Intracellular Na + /H + (NHX) antiporters have important roles in cellular pH and Na + , K + homeostasis. The six Arabidopsis thaliana intracellular NHX members are divided into two groups, endosomal (NHX5 and NHX6) and vacuolar (NHX1 to NHX4). Of the vacuolar members, NHX1 has been characterized functionally, but the remaining members have largely unknown roles. Using reverse genetics, we show that, unlike the single knockouts nhx1 or nhx2, the double knockout nhx1 nhx2 had significantly reduced growth, smaller cells, shorter hypocotyls in etiolated seedlings and abnormal stamens in mature flowers. Filaments of nhx1 nhx2 did not elongate and lacked the ability to dehisce and release pollen, resulting in a near lack of silique formation. Pollen viability and germination was not affected. Quantification of vacuolar pH and intravacuolar K + concentrations indicated that nhx1 nhx2 vacuoles were more acidic and accumulated only 30% of the wild-type K + concentration, highlighting the roles of NHX1 and NHX2 in mediating vacuolar K + /H + exchange. Growth under added Na + , but not K + , partly rescued the flower and growth phenotypes. Our results demonstrate the roles of NHX1 and NHX2 in regulating intravacuolar K + and pH, which are essential to cell expansion and flower development.

Recent evidence highlights novel roles for intracellular Na(+)/H(+) antiporters (NHXs) in plants. The availability of knockouts and overexpressors of specific NHX isoforms has provided compelling genetic evidence to support earlier physiological and biochemical data which suggested the involvement of NHX antiporters in ion and pH regulation. Most plants sequenced to date contain multiple NHX members and, based on their sequence identity and localization, can be grouped into three distinct functional classes: plasma membrane, vacuolar, and endosomal associated. Orthologues of each functional class are represented in all sequenced plant genomes, suggesting conserved and fundamental roles across taxa. In this review we seek to highlight recent findings which demonstrate that intracellular NHX antiporters (i.e. vacuolar and endosomal isoforms) play roles in growth and development, including cell expansion, cell volume regulation, ion homeostasis, osmotic adjustment, pH regulation, vesicular trafficking, protein processing, cellular stress responses, as well as flowering. A significant new discovery demonstrated that in addition to the better known vacuolar NHX isoforms, plants also contain endosomal NHX isoforms that regulate protein processing and trafficking of cellular cargo. We draw parallels from close orthologues in yeast and mammals and discuss distinctive NHX functions in plants.

Carotenoids perform many critical functions in plants, animals, and humans. It is therefore important to understand carotenoid biosynthesis and its regulation in plants. Phytoene synthase (PSY) catalyzes the first committed and rate-limiting step in carotenoid biosynthesis. While PSY is present as a single copy gene in Arabidopsis, duplicated PSY genes have been identified in many economically important monocot and dicot crops. CmPSY1 was previously identified from melon (Cucumis melo L.), but was not functionally characterized. We isolated a second PSY gene, CmPSY2, from melon in this work. CmPSY2 possesses a unique intron/exon structure that has not been observed in other plant PSYs. Both CmPSY1 and CmPSY2 are functional in vitro, but exhibit distinct expression patterns in different melon tissues and during fruit development, suggesting differential regulation of the duplicated melon PSY genes. In vitro chloroplast import assays verified the plastidic localization of CmPSY1 and CmPSY2 despite the lack of an obvious plastid target peptide in CmPSY2. Promoter motif analysis of the duplicated melon and tomato PSY genes and the Arabidopsis PSY revealed distinctive cis-regulatory structures of melon PSYs and identified gibberellin-responsive motifs in all PSYs except for SlPSY1, which has not been reported previously. Overall, these data provide new insights into the evolutionary history of plant PSY genes and the regulation of PSY expression by developmental and environmental signals that may involve different regulatory networks.

Tissue-resident macrophages (ResMf) play important roles in the normal development and physiological functions as well as tissue repair and immune/inflammatory response to both internal and external insults. In cornea, ResMf are critical to the homeostasis and maintenance, wound healing, ocular immune privilege, and immune/inflammatory response to injury and microbial infection. However, the roles of microRNAs in corneal ResMf are utterly unknown. Previously, we demonstrated that the conserved miR-183/ 96/182 cluster (miR-183/96/182) plays important roles in sensory neurons and subgroups of both innate and adaptive immune cells and modulates corneal response to bacterial infection. In this study, we provide direct evidence that the mouse corneal ResMf constitutively produce both IL-17f and IL-10. This function is regulated by miR-183/96/182 through targeting Runx1 and Maf, key transcriptional regulators for IL-17f and IL-10 expression, respectively. In addition, we show that miR-183/96/182 has a negative feedback regulation on the TLR4 pathway in mouse corneal ResMf. Furthermore, miR-183/96/182 regulates the number of corneal ResMf. Inactivation of miR-183/96/182 in mouse results in more steady-state corneal resident immune cells, including ResMf, and leads to a simultaneous early upregulation of innate IL-17f and IL-10 production in the cornea after Pseudomonas aeruginosa infection. Its multiplex regulations on the simultaneous production of IL-17f and IL-10, TLR4 signaling pathway and the number of corneal ResMf place miR-183/96/182 in the center of corneal innate immunity, which is key to the homeostasis of the cornea, ocular immune privilege, and the corneal response to microbial infections. ImmunoHorizons, 2020, 4: 729-744.

A comprehensive understanding of the lipid and fatty acid metabolic machinery is needed for optimizing production of oils and fatty acids for fuel, industrial feedstocks and nutritional improvement in plants. T-DNA mutants in the poorly annotated Arabidopsis thaliana gene At1g08640 were identified as containing moderately high levels (50–100%) of 16∶1Δ7 and 18∶1Δ9 leaf fatty acids and subtle decreases (5–30%) of 16∶3 and 18∶3 (http://www.plastid.msu.edu/). TLC separation of fatty acids in the leaf polar lipids revealed that the chloroplastic galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were the main lipid types affected by this mutation. Analysis of the inferred amino acid sequence of At1g08640 predicted the presence of a transit peptide, three transmembrane domains and an N-terminal J-like domain, and the gene was named CJD1 for Chloroplast J-like Domain 1. GFP reporter experiments and in vitro chloroplast import assays demonstrated CJD1 is a chloroplast membrane protein. Screening of an Arabidopsis cDNA library by yeast-2-hybrid (Y2H) using the J-like domain of CJD1 as bait identified a plastidial inner envelope protein (Accumulation and Replication of Chloroplasts 6, ARC6) as the primary interacting partner in the Y2H assay. ARC6 plays a central role in chloroplast division and binds CJD1 via its own J-like domain along with an adjacent conserved region whose function is not fully known. These results provide a starting point for future investigations of how mutations in CJD1 affect lipid composition.