Meta-Analyses of Microarrays of Arabidopsis asymmetric leaves1 (as1), as2 and Their Modifying Mutants Reveal a Critical Role for the ETT Pathway in Stabilization of Adaxial–Abaxial Patterning and Cell Division During Leaf Development

Abstract

It is necessary to use algorithms to analyze gene expression data from DNA microarrays, such as in clustering and machine learning. Previously, we developed the knowledge-based fuzzy adaptive resonance theory (KB-FuzzyART), a clustering algorithm suitable for analyzing gene expression data, to find clues for identifying gene networks. Leaf primordia form around the shoot apical meristem (SAM), which consists of indeterminate stem cells. Upon initiation of leaf development, adaxial–abaxial patterning is crucial for lateral expansion, via cellular proliferation, and the formation of flat symmetric leaves. Many regulatory genes that specify such patterning have been identified. Analysis by the KB-FuzzyART and subsequent molecular and genetic analyses previously showed that ASYMMETRIC LEAVES1 (AS1) and AS2 repress the expression of some abaxial-determinant genes, such as AUXIN RESPONSE FACTOR3 (ARF3)/ETTIN (ETT) and ARF4, which are responsible for defects in leaf adaxial–abaxial polarity in as1 and as2. In the present study, genetic analysis revealed that ARF3/ETT and ARF4 were regulated by modifier genes, BOBBER1 (BOB1) and ELONGATA3 (ELO3), together with AS1–AS2. We analyzed expression arrays with as2 elo3 and as2 bob1, and extracted genes downstream of ARF3/ETT by using KB-FuzzyART and molecular analyses. The results showed that expression of Kip-related protein (KRP) (for inhibitors of cyclin-dependent protein kinases) and Isopentenyltransferase (IPT) (for biosynthesis of cytokinin) genes were controlled by AS1–AS2 through ARF3/ETT and ARF4 functions, which suggests that the AS1–AS2–ETT pathway plays a critical role in controlling the cell division cycle and the biosynthesis of cytokinin around SAM to stabilize leaf development in Arabidopsis thaliana.

Dual regulation of ETTIN (ARF3) gene expression by AS1-AS2, which maintains the DNA methylation level, is involved in stabilization of leaf adaxial-abaxial partitioning in Arabidopsis

“…1B; supplementary material Fig. S1B) (Takahashi et al, 2008;Kojima et al, 2011;Takahashi et al, 2013). Cluster 3 included ETT and BP, the latter of which has been reported to be a direct target of AS1 (Guo et al, 2008), and cluster 6 included some of the abaxial determinant genes, such as KAN2.…”

ASYMMETRIC LEAVES2 and FASCIATA2 cooperatively regulate the formation of leaf adaxial-abaxial polarity in Arabidopsis thaliana

“…These results are consistent with a conclusion that the filamentous leaves in the as2-1 fas2-2 double mutants were abaxialized. To date, it has been reported that the formation of filamentous leaves in several double mutants combined with the as2-1 mutant are, at least partially, due to increased mRNA levels of ETT and ARF4 (Ishibashi et al 2012;Takahashi et al 2013). The increased levels of ETT and ARF4 transcripts in the as2-1 fas2-2 double mutant might be responsible for the formation of the filamentous leaves.…”

“…Takahashi et al (2013) show that the mRNA levels of genes for the CDK inhibitor, Kip-related Proteins (KRPs), are increased, in common, in the as2 eal and as2 elo3 double mutants exhibiting abaxialized filamentous leaves, and that the increased levels of KRP transcripts are due to the upregulation of ETT/ARF3 in the as2 eal double mutant. We investigated the mRNA levels of KRP genes in the as2-1 fas2-2 double mutants.…”

A genetic link between epigenetic repressor AS1–AS2 and DNA replication factors in establishment of adaxial–abaxial leaf polarity of <i>Arabidopsis</i>

“…AS1 and AS2 encode nuclear proteins and form a complex (designated AS1-AS2) (Guo et al 2008;Luo et al 2012;Xu et al 2003;Yang et al 2008). Mutations in these genes are associated with pleiotropic abnormalities in leaves along the three developmental axes described above (Byrne et al 2000;Iwakawa et al 2002Matsumura et al 2009;Ori et al 2000;Rédei and Hirono 1964;Semiarti et al 2001;Tsukaya and Uchimiya 1997), suggesting that AS1-AS2 regulates multiple genes Machida et al 2015;Takahashi et al 2008;Takahashi et al 2013) that might be involved in leaf formation along the three axes.…”