Homeodomain-leucine Zipper Research Articles

START domains generate paralog-specific regulons from a single network architecture.

Functional divergence of transcription factors (TFs) has driven cellular and organismal complexity throughout evolution, but its mechanistic drivers remain poorly understood. Here we test for new mechanisms using CORONA (CNA) and PHABULOSA (PHB), two functionally diverged paralogs in the CLASS III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIPIII) family of TFs. We show that virtually all genes bound by PHB ( ~ 99%) are also bound by CNA, ruling out occupation of distinct sets of genes as a mechanism of functional divergence. Further, genes bound and regulated by both paralogs are almost always regulated in the same direction, ruling out opposite regulation of shared targets as a mechanistic driver. Functional divergence of CNA and PHB instead results from differential usage of shared binding sites, with hundreds of uniquely regulated genes emerging from a commonly bound genetic network. Regulation of a given gene by CNA or PHB is thus a function of whether a bound site is considered 'responsive' versus 'non-responsive' by each paralog. Discrimination between responsive and non-responsive sites is controlled, at least in part, by their lipid binding START domain. This suggests a model in which HD-ZIPIII TFs use information integrated by their START domain to generate paralog-specific transcriptional outcomes from a shared network architecture. Taken together, our study identifies a mechanism of HD-ZIPIII TF paralog divergence and proposes the ubiquitously distributed START evolutionary module as a driver of functional divergence.

FaTEDT1L of Octoploid Cultivated Strawberry Functions as a Transcriptional Activator and Enhances Abiotic Stress Tolerance in Transgenic Arabidopsis.

Plants may encounter abiotic stresses, such as drought, flooding, salinity, and extreme temperatures, thereby negatively affecting their growth, development, and reproduction. In order to enhance their tolerance to such stresses, plants have developed intricate signaling networks that regulate stress-responsive gene expression. For example, Arabidopsis Enhanced Drought Tolerance1/HOMEODOMAIN GLABROUS 11 (AtEDT1/HDG11), one of the transcription factor genes from the group IV of homeodomain-leucine zipper (HD-ZIP) gene family, has been shown to increase drought tolerance in various transgenic plants. However, the underlying molecular mechanisms of enhanced stress tolerance remain unclear. In this study, we identified a homologous gene related to AtEDT1/HDG11, named FaTEDT1L, from the transcriptome sequencing database of cultivated strawberry. Phylogenetic analysis revealed the close relationship of FaTEDT1L with AtEDT1/HDG11, which is one of the group IV members of the HD-ZIP gene family. Yeast one-hybrid analysis showed that FaTEDT1L functions as a transcriptional activator. Transgenic Arabidopsis plants overexpressing FaTEDT1L under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited significantly enhanced tolerance to osmotic stress (both drought and salinity) when compared to the wild-type (WT) plants. Under osmotic stress, the average root length was 3.63 ± 0.83 cm, 4.20 ± 1.03 cm, and 4.60 ± 1.14 cm for WT, 35S::FaTEDT1L T2 #3, and 35S:: FaTEDT1L T2 #5, respectively. Substantially increased root length in 35S::FaTEDT1L T2 #3 and 35S::FaTEDT1L T2 #5 was noted when compared to the WT. In addition, the average water loss rates were 64%, 57.1%, and 55.6% for WT, 35S::FaTEDT1L T2 #3, and 35S::FaTEDT1L T2 #5, respectively, after drought treatment, indicating a significant decrease in water loss rate of 35S:: FaTEDT1L T2 #3 and 35S::FaTEDT1L T2 #5 is a critical factor in enhancing plant drought resistance. These findings thus highlight the crucial role of FaTEDT1L in mitigating drought and salt stresses and regulating plant osmotic stress tolerance. Altogether, FaTEDT1L shows its potential usage as a candidate gene for strawberry breeding in improving crop resilience and increasing agricultural productivity under adverse environmental conditions.

A Bamboo HD-Zip Transcription Factor PeHDZ72 Conferred Drought Tolerance by Promoting Sugar and Water Transport.

Drought drastically affects plant growth, development and productivity. Plants respond to drought stress by enhancing sugar accumulation and water transport. Homeodomain-leucine zipper (HD-Zip) transcription factors (TFs) participate in various aspects of plant growth and stress response. However, the internal regulatory mechanism of HD-Zips in moso bamboo (Phyllostachys edulis) remains largely unknown. In this study, we identified an HD-Zip member, PeHDZ72, which was highly expressed in bamboo shoots and roots and was induced by drought. Furthermore, PeSTP_46019, PeSWEET_23178 and PeTIP4-3 were identified as downstream genes of PeHDZ72 in moso bamboo by DAP-seq. The expressions of these three genes were all induced by drought stress. Y1H, DLR and GUS activity assays demonstrated that PeHDZ72 could bind to three types of HD-motifs in the promoters of these three genes. Overexpression of PeHDZ72 led to a remarkable enhancement in drought tolerance in transgenic rice, with significantly improved soluble sugar and sucrose contents. Meanwhile, the expressions of OsSTPs, OsSWEETs and OsTIP were all upregulated in transgenic rice under drought stress. Overall, our results indicate that drought stress might induce the expression of PeHDZ72, which in turn activated downstream genes PeSTP_46019, PeSWEET_23178 and PeTIP4-3, contributing to the improvement of cellular osmotic potential in moso bamboo in response to drought stress.

An HD-Zip III transcription factor, BjPHVa, negatively regulates non-glandular trichome formation in Brassica juncea.

Trichomes are specialized structures derived from epidermal cells. Non-glandular trichomes primarily protect plants from herbivores and intense ultraviolet radiation by acting as a physical barrier. Recent research has highlighted the importance of homeodomain leucine zipper (HD-Zip) IV transcription factors (TFs) in promoting trichome development. In this study, an HD-Zip III TF called PHAVOLUTA (BjPHVa) was identified as a negative regulator of non-glandular trichome initiation in Brassica juncea. Genome editing of BjPHVa resulted in a significant increase in trichome number in B. juncea. Co-expression networks revealed a strong association between trichome development and the HD-Zip family, which was supported by transcriptomic analysis findings. An R2R3-MYB TF, BjGL1a, a key regulator of trichome development, was found to be associated with BjPHVa-regulated trichome development. Knockdown of BjGL1a expression resulted in reduced trichome number in B. juncea. BjPHVa was observed to interact directly with BjGL1a while binding to the BjGL1a promoter, resulting in the inhibition of BjGL1a transcription. These results provide new insights into the identification of regulators involved in trichome development and offer new opportunities to enhance resistance to predicted stresses through genome editing targeting PHVa within Brassicaceae.

Homeodomain leucine zipper (HD-ZIP) gene family in Apostasia shenzhenica Z. J. Liu & L. J. Chen (Orchidaceae): In silico identification and characterization

The homeodomain leucine zipper (HD-ZIP) gene family constitutes an important class of transcription factors found in plants that play crucial roles in various aspects of growth, development and stress regulation. Despite this, there have been minuscule reports on identification and characterization of HD-ZIP gene family in orchids. The present study identifies 31 putative genes (AsHD-ZIP) in Apostasia shenzhenica, using in silico approaches. All of the identified transcription factors were grouped into four subfamilies (HD-ZIP I to IV) based on the distribution of various conserved domains and motifs i.e., HD-ZIP I (homeodomain and leucine zipper), HD-ZIP II (homeodomain, leucine zipper and CPSCE), HD-ZIP III (homeodomain, leucine zipper, MEKHLA and START) and HD-ZIP IV (homeodomain, leucine zipper and START). Prediction of physicochemical properties, protein structures, protein-protein interactions, gene ontology, and gene structures further validated this classification. Phylogenetic analysis showed clustering of all subgroups separately along with their orthologs in Arabidopsis thaliana, Oryza sativa, Dendrobium catenatum and Phalaenopsis equestris. Furthermore, spatio-temporal expression profiling and presence of cis-regulatory elements confirmed tissue specific expression. As HD-ZIP genes are vital players in regulation of plant growth and development as well as in biotic and abiotic stress responses, this study provides a foundation for genetic improvement in orchids through functional validation and characterization of HD-ZIP gene family.

Cotton HD-Zip I transcription factor GhHB4-like regulates the plant response to salt stress

Soil salinity is a major environmental constraint to plant production. The homeodomain-leucine zipper I (HD-Zip I) transcription factors play a crucial role in growth, development and defence responses of plants. However, the function and underlying mechanism of HD-Zip I in cotton remain unexplored. This study investigated the role of GhHB4-like, a cotton HD-Zip I gene, in plant tolerance to salt stress. Ectopic expression of GhHB4-like gene enhanced, while its silencing impaired the salt tolerance in Arabidopsis. Y1H and effector-reporter assays revealed that GhHB4-like activated the expression of GhNAC007, which is essential for salt resistance. Knock-down of GhNAC007 also impaired salt resistance of cotton plants. In addition, GhHB4-like-GhNAC007 might have positively regulated the expression of GhMYB96 and ABA signalling-related genes, thereby leading to enhanced salt resistance. Interestingly, deleting motifs 3 and 5 near the 3′-end of GhHB4-like significantly enhanced GhNAC007 activation, indicating that both motifs acted as transcriptional activation inhibitory domains. The results suggest that GhHB4-like-GhNAC007 regulated plant response to salt stress, potentially by modulating GhMYB96 and ABA signalling-related genes.

Genome-wide analysis of the HD-Zip IV gene family in Camellia sinensis and functional characterization of CsHDZIV3 under trichome development

The HD-Zip IV transcription factors, which belong to the homeodomain leucine zipper (HD-ZIP) family, are essential for the development of epidermal cells in plants. However, there is limited information about HD-Zip IV in tea plant (Camellia sinensis L.). Here we identified 10 HD-Zip IV members (CsHDZIV1∼10) in tea plant, which are distributed in 8 chromosomes with two segmentally duplicated gene pairs. The exon number of CsHDZIVs ranges from 9 to 11, with most genes having two conserved motifs in the 3’UTR. CsHDZIV1∼9 have almost identical motifs and tertiary structures. The promoter region of CsHDZIVs was predicted to harbor light, phytohormone, stress, growth and developmental elements. 9 CsHDZIVs are divided into 5 subfamilies and located in the nucleus. They exhibited expression trends across 12 tea plant tissues and responded to PEG, cold, NaCl and shading treatments. CsHDZIV3 overexpression partially and completely restored trichome development defects of gl2 leaves and stems, respectively. CsHDZIV3 showed histochemical staining in Arabidopsis trichomes and protein interaction with itself. It might regulate trichome development through forming homodimers. These findings contribute to functional study of tea plant HD-Zip IV and provide guiding significance for the investigation of trichome development and the improvement of trichome traits.

Adaxial-abaxial bipolar leaf genes encode a putative cytokinin receptor and HD-Zip III, and control the formation of ectopic shoot meristems in rice.

Shoot apical meristems (SAMs) continuously initiate organ formation and maintain pluripotency through dynamic genetic regulations and cell-to-cell communications. The activity of meristems directly affects the plant's structure by determining the number and arrangement of organs and tissues. We have taken a forward genetic approach to dissect the genetic pathway that controls cell differentiation around the SAM. The rice mutants, adaxial-abaxial bipolar leaf 1 and 2 (abl1 and abl2), produce an ectopic leaf that is fused back-to-back with the fourth leaf, the first leaf produced after embryogenesis. The abaxial-abaxial fusion is associated with the formation of an ectopic shoot meristem at the adaxial base of the fourth leaf primordium. We cloned the ABL1 and ABL2 genes of rice by mapping their chromosomal positions. ABL1 encodes OsHK6, a histidine kinase, and ABL2 encodes a transcription factor, OSHB3 (Class III homeodomain leucine zipper). Expression analyses of these mutant genes as well as OSH1, a rice ortholog of the Arabidopsis STM gene, unveiled a regulatory circuit that controls the formation of an ectopic meristem near the SAM at germination.

Nuclear localization of Arabidopsis HD-Zip IV transcription factor GLABRA2 is driven by importin α.

GLABRA2 (GL2), a class IV homeodomain leucine-zipper (HD-Zip IV) transcription factor from Arabidopsis, is a developmental regulator of specialized cell types in the epidermis. GL2 contains a monopartite nuclear localization sequence (NLS) that is conserved in most HD-Zip IV members across the plants. We demonstrate that NLS mutations affect nuclear transport and result in a loss-of-function phenotypes. NLS fusions to enhanced yellow fluorescent protein (EYFP) show that it is sufficient for nuclear localization in roots and trichomes. Despite partial overlap of the NLS with the homeodomain, genetic dissection indicates that nuclear localization and DNA binding are separable functions. Affinity purification of GL2 from plants followed by MS-based proteomics identified importin α (IMPα) isoforms as potential GL2 interactors. NLS structural prediction and molecular docking studies with IMPα-3 revealed major interacting residues. Cytosolic yeast two-hybrid assays and co-immunoprecipitation experiments with recombinant proteins verified NLS-dependent interactions between GL2 and several IMPα isoforms. IMPα triple mutants (impα-1,2,3) exhibit abnormal trichome formation and defects in GL2 nuclear localization in trichomes, consistent with tissue-specific and redundant functions of IMPα isoforms. Taken together, our findings provide mechanistic evidence for IMPα-dependent nuclear localization of GL2 in Arabidopsis, a process that is critical for cell type differentiation of the epidermis.

The HD-ZIP IV transcription factor GLABRA2 acts as an activator for proanthocyanidin biosynthesis in Medicago truncatula seed coat.

Proanthocyanidins (PAs), a group of flavonoids, are found in leaves, flowers, fruits, and seed coats of many plant species. PAs are primarily composed of epicatechin units in the seed coats of the model legume species, Medicago truncatula. It can be synthesized from two separate pathways, the leucoanthocyanidin reductase (MtLAR) pathway and the anthocyanidin synthase (MtANS) pathway, which produce epicatechin through anthocyanidin reductase (MtANR). These pathways are mainly controlled by the MYB-bHLH-WD40 (MBW) ternary complex. Here, we characterize a class IV homeodomain-leucine zipper (HD-ZIP IV) transcription factor, GLABRA2 (MtGL2), which contributes to PA biosynthesis in the seed coat of M. truncatula. Null mutation of MtGL2 results in dark brown seed coat, which is accompanied by reduced PAs accumulation and increased anthocyanins content. The MtGL2 gene is predominantly expressed in the seed coat during the early stages of seed development. Genetic and molecular analyses indicate that MtGL2 positively regulates PA biosynthesis by directly activating the expression of MtANR. Additionally, our results show that MtGL2 is strongly induced by the MBW activator complexes that are involved in PA biosynthesis. Taken together, our results suggest that MtGL2 acts as a novel positive regulator in PA biosynthesis, expanding the regulatory network and providing insights for genetic engineering of PA production.

Passion fruit HD-ZIP genes: Characterization, expression variance, and overexpression PeHB31 enhanced drought tolerance via lignin pathway

The HD-ZIP (homeodomain-leucine zipper) genes hold significant importance in transcriptional regulation, especially in plant development and responses to abiotic stresses. However, a comprehensive study targeting HD-ZIP family members in passion fruit has been absent. In our current research, 34 HD-ZIP family members (PeHBs) were identified by bioinformatics analysis. Transcriptome analysis revealed that PeHBs exhibited distinct expression patterns when subjected to the four different abiotic stresses, and significant differential expression of PeHBs was also found among the three developmental stages of the fruit and between the purple and yellow genotype passion fruit leaves. An integrated metabolome and transcriptome analysis further revealed that the HD-ZIP III class gene PeHB31 (homologous to ATHB8), was co-upexpressed with lignans in yellow fruit P. edulis (commonly used as a resistance rootstock) when compared to purple fruit P. edulis. The transformation of Arabidopsis and yeast with the PeHB31 gene showed an enhancement in their capacity to withstand drought conditions. Notably, the transgenic Arabidopsis plants exhibited an increase in lignin content within the vascular tissues of their stems. This research lays the groundwork for future studies on the control mechanisms of lignin biosynthesis by HD-ZIP genes (especially HD-ZIP classes III and I) involved in drought tolerance.

Arabidopsis PROTODERMAL FACTOR2 binds lysophosphatidylcholines and transcriptionally regulates phospholipid metabolism.

Plant homeodomain leucine zipper IV (HD-Zip IV) transcription factors (TFs) contain an evolutionarily conserved steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. While the START domain is required for TF activity, its presumed role as a lipid sensor is not clear. Here we used tandem affinity purification from Arabidopsis cell cultures to demonstrate that PROTODERMAL FACTOR2 (PDF2), a representative member that controls epidermal differentiation, recruits lysophosphatidylcholines (LysoPCs) in a START-dependent manner. Microscale thermophoresis assays confirmed that a missense mutation in a predicted ligand contact site reduces lysophospholipid binding. We additionally found that PDF2 acts as a transcriptional regulator of phospholipid- and phosphate (Pi) starvation-related genes and binds to a palindromic octamer with consensus to a Pi response element. Phospholipid homeostasis and elongation growth were altered in pdf2 mutants according to Pi availability. Cycloheximide chase experiments revealed a role for START in maintaining protein levels, and Pi starvation resulted in enhanced protein destabilization, suggesting a mechanism by which lipid binding controls TF activity. We propose that the START domain serves as a molecular sensor for membrane phospholipid status in the epidermis. Our data provide insights toward understanding how the lipid metabolome integrates Pi availability with gene expression.

Genome-wide identification and analysis of the HD-Zip transcription factors in perennial ryegrass highlight LpHOX22 and LpHOX24 as negative regulators of osmotic and heat stresses

The homeodomain leucine zipper (HD-Zip) transcription factors are involved in plant responses to abiotic stresses. In this study, we have identified a total of 52 HD-Zip genes from the released genome of perennial ryegrass (Lolium perenne), a cool-season turfgrass and forage species, which is relatively sensitive to heat and drought stresses. These HD-Zip genes are classified into classes I-IV, of which, 49 are evenly distributed on all seven chromosomes of perennial ryegrass whereas other 3 are on scaffolds. Cis-elements analysis showed that phytohormone, light signaling, and stress responsive elements are abundant in the promoter region of the HD-Zip genes. Expression analysis indicated that LpHOX24 and its paralog LpHOX22 were significantly induced by heat stress treatment. Ectopic overexpressing (OE) of LpHOX22 and LpHOX24 in Arabidopsis compromised plant osmotic stress tolerance as evidenced by declined survival rate with elevated electrolyte leakage (EL) and malondialdehyde (MDA) accumulation. Expression levels of several osmotic responsive genes were downregulated in OE lines. Additionally, heat stress tolerance was impaired in LpHOX22 and LpHOX24 overexpression transformants, along with increased EL and MDA content. Further analysis revealed that overexpression of LpHOX22 and LpHOX24 repressed the expressions of AtHSFAs and AtDREB2A when compared to wild type after heat stress treatment. In summary, LpHOX22 and LpHOX24 potentially functioned as negative regulators in perennial ryegrass in response to osmotic and heat stresses.

Genetic and Molecular Regulation of Cotton Fiber Initiation and Elongation

Cotton fiber, a crucial and sustainable resource for global textile production, undergoes a complex five-stage developmental process, encompassing initiation, elongation, transition, secondary cell wall biosynthesis, and maturation. These elongated single-cell fibers originate from the outer ovule epidermis. The development of cotton fibers involves intricate changes in gene expression and physiological processes, resulting in a nearly pure cellulose product that is vital for the global cotton industry. Decoding the genes associated with fiber development enhances our understanding of cotton fiber mechanisms and facilitates the cultivation of varieties with enhanced quality. In recent decades, advanced omics approaches, including genomics, transcriptomics, and proteomics, have played a pivotal role in identifying the genes and gene products linked to cotton fiber development, including the MYB transcription factor family, which coordinates cotton fiber development. Molecular studies have revealed the transcription factors, like MYB, WRKY, Homeodomain Leucine Zipper (HD-ZIP), and basic helix–loop–helix (bHLH), influencing fiber initiation and elongation. The intricate interplay of phytohormones, like auxin, gibberellic acid (GA), brassinosteroids (BRs), jasmonic acid (JA), ethylene, abscisic acid (ABA), and cytokinin, is explored, providing a comprehensive perspective on the shaping of cotton fibers. Numerous candidate genes and cellular processes affecting various aspects of fiber development hold promise for genetic engineering or marker-assisted breeding to improve fiber quality. This review presents a comprehensive overview of key achievements in cotton molecular biology, with a specific emphasis on recent advancements in understanding the transcription factors and phytohormones involved in cotton fiber initiation and elongation.

Optimizing Promoters and Subcellular Localization for Constitutive Transgene Expression in Marchantia polymorpha.

Marchantia polymorpha has become an important model system for comparative studies and synthetic biology. The systematic characterization of genetic elements would make heterologous gene expression more predictable in this test bed for gene circuit assembly and bioproduction. Yet, the toolbox of genetic parts for Marchantia includes only a few constitutive promoters that need benchmarking to assess their utility. We compared the expression patterns of previously characterized and new constitutive promoters. We found that driving expression with the double enhancer version of the cauliflower mosaic virus 35S promoter (pro35S × 2) provided the highest yield of proteins, although it also inhibits the growth of transformants. In contrast, promoters derived from the Marchantia genes for ETHYLENE RESPONSE FACTOR 1 and the CLASS II HOMEODOMAIN-LEUCINE ZIPPER protein drove expression to higher levels across all tissues without a growth penalty and can provide intermediate levels of gene expression. In addition, we showed that the cytosol is the best subcellular compartment to target heterologous proteins for higher levels of expression without a significant growth burden. To demonstrate the potential of these promoters in Marchantia, we expressed RUBY, a polycistronic betalain synthesis cassette linked by P2A sequences, to demonstrate coordinated expression of metabolic enzymes. A heat-shock-inducible promoter was used to further mitigate growth burdens associated with high amounts of betalain accumulation. We have expanded the existing tool kit for gene expression in Marchantia and provided new resources for the Marchantia research community.

Transcriptional modules and hormonal metabolic pathways reveal the critical role of TgHB12-like in the regulation of flower opening and petal senescence in Tulipa gesneriana

Tulips (Tulipa gesneriana) are one of the most widely cultivated bulbous plants with substantial ornamental value. However, the lack of well-documented reference genomes has limited the research progress and molecular breeding of tulips. In the present study, a full-length transcriptome of a commercial tulip cultivar was obtained using single-molecule long-read sequencing (PacBio Iso-Seq). In total, 244,357 full-length transcripts were identified, which had an average length of 2,044 bp and an N50 value of 3,861; 67,350 of these were annotated to databases. An inaugural integrated analysis of the transcriptome and phytohormone profiles during flower opening and petal senescence was performed using Illumina RNA-seq, coupled with Mfuzz (an R pakage, http://mfuzz.sysbiolab.eu) and weighted gene coexpression network analysis (WGCNA). A total of 16 gene coexpression and six transcription factor (TF) modules were constructed. Additionally, 26 hormone analogs were comprehensively profiled. Finally, a prominently novel gene, Tulipa gesneriana Homeobox12-like (TgHB12-like), which encodes an homeodomain–leucine zipper (HD-zip) TF, was identified as a pivotal regulator of petal senescence. Overall, this work facilitates the identification of hormones and TFs in plants related to flower opening and senescence in tulips. It also provides an important and valuable genetic basis for further research in them.

The Ins and Outs of Homeodomain-Leucine Zipper/Hormone Networks in the Regulation of Plant Development.

The generation of complex plant architectures depends on the interactions among different molecular regulatory networks that control the growth of cells within tissues, ultimately shaping the final morphological features of each structure. The regulatory networks underlying tissue growth and overall plant shapes are composed of intricate webs of transcriptional regulators which synergize or compete to regulate the expression of downstream targets. Transcriptional regulation is intimately linked to phytohormone networks as transcription factors (TFs) might act as effectors or regulators of hormone signaling pathways, further enhancing the capacity and flexibility of molecular networks in shaping plant architectures. Here, we focus on homeodomain-leucine zipper (HD-ZIP) proteins, a class of plant-specific transcriptional regulators, and review their molecular connections with hormonal networks in different developmental contexts. We discuss how HD-ZIP proteins emerge as key regulators of hormone action in plants and further highlight the fundamental role that HD-ZIP/hormone networks play in the control of the body plan and plant growth.

ALTERED MERISTEM PROGRAM1 sustains cellular differentiation by limiting HD-ZIP III transcription factor gene expression.

Plants show remarkable developmental and regenerative plasticity through the sustained activity of stem cells in meristems. Under certain conditions, pluripotency can even be reestablished in cells that have already entered differentiation. Mutation of the putative carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) in Arabidopsis (Arabidopsis thaliana) causes a set of hypertrophic phenotypes, indicating a defect in the suppression of pluripotency. A role of AMP1 in the miRNA-mediated inhibition of translation has previously been reported; however, how this activity is related to its developmental functions is unclear. Here, we examined the functional interaction between AMP1 and the Class III homeodomain-leucine zipper (HD-ZIP III) transcription factors, which are miRNA-controlled determinants of shoot meristem specification. We found that the HD-ZIP III transcriptional output is enhanced in the amp1 mutant and that plant lines with increased HD-ZIP III activity not only developed amp1 mutant-like phenotypes but also showed a synergistic genetic interaction with the mutant. Conversely, the reduction of HD-ZIP III function suppressed the shoot hypertrophy defects of the amp1 mutant. We further provide evidence that the expression domains of HD-ZIP III family members are expanded in the amp1 mutant and that this misexpression occurs at the transcriptional level and does not involve the function of miRNA165/166. Finally, amp1 mutant-specific phenotypes cannot be mimicked by a general inhibition of miRNA function in the AMP1 expression domain. These findings lead us to a model in which AMP1 restricts cellular pluripotency upstream of HD-ZIP III proteins, and this control appears to be not directly mediated by the canonical miRNA pathway.

Functional and regulatory diversity of homeobox-leucine zipper transcription factors BnaHB6 under dehydration and salt stress in Brassica napus L.

The plant-specific homeodomain-leucine zipper I subfamily is involved in the regulation of various biological processes, particularly growth, development and stress response. In the present study, we characterized four BnaHB6 homologues from Brassica napus. All BnaHB6 proteins have transcriptional activation activity. Structural and functional data indicate the complex role of BnaHB6 genes in regulating biological processes, with some functions conserved and others diverged. Transcriptional analyzes revealed that they are induced in a similar manner in different tissues but show different expression patterns in response to stress and circadian rhythm. Only the BnaA09HB6 and BnaC08HB6 genes are expressed under dehydration and salt stress, and in darkness. The partial transcriptional overlap of BnaHB6s with the evolutionarily related genes BnaHB5 and BnaHB16 was also observed. Transgenic Arabidopsis thaliana plants expressing a single proBnaHB6::GUS partially confirmed the expression results. Bioinformatic analysis allowed the identification of TF-binding sites in the BnaHB6 promoters that may control their expression under stress and circadian rhythm. ChIP-qPCR analysis revealed that BnaA09HB6 and BnaC08HB6 bind directly to the promoters of the target genes BnaABF4 and BnaDREB2A. Comparison of their expression patterns in the WT plants and the bnac08hb6 mutant showed that BnaC08HB6 positively regulates the expression of the BnaABF4 and BnaDREB2A genes under dehydration and salt stress. We conclude that four BnaHB6 homologues have distinct functions in response to stress despite high sequence similarity, possibly indicating different binding preferences with BnaABF4 and BnaDREB2A. We hypothesize that BnaC08HB6 and BnaA09HB6 function in a complex regulatory network under stress.

Genome-wide characterization and expression profiling of the HD-ZIP gene family in Acoraceae under salinity and cold stress.

The Homeodomain-Leucine Zipper (HD-ZIP) transcription factors play a pivotal role in governing various aspects of plant growth, development, and responses to abiotic stress. Despite the well-established importance of HD-ZIPs in many plants, their functions in Acoraceae, the basal lineage of monocots, remain largely unexplored. Using recently published whole-genome data, we identified 137 putative HD-ZIPs in two Acoraceae species, Acorus gramineus and Acorus calamus. These HD-ZIP genes were further classified into four subfamilies (I, II, III, IV) based on phylogenetic and conserved motif analyses, showcasing notable variations in exon-intron patterns among different subfamilies. Two microRNAs, miR165/166, were found to specifically target HD-ZIP III genes with highly conserved binding sites. Most cis-acting elements identified in the promoter regions of Acoraceae HD-ZIPs are involved in modulating light and phytohormone responsiveness. Furthermore, our study revealed an independent duplication event in Ac. calamus and a one-to-multiple correspondence between HD-ZIP genes of Ac. calamus and Ac. gramineus. Expression profiles obtained from qRT-PCR demonstrated that HD-ZIP I genes are strongly induced by salinity stress, while HD-ZIP II members have contrasting stress responses in two species. HD-ZIP III and IV genes show greater sensitivity in stress-bearing roots. Taken together, these findings contribute valuable insights into the roles of HD-ZIP genes in stress adaptation and plant resilience in basal monocots, illuminating their multifaceted roles in plant growth, development, and response to abiotic stress.