The flowering hormone florigen accelerates secondary cell wall biogenesis to harmonize vascular maturation with reproductive development.

Florigen, a proteinaceous hormone, functions as a universal long-range promoter of flowering and concurrently as a generic growth-attenuating hormone across leaf and stem meristems. In flowering plants, the transition from the vegetative phase to the reproductive phase entails the orchestration of new growth coordinates and a global redistribution of resources, signals, and mechanical loads among organs. However, the ultimate cellular processes governing the adaptation of the shoot system to reproduction remain unknown. We hypothesized that if the mechanism for floral induction is universal, then the cellular metabolic mechanisms underlying the conditioning of the shoot system for reproduction would also be universal and may be best regulated by florigen itself. To understand the cellular basis for the vegetative functions of florigen, we explored the radial expansion of tomato stems. RNA-Seq and complementary genetic and histological studies revealed that florigen of endogenous, mobile, or induced origins accelerates the transcription network navigating secondary cell wall biogenesis as a unit, promoting vascular maturation and thereby adapting the shoot system to the developmental needs of the ensuing reproductive phase it had originally set into motion. We then demonstrated that a remarkably stable and broadly distributed florigen promotes MADS and MIF genes, which in turn regulate the rate of vascular maturation and radial expansion of stems irrespective of flowering or florigen level. The dual acceleration of flowering and vascular maturation by florigen provides a paradigm for coordinated regulation of independent global developmental programs.

Alendronate affects calcium dynamics in cardiomyocytes in vitro.

Therapy with bisphosphonates, including alendronate (ALN), is considered a safe and effective treatment for osteoporosis. However, recent studies have reported an unexpected increase in serious atrial fibrillation (AF) in patients treated with bisphosphonates. The mechanism that explains this side effect remains unknown. Since AF is associated with an altered sarcoendoplasmic reticulum calcium load, we studied how ALN affects cardiomyocyte calcium homeostasis and protein isoprenylation in vitro. Acute and long-term (48h) treatment of atrial and ventricular cardiomyocytes with ALN (10(-8)-10(-6)M) was performed. Changes in calcium dynamics were determined by both fluorescence measurement of cytosolic free Ca(2+) concentration and western blot analysis of calcium-regulating proteins. Finally, effect of ALN on protein farnesylation was also identified. In both atrial and ventricular cardiomyocytes, ALN treatment delayed and diminished calcium responses to caffeine. Only in atrial cells, long-term exposure to ALN-induced transitory calcium oscillations and led to the development of oscillatory component in calcium responses to caffeine. Changes in calcium dynamics were accompanied by changes in expression of proteins controlling sarcoendoplasmic reticulum calcium. In contrast, ALN minimally affected protein isoprenylation in these cells. In summary, treatment of atrial cardiomyocytes with ALN-induced abnormalities in calcium dynamics consistent with induction of a self-stimulatory, pacemaker-like behavior, which may contribute to the development of cardiac side effects associated with these drugs.

Egr-1 negatively regulates calsequestrin expression and calcium dynamics in ventricular cells.

AIMS: The transcription factor early growth response-1 (Egr-1) is increased in models of cardiac pathology; however, it is unclear how Egr-1 impacts the heart. We sought to identify how Egr-1 regulates expression of proteins involved in cardiomyocyte calcium homeostasis. METHODS: Protein expression was measured by immunoblotting in control cardiac differentiated H9c2 cells or in H9c2 cells overexpressing wild-type Egr-1 (Egr-1) or an Egr-1 (I293F) mutant. Microspectrofluorimetry of fura-2-loaded cells was used to study calcium dynamics. Chromatin immunoprecipitation with anti-Egr-1 antibody was used to identify Egr-1-associated DNA. RESULTS: Calsequestrin (CSQ) expression was reduced in Egr-1- and profoundly reduced in I293F-expressing cells. Calreticulin, triadin, sarcoendoplasmic reticulum ATPase 2a, phospholamban, and phosphoserine 16-phospholamban expression was unaffected. Calcium release from CSQ-dependent ryanodine-sensitive stores was reduced in Egr-1 and absent in I293F-expressing cells. In contrast, calcium release from calreticulin-dependent inositol 1,4,5-trisphosphate stores was unaffected. In vivo and in vitro chromatin immunoprecipitation demonstrated Egr-1 binding to the CSQ2 promoter. The Egr-1-binding region contains overlapping Egr-1, SP1, and nuclear factor of activated T-cells (NFAT) sites and a CpG island. Reciprocal immunoprecipitation coupled to immunoblots indicated Egr-1:NFAT3 binding was present in all cells lines. Treatment with cyclosporin A, inhibition of DNA methylation using 5-azadeoxycytidine, or inhibition of protein acetylation using sodium butyrate reduced CSQ expression. CONCLUSION: Our data suggest that Egr-1:DNA binding at the promoter, DNA methylation, and protein acetylation are important in CSQ repression. Moreover, we demonstrate that a reduction in CSQ protein is associated with abnormal calcium dynamics. We conclude that Egr-1 acts as a transcriptional repressor at the CSQ promoter, resulting in downregulation of CSQ, the major calcium storage protein that links excitation-contraction coupling in the cardiac sarcoendoplasmic reticulum.