Qingyun Bu

Role of the Arabidopsis thaliana NAC transcription factors ANAC019 and ANAC055 in regulating jasmonic acid-signaled defense responses.

Jasmonic acid (JA) is an important phytohormone that regulates plant defense responses against herbivore attack, pathogen infection and mechanical wounding. In this report, we provided biochemical and genetic evidence to show that the Arabidopsis thaliana NAC family proteins ANAC019 and ANAC055 might function as transcription activators to regulate JA-induced expression of defense genes. The role of the two NAC genes in JA signaling was examined with the anac019 anac055 double mutant and with transgenic plants overexpressing ANAC019 or ANAC055. The anac019 anac055 double mutant plants showed attenuated JA-induced VEGETATIVE STORAGE PROTEIN1 (VSP1) and LIPOXYGENASE2 (LOX2) expression, whereas transgenic plants overexpressing the two NAC genes showed enhanced JA-induced VSP1 and LOX2 expression. That the JA-induced expression of the two NAC genes depends on the function of COI1 and AtMYC2, together with the finding that overexpression of ANAC019 partially rescued the JA-related phenotype of the atmyc2-2 mutant, has led us to a hypothesis that the two NAC proteins act downstream of AtMYC2 to regulate JA-signaled defense responses. Further evidence to substantiate this idea comes from the observation that the response of the anac019 anac055 double mutant to a necrotrophic fungus showed high similarity to that of the atmyc2-2 mutant.

Mutation of the Rice Narrow leaf1 Gene, Which Encodes a Novel Protein, Affects Vein Patterning and Polar Auxin Transport

The size and shape of the plant leaf is an important agronomic trait. To understand the molecular mechanism governing plant leaf shape, we characterized a classic rice (Oryza sativa) dwarf mutant named narrow leaf1 (nal1), which exhibits a characteristic phenotype of narrow leaves. In accordance with reduced leaf blade width, leaves of nal1 contain a decreased number of longitudinal veins. Anatomical investigations revealed that the culms of nal1 also show a defective vascular system, in which the number and distribution pattern of vascular bundles are altered. Map-based cloning and genetic complementation analyses demonstrated that Nal1 encodes a plant-specific protein with unknown biochemical function. We provide evidence showing that Nal1 is richly expressed in vascular tissues and that mutation of this gene leads to significantly reduced polar auxin transport capacity. These results indicate that Nal1 affects polar auxin transport as well as the vascular patterns of rice plants and plays an important role in the control of lateral leaf growth.

The Arabidopsis RING Finger E3 Ligase RHA2a Is a Novel Positive Regulator of Abscisic Acid Signaling during Seed Germination and Early Seedling Development

The phytohormone abscisic acid (ABA) is well known for its regulatory roles in integrating environmental constraints with the developmental programs of plants. Here, we characterize the biological function of the Arabidopsis (Arabidopsis thaliana) RING-H2 protein RHA2a in ABA signaling. The rha2a mutant is less sensitive to ABA than the wild type during seed germination and early seedling development, whereas transgenic plants overexpressing RHA2a are hypersensitive, indicating that RHA2a positively regulates ABA-mediated control of seed germination and early seedling development. Double mutant analyses of rha2a with several known ABA-insensitive mutants suggest that the action of RHA2a in ABA signaling is independent of that of the transcription factors ABI3, ABI4, and ABI5. We provide evidence showing that RHA2a also positively regulates plant responses to salt and osmotic stresses during seed germination and early seedling development. RHA2a is a functional E3 ubiquitin ligase, and its conserved RING domain is likely important for the biological function of RHA2a in ABA signaling. Together, these results suggest that the E3 ligase RHA2a is an important regulator of ABA signaling during seed germination and early seedling development.Jasmonate- and ABA-mediated signalings are involved in the activation of defense responses of plants to biotic and abiotic stresses. Accumulating evidence has suggested the existence of comprehensive synergistic or antagonistic cross-talks between these two signaling pathways. However, relatively little is known about how these cross-talks are executed at the molecular level. Our recent works have implied that, ANAC019 and ANAC055, two highly related NAC family transcription factors in Arabidopsis, may play a dual role in regulating jasmonate response and ABA response.

Regulation of Drought Tolerance by the F-Box Protein MAX2 in Arabidopsis

An F-box protein regulates drought and osmotic stress responses in a strigolactone-independent manner in Arabidopsis. MAX2 (for MORE AXILLARY GROWTH2) has been shown to regulate diverse biological processes, including plant architecture, photomorphogenesis, senescence, and karrikin signaling. Although karrikin is a smoke-derived abiotic signal, a role for MAX2 in abiotic stress response pathways is least investigated. Here, we show that the max2 mutant is strongly hypersensitive to drought stress compared with wild-type Arabidopsis (Arabidopsis thaliana). Stomatal closure of max2 was less sensitive to abscisic acid (ABA) than that of the wild type. Cuticle thickness of max2 was significantly thinner than that of the wild type. Both of these phenotypes of max2 mutant plants correlate with the increased water loss and drought-sensitive phenotype. Quantitative real-time reverse transcription-polymerase chain reaction analyses showed that the expression of stress-responsive genes and ABA biosynthesis, catabolism, transport, and signaling genes was impaired in max2 compared with wild-type seedlings in response to drought stress. Double mutant analysis of max2 with the ABA-insensitive mutants abi3 and abi5 indicated that MAX2 may function upstream of these genes. The expression of ABA-regulated genes was enhanced in imbibed max2 seeds. In addition, max2 mutant seedlings were hypersensitive to ABA and osmotic stress, including NaCl, mannitol, and glucose. Interestingly, ABA, osmotic stress, and drought-sensitive phenotypes were restricted to max2, and the strigolactone biosynthetic pathway mutants max1, max3, and max4 did not display any defects in these responses. Taken together, these results uncover an important role for MAX2 in plant responses to abiotic stress conditions.

The Arabidopsis RING Finger E3 Ligase RHA2b Acts Additively with RHA2a in Regulating Abscisic Acid Signaling and Drought Response

We have previously shown that the Arabidopsis (Arabidopsis thaliana) RING-H2 E3 ligase RHA2a positively regulates abscisic acid (ABA) signaling during seed germination and postgerminative growth. Here, we report that RHA2b, the closest homolog of RHA2a, is also an active E3 ligase and plays an important role in ABA signaling. We show that RHA2b expression is induced by ABA and that overexpression of RHA2b leads to ABA-associated phenotypes such as ABA hypersensitivity in seed germination and seedling growth, enhanced stomatal closure, reduced water loss, and, therefore, increased drought tolerance. On the contrary, the rha2b-1 mutant shows ABA-insensitive phenotypes and reduced drought tolerance. We provide evidence showing that a rha2a rha2b-1 double mutant generally enhances ABA insensitivity of rha2b-1 in seed germination, seedling growth, and stomatal closure, suggesting that RHA2b and RHA2a act redundantly in regulating ABA responses. Genetic analyses support that, like RHA2a, the RHA2b action in ABA signaling is downstream of a protein phosphatase 2C, ABA-INSENSITIVE2 (ABI2), and in parallel with that of the ABI transcription factors ABI3/4/5. We speculate that RHA2b and RHA2a may have redundant yet distinguishable functions in the regulation of ABA responses.

MAX2 Affects Multiple Hormones to Promote Photomorphogenesis

Ubiquitin-26S proteasome system (UPS) has been shown to play central roles in light and hormone-regulated plant growth and development. Previously, we have shown that MAX2, an F-box protein, positively regulates facets of photomorphogenic development in response to light. However, how MAX2 controls these responses is still unknown. Here, we show that MAX2 oppositely regulates GA and ABA biosynthesis to optimize seed germination in response to light. Dose-response curves showed that max2 seeds are hyposensitive to GA and hypersensitive to ABA in seed germination responses. RT-PCR assays demonstrated that the expression of GA biosynthetic genes is down-regulated, while the expression of GA catabolic genes is up-regulated in the max2 seeds compared to wild-type. Interestingly, expression of both ABA biosynthetic and catabolic genes is up-regulated in the max2 seeds compared to wild-type. Treatment with an auxin transport inhibitor, NPA, showed that increased auxin transport in max2 seedlings contributes to the long hypocotyl phenotype under light. Moreover, light-signaling phenotypes are restricted to max2, as the biosynthetic mutants in the strigolactone pathway, max1, max3, and max4, did not display any defects in seed germination and seedling de-etiolation compared to wild-type. Taken together, these data suggest that MAX2 modulates multiple hormone pathways to affect photomorphogenesis.

Phosphorylation by CK2 Enhances the Rapid Light-induced Degradation of Phytochrome Interacting Factor 1 in Arabidopsis

The phytochrome family of sensory photoreceptors interacts with phytochrome interacting factors (PIFs), repressors of photomorphogenesis, in response to environmental light signals and induces rapid phosphorylation and degradation of PIFs to promote photomorphogenesis. However, the kinase that phosphorylates PIFs is still unknown. Here we show that CK2 directly phosphorylates PIF1 at multiple sites. α1 and α2 subunits individually phosphorylated PIF1 weakly in vitro. However, each of four β subunits strongly stimulated phosphorylation of PIF1 by α1 or α2. Mapping of the phosphorylation sites identified seven Ser/Thr residues scattered throughout PIF1. Ser/Thr to Ala scanning mutations at all seven sites eliminated CK2-mediated phosphorylation of PIF1 in vitro. Moreover, the rate of degradation of the Ser/Thr to Ala mutant PIF1 was significantly reduced compared with wild-type PIF1 in transgenic plants. In addition, hypocotyl lengths of the mutant PIF1 transgenic plants were much longer than the wild-type PIF1 transgenic plants under light, suggesting that the mutant PIF1 is suppressing photomorphogenesis. Taken together, these data suggest that CK2-mediated phosphorylation enhances the light-induced degradation of PIF1 to promote photomorphogenesis.

PHYTOCHROME INTERACTING FACTOR1 Enhances the E3 Ligase Activity of CONSTITUTIVE PHOTOMORPHOGENIC1 to Synergistically Repress Photomorphogenesis in Arabidopsis

This study shows that PHYTOCHROME INTERACTING FACTOR1 forms a complex with CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1)/SUPPRESSOR OF PHYA and enhances the substrate recruitment and autoubiquitylation and transubiquitylation activity of COP1. The data support a genetic and biochemical model in light signaling pathways and expand the regulatory roles of bHLH factors as potential cofactors of E3 ligases. CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is a RING/WD40 repeat–containing ubiquitin E3 ligase that is conserved from plants to humans. COP1 forms complexes with SUPPRESSOR OF PHYTOCHROME A (SPA) proteins, and these complexes degrade positively acting transcription factors in the dark to repress photomorphogenesis. Phytochrome-interacting basic helix-loop-helix transcription factors (PIFs) also repress photomorphogenesis in the dark. In response to light, the phytochrome family of sensory photoreceptors simultaneously inactivates COP1-SPA complexes and induces the rapid degradation of PIFs to promote photomorphogenesis. However, the functional relationship between PIFs and COP1-SPA complexes is still unknown. Here, we present genetic evidence that the pif and cop1/spa Arabidopsis thaliana mutants synergistically promote photomorphogenesis in the dark. LONG HYPOCOTYL5 (HY5) is stabilized in the cop1 pif1, spa123 pif1, and pif double, triple, and quadruple mutants in the dark. Moreover, the hy5 mutant suppresses the constitutive photomorphogenic phenotypes of the pifq mutant in the dark. PIF1 forms complexes with COP1, HY5, and SPA1 and enhances the substrate recruitment and autoubiquitylation and transubiquitylation activities of COP1. These data uncover a novel function of PIFs as the potential cofactors of COP1 and provide a genetic and biochemical model of how PIFs and COP1-SPA complexes synergistically repress photomorphogenesis in the dark.

Casein kinase II α subunits affect multiple developmental and stress‐responsive pathways in Arabidopsis

Casein kinase II (formerly known as CK2), a ubiquitous Ser/Thr kinase, plays critical roles in all higher organisms including plants. The CK2 holoenzyme consists of two catalytic α subunits and two regulatory β subunits. The Arabidopsis genome has four α subunit and four β subunit genes, and members of both the α and β subunit families have been shown to be localized in the cytoplasm, nucleus and also in chloroplasts. However, the biological roles of CK2 subunits have not been fully characterized yet. Here we identified T-DNA insertion mutants in three α subunit genes (α1, α2 and α3) and made double and triple mutants. The CK2 α1α2α3 triple mutants displayed reduced CK2 activity compared with wild-type seedlings. Phenotypic characterization showed that CK2 α1α2α3 triple mutants are late flowering under both long- and short-day conditions. Genes encoding floral integrators are differentially regulated in the triple mutant compared with the wild-type plants. CK2 α1α2α3 triple mutants also displayed reduced hypocotyl growth, smaller cotyledon size and a reduced number of lateral roots compared with wild-type seedlings under light. Abscisic acid-induced blockage of seed germination and cotyledon greening is reduced in CK2 α subunit mutants in an additive manner. Moreover, CK2 α subunit mutants are also hyposensitive to a NaCl-induced blockage of seed germination. Taken together, these data suggest that CK2 α subunits affect diverse developmental and stress responsive pathways in Arabidopsis.

CUL4 forms an E3 ligase with COP1 and SPA to promote light-induced degradation of PIF1

Plants undergo contrasting developmental programs in dark and light. Photomorphogenesis, a light-adapted programme is repressed in the dark by the synergistic actions of CUL4(COP1-SPA) E3 ubiquitin ligase and a subset of basic helix-loop-helix transcription factors called phytochrome interacting factors (PIFs). To promote photomorphogenesis, light activates the phytochrome family of sensory photoreceptors, which inhibits these repressors by poorly understood mechanisms. Here, we show that the CUL4(COP1-SPA) E3 ubiquitin ligase is necessary for the light-induced degradation of PIF1 in Arabidopsis. The light-induced ubiquitylation and subsequent degradation of PIF1 is reduced in the cop1, spaQ and cul4 backgrounds. COP1, SPA1 and CUL4 preferentially form complexes with the phosphorylated forms of PIF1 in response to light. The cop1 and spaQ seeds display strong hyposensitive response to far-red light-mediated seed germination and light-regulated gene expression. These data show a mechanism by which an E3 ligase attenuates its activity by degrading its cofactor in response to light.