Alison DeLong

Reproductive success of angiosperms depends on the ability of pollen to build a highly polarized cellular extension (the pollen tube) that delivers sperm to female gametes for fertilization. This process is highly temperature sensitive, but we lack information about how heat stress affects pollen function and the molecular pathways that impart thermotolerance. Here we show that maintaining cellular integrity during pollen tube germination and extension is the pollen performance parameter that distinguishes thermosensitive from thermotolerant tomato cultivars and that loss-of-function of one of four pollen-expressed Rapid Alkalinization Factor (RALF) signaling peptides can enhance thermotolerance. Moreover, we found that heat stress antagonizes RALF signaling required to maintain patterns of pectin distribution in the pollen tube cell wall to ensure integrity. These findings establish a molecular framework for understanding how pollen tube growth is modulated in response to the environment and show that RALF signaling can be altered to achieve thermotolerant pollen tube growth.

Significance: The innovation of stomatal pores in plant evolution enables the success and diversification of land plants over the past 400 million years. The production and patterning of stomata are regulated by a group of deeply conserved bHLH transcription factors in land plants, with the founding member SPEECHLESS (SPCH) playing a pivotal role in the initiation of stomatal lineage. The protein stability and function of SPCH are tightly regulated by protein phosphorylation mediated by multiple kinases in Arabidopsis . Here, we establish the highly conserved PP2A protein phosphatases as positive regulators of SPCH and thus stomatal production, underscoring a key function of PP2A in fine-tuning stomatal development, a highly plastic biological process that influences many plant responses to changing environmental conditions.

A versatile hub for cellular control, the eukaryotic protein phosphatase 2A (PP2A) enzyme family is thought to achieve specificity through combinatorial complexity. Phylogenetic analysis has revealed that expansion of PP2A gene families resulted from whole genome duplications followed by non-random gene loss, and selection analysis suggests that retention of B56/PPP2R5 gene family members after genome duplication events was driven by functional diversification. Here we identify the sites at which positive selection is detected in the plant B56 gene family, and discuss the significance of selection at these positions in the context of PP2A holoenzyme structure. The pattern of positive selection observed in the B11 subclade is distinctive, and suggests selective pressure on interactions with substrates and the enzymatic core.

Whole-genome duplications have driven expansion of PP2A gene families; however, distinct subclades exhibit reduced rates of expansion and may perform specialize

Strictly controlled production of ethylene gas lies upstream of the signaling activities of this crucial regulator throughout the plant life cycle. Although the biosynthetic pathway is enzymatically simple, the regulatory circuits that modulate signal production are fine tuned to allow integration of responses to environmental and intrinsic cues. Recently identified posttranslational mechanisms that control ethylene production converge on one family of biosynthetic enzymes and overlay several independent reversible phosphorylation events and distinct mediators of ubiquitin-dependent protein degradation. Although the core pathway is conserved throughout seed plants, these posttranslational regulatory mechanisms may represent evolutionarily recent innovations. The evolutionary origins of the pathway and its regulators are not yet clear; outside the seed plants, numerous biochemical and phylogenetic questions remain to be addressed.

The gaseous hormone ethylene is one of the master regulators of development and physiology throughout the plant life cycle. Ethylene biosynthesis is stringently regulated to permit maintenance of low levels during most phases of vegetative growth but to allow for rapid peaks of high production at developmental transitions and under stress conditions. In most tissues ethylene is a negative regulator of cell expansion, thus low basal levels of ethylene biosynthesis in dark-grown seedlings are critical for optimal cell expansion during early seedling development. The committed steps in ethylene biosynthesis are performed by the enzymes 1-aminocyclopropane 1-carboxylate synthase (ACS) and 1-aminocyclopropane 1-carboxylate oxidase (ACO). The abundance of different ACS enzymes is tightly regulated both by transcriptional control and by post-translational modifications and proteasome-mediated degradation. Here we show that specific ACS isozymes are targets for regulation by protein phosphatase 2A (PP2A) during Arabidopsis thaliana seedling growth and that reduced PP2A function causes increased ACS activity in the roots curl in 1-N-naphthylphthalamic acid 1 (rcn1) mutant. Genetic analysis reveals that ethylene overproduction in PP2A-deficient plants requires ACS2 and ACS6, genes that encode ACS proteins known to be stabilized by phosphorylation, and proteolytic turnover of the ACS6 protein is retarded when PP2A activity is reduced. We find that PP2A and ACS6 proteins associate in seedlings and that RCN1-containing PP2A complexes specifically dephosphorylate a C-terminal ACS6 phosphopeptide. These results suggest that PP2A-dependent destabilization requires RCN1-dependent dephosphorylation of the ACS6 C-terminus. Surprisingly, rcn1 plants exhibit decreased accumulation of the ACS5 protein, suggesting that a regulatory phosphorylation event leads to ACS5 destabilization. Our data provide new insight into the circuitry that ensures dynamic control of ethylene synthesis during plant development, showing that PP2A mediates a finely tuned regulation of overall ethylene production by differentially affecting the stability of specific classes of ACS enzymes. Like animals, plants produce a number of substances that regulate growth and coordinate developmental transitions and responses to environmental signals. Ethylene gas is one such regulator of the plant life cycle, playing important roles in fruit ripening, pathogen defenses, and the regulation of cell expansion. Because overall plant form is determined largely by the degree and directionality of cell expansion, ethylene is a crucial regulator of morphology, and ethylene production must be maintained at low levels during phases of rapid cell expansion, such as early seedling growth. Recent work has identified molecular mechanisms that target ethylene biosynthetic enzymes for proteolytic degradation; this degradation plays a key role in controlling ethylene production. Here we exploit the molecular genetic resources available in the Arabidopsis thaliana system to identify a highly conserved protein complex that dephosphorylates target proteins as a new component of the mechanism that regulates degradation of ethylene-producing enzymes. Our findings show that protein phosphatase 2A plays a nuanced role in this regulatory circuit, with both positive and negative inputs into the stability of specific proteins that drive ethylene biosynthesis. This work enhances our understanding of the mechanisms that enforce adaptive levels of hormone production in plants.

Protein dephosphorylation by the serine/threonine protein phosphatase 2A (PP2A) modulates a broad array of cellular functions. PP2A normally acts as a heterotrimeric holoenzyme complex comprising a catalytic subunit bound by regulatory A and B subunits. Characterization of the regulatory A subunit isoforms (ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 [RCN1], PP2AA2, and PP2AA3) of Arabidopsis thaliana PP2A has shown that RCN1 plays a primary role in controlling root and hypocotyl PP2A activity in seedlings. Here we show that hypocotyl and root growth exhibit different requirements for RCN1-mediated regulation of PP2A activity. Roots of rcn1 mutant seedlings exhibit characteristic abnormalities in cell division patterns at the root apical meristem, as well as reduced growth under ionic, osmotic, and oxidative stress conditions. We constructed chimeric A subunit genes and found that restoration of normal root tip development in rcn1 plants requires both regulatory and coding sequences of RCN1, whereas the hypocotyl elongation defect of rcn1 plants can be complemented by either RCN1 or PP2AA3 transgenes. Furthermore, the RCN1 and PP2AA3 proteins exhibit ubiquitous subcellular localization patterns in seedlings and both associate with membrane compartments. Together, these results show that RCN1-containing PP2A has unique functions that cannot be attributed to isoform-specific expression and localization patterns. Postembryonic RCN1 function is required to maintain normal auxin distribution and stem cell function at the root apex. Our data show that RCN1-regulated phosphatase activity plays a unique role in regulating postembryonic root development and stress response.

Activity of protein phosphatase 2A (PP2A) is tightly regulated and performs a diverse repertoire of cellular functions. Previously we isolated a dominant-negative active site mutant of the PP2A catalytic (C) subunit using a yeast complementation assay. We have established stable fibroblastic cell lines expressing epitope-tagged versions of the wild-type and H118N mutant C subunits and have used these cells to investigate mechanisms that regulate PP2A activity. Cells expressing the mutant C subunit exhibit a decreased growth rate and a prolonged G1 cell cycle phase. The mutant protein is enzymatically inactive, but extracts made from cells expressing the H118N C subunit show normal levels of total PP2A activity in vitro. The H118N mutant shows reduced binding to the regulatory A subunit, but binds normally to the α4 protein, a non-canonical regulator of PP2A. Expression of the H118N mutant interferes with the normal control of C subunit abundance, causing accumulation of the endogenous wild-type protein as well as the mutant transgene product. Our results indicate that the H118N mutant isoform retards C subunit turnover and suggest that PP2A C subunit turnover may be important for normal cell cycle progression. J. Cell. Biochem. 103: 1309–1325, 2008.