Many biochemical approaches show functions of calcium-dependent protein kinases (CDPKs) in abscisic acid (ABA) signal transduction, but molecular genetic evidence linking defined CDPK genes with ABA-regulated biological functions at the whole-plant level has been lacking. Here, we report that ABA stimulated two homologous CDPKs in Arabidopsis thaliana, CPK4 and CPK11. Loss-of-function mutations of CPK4 and CPK11 resulted in pleiotropic ABA-insensitive phenotypes in seed germination, seedling growth, and stomatal movement and led to salt insensitivity in seed germination and decreased tolerance of seedlings to salt stress. Double mutants of the two CDPK genes had stronger ABA-and salt-responsive phenotypes than the single mutants. CPK4-or CPK11-overexpressing plants generally showed inverse ABA-related phenotypes relative to those of the loss-of-function mutants. Expression levels of many ABA-responsive genes were altered in the loss-of-function mutants and overexpression lines. The CPK4 and CPK11 kinases both phosphorylated two ABA-responsive transcription factors, ABF1 and ABF4, in vitro, suggesting that the two kinases may regulate ABA signaling through these transcription factors. These data provide in planta genetic evidence for the involvement of CDPK/calcium in ABA signaling at the whole-plant level and show that CPK4 and CPK11 are two important positive regulators in CDPK/calcium-mediated ABA signaling pathways.

Abscisic acid (ABA) is a vital phytohormone that regulates mainly stomatal aperture and seed development, but ABA receptors involved in these processes have yet to be determined. We previously identified from broad bean an ABA-binding protein (ABAR) potentially involved in stomatal signalling, the gene for which encodes the H subunit of Mg-chelatase (CHLH), which is a key component in both chlorophyll biosynthesis and plastid-to-nucleus signalling. Here we show that Arabidopsis ABAR/CHLH specifically binds ABA, and mediates ABA signalling as a positive regulator in seed germination, post-germination growth and stomatal movement, showing that ABAR/CHLH is an ABA receptor. We show also that ABAR/CHLH is a ubiquitous protein expressed in both green and non-green tissues, indicating that it might be able to perceive the ABA signal at the whole-plant level.

Plant responses to ethylene are mediated by regulation of EBF1/2-dependent degradation of the ETHYLENE INSENSITIVE3 (EIN3) transcription factor. Here, we report that the level of EIL1 protein is upregulated by ethylene through an EBF1/2-dependent pathway. Genetic analysis revealed that EIL1 and EIN3 cooperatively but differentially regulate a wide array of ethylene responses, with EIL1 mainly inhibiting leaf expansion and stem elongation in adult plants and EIN3 largely regulating a multitude of ethylene responses in seedlings. When EBF1 and EBF2 are disrupted, EIL1 and EIN3 constitutively accumulate in the nucleus and remain unresponsive to exogenous ethylene application. Further study revealed that the levels of EBF1 and EBF2 proteins are downregulated by ethylene and upregulated by silver ion and MG132, suggesting that ethylene stabilizes EIN3/EIL1 by promoting EBF1 and EBF2 proteasomal degradation. Also, we found that EIN2 is indispensable for mediating ethylene-induced EIN3/EIL1 accumulation and EBF1/2 degradation, whereas MKK9 is not required for ethylene signal transduction, contrary to a previous report. Together, our studies demonstrate that ethylene similarly regulates EIN3 and EIL1, the two master transcription factors coordinating myriad ethylene responses, and clarify that EIN2 but not MKK9 is required for ethylene-induced EIN3/EIL1 stabilization. Our results also reveal that EBF1 and EBF2 act as essential ethylene signal transducers that by themselves are subject to proteasomal degradation.

It remains unclear whether the phloem unloading pathway alters to adapt to developmental transition in fleshy fruits that accumulate high level of soluble sugars. Using a combination of electron microscopy, transport of the phloem-mobile symplasmic tracer carboxyfluorescein, movement of the companion cell-expressed and the green fluorescent protein-tagged viral movement protein, and assays of the sucrose cleavage enzymes, the pathway of phloem unloading was studied in the berries of a hybrid grape (Vitis vinifera 3 Vitis labrusca). Structural investigations showed that the sieve element-companion cell complex is apparently symplasmically connected through plasmodesmata with surrounding parenchyma cells throughout fruit development, though a small portion of plasmodesmata are apparently blocked in the ripening stage. Both carboxyfluorescein and the green fluorescent protein-tagged viral movement protein were released from the functional phloem strands during the early and middle stages of fruit development, whereas the two symplasmic tracers were confined to the phloem strands during the late stage. This reveals a shift of phloem unloading from symplasmic to apoplasmic pathway during fruit development. The turning point of the phloem unloading pathways was further shown to be at or just before onset of ripening, an important developmental checkpoint of grape berry. In addition, the levels of both the expression and activities of cell wall acid invertase increased around the onset of ripening and reached a high level in the late stage, providing further evidence for an operation of the apoplasmic unloading pathway after onset of ripening. These data demonstrate clearly the occurrence of an adaptive shift of phloem unloading pathway to developmental transition from growing phase to ripening in grape berry.

Abscisic acid (ABA) is an essential hormone for plant development and stress responses. ABA signaling is suppressed by clade A PP2C phosphatases, which function as key repressors of this pathway through inhibiting ABA-activated SnRK2s (SNF1-related protein kinases). Upon ABA perception, the PYR/PYL/RCAR ABA receptors bind to PP2Cs with high affinity and biochemically inhibit their activity. While this mechanism has been extensively studied, how PP2Cs are regulated at the protein level is only starting to be explored. RING DOMAIN LIGASE5 (RGLG5) belongs to a five-member E3 ubiquitin ligase family whose target proteins remain unknown. We report that RGLG5, together with RGLG1, releases the PP2C blockade of ABA signaling by mediating PP2CA protein degradation. ABA promotes the interaction of PP2CA with both E3 ligases, which mediate ubiquitination of PP2CA and are required for ABA-dependent PP2CA turnover. Downregulation of and stabilizes endogenous PP2CA and diminishes ABA-mediated responses. Moreover, the reduced response to ABA in germination assays is suppressed in the (artificial microRNA) triple mutant, supporting a functional link among these loci. Overall, our data indicate that RGLG1 and RGLG5 are important modulators of ABA signaling, and they unveil a mechanism for activation of the ABA pathway by controlling PP2C half-life.