Alkaline/neutral invertase (A/N-INV), an enzyme that hydrolyzes sucrose irreversibly into glucose and fructose, is essential for normal plant growth,development, and stress tolerance. However, the physiological and/or molecular mechanism underpinning the role of A/N-INV in abiotic stress tolerance is poorly understood. In this report, an A/N-INV gene (PtrA/NINV) was isolated from Poncirus trifoliata, a cold-hardy relative of citrus, and functionally characterized. PtrA/NINV expression levels were induced by cold, salt, dehydration, sucrose, and ABA, but decreased by glucose. PtrA/NINV was found to localize in both chloroplasts and mitochondria. Overexpression of PtrA/NINV conferred enhanced tolerance to multiple stresses, including cold, high salinity, and drought, as supported by lower levels of reactive oxygen species (ROS), reduced oxidative damages, decreased water loss rate, and increased photosynthesis efficiency, relative to wild-type (WT). The transgenic plants exhibited higher A/N-INV activity and greater reducing sugar content under normal and stress conditions. PtrA/NINV is an important gene implicated in sucrose decomposition, and plays a positive role in abiotic stress tolerance by promoting osmotic adjustment, ROS detoxification and photosynthesis efficiency. Thus, PtrA/NINV has great potential to be used in transgenic breeding for improvement of stress tolerance.

Summary: Ethylene‐responsive factors (ERFs) have been revealed to play essential roles in a variety of physiological and biological processes in higher plants. However, functions and regulatory pathways of most ERFs in cold stress remain largely unclear. Here, we identified PtrERF109 of trifoliate orange (Poncirus trifoliata (L.) Raf.) and deciphered its role in cold tolerance. PtrERF109 was drastically up‐regulated by cold, ethylene and dehydration, but repressed by salt. PtrERF109 was localized in the nucleus and displayed transcriptional activity, and the C terminus is required for the activation. Overexpression of PtrERF109 conferred enhanced cold tolerance in transgenic tobacco and lemon plants, whereas VIGS (virus‐induced gene silencing)‐mediated suppression of PtrERF109 in trifoliate orange led to increased cold susceptibility. PtrERF109 overexpression caused extensive transcriptional reprogramming of several suites of stress‐responsive genes. Prx1 encoding class III peroxidase (POD) was one of the antioxidant genes exhibiting the greatest induction. PtrERF109 was shown to directly bind to the promoter of PtrPrx1 (trifoliate orange Prx1 homologue) and positively activated its expression. In addition, the PtrERF109‐overexpressing plants exhibited significantly higher POD activity and accumulated dramatically less H₂O₂ and were more tolerant to oxidative stress, whereas the VIGS plants exhibited opposite trends, in comparison with wild type. Taken together, these results indicate that PtrERF109 as a positive regulator contributes to imparting cold tolerance by, at least partly, directly regulating the POD‐encoding gene to maintain a robust antioxidant capacity for effectively scavenging the reactive oxygen species. Our findings gain insight into better understanding of transcriptional regulation of antioxidant genes in response to cold stress.

Citrus plants routinely experience a broad range of biotic and abiotic stress that occur either concurrently or sequentially in the field, causing huge losses in yield. Hence, cultivated citrus plants that tolerate only one type of abiotic stress are insufficient to maintain citrus productivity as the climate changes. Plants primarily develop delicate regulatory mechanisms to detect, transduce and respond to adverse environmental conditions. Therefore, elucidating the physiological, biochemical and molecular mechanisms underlying the dynamic response to multiple abiotic stresses is a prerequisite for determining the targets for genetic improvement programs of citrus. In this review, we pinpoint the current understanding of the physiological basis of citrus tolerance to abiotic stress. Then, we discuss recent insights into the integrated molecular mechanisms that are involved in the citrus response to multiple types of abiotic stress. Finally, we discuss recent prospects for using modern molecular technologies to facilitate the genetic improvement of citrus.