ATML1 Regulates the Differentiation of ER Body-Containing Large Pavement Cells in Rosette Leaves of Brassicaceae Plants.

Endoplasmic reticulum (ER)-derived organelles, ER bodies, participate in the defense against herbivores in Brassicaceae plants. ER bodies accumulate ß-glucosidases, which hydrolyze specialized thioglucosides known as glucosinolates to generate bioactive substances. In Arabidopsis thaliana, the leaf ER (LER) bodies are formed in large pavement cells, which are found in the petioles, margins and blades of rosette leaves. However, the regulatory mechanisms involved in establishing large pavement cells are unknown. Here, we show that the ARABIDOPSIS THALIANA MERISTEM L1 LAYER (ATML1) transcription factor regulates the formation of LER bodies in large pavement cells of rosette leaves. Overexpression of ATML1 enhanced the expression of LER body-related genes and the number of LER body-containing large pavement cells, whereas its knock-out resulted in opposite effects. ATML1 enhances endoreduplication and cell size through LOSS OF GIANT CELLS FROM ORGANS (LGO). Although the overexpression and knock-out of LGO affected the appearance of large pavement cells in Arabidopsis, the effect on LER body-related gene expression and LER body formation was weak. LER body-containing large pavement cells were also found in Eutrema salsugineum, another Brassicaceae species. Our results demonstrate that ATML1 establishes large pavement cells to induce LER body formation in Brassicaceae plants and thereby possibly contribute to the defense against herbivores.

NAI2 and TSA1 Drive Differentiation of Constitutive and Inducible ER Body Formation in Brassicaceae.

Brassicaceae and closely related species develop unique endoplasmic reticulum (ER)-derived structures called ER bodies, which accumulate ß-glucosidases/myrosinases that are involved in chemical defense. There are two different types of ER bodies: ER bodies constitutively present in seedlings (cER bodies) and ER bodies in rosette leaves induced by treatment with the wounding hormone jasmonate (JA) (iER bodies). Here, we show that At-α whole-genome duplication (WGD) generated the paralogous genes NAI2 and TSA1, which consequently drive differentiation of cER bodies and iER bodies in Brassicaceae plants. In Arabidopsis, NAI2 is expressed in seedlings where cER bodies are formed, whereas TSA1 is expressed in JA-treated leaves where iER bodies are formed. We found that the expression of NAI2 in seedlings and the JA inducibility of TSA1 are conserved across other Brassicaceae plants. The accumulation of NAI2 transcripts in Arabidopsis seedlings is dependent on the transcription factor NAI1, whereas the JA induction of TSA1 in rosette leaves is dependent on MYC2, MYC3 and MYC4. We discovered regions of microsynteny, including the NAI2/TSA1 genes, but the promoter regions are differentiated between TSA1 and NAI2 genes in Brassicaceae. This suggests that the divergence of function between NAI2 and TSA1 occurred immediately after WGD in ancestral Brassicaceae plants to differentiate the formation of iER and cER bodies. Our findings indicate that At-α WGD enabled diversification of defense strategies, which may have contributed to the massive diversification of Brassicaceae plants.