Knockout of the tomatoHAIRY MERISTEM 4alters phloem-characteristics and impairs development

Abstract

AbstractThe HAIRY MERISTEM (HAM) gene family encodes Type I and II GRAS domain transcription factors in plants. Type II HAMs, predominantly expressed in meristems and regulated by microRNA171, are essential for maintaining undifferentiated meristems, a role conserved across various species. Conversely, the functions of Type I HAMs have been less characterized. In this study, we investigated the role of SlHAM4, a Type I HAM in tomato. Using publicly available expression data and a GUS reporter gene driven by the nativeSlHAM4promoter, we determined thatSlHAM4is predominantly expressed in phloem tissues. CRISPR-inducedSlHAM4loss-of- function mutations (slham4CR) resulted in a range of shoot and fruit abnormalities, which were fully reversed by reintroducingSlHAM4under its native promoter in the mutant background. Mutant abnormalities included increased anthocyanin pigmentation in the leaf and sepal primordia, reminiscent of the phenotypes observed in certain Arabidopsis mutants with compromised phloem, and development of simpler leaves, which was associated with reduction in external phloem area in the leaf rachis. In addition,slham4CRplants produced significantly smaller fruits of which a fraction of them exhibited catface-like scars, attributed to tears which occurred in the pericarp of mutant ovaries following fruit set. Transcriptome analysis of the wild-type looking mutant ovaries at anthesis revealed specific downregulation of genes implicated in phloem development and functions, in particular those expressed in companion cells (CC). We propose that SlHAM4 is necessary for proper phloem function in part by regulating the expression of a suite of CCs genes that encode essential phloem proteins.One-sentence summarySlHAM4is predominantly expressed in the phloem and its knockout alters phloem- characteristics and impair development highlighting its requirement for proper phloem functionality.