ABA guides stomatal proliferation and patterning through the EPF-SPCH signaling pathway in Arabidopsis thaliana.

Adaptation to dehydration stress requires plants to coordinate environmental and endogenous signals to inhibit stomatal proliferation and modulate their patterning. The stress hormone abscisic acid (ABA) induces stomatal closure and restricts stomatal lineage to promote stress tolerance. Here, we report that mutants with reduced ABA levels, xer-1, xer-2 and aba2-2, developed stomatal clusters. Similarly, the ABA signaling mutant snrk2.2/2.3/2.6, which lacks core ABA signaling kinases, also displayed stomatal clusters. Exposure to ABA or inhibition of ABA catabolism rescued the increased stomatal density and spacing defects observed in xer and aba2-2, suggesting that basal ABA is required for correct stomatal density and spacing. xer-1 and aba2-2 displayed reduced expression of EPF1 and EPF2, and enhanced expression of SPCH and MUTE. Furthermore, ABA suppressed elevated SPCH and MUTE expression in epf2-1 and epf1-1, and partially rescued epf2-1 stomatal index and epf1-1 clustering defects. Genetic analysis demonstrated that XER acts upstream of the EPF2-SPCH pathway to suppress stomatal proliferation, and in parallel with EPF1 to ensure correct stomatal spacing. These results show that basal ABA and functional ABA signaling are required to fine-tune stomatal density and patterning.

CBF4/DREB1D represses XERICO to attenuate ABA, osmotic and drought stress responses in Arabidopsis.

Water stress can severely impact plant growth, productivity and yield. Consequently, plants have evolved various strategies through which they can respond and adapt to their environment. XERICO (XER) is a stress-responsive RING E3 ubiquitin ligase that modulates abscisic acid (ABA) levels and promotes drought tolerance when overexpressed. To better understand the biological role of XER in stress responses, we characterized a xer-1 hypomorphic mutant and a CRISPR/Cas9-induced xer-2 null mutant in Arabidopsis. Both xer mutant alleles exhibited increased drought sensitivity, supporting the results from overexpression studies. Furthermore, we discovered that both xer mutants have greater stomatal indices and that XER is expressed in epidermal cells, indicating that XER functions in the epidermis to repress stomatal development. To explore XER spatiotemporal and stress-dependent regulation, we conducted a yeast one-hybrid screen and found that CBF4/DREB1D associates with the XER 5' untranslated region (5'-UTR). We generated three cbf4 null mutants with CRISPR/Cas9 and showed that CBF4 negatively regulates ABA responses, promotes stomatal development and reduces drought tolerance, in contrast to the roles shown for XER. CBF4 is induced by ABA and osmotic stress, and localizes to the nucleus where it downregulates XER expression via the DRE element in its 5'-UTR. Lastly, genetic interaction studies confirmed that xer is epistatic to cbf4 in stomatal development and in ABA, osmotic and drought stress responses. We propose that the repression of XER by CBF4 functions to attenuate ABA signaling and stress responses to maintain a balance between plant growth and survival under adverse environmental conditions.

Spatiotemporal Restriction of FUSCA3 Expression by Class I BPCs Promotes Ovule Development and Coordinates Embryo and Endosperm Growth.

Spatiotemporal regulation of gene expression is critical for proper developmental timing in plants and animals. The transcription factor FUSCA3 (FUS3) regulates developmental phase transitions by acting as a link between hormonal pathways in Arabidopsis (Arabidopsis thaliana). However, the mechanisms governing its spatiotemporal expression pattern are poorly understood. Here, we show that FUS3 is repressed in the ovule integuments and seed endosperm. FUS3 repression requires class I BASIC PENTACYSTEINE (BPC) proteins, which directly bind GA/CT cis-elements in FUS3 and restrict its expression pattern. During vegetative and reproductive development, FUS3 derepression in bpc1-1 bpc2 (bpc1/2) double mutant or misexpression in ProML1:FUS3 lines causes dwarf plants carrying defective flowers and aborted ovules. After fertilization, ectopic FUS3 expression in bpc1/2 endosperm or ProML1:FUS3 endosperm and endothelium increases endosperm nuclei proliferation and seed size, causing delayed or arrested embryo development. These phenotypes are rescued in bpc1/2 fus3-3 Finally, class I BPCs interact with FIS-PRC2 (FERTILIZATION-INDEPENDENT SEED-Polycomb Repressive Complex2), which represses FUS3 in the endosperm during early seed development. We propose that BPC1 and 2 promote the transition from reproductive to seed development by repressing FUS3 in ovule integuments. After fertilization, BPC1 and 2 and FIS-PRC2 repress FUS3 in the endosperm to coordinate early endosperm and embryo growth.