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Mutations in tetrapyrrole biosynthesis pathway uncouple nuclear WUSCHEL expression from de novo shoot development in Arabidopsis

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Plant de novo organogenesis in tissue culture systems has long been exploited to study the plasticity of pluripotency. External application of high cytokinin-to-auxin ratio in cultured medium stimulates greening of tissue and promotes nascent shoot apical meristem (SAM) formation. The stem cell niche in SAM is maintained by a negative feedback loop between CLAVATA and WUSCHEL (WUS) signaling. The fact that cytokinin is able to induce expression of SAM master regulator WUS suggests that the capacity of de novo shoot development is largely dependent on WUS activity. However, the molecular mechanism of WUS expression remains obscure. Here we found that WUS expression during de novo SAM formation is affected by the altered tetrapyrrole metabolism catalyzed in the plastid. Loss-of-function mutations in HEME OXYGENASE/LONG HYPOCOTYL 1 (hy1), Mg-CHELATASE H (chlh), Mg-CHELATASE I1 (chli1), and the regulator of Mg-CHELATASE, GENOME-UNCOUPLED 4 (gun4), result in elevated WUS expression but shoot regeneration efficiency is decreased whereas loss-of-function mutation in PROTOPORPHYRIN IX FERROCHELATASE 2 (fc2) exhibits compromised WUS expression with a reduced number of shoots when mutant root explants are cultured on shoot induction medium. Our genetic results show that nuclear WUS expression is affected by tetrapyrrole intermediate(s) under the varying plastid status stimulated by external cytokinin treatment during de novo organogenesis.

Key Message

We propose novel regulatory mechanism of nuclear WUS expression that is likely modulated by tetrapyrrole intermediate(s) under the varying plastid status stimulated by external cytokinin treatment during de novo organogenesis.

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Fig. 1
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Arabidopsis histidine kinase 3


Cytokinin hypersensitive 2


Cytokinin response 1


Protoporphyrin IX ferrochelatase 2


Genome uncoupled


Long hypocotyl




Shoot induction medium




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This work was supported by Grant Agency in the Czech Republic (GAČR17-23702S and GAČR 18-23972Y) and the European Regional Development Fund (ERDF) Project (No. CZ.02.1.01/0.0/0.0/16_019/0000827). We thank European Arabidopsis Stock Centre (NASC) and Tatsuo Kakimoto for providing cre1-2 mutant seeds.

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IK, DZ, AM, and YI carried out experiments and wrote the manuscript.

Correspondence to Yoshihisa Ikeda.

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Communicated by Jochen Kumlehn.

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Kubalová, I., Zalabák, D., Mičúchová, A. et al. Mutations in tetrapyrrole biosynthesis pathway uncouple nuclear WUSCHEL expression from de novo shoot development in Arabidopsis. Plant Cell Tiss Organ Cult 139, 395–401 (2019). https://doi.org/10.1007/s11240-019-01680-w

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  • Arabidopsis thaliana
  • Cytokinin
  • Retrograde signaling
  • Shoot apical meristem
  • Tetrapyrrole biosynthesis