Homeodomain-leucine Zipper Proteins Research Articles

Zoom-in to molecular mechanisms underlying root growth and function under heterogeneous soil environment and abiotic stresses.

The review describes tissue-specific and non-cell autonomous molecular responses regulating the root system architecture and function in plants. Phenotypic plasticity of roots relies on specific molecular and tissue specific responses towards local and microscale heterogeneity in edaphic factors. Unlike gravitropism, hydrotropism in Arabidopsis is regulated by MIZU KUSSIE1 (MIZ1)-dependent asymmetric distribution of cytokinin and activation of Arabidopsis response regulators, ARR16 and ARR17 on the lower water potential side of the root leading to higher cell division and root bending. The cortex specific role of Abscisic acid (ABA)-activated SNF1-related protein kinase 2.2 (SnRK2.2) and MIZ1 in elongation zone is emerging for hydrotropic curvature. Halotropism involves clathrin-mediated internalization of PIN FORMED 2 (PIN2) proteins at the side facing higher salt concentration in the root tip, and ABA-activated SnRK2.6 mediated phosphorylation of cortical microtubule-associated protein Spiral2-like (SP2L) in the root transition zone, which results in anisotropic cell expansion and root bending away from higher salt. In hydropatterning, Indole-3-acetic acid 3 (IAA3) interacts with SUMOylated-ARF7 (Auxin response factor 7) and prevents expression of Lateral organ boundaries-domain 16 (LBD16) in air-side of the root, while on wet side of the root, IAA3 cannot repress the non-SUMOylated-ARF7 thereby leading to LBD16 expression and lateral root development. In root vasculature, ABA induces expression of microRNA165/microRNA166 in endodermis, which moves into the stele to target class III Homeodomain leucine zipper protein (HD-ZIP III) mRNA in non-cell autonomous manner. The bidirectional gradient of microRNA165/6 and HD-ZIP III mRNA regulates xylem patterning under stress. Understanding the tissue specific molecular mechanisms regulating the root responses under heterogeneous and stress environments will help in designing climate-resilient crops.

ClSnRK2.3 negatively regulates watermelon fruit ripening and sugar accumulation.

Watermelon (Citrullus lanatus) as non-climacteric fruit is domesticated from the ancestors with inedible fruits. We previously revealed that the abscisic acid (ABA) signaling pathway gene ClSnRK2.3 might influence watermelon fruit ripening. However, the molecular mechanisms are unclear. Here, we found that the selective variation of ClSnRK2.3 resulted in lower promoter activity and gene expression level in cultivated watermelons than ancestors, which indicated ClSnRK2.3 might be a negative regulator in fruit ripening. Overexpression (OE) of ClSnRK2.3 significantly delayed watermelon fruit ripening and suppressed the accumulation of sucrose, ABA and gibberellin GA4 . Furthermore, we determined that the pyrophosphate-dependent phosphofructokinase (ClPFP1) in sugar metabolism pathway and GA biosynthesis enzyme GA20 oxidase (ClGA20ox) could be phosphorylated by ClSnRK2.3 and thereby resulting in accelerated protein degradation in OE lines and finally led to low levels of sucrose and GA4 . Besides that, ClSnRK2.3 phosphorylated homeodomain-leucine zipper protein (ClHAT1) and protected it from degradation to suppress the expression of the ABA biosynthesis gene 9'-cis-epoxycarotenoid dioxygenase 3 (ClNCED3). These results indicated that ClSnRK2.3 negatively regulated watermelon fruit ripening by manipulating the biosynthesis of sucrose, ABA and GA4 . Altogether, these findings revealed a novel regulatory mechanism in non-climacteric fruit development and ripening.

HD-ZIP III-dependent local promotion of brassinosteroid synthesis suppresses vascular cell division in Arabidopsis root apical meristem

Spatiotemporal control of cell division in the meristem is vital for plant growth. In the stele of the root apical meristem (RAM), procambial cells divide periclinally to increase the number of vascular cell files. Class III homeodomain leucine zipper (HD-ZIP III) proteins are key transcriptional regulators of RAM development and suppress the periclinal division of vascular cells in the stele; however, the mechanism underlying the regulation of vascular cell division by HD-ZIP III transcription factors (TFs) remains largely unknown. Here, we performed transcriptome analysis to identify downstream genes of HD-ZIP III and found that HD-ZIP III TFs positively regulate brassinosteroid biosynthesis-related genes, such as CONSTITUTIVE PHOTOMORPHOGENIC DWARF (CPD), in vascular cells. Introduction of pREVOLUTA::CPD in a quadruple loss-of-function mutant of HD-ZIP III genes partly rescued the phenotype in terms of the vascular defect in the RAM. Treatment of a quadruple loss-of-function mutant, a gain-of-function mutant of HD-ZIP III, and the wild type with brassinosteroid and a brassinosteroid synthesis inhibitor also indicated that HD-ZIP III TFs act together to suppress vascular cell division by increasing brassinosteroid levels. Furthermore, brassinosteroid application suppressed the cytokinin response in vascular cells. Together, our findings suggest that the suppression of vascular cell division by HD-ZIP III TFs is caused, at least in part, by the increase in brassinosteroid levels through the transcriptional activation of brassinosteroid biosynthesis genes in the vascular cells of the RAM. This elevated brassinosteroid level suppresses cytokinin response in vascular cells, inhibiting vascular cell division in the RAM.

Genome-Wide Identification of Homeodomain Leucine Zipper (HD-ZIP) Transcription Factor, Expression Analysis, and Protein Interaction of HD-ZIP IV in Oil Palm Somatic Embryogenesis

Understanding the molecular mechanisms underlying somatic embryogenesis is essential for resolving the problems related to the long duration of the process and a low rate of somatic embryo induction in oil palm tissue culture. In this study, we conducted genome-wide identification of the oil palm homeodomain leucine zipper (EgHD-ZIP) family, which is one of the plant-specific transcription factors reported to be involved in embryogenesis. EgHD-ZIP proteins can be divided into four subfamilies, which have similarities in gene structure and protein-conserved motifs within a group. In silico expression analysis showed that the expression of EgHD-ZIP gene members in the EgHD-ZIP I and II families, as well as most members in the EgHD-ZIP IV family, were up-regulated during the zygotic and somatic embryo developmental stages. In contrast, the expression of EgHD-ZIP gene members in the EgHD-ZIP III family was down-regulated during zygotic embryo development. Moreover, the expression of EgHD-ZIP IV genes was validated in the oil palm callus and at the somatic embryo stages (globular, torpedo, and cotyledon). The results revealed that EgHD-ZIP IV genes were up-regulated at the late stages of somatic embryogenesis (torpedo and cotyledon). While BABY BOOM (BBM) gene was up-regulated at the early stage of somatic embryogenesis (globular). In addition, the Yeast-two hybrid assay revealed the direct binding between all members of the oil palm HD-ZIP IV subfamily (EgROC2, EgROC3, EgROC5, EgROC8, and EgBBM). Our findings suggested that the EgHD-ZIP IV subfamily and EgBBM work together to regulate somatic embryogenesis in oil palms. This process is important because it is widely used in plant biotechnology to produce large quantities of genetically identical plants, which can be used for oil palm tissue culture improvement.

Genome-Wide Identification and Expression Profile of the HD-Zip Transcription Factor Family Associated with Seed Germination and Abiotic Stress Response in Miscanthus sinensis.

Miscanthus sinensis is an ornamental grass, non-food bioenergy crop, and forage with high feeding value. It can adapt to many kinds of soil conditions due to its high level of resistance to various abiotic stresses. However, a low level of seed germination restricts the utilization and application of M. sinensis. It is reported that the Homeodomain-leucine zipper (HD-Zip) gene family participates in plant growth and development and ability to cope with outside environment stresses, which may potentially regulate seed germination and stress resistance in M. sinensis. In this study, a complete overview of M. sinensis HD-Zip genes was conducted, including gene structure, conserved motifs, chromosomal distribution, and gene duplication patterns. A total of 169 members were identified, and the HD-Zip proteins were divided into four subgroups. Inter-chromosomal evolutionary analysis revealed that four pairs of tandem duplicate genes and 72 segmental duplications were detected, suggesting the possible role of gene replication events in the amplification of the M. sinensis HD-Zip gene family. There was an uneven distribution of HD-Zip genes on 19 chromosomes of M. sinensis. Also, evolutionary analysis showed that M. sinensis HD-Zip gene family members had more collinearity with monocotyledons and less with dicotyledons. The gene structure analysis showed that there were 93.5% of proteins with motif 1 and motif 4, while motif 10 was only found in group IV. Based on the cis-elements analysis, it appeared that most of the genes were related to plant growth and development, various hormones, and abiotic stress. Furthermore, qRT-PCR analysis showed that Misin06G303300.1 was significantly expressed in seed germination and Misin05G030000.1 and Misin06G303300.1 were highly expressed under chromium, salt, and drought stress. Results in this study will provide a basis for further exploring the potential role of HD-Zip genes in stress responses and genetic improvement of M. sinensis seed germination.

Overexpression of Pyrus sinkiangensis HAT5 enhances drought and salt tolerance, and low-temperature sensitivity in transgenic tomato.

The homeodomain-leucine zipper protein HAT belongs to the homeodomain leucine zipper subfamily (HD-Zip) and is important for regulating plant growth and development and stress tolerance. To investigate the role of HAT5 in tolerance to drought, salt, and low temperature stress, we selected a HAT gene from Pyrus sinkiangensis Yü (Pyrus sinkiangensis T.T. Yu). The sequences were analyzed using ioinformatics, and the overexpressed tomato lines were obtained using molecular biology techniques. The phenotypes, physiological, and biochemical indexes of the wild-type and transgenic tomato lines were observed under different stress conditions. We found that the gene had the highest homology with PbrHAT5. Under drought and NaCl stress, osmotic regulatory substances (especially proline) were significantly accumulated, and antioxidant enzyme activities were enhanced. The malondialdehyde level and relative electrical conductivity of transgenic tomatoes under low temperature (freezing) stress were significantly higher than those of wild-type tomatoes. The reactive oxygen species scavenging system was unbalanced. This study found that PsHAT5 improved the tolerance of tomatoes to drought and salt stress by regulating proline metabolism and oxidative stress ability, reducing the production of reactive oxygen species, and maintaining normal cell metabolism. In conclusion, the PsHAT5 transcription factor has great potential in crop resistance breeding, which lays a theoretical foundation for future excavation of effective resistance genes of the HD-Zip family and experimental field studies.

The roles of HD-ZIP proteins in plant abiotic stress tolerance.

Homeodomain leucine zipper (HD-ZIP) proteins are plant-specific transcription factors that contain a homeodomain (HD) and a leucine zipper (LZ) domain. The highly conserved HD binds specifically to DNA and the LZ mediates homodimer or heterodimer formation. HD-ZIP transcription factors control plant growth, development, and responses to abiotic stress by regulating downstream target genes and hormone regulatory pathways. HD-ZIP proteins are divided into four subclasses (I–IV) according to their sequence conservation and function. The genome-wide identification and expression profile analysis of HD-ZIP proteins in model plants such as Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) have improved our understanding of the functions of the different subclasses. In this review, we mainly summarize and discuss the roles of HD-ZIP proteins in plant response to abiotic stresses such as drought, salinity, low temperature, and harmful metals. HD-ZIP proteins mainly mediate plant stress tolerance by regulating the expression of downstream stress-related genes through abscisic acid (ABA) mediated signaling pathways, and also by regulating plant growth and development. This review provides a basis for understanding the roles of HD-ZIP proteins and potential targets for breeding abiotic stress tolerance in plants.

The miR166-mRNA network regulates vascular tissue differentiation in Moso bamboo.

miR166s play an important role in plant tissue differentiation. However, the functions of miR166s in the differentiation of vascular tissue in bamboo have not yet been elucidated. Here, we showed that five miR166s are overexpressed (tags per million reads > 2,000) in underground shoot samples of wild-type (WT) Moso bamboo (Phyllostachys edulis) and a thick-walled variant (P. edulis “Pachyloen”) throughout the developmental process. Potential targets of these miR166s include some genes encoding homeodomain-leucine zipper (HD-ZIP) transcription factors and protein kinases. Cleavage sites for miR166s were identified in seven PeHD-ZIP homologs and a protein kinase gene via degradome sequencing (p < 0.05). Dual-luciferase and transient expression assays confirmed the binding of miR166s to PeHOXs. Fluorescence in situ hybridization revealed that miR166s were localized to the xylem of the leaf, root, and internode of 2-month-old pot seedlings of WT Moso bamboo. Overall, these findings reveal that miR166s are regulators of vascular tissue differentiation in bamboo. The miR166s identified in our study provide novel targets for bamboo breeding.

Identification and analysis of HD‐Zip genes involved in the leaf development of Liriodendron chinense using multidimensional analysis

Homeodomain-leucine zipper (HD-Zip) proteins are plant-specific transcription factors that play important roles in different biological processes, especially leaf development. However, no studies to date have identified the HD-Zip genes in Liriodendron chinense nor characterized their functions. We identified the HD-Zip genes in L. chinense by analysing the phylogeny, chromosome location, structure, conserved motif, cis-regulatory elements, synteny, post-transcriptional regulation and expression patterns of these genes during leaf development. A total of 36 LcHD-Zip genes were identified and divided into four subfamilies (HD-Zip I to IV). Synteny analysis revealed that segmental duplication was the main force driving the expansion of LcHD-Zip genes. These 36 LcHD-Zip genes exhibited 11 different expression patterns. Pattern 1, 2, 3, 4, 6, 7, 8 and 9 genes may play important roles in leaf development, such as leaf initiation, leaf polarity establishment, leaf shape development, phytohormone-mediated leaf growth and leaf epidermal structure formation. Four HD-Zip III genes were targeted by microRNAs (miRNAs), and the miR165/166a-HD-Zip regulatory module formed regulated leaf initiation and leaf polarity establishment. Overall, LcHD-Zip genes play key roles in leaf development of L. chinense. This work provides a foundation for the functional verification of HD-Zip genes identified in this study.

Genome-wide identification and expression analyses of homeodomain-leucine zipper family genes reveal their involvement in stress response in apple (Malus × domestica)

The homeodomain-leucine zipper (HD-Zip) family has been shown to perform a multitude of functions during plant development and stress responses; however, the family members and functions have not been identified in apple (Malus × domestica). In this study, 83 HD-Zips (MdHDZs) were identified in the apple genome. They were assembled into four subgroups according to the classification in Arabidopsis, where MdHDZs in the same subgroup had similar gene structures and conserved protein motifs. Putative cis-element analysis of MdHDZs promoter regions uncovered numerous elements related to the response of stress and plant hormones. In addition, twelve transcripts of the MdHDZs showed different expression patterns under salt, drought, low temperature and ABA stresses by quantitative reverse transcription-PCR (qRT-PCR) assay. To further explore the function of MdHDZs in apple, MdHDZ3 was selected to verify its function under salt, low temperature and ABA stresses; and genetic transformation was used to obtain MdHDZ3 transgenic apple calli. The results demonstrated that MdHDZ3 increased sensitivity to salt, low temperature and abscisic acid in apple calli, suggesting that MdHDZ3 plays an important role in response to stresses. Subcellular localization and three-dimensional structural analysis revealed that MdHDZ3 was a nuclear-localized protein. Taken together, these findings provide potential information for further identification of HD-Zip proteins in apple.

Identification and characterization of HD-Zip genes reveals their roles in stresses responses and facultative crassulacean acid metabolism in Dendrobium catenatum

Dendrobium catenatum possesses special survival strategy including facultative crassulacean acid metabolism (CAM) to cope with various environmental stresses. The homeodomain-leucine zipper (HD-Zip) proteins had been reported to play essential roles in adaption to the environment. However, the role of the HD-Zip gene family in stresses responses and drought-induced facultative CAM in D. catenatum was largely unknown. In this study, 35 HD-Zip genes were identified in D. catenatum (DcHDZs), and were phylogenetically grouped into four subfamilies, which was further supported by the analysis of their conserved motifs and structures. Expression profile analysis of DcHD-Zip genes based on public transcriptomic data and qRT-PCR showed that most DcHD-Zip genes showed biased expression in leaf and flower than in stem and root. Three DcHD-Zip genes denominated as DcHDZ04, DcHDZ20 and DcHDZ22 belong to HD-Zip I and II subfamilies, were induced significantly under high light, low light, salt and heat stresses. Moreover, we found that the plant hormone biosynthesis and signal transduction played a vital role in facultative CAM of D. catenatum under drought, especially ABA. Nine DcHD-Zip genes were identified as DEGs and showed close relationship with several genes involved in plant hormone biosynthesis and signal transduction, suggesting their important roles in drought-induced facultative CAM of D. catenatum. This systematic analysis provided a foundation for further functional characterization of HD-Zip genes and also revealed an important regulation mechanism of facultative CAM in D. catenatum.

Identification of microRNAs and their gene targets in cytoplasmic male sterile and fertile maintainer lines of pigeonpea.

Comparative analysis of genome-wide miRNAs and their gene targets between cytoplasmic male sterile (CMS) and fertile lines of pigeonpea suggests a possible role of miRNA-regulated pathways in reproductive development. Exploitation of hybrid vigor using CMS technology has delivered nearly 50% yield gain in pigeonpea. Among various sterility-inducing cytoplasms (A1-A9) reported so far in pigeonpea, A2 and A4 are the two major sources that facilitate hybrid seed production. Recent evidence suggests involvement of micro RNA in vast array of biological processes including plant reproductive development. In pigeonpea, information about the miRNAs is insufficient. In view of this, we sequenced six small RNA libraries of CMS line UPAS 120A and isogenic fertile line UPAS 120B using Illumina technology. Results revealed 316 miRNAs including 248 known and 68 novel types. A total of 637 gene targets were predicted for known miRNAs, while 324 genes were associated with novel miRNAs. Degradome analysis revealed 77 gene targets of predicted miRNAs, which included a variety of transcription factors playing key roles in plant reproduction such as F-box family proteins, apetala 2, auxin response factors, ethylene-responsive factors, homeodomain-leucine zipper proteins etc. Differential expression of both known and novel miRNAs implied roles for both conserved as well as species-specific players. We also obtained several miRNA families such as miR156, miR159, miR167 that are known to influence crucial aspects of plant fertility. Gene ontology and pathway level analyses of the target genes showed their possible implications for crucial events during male reproductive development such as tapetal degeneration, pollen wall formation, retrograde signaling etc. To the best of our knowledge, present study is first to combine deep sequencing of small RNA and degradome for elucidating the role of miRNAs in flower and male reproductive development in pigeonpea.

Betula platyphylla BpHOX2 transcription factor binds to different cis‐acting elements and confers osmotic tolerance

The homeodomain-leucine zipper (HD-Zip) proteins play crucial roles in plant developmental and environmental responses. However, how they mediate gene expression to facilitate abiotic stress tolerance remains unknown. In the present study, we characterized BpHOX2 (encoding a HD-Zip I family protein) from birch (Betula platyphylla). BpHOX2 is predominately expressed in mature stems and leaves, but expressed at a low level in apical buds and roots, suggesting that it has tissue-specific characteristics. BpHOX2 expression was highly induced by osmotic and salt, but only slightly induced by abscisic acid. Overexpression of BpHOX2 markedly improved osmotic tolerance, while knockdown of BpHOX2 increased sensitivity to osmotic stress. BpHOX2 could induce the expression of pyrroline-5-carboxylate synthase, peroxidase, and superoxide dismutase genes to improve proline levels and the reactive oxygen species scavenging capability. Chromatin immunoprecipitation sequencing combined with RNA sequencing showed that BpHOX2 could bind to at least four cis-acting elements, including dehydration-responsive element "RCCGAC", Myb-p binding box "CCWACC," and two novel cis-acting elements with the sequences of "AAGAAG" and "TACGTG" (termed HBS1 and HBS2, respectively) to regulate gene expression. Our results suggested that BpHOX2 is a transcription factor that binds to different cis-acting elements to regulate gene expression, ultimately improving osmotic tolerance in birch.

Four HD-ZIPs are involved in banana fruit ripening by activating the transcription of ethylene biosynthetic and cell wall-modifying genes.

Four MaHDZs are possibly involved in banana fruit ripening by activating the transcription of genes related to ethylene biosynthesis and cell wall degradation, such as MaACO5,MaEXP2, MaEXPA10, MaPG4 and MaPL4. The homeodomain-leucine zipper (HD-ZIP) proteins represent plant-specific transcription factors, which contribute to various plant physiological processes. However, little information is available regarding the association of HD-ZIPs with banana fruit ripening. In this study, we identified a total of 96 HD-ZIP genes in banana genome, which were divided into four different groups consisting of 35, 31, 9 and 21 members in the I, II, III and IV subfamilies, respectively. The expression patterns of MaHDZ genes during fruit ripening showed that MaHDZI.19, MaHDZI.26, MaHDZII.4 and MaHDZII.7 were significantly up-regulated in the ripening stage and thus suggested to be potential regulators of banana fruit ripening. Furthermore, MaHDZI.19, MaHDZI.26, MaHDZII.4 and MaHDZII.7 were found to localize exclusively in the nucleus and exhibit transcriptional activation capacities. Importantly, MaHDZI.19, MaHDZI.26, MaHDZII.4 and MaHDZII.7 stimulated the transcription of several ripening-related genes including MaACO5 related to ethylene biosynthesis, MaEXP2, MaEXPA10, MaPG4 and MaPL4 were associated with cell wall degradation, through directly binding to their promoters. Taken together, our findings expand the functions of HD-ZIP transcription factors and identify four MaHDZs likely involved in regulating banana fruit ripening by activating the expression of genes related to ethylene biosynthesis and cell wall modification, which may have potential application in banana molecular breeding.

Overexpression of OsHox-6 Gene Enhanced Tiller Number in Rice But Induced Yield Penalty

OsHox-6, belongs to the transcription factor homeodomain leucine zipper (HD-Zip) protein sub-family I, has unknown function. This study was aimed to characterize the phenotypes of two homozygous transgenic rice lines (S29-62-2 and S.40.4-158-1) containing an extra copy of OsHox-6 gene under the control of a rice constitutive promoter, OsLEA3, and to evaluate their tolerance to water stress. A real-time quantitative PCR (qRT-PCR) showed that the transcript expression of OsHox-6 gene in the transgenic lines increased 5-10 folds under a normal irrigation and 10-20 folds after exposure to water stress conditions as compared to its wild type control. Transgenic plants overexpressing OsHox-6 exhibited phenotypic alteration at the normal irrigation by inducing tiller formation, suggesting a decrease in the apical dominance. Transgenic plants also showed significant enhancement in the total grain number, however, the number of empty grains also increased significantly (~16-22%). After imposed to the water stress, the number of empty grains in the transgenic lines was even higher (up to 83% in average). Furthermore, observations on the water loss rates, relative water contents and drought resistance indices (DRI) suggested that the overexpression of OsHox-6 did not significantly increase tolerance to water stress. Further research is required to reveal the detailed mechanisms of OsHox-6 in response to water and other abiotic stresses.

Genome-wide characterization and expression analysis of the HD-Zip gene family in response to drought and salinity stresses in sesame

BackgroundThe homeodomain-leucine zipper (HD-Zip) gene family is one of the plant-specific transcription factor families, involved in plant development, growth, and in the response to diverse stresses. However, comprehensive analysis of the HD-Zip genes, especially those involved in response to drought and salinity stresses is lacking in sesame (Sesamum indicum L.), an important oil crop in tropical and subtropical areas.ResultsIn this study, 45 HD-Zip genes were identified in sesame, and denominated as SiHDZ01-SiHDZ45. Members of SiHDZ family were classified into four groups (HD-Zip I-IV) based on the phylogenetic relationship of Arabidopsis HD-Zip proteins, which was further supported by the analysis of their conserved motifs and gene structures. Expression analyses of SiHDZ genes based on transcriptome data showed that the expression patterns of these genes were varied in different tissues. Additionally, we showed that at least 75% of the SiHDZ genes were differentially expressed in responses to drought and salinity treatments, and highlighted the important role of HD-Zip I and II genes in stress responses in sesame.ConclusionsThis study provides important information for functional characterization of stress-responsive HD-Zip genes and may contribute to the better understanding of the molecular basis of stress tolerance in sesame.

Small Grain and Dwarf 2, encoding an HD-Zip II family transcription factor, regulates plant development by modulating gibberellin biosynthesis in rice

Homeodomain leucine zipper (HD-Zip) proteins are transcription factors that regulate plant development. Bioactive gibberellin (GA) is a key endogenous hormone that participates in plant growth. However, the relationship between HD-Zip genes and modulation of GA biosynthesis in rice remains elusive. Here, we identified a rice mutant, designated as small grain and dwarf 2 (sgd2), which had reduced height and grain size compared with the wild type. Cytological observations indicated that the defective phenotype was mainly due to decreased cell length. Map-based cloning and complementation tests demonstrated that a 9 bp deletion in a homeodomain leucine zipper (HD-Zip) II family transcription factor was responsible for the sgd2 mutant phenotype. Expression of SGD2 was pronounced in developing panicles, and its protein was localized in nucleus. Luciferase reporter system and transactivation assays in yeast suggested that SGD2 functioned as a transcriptional repressor. High performance liquid chromatography assays showed that the endogenous GA1 level in the sgd2 mutant was dramatically decreased, and exogenous GA3 recovered the second leaf sheath to normal length. Results of qRT-PCR showed that the expression levels of genes positively regulating GA-biosynthesis were mostly down-regulated in the mutant. Our data identified the role of an HD-Zip transcription factor that affects rice plant development by modulating gibberellin biosynthesis.

AtHB2, a class II HD-ZIP protein, negatively regulates the expression of CsANS, which encodes a key enzyme in Camellia sinensis catechin biosynthesis.

Tea (Camellia sinensis) is an important cash crop that is beneficial to human health because of its remarkable content of catechins. The biosynthesis of catechins follows the flavonoid pathway, which is highly branched. Among the enzymes involved in catechin biosynthesis, ANTHOCYANIDIN SYNTHASE (CsANS) functions at a branch point and play a critical role. Our previous work has showed that the gene encoding CsANS is regulated by light signals; however, the molecular mechanism behind remains unclear. Here, we cloned a full-length CsANS promoter and found that it contained a cis-element recognized by Arabidopsis thaliana HOMEOBOX2 (AtHB2). AtHB2 constitutes one of the class II HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP) proteins, which accumulate in the dark and mediate the shade avoidance response in most angiosperms. To analyze the transcription of CsANS in vivo, β-glucuronidase and luciferase reporter genes driven by the obtained promoter were introduced into A. thaliana and Nicotiana attenuata, respectively. In both expression systems there were indications that the A. thaliana PRODUCTION OF ANTHOCYANIN PIGMENT1 (AtPAP1), a MYB transcription factor of flavonoid biosynthesis, increased the activity of the CsANS promoter, while AtHB2 could significantly undermine the effect of AtPAP1. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that AtHB2 interacted with the A. thaliana TRANSPARENT TESTA GLABRA 1 (AtTTG1). A yeast three-hybrid assay further suggested that AtHB2 represses the expression of CsANS and regulates its response to light signals through competitive interactions with AtTTG1. These results show that HD-ZIP II proteins participate in light regulation of flavonoid biosynthesis.

Genomic and transcriptomic analyses of HD-Zip family transcription factors and their responses to abiotic stress in tea plant (Camellia sinensis)

Tea plant (Camellia sinensis (L.) O. Kuntze) is a perennial evergreen woody plant, and its leaves contain various beneficial ingredients and have healthy efficacy. HD-Zip (homeodomain-leucine zipper) transcription factors (TFs) are widely distributed in plants and play an important role in plant growth and environmental response. To date, knowledge on HD-Zip gene family in tea plant is still limited. In this study, 33 HD-Zip TFs were selected based on the genomic and transcriptomic databases of tea plant. The conserved domains and common motifs of these TFs were predicted and analyzed. These 33 Cshdz TFs were divided into four groups (HD-Zip I, HD-Zip II, HD-Zip III, and HD-Zip IV). The interaction network of the HD-Zip proteins of tea plant was established based on the data of Arabidopsis. In addition, the expression levels of these Cshdz genes in tea plant cv. ‘Longjing43’ were detected and analyzed under five abiotic stress treatments. Results showed that the different expression profiles of Cshdz genes were associated with different abiotic stress treatments. Our findings suggested a potential relationship between the resistance of tea plant and its Cshdz genes.

PpHB22, a member of HD-Zip proteins, activates PpDAM1 to regulate bud dormancy transition in ‘Suli’ pear (Pyrus pyrifolia White Pear Group)

Homeodomain-leucine zipper (HD-Zip) proteins, which form one of the largest and most diverse families, regulate many biological processes in plants, including differentiation, flowering, vascular development, and stress signaling. Abscisic acid (ABA) has been proved to be one of the key regulators of bud dormancy and to influence several HD-Zip genes expression. However, the role of HD-Zip genes in regulating bud dormancy remains unclear. We identified 47 pear (P. pyrifolia White Pear Group) HD-Zip genes, which were classified into four subfamilies (HD-Zip I–IV). We further revealed that gene expression levels of some HD-Zip members were closely related to ABA concentrations in flower buds during dormancy transition. Exogenous ABA treatment confirmed that PpHB22 and several other HD-Zip genes responded to ABA. Yeast one-hybrid and dual luciferase assay results combining subcellular localization showed that PpHB22 was present in nucleus and directly induced PpDAM1 (dormancy associated MADS-box 1) expression. Thus, PpHB22 is a negative regulator of plant growth associated with the ABA response pathway and functions upstream of PpDAM1. These findings enrich our understanding of the function of HD-Zip genes related to the bud dormancy transition.