Excess Pyrophosphate Restrains Pavement Cell Morphogenesis and Alters Organ Flatness in Arabidopsis thaliana

摘要

In Arabidopsis thaliana , 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 fugu5 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 fugu5 pavement cells exhibited defective puzzle-cell formation. Importantly, removal of PPi from fugu5 background by the yeast cytosolic PPase IPP1, in fugu5-1 AVP1 _(pro)::IPP1 transgenic lines, restored the phenotypic aberrations of fugu5 pavement cells. Surprisingly, pavement cells in mutants with defects in gluconeogenesis ( pck1-2 ) or the glyoxylate cycle ( icl-2 ; mls-2 ) showed no phenotypic alteration, indicating that reduced sucrose production from seed storage lipids is not the cause of fugu5 epidermal phenotype. fugu5 had oblong cotyledons similar to those of angustifolia-1 ( an-1 ), 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, an-1 fugu5-1 was analyzed. Surprisingly, epidermis developmental defects were synergistically enhanced in the double mutant. In fact, an-1 fugu5-1 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 an-1 fugu5-1 AVP1 _(pro)::IPP1 . 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 in vitro 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 an-1 fugu5-1 . Together, our data suggest that PPi homeostasis is a prerequisite for proper pavement cell morphogenesis, epidermal growth and development, and organ flattening.