HD-Zip Genes Research Articles

Challenging the paradigms of leaf evolution: Class III HD-Zips in ferns and lycophytes.

Despite the extraordinary significance leaves have for life on Earth, their origin and development remain vigorously debated. More than a century of paleobotanical, morphological, and phylogenetic research has still not resolved fundamental questions about leaves. Developmental genetic data are sparse in ferns, and comparative studies of lycophytes and seed plants have reached opposing conclusions on the conservation of a leaf developmental program. We performed phylogenetic and expression analyses of a leaf developmental regulator (Class III HD-Zip genes; C3HDZs) spanning lycophytes and ferns. We show that a duplication and neofunctionalization of C3HDZs probably occurred in the ancestor of euphyllophytes, and that there is a common leaf developmental mechanism conserved between ferns and seed plants. We show C3HDZ expression in lycophyte and fern sporangia and show that C3HDZs have conserved expression patterns during initiation of lateral primordia (leaves or sporangia). This expression is maintained throughout sporangium development in lycophytes and ferns and indicates an ancestral role of C3HDZs in sporangium development. We hypothesize that there is a deep homology of all leaves and that a sporangium-specific developmental program was coopted independently for the development of lycophyte and euphyllophyte leaves. This provides molecular genetic support for a paradigm shift in theories of lycophyte leaf evolution.

Class III HD-Zip activity coordinates leaf development in Physcomitrella patens

Land plant bodies develop from meristems, groups of pluripotent stem cells, which may persist throughout the life of a plant or, alternatively, have a transitory existence. Early diverging land plants exhibit indeterminate (persistent) growth in their haploid gametophytic generation, whereas later diverging lineages exhibit indeterminate growth in their diploid sporophytic generation, raising the question of whether genetic machinery directing meristematic functions was co-opted between generations. Class III HD-Zip (C3HDZ) genes are required for the establishment and maintenance of shoot apical meristems in flowering plants. We demonstrate that in the moss Physcomitrella patens, C3HDZ genes are expressed in transitory meristems in both the gametophytic and sporophytic generations, but not in the persistent shoot meristem of the gametyphyte. Loss-of-function of P. patens C3HDZ was engineered using ectopic expression of miR166, an endogenous regulator of C3HDZ gene activity. Loss of C3HDZ gene function impaired the function of gametophytic transitory meristematic activity but did not compromise the functioning of the persistent shoot apical meristem during the gametophyte generation. These results argue against a wholesale co-option of meristematic gene regulatory networks from the gametophyte to the sporophyte during land plant evolution, instead suggesting that persistent meristems with a single apical cell in P. patens and persistent complex meristems in flowering plants are regulated by different genetic programs.

A genome-wide survey of homeodomain-leucine zipper genes and analysis of cold-responsive HD-Zip I members' expression in tomato.

Homeodomain-leucine zipper (HD-Zip) proteins are a kind of transcriptional factors that play a vital role in plant growth and development. However, no detailed information of HD-Zip family in tomato has been reported till now. In this study, 51 HD-Zip genes (SlHZ01-51) in this family were identified and categorized into 4 classes by exon-intron and protein structure in tomato (Solanum lycopersicum) genome. The synthetical phylogenetic tree of tomato, Arabidopsis and rice HD-Zip genes were established for an insight into their evolutionary relationships and putative functions. The results showed that the contribution of segmental duplication was larger than that of tandem duplication for expansion and evolution of genes in this family of tomato. The expression profile results under abiotic stress suggested that all SlHZ I genes were responsive to cold stress. This study will provide a clue for the further investigation of functional identification and the role of tomato HD-Zip I subfamily in plant cold stress responses and developmental events.

Genome-wide identification and expression profile of homeodomain-leucine zipper Class I gene family in Cucumis sativus

The HD-Zip proteins comprise one of the largest families of transcription factors in plants. HD-Zip genes have been grouped into four different classes: HD-Zip I to IV. In this study, we described the identification and structural characterization of Class I HD-Zip genes in cucumber. A complete set of 13 HD-Zip I genes were identified in the cucumber genome using Blast search tools and phylogeny. The cucumber HD-Zip I family contained a smaller number of identified genes compared to other higher plants such as Arabidopsis and maize due to the absence of recent gene duplication events. Chromosomal location of these genes revealed that they are distributed unevenly across 5 of 7 chromosomes. Tissue-specific expression profiles showed that 13 cucumber HD-Zip I genes were expressed in at least one of the tissues, which suggested that cucumber HD-Zip I genes took part in many cellular processes. The transcript abundance level analysis during abiotic stress conditions (NaCl, ABA and low temperature treatments) identified a group of HD-Zip I genes that responded to one or more treatments.

Formation of woody biomass is regulated by class III HD Zip genes

Secondary growth and the development of woody tissue is a key process in the formation of woody biomass. The gene family of Class III HDZip genes has been shown in the herbaceous Arabidopsis model to play a central role in regulating polarity and vascular development. While Arabidopis is a poor model for investigating processes of wood formation, in this project all poplar Class III HDZip genes were cloned and expressed in hybrid aspen as a tree model system. To circumvent an endogenous regulation mechanism involving microRNAs the sequences were also mutated to render them microRNA resistant. Lines expressing the mutated Poplar ortholog of the Arabidopsis Revoluta gene (Populus Revoluta PRE) show a spectacular phenotype with stunted growth, radialized and rolled leaves, and a double and at times triplication or quadruple layer of the xylem, suggesting the formation of multiple layers of cambium. ClassIII HDZip genes have thus been shown to be crucial for the formation of lignified tissue in trees.

Interplay of auxin, KANADI and Class III HD-ZIP transcription factors in vascular tissue formation

Class III HD-ZIP and KANADI gene family members have complementary expression patterns in the vasculature and their gain-of-function and loss-of-function mutants have complementary vascular phenotypes. This suggests that members of the two gene families are involved in the establishment of the spatial arrangement of phloem, cambium and xylem. In this study, we have investigated the role of these two gene families in vascular tissue differentiation, in particular their interactions with the plant hormone auxin. We have analyzed the vasculature of plants that have altered expression levels of Class III HD-ZIP and KANADI transcription factors in provascular cells. Removal of either KANADI or Class III HD-ZIP expression in procambium cells led to a wider distribution of auxin in internal tissues, to an excess of procambium cell recruitment and to increased cambium activity. Ectopic expression of KANADI1 in provascular cells inhibited procambium cell recruitment due to negative effects of KANADI1 on expression and polar localization of the auxin efflux-associated protein PIN-FORMED1. Ectopic expression of Class III HD-ZIP genes promoted xylem differentiation. We propose that Class III HD-ZIP and KANADI transcription factors control cambium activity: KANADI proteins by acting on auxin transport, and Class III HD-ZIP proteins by promoting axial cell elongation and xylem differentiation.

Transformasi padi indica kultivar batutegi dan kasalath dengan gen regulator hd-zip untuk perakitan varietas toleran kekeringan

water deficiency. Genetic engineering at the level of transcription factors (TF) is particulary a promising strategy in developing drought tolerant rice cultivar. HD-Zip genes are TF that function in plant adaptation to some environmental stresses including water deficit. The recombinant plasmid pC1301H Oshox6 which contained HD-Zip Oshox6 gene was placed under a drought inducible promoter called LEA promoter, gusA and hpt genes were driven with CaMV promoter. The aim of research was to obtain indica rice transgenic plants of Batutegi and Kasalath cultivars using pC1301H Oshox6 plasmid. Recombinant plasmid was transformed into immature rice embryos using Agrobacterium tumefaciens . Kasalath cultivar showed a better capacity to form embryogenic calli compared to Batutegi. Transformation efficiency of Batutegi is lower (1.5 - 0.3%) than Kasalath (2.2-28.3%). Regeneration efficiency is 25-83.3% and 7.7-100% for Batutegi and Kasalath, respectively. Number of putative transformant plantlets of Batutegi and Kasalath are 63 and 48 plantlets, respectively. Southern blot analysis (using hpt probe) on 12 independent lines of each Batutegi and Kasalath cultivars showed different gene copy number, ranging from one to four copies of gene. Keywords: agrobacterium tumefaciens, LEA promoter, HD-Zip Oshox6, rice

Developmental Role and Auxin Responsiveness of Class III Homeodomain Leucine Zipper Gene Family Members in Rice

Members of the Class III homeodomain leucine zipper (Class III HD-Zip) gene family are central regulators of crucial aspects of plant development. To better understand the roles of five Class III HD-Zip genes in rice (Oryza sativa) development, we investigated their expression patterns, ectopic expression phenotypes, and auxin responsiveness. Four genes, OSHB1 to OSHB4, were expressed in a localized domain of the shoot apical meristem (SAM), the adaxial cells of leaf primordia, the leaf margins, and the xylem tissue of vascular bundles. In contrast, expression of OSHB5 was observed only in phloem tissue. Plants ectopically expressing microRNA166-resistant versions of the OSHB3 gene exhibited severe defects, including the ectopic production of leaf margins, shoots, and radialized leaves. The treatment of seedlings with auxin quickly induced ectopic OSHB3 expression in the entire region of the SAM, but not in other tissues. Furthermore, this ectopic expression of OSHB3 was correlated with leaf initiation defects. Our findings suggest that rice Class III HD-Zip genes have conserved functions with their homologs in Arabidopsis (Arabidopsis thaliana), but have also acquired specific developmental roles in grasses or monocots. In addition, some Class III HD-Zip genes may regulate the leaf initiation process in the SAM in an auxin-dependent manner.

MicroRNA166 controls root and nodule development in Medicago truncatula

Legume root architecture is characterized by the development of two de novo meristems, leading to the formation of lateral roots or symbiotic nitrogen-fixing nodules. Organogenesis involves networks of transcription factors, the encoding mRNAs of which are frequently targets of microRNA (miRNA) regulation. Most plant miRNAs, in contrast with animal miRNAs, are encoded as single entities in an miRNA precursor. In the model legume Medicago truncatula, we have identified the MtMIR166a precursor containing tandem copies of MIR166 in a single transcriptional unit. These miRNAs post-transcriptionally regulate a new family of transcription factors associated with nodule development, the class-III homeodomain-leucine zipper (HD-ZIP III) genes. In situ expression analysis revealed that these target genes are spatially co-expressed with MIR166 in vascular bundles, and in apical regions of roots and nodules. Overexpression of the tandem miRNA precursor correlated with MIR166 accumulation and the downregulation of several class-III HD-ZIP genes, indicating its functionality. MIR166 overexpression reduced the number of symbiotic nodules and lateral roots, and induced ectopic development of vascular bundles in these transgenic roots. Hence, plant polycistronic miRNA precursors, although rare, can be processed, and MIR166-mediated post-transcriptional regulation is a new regulatory pathway involved in the regulation of legume root architecture.

KANADI and Class III HD-Zip Gene Families Regulate Embryo Patterning and Modulate Auxin Flow during Embryogenesis inArabidopsis

Embryo patterning in Arabidopsis thaliana is highly affected when KANADI or Class III HD-Zip genes are compromised. Triple loss-of-function kan1 kan2 kan4 embryos exhibit striking defects in the peripheral-central axis, developing lateral leaf-like organs from the hypocotyls, whereas loss of Class III HD-Zip gene activity results in a loss of bilateral symmetry. Loss of KANADI activity in a Class III HD-Zip mutant background mitigates the defects in bilateral symmetry, implying that the two gene families act antagonistically during embryonic pattern formation. Dynamic patterns of auxin concentration and flux contribute to embryo patterning. Polar cellular distribution of PIN-FORMED1 (PIN1) mediates auxin flow throughout embryogenesis and is required for establishment of the apical-basal axis and bilateral symmetry. Defects in the pattern of PIN1 expression are evident when members of either the KANADI or Class III HD-Zip gene families are compromised. Abnormal expression patterns of PIN1 in KANADI or Class III HD-Zip multiple mutants and the phenotype of plants in which members of both gene families are mutated suggest that pattern formation along the central-peripheral axis results from interplay between auxin and the KANADI and Class III HD-Zip transcription factors, whose defined spatial and temporal expression patterns may also be influenced by auxin.

Distinct Developmental Mechanisms Reflect the Independent Origins of Leaves in Vascular Plants

Vascular plants diverged more than 400 million years ago into two lineages, the lycophytes and the euphyllophytes . Leaf-like organs evolved independently in these two groups . Microphylls in lycophytes are hypothesized to have originated as lateral outgrowths of tissue that later became vascularized (the enation theory) or through the sterilization of sporangia (the sterilization hypothesis) . Megaphylls in euphyllophytes are thought to represent modified lateral branches . The fossil record also indicates that the seed plant megaphyll evolved uniquely in the ancestor of seed plants, independent of megaphylls in ferns, because seed plants evolved from leafless progymnosperm ancestors . Surprisingly, a recent study of KNOX and ARP gene expression in a lycophyte was reported to indicate recruitment of a similar mechanism for determinacy in both types of leaves . We examined the expression of Class III HD-Zip genes in the lycophyte Selaginella kraussiana and in two gymnosperms, Ginkgo and Pseudotsuga. Our data indicate that mechanisms promoting leaf initiation, vascularization, and polarity are quite different in lycophytes and seed plants, consistent with the hypotheses that megaphylls originated as lateral branches whereas microphylls originated as tissue outgrowths.

Shoot Meristem Function and Leaf Polarity: The Role of Class III HD–ZIP Genes

The shoot apical meristem comprises an organized cluster of cells with a central region population of self-maintaining stem cells providing peripheral region cells that are recruited to form differentiated lateral organs. Leaves, the principal lateral organ of the shoot, develop as polar structures typically with distinct dorsoventrality. Interdependent interactions between the meristem and developing leaf provide essential cues that serve both to maintain the meristem and to pattern dorsoventrality in the initiating leaf. A key component of both processes are the class III HD–ZIP genes. Current findings are defining the developmental role of members of this family and are identifying multiple mechanisms controlling expression of these genes.

Evolution of class III homeodomain-leucine zipper genes in streptophytes.

Land plants underwent tremendous evolutionary change following the divergence of the ancestral lineage from algal relatives. Several important developmental innovations appeared as the embryophyte clade diversified, leading to the appearance of new organs and tissue types. To understand how these changes came about, we need to identify the fundamental genetic developmental programs that are responsible for growth, patterning, and differentiation and describe how these programs were modified and elaborated through time to produce novel morphologies. Class III homeodomain-leucine zipper (class III HD-Zip) genes, identified in the model plant Arabidopsis thaliana, provide good candidates for basic land plant patterning genes. We show that these genes may have evolved in a common ancestor of land plants and their algal sister group and that the gene family has diversified as land plant lineages have diversified. Phylogenetic analysis, expression data from nonflowering lineages, and evidence from Arabidopsis and other flowering plants indicate that class III HD-Zip genes acquired new functions in sporophyte apical growth, vascular patterning and differentiation, and leaf development. Modification of expression patterns that accompanied diversification of class III HD-Zip genes likely played an important role in the evolution of land plant form.

Vascular development: the long and winding road

Vascular development involves the specification of distinct meristematic cells that proliferate and then differentiate into two separate multicellular tissues: xylem and phloem. Organ-specific patterning, which requires the co-ordination of vascular development with organogenesis, introduces another layer of complexity to the development of vascular tissues. Because vascular tissues develop internally, analyses of their development are technically challenging. Nevertheless, the combined use of genetic and genomic approaches has provided significant insight into the mechanisms of vascular development. Notable highlights include the identification of class III HD-ZIP genes as regulators of both (pro)cambial activity and vascular tissue specification, the characterization of vesicle-trafficking components (SCARFACE [SFC]/VAN3) as being necessary for axial vein pattern, the genetic characterization of xylogen, and the identification of transcription factors and hormone signals that regulate vascular cell identity.

Phloem and xylem specification: pieces of the puzzle emerge

The plant vascular system is composed of two tissue types, xylem and phloem, which originate from the vascular meristem, the procambium. Recently, several regulatory mechanisms that control the specification of these two tissue types have been uncovered. These include the asymmetric patterning of xylem and phloem in the vascular bundle by the class III HD-ZIP and KANADI genes, the tissue-type-specific control of vascular cell proliferation by brassinosteroids and class III HD-ZIP genes, the regulation of vascular tissue identity by the MYB-like transcription factor APL, and inductive signalling during xylem differentiation by xylogen. These findings define an emerging developmental framework for the control of vascular tissue specification.

Roles for Class III HD-Zip and KANADI genes in Arabidopsis root development.

Meristems within the plant body differ in their structure and the patterns and identities of organs they produce. Despite these differences, it is becoming apparent that shoot and root apical and vascular meristems share significant gene expression patterns. Class III HD-Zip genes are required for the formation of a functional shoot apical meristem. In addition, Class III HD-Zip and KANADI genes function in patterning lateral organs and vascular bundles produced from the shoot apical and vascular meristems, respectively. We utilize both gain- and loss-of-function mutants and gene expression patterns to analyze the function of Class III HD-Zip and KANADI genes in Arabidopsis roots. Here we show that both Class III HD-Zip and KANADI genes play roles in the ontogeny of lateral roots and suggest that Class III HD-Zip gene activity is required for meristematic activity in the pericycle analogous to its requirement in the shoot apical meristem.

Radial Patterning of Arabidopsis Shoots by Class III HD-ZIP and KANADI Genes

Background: Shoots of all land plants have a radial pattern that can be considered to have an adaxial (central)-abaxial (peripheral) polarity. In Arabidopsis, gain-of-function alleles of PHAVOLUTA and PHABULOSA, members of the class III HD-ZIP gene family, result in adaxialization of lateral organs. Conversely, loss-of-function alleles of the KANADI genes cause an adaxialization of lateral organs. Thus, the class III HD-ZIP and KANADI genes comprise a genetic system that patterns abaxial-adaxial polarity in lateral organs produced from the apical meristem.Results: We show that gain-of-function alleles of REVOLUTA, another member of the class III HD-ZIP gene family, are characterized by adaxialized lateral organs and alterations in the radial patterning of vascular bundles in the stem. The gain-of-function phenotype can be obtained by changing only the REVOLUTA mRNA sequence and without changing the protein sequence; this finding indicates that this phenotype is likely mediated through an interference with microRNA binding. Loss of KANADI activity results in similar alterations in vascular patterning as compared to REVOLUTA gain-of-function alleles. Simultaneous loss-of-function of PHABULOSA, PHAVOLUTA, and REVOLUTA abaxializes cotyledons, abolishes the formation of the primary apical meristem, and in severe cases, eliminates bilateral symmetry; these phenotypes implicate these three genes in radial patterning of both embryonic and postembryonic growth.Conclusions: Based on complementary vascular and leaf phenotypes of class III HD-ZIP and KANADI mutants, we propose that a common genetic program dependent upon miRNAs governs adaxial-abaxial patterning of leaves and radial patterning of stems in the angiosperm shoot. This finding implies that a common patterning mechanism is shared between apical and vascular meristems.

Genetic Transformation of Transcription Factor (35S-oshox4) Gene into Rice Genome and Transformant Analysis of hpt Gene by PCR and Hygromycin Resistance Test

Global warming, climate change and crop extensification in marginal dryland areas are related to long dry season and waterdeficit. The water availability is an important factor in improving plant production. Application of drought tolerant rice cultivars is one of several options that might be used. Genetic engineering at the level of transcription factors is particularly promising strategy to develop drought tolerant rice varieties. Transcription factors regulate a wide range of target genes in which of them contribute to stress tolerance. HD Zip genes are transcription factor that potential in the adaptation of plants to some environment stresses including water deficit. HD-ZIP oshox4 (oryza sativa homeobox) gene controlled by 35S promotor is inserted into pCAMBIA 1300 vector with hpt (hygromycin) gene as a selectable marker. The aim of this research is to obtain transgenic rice plant from transformation with 35S-oshox4 plasmid, segregation analysis of marker gene (hpt) by PCR method at T0 and T1 generation, and hygromycin resistance analysis of seeds. Recombinant plasmid was transformed into rice genome of IRAT 112 and rojolele cultivars using Agrobacterium tumefaciens. The results showed thattransformation efficiency of IRAT 112 was 5.7-13.6% and 26-66,7% for rojolele. While regeneration efficiency for IRAT 112 is 4.7-43.7% and 23-44.1% for rojolele. The result of hygromycin resistance test at T1 seeds were obtained 14 lines cv. rojolele segregation Mendelian for hpt gene. The PCR analysis using specific primers for hpt gene at the parent (T0) from 14 lines showed that 7 lines contain the gene. At the second generation (T1), PCR analysis using hpt primers showed that 3 from 4 lines were followed Mendelian segregation pattern by the presence of specific band.Key words: HD-Zip oshox (oryza sativa homeobox), 35S-oshox4, hpt, Agrobacterium tumefaciens, PCR.