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Rewinding evolution in planta: A Rubisco-null platform validates high-performance ancestral enzymes.

Vishalsingh R Chaudhari1, Myat T Lin1, Kevin M Hines1

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Researchers created a Rubisco-null tobacco platform to evaluate engineered photosynthetic enzymes. This system enabled the assessment of resurrected ancestral Rubiscos, revealing enhanced catalytic efficiency for improving C3 crop productivity.

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Area of Science:

  • Plant Biology
  • Biochemistry
  • Genetics

Background:

  • Improving Rubisco, a key photosynthetic enzyme, is crucial for enhancing C3 crop productivity.
  • Evaluating engineered Rubisco variants in planta is challenging due to native enzyme interference.

Purpose of the Study:

  • To develop a Rubisco-null platform in Nicotiana tabacum for in planta evaluation of engineered Rubisco variants.
  • To assess the performance of resurrected ancestral Rubiscos using this novel system.

Main Methods:

  • CRISPR-Cas9 was used to knock out all 11 nuclear-encoded small subunit (rbcS) genes in Nicotiana tabacum.
  • A chloroplast expression system was developed for coexpressing cyanobacterial Rubisco subunits.
  • Two resurrected ancestral Solanaceae Rubiscos were expressed and kinetically analyzed.

Main Results:

  • A viable Rubisco-null Nicotiana tabacum platform was successfully created.
  • Transgenic plants expressing ancestral Rubiscos were phenotypically normal and accumulated Rubisco to wild-type levels.
  • Resurrected ancestral Rubiscos exhibited 16-20% higher catalytic efficiency under ambient conditions due to faster turnover rates.

Conclusions:

  • The developed platform allows for robust in vivo assessment of novel Rubisco variants.
  • Ancestral enzyme reconstruction is a powerful strategy for identifying superior Rubiscos.
  • This approach holds promise for improving photosynthesis and productivity in C3 crops.