin (promotes buds)

🧬 OsAAP7 transports basic & neutral amino acids ⬇ 📈 Accumulation of Phe, Lys, Arg ⬇ ⚖ Alters nitrogen status ⬇ 🔁 Shifts auxin/cytokinin balance ⬇ ✋ Suppresses axillary bud outgrowth ⬇ 🌾 Reduced tillering & yield

✅ Final Conclusions

OsAAP7 is an ER-localized amino acid transporter
It negatively regulates:
- Tillering
- Yield
- Nitrogen use efficiency
CRISPR knockout of OsAAP7 is beneficial
OsAAP7 is a strong candidate for high-yield rice breeding

Quiz

Score: 0/49 (0%)

Q0. What is the primary form of nitrogen transported and allocated within rice plants, as emphasized in the paper?

Nitrate

Ammonium

Amino acids

Urea

Q1. OsAAP7 belongs to which transporter superfamily?

APC superfamily

AAAP superfamily

ABC transporter family

MFS transporter family

Q2. True or False: The study reports that OsAAP7 expression is negatively correlated with tillering and grain yield per plant.

True

False

Q3. In the 521-variety haplotype analysis, Hap1 of the OsAAP7 promoter was predominantly found in which subspecies?

Japonica

Indica

Both equally

Wild rice only (not cultivated)

Q4. True or False: Hap2 varieties generally showed higher OsAAP7 expression than Hap1 varieties.

True

False

Q5. What subcellular compartment is OsAAP7 primarily localized to according to GFP co-localization?

Plasma membrane

Nucleus

Endoplasmic reticulum membrane

Chloroplast envelope

Q6. True or False: During the vegetative stage, OsAAP7 expression was high in lateral roots and leaf tissues but low in root tips and stems.

True

False

Q7. Which assay directly tested whether OsAAP7 can transport specific amino acids using a mutant background?

Yeast complementation assay

ELISA

Southern blot

ChIP-seq

Q8. Which set of amino acids was supported by the yeast transport results and/or prior substrate testing for OsAAP7?

Asp, Glu, Asn

Phe, Lys, Leu, Arg

Val, Pro, Thr only

Gly, Ser, Ala

Q9. True or False: CRISPR/Cas9 knockout of OsAAP7 increased tiller number and grain yield compared to wild-type ZH11.

True

False

Q10. Which promoter was used to drive OsAAP7 overexpression in rice?

OsAAP7 native promoter

35S promoter

Ubiquitin promoter only

Actin promoter only

Q11. True or False: Overexpression of OsAAP7 increased nitrogen utilization efficiency (NUtE) relative to ZH11.

True

False

Q12. Which experimental method quantified free amino acids in plant tissues?

HPLC

SDS-PAGE

Western blot

Flow cytometry

Q13. True or False: OsAAP7 overexpression lines accumulated higher levels of Phe, Lys, and Arg than wild-type in multiple tissues/stages.

True

False

Q14. What was the purpose of FITC-labeled amino acid uptake experiments?

Measure Cas9 off-target edits

Directly visualize/quantify amino acid uptake/transport

Quantify DNA methylation

Measure chlorophyll fluorescence

Q15. True or False: Under identical FITC-amino-acid treatments, OsAAP7 OE lines showed stronger fluorescence signals than ZH11, while knockout lines showed weaker signals.

True

False

Q16. In hydroponic experiments, what general effect did high exogenous concentrations of Phe/Lys/Arg have on OsAAP7 OE lines?

Strongly promoted axillary bud elongation

Strongly suppressed axillary bud growth

Had no effect

Only affected primary roots

Q17. True or False: In hydroponic experiments, mutant (OsAAP7 knockout) lines tended to show the opposite axillary-bud response compared to OE lines under amino-acid treatments.

True

False

Q18. Which tissue was used for RNA-seq to study OsAAP7’s mechanism on tillering?

Mature grains

Flag leaves

Axillary buds

Root tips only

Q19. True or False: The paper used PCA (principal component analysis) to demonstrate transcriptome separation among ZH11, OE, and CRISPR samples.

True

False

Q20. What thresholds were used to call DEGs in the RNA-seq analysis described in Methods?

p < 0.05 and fold change > 1.4

p < 0.01 and fold change > 10

p < 0.001 only

FDR < 0.25 only

Q21. True or False: GO/KEGG enrichment suggested DEGs were enriched in processes including nitrogen compound processes and phenylpropanoid/lignin-related pathways.

True

False

Q22. Which change in nitrogen transporter gene expression pattern was reported in OsAAP7 knockout lines relative to ZH11?

Lower expression of ammonium/nitrate transporters

Higher expression of ammonium/nitrate transporters

No change in transporters

Transporter genes were not analyzed

Q23. True or False: Auxin pathway genes (e.g., OsIAA17/21/30) were upregulated in OE lines and downregulated in CRISPR lines relative to ZH11.

True

False

Q24. Which cytokinin-related gene trend was reported for knockout lines compared to OE lines?

LOG/LOGL genes up in knockouts, down in OE

LOG/LOGL genes down in knockouts, up in OE

LOG/LOGL unchanged in all lines

Cytokinin genes were not measured

Q25. True or False: OsAAP7 overexpression increased tiller number because it increased cytokinin activation genes (LOG).

True

False

Q26. What is the most direct agronomic interpretation proposed by the authors?

Overexpress OsAAP7 to increase yield

Knock out OsAAP7 to promote tillering and yield

Silence all AAP genes for best yield

Only modify nitrate transporters; AAPs are irrelevant

Q27. True or False: The authors suggest OsAAP7 provides a novel genetic resource for high-yield breeding using gene editing.

True

False

Q28. Which best describes the relationship between tillering and yield described in the Introduction?

Tillering reduces panicle number and yield

Tillering increases panicle number, affecting yield

Tillering only affects root growth

Tillering is unrelated to yield components

Q29. True or False: Plants can uptake both inorganic nitrogen (nitrate/ammonium) and organic nitrogen (amino acids) from soil.

True

False

Q30. Into which two major superfamilies are plant amino acid transporters (AATs) divided in the paper?

AAAP and APC

MFS and ABC

PIN and AUX/LAX

NRT1 and NRT2

Q31. True or False: The APC superfamily includes subfamilies such as CATs, ACTs, and PHSs.

True

False

Q32. How many AAP genes are identified in rice according to the paper?

Q33. True or False: The paper describes CRISPR/Cas9 as involving sgRNA targeting followed by Cas9 cutting at the intended locus.

True

False

Q34. Which resource/database did the authors use to extract OsAAP7 promoter SNP variants for haplotype analysis?

NCBI GenBank

RiceVarMap 2.0

Ensembl Fungi

TAIR

Q35. True or False: The promoter region examined for haplotypes was approximately 2600 bp upstream of the ATG start codon.

True

False

Q36. For the pOsAAP7-GUS construct, what length promoter fragment was cloned upstream of GUS?

500 bp

2113 bp

10,000 bp

1491 bp

Q37. True or False: OsAAP7 cDNA used for overexpression was 1491 bp long.

True

False

Q38. Which transformation method was used to generate transgenic rice lines?

Biolistic gene gun only

Agrobacterium-mediated callus infection

Electroporation of seeds

Viral vector infection

Q39. True or False: The T2 generation was selected for subsequent experiments in this study.

True

False

Q40. In yeast assays, what was the purpose of using uracil-free SD/YNB media?

To select against bacteria

To select for plasmid-containing yeast transformants

To induce sporulation

To inhibit amino acid uptake

Q41. True or False: In the yeast spot assay, amino acids (e.g., Phe, Lys, Leu) were provided as the sole nitrogen source to test transporter function.

True

False

Q42. Which instrument was used to detect fluorescence in the fluorescent amino acid uptake experiment?

Flow cytometer

Chemiluminescence apparatus

qPCR machine

Spectrophotometer at 750 nm

Q43. True or False: Hydroponic culture conditions included a phytotron regime of 30°C for 14 h and 25°C for 10 h.

True

False

Q44. How was nitrogen utilization efficiency (NUtE) calculated in the study?

Grain yield / total plant dry weight × 100

Grain yield / (grain N + straw N) × 100

Straw yield / grain yield × 100

Total N / chlorophyll content × 100

Q45. True or False: Differentially expressed genes were identified using featureCounts for alignment and DESeq for differential analysis.

True

False

Q46. Which rice reference genome build did the RNA-seq reads align to?

MSU7

IRGSP-1.0

TAIR10

B73 RefGen v4

Q47. True or False: The study reports that knockouts of OsAAP7 had higher NUtE than ZH11 while OE lines had lower NUtE.

True

False

Q48. Which statement best matches the paper’s final conclusion about OsAAP7 function?

OsAAP7 increases tillering by exporting auxin

OsAAP7 mainly transports Phe, Lys, Leu, and Arg and negatively regulates tillering and yield

OsAAP7 only affects panicle branching, not tillers

OsAAP7 is unrelated to nitrogen and hormone pathways

Exam Article

🌾 Big Picture: What Is This Paper About?

🌱 Background & Biological Context

🌾 Why Tillering Matters

🧪 Nitrogen in Plants

🚚 Amino Acid Transporters (AATs)

1️⃣ APC superfamily

2️⃣ AAAP superfamily

🧬 What Was Known Before?

🧠 Study Goals

🧬 Natural Variation: Indica vs Japonica

🔍 Promoter Haplotype Analysis (521 varieties)

📊 Key Findings

🧫 Expression Pattern of OsAAP7

🧪 GUS Reporter Analysis

🧬 Subcellular Localization

🧪 What Does OsAAP7 Transport?

🧫 Yeast Complementation Assays

🌈 Fluorescent Amino Acid Uptake

✂️ Genetic Engineering Approaches

🔼 Overexpression Lines (OE)

🔽 Knockout Lines (CRISPR/Cas9)

🌾 Field Phenotypes

🌱 Tillering

🌾 Grain Yield

⚡ Nitrogen Use Efficiency (NUtE)

🧪 Amino Acid Accumulation (HPLC)

📈 Overexpression Lines

📉 Knockout Lines

🌿 Hydroponic Experiments: Axillary Bud Growth

🧫 Low amino acid concentrations

🧪 High amino acid concentrations

🧠 RNA-seq: Molecular Mechanism

🔬 Differential Expression

🧬 Affected Pathways

🧠 Final Model (Conceptual)

✅ Final Conclusions

Quiz