CLE peptides have been implicated in various developmental processes of plants and mediate their responses to environmental stimuli. However, the biological relevance of most CLE genes remains to be functionally characterized. Here, we report that CLE9, which is expressed in stomata, acts as an essential regulator in the induction of stomatal closure. Exogenous application of CLE9 peptides or overexpression of CLE9 effectively led to stomatal closure and enhanced drought tolerance, whereas CLE9 loss-of-function mutants were sensitivity to drought stress. CLE9-induced stomatal closure was impaired in abscisic acid (ABA)-deficient mutants, indicating that ABA is required for CLE9-medaited guard cell signalling. We further deciphered that two guard cell ABA-signalling components, OST1 and SLAC1, were responsible for CLE9-induced stomatal closure. MPK3 and MPK6 were activated by the CLE9 peptide, and CLE9 peptides failed to close stomata in mpk3 and mpk6 mutants. In addition, CLE9 peptides stimulated the induction of hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO) synthesis associated with stomatal closure, which was abolished in the NADPH oxidase-deficient mutants or nitric reductase mutants, respectively. Collectively, our results reveal a novel ABA-dependent function of CLE9 in the regulation of stomatal apertures, thereby suggesting a potential role of CLE9 in the stress acclimatization of plants.

As sessile organisms, plants are continuously exposed to a wide range of environmental stress. In addition to their crucial roles in plant growth and development, small signaling peptides are also implicated in sensing environmental stimuli. Notably, recent studies in plants have revealed that small signaling peptides are actively involved in controlling stomatal aperture to defend against biotic and abiotic stress. This review illustrates our growing knowledge of small signaling peptides in the modulation of stomatal aperture and highlights future challenges to decipher peptide signaling pathways in guard cells.

Summary Known for their regulatory roles in stem cell homeostasis, CLAVATA3/ESR‐RELATED (CLE) peptides also function as mediators of external stimuli such as hormones. De novo shoot regeneration, representing the remarkable plant cellular plasticity, involves reconstitution of stem cells under control of stem‐cell regulators. Yet whether and how stem cell‐regulating CLE peptides are implicated in plant regeneration remains unknown. By CRISPR/Cas9‐induced loss‐of‐function studies, peptide application, precursor overexpression, and expression analyses, the role of CLE1–CLE7 peptides and their receptors in de novo shoot regeneration was studied in Arabidopsis thaliana. CLE1–CLE7 are induced by callus‐induction medium and dynamically expressed in pluripotent callus. Exogenously‐applied CLE1–CLE7 peptides or precursor overexpression effectively leads to shoot regeneration suppression, whereas their simultaneous mutation results in enhanced regenerative capacity, demonstrating that CLE1–CLE7 peptides redundantly function as negative regulators of de novo shoot regeneration. CLE1–CLE7‐mediated shoot regeneration suppression is impaired in loss‐of‐function mutants of callus‐expressed CLAVATA1 (CLV1) and BARELY ANY MERISTEM1 (BAM1) genes, indicating that CLV1/BAM1 are required for CLE1–CLE7‐mediated shoot regeneration signaling. CLE1–CLE7 signaling resulted in transcriptional repression of WUSCHEL (WUS), a stem cell‐promoting transcription factor known as a principal regulator of plant regeneration. Our results indicate that functionally‐redundant CLE1–CLE7 peptides genetically act through CLV1/BAM1 receptors and repress WUS expression to modulate shoot‐regeneration capacity, establishing the mechanistic basis for CLE1–CLE7‐mediated shoot regeneration and a novel role for CLE peptides in hormone‐dependent developmental plasticity.

Strigolactones (SLs) are known to mediate plant acclimation to environmental stress. We recently reported that SLs acted as prominent regulators in promotion of stomatal closure. However, the detailed mechanism by which SLs induce stomatal closure requires further investigation. Here we studied the essential role of the calcium (Ca 2+) signal mediating by the calcium-dependent protein kinase (CPK) in SL-induced stomatal closure. SL-induced stomatal closure was strongly inhibited by a Ca 2+ chelator and Ca 2+ channel blockers, indicating that Ca 2+ functions in SL promotion of stomatal closure. Through examining a collection of cpk mutants, we identified CPK33, potentially acting as a Ca 2+ transducer, which is implicated in guard cell SL signaling. SL-and Ca 2+-induced stomatal closure were impaired in cpk33 mutants. CPK33 kinase activity is essential for SL induction of stomatal closure as SL-induced stomatal closure is blocked in the dead kinase mutant of CPK33. The cpk33 mutant is impaired in H 2 O 2-induced stomatal closure, but not in SLmediated H 2 O 2 production. Our study thus uncovers an important player CPK33 which functions as an essential Ca 2+ signals mediator in Arabidopsis guard cell SL signaling.