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.

SAUR proteins and PP2C.D phosphatases regulate H+-ATPases and K+ channels to control stomatal movements.

Activation of plasma membrane (PM) H+-ATPase activity is crucial in guard cells to promote light-stimulated stomatal opening, and in growing organs to promote cell expansion. In growing organs, SMALL AUXIN UP RNA (SAUR) proteins inhibit the PP2C.D2, PP2C.D5, and PP2C.D6 (PP2C.D2/5/6) phosphatases, thereby preventing dephosphorylation of the penultimate phosphothreonine of PM H+-ATPases and trapping them in the activated state to promote cell expansion. To elucidate whether SAUR-PP2C.D regulatory modules also affect reversible cell expansion, we examined stomatal apertures and conductances of Arabidopsis thaliana plants with altered SAUR or PP2C.D activity. Here, we report that the pp2c.d2/5/6 triple knockout mutant plants and plant lines overexpressing SAUR fusion proteins exhibit enhanced stomatal apertures and conductances. Reciprocally, saur56 saur60 double mutants, lacking two SAUR genes normally expressed in guard cells, displayed reduced apertures and conductances, as did plants overexpressing PP2C.D5. Although altered PM H+-ATPase activity contributes to these stomatal phenotypes, voltage clamp analysis showed significant changes also in K+ channel gating in lines with altered SAUR and PP2C.D function. Together, our findings demonstrate that SAUR and PP2C.D proteins act antagonistically to facilitate stomatal movements through a concerted targeting of both ATP-dependent H+ pumping and channel-mediated K+ transport.

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

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. PP2C.D genes are broadly expressed and are crucial for diverse plant growth and developmental processes, including apical hook development, phototropism, and organ growth. GFP-SAUR19 overexpression suppresses the growth defects conferred by PP2C.D5 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 PP2C.D 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.

Mutation of a Conserved Motif of PP2C.D Phosphatases Confers SAUR Immunity and Constitutive Activity.

The phytohormone auxin promotes the growth of plant shoots by stimulating cell expansion via plasma membrane (PM) H+-ATPase activation, which facilitates cell wall loosening and solute uptake. Mechanistic insight was recently obtained by demonstrating that auxin-induced SMALL AUXIN UP RNA (SAUR) proteins inhibit D-CLADE TYPE 2C PROTEIN PHOSPHATASE (PP2C.D) activity, thereby trapping PM H+-ATPases in the phosphorylated, activated state, but how SAURs bind PP2C.D proteins and inhibit their activity is unknown. Here, we identified a highly conserved motif near the C-terminal region of the PP2C.D catalytic domain that is required for SAUR binding in Arabidopsis (Arabidopsis thaliana). Missense mutations in this motif abolished SAUR binding but had no apparent effect on catalytic activity. Consequently, mutant PP2C.D proteins that could not bind SAURs exhibited constitutive activity, as they were immune to SAUR inhibition. In planta expression of SAUR-immune pp2c.d2 or pp2c.d5 derivatives conferred severe cell expansion defects and corresponding constitutively low levels of PM H+-ATPase phosphorylation. These growth defects were not alleviated by either auxin treatment or 35S:StrepII-SAUR19 coexpression. In contrast, a PM H+-ATPase gain-of-function mutation that results in a constitutively active H+ pump partially suppressed SAUR-immune pp2c.d5 phenotypes, demonstrating that impaired PM H+-ATPase function is largely responsible for the reduced growth of the SAUR-immune pp2c.d5 mutant. Together, these findings provide crucial genetic support for SAUR-PP2C.D regulation of cell expansion via modulation of PM H+-ATPase activity. Furthermore, SAUR-immune pp2c.d derivatives provide new genetic tools for elucidating SAUR and PP2C.D functions and manipulating plant organ growth.

Basic Proline-Rich Protein-Mediated Microtubules Are Essential for Lobe Growth and Flattened Cell Geometry.

Complex cell shapes are generated first by breaking symmetry, and subsequent polar growth. Localized bending of anticlinal walls initiates lobe formation in the epidermal pavement cells of cotyledons and leaves, but how the microtubule cytoskeleton mediates local cell growth, and how plant pavement cells benefit from adopting jigsaw puzzle-like shapes, are poorly understood. In Arabidopsis (Arabidopsis thaliana), the basic Pro-rich protein (BPP) microtubule-associated protein family comprises seven members. We analyzed lobe morphogenesis in cotyledon pavement cells of a BPP1;BPP2;BPP5 triple knockout mutant. New image analysis methods (MtCurv and BQuant) showed that anticlinal microtubule bundles were significantly reduced and cortical microtubules that fan out radially across the periclinal wall did not enrich at the convex side of developing lobes. Despite these microtubule defects, new lobes were initiated at the same frequency as in wild-type cells, but they did not expand into well-defined protrusions. Eventually, mutant cells formed nearly polygonal shapes and adopted concentric microtubule patterns. The mutant periclinal cell wall bulged outward. The radius of the calculated inscribed circle of the pavement cells, a proposed proxy for maximal stress in the cell wall, was consistently larger in the mutant cells during cotyledon development, and correlated with an increase in cell height. These bpp mutant phenotypes provide genetic and cell biological evidence that initiation and growth of lobes are distinct morphogenetic processes, and that interdigitated cell geometry effectively suppresses large outward bulging of pavement cells.

Novel Arabidopsis microtubule-associated proteins track growing microtubule plus ends.

BACKGROUND: Microtubules (MTs) are polarized polymers with highly dynamic plus ends that stochastically switch between growth and shrinkage phases. In eukaryotic cells, a plethora of MT-associated proteins (MAPs) regulate the dynamics and higher-order organization of MTs to mediate distinct cellular functions. Plus-end tracking proteins (+TIPs) are a group of MAPs that specifically accumulate at the growing MT plus ends, where they modulate the behavior of the MT plus ends and mediate interactions with cellular targets. Although several functionally important + TIP proteins have been characterized in yeast and animals, little is known about this group of proteins in plants. RESULTS: We report here that two homologous MAPs from Arabidopsis thaliana, Growing Plus-end Tracking 1 (GPT1) and GPT2 (henceforth GPT1/2), contain basic MT-binding regions at their central and C-terminal regions, and bind directly to MTs in vitro. Interestingly, GPT1/2 preferentially accumulated at the growing plus ends of cortical MTs in interphase Arabidopsis cells. When the GPT1/12-decorated growing plus ends switched to rapid depolymerization, GPT1/2 dissociated from the MT plus ends. Conversely, when the depolymerizing ends were rescued and started to polymerize again, GPT1/2 were immediately recruited to the growing MT tips. This tip tracking behavior of GPT proteins does not depend on the two established plant + TIPs, End-Binding protein 1 (EB1) and SPIRAL1 (SPR1). CONCLUSIONS: The Arabidopsis MAPs GPT1 and GPT2 bind MTs directly through their basic regions. These MAPs track the plus ends of growing MTs independently of EB1 and SPR1 and represent a novel plant-specific + TIP family.

The PAL2 promoter activities in relation to structural development and adaptation in Arabidopsis thaliana.

Phenylalanine ammonia lyase (PAL) plays a major role in plant growth, development and adaptation. In Arabidopsis thaliana, the enzyme is encoded by four genes, namely PAL1, PAL2, PAL3, and PAL4 with PAL1 and PAL2 being closely related phylogenetically and functionally. PAL1 promoter activities are associated with plant development and are inducible by various stress agents. However, PAL2 promoter activities have not been functionally analysed. Here, we show that the PAL2 promoter activities are associated with the structural development of a plant and its organs. This function was inducible in an organ-specific manner by the avirulent strain of Pseudomonas syringae pv. tomato (JL1065). The PAL2 promoter was active throughout the course of the plant development particularly in the root, rosette leaf, and inflorescence stem that provide the plant with structural support. In aerial organs, the levels of PAL2 promoter activities were negatively correlated with relative positions of the organs to the rosette leaves. The promoter was inducible in the root following an inoculation by JL1065 in the leaf suggesting PAL2 to be part of an induced defence system. Our results demonstrate how the PAL2 promoter activities are being coordinated and synchronised for the structural development of the plant and its organs based on the developmental programme. Under certain stress conditions the activity may be induced in favour of certain organs.

Biphasic control of cell expansion by auxin coordinates etiolated seedling development.

Seedling emergence is critical for food security. It requires rapid hypocotyl elongation and apical hook formation, both of which are mediated by regulated cell expansion. How these events are coordinated in etiolated seedlings is unclear. Here, we show that biphasic control of cell expansion by the phytohormone auxin underlies this process. Shortly after germination, high auxin levels restrain elongation. This provides a temporal window for apical hook formation, involving a gravity-induced auxin maximum on the eventual concave side of the hook. This auxin maximum induces PP2C.D1 expression, leading to asymmetrical H+-ATPase activity across the hypocotyl that contributes to the differential cell elongation underlying hook development. Subsequently, auxin concentrations decline acropetally and switch from restraining to promoting elongation, thereby driving hypocotyl elongation. Our findings demonstrate how differential auxin concentrations throughout the hypocotyl coordinate etiolated development, leading to successful soil emergence.