SAUR63 stimulates cell growth at the plasma membrane

In plants, regulated cell expansion determines organ size and shape. Several members of the family of redundantly acting Small Auxin Up RNA (SAUR) proteins can stimulate plasma membrane (PM) H+-ATPase proton pumping activity by inhibiting PM-associated PP2C.D phosphatases, thereby increasing the PM electrochemical potential, acidifying the apoplast, and stimulating cell expansion. Similarly, Arabidopsis thaliana SAUR63 was able to increase growth of various organs, antagonize PP2C.D5 phosphatase, and increase H+-ATPase activity. Using a gain-of-function approach to bypass genetic redundancy, we dissected structural requirements for SAUR63 growth-promoting activity. The divergent N-terminal domain of SAUR63 has a predicted basic amphipathic α-helix and was able to drive partial PM association. Deletion of the N-terminal domain decreased PM association of a SAUR63 fusion protein, as well as decreasing protein level and eliminating growth-promoting activity. Conversely, forced PM association restored ability to promote H+-ATPase activity and cell expansion, indicating that SAUR63 is active when PM-associated. Lipid binding assays and perturbations of PM lipid composition indicate that the N-terminal domain can interact with PM anionic lipids. Mutations in the conserved SAUR domain also reduced PM association in root cells. Thus, both the N-terminal domain and the SAUR domain may cooperatively mediate the SAUR63 PM association required to promote growth

アラビドプシス ノ ビショウカン フズイ タンパクシツ ノ カイセキ

博第1367号甲第1367号甲第1367号博士(バイオサイエンス)奈良先端科学技術大学院大

Functional Analysis of PAL2 Gene Promoter in Arabidopsis Thaliana (L.) Heynh. During Plant Development Exposed to Biotic and Abiotic Stresses

Phenylalanine ammonia-lyase (PAL; E.C.4.3.1.5) enzyme is essential for plant normal growth, development and adaptation to different environmental stresses. In Arabidopsis thaliana, the PAL enzyme is encoded by four gene isoforms which are designated as PAL1 (AT2G37040), PAL2 (AT3G53260), PAL3 (AT5G04230), and PAL4 (AT3G10340) respectively. PAL1 and PAL2 genes are closely related to each other phylogenetically and functionally. PAL1 promoter is involved in plant development and also plant response under induction of a myriad of stresses. However, functional analysis on the PAL2 promoter of A. thaliana has not been carried out. The PAL2 promoter activities were investigated by fusing 1.8-kb 5’ upstream of the translation start site with a β-glucuronidase (GUS) gene in transgenic A. thaliana. The PAL2 promoter activities were associated with the structural development of the plant and its organs and avirulent Pseudomonas syringae pv. tomato JL1065 induced the promoter response in an organ-specific manner. In the context of plant development, the PAL2 promoter was active from the germination of young seedling to the reproductive stage, particularly in the rosette leaf, root, and inflorescence stem which are the major structural organs supporting the floral organs particularly bud, flower, and silique. The rosette leaf, root and stem are considered as the major structural organs as they provide the mechanical strength to support vertical position of the whole plant. The PAL2 promoter activities in both rosette leaf and root were roughly 3-fold, and stem 2-fold higher than the floral organs and silique. The PAL2 promoter activities displayed decreasing trend in the aerial organs with position further from the rosette leaves. In the context of plant adaptation, PAL2 promoter activities was induced in the distal root with roughly 2-fold increase after 4-day post-inoculation with avirulent JL1065 in the aerial organs, suggesting PAL2 promoter was involved in induced defence system. During moderate water deficit stress mediated by sodium chloride and polyethylene glycol solution for short-term period, PAL2 promoter activities were not significantly induced for water stress-responsiveness. These findings imply that PAL2 promoter maybe regulated transcriptionally for the normal plant structural development, and plant adaptation to avirulent Pseudomonas infection

A subset of plasma membrane-localized PP2C.D phosphatases negatively regulate SAUR-mediated cell expansion in Arabidopsis

<div><p>The plant hormone auxin regulates numerous growth and developmental processes throughout the plant life cycle. One major function of auxin in plant growth and development is the regulation of cell expansion. Our previous studies have shown that SMALL AUXIN UP RNA (SAUR) proteins promote auxin-induced cell expansion via an acid growth mechanism. These proteins inhibit the PP2C.D family phosphatases to activate plasma membrane (PM) H⁺-ATPases and thereby promote cell expansion. However, the functions of individual PP2C.D phosphatases are poorly understood. Here, we investigated PP2C.D-mediated control of cell expansion and other aspects of plant growth and development. The nine PP2C.D family members exhibit distinct subcellular localization patterns. Our genetic findings demonstrate that the three plasma membrane-localized members, PP2C.D2, PP2C.D5, and PP2C.D6, are the major regulators of cell expansion. These phosphatases physically interact with SAUR19 and PM H⁺-ATPases, and inhibit cell expansion by dephosphorylating the penultimate threonine of PM H⁺-ATPases. <i>PP2C</i>.<i>D</i> genes are broadly expressed and are crucial for diverse plant growth and developmental processes, including apical hook development, phototropism, and organ growth. <i>GFP-SAUR19</i> overexpression suppresses the growth defects conferred by <i>PP2C</i>.<i>D5</i> overexpression, indicating that SAUR proteins antagonize the growth inhibition conferred by the plasma membrane-localized PP2C.D phosphatases. Auxin and high temperature upregulate the expression of some <i>PP2C</i>.<i>D</i> family members, which may provide an additional layer of regulation to prevent plant overgrowth. Our findings provide novel insights into auxin-induced cell expansion, and provide crucial loss-of-function genetic support for SAUR-PP2C.D regulatory modules controlling key aspects of plant growth.</p></div

Auxin induces <i>PP2C</i>.<i>D</i> gene expression, and high temperature upregulates PP2C.D2 protein levels.

<p>(A) GUS-stained shoots of 5-day-old light-grown seedlings treated with 10 μM IAA for 4 h. Scale bars = 1 mm. (B) GUS-stained roots of 5-day-old light-grown seedlings treated with 10 μM IAA for 4 h. Scale bars = 1 mm. (C) qRT-PCR analyses of <i>SAUR</i> and <i>PP2C</i>.<i>D</i> gene expression. RNA was prepared from 3-day-old light-grown seedlings treated with 5 μM NAA or solvent control for various times. Relative expression represents expression value of NAA/expression value of mock. qRT-PCR results were based on three biological replicates. <i>S19</i>, <i>SAUR19</i>; <i>S23</i>, <i>SAUR23</i>; <i>S9</i>, <i>SAUR9</i>. Error bars = SD. (D) GUS-stained shoots of light-grown seedlings. Two or four-day-old <i>PP2C</i>.<i>D2-GUS</i> seedlings grown at 20 ^oC were shifted to 28 ^oC for 6 h, 24 h, or 5 d. GUS staining was performed at 37 ^oC for 2 h (6 h, 24 h) or 1 h (5 d). (E) Western blot analysis of PP2C.D2-GFP protein expression. Two-day-old <i>PP2C</i>.<i>D2-GFP</i> seedlings grown at 20 ^oC were shifted to 28 ^oC for 5 days. 25 micrograms of total proteins from shoots were loaded for western blot analyses using anti-GFP and anti-SEC12 antibodies. (F) qRT-PCR analyses of <i>SAUR</i> and <i>PP2C</i>.<i>D</i> gene expression. RNA was prepared from light-grown seedlings that were grown at 20 ^oC and shifted to 28 ^oC for various times. Relative expression represents expression value of 28 ^oC/expression value of 20 ^oC. qRT-PCR results were based on three biological replicates. Error bars = SD. (G) Relative hypocotyl length of 7-day-old light-grown seedlings. Relative hypocotyl length represents length value of 28 ^oC/length value of 20 ^oC. Error bars = SEM (n ≥ 22).</p

Expression patterns of <i>PP2C</i>.<i>D-GUS</i> reporters.

<p><b>(</b>A) Shoots of 5-day-old light-grown seedlings. (B) Roots of 5-day-old light-grown seedlings. (C) Shoots of 3-day-old etiolated seedlings. (D) Apical hook of a 2-day-old etiolated seedling. GUS staining was performed at 37 ^oC for 24 (A-C) or 4 h (D). Scale bars = 1 mm (A-C) or 0.5 mm (D).</p

PP2C.D phosphatases interact with SAUR19 and plasma membrane H⁺-ATPases.

<p><b>(</b>A) Yeast two-hybrid assay demonstrating PP2C.D2 and SAUR19 protein interaction. Cells were plated onto appropriate selection media and grown at room temperature for 3 to 6 days. (B) Co-IP assays detecting PP2C.D5-HA and GFP-SAUR19 protein interaction. Microsomal proteins were prepared from 6-day-old etiolated seedlings. Pre-immune bleed serum (pre) or anti-GFP antibody were used for the co-IP assays. PP2C.D5-HA and GFP-SAUR19 were detected by anti-HA and anti-GFP antibodies, respectively. (C) Co-IP assays detecting PP2C.D and PM H⁺-ATPase protein interactions. Microsomal proteins were prepared from 8-day-old light-grown seedlings. Pre-immune bleed serum (pre) or anti-AHA antibody were used for the co-IP assays. PP2C.D2-GFP, PP2C.D5-GFP, PP2C.D8-GFP, and PP2C.D6-HA proteins were detected by anti-GFP and anti-HA antibodies, respectively. (D) PP2C.D expression abolishes AHA2 complementation of PM H⁺-ATPase activity in yeast. Gal, galactose; Glu, glucose; Vec, empty vector. (E). <i>In vitro</i> AHA2 dephosphorylation assays examining the dephosphorylation of AHA2-Thr^947P expressed in yeast. (B and C) 300–400 μg and 10–20 μg of microsomal proteins were used for co-IP and western blots, respectively.</p

Differential localization of PP2C.D-GFP fusion proteins.

<p>(A) Localization of PP2C.D-GFP fusion proteins in the root tips of 5-day-old seedlings. <i>PP2C</i>.<i>D1-GFP</i> seedlings were treated with 10 μM IAA for 4 h to increase expression to detectable levels. (B) Localization of PP2C.D1-GFP fusion protein in the apical hooks of 2-day-old etiolated seedlings counter-stained with 10 μg/ml propidium iodide (PI) for 30 min. (C) Localization of PP2C.D8-GFP fusion protein in the root tips of 5-day-old seedlings. Seedlings were counter-stained with 0.5 μM MitoTracker Red CMXRos (Invitrogen) for 20 min. (A—C) Root tips and apical hooks were observed under a Nikon A1 spectral confocal microscope. Scale bars = 50 μm (A), 25 μm (B), or 10 μm (C).</p