SUPPRESSOR OF PHYTOCHROME B-4 #3 reduces the expression of PIF-activated genes and increases expression of growth repressors to regulate hypocotyl elongation in short days.

SUPPRESSOR OF PHYTOCHROME B-4 #3 (SOB3) is a member of the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) family of transcription factors that are involved in light-mediated growth in Arabidopsis thaliana, affecting processes such as hypocotyl elongation. The majority of the research on the AHLs has been conducted in continuous light. However, there are unique molecular events that promote growth in short days (SD) compared to constant light conditions. Therefore, we investigated how AHLs affect hypocotyl elongation in SD. Firstly, we observed that AHLs inhibit hypocotyl growth in SD, similar to their effect in constant light. Next, we identified AHL-regulated genes in SD-grown seedlings by performing RNA-seq in two sob3 mutants at different time points. Our transcriptomic data indicate that PHYTOCHROME INTERACTING FACTORS (PIFs) 4, 5, 7, and 8 along with PIF-target genes are repressed by SOB3 and/or other AHLs. We also identified PIF target genes that are repressed and have not been previously described as AHL-regulated, including PRE1, PIL1, HFR1, CDF5, and XTR7. Interestingly, our RNA-seq data also suggest that AHLs activate the expression of growth repressors to control hypocotyl elongation, such as HY5 and IAA17. Notably, many growth-regulating and other genes identified from the RNA-seq experiment were differentially regulated between these two sob3 mutants at the time points tested. Surprisingly, our ChIP-seq data suggest that SOB3 mostly binds to similar genes throughout the day. Collectively, these data suggest that AHLs affect gene expression in a time point-specific manner irrespective of changes in binding to DNA throughout SD.

Two ATAF transcription factors ANAC102 and ATAF1 contribute to the suppression of cytochrome P450-mediated brassinosteroid catabolism in Arabidopsis.

PHYB ACTIVATION TAGGED SUPPRESSOR 1 (BAS1) and SUPPRESSOR OF PHYB-4 7 (SOB7) are two cytochrome P450 enzymes that inactivate brassinosteroids (BRs) in Arabidopsis. The NAC transcription factor (TF) ATAF2 (ANAC081) and the core circadian clock regulator CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) both suppress the expression of BAS1 and SOB7 via direct promoter binding. Additionally, BRs cause feedback suppression on ATAF2 expression. Here, we report that two ATAF-subgroup TFs, ANAC102 and ATAF1 (ANAC002), also contribute to the transcriptional suppression of BAS1 and SOB7. ANAC102 and ATAF1 gene-knockout mutants exhibit elevated expression of both BAS1 and SOB7, expanded tissue-level accumulation of their protein products and reduced hypocotyl growth in response to exogenous BR treatments. Similar to ATAF2, both ANAC102 and ATAF1 are transcriptionally suppressed by BRs and white light. Neither BAS1 nor SOB7 expression is further elevated in ATAF double or triple mutants, suggesting that the suppression effect of these three ATAFs is not additive. In addition, ATAF single, double, and triple mutants have similar levels of BR responsiveness with regard to hypocotyl elongation. ATAF2, ANAC102, ATAF1, and CCA1 physically interact with itself and each other, suggesting that they may coordinately suppress BAS1 and SOB7 expression via protein-protein interactions. Despite the absence of CCA1-binding elements in their promoters, ANAC102 and ATAF1 have similar transcript circadian oscillation patterns as that of CCA1, suggesting that these two ATAF genes may be indirectly regulated by the circadian clock.

Pistil Mating Type and Morphology Are Mediated by the Brassinosteroid Inactivating Activity of the S-Locus Gene BAHD in Heterostylous Turnera Species.

Heterostyly is a breeding system that promotes outbreeding through a combination of morphological and physiological floral traits. In Turnera these traits are governed by a single, hemizygous S-locus containing just three genes. We report that the S-locus gene, BAHD, is mutated and encodes a severely truncated protein in a self-compatible long homostyle species. Further, a self-compatible long homostyle mutant possesses a T. krapovickasii BAHD allele with a point mutation in a highly conserved domain of BAHD acyl transferases. Wild type and mutant TkBAHD alleles were expressed in Arabidopsis to assay for brassinosteroid (BR) inactivating activity. The wild type but not mutant allele caused dwarfism, consistent with the wild type possessing, but the mutant allele having lost, BR inactivating activity. To investigate whether BRs act directly in self-incompatibility, BRs were added to in vitro pollen cultures of the two mating types. A small morph specific stimulatory effect on pollen tube growth was found with 5 µM brassinolide, but no genotype specific inhibition was observed. These results suggest that BAHD acts pleiotropically to mediate pistil length and physiological mating type through BR inactivation, and that in regard to self-incompatibility, BR acts by differentially regulating gene expression in pistils, rather than directly on pollen.

Overexpression of AtAHL20 causes delayed flowering in Arabidopsis via repression of FT expression.

BACKGROUND: The 29-member Arabidopsis AHL gene family is classified into three main classes based on nucleotide and protein sequence evolutionary differences. These differences include the presence or absence of introns, type and/or number of conserved AT-hook and PPC domains. AHL gene family members are divided into two phylogenetic clades, Clade-A and Clade-B. A majority of the 29 members remain functionally uncharacterized. Furthermore, the biological significance of the DNA and peptide sequence diversity, observed in the conserved motifs and domains found in the different AHL types, is a subject area that remains largely unexplored. RESULTS: Transgenic plants overexpressing AtAHL20 flowered later than the wild type under both short and long days. Transcript accumulation analyses showed that 35S:AtAHL20 plants contained reduced FT, TSF, AGL8 and SPL3 mRNA levels. Similarly, overexpression of AtAHL20's orthologue in Camelina sativa, Arabidopsis' closely related Brassicaceae family member species, conferred a late-flowering phenotype via suppression of CsFT expression. However, overexpression of an aberrant AtAHL20 gene harboring a missense mutation in the AT-hook domain's highly conserved R-G-R core motif abolished the late-flowering phenotype. Data from targeted yeast-two-hybrid assays showed that AtAHL20 interacted with itself and several other Clade-A Type-I AHLs which have been previously implicated in flowering-time regulation: AtAHL19, AtAHL22 and AtAHL29. CONCLUSION: We showed via gain-of-function analysis that AtAHL20 is a negative regulator of FT expression, as well as other downstream flowering time regulating genes. A similar outcome in Camelina sativa transgenic plants overexpressing CsAHL20 suggest that this is a conserved function. Our results demonstrate that AtAHL20 acts as a photoperiod-independent negative regulator of transition to flowering.

Self-transcriptional repression of the Arabidopsis NAC transcription factor ATAF2 and its genetic interaction with phytochrome A in modulating seedling photomorphogenesis.

MAIN CONCLUSION: The NAC transcription factor ATAF2 suppresses its own transcription via self-promoter binding. ATAF2 genetically interacts with the circadian regulator CCA1 and phytochrome A to modulate seedling photomorphogenesis in Arabidopsis thaliana. ATAF2 (ANAC081) is a NAC (NAM, ATAF and CUC) transcription factor (TF) that participates in the regulation of disease resistance, stress tolerance and hormone metabolism in Arabidopsis thaliana. We previously reported that ATAF2 promotes Arabidopsis hypocotyl growth in a light-dependent manner via transcriptionally suppressing the brassinosteroid (BR)-inactivating cytochrome P450 genes BAS1 (CYP734A1, formerly CYP72B1) and SOB7 (CYP72C1). Assays using low light intensities suggest that the photoreceptor phytochrome A (PHYA) may play a more critical role in ATAF2-regulated photomorphogenesis than phytochrome B (PHYB) and cryptochrome 1 (CRY1). In addition, ATAF2 is also regulated by the circadian clock. The core circadian TF CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) physically interacts with ATAF2 at the DNA-protein and protein-protein levels, and both differentially suppress BAS1- and SOB7-mediated BR catabolism. In this research, we show that ATAF2 can bind its own promoter as a transcriptional self-repressor. This self-feedback-suppression loop is a typical feature of multiple circadian-regulated genes. Additionally, ATAF2 and CCA1 synergistically suppress seedling photomorphogenesis as reflected by the light-dependent hypocotyl growth analysis of their single and double gene knock-out mutants. Similar fluence-rate response assays using ATAF2 and photoreceptor (PHYB, CRY1 and PHYA) knock-out mutants demonstrate that PHYA is required for ATAF2-regulated photomorphogenesis in a wide range of light intensities. Furthermore, disruption of PHYA can suppress the BR-insensitive hypocotyl-growth phenotype of ATAF2 loss-of-function seedlings in the light, but not in darkness. Collectively, our results provide a genetic interaction synopsis of the circadian-clock-photomorphogenesis-BR integration node involving ATAF2, CCA1 and PHYA.

CIRCADIAN CLOCK ASSOCIATED 1 and ATAF2 differentially suppress cytochrome P450-mediated brassinosteroid inactivation.

Brassinosteroids (BRs) are a group of steroid hormones regulating plant growth and development. Since BRs do not undergo transport among plant tissues, their metabolism is tightly regulated by transcription factors (TFs) and feedback loops. BAS1 (CYP734A1, formerly CYP72B1) and SOB7 (CYP72C1) are two BR-inactivating cytochrome P450s identified in Arabidopsis thaliana. We previously found that a TF ATAF2 (ANAC081) suppresses BAS1 and SOB7 expression by binding to the Evening Element (EE) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)-binding site (CBS) on their promoters. Both the EE and CBS are known binding targets of the circadian regulatory protein CCA1. Here, we confirm that CCA1 binds the EE and CBS motifs on BAS1 and SOB7 promoters, respectively. Elevated accumulations of BAS1 and SOB7 transcripts in the CCA1 null mutant cca1-1 indicate that CCA1 is a repressor of their expression. When compared with either cca1-1 or the ATAF2 null mutant ataf2-2, the cca1-1 ataf2-2 double mutant shows higher SOB7 transcript accumulations and a stronger BR-insensitive phenotype of hypocotyl elongation in white light. CCA1 interacts with ATAF2 at both DNA-protein and protein-protein levels. ATAF2, BAS1, and SOB7 are all circadian regulated with distinct expression patterns. These results demonstrate that CCA1 and ATAF2 differentially suppress BAS1- and SOB7-mediated BR inactivation.

Improving seed size, seed weight and seedling emergence in Camelina sativa by overexpressing the Atsob3-6 gene variant.

Seedling stand establishment is a critical factor affecting crop yield in low-precipitation agricultural regions. This is especially true for small seeded crops, such as Camelina (Camelina sativa) and canola (Brassica napus), that need to be planted shallow. Deeper planting would be desirable so that seeds can access soil moisture and bigger seeds could improve emergence and stand establishment by providing the energy necessary for seedling elongation. AHL (AT-Hook Containing, Nuclear Localized) genes play an important role in seedling growth and development. AHL proteins contain two structural units, the DNA-binding AT-hook motif and the Plant and Prokaryote Conserved (PPC) domain, required for protein-protein interactions. Our previous studies demonstrate that AtAHL29/SOB3 (Suppressor of phytochrome B-4 #3) regulates seedling development in Arabidopsis (Arabidopsis thaliana). Activation-tagged overexpression of AtSOB3 (Atsob3-D) represses the long-hypocotyl phenotype of an Arabidopsis phytochrome B mutant. In contrast, overexpression of the Atsob3-6 variant (Atsob3-6-OX), with a non-functional AT-hook, confers a long-hypocotyl phenotype. In this study, we demonstrate the role of Atsob3-D and Atsob3-6-OX in modulating seed size and hypocotyl length in the brassicas Arabidopsis and Camelina. In Arabidopsis, Atsob3-D reduces seed weight whereas Atsob3-6-OX increases seed weight and size when compared to the wild type. Similarly, Atsob3-6-OX transgenic Camelina seedlings are taller than the wild type, and produce larger and heavier seeds. These larger Atsob3-6-OX Camelina seeds also confer better emergence in deep-soil planting when compared to the wild type. Taken together, Atsob3-6-OX increases seed size, seed weight, seedling hypocotyl length and stand establishment in the oilseed crop Camelina.

Brassinosteroid signaling converges with SUPPRESSOR OF PHYTOCHROME B4-#3 to influence the expression of SMALL AUXIN UP RNA genes and hypocotyl growth.

Interactions between signaling pathways help guide plant development. In this study, we found that brassinosteroid (BR) signaling converges with SUPPRESSOR OF PHYTOCHROME B4-#3 (SOB3) to influence both the transcription of genes involved in cell elongation and hypocotyl growth. Specifically, SOB3 mutant hypocotyl phenotypes, which are readily apparent when the seedlings are grown in dim white light, were attenuated by treatment with either brassinolide (BL) or the BR biosynthesis inhibitor brassinazole (BRZ). Hypocotyls of SOB3 mutant seedlings grown in white light with a higher fluence rate also exhibited altered sensitivities to BL, further suggesting a connection to BR signaling. However, the impact of BL treatment on SOB3 mutants grown in moderate-intensity white light was reduced when polar auxin transport was inhibited. BL treatment enhanced transcript accumulation for all six members of the SMALL AUXIN UP RNA19 (SAUR19) subfamily, which promote cell expansion, are repressed by SOB3 and light, and are induced by auxin. Conversely, BRZ inhibited the expression of SAUR19 and its homologs. Expression of these SAURs was also enhanced in lines expressing a constitutively active form of the BR signaling component BZR1, further indicating that the transcription of SAUR19 subfamily members are influenced by this hormone signaling pathway. Taken together, these results indicate that SOB3 and BR signaling converge to influence the transcription of hypocotyl growth-promoting SAUR19 subfamily members.

ATAF2 integrates Arabidopsis brassinosteroid inactivation and seedling photomorphogenesis.

The Arabidopsis thaliana hypocotyl is a robust system for studying the interplay of light and plant hormones, such as brassinosteroids (BRs), in the regulation of plant growth and development. Since BRs cannot be transported between plant tissues, their cellular levels must be appropriate for given developmental fates. BR homeostasis is maintained in part by transcriptional feedback regulation loops that control the expression of key metabolic enzymes, including the BR-inactivating enzymes BAS1 (CYP734A1, formerly CYP72B1) and SOB7 (CYP72C1). Here, we find that the NAC transcription factor (TF) ATAF2 binds the promoters of BAS1 and SOB7 to suppress their expression. ATAF2 restricts the tissue-specific expression of BAS1 and SOB7 in planta. ATAF2 loss- and gain-of-function seedlings have opposite BR-response phenotypes for hypocotyl elongation. ATAF2 modulates hypocotyl growth in a light-dependent manner, with the photoreceptor phytochrome A playing a major role. The photomorphogenic phenotypes of ATAF2 loss- and gain-of-function seedlings are suppressed by treatment with the BR biosynthesis inhibitor brassinazole. Moreover, the disruption of BAS1 and SOB7 abolishes the short-hypocotyl phenotype of ATAF2 loss-of-function seedlings in low fluence rate white light, demonstrating an ATAF2-mediated connection between BR catabolism and photomorphogenesis. ATAF2 expression is suppressed by both BRs and light, which demonstrates the existence of an ATAF2-BAS1/SOB7-BR-ATAF2 feedback regulation loop, as well as a light-ATAF2-BAS1/SOB7-BR-photomorphogenesis pathway. ATAF2 also modulates root growth by regulating BR catabolism. As it is known to regulate plant defense and auxin biosynthesis, ATAF2 therefore acts as a central regulator of plant defense, hormone metabolism and light-mediated seedling development.

SUPPRESSOR OF PHYTOCHROME B4-#3 Represses Genes Associated with Auxin Signaling to Modulate Hypocotyl Growth.

Developing seedlings are well equipped to alter their growth in response to external factors in order to maximize their chances of survival. SUPPRESSOR OF PHYTOCHROME B4-#3 (SOB3) and other members of the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) family of transcription factors modulate the development of Arabidopsis (Arabidopsis thaliana) by repressing hypocotyl elongation in young seedlings growing in light. However, the molecular mechanism behind how AHLs influence seedling development is largely unknown. We have identified genes associated with auxin-mediated hypocotyl elongation as downstream targets of SOB3. We found that YUCCA8 (YUC8) as well as members of the SMALL AUXIN UP-REGULATED RNA19 (SAUR19) subfamily were down-regulated in the short-hypocotyl, gain-of-function SOB3-D mutant and up-regulated in the dominant-negative, tall-hypocotyl sob3-6 mutant. SOB3-D and sob3-6 hypocotyls also exhibited altered sensitivity to the polar auxin transport inhibitor N-1-napthylphthalamic acid, suggesting a critical connection between auxin and the modulation of seedling elongation by SOB3 Finally, we found that overexpression of GREEN FLUORESCENT PROTEIN-SAUR19 in the SOB3-D line partially rescued defects in hypocotyl elongation, and SOB3 bound directly to the promoters of YUC8 and SAUR19 subfamily members. Taken together, these data indicate that SOB3 modulates hypocotyl elongation in young seedlings by directly repressing the transcription of genes associated with auxin signaling.

Arabidopsis thaliana AHL family modulates hypocotyl growth redundantly by interacting with each other via the PPC/DUF296 domain.

The Arabidopsis thaliana genome encodes 29 AT-hook motif containing nuclear localized (AHL) genes, which evolved into two phylogenic clades. The AHL proteins contain one or two AT-hook motif(s) and one plant and prokaryote conserved (PPC)/domain of unknown function #296 (DUF296) domain. Seedlings lacking both SOB3/AHL29 and ESC/AHL27 confer a subtle long-hypocotyl phenotype compared with the WT or either single-null mutant. In contrast, the missense allele sob3-6 confers a dramatic long-hypocotyl phenotype in the light. In this study, we examined the dominant-negative feature of sob3-6 and found that it encodes a protein with a disrupted AT-hook motif that abolishes binding to AT-rich DNA. A loss-of-function approach demonstrated different, yet redundant, contributions of additional AHL genes in suppressing hypocotyl elongation in the light. We showed that AHL proteins interact with each other and themselves via the PPC/DUF296 domain. AHLs also share interactions with other nuclear proteins, such as transcription factors, suggesting that these interactions also contribute to the functional redundancy within this gene family. The coordinated action of AHLs requires an AT-hook motif capable of binding AT-rich DNA, as well as a PPC/DUF296 domain containing a conserved Gly-Arg-Phe-Glu-Ile-Leu region. Alteration of this region abolished SOB3/AHL29's physical interaction with transcription factors and resulted in a dominant-negative allele in planta that was phenotypically similar to sob3-6. We propose a molecular model where AHLs interact with each other and themselves, as well as other nuclear proteins, to form complexes which modulate plant growth and development.

Arabidopsis lateral organ boundaries negatively regulates brassinosteroid accumulation to limit growth in organ boundaries.

Leaves and flowers begin life as outgrowths from the edges of shoot apical meristems. Stem cell divisions in the meristem center replenish cells that are incorporated into organ primordia at the meristem periphery and leave the meristem. Organ boundaries, regions of limited growth that separate forming organs from the meristem, serve to isolate these two domains and are critical for coordination of organogenesis and meristem maintenance. Boundary formation and maintenance are poorly understood processes, despite the identification of a number of boundary-specific transcription factors. Here we provide genetic and biochemical evidence that the Arabidopsis thaliana transcription factor lateral organ boundaries (LOB) negatively regulates accumulation of the plant steroid hormone brassinosteroid (BR) in organ boundaries. We found that ectopic expression of LOB results in reduced BR responses. We identified BAS1, which encodes a BR-inactivating enzyme, as a direct target of LOB transcriptional activation. Loss-of-function lob mutants exhibit organ fusions, and this phenotype is suppressed by expression of BAS1 under the LOB promoter, indicating that BR hyperaccumulation contributes to the lob mutant phenotype. In addition, LOB expression is BR regulated; therefore, LOB and BR form a feedback loop to modulate local BR accumulation in organ boundaries to limit growth in the boundary domain.

Insights into the evolution and diversification of the AT-hook Motif Nuclear Localized gene family in land plants.

BACKGROUND: Members of the ancient land-plant-specific transcription factor AT-Hook Motif Nuclear Localized (AHL) gene family regulate various biological processes. However, the relationships among the AHL genes, as well as their evolutionary history, still remain unexplored. RESULTS: We analyzed over 500 AHL genes from 19 land plant species, ranging from the early diverging Physcomitrella patens and Selaginella to a variety of monocot and dicot flowering plants. We classified the AHL proteins into three types (Type-I/-II/-III) based on the number and composition of their functional domains, the AT-hook motif(s) and PPC domain. We further inferred their phylogenies via Bayesian inference analysis and predicted gene gain/loss events throughout their diversification. Our analyses suggested that the AHL gene family emerged in embryophytes and further evolved into two distinct clades, with Type-I AHLs forming one clade (Clade-A), and the other two types together diversifying in another (Clade-B). The two AHL clades likely diverged before the separation of Physcomitrella patens from the vascular plant lineage. In angiosperms, Clade-A AHLs expanded into 5 subfamilies; while, the ones in Clade-B expanded into 4 subfamilies. Examination of their expression patterns suggests that the AHLs within each clade share similar expression patterns with each other; however, AHLs in one monophyletic clade exhibit distinct expression patterns from the ones in the other clade. Over-expression of a Glycine max AHL PPC domain in Arabidopsis thaliana recapitulates the phenotype observed when over-expressing its Arabidopsis thaliana counterpart. This result suggests that the AHL genes from different land plant species may share conserved functions in regulating plant growth and development. Our study further suggests that such functional conservation may be due to conserved physical interactions among the PPC domains of AHL proteins. CONCLUSIONS: Our analyses reveal a possible evolutionary scenario for the AHL gene family in land plants, which will facilitate the design of new studies probing their biological functions. Manipulating the AHL genes has been suggested to have tremendous effects in agriculture through increased seedling establishment, enhanced plant biomass and improved plant immunity. The information gleaned from this study, in turn, has the potential to be utilized to further improve crop production.

The ability of Arabidopsis to recover from Basta and its application in isolating Cas9-free mutants.

After successfully performing Agrobacterium-mediated CRISPR-Cas9-based gene editing in plants, isolation of the Cas9 T-DNA is essential for the stable inheritance of induced mutations. Here, we report a simple technique that allows the isolation of Cas9-free mutants, eliminating the need for outcrossing or other intricate methods. This method is based on the ability of Basta-sensitive Arabidopsis thaliana seedlings, which generally perish, to recover and grow once transplanted to Basta-free growth media. By growing gene-edited heterozygous populations of single-locus insertion Basta-resistant plants on Basta selection media, plants lacking the Cas9 T-DNA can be identified. These pale-looking plants lacking Cas9 are then rescued on media lacking the Basta to recover Cas9-free plants. The ability of seedlings to recover from Basta selection was also studied in camelina, canola, and wheat. All three crops showed different recovery rates, with wheat demonstrating the highest recovery once transplanted from Basta to normal growth media. In summary, our findings demonstrate that by harnessing the recovery capability of Basta-sensitive seedlings, we can effectively identify and rescue plants lacking the Cas9 T-DNA, enabling the isolation of Cas9-free mutants in Arabidopsis and potentially extending to other crops.

The NAC transcription factor ATAF2 promotes ethylene biosynthesis and response in Arabidopsis thaliana seedlings.

Arabidopsis thaliana activating factor 2 (ATAF2) plays extensive regulatory roles in pathogenesis, seedling development, and stress responses. Here, we performed transcriptome analysis on ATAF2 loss- and gain-of-function mutants to identify differentially expressed genes (DEGs). Gene ontology analyses on DEGs reveal that ATAF2 enhances seedling responses to multiple hormone and stress signals. In particular, our transcriptome analysis suggests that ATAF2 promotes ethylene biosynthesis and responses via activating relevant genes. This novel role of ATAF2 was further demonstrated by using multiple ATAF2-null and overexpression lines for reverse transcription quantitative PCR verification, ethylene production measurements, and assays of seedlings growth responses to the ethylene immediate biosynthetic precursor 1-aminocyclopropane-1-carboxylic acid (ACC). ACC suppresses ATAF2 expression to form a negative feedback regulation loop.

Rice CYP734A cytochrome P450s inactivate brassinosteroids in Arabidopsis.

Endogenous brassinosteroid concentrations are an important target for optimizing the growth of crop plants because these hormones influence yield and stress tolerance. The CYP734A subfamily of cytochrome P450 enzymes has been shown to inactivate brassinosteroid hormones in Arabidopsis and tomato. Rice has three genes for CYP734A enzymes whose expression appears to be up-regulated by exogenous brassinolide. The amino acids predicted to be in the active site of the rice enzymes vary when compared with the Arabidopsis protein sequence, suggesting that there could be differences in their ability to inactivate the hormone. We have cloned three CYP734A rice genes and expressed them in Arabidopsis to assess their efficacy as brassinosteroid-inactivating enzymes. We found that incorrect transcript splicing can complicate the expression of monocot genomic clones in a eudicot. However, the Arabidopsis system allowed us to characterize an atypical splice variant in one of the rice genes. cDNA clones produced high levels of expression and conferred the brassinosteroid inactivation phenotype. This study shows that Arabidopsis is a useful heterologous system for testing plant genes predicted to act in biochemical pathways that are conserved between monocots and eudicots.

Integration of light and abscisic acid signaling during seed germination and early seedling development.

Seed germination is regulated by endogenous hormonal cues and external environmental stimuli such as water, low temperature, and light. After germination, the young seedling must rapidly establish its root system and the photoautotrophic capability appropriate to its surrounding environment. Light and the phytohormone abscisic acid (ABA) both regulate seed germination and seedling development, although how light and ABA signals are integrated at the molecular level is not understood. Here, we found that the previously described light-signaling component HY5 also mediates ABA response in seed germination, early seedling growth, and root development in Arabidopsis. HY5 binds to the promoter of the transcription factor ABI5 gene with high affinity and is required for the expression of ABI5 and ABI5-targeted late embryogenesis-abundant genes in seeds. Chromatin immunoprecipitation also indicated that the binding of HY5 to the ABI5 promoter is significantly enhanced by ABA. Overexpression of ABI5 restores ABA sensitivity in hy5 and results in enhanced light responses and shorter hypocotyls in the wild type. Our studies identified an unexpected mode of light and ABA signal integration that may help young seedlings better adapt to environmental stresses.

Arabidopsis CYP72C1 is an atypical cytochrome P450 that inactivates brassinosteroids.

Cytochrome P450 monooxygenases (P450s) are a diverse family of proteins that have specialized roles in secondary metabolism and in normal cell development. Two P450s in particular, CYP734A1 and CYP72C1, have been identified as brassinosteroid-inactivating enzymes important for steroid-mediated signal transduction in Arabidopsis thaliana. Genetic analyses have demonstrated that these P450s modulate growth throughout plant development. While members of the CYP734A subfamily inactivate brassinosteroids through C-26 hydroxylation, the biochemical activity of CYP72C1 is unknown. Because CYP734A1 and CYP72C1 in Arabidopsis diverge more than brassinosteroid inactivating P450s in other plants, this study examines the structure and biochemistry of each enzyme. Three-dimensional models were generated to examine the substrate binding site structures and determine how they might affect the function of each P450. These models have indicated that the active site of CYP72C1 does not contain several conserved amino acids typically needed for substrate hydroxylation. Heterologous expression of these P450s followed by substrate binding analyses have indicated that CYP734A1 binds active brassinosteroids, brassinolide and castasterone, as well as their upstream precursors whereas CYP72C1 binds precursors more effectively. Seedling growth assays have demonstrated that the genetic state of CYP734A1, but not CYP72C1, affected responsiveness to high levels of exogenous brassinolide supporting our observations that CYP72C1 acts on brassinolide precursors. Although there may be some overlap in their physiological function, the distinct biochemical functions of these proteins in Arabidopsis has significant potential to fine-tune the levels of different brassinosteroid hormones throughout plant growth and development.

Emerging Molecular Links Between Plant Photomorphogenesis and Virus Resistance.

Photomorphogenesis refers to photoreceptor-mediated morphological changes in plant development that are triggered by light. Multiple photoreceptors and transcription factors (TFs) are involved in the molecular regulation of photomorphogenesis. Likewise, light can also modulate the outcome of plant-virus interactions since both photosynthesis and many viral infection events occur in the chloroplast. Despite the apparent association between photosynthesis and virus infection, little is known about whether there are also interplays between photomorphogenesis and plant virus resistance. Recent research suggests that plant-virus interactions are potentially regulated by several photoreceptors and photomorphogenesis regulators, including phytochromes A and B (PHYA and PHYB), cryptochromes 2 (CRY2), phototropin 2 (PHOT2), the photomorphogenesis repressor constitutive photomorphogenesis 1 (COP1), the NAM, ATAF, and CUC (NAC)-family TF ATAF2, the Aux/IAA protein phytochrome-associated protein 1 (PAP1), the homeodomain-leucine zipper (HD-Zip) TF HAT1, and the core circadian clock component circadian clock associated 1 (CCA1). Particularly, the plant growth promoting brassinosteroid (BR) hormones play critical roles in integrating the regulatory pathways of plant photomorphogenesis and viral defense. Here, we summarize the current understanding of molecular mechanisms linking plant photomorphogenesis and defense against viruses, which represents an emerging interdisciplinary research topic in both molecular plant biology and virology.

The Turnera Style S-Locus Gene TsBAHD Possesses Brassinosteroid-Inactivating Activity When Expressed in Arabidopsis thaliana.

Heterostyly distinct hermaphroditic floral morphs enforce outbreeding. Morphs differ structurally, promote cross-pollination, and physiologically block self-fertilization. In Turnera the self-incompatibility (S)-locus controlling heterostyly possesses three genes specific to short-styled morph genomes. Only one gene, TsBAHD, is expressed in pistils and this has been hypothesized to possess brassinosteroid (BR)-inactivating activity. We tested this hypothesis using heterologous expression in Arabidopsis thaliana as a bioassay, thereby assessing growth phenotype, and the impacts on the expression of endogenous genes involved in BR homeostasis and seedling photomorphogenesis. Transgenic A. thaliana expressing TsBAHD displayed phenotypes typical of BR-deficient mutants, with phenotype severity dependent on TsBAHD expression level. BAS1, which encodes an enzyme involved in BR inactivation, was downregulated in TsBAHD-expressing lines. CPD and DWF, which encode enzymes involved in BR biosynthesis, were upregulated. Hypocotyl growth of TsBAHD dwarfs responded to application of brassinolide in light and dark in a manner typical of plants over-expressing genes encoding BR-inactivating activity. These results provide empirical support for the hypothesis that TsBAHD possesses BR-inactivating activity. Further this suggests that style length in Turnera is controlled by the same mechanism (BR inactivation) as that reported for Primula, but using a different class of enzyme. This reveals interesting convergent evolution in a biochemical mechanism to regulate floral form in heterostyly.