Excess Pyrophosphate Restrains Pavement Cell Morphogenesis and Alters Organ Flatness in .

In , the vacuolar proton-pumping pyrophosphatase (H-PPase) is highly expressed in young tissues, which consume large amounts of energy in the form of nucleoside triphosphates and produce pyrophosphate (PPi) as a byproduct. We reported that excess PPi in the H-PPase loss-of-function mutant severely compromised gluconeogenesis from seed storage lipids, arrested cell division in cotyledonary palisade tissue, and triggered compensated cell enlargement; this phenotype was recovered upon sucrose supply. Thus, we provided evidence that the hydrolysis of inhibitory PPi, rather than vacuolar acidification, is the major contribution of H-PPase during seedling establishment. Here, examination of the epidermis revealed that pavement cells exhibited defective puzzle-cell formation. Importantly, removal of PPi from background by the yeast cytosolic PPase IPP1, in transgenic lines, restored the phenotypic aberrations of pavement cells. Surprisingly, pavement cells in mutants with defects in gluconeogenesis () or the glyoxylate cycle (; ) showed no phenotypic alteration, indicating that reduced sucrose production from seed storage lipids is not the cause of epidermal phenotype. had oblong cotyledons similar to those of (), whose leaf pavement cells display an abnormal arrangement of cortical microtubules (MTs). To gain insight into the genetic interaction between ANGUSTIFOLIA and H-PPase in pavement cell differentiation, was analyzed. Surprisingly, epidermis developmental defects were synergistically enhanced in the double mutant. In fact, pavement cells showed a striking three-dimensional growth phenotype on both abaxial and adaxial sides of cotyledons, which was recovered by hydrolysis of PPi in . Live imaging revealed that cortical MTs exhibited a reduced velocity, were slightly fragmented and sparse in the above lines compared to the WT. Consistently, addition of PPi led to a dose-dependent delay of tubulin polymerization, thus supporting a link between PPi and MT dynamics. Moreover, mathematical simulation of three-dimensional growth based on cotyledon proximo-distal and medio-lateral phenotypic quantification implicated restricted cotyledon expansion along the medio-lateral axis in the crinkled surface of . Together, our data suggest that PPi homeostasis is a prerequisite for proper pavement cell morphogenesis, epidermal growth and development, and organ flattening.