In plants, innate immune responses are initiated by plasma membrane-located pattern recognition receptors (PRRs) upon recognition of elicitors, including exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs). Arabidopsis thaliana produces more than 1000 secreted peptide candidates, but it has yet to be established whether any of these act as elicitors. Here we identified an A. thaliana gene family encoding precursors of PAMP-induced secreted peptides (prePIPs) through an in-silico approach. The expression of some members of the family, including prePIP1 and prePIP2, is induced by a variety of pathogens and elicitors. Subcellular localization and proteolytic processing analyses demonstrated that the prePIP1 product is secreted into extracellular spaces where it is cleaved at the C-terminus. Overexpression of prePIP1 and prePIP2, or exogenous application of PIP1 and PIP2 synthetic peptides corresponding to the C-terminal conserved regions in prePIP1 and prePIP2, enhanced immune responses and pathogen resistance in A. thaliana. Genetic and biochemical analyses suggested that the receptor-like kinase 7 (RLK7) functions as a receptor of PIP1. Once perceived by RLK7, PIP1 initiates overlapping and distinct immune signaling responses together with the DAMP PEP1. PIP1 and PEP1 cooperate in amplifying the immune responses triggered by the PAMP flagellin. Collectively, these studies provide significant insights into immune modulation by Arabidopsis endogenous secreted peptides. Both animals and plants have evolved mechanisms to trigger innate immunity through perception of exogenous and endogenous molecules. In the model plant Arabidopsis thaliana, endogenous molecules such as the peptide elicitor PEP1 activate the immune response by means of cell surface-located receptors. Here we describe a new gene family in A. thaliana named prePIPs, whose members encode secreted peptide precursors, and show that one of its members, prePIP1, is secreted into extracellular space and cleaved at the C-terminus. Exogenous application of PIP1, the synthetic 13-amino acid peptide corresponding to the conserved C-terminal region of prePIP1, triggered immune responses and led to enhanced pathogen resistance in A. thaliana. We further provide evidence showing that PIP1 signals via the receptor-like kinase 7 (RLK7) and employs both shared and distinct components with the PEP1 signaling pathway. Both PIP1 and PEP1 cooperatively amplify the immune response triggered by flg22, the active epitope of bacterial flagellin.

Thiamine is required for both plant growth and development. Here, the involvement of a thiamine thiazole synthase, THI1, has been demonstrated in both guard cell abscisic acid (ABA) signaling and the drought response in Arabidopsis (Arabidopsis thaliana). THI1 overexpressors proved to be more sensitive to ABA than the wild type with respect to both the activation of guard cell slow type anion channels and stomatal closure; this effectively reduced the rate of water loss from the plant and thereby enhanced its level of drought tolerance. A yeast two-hybrid strategy was used to screen a cDNA library from epidermal strips of leaves for THI1 regulatory factors, and identified CPK33, a Ca²⁺-dependent protein kinase, as interactor with THI1 in a plasma membrane-delimited manner. Loss-of-function cpk33 mutants were hypersensitive to ABA activation of slow type anion channels and ABA-induced stomatal closure, while the CPK33 overexpression lines showed opposite phenotypes. CPK33 kinase activity was essential for ABA-induced stomatal closure. Consistent with their contrasting regulatory role over stomatal closure, THI1 suppressed CPK33 kinase activity in vitro. Together, our data reveal a novel regulatory role of thiamine thiazole synthase to kinase activity in guard cell signaling.

Guard cells shrink in response to drought and abscisic acid (ABA), which is caused by efflux of ions that in turn reduces stomatal aperture and improves the plant's ability to retain moisture. Cytosolic free calcium is an essential secondary messenger in guard cell ABA signaling, but the details of this regulatory pathway remain sketchy. Here, the calmodulin-like protein CML20, which has four EF-hand domains and calcium-binding activity in vitro, was found to be a negative regulator of ABA-induced stomatal movement in Arabidopsis. The guard cells of cml20 loss-of-function mutant plants were hypersensitive to both ABA-activated S-type anion currents, and ABA inhibited inward K+ currents than those of wild type. Additional, due to smaller stomatal aperture, cml20 showed less water loss from the leaves than wild type. These phenotypes of CML20 overexpressing plants contrasted with wild type in the opposite direction. In the cml20 mutant, the transcripts of stress responsive genes, such as MYB2, RAB18, ERD10, COR47 and RD29A were up-regulated in response to drought and ABA, while down-regulated of APX2 transcription and higher reactive oxygen species (ROS) accumulation. These observations support the CML20, a functional Ca2+ sensor, is a negative regulator in guard cell ABA signaling.