AUTHOR=Soda Midori N. , Hayashi Yuki , Takahashi Koji , Kinoshita Toshinori 

TITLE=Tryptophan synthase ß subunit 1 affects stomatal phenotypes in Arabidopsis thaliana

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.1011360

DOI=10.3389/fpls.2022.1011360

ISSN=1664-462X

ABSTRACT=<p>Stomata open in response to several environmental stimuli, such as light and low CO<sub>2</sub>. Plasma membrane (PM) H<sup>+</sup>-ATPase in guard cells plays a pivotal role for light-induced stomatal opening. In contrast, stomata close in response to the dark or plant hormone abscisic acid (ABA). However, molecular mechanisms of stomatal movements remain unclear. To elucidate the molecular mechanism of stomatal movements, we performed a genetic screen based on stomatal aperture-dependent weight decrease of detached leaves from EMS-treated <italic>Arabidopsis thaliana</italic> and isolated a <italic>rapid transpiration in detached leaves 2</italic> (<italic>rtl2</italic>). The <italic>rtl2</italic> mutant showed constitutive open-stomata phenotype with lower leaf temperature. ABA had no effect on stomatal aperture in <italic>rtl2</italic>. The <italic>rtl2</italic> mutant also showed increased stomatal density, severe dwarf phenotype with pale green leaves and dark veins. Map-based analysis of the <italic>RTL2</italic> locus revealed that the <italic>rtl2</italic> mutant possesses a single nucleotide substitution, which induces amino acid substitution Gly162 to Glu in the tryptophan synthase ß subunit 1 (TSB1). The <italic>TSB1</italic> encodes an enzyme in tryptophan (Trp) biosynthetic pathway. Amount of TSB1 protein was drastically reduced in <italic>rtl2</italic> mutant. A different allele of <italic>tsb1</italic> mutant (<italic>tsb1-1</italic>) also showed constitutive open-stomata phenotype with reduced TSB1 protein as in <italic>rtl2</italic>. Analyses of test-crossed plants of <italic>rtl2</italic> and <italic>tsb1-1</italic> showed open-stomata and dwarf phenotypes. These results indicate that a responsible gene for <italic>rtl2</italic> is <italic>TSB1</italic>. We further investigated stomatal phenotype in mutants from Trp biosynthetic pathway, such as <italic>wei2-1 wei7-1</italic>, <italic>trp3-1</italic>, and <italic>tsb2-1</italic>. The <italic>trp3-1</italic> mutant showed significant wider stomatal aperture as well as <italic>tsb1-1</italic>. Trp biosynthetic pathway closely relates to auxin biosynthesis. Then, we investigated auxin responsible genes and found that an expression of <italic>AUR3</italic> was up in <italic>rtl2</italic>. In contrast, auxin had no effect on stomatal aperture in Arabidopsis and the phosphorylation status of PM H<sup>+</sup>-ATPase in guard cell protoplasts from <italic>Vicia faba</italic>. In addition, auxin antagonist had no effect on stomatal aperture. Interestingly, <italic>tsb1-1</italic> grown under hydroponic culture system showed normal stomatal aperture by exogenously application of Trp. These results suggest that open stomata phenotype in <italic>tsb1-1</italic> is due to Trp deficiency but not auxin.</p>