Citrus transcription factor CsHB5 regulates abscisic acid biosynthetic genes and promotes senescence.

Senescence is a gradual physiological process involving the integration of numerous internal and environmental signals. Abscisic acid (ABA) is a well-known inducer of senescence. However, the regulatory mechanisms underlying ABA-mediated senescence remain largely unknown. Here, we report that the citrus homeodomain leucine zipper I (HD-ZIP I) transcription factor CsHB5 functions as a regulator of ABA-triggered senescence. CsHB5 acts as a nucleus-localized transcriptional activator, the expression of which appeared to be closely associated with citrus senescence. Overexpression of CsHB5 in citrus calli upregulated the expression of ABA- and reactive oxygen species (ROS)-related genes, and significantly increased the content of ABA and hydrogen peroxide (H2 O2 ), whereas silencing CsHB5 in citrus calli downregulated the expression of ABA-related genes. Additionally, heterogenous overexpression of CsHB5 in Solanum lycopersicum (tomato) and Arabidopsis thaliana (Arabidopsis) leads to early leaf yellowing under dark-induced senescence conditions. Meanwhile, the levels of ABA and H2 O2 in transgenic tomatoes increased significantly and the lycopene content decreased. Transcriptome analysis of CsHB5-overexpressing citrus calli and tomato showed that CsHB5 was involved in multiple senescence-associated processes, including chlorophyll degradation, nutrient compound biosynthesis and transport, as well as ABA and ROS signal transduction. The results of yeast one-hybrid assays, electrophoretic mobility shift assays and dual luciferase assays indicated that CsHB5 directly binds to the promoters of ABA biosynthetic genes, including ß-carotene hydroxylase 1 (BCH1) and 9-cis-epoxycarotenoid dioxygenase 2 (NCED2), thereby activating their transcription. Our findings revealed that CsHB5 participates in senescence, at least partly, by directly controlling ABA accumulation. Our work provides insight into the regulatory mechanisms underlying ABA-mediated senescence.

The underground life of homeodomain-leucine zipper transcription factors.

Roots are the anchorage organs of plants, responsible for water and nutrient uptake, exhibiting high plasticity. Root architecture is driven by the interactions of biomolecules, including transcription factors and hormones that are crucial players regulating root plasticity. Multiple transcription factor families are involved in root development; some, such as ARFs and LBDs, have been well characterized, whereas others remain less well investigated. In this review, we synthesize the current knowledge about the involvement of the large family of homeodomain-leucine zipper (HD-Zip) transcription factors in root development. This family is divided into four subfamilies (I-IV), mainly according to structural features, such as additional motifs aside from HD-Zip, as well as their size, gene structure, and expression patterns. We explored and analyzed public databases and the scientific literature regarding HD-Zip transcription factors in Arabidopsis and other species. Most members of the four HD-Zip subfamilies are expressed in specific cell types and several individuals from each group have assigned functions in root development. Notably, a high proportion of the studied proteins are part of intricate regulation pathways involved in primary and lateral root growth and development.

The AtHB1 Transcription Factor Controls the miR164-CUC2 Regulatory Node to Modulate Leaf Development.

The presence of small tooth-like indentations, or serrations, characterizes leaf margins of Arabidopsis thaliana plants. The NAC family member CUP-SHAPED COTYLEDON 2 (CUC2), which undergoes post-transcriptional gene silencing by three micro-RNA genes (MIR164A, B and C), controls the extension of leaf serration. Here, we analyzed the role of AtHB1, a transcription factor (TF) belonging to the homeodomain-leucine zipper subfamily I, in shaping leaf margins. Using mutants with an impaired silencing pathway as background, we obtained transgenic plants expressing AtHB1 over 100 times compared to controls. These plants presented an atypical developmental phenotype characterized by leaves with deep serration. Transcript measurements revealed that CUC2 expression was induced in plants overexpressing AtHB1 and repressed in athb1 mutants, indicating a positive regulation exerted by this TF. Moreover, molecular analyses of AtHB1 overexpressing and mutant plants revealed that AtHB1 represses MIR164 transcription. We found that overexpression of MIR164B was able to reverse the serration phenotype of plants overexpressing AtHB1. Finally, chromatin immunoprecipitation assays revealed that AtHB1 was able to bind in vivo the promoter regions of all three MIR164 encoding loci. Altogether, our results indicate that AtHB1 directly represses MIR164 expression to enhance leaf serration by increasing CUC2 levels.

Preferential insertion of a Ty1 LTR-retrotransposon into the A sub-genome's HD1 gene significantly correlated with the reduction in stem trichomes of tetraploid cotton.

Stem trichomes and seed fibers originate from epidermal cells and partially share a regulatory pathway at the molecular level. In Gossypium barbadense, two insertions of a Ty1 long-terminal repeat-retrotransposon [transposable element TE1 and TE2] in a homeodomain-leucine zipper gene (HD1) result in glabrous stems. The primers used to identify the TE insertions in G. barbadense were applied to screen for the same events in 81 modern G. hirsutum varieties and 31 wild races. Three wild races were found carrying the same TEs as G. barbadense. However, the TE insertions in two of these wild races occurred at different sites (4th exon), therefore, named TE3, while the TE in the other wild race occurred at the same site as TE2. An RNA sequencing and qRT-PCR analysis indicated that the loss of HD1 function was caused by the TE insertion. Genetic mapping revealed a strong association between glabrous stems and TE3 insertions, confirming that HD1 is a critical gene for stem trichome initiation in G. hirsutum, as in G. barbadense. Using the long-terminal repeat sequence as a query to search against the Texas Marker-1 reference genome sequence, we found that the TE occurred after tetraploid cotton formation and evolved at different rates in G. hirsutum and G. barbadense. Interestingly, at least three independent insertion events of the same retrotransposon occurred preferentially in the A sub-genome's HD1 gene, but not in the D sub-genome of G. hirsutum or G. barbadense, suggesting that an unknown TE insertion mechanism and resultant gene function changes may have hastened cotton speciation.

Key role of the motor protein Kinesin 13B in the activity of homeodomain-leucine zipper I transcription factors.

The sunflower (Helianthus annuus) homeodomain-leucine zipper I transcription factor HaHB11 conferred differential phenotypic features when it was expressed in Arabidopsis, alfalfa, and maize plants. Such differences were increased biomass, seed yield, and tolerance to flooding. To elucidate the molecular mechanisms leading to such traits and identify HaHB11-interacting proteins, a yeast two-hybrid screening of an Arabidopsis cDNA library was carried out using HaHB11 as bait. The sole protein identified with high confidence as interacting with HaHB11 was Kinesin 13B. The interaction was confirmed by bimolecular fluorescence complementation and by yeast two-hybrid assay. Kinesin 13B also interacted with AtHB7, the Arabidopsis closest ortholog of HaHB11. Histochemical analyses revealed an overlap between the expression patterns of the three genes in hypocotyls, apical meristems, young leaves, vascular tissue, axillary buds, cauline leaves, and cauline leaf nodes at different developmental stages. AtKinesin 13B mutants did not exhibit a differential phenotype when compared with controls; however, both HaHB11 and AtHB7 overexpressor plants lost, partially or totally, their differential phenotypic characteristics when crossed with such mutants. Altogether, the results indicated that Kinesin 13B is essential for the homeodomain-leucine zipper transcription factors I to exert their functions, probably via regulation of the intracellular distribution of these transcription factors by the motor protein.

Leaf primordium size specifies leaf width and vein number among row-type classes in barley.

Exploring genes with impact on yield-related phenotypes is the preceding step to accomplishing crop improvements while facing a growing world population. A genome-wide association scan on leaf blade area (LA) in a worldwide spring barley collection (Hordeum vulgare L.), including 125 two- and 93 six-rowed accessions, identified a gene encoding the homeobox transcription factor, Six-rowed spike 1 (VRS1). VRS1 was previously described as a key domestication gene affecting spike development. Its mutation converts two-rowed (wild-type VRS1, only central fertile spikelets) into six-rowed spikes (mutant vrs1, fully developed fertile central and lateral spikelets). Phenotypic analyses of mutant and wild-type leaves revealed that mutants had an increased leaf width with more longitudinal veins. The observed significant increase of LA and leaf nitrogen (%) during pre-anthesis development in vrs1 mutants also implies a link between wider leaf and grain number, which was validated from the association of vrs1 locus with wider leaf and grain number. Histological and gene expression analyses indicated that VRS1 might influence the size of leaf primordia by affecting cell proliferation of leaf primordial cells. This finding was supported by the transcriptome analysis of mutant and wild-type leaf primordia where in the mutant transcriptional activation of genes related to cell proliferation was detectable. Here we show that VRS1 has an independent role on barley leaf development which might influence the grain number.

A novel HD-ZIP IV/MIXTA complex promotes glandular trichome initiation and cuticle development in Artemisia annua.

Glandular trichomes and cuticles are both specialized structures that cover the epidermis of aerial plant organs. The former are commonly regarded as 'biofactories' for producing valuable natural products. The latter are generally considered as natural barriers for defending plants against abiotic and biotic stresses. However, the regulatory network for their formation and relationship remains largely elusive. Here we identify a homeodomain-leucine zipper (HD-ZIP) IV transcription factor, AaHD8, directly promoting the expression of AaHD1 for glandular trichome initiation in Artemisia annua. We found that AaHD8 positively regulated leaf cuticle development in A. annua via controlling the expression of cuticle-related enzyme genes. Furthermore, AaHD8 interacted with a MIXTA-like protein AaMIXTA1, a positive regulator of trichome initiation and cuticle development, forming a regulatory complex and leading to enhanced transcriptional activity in regulating the expression of AaHD1 and cuticle development genes. Our results reveal a molecular mechanism by which a novel HD-ZIP IV/MIXTA complex plays a significant role in regulating epidermal development, including glandular trichome initiation and cuticle formation.

Plant transcription factors from the homeodomain-leucine zipper family I. Role in development and stress responses.

In front of stressful conditions plants display adaptation mechanisms leading to changes in their morphology, physiology, development and molecular composition. Transcription factors (TFs) play crucial roles in these complex adaptation processes. This work is focused in the homeodomain-leucine zipper I (HD-Zip I) family of TFs, unique to plants. First discovered in 1991, they were identified and isolated from monocotyledonous and dicotyledonous plants showing high structural similarity and diversified functions. These TFs have, besides the homeodomain and leucine zipper, conserved motifs in their carboxy-termini allowing the interaction with the basal machinery and with other regulatory proteins. The model dicotyledonous plant Arabidopsis thaliana has 17 HD-Zip I members; most of them regulated by external stimuli and hormones. These TFs are involved in key developmental processes like root and stem elongation, rosette leaves morphology determination, inflorescence stem branching, flowering and pollen hydration. Moreover, they are key players in responses to environmental stresses and illumination conditions. Several HD-Zip I encoding genes from different species were protected in patents because their overexpression or mutation generates improved agronomical phenotypes. Here we discuss many aspects about these TFs including structural features, biological functions and their utilization as biotechnological tools to improve crops. © 2017 IUBMB Life, 69(5):280-289, 2017.

Regulatory function of homeodomain-leucine zipper (HD-ZIP) family proteins during embryogenesis.

Homeodomain-leucine zipper proteins (HD-ZIPs) form a plant-specific family of transcription factors functioning as homo- or heterodimers. Certain members of all four classes of this family are involved in embryogenesis, the focus of this review. They support auxin biosynthesis, transport and response, which are in turn essential for the apical-basal patterning of the embryo, radicle formation and outgrowth of the cotyledons. They transcriptionally regulate meristem regulators to maintain the shoot apical meristem once it is initiated. Some members are specific to the protoderm, the outermost layer of the embryo, and play a role in shoot apical meristem function. Within classes, homeodomain-leucine zippers tend to act redundantly during embryo development, and there are many examples of regulation within and between classes of homeodomain-leucine zippers. This indicates a complex network of regulation that awaits future experiments to uncover.

A non-DNA-binding activity for the ATHB4 transcription factor in the control of vegetation proximity.

In plants, perception of vegetation proximity by phytochrome photoreceptors activates a transcriptional network that implements a set of responses to adapt to plant competition, including elongation of stems or hypocotyls. In Arabidopsis thaliana, the homeodomain-leucine zipper (HD-Zip) transcription factor ARABIDOPSIS THALIANA HOMEOBOX 4 (ATHB4) regulates this and other responses, such as leaf polarity. To better understand the shade regulatory transcriptional network, we have carried out structure-function analyses of ATHB4 by overexpressing a series of truncated and mutated forms and analyzing three different responses: hypocotyl response to shade, transcriptional activity and leaf polarity. Our results indicated that ATHB4 has two physically separated molecular activities: that performed by HD-Zip, which is involved in binding to DNA-regulatory elements, and that performed by the ETHYLENE-RESPONSIVE ELEMENT BINDING FACTOR-associated amphiphilic repression (EAR)-containing N-terminal region, which is involved in protein-protein interaction. Whereas both activities are required to regulate leaf polarity, DNA-binding activity is not required for the regulation of the seedling responses to plant proximity, which indicates that ATHB4 works as a transcriptional cofactor in the regulation of this response. These findings suggest that transcription factors might employ alternative mechanisms of action to regulate different developmental processes.

Molecular interactions of the γ-clade homeodomain-leucine zipper class I transcription factors during the wheat response to water deficit.

The γ-clade of class I homeodomain-leucine zipper (HD-Zip I) transcription factors (TFs) constitute members which play a role in adapting plant growth to conditions of water deficit. Given the importance of wheat (Triticum aestivum L.) as a global food crop and the impact of water deficit upon grain yield, we focused on functional aspects of wheat drought responsive HD-Zip I TFs. While the wheat γ-clade HD-Zip I TFs share significant sequence similarities with homologous genes from other plants, the clade-specific features in transcriptional response to abiotic stress were detected. We demonstrate that wheat TaHDZipI-3, TaHDZipI-4, and TaHDZipI-5 genes respond differentially to a variety of abiotic stresses, and that proteins encoded by these genes exhibit pronounced differences in oligomerisation, strength of DNA binding, and trans-activation of an artificial promoter. Three-dimensional molecular modelling of the protein-DNA interface was conducted to address the ambiguity at the central nucleotide in the pseudo-palindromic cis-element CAATNATTG that is recognised by all three HD-Zip I proteins. The co-expression of these genes in the same plant tissues together with the ability of HD-Zip I TFs of the γ-clade to hetero-dimerise suggests a role in the regulatory mechanisms of HD-Zip I dependent transcription. Our findings highlight the complexity of TF networks involved in plant responses to water deficit. A better understanding of the molecular complexity at the protein level during crop responses to drought will enable adoption of efficient strategies for production of cereal plants with enhanced drought tolerance.

Identification of miRNAs and Their Targets in the Liverwort Marchantia polymorpha by Integrating RNA-Seq and Degradome Analyses.

Bryophytes (liverworts, hornworts and mosses) comprise the three earliest diverging lineages of land plants (embryophytes). Marchantia polymorpha, a complex thalloid Marchantiopsida liverwort that has been developed into a model genetic system, occupies a key phylogenetic position. Therefore, M. polymorpha is useful in studies aiming to elucidate the evolution of gene regulation mechanisms in plants. In this study, we used computational, transcriptomic, small RNA and degradome analyses to characterize microRNA (miRNA)-mediated pathways of gene regulation in M. polymorpha. The data have been integrated into the open access ContigViews-miRNA platform for further reference. In addition to core components of the miRNA pathway, 129 unique miRNA sequences, 11 of which could be classified into seven miRNA families that are conserved in embryophytes (miR166a, miR390, miR529c, miR171-3p, miR408a, miR160 and miR319a), were identified. A combination of computational and degradome analyses allowed us to identify and experimentally validate 249 targets. In some cases, the target genes are orthologous to those of other embryophytes, but in other cases, the conserved miRNAs target either paralogs or members of different gene families. In addition, the newly discovered Mpo-miR11707.1 and Mpo-miR11707.2 are generated from a common precursor and target MpARGONAUTE1 (LW1759). Two other newly discovered miRNAs, Mpo-miR11687.1 and Mpo-miR11681.1, target the MADS-box transcription factors MpMADS1 and MpMADS2, respectively. Interestingly, one of the pentatricopeptide repeat (PPR) gene family members, MpPPR_66 (LW9825), the protein products of which are generally involved in various steps of RNA metabolism, has a long stem-loop transcript that can generate Mpo-miR11692.1 to autoregulate MpPPR_66 (LW9825) mRNA. This study provides a foundation for further investigations of the RNA-mediated silencing mechanism in M. polymorpha as well as of the evolution of this gene silencing pathway in embryophytes.

The homeodomain transcription factor TaHDZipI-2 from wheat regulates frost tolerance, flowering time and spike development in transgenic barley.

Homeodomain leucine zipper class I (HD-Zip I) transcription factors (TFs) play key roles in the regulation of plant growth and development under stresses. Functions of the TaHDZipI-2 gene isolated from the endosperm of developing wheat grain were revealed. Molecular characterization of TaHDZipI-2 protein included studies of its dimerisation, protein-DNA interactions and gene activation properties using pull-down assays, in-yeast methods and transient expression assays in wheat cells. The analysis of TaHDZipI-2 gene functions was performed using transgenic barley plants. It included comparison of developmental phenotypes, yield components, grain quality, frost tolerance and the levels of expression of potential target genes in transgenic and control plants. Transgenic TaHDZipI-2 lines showed characteristic phenotypic features that included reduced growth rates, reduced biomass, early flowering, light-coloured leaves and narrowly elongated spikes. Transgenic lines produced 25-40% more seeds per spike than control plants, but with 50-60% smaller grain size. In vivo lipid imaging exposed changes in the distribution of lipids between the embryo and endosperm in transgenic seeds. Transgenic lines were significantly more tolerant to frost than control plants. Our data suggest the role of TaHDZipI-2 in controlling several key processes underlying frost tolerance, transition to flowering and spike development.

Integrated mapping and characterization of the gene underlying the okra leaf trait in Gossypium hirsutum L.

Diverse leaf morphology has been observed among accessions of Gossypium hirsutum, including okra leaf, which has advantages and disadvantages in cotton production. The okra leaf locus has been mapped to chromosome 15 of the Dt subgenome, but the underlying gene has yet to be identified. In this study, we used a combination of targeted association analysis, F2 population-based fine mapping, and comparative sequencing of orthologues to identify a candidate gene underlying the okra leaf trait in G. hirsutum. The okra leaf gene identified, GhOKRA, encoded a homeodomain leucine-zipper class I protein, whose closely related genes in several other plant species have been shown to be involved in regulating leaf morphology. The transcript levels of GhOKRA in shoot apices were positively correlated with the phenotypic expression of the okra leaf trait. Of the multiple sequence variations observed in the coding region among GrOKRA of Gossypium raimondii and GhOKRA-Dt of normal and okra/superokra leaf G. hirsutum accessions, a non-synonymous substitution near the N terminus and the variable protein sequences at the C terminus may be related to the leaf shape difference. Our results suggest that both transcription and protein activity of GhOKRA may be involved in regulating leaf shape. Furthermore, we found that non-reciprocal homoeologous recombination, or gene conversion, may have played a role in the origin of the okra leaf allele. Our results provided tools for further investigating and understanding the fundamental biological processes that are responsible for the cotton leaf shape variation and will help in the design of cotton plants with an ideal leaf shape for enhanced cotton production.

Functional characterization of the homeodomain leucine zipper I transcription factor AtHB13 reveals a crucial role in Arabidopsis development.

AtHB13 is a homeodomain leucine zipper I transcription factor whose function in development is largely unknown. AtHB13 and AtHB23 mutant and silenced lines were characterized by expression studies, reciprocal crosses, complementation, molecular analyses, and developmental phenotypes. The athb13-1 and athb13-2 mutants, athb23 silenced, and athb13/athb23 double-silenced plants exhibited faster elongation rates of their inflorescence stems, whereas only athb13-1 and the double-knockdown athb13/athb23 exhibited shorter siliques, fewer seeds, and unfertilized ovules compared with the wild type (WT). The cell sizes of mutant and WT plants were similar, indicating that these transcription factors probably affect cell division. Reciprocal crosses between athb13-1 and the WT genotype indicated that the silique defect was male specific. Pollen hydration assays indicated that the pollen grains of the athb13-1 mutant were unable to germinate on stigmas. AtHB23-silenced plants exhibited normal siliques, whereas double-knockdown athb13/athb23 plants were similar to athb13-1 plants. Both AtHB13 and AtHB23 were able to rescue the abnormal silique phenotype. AtHB23 was upregulated in athb13-2 plants, whereas its transcript levels in athb13-1 mutants were not significantly increased. Transcriptome analysis comparing athb13-1 and WT inflorescences revealed that a large number of genes, including several involved in pollen coat formation, are regulated by AtHB13. Finally, athb13-1 complementation with mutated versions of AtHB13 confirmed that two different tryptophans in its C terminus are essential. We conclude that AtHB13 and AtHB23 play independent, negative developmental roles in stem elongation, whereas only AtHB13 is crucial for pollen germination. Furthermore, AtHB23, which does not normally exert a functional role in pollen, can act as a substitute for AtHB13.

Thermospermine levels are controlled by an auxin-dependent feedback loop mechanism in Populus xylem.

Polyamines are small polycationic amines that are widespread in living organisms. Thermospermine, synthesized by thermospermine synthase ACAULIS5 (ACL5), was recently shown to be an endogenous plant polyamine. Thermospermine is critical for proper vascular development and xylem cell specification, but it is not known how thermospermine homeostasis is controlled in the xylem. We present data in the Populus model system supporting the existence of a negative feedback control of thermospermine levels in stem xylem tissues, the main site of thermospermine biosynthesis. While over-expression of the ACL5 homologue in Populus, POPACAULIS5, resulted in strong up-regulation of ACL5 expression and thermospermine accumulation in leaves, the corresponding levels in the secondary xylem tissues of the stem were similar or lower than those in the wild-type. POPACAULIS5 over-expression had a negative effect on accumulation of indole-3-acetic acid, while exogenous auxin had a positive effect on POPACAULIS5 expression, thus promoting thermospermine accumulation. Further, over-expression of POPACAULIS5 negatively affected expression of the class III homeodomain leucine zipper (HD-Zip III) transcription factor gene PttHB8, a homologue of AtHB8, while up-regulation of PttHB8 positively affected POPACAULIS5 expression. These results indicate that excessive accumulation of thermospermine is prevented by a negative feedback control of POPACAULIS5 transcript levels through suppression of indole-3-acetic acid levels, and that PttHB8 is involved in the control of POPACAULIS5 expression. We propose that this negative feedback loop functions to maintain steady-state levels of thermospermine, which is required for proper xylem development, and that it is dependent on the presence of high concentrations of endogenous indole-3-acetic acid, such as those present in the secondary xylem tissues.

Evolution of the class IV HD-zip gene family in streptophytes.

Class IV homeodomain leucine zipper (C4HDZ) genes are plant-specific transcription factors that, based on phenotypes in Arabidopsis thaliana, play an important role in epidermal development. In this study, we sampled all major extant lineages and their closest algal relatives for C4HDZ homologs and phylogenetic analyses result in a gene tree that mirrors land plant evolution with evidence for gene duplications in many lineages, but minimal evidence for gene losses. Our analysis suggests an ancestral C4HDZ gene originated in an algal ancestor of land plants and a single ancestral gene was present in the last common ancestor of land plants. Independent gene duplications are evident within several lineages including mosses, lycophytes, euphyllophytes, seed plants, and, most notably, angiosperms. In recently evolved angiosperm paralogs, we find evidence of pseudogenization via mutations in both coding and regulatory sequences. The increasing complexity of the C4HDZ gene family through the diversification of land plants correlates to increasing complexity in epidermal characters.

Origin of a novel regulatory module by duplication and degeneration of an ancient plant transcription factor.

It is commonly believed that gene duplications provide the raw material for morphological evolution. Both the number of genes and size of gene families have increased during the diversification of land plants. Several small proteins that regulate transcription factors have recently been identified in plants, including the LITTLE ZIPPER (ZPR) proteins. ZPRs are post-translational negative regulators, via heterodimerization, of class III Homeodomain Leucine Zipper (C3HDZ) proteins that play a key role in directing plant form and growth. We show that ZPR genes originated as a duplication of a C3HDZ transcription factor paralog in the common ancestor of euphyllophytes (ferns and seed plants). The ZPRs evolved by degenerative mutations resulting in loss all of the C3HDZ functional domains, except the leucine zipper that modulates dimerization. ZPRs represent a novel regulatory module of the C3HDZ network unique to the euphyllophyte lineage, and their origin correlates to a period of rapid morphological changes and increased complexity in land plants. The origin of the ZPRs illustrates the significance of gene duplications in creating developmental complexity during land plant evolution that likely led to morphological evolution.

In silico genome-wide analysis of homeodomain-leucine zipper transcription factors in Cannabis sativa L.

HD-Zip (Homeodomain-Leucine Zipper) is a family of transcription factors unique to higher plants and plays a vital role in plant growth and development. Increasing research results show that HD-Zip transcription factors are widely involved in many life processes in plants. However, the HD-Zip transcription factor for cannabis, a valuable crop, has not yet been identified. The sequence characteristics, chromosome localization, system evolution, conservative motif, gene structure, and gene expression of the HD-Zip transcription factor in the cannabis genome were systematically studied. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to verify its function. The results showed that cannabis contained 33 HD-Zip gene members. The number of amino acids is 136-849aa, the isoelectric point is 4.54-9.04, and the molecular weight is 23264.32-93147.87Da. Many cis-acting elements are corresponding to hormone and abiotic stress in the HD-Zip family promoter area of cannabis. Sequencing of the transcriptome at 5 tissue sites of hemp, stems, leaves, bracts, and seeds showed similar levels of expression of 33 members of the HD-Zip gene family at 5 tissue sites. Bioinformatics results show that HD-Zip expression is tissue-specific and may be influenced by hormones and environmental factors. This lays a foundation for further research on the gene function of HD-Zip.

Capsicum annuum homeobox 1 (CaHB1) is a nuclear factor that has roles in plant development, salt tolerance, and pathogen defense.

Homeodomain-leucine zipper (HD-Zip) family proteins are unique to plants, but little is known about their role in defense responses. CaHB1 is a nuclear factor in peppers, belonging to subfamily II of HD-Zip proteins. Here, we determined the role of CaHB1 in the defense response. CaHB1 expression was induced when pepper plants were challenged with Phytophthora capsici, a plant pathogen to which peppers are susceptible, or environmental stresses such as drought and salt stimuli. CaHB1 was also highly expressed in pepper leaves following application of SA, whereas ethephon and MeJA had a moderate effect. To further investigate the function of CaHB1 in plants, we performed gain-of-function study by overexpression of CaHB1 in tomato. CaHB1-transgenic tomatoes showed significant growth enhancement including increased leaf thickness and enlarged cell size (1.8-fold larger than control plants). Microscopic analysis revealed that leaves from CaHB1-transgenic plants had thicker cell walls and cuticle layers than those from controls. Moreover, CaHB1-transgenic plants displayed enhanced resistance against Phytophthora infestans and increased tolerance to salt stress. Additionally, RT-PCR analysis of CaHB1-transgenic tomatoes revealed constitutive up-regulation of multiple genes involved in plant defense and osmotic stress. Therefore, our findings suggest roles for CaHB1 in development, salt stress, and pathogen defense.