Hylocereus polyrhizus, also known as pitaya or dragon fruit, is a climbing cactus grown worldwide because of its excellent performance under drought stress and appealing red-purple fruits. In practice, accelerating flower formation and inducing more flowers usually result in higher yield. However, the genes for this purpose have not been well characterized in pitaya. Previously, FLOWERING BHLHs (FBHs) have been identified as positive regulators of flower formation. In the present work, a total of eight FBHs were identified in pitaya. This is a greater number than in beet and spinach, possibly because of the recent whole-genome duplication that occurred in the pitaya genome. The phylogenetic tree indicated that the FBHs could be divided into three groups. In TYPE II, the genes of Caryophyllales encode atypical FBHs and are generated by dispersed duplication. The K_a/K_s ratios indicated that HpFBHs are under purifying selection. Promoter and expression analysis of HpFBHs revealed that they are spatiotemporally activated in flower-related tissues and responsive to multiple abiotic stresses. These results indicated that HpFBHs are involved in the flower formation of pitaya. Therefore, typical HpFBH1/3 from TYPE III and an atypical HpFBH8 from TYPE II were selected for functional verification. HpFBH3 was found to heterodimerize with HpFBH1 in the nucleus using subcellular localization, yeast two-hybrid and luciferase complementation assays. With bioinformatic analysis, all HpFBHs were predicted to transactivate downstream genes via binding to the E-boxes, which were frequently detected in the promoters of HpCOs, HpFTs and HpSOC1s. RNA-Seq datasets showed that these flowering accelerators were expressed in coordination with HpFBH3. Yeast one-hybrid and dual-luciferase reporter assays further verified that HpFBH3 transactivated HpCO7 by selectively binding to the E-boxes in the promoter. Moreover, ectopic overexpression of HpFBH3 accelerated flower formation in Arabidopsis. In summary, this study systematically characterized the typical HpFBHs, especially HpFBH3, as positive regulators of flower formation, which could be target genes for the genetic improvement of pitaya.

Phosphorus (Pi) plays a crucial role in the growth and development of plants. Membrane lipid regulation is one of the main mechanisms underlying plant adaptation to Pi deficiency. Previously, the high tolerance to low-Pi stress was justified in an elite line, MSDZ 109, which was obtained from Malus mandshurica. To better understand the mechanism underlying high adaptation to low-Pi stress, currently, lipidomic and transcriptomic analysis, as well as CRISPR/Cas9 and MmGDPD1-overexpressing methodologies were comprehensively integrated into a strategy for elucidating the high tolerance to low-Pi stress. Totally, 770 differential metabolites were identified from the roots between the low-Pi and stress-free, belonging to 21 sub-classes of lipid compounds. Fatty acids (FA) constituted the predominant lipid component, accounting for approximately 50%–60% of the total lipids, and triglycerides (TAG) ranked the second, comprising around 12% of the total, consecutively followed by phosphatidylcholine (PC) and diacylglycerol (DAG) with approximately 10% and 8% of the total, respectively. The synchronous transcriptomic analysis revealed a significant up-regulation of genes related to glycerophospholipid and glycerolipid metabolism, specifically those (e.g., MmGDPD1, MmDGDG1, MmMGDG1, MmSQDG, etc.) involved in phospholipid and galactosyl synthesis in response to low-Pi stress. GUS fusing reporter assay showed that MmGDPD1 promoter induced GUS gene expression and demonstrated initiation activity. Based on expression analysis, a dual-luciferase reporter assay, as well as yeast one-hybrid (Y1H) identification, MmPHR1 was justified to bind with the MmGDPD1 promoter and positively regulate plant tolerance to low-Pi stress. To further elucidate the role of MmGDPD1, CRISPR/Cas9 and MmGDPD1-overexpressing vectors were successfully introduced into apple (‘Royal Gala’) calli. Interestingly, the MmGDPD1-KO line calli exhibited the remarkable decreases in the contents of phosphodiesterase (PDE), activity, as well as the contents of total Pi, and Pi in comparison with those of the wild type. Conversely, MmGDPD1-OE ones demonstrated the significant elevation in Pi accumulations, further justifying its potential role in Pi remobilization in apple. Therefore, MmGDPD1 substantially involves elevating low-Pi tolerance via promoting Pi release in M. mandshurica.