Summary: Plant ethylene‐responsive factors (ERFs) play essential roles in cold stress response, but the molecular mechanisms underlying this process remain poorly understood. In this study, we characterized PtrERF9 from trifoliate orange (Poncirus trifoliata (L.) Raf.), a cold‐hardy plant. PtrERF9 was up‐regulated by cold in an ethylene‐dependent manner. Overexpression of PtrERF9 conferred prominently enhanced freezing tolerance, which was drastically impaired when PtrERF9 was knocked down by virus‐induced gene silencing. Global transcriptome profiling indicated that silencing of PtrERF9 resulted in substantial transcriptional reprogramming of stress‐responsive genes involved in different biological processes. PtrERF9 was further verified to directly and specifically bind with the promoters of glutathione S‐transferase U17 (PtrGSTU17) and ACC synthase1 (PtrACS1). Consistently, PtrERF9‐overexpressing plants had higher levels of PtrGSTU17 transcript and GST activity, but accumulated less ROS, whereas the silenced plants showed the opposite changes. Meanwhile, knockdown of PtrERF9 decreased PtrACS1 expression, ACS activity and ACC content. However, overexpression of PtrERF9 in lemon, a cold‐sensitive species, caused negligible alterations of ethylene biosynthesis, which was attributed to perturbed interaction between PtrERF9, along with lemon homologue ClERF9, and the promoter of lemon ACS1 gene (ClACS1) due to mutation of the cis‐acting element. Taken together, these results indicate that PtrERF9 acts downstream of ethylene signalling and functions positively in cold tolerance via modulation of ROS homeostasis by regulating PtrGSTU17. In addition, PtrERF9 regulates ethylene biosynthesis by activating PtrACS1 gene, forming a feedback regulation loop to reinforce the transcriptional regulation of its target genes, which may contribute to the elite cold tolerance of Poncirus trifoliata.

In plant-microbe interactions, plant sugars produced by photosynthesis are not only a carbon source for pathogens, but may also act as signals that modulate plant defense responses. Here, we report that decreasing sorbitol synthesis in apple (Malus domestica) leaves by antisense suppression of ALDOSE-6-PHOSPHATE REDUCTASE (A6PR) leads to downregulation of 56 NUCLEOTIDE BINDING/LEUCINE-RICH REPEAT (NLR) genes and converts the phenotypic response to Alternaria alternata from resistant to susceptible. We identified a resistance protein encoded by the apple MdNLR16 gene and a small protein encoded by the fungal HRIP1 gene that interact in both a yeast two-hybrid assay and a bimolecular fluorescence complementation assay. Deletion of HRIP1 in A. alternata enables gain of virulence on the wild-type control plant. Overexpression of MdNLR16 in two antisense A6PR lines increases resistance, whereas RNAi suppression of MdNLR16 in the wild-type control decreases resistance against A. alternata. MdWRKY79 transcriptionally regulates MdNLR16 by binding to the promoter of MdNLR16 in response to sorbitol, and exogenous sorbitol feeding partially restores resistance of the antisense A6PR lines to A. alternata. These findings indicate that sorbitol modulates resistance to A. alternata via the MdNLR16 protein that interacts with the fungal effector in a classic gene-for-gene manner in apple.

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.

Malate accumulation in the vacuole largely determines apple (Malus domestica) fruit acidity, and low fruit acidity is strongly associated with truncation of Ma1, an ortholog of ALUMINUM-ACTIVATED MALATE TRANSPORTER9 (ALMT9) in Arabidopsis (Arabidopsis thaliana). A mutation at base 1,455 in the open reading frame of Ma1 leads to a premature stop codon that truncates the protein by 84 amino acids at its C-terminal end. Here, we report that both the full-length protein, Ma1, and its naturally occurring truncated protein, ma1, localize to the tonoplast; when expressed in Xenopus laevis oocytes and Nicotiana benthamiana cells, Ma1 mediates a malate-dependent inward-rectifying current, whereas the ma1-mediated transmembrane current is much weaker, indicating that ma1 has significantly lower malate transport activity than Ma1. RNA interference suppression of Ma1 expression in ‘McIntosh’ apple leaves, ‘Empire’ apple fruit, and ‘Orin’ apple calli results in a significant decrease in malate level. Genotyping and phenotyping of 186 apple accessions from a diverse genetic background of 17 Malus species combined with the functional analyses described above indicate that Ma1 plays a key role in determining fruit acidity and that the truncation of Ma1 to ma1 is genetically responsible for low fruit acidity in apple. Furthermore, we identified a C-terminal domain conserved in all tonoplast-localized ALMTs essential for Ma1 function; protein truncations into this conserved domain significantly lower Ma1 transport activity. We conclude that the truncation of Ma1 to ma1 reduces its malate transport function by removing a conserved C-terminal domain, leading to low fruit acidity in apple.