Luis Lopez-Molina

A postgermination developmental arrest checkpoint is mediated by abscisic acid and requires the ABI5 transcription factor in Arabidopsis

Seed dormancy is a trait of considerable adaptive significance because it maximizes seedling survival by preventing premature germination under unfavorable conditions. Understanding how seeds break dormancy and initiate growth is also of great agricultural and biotechnological interest. Abscisic acid (ABA) plays primary regulatory roles in the initiation and maintenance of seed dormancy. Here we report that the basic leucine zipper transcription factor ABI5 confers an enhanced response to exogenous ABA during germination, and seedling establishment, as well as subsequent vegetative growth. These responses correlate with total ABI5 levels. We show that ABI5 expression defines a narrow developmental window following germination, during which plants monitor the environmental osmotic status before initiating vegetative growth. ABI5 is necessary to maintain germinated embryos in a quiescent state thereby protecting plants from drought. As expected for a key player in ABA-triggered processes, ABI5 protein accumulation, phosphorylation, stability, and activity are highly regulated by ABA during germination and early seedling growth.

The DBP gene is expressed according to a circadian rhythm in the suprachiasmatic nucleus and influences circadian behavior

DBP, a PAR leucine zipper transcription factor, accumulates according to a robust circadian rhythm in liver and several other tissues of mouse and rat. Here we report that DBP mRNA levels also oscillate strongly in the suprachiasmatic nucleus (SCN) of the hypothalamus, believed to harbor the central mammalian pacemaker. However, peak and minimum levels of DBP mRNA are reached about 4 h earlier in the SCN than in liver, suggesting that circadian DBP expression is controlled by different mechanisms in SCN and in peripheral tissues. Mice homozygous for a DBP‐null allele display less locomotor activity and free‐run with a shorter period than otherwise isogenic wild‐type animals. The altered locomotor activity in DBP mutant mice and the highly rhythmic expression of the DBP gene in SCN neurons suggest that DBP is involved in controlling circadian behavior. However, since DBP−/− mice are still rhythmic and since DBP protein is not required for the circadian expression of its own gene, dbp is more likely to be a component of the circadian output pathway than a master gene of the clock.

The Gibberellic Acid Signaling Repressor RGL2 Inhibits Arabidopsis Seed Germination by Stimulating Abscisic Acid Synthesis and ABI5 Activity

Seed germination is antagonistically controlled by the phytohormones gibberellic acid (GA) and abscisic acid (ABA). GA promotes seed germination by enhancing the proteasome-mediated destruction of RGL2 (for RGA-LIKE2), a key DELLA factor repressing germination. By contrast, ABA blocks germination by inducing ABI5 (for ABA-INSENSITIVE5), a basic domain/leucine zipper transcription factor repressing germination. Decreased GA synthesis leads to an increase in endogenous ABA levels through a stabilized RGL2, a process that may involve XERICO, a RING-H2 zinc finger factor promoting ABA synthesis. In turn, increased endogenous ABA synthesis is necessary to elevate not only ABI5 RNA and protein levels but also, critically, those of RGL2. Increased ABI5 protein is ultimately responsible for preventing seed germination when GA levels are reduced. However, overexpression of ABI5 was not sufficient to repress germination, as ABI5 activity requires phosphorylation. The endogenous ABI5 phosphorylation and inhibition of germination could be recapitulated by the addition of a SnRK2 protein kinase to the ABI5 overexpression line. In sleepy1 mutant seeds, RGL2 overaccumulates; germination of these seeds can occur under conditions that produce low ABI5 expression. These data support the notion that ABI5 acts as the final common repressor of germination in response to changes in ABA and GA levels.

Circadian expression of the steroid 15 alpha-hydroxylase (Cyp2a4) and coumarin 7-hydroxylase (Cyp2a5) genes in mouse liver is regulated by the PAR leucine zipper transcription factor DBP.

ABSTRACT To study the molecular mechanisms of circadian gene expression, we have sought to identify genes whose expression in mouse liver is regulated by the transcription factor DBP (albumin D-site-binding protein). This PAR basic leucine zipper protein accumulates according to a robust circadian rhythm in nuclei of hepatocytes and other cell types. Here, we report that the Cyp2a4 gene, encoding the cytochrome P450 steroid 15α-hydroxylase, is a novel circadian expression gene. This enzyme catalyzes one of the hydroxylation reactions leading to further metabolism of the sex hormones testosterone and estradiol in the liver. Accumulation of CYP2A4 mRNA in mouse liver displays circadian kinetics indistinguishable from those of the highly related CYP2A5 gene. Proteins encoded by both theCyp2a4 and Cyp2a5 genes also display daily variation in accumulation, though this is more dramatic for CYP2A4 than for CYP2A5. Biochemical evidence, including in vitro DNase I footprinting on the Cyp2a4 and Cyp2a5 promoters and cotransfection experiments with the human hepatoma cell line HepG2, suggests that the Cyp2a4 and Cyp2a5 genes are indeed regulated by DBP. These conclusions are corroborated by genetic studies, in which the circadian amplitude of CYP2A4 and CYP2A5 mRNAs and protein expression in the liver was significantly impaired in a mutant mouse strain homozygous for a dbp null allele. These experiments strongly suggest that DBP is a major factor controlling circadian expression of the Cyp2a4 and Cyp2a5genes in the mouse liver.

A seed coat bedding assay shows that RGL2-dependent release of abscisic acid by the endosperm controls embryo growth in Arabidopsis dormant seeds

Seed dormancy is an ecologically important adaptive trait in plants whereby germination is repressed even under favorable germination conditions such as imbibition with water. In Arabidopsis and most plant species, dormancy absolutely requires an unidentified seed coat germination-repressive activity and constitutively higher abscisic acid (ABA) levels upon seed imbibition. The mechanisms underlying these processes and their possible relationship are incompletely understood. We developed a “seed coat bedding” assay monitoring the growth of dissected embryos cultured on a layer of seed coats, allowing combinatorial experiments using dormant, nondormant, and various genetically modified seed coat and embryonic materials. This assay, combined with direct ABA measurements, revealed that, upon imbibition, dormant coats, unlike nondormant coats, actively produce and release ABA to repress embryo germination, whatever the embryo origin, i.e., from dormant, nondormant, or never dormant aba seeds, unable to synthesize ABA. The persistent high ABA levels in imbibed dormant seeds requires the permanent expression of the DELLA gene RGL2, where it remains insensitive to gibberellins (GA) unlike in nondormant seeds. These findings present the seed coat as an organ actively controlling germination upon seed imbibition and provide a framework to investigate how environmental factors break seed dormancy.

Far-red light inhibits germination through DELLA-dependent stimulation of ABA synthesis and ABI3 activity

Under the canopy, far‐red (FR) light represses seed germination by inactivating phytochrome photoreceptors. This elicits a decrease in gibberellins (GA) levels and an increase in abscisic acid (ABA) levels. GA promotes germination by enhancing the proteasome‐mediated destruction of DELLA repressors. ABA prevents germination by stimulating the expression of ABI repressors. How phytochromes elicit changes in hormone levels or how GA‐ and ABA‐dependent signals are coordinated to repress germination remains poorly understood. We show that repression of germination by FR light involves stabilized DELLA factors GAI, RGA and RGL2 that stimulate endogenous ABA synthesis. In turn, ABA blocks germination through the transcription factor ABI3. The role of PIL5, a basic helix‐loop‐helix transcription factor stimulating GAI and RGA expression, is significant, provided GA synthesis is high enough; otherwise, high GAI and RGA protein levels persist to block germination. Under white light, GAI and RGA driven by the RGL2 promoter can substitute for RGL2 to promote ABA synthesis and repress germination, consistent with the recent findings with RGL2. The three DELLA factors inhibit testa rupture whereas ABI3 blocks endosperm rupture.

Abscisic Acid Represses Growth of the Arabidopsis Embryonic Axis after Germination by Enhancing Auxin Signaling

Under unfavorable environmental conditions, the stress phytohormone ABA inhibits the developmental transition from an embryo in a dry seed into a young seedling. We developed a genetic screen to isolate Arabidopsis thaliana mutants whose early seedling development is resistant to ABA. Here, we report the identification of a recessive mutation in AUXIN RESISTANT1 (AUX1), encoding a cellular auxin influx carrier. Although auxin is a major morphogenesis hormone in plants, little is known about ABA–auxin interactions during early seedling growth. We show that aux1 and pin2 mutants are insensitive to ABA-dependent repression of embryonic axis (hypocotyl and radicle) elongation. Genetic and physiological experiments show that this involves auxin transport to the embryonic axis elongation zone, where ABA enhances the activity of an auxin-responsive promoter. We propose that ABA represses embryonic axis elongation by potentiating auxin signaling in its elongation zone. This involves repression of the AUXIN INDUCIBLE (Aux/IAA) gene AXR2/IAA7, encoding a key component of ABA- and auxin-dependent responses during postgerminative growth.

Spatially and genetically distinct control of seed germination by phytochromes A and B

Phytochromes phyB and phyA mediate a remarkable developmental switch whereby, early upon seed imbibition, canopy light prevents phyB-dependent germination, whereas later on, it stimulates phyA-dependent germination. Using a seed coat bedding assay where the growth of dissected embryos is monitored under the influence of dissected endosperm, allowing combinatorial use of mutant embryos and endosperm, we show that canopy light specifically inactivates phyB activity in the endosperm to override phyA-dependent signaling in the embryo. This interference involves abscisic acid (ABA) release from the endosperm and distinct spatial activities of phytochrome signaling components. Under the canopy, endospermic ABA opposes phyA signaling through the transcription factor (TF) ABI5, which shares with the TF PIF1 several target genes that negatively regulate germination in the embryo. ABI5 enhances the expression of phytochrome signaling genes PIF1, SOMNUS, GAI, and RGA, but also of ABA and gibberellic acid (GA) metabolic genes. Over time, weaker ABA-dependent responses eventually enable phyA-dependent germination, a distinct type of germination driven solely by embryonic growth.

The GA-signaling repressor RGL3 represses testa rupture in response to changes in GA and ABA levels

We recently reported that the DELLA factor RGL2 represses testa rupture in response to changes in ABA and GA levels. Here, we provide genetic evidence that this observation extends to RGL3, another DELLA factor whose function was not previously characterized. However, RGL3’s repressive activity was seen only in an rgl2 genetic background. This may be explained by the observation that RGL3’s mRNA levels are positively regulated by ABA and low GA but to a lesser extent than those of RGL2. This could ensure that RGL2’s repressive activity dominates relative to that of RGL3 under most germination conditions.

Abscisic acid transporters cooperate to control seed germination

Seed germination is a key developmental process that has to be tightly controlled to avoid germination under unfavourable conditions. Abscisic acid (ABA) is an essential repressor of seed germination. In Arabidopsis, it has been shown that the endosperm, a single cell layer surrounding the embryo, synthesizes and continuously releases ABA towards the embryo. The mechanism of ABA transport from the endosperm to the embryo was hitherto unknown. Here we show that four AtABCG transporters act in concert to deliver ABA from the endosperm to the embryo: AtABCG25 and AtABCG31 export ABA from the endosperm, whereas AtABCG30 and AtABCG40 import ABA into the embryo. Thus, this work establishes that radicle extension and subsequent embryonic growth are suppressed by the coordinated activity of multiple ABA transporters expressed in different tissues.