RESUMEN

HaHB4 is a sunflower transcription factor belonging to the homeodomain-leucine zipper I family whose ectopic expression in Arabidopsis triggers drought tolerance. The use of PCR to clone the HaHB4 coding sequence for wheat transformation caused unprogrammed mutations producing subtle differences in its activation ability in yeast. Transgenic wheat plants carrying a mutated version of HaHB4 were tested in 37 field experiments. A selected transgenic line yielded 6% more (P<0.001) and had 9.4% larger water use efficiency (P<0.02) than its control across the evaluated environments. Differences in grain yield between cultivars were explained by the 8% improvement in grain number per square meter (P<0.0001), and were more pronounced in stress (16% benefit) than in non-stress conditions (3% benefit), reaching a maximum of 97% in one of the driest environments. Increased grain number per square meter of transgenic plants was accompanied by positive trends in spikelet numbers per spike, tillers per plant, and fertile florets per plant. The gene transcripts associated with abiotic stress showed that HaHB4's action was not dependent on the response triggered either by RD19 or by DREB1a, traditional candidates related to water deficit responses. HaHB4 enabled wheat to show some of the benefits of a species highly adapted to water scarcity, especially in marginal regions characterized by frequent droughts.

Asunto(s)

Helianthus/genética , Proteínas de Homeodominio/genética , Proteínas de Plantas/genética , Factores de Transcripción/genética , Triticum/crecimiento & desarrollo , Proteínas de Homeodominio/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Factores de Transcripción/metabolismo , Triticum/genética

RESUMEN

AtHB1 is an Arabidopsis (Arabidopsis thaliana) homeodomain-leucine zipper transcription factor that participates in hypocotyl elongation under short-day conditions. Here, we show that its expression is posttranscriptionally regulated by an upstream open reading frame (uORF) located in its 5' untranslated region. This uORF encodes a highly conserved peptide (CPuORF) that is present in varied monocot and dicot species. The Arabidopsis uORF and its maize (Zea mays) homolog repressed the translation of the main open reading frame in cis, independent of the sequence of the latter. Published ribosome footprinting results and the analysis of a frame-shifted uORF, in which the repression capability was lost, indicated that the uORF causes ribosome stalling. The regulation exerted by the CPuORF was tissue specific and did not act in the absence of light. Moreover, a photosynthetic signal is needed for the CPuORF action, since plants with uncoupled chloroplasts did not show uORF-dependent repression. Plants transformed with the native AtHB1 promoter driving AtHB1 expression did not show differential phenotypes, whereas those transformed with a construct in which the uORF was mutated exhibited serrated leaves, compact rosettes, and, most significantly, short nondehiscent anthers and siliques containing fewer or no seeds. Thus, we propose that the uncontrolled expression of AtHB1 is deleterious for the plant and, hence, finely repressed by a translational mechanism.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Sistemas de Lectura Abierta/genética , Especificidad de Órganos/genética , Factores de Transcripción/metabolismo , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Codón/genética , Secuencia Conservada/genética , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Homocigoto , Mutación/genética , Especificidad de Órganos/efectos de la radiación , Fenotipo , Biosíntesis de Proteínas , Ribosomas/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Zea mays/genética

RESUMEN

Plant responses to water deficit involve complex molecular mechanisms in which transcription factors have key roles. Previous reports ectopically overexpressed a few members of the homeodomain-leucine zipper I (HD-Zip I) family of transcription factors from different species, and the obtained transgenic plants exhibited drought tolerance which extent depended on the level of overexpression, triggering diverse molecular and physiological pathways. Here we show that most HD-Zip I genes are regulated by drought in the vegetative and/or reproductive stages. Moreover, uncharacterized members of this family were expressed as transgenes both in Col-0 and rdr6-12 backgrounds and were able to enhance drought tolerance in host plants. The extent of such tolerance depended on the expression level of the transgene and was significantly higher in transgenic rdr6-12 than in Col-0. Comparative transcriptome analyses of Arabidopsis thaliana plants overexpressing HD-Zip I proteins indicated that many members have common targets. Moreover, the water deficit tolerance exhibited by these plants is likely due to the induction and repression of certain of these common HD-Zip I-regulated genes. However, each HD-Zip I member regulates other pathways, which, in some cases, generate differential and potentially undesirable traits in addition to drought tolerance. In conclusion, only a few members of this family could become valuable tools to improve drought-tolerance.

Asunto(s)

Arabidopsis/fisiología , Proteínas de Homeodominio/fisiología , Proteínas de Plantas/fisiología , Plantas Modificadas Genéticamente/fisiología , Factores de Transcripción/fisiología , Agua/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Sequías , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Leucina Zippers , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Transducción de Señal , Estrés Fisiológico , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

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.

Asunto(s)

Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulación del Desarrollo de la Expresión Génica , Germinación , Proteínas de Homeodominio/metabolismo , Óvulo Vegetal/genética , Óvulo Vegetal/crecimiento & desarrollo , Óvulo Vegetal/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Semillas/genética , Semillas/crecimiento & desarrollo , Semillas/metabolismo

RESUMEN

Arabidopsis thaliana HomeoBox 1 (AtHB1) is a homeodomain-leucine zipper transcription factor described as a transcriptional activator with unknown function. Its role in A. thaliana development was investigated. AtHB1 expression was analyzed in transgenic plants bearing its promoter region fused to reporter genes. Knock-down mutant and overexpressor plant phenotypes were analyzed in different photoperiod regimes. AtHB1 was mainly expressed in hypocotyls and roots and up-regulated in seedlings grown under a short-day photoperiod. AtHB1 knock-down mutants and overexpressors showed shorter and longer hypocotyls, respectively, than wild type (WT). AtHB1 transcript levels were lower in PHYTOCHROME-INTERACTING FACTOR 1 (PIF1) mutants than in controls, suggesting that AtHB1 is regulated by PIF1 in hypocotyls. ß-glucuronidase (GUS) activity in Nicotiana benthamiana leaves cotransformed with PromAtHB1::GUS and 35S::PIF1 indicated that PIF1 induces AtHB1 expression. Hypocotyl lenght was measured in seedlings of athb1, pif1, or double athb1/pif1 mutants and PIF1 or AtHB1 overexpressors in WT, athb1 or pif1 backgrounds, both in short- or long-day. These analyses allowed us to determine that AtHB1 is a factor acting downstream of PIF1. Finally, a transcriptome analysis of athb1 mutant hypocotyls revealed that AtHB1 regulates genes involved in cell wall composition and elongation. The results suggest that AtHB1 acts downstream of PIF1 to promote hypocotyl elongation, especially in response to short-day photoperiods.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hipocótilo/crecimiento & desarrollo , Hipocótilo/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Hipocótilo/genética , Modelos Biológicos , Datos de Secuencia Molecular , Raíces de Plantas/metabolismo , Factores de Transcripción/genética

RESUMEN

Different members of the HD-Zip I family of transcription factors exhibit differential AHA-like activation motifs, able to interact with proteins of the basal transcriptional machinery. Homeodomain-leucine zipper proteins are transcription factors unique to plants, classified in four subfamilies. Subfamily I members have been mainly associated to abiotic stress responses. Several ones have been characterized using knockout or overexpressors plants, indicating that they take part in different signal transduction pathways even when their expression patterns are similar and they bind the same DNA sequence. A bioinformatic analysis has revealed the existence of conserved motifs outside the HD-Zip domain, including transactivation AHA motifs. Here, we demonstrate that these putative activation motifs are functional. Four members of the Arabidopsis family were chosen: AtHB1, AtHB7, AtHB12 and AtHB13. All of them exhibited activation activity in yeast and in plants but with different degrees. The protein segment necessary for such activation was different for these four transcription factors as well as the role of the tryptophans they present. When interaction with components of the basal transcription machinery was tested, AtHB1 was able to interact with TBP, AtHB12 interacted with TFIIB, AtHB7 interacted with both, TBP and TFIIB while AtHB13 showed weak interactions with any of them, in yeast two-hybrid as well as in pull-down assays. Transient transformation of Arabidopsis seedlings confirmed the activation capacity and specificity of these transcription factors and showed some differences with the results obtained in yeast. In conclusion, the differential activation functionality of these transcription factors adds an important level of functional divergence of these proteins, and together with their expression patterns, these differences could explain, at least in part, their functional divergence.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factores de Transcripción/genética , Activación Transcripcional , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Secuencia de Bases , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Leucina Zippers , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Plantones/genética , Plantones/metabolismo , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factor de Transcripción TFIIB/genética , Factor de Transcripción TFIIB/metabolismo , Factores de Transcripción/metabolismo , Técnicas del Sistema de Dos Híbridos

RESUMEN

BACKGROUND: Plant HD-Zip transcription factors are modular proteins in which a homeodomain is associated to a leucine zipper. Of the four subfamilies in which they are divided, the tested members from subfamily I bind in vitro the same pseudopalindromic sequence CAAT(A/T)ATTG and among them, several exhibit similar expression patterns. However, most experiments in which HD-Zip I proteins were over or ectopically expressed under the control of the constitutive promoter 35S CaMV resulted in transgenic plants with clearly different phenotypes. Aiming to elucidate the structural mechanisms underlying such observation and taking advantage of the increasing information in databases of sequences from diverse plant species, an in silico analysis was performed. In addition, some of the results were also experimentally supported. RESULTS: A phylogenetic tree of 178 HD-Zip I proteins together with the sequence conservation presented outside the HD-Zip domains allowed the distinction of six groups of proteins. A motif-discovery approach enabled the recognition of an activation domain in the carboxy-terminal regions (CTRs) and some putative regulatory mechanisms acting in the amino-terminal regions (NTRs) and CTRs involving sumoylation and phosphorylation. A yeast one-hybrid experiment demonstrated that the activation activity of ATHB1, a member of one of the groups, is located in its CTR. Chimerical constructs were performed combining the HD-Zip domain of one member with the CTR of another and transgenic plants were obtained with these constructs. The phenotype of the chimerical transgenic plants was similar to the observed in transgenic plants bearing the CTR of the donor protein, revealing the importance of this module inside the whole protein. CONCLUSIONS: The bioinformatical results and the experiments conducted in yeast and transgenic plants strongly suggest that the previously poorly analyzed NTRs and CTRs of HD-Zip I proteins play an important role in their function, hence potentially constituting a major source of functional diversity among members of this subfamily.

Asunto(s)

Proteínas de Homeodominio/metabolismo , Leucina Zippers , Filogenia , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Biología Computacional , Secuencia Conservada , ADN de Plantas/genética , Proteínas de Homeodominio/genética , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Análisis de Secuencia de Proteína , Factores de Transcripción/genética