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.

Intracellular Na + /H + antiporters (NHXs) play important roles in cellular pH and Na + and K + homeostasis in all eukaryotes. Based on sequence similarity, the six intracellular Arabidopsis thaliana members are divided into two groups. Unlike the vacuolar NHX1-4, NHX5 and NHX6 are believed to be endosomal; however, little data exist to support either their function or localization. Using reverse genetics, we show that whereas single knockouts nhx5 or nhx6 did not differ from the wild type, the double knockout nhx5 nhx6 showed reduced growth, with smaller and fewer cells and increased sensitivity to salinity. Reduced growth of nhx5 nhx6 was due to slowed cell expansion. Transcriptome analysis indicated that nhx5, nhx6, and the wild type had similar gene expression profiles, whereas transcripts related to vesicular trafficking and abiotic stress were enriched in nhx5 nhx6. We show that unlike other intracellular NHX proteins, NHX5 and NHX6 are associated with punctate, motile cytosolic vesicles, sensitive to Brefeldin A, that colocalize to known Golgi and trans-Golgi network markers. We provide data to show that vacuolar trafficking is affected in nhx5 nhx6. Possible involvements of NHX5 and NHX6 in maintaining organelle pH and ion homeostasis with implications in endosomal sorting and cellular stress responses are discussed.

We report the phased genome sequence of an interspecific hybrid, the flowering cherry ‘Somei-Yoshino’ (Cerasus × yedoensis). The sequence data were obtained by single-molecule real-time sequencing technology, split into two subsets based on genome information of the two probable ancestors, and assembled to obtain two haplotype phased genome sequences of the interspecific hybrid. The resultant genome assembly consisting of the two haplotype sequences spanned 690.1 Mb with 4,552 contigs and an N50 length of 1.0 Mb. We predicted 95,076 high-confidence genes, including 94.9% of the core eukaryotic genes. Based on a high-density genetic map, we established a pair of eight pseudomolecule sequences, with highly conserved structures between the two haplotype sequences with 2.4 million sequence variants. A whole genome resequencing analysis of flowering cherries suggested that ‘Somei-Yoshino’ might be derived from a cross between C. spachiana and either C. speciosa or its relatives. A time-course transcriptome analysis of floral buds and flowers suggested comprehensive changes in gene expression in floral bud development towards flowering. These genome and transcriptome data are expected to provide insights into the evolution and cultivation of flowering cherry and the molecular mechanism underlying flowering.

Flowering is an essential stage of fruit production. To understand the molecular mechanisms controlling flowering in maloid fruit tree species, we isolated and analyzed genes homologous to Arabidopsis LEAFY (LFY; flower meristem identity gene) and TERMINAL FLOWER 1 (TFL1; inflorescence meristem identity gene) from six fruit tree species in the subfamily Maloideae of the Rosaceae; apple (Malus · domestica), Japanese pear (Pyrus pyrifolia), European pear (Pyrus communis), quince (Cydonia oblonga), Chinese quince (Chaenomeles sinensis), and loquat (Eriobotrya japonica). Two LFY homologues and two TFL1 homologues were cloned from all six maloid species by rapid amplification of 3¢ and 5¢ cDNA ends, reverse transcription-PCR, and PCR with genomic DNA. Phylogenetic analysis by the neighbor-joining method showed that the two LFY homologues and two TFL1 homologues were classified into two distinct clades. The presence of multiple copies of LFY and TFL1 homologues is discussed with reference to the polyploid origin of the subfamily Maloideae.