Knock-Down of Arabidopsis PLC5 Reduces Primary Root Growth and Secondary Root Formation While Overexpression Improves Drought Tolerance and Causes Stunted Root Hair Growth

Abstract

Phospholipase C (PLC) is a well-known signaling enzyme in metazoans that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate and diacylglycerol as second messengers involved in mutiple processes. Plants contain PLC too, but relatively little is known about its function there. The model system Arabidopsis thaliana contains nine PLC genes. Reversed genetics have implicated several roles for PLCs in plant development and stress signaling. Here, PLC5 is functionally addressed. Promoter-β-glucuronidase (GUS) analyses revealed expression in roots, leaves and flowers, predominantly in vascular tissue, most probably phloem companion cells, but also in guard cells, trichomes and root apical meristem. Only one plc5-1 knock-down mutant was obtained, which developed normally but grew more slowly and exhibited reduced primary root growth and decreased lateral root numbers. These phenotypes could be complemented by expressing the wild-type gene behind its own promoter. Overexpression of PLC5 (PLC5-OE) using the UBQ10 promoter resulted in reduced primary and secondary root growth, stunted root hairs, decreased stomatal aperture and improved drought tolerance. PLC5-OE lines exhibited strongly reduced phosphatidylinositol 4-monophosphate (PIP) and PIP2 levels and increased amounts of phosphatidic acid, indicating enhanced PLC activity in vivo. Reduced PIP2 levels and stunted root hair growth of PLC5-OE seedlings could be recovered by inducible overexpression of a root hair-specific PIP 5-kinase, PIP5K3. Our results show that PLC5 is involved in primary and secondary root growth and that its overexpression improves drought tolerance. Independently, we provide new evidence that PIP2 is essential for the polar tip growth of root hairs.

Role for Arabidopsis PLC7 in Stomatal Movement, Seed Mucilage Attachment, and Leaf Serration

“…For plc7-4 identification, forward primer 5′-TCCTTCCTGTTATCCATGACG-3′; reverse primer 5′-TTGAAGAAAGCATCAAGGTGG-3′) and left border primer LBb1.3 (in combination with the reverse primer) were used. To generate a plc5/7- double mutant, plc7-3 was crossed with plc5-1 (SALK_144469), a T-DNA insertion KD mutant that was functionally complemented ( Zhang et al, 2018b ).…”

“…GUS staining was performed as described previously ( Zhang et al, 2018a , b ). Briefly, transgenic plants carrying pPLC7::GUS - SYFP were grown for indicated times, after which specific tissues were taken and incubated in an X-Gluc reaction solution containing 1 mg/ml 5-bromo-4-chloro-3 indolyl-β-D-glucuronic acid (X-gluc), 50 mM phosphate buffer (pH 7.0), and 0.1% TX-100.…”

“…Drought assays were performed as described ( Zhang et al, 2018a , b ). In brief, seeds were stratified at 4°C in the dark and sowed in pots (4.5 cm × 4.5 cm × 7.5 cm) containing equal amounts (80 g) of soil.…”

“…In petunia and tobacco, PLC has been shown to regulate the tip growth of pollen tubes ( Dowd et al, 2006 ; Helling et al, 2006 ). Arabidopsis T-DNA insertion mutants for PLC3 , PLC5 and PLC9 have revealed roles for PLC in seed germination, primary- and lateral root development, ABA signaling and heat stress tolerance ( Zheng et al, 2012 ; Gao et al, 2014 ; Zhang et al, 2018a , b ), while PLC2 -insertion mutants showed defects in female gametogenesis and embryo development ( Li et al, 2015 ; Di Fino et al, 2017 ). Overexpression of PLC has been shown to increase the drought tolerance of maize, canola, tobacco and Arabidopsis ( Wang et al, 2008 ; Georges et al, 2009 ; Tripathy et al, 2011 ; Zhang et al, 2018a , b ).…”

“…The Arabidopsis genome encodes 9 PLC genes, which are subdivided into four clades ( Hunt et al, 2004 ; Tasma et al, 2008 ; Munnik, 2014 ; Pokotylo et al, 2014 ). Earlier, we found that knock-out (KO) mutants of PLC3 and a knock-down (KD) mutant of PLC5 , exhibited small defects in root development ( Zhang et al, 2018a , b ). Interestingly, plc3plc5- double mutants did not intensify the phenotype, even though they belong to different clades, indicating that another PLC could be involved.…”