Calcium (Ca²⁺) plays an important role in determining plant growth and development because it maintains cell wall and
membrane integrity. Therefore, understanding the role of Ca2+ in carbon and lipid metabolism could provide insights
into the dynamic changes in cell membranes and cell walls during the rapid elongation of cotton fibers. In the present
study, we found that the lack of Ca²⁺ promoted fiber elongation and rapid ovule expansion, but it also caused tissue
browning in the ovule culture system. RNA-sequencing revealed that Ca²⁺ deficiency induced cells to be highly oxidized,
and the expression of genes related to carbon metabolism and lipid metabolism was activated significantly. All gene
members of nine key enzymes involved in glycolysis were up-regulated, and glucose was significantly reduced in Ca²⁺
deficiency-treated tissues. Ca²⁺ deficiency adjusted the flowing of glycolysis metabolic. However, low K⁺ recovered
the expression levels of glycolysis genes and glucose content caused by Ca2+ deficiency. Electrospray ionizationtandem
mass spectrometry technology was applied to uncover the dynamic profile of lipidome under Ca²⁺ and K⁺
interacted conditions. Ca²⁺ deficiency led to the decrease of fatty acid (FA), diacylglycerol (DAG), glycolipid and the
significant increase of triacylglycerol (TAG), phospholipid phosphatidylethanolamine (PE), phosphatidylglycerol (PG),
and PC (phosphatidylcholine). Low K⁺ restored the contents of FA, phospholipids, and glycolipids, effectively relieved
the symptoms caused by Ca²⁺ deficiency, and recovered the development of fiber cells. This study revealed dynamic
changes in transcript and metabolic levels and uncovered the signaling interaction of Ca²⁺ deficiency and low K⁺ in
glycolysis and lipid metabolism during fiber development.