AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED Research Articles

Identification of tomato AHL gene families and functional analysis their roles in fruit development and abiotic stress response

The AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) transcription factors play important roles in regulating plant development and stress response. However, the AHL family genes have not been identified in tomato (Solanum lycopersicum) and their biological functions have not been elucidated. In this work, the gene families encoding AHLs were identified in tomato genome, and their physical and chemical characteristics, subcellular localization, gene expression profiles during fruit development and upon abiotic stimulus were investigated. Overall, a total of 18 AHL members were identified in tomato genome, phylogenetic analysis classified these SlAHL members into two clades, clade A (SlAHL1-8) and clade B (SlAHL9-18). Six clade A SlAHLs were detected to be subcellular localized in the nucleus. The transcripts of the representative clade A SlAHLs predominantly accumulated 10 days post anthesis (dpa) in tomato fruits, revealing an involvement of these SlAHLs in early fruit development. Furthermore, compared with clade B members, the transcripts of the clade A SlAHLs were more responsive to heat, drought, cold and salt stresses, suggesting that these SlAHLs may play major roles in response to abiotic stresses. Moreover, overexpression of SlAHL1 and SlAHL7 in Arabidopsis increased the sensitivity to ABA during seed germination and seedling stages. Overexpression of SlAHL1 inhibited seed germination while increased primary root elongation upon salt and drought stresses. Together, our work suggested that the clade A SlAHL genes may play an important role in response to abiotic stresses, which paving the way for future functional analysis of AHL genes in tomato and other Solanaceae species.

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 AT-Hook proteins regulate A/NINV7 expression to modulate sucrose catabolism for cold tolerance in Poncirus trifoliata.

Invertase (INV)-mediated sucrose (Suc) hydrolysis, leading to the irreversible production of glucose (Glc) and fructose (Frc), plays an essential role in abiotic stress tolerance of plants. However, the regulatory network associated with the Suc catabolism in response to cold environment remains largely elusive. Herein, the cold-induced alkaline/neutral INV gene PtrA/NINV7 of trifoliate orange (Poncirus trifoliata (L.) Raf.) was shown to function in cold tolerance via mediating the Suc hydrolysis. Meanwhile, a nuclear matrix-associated region containing A/T-rich sequences within its promoter was indispensable for the cold induction of PtrA/NINV7. Two AT-Hook Motif Containing Nuclear Localized (AHL) proteins, PtrAHL14 and PtrAHL17, were identified as upstream transcriptional activators of PtrA/NINV7 by interacting with the A/T-rich motifs. PtrAHL14 and PtrAHL17 function positively in the cold tolerance by modulating PtrA/NINV7-mediated Suc catabolism. Furthermore, both PtrAHL14 and PtrAHL17 could form homo- and heterodimers between each other, and interacted with two histone acetyltransferases (HATs), GCN5 and TAF1, leading to elevated histone3 acetylation level under the cold stress. Taken together, our findings unraveled a new cold-responsive signaling module (AHL14/17-HATs-A/NINV7) for orchestration of Suc catabolism and cold tolerance, which shed light on the molecular mechanisms underlying Suc catabolism catalyzed by A/NINVs under cold stress.

Control of cambium initiation and activity in Arabidopsis by the transcriptional regulator AHL15

Plant secondary growth, which is the basis of wood formation, includes the production of secondary xylem, which is derived from meristematic cambium cells embedded in vascular tissue. Here, we identified an important role for the Arabidopsis thaliana (Arabidopsis) AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED 15 (AHL15) transcriptional regulator in controlling vascular cambium activity. The limited secondary xylem development in inflorescence stems of herbaceous Arabidopsis plants was significantly reduced in ahl15 loss-of-function mutants, whereas constitutive or vascular meristem-specific AHL15 overexpression produced woody inflorescence stems. AHL15 was required for enhanced secondary xylem formation in the woody suppressor of overexpression of constans 1 (soc1) fruitfull (ful) double loss-of-function mutant. Moreover, we found that AHL15 induces vascular cambium activity downstream of the repressing SOC1 and FUL transcription factors, most likely similar to how it enhances lateral branching by promoting biosynthesis of the hormone cytokinin. Our results uncover a novel pathway driving cambium development, in which AHL15 expression levels act in parallel to and are dependent on the well-established TDIF-PXY-WOX pathway to differentiate between herbaceous and woody stem growth.

Characteristics of the AT-Hook Motif Containing Nuclear Localized (AHL) Genes in Carrot Provides Insight into Their Role in Plant Growth and Storage Root Development.

The AT-hook motif containing nuclear localized (AHL) gene family, controlling various developmental processes, is conserved in land plants. They comprise Plant and Prokaryote Conserved (PPC) domain and one or two AT-hook motifs. DcAHLc1 has been proposed as a candidate gene governing the formation of the carrot storage root. We identified and in-silico characterized carrot AHL proteins, performed phylogenetic analyses, investigated their expression profiles and constructed gene coexpression networks. We found 47 AHL genes in carrot and grouped them into two clades, A and B, comprising 29 and 18 genes, respectively. Within Clade-A, we distinguished three subclades, one of them grouping noncanonical AHLs differing in their structure (two PPC domains) and/or cellular localization (not nucleus). Coexpression network analysis attributed AHLs expressed in carrot roots into four of the 72 clusters, some of them showing a large number of interactions. Determination of expression profiles of AHL genes in various tissues and samples provided basis to hypothesize on their possible roles in the development of the carrot storage root. We identified a group of rapidly evolving noncanonical AHLs, possibly differing functionally from typical AHLs, as suggested by their expression profiles and their predicted cellular localization. We pointed at several AHLs likely involved in the development of the carrot storage root.

An Arabidopsis AT-hook motif nuclear protein mediates somatic embryogenesis and coinciding genome duplication

Plant somatic cells can be reprogrammed into totipotent embryonic cells that are able to form differentiated embryos in a process called somatic embryogenesis (SE), by hormone treatment or through overexpression of certain transcription factor genes, such as BABY BOOM (BBM). Here we show that overexpression of the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED 15 (AHL15) gene induces formation of somatic embryos on Arabidopsis thaliana seedlings in the absence of hormone treatment. During zygotic embryogenesis, AHL15 expression starts early in embryo development, and AH15 and other AHL genes are required for proper embryo patterning and development beyond the globular stage. Moreover, AHL15 and several of its homologs are upregulated and required for SE induction upon hormone treatment, and they are required for efficient BBM-induced SE as downstream targets of BBM. A significant number of plants derived from AHL15 overexpression-induced somatic embryos are polyploid. Polyploidisation occurs by endomitosis specifically during the initiation of SE, and is caused by strong heterochromatin decondensation induced by AHL15 overexpression.

Chromatin phosphoproteomics unravels a function for AT-hook motif nuclear localized protein AHL13 in PAMP-triggered immunity

In many eukaryotic systems during immune responses, mitogen-activated protein kinases (MAPKs) link cytoplasmic signaling to chromatin events by targeting transcription factors, chromatin remodeling complexes, and the RNA polymerase machinery. So far, knowledge on these events is scarce in plants and no attempts have been made to focus on phosphorylation events of chromatin-associated proteins. Here we carried out chromatin phosphoproteomics upon elicitor-induced activation of Arabidopsis The events in WT were compared with those in mpk3, mpk4, and mpk6 mutant plants to decipher specific MAPK targets. Our study highlights distinct signaling networks involving MPK3, MPK4, and MPK6 in chromatin organization and modification, as well as in RNA transcription and processing. Among the chromatin targets, we characterized the AT-hook motif containing nuclear localized (AHL) DNA-binding protein AHL13 as a substrate of immune MAPKs. AHL13 knockout mutant plants are compromised in pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species production, expression of defense genes, and PAMP-triggered immunity. Transcriptome analysis revealed that AHL13 regulates key factors of jasmonic acid biosynthesis and signaling and affects immunity toward Pseudomonas syringae and Botrytis cinerea pathogens. Mutational analysis of the phosphorylation sites of AHL13 demonstrated that phosphorylation regulates AHL13 protein stability and thereby its immune functions.

Transcriptional Regulation of Lipid Catabolism during Seedling Establishment

Lipid catabolism in germinating seeds provides energy and substrates for initial seedling growth, but how this process is regulated is not well understood. Here, we show that an AT-hook motif-containing nuclear localized (AHL) protein regulates lipid mobilization and fatty acid β-oxidation during seed germination and seedling establishment. AHL4 was identified to directly interact with the lipid mediator phosphatidic acid (PA). Knockout (KO) of AHL4 enhanced, but overexpression (OE) of AHL4 attenuated, triacylglycerol (TAG) degradation and seedling growth. Normal seedling growth of the OE lines was restored by sucrose supplementation to the growth medium. AHL4-OE seedlings displayed decreased expression of genes involved in TAG hydrolysis and fatty acid oxidation, whereas the opposite was observed in AHL4-KOs. These genes contained AHL4-binding cis elements, and AHL4 was shown to bind to the promoter regions of genes encoding the TAG lipases SDP1 and DALL5 and acyl-thioesterase KAT5. These AHL4–DNA interactions were suppressed by PA species that bound to AHL4. These results indicate that AHL4 suppresses lipid catabolism by repressing the expression of specific genes involved in TAG hydrolysis and fatty acid oxidation, and that PA relieves AHL4-mediated suppression and promotes TAG degradation. Thus, AHL4 and PA together regulate lipid degradation during seed germination and seedling establishment.

Genome-wide identification and analyses of the AHL gene family in cotton (Gossypium)

BackgroundMembers of the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) family are involved in various plant biological processes via protein-DNA and protein-protein interaction. However, no the systematic identification and analysis of AHL gene family have been reported in cotton.ResultsTo investigate the potential functions of AHLs in cotton, genome-wide identification, expressions and structure analysis of the AHL gene family were performed in this study. 48, 51 and 99 AHL genes were identified from the G.raimondii, G.arboreum and G.hirsutum genome, respectively. Phylogenetic analysis revealed that the AHLs in cotton evolved into 2 clades, Clade-A with 4–5 introns and Clade-B with intronless (excluding AHL20–2). Based on the composition of the AT-hook motif(s) and PPC/DUF 296 domain, AHL proteins were classified into three types (Type-I/−II/−III), with Type-I AHLs forming Clade-B, and the other two types together diversifying in Clade-A. The detection of synteny and collinearity showed that the AHLs expanded with the specific WGD in cotton, and the sequence structure of AHL20–2 showed the tendency of increasing intron in three different Gossypium spp. The ratios of non-synonymous (Ka) and synonymous (Ks) substitution rates of orthologous gene pairs revealed that the AHL genes of G.hirsutum had undergone through various selection pressures, purifying selection mainly in A-subgenome and positive selection mainly in D-subgenome. Examination of their expression patterns showed most of AHLs of Clade-B expressed predominantly in stem, while those of Clade-A in ovules, suggesting that the AHLs within each clade shared similar expression patterns with each other. qRT-PCR analysis further confirmed that some GhAHLs higher expression in stems and ovules.ConclusionIn this study, 48, 51 and 99 AHL genes were identified from three cotton genomes respectively. AHLs in cotton were classified into two clades by phylogenetic relationship and three types based on the composition of motif and domain. The AHLs expanded with segmental duplication, not tandem duplication. The expression profiles of GhAHLs revealed abundant differences in expression levels in various tissues and at different stages of ovules development. Our study provided significant insights into the potential functions of AHLs in regulating the growth and development in cotton.

MQTL-seq and classical mapping implicates the role of an AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) family gene in Ascochyta blight resistance of chickpea.

Ascochyta blight (AB) caused by the fungal pathogen Ascochyta rabiei is a serious foliar disease of chickpea (Cicer arietinum L.). Despite many genetic studies on chickpea-Ascochyta interaction, genome-wide scan of chickpea for the identification of AB-associated quantitative trait loci (QTLs) and their gene(s) has not been accomplished. To elucidate narrow QTLs for AB resistance, here, we report the use of multiple QTL-sequencing approach on 2 sets of extreme AB phenotype bulks derived from Cicer intraspecific and interspecific crosses. Two major QTLs, qABR4.1 and qABR4.2, and a minor QTL, qABR4.3, were identified on assembled chickpea pseudomolecule 4. We narrowed qABR4.1 to a "robust region" at 4.568-4.618Mb through mapping on a larger intraspecific cross-derived population and comparative analysis. Among 4 genes, the CaAHL18 gene showed higher expression under Ascochyta stress in AB resistant parent suggesting that it is the candidate gene under "robust qABR4.1." Dual-luciferase assay with CaAHL18 polymorphic cis-regulatory sequences showed that allelic variation is associated with higher expression. Thus, our findings on chickpea-Ascochyta interaction have narrowed down AB resistance associated QTLs on chickpea physical map. The narrowed QTLs and gene-associated markers will help in biotechnological and breeding programs for chickpea improvement.

Coordination of matrix attachment and ATP-dependent chromatin remodeling regulate auxin biosynthesis and Arabidopsis hypocotyl elongation.

Hypocotyl elongation is extensively controlled by hormone signaling networks. In particular, auxin metabolism and signaling play key roles in light-dependent hypocotyl growth. The nuclear matrix facilitates organization of DNA within the nucleus, and dynamic interactions between nuclear matrix and DNA are related to gene regulation. Conserved scaffold/matrix attachment regions (S/MARs) are anchored to the nuclear matrix by the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) proteins in Arabidopsis. Here, we found that ESCAROLA (ESC)/AHL27 and SUPPRESSOR OF PHYTOCHROME B-4 #3 (SOB3)/AHL29 redundantly regulate auxin biosynthesis in the control of hypocotyl elongation. The light-inducible AHL proteins bind directly to an S/MAR region of the YUCCA 9 (YUC9) promoter and suppress its expression to inhibit hypocotyl growth in light-grown seedlings. In addition, they recruit the SWI2/SNF2-RELATED 1 (SWR1) complex and promote exchange of H2A with the histone variant H2A.Z at the YUC9 locus to further elaborately control auxin biosynthesis. Consistent with these results, the long hypocotyl phenotypes of light-grown genetic mutants of the AHLs and H2A.Z-exchanging components were suppressed by potent chemical inhibitors of auxin transport and YUC enzymes. These results suggest that the coordination of matrix attachment and chromatin modification underlies auxin biosynthesis in light-dependent hypocotyl growth.

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.