Steven Penfield

Cold and light control seed germination through the bHLH transcription factor SPATULA

BACKGROUND Plants integrate signals from the environment and use these to modify the timing of development according to seasonal cues. Seed germination is a key example of this phenomenon and in Arabidopsis is promoted by the synergistic interaction of light and low temperatures in dormant seeds. This signaling pathway is known to converge on the regulation of the gibberellin (GA) biosynthetic genes GA3 oxidase (GA3ox), whose expression is transcriptionally induced by light and cold in imbibed seeds. However, the molecular basis of this response has until now been unknown. RESULTS Here we show that the bHLH transcription factor SPATULA is a light-stable repressor of seed germination and mediates the germination response to temperature. Furthermore, SPT is required in dormant seeds for maintaining the repression of GA3ox transcription. We also show that the related protein PIL5 represses seed germination and GA3ox expression in the dark. CONCLUSIONS We conclude that SPT and PIL5 form part of a regulatory network coupling seed germination and GA3ox expression to light and temperature signaling in the seed.

Temperature perception and signal transduction in plants

Plants can show remarkable responses to small changes in temperature, yet one of the great unknowns in plant science is how that temperature signal is perceived. The identity of the early components of the temperature signal transduction pathway also remains a mystery. To understand the consequences of anthropogenic environmental change we will have to learn much more about the basic biology of how plants sense temperature. Recent advances show that many known plant-temperature responses share common signalling components, and suggest ways in which these might be linked to form a plant temperature signalling network.

Arabidopsis ABA INSENSITIVE4 Regulates Lipid Mobilization in the Embryo and Reveals Repression of Seed Germination by the Endosperm

Regulation of seed germination requires coordinate action by the embryo and surrounding endosperm. We used Arabidopsis thaliana to establish the relative roles of embryo and endosperm in the control of seed germination and seedling establishment. We previously showed that endospermic oil reserves are used postgerminatively via gluconeogenesis to fuel seedling establishment and that lipid breakdown is repressed by abscisic acid (ABA) in embryo but not endosperm tissues. Here, we use RNA amplification to describe the transcriptome of the endosperm and compare the hormone responses of endosperm and embryo tissues. We show that the endosperm responds to both ABA and gibberellin but that ABA in particular regulates nuclear but not plastid-encoded photosynthetic gene expression in the embryo. We also show that ABA INSENSITIVE4 (ABI4) expression is confined to the embryo, accounts for the major differences in embryo response to ABA, and defines a role for ABI4 as a repressor of lipid breakdown. Furthermore, ABI5 expression in the endosperm defines a second region of altered ABA signaling in the micropylar endosperm cap. Finally, embryo and endosperm ABA signaling mutants demonstrate the spatial specificity of ABA action in seed germination. We conclude that the single cell endosperm layer plays an active role in the regulation of seed germination in Arabidopsis.

Reserve Mobilization in the Arabidopsis Endosperm Fuels Hypocotyl Elongation in the Dark, Is Independent of Abscisic Acid, and Requires PHOSPHOENOLPYRUVATE CARBOXYKINASE1

Arabidopsis thaliana is used as a model system to study triacylglycerol (TAG) accumulation and seed germination in oilseeds. Here, we consider the partitioning of these lipid reserves between embryo and endosperm tissues in the mature seed. The Arabidopsis endosperm accumulates significant quantities of storage lipid, and this is effectively catabolized upon germination. This lipid differs in composition from that in the embryo and has a specific function during germination. Removing the endosperm from the wild-type seeds resulted in a reduction in hypocotyl elongation in the dark, demonstrating a role for endospermic TAG reserves in fueling skotomorphogenesis. Seedlings of two allelic gluconeogenically compromised phosphoenolpyruvate carboxykinase1 (pck1) mutants show a reduction in hypocotyl length in the dark compared with the wild type, but this is not further reduced by removing the endosperm. The short hypocotyl phenotypes were completely reversed by the provision of an exogenous supply of sucrose. The PCK1 gene is expressed in both embryo and endosperm, and the induction of PCK1:β-glucuronidase at radicle emergence occurs in a robust, wave-like manner around the embryo suggestive of the action of a diffusing signal. Strikingly, the induction of PCK1 promoter reporter constructs and measurements of lipid breakdown demonstrate that whereas lipid mobilization in the embryo is inhibited by abscisic acid (ABA), no effect is seen in the endosperm. This insensitivity of endosperm tissues is not specific to lipid breakdown because hydrolysis of the seed coat cell walls also proceeded in the presence of concentrations of ABA that effectively inhibit radicle emergence. Both processes still required gibberellins, however. These results suggest a model whereby the breakdown of seed carbon reserves is regulated in a tissue-specific manner and shed new light on phytohormonal regulation of the germination process.

Function search in a large transcription factor gene family in Arabidopsis: assessing the potential of reverse genetics to identify insertional mutations in R2R3 MYB genes.

More than 92 genes encoding MYB transcription factors of the R2R3 class have been described in Arabidopsis. The functions of a few members of this large gene family have been described, indicating important roles for R2R3 MYB transcription factors in the regulation of secondary metabolism, cell shape, and disease resistance, and in responses to growth regulators and stresses. For the majority of the genes in this family, however, little functional information is available. As the first step to characterizing these genes functionally, the sequences of >90 family members, and the map positions and expression profiles of >60 members, have been determined previously. An important second step in the functional analysis of the MYB family, through a process of reverse genetics that entails the isolation of insertion mutants, is described here. For this purpose, a variety of gene disruption resources has been used, including T-DNA–insertion populations and three distinct populations that harbor transposon insertions. We report the isolation of 47 insertions into 36 distinct MYB genes by screening a total of 73 genes. These defined insertion lines will provide the foundation for subsequent detailed functional analyses for the assignment of specific functions to individual members of the R2R3 MYB gene family.

Induction of Dormancy in Arabidopsis Summer Annuals Requires Parallel Regulation of DOG1 and Hormone Metabolism by Low Temperature and CBF Transcription Factors

In plants grown under field conditions, seed dormancy varies considerably with the time of year the seed is set, and this variation is important for the coordination of plant life history and seed quality. This work elucidates the mechanisms through which environmental temperature during seed set affects the germination behavior of seeds when released from the mother plant. Summer annuals overwinter as seeds in the soil seed bank. This is facilitated by a cold-induced increase in dormancy during seed maturation followed by a switch to a state during seed imbibition in which cold instead promotes germination. Here, we show that the seed maturation transcriptome in Arabidopsis thaliana is highly temperature sensitive and reveal that low temperature during seed maturation induces several genes associated with dormancy, including DELAY OF GERMINATION1 (DOG1), and influences gibberellin and abscisic acid levels in mature seeds. Mutants lacking DOG1, or with altered gibberellin or abscisic acid synthesis or signaling, in turn show reduced ability to enter the deeply dormant states in response to low seed maturation temperatures. In addition, we find that DOG1 promotes gibberellin catabolism during maturation. We show that C-REPEAT BINDING FACTORS (CBFs) are necessary for regulation of dormancy and of GA2OX6 and DOG1 expression caused by low temperatures. However, the temperature sensitivity of CBF transcription is markedly reduced in seeds and is absent in imbibed seeds. Our data demonstrate that inhibition of CBF expression is likely a critical feature allowing cold to promote rather than inhibit germination and support a model in which CBFs act in parallel to a low-temperature signaling pathway in the regulation of dormancy.

A Role for Multiple Circadian Clock Genes in the Response to Signals That Break Seed Dormancy in Arabidopsis

Plant seeds can sense diverse environmental signals and integrate the information to regulate developmental responses, such as dormancy and germination. The circadian clock confers a growth advantage on plants and uses environmental information for entrainment. Here, we show that normal circadian clock gene function is essential for the response to dormancy-breaking signals in seeds. We show that mutations in the clock genes LATE ELONGATED HYPOCOTYL, CIRCADIAN CLOCK ASSOCIATED1 (CCA1), and GIGANTEA (GI) cause germination defects in response to low temperature, alternating temperatures, and dry after-ripening. We demonstrate that the transcriptional clock is arrested in an evening-like state in dry seeds but rapidly entrains to light/dark cycles in ambient temperatures upon imbibition. Consistent with a role for clock genes in seed dormancy control, CCA1 expression is transcriptionally induced in response to dry after-ripening and that after-ripening affects the amplitude of subsequent transcriptional clock gene oscillations. Control of abscisic acid- and gibberellin-related gene expression in seeds requires normal circadian function, and GI and TIMING OF CAB EXPRESSION1 regulate the response to ABA and GA in seeds. We conclude that circadian clock genes play a key role in the integration of environmental signaling controlling dormancy release in plants.

Storage reserve mobilization in germinating oilseeds: Arabidopsis as a model system

Germinating oilseeds break down fatty acids through peroxisomal beta-oxidation and convert the carbon into soluble carbohydrates through the glyoxylate cycle and gluconeogenesis. This interconversion is unique among higher eukaryotes. Using a combination of forward and reverse genetic screens, we have isolated mutants that compromise fatty acid breakdown at each step. These mutants exhibit characteristic, yet nonidentical, seedling establishment phenotypes that can be rescued by the provision of an alternative carbon source. In addition, we have recently shown that Arabidopsis seeds lipid breakdown occurs in two distinct tissues, the embryo and endosperm. The utilization of endospermic lipid reserves requires gluconeogenesis and transport of the resulting sugars to the germinating embryo. We discuss the potential of the Arabidopsis endosperm tissue as a simplified model system for the study of germination and lipid breakdown in germinating oilseeds.

DELLA-mediated cotyledon expansion breaks coat-imposed seed dormancy

Seed dormancy is a key adaptive trait in plants responsible for the soil seed bank. The long established hormone-balance theory describes the antagonistic roles of the dormancy promoting plant hormone abscisic acid (ABA), and the germination promoting hormone gibberellin (GA) in dormancy control. Light, temperature, and other dormancy-breaking signals function to modulate the synthesis and perception of these hormones in the seed. However, the way in which these hormones control dormancy in the imbibed seed remains unknown. Here, we show that the DELLA protein regulators of the GA response are required for dormancy and describe a model through which hormone signal integration and dormancy regulation is achieved. We demonstrate that cotyledon expansion precedes radicle emergence during Arabidopsis seed germination and that a striking correlation exists between final seedling cotyledon size and seed dormancy in the DELLA mutants. Furthermore, twelve previously characterized seed-dormancy mutants are also defective in the control of cotyledon size in a manner consistent with their effect on germination potential. We propose that DELLA-mediated, light-, temperature-, and hormone-responsive cotyledon expansion prior to radicle emergence overcomes dormancy imposed by the seed coat and underlies seed-dormancy control in Arabidopsis.

A role for an alternative splice variant of PIF6 in the control of Arabidopsis primary seed dormancy

Phytochrome interacting factor (PIF) transcription factors have been shown to be important in the regulation of seed dormancy and germination by environmental cues. Many PIF-family transcription factors are expressed in seeds but only PIF1 and SPATULA (SPT) have been tested for a role in germination control. Here we show that PIF6 is expressed strongly during seed development, and that two splice variants exist, one full length (the α form), and a second, the β form, in which a cryptic intron containing the potential DNA binding domain is spliced out, predicted to lead to the generation of a premature stop codon. Loss of PIF6 increases primary seed dormancy, whereas overexpression of the β form, but not the α form, reduce dormancy. Our data show the potential for natural splice variants of PIF transcription factors to be important in the evolution of the control of environmental signalling in plants.