Protocol for screening and expression studies of T-DNA and tagging-based insertional knox mutants in Arabidopsis thaliana.

KNOTTED1-like homeobox (KNOX) genes serve important roles in meristem function and many developmental processes in all higher plants. In Arabidopsis, studies of KNOX genes especially among members of class II KNOX genes remain limited and functional data are largely lacking. In the present study, we established a reproducible protocol that is important for genetic studies of KNOX genes using Arabidopsis insertional mutants. This protocol contains a reproducible and serial procedure containing detailed and step-by-step laboratory and field works covering all experiment steps from the screening of homozygous mutant lines to the KNOX expression analysis using qRT-PCR in a single paper. The troubleshooting and challenges that might occur are also presented and discussed. T-DNA insertion mutants for all Arabidopsis KNOX genes (except for knat4) were isolated based on kanamycin screening, phenotype selection, and PCR genotyping. Surprisingly, the insertions resulted in strong repression of the respective KNOX genes. However, no gene suppression was observed for the positively selected knat5 mutant. Moreover, qRT-PCR was effective for transcript analysis among the knox mutant samples. The use of different relative expression quantification produces a similar indication of expression level. Overall, the proposed procedure is highly effective for expression studies of KNOX genes in Arabidopsis mutants and will serve as a fundamental work protocol to open opportunities for genetic studies of genes involving insertional mutants in Arabidopsis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02868-8.

Class I KNOX transcription factors promote differentiation of cambial derivatives into xylem fibers in the Arabidopsis hypocotyl.

The class I KNOX transcription factors SHOOT MERISTEMLESS (STM) and KNAT1 are important regulators of meristem maintenance in shoot apices, with a dual role of promoting cell proliferation and inhibiting differentiation. We examined whether they control stem cell maintenance in the cambium of Arabidopsis hypocotyls, a wood-forming lateral meristem, in a similar fashion as in the shoot apical meristem. Weak loss-of-function alleles of KNAT1 and STM led to reduced formation of xylem fibers - highly differentiated cambial derivatives - whereas cell proliferation in the cambium was only mildly affected. In a knat1;stm double mutant, xylem fiber differentiation was completely abolished, but residual cambial activity was maintained. Expression of early and late markers of xylary cell differentiation was globally reduced in the knat1;stm double mutant. KNAT1 and STM were found to act through transcriptional repression of the meristem boundary genes BLADE-ON-PETIOLE 1 (BOP1) and BOP2 on xylem fiber differentiation. Together, these data indicate that, in the cambium, KNAT1 and STM, contrary to their function in the shoot apical meristem, promote cell differentiation through repression of BOP genes.