A complex tissue-specific interplay between the Arabidopsis transcription factors AtMYB68, AtHB23, and AtPHL1 modulates primary and lateral root development and adaptation to salinity.

Adaptation to different soil conditions is a well-regulated process vital for plant life. AtHB23 is a homeodomain-leucine zipper I transcription factor (TF) that was previously revealed as crucial for plant survival under salinity conditions. We wondered whether this TF has partners to perform this essential function. Therefore, TF cDNA library screening, yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays were complemented with expression analyses and phenotypic characterization of silenced, mutant, overexpression, and crossed plants in normal and salinity conditions. We revealed that AtHB23, AtPHL1, and AtMYB68 interact with each other, modulating root development and the salinity response. The encoding genes are coexpressed in specific root tissues and at specific developmental stages. In normal conditions, amiR68 silenced plants have fewer initiated roots, the opposite phenotype to that shown by amiR23 plants. AtMYB68 and AtPHL1 play opposite roles in lateral root elongation. Under salinity conditions, AtHB23 plays a crucial positive role in cooperating with AtMYB68, whereas AtPHL1 acts oppositely by obstructing the function of the former, impacting the plant's survival ability. Such interplay supports the complex interaction between these TF in primary and lateral roots. The root adaptation capability is associated with the amyloplast state. We identified new molecular players that through a complex relationship determine Arabidopsis root architecture and survival in salinity conditions.

Expressing the sunflower transcription factor HaHB11 in maize improves waterlogging and defoliation tolerance.

The sunflower (Helianthus annuus) transcription factor HaHB11 (H. annuus  Homeobox 11) belongs to the homeodomain-leucine zipper family and confers improved yield to maize (Zea mays) hybrids (HiII × B73) and lines. Here we report that transgenic maize lines expressing HaHB11 exhibited better performance under waterlogging, both in greenhouse and field trials carried out during three growth cycles. Transgenic plants had increased chlorophyll content, wider stems, more nodal roots, greater total aerial biomass, a higher harvest index, and increased plant grain yield. Under severe defoliation caused by a windstorm during flowering, transgenic genotypes were able to set more grains than controls. This response was confirmed in controlled defoliation assays. Hybrids generated by crossing B73 HaHB11 lines with the contrasting Mo17 lines were also tested in the field and exhibited the same beneficial traits as the parental lines, compared with their respective controls. Moreover, they were less penalized by stress than commercial hybrids. Waterlogging tolerance increased via improvement of the root system, including more xylem vessels, reduced tissue damage, less superoxide accumulation, and altered carbohydrate metabolism. Multivariate analyses corroborated the robustness of the differential traits observed. Furthermore, canopy spectral reflectance data, computing 29 vegetation indices associated with biomass, chlorophyll, and abiotic stress, helped to distinguish genotypes as well as their growing conditions. Altogether the results reported here indicate that this sunflower gene constitutes a suitable tool to improve maize plants for environments prone to waterlogging and/or wind defoliation.

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.

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.

Field-grown transgenic wheat expressing the sunflower gene HaHB4 significantly outyields the wild type.

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.

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.

The rice transcription factor OsWRKY47 is a positive regulator of the response to water deficit stress.

OsWRKY47 is a divergent rice transcription factor belonging to the group II of the WRKY family. A transcriptomic analysis of the drought response of transgenic rice plants expressing P SARK ::IPT, validated by qPCR, indicated that OsWRKY47 expression was induced under drought stress in P SARK ::IPT plants. A PCR-assisted site selection assay (SELEX) of recombinant OsWRKY47 protein showed that the preferred sequence bound in vitro is (G/T)TTGACT. Bioinformatics analyses identified a number of gene targets of OsWRKY47; among these two genes encode a Calmodulin binding protein and a Cys-rich secretory protein. Using Oswrk47 knockout mutants and transgenic rice overexpressing OsWRKY47 we show that the transcription of these putative targets were regulated by OsWRKY47. Phenotypic analysis carried out with transgenic rice plants showed that Oswrky47 mutants displayed higher sensitivity to drought and reduced yield, while plants overexpressing OsWRKY47 were more tolerant.

The Transcription Factor HaHB11 Boosts Grain Set and Yield in Rice Plants, Allowing Them to Approach Their Ideal Phenotype.

The ideal rice phenotype is that of plants exhibiting fewer panicles with high biomass, large grain number, flag leaf area with small insertion angles, and an erected morphology improving light interception. The sunflower transcription factor HaHB11, homeodomain-leucine zipper I, confers increased seed yield and abiotic stress tolerance to Arabidopsis and maize. Here, we report the obtaining and characterization of rice plants expressing HaHB11 driven by its promoter or the 35S constitutive one. Transgenic p35S:HaHB11 plants closely resembled the ideal high-yield phenotype, whereas those carrying the pHaHB11:HaHB11 construct were hard to distinguish from the wild type. The former had an erected architecture, enhanced vegetative leaf biomass, rolled flag leaves with a larger surface, sharper insertion angles insensitive to brassinosteroids, and higher harvest index and seed biomass than the wild type. The combination of the distinct features exhibited by p35S:HaHB11 plants, including the increased number of set grains per panicle, supports the high-yield phenotype. We wondered where HaHB11 has to be expressed to achieve the high-yield phenotype and evaluated HaHB11 expression levels in all tissues. The results indicate that its expression is particularly necessary in the flag leaf and panicle to produce the ideal phenotype.

AtHB40 modulates primary root length and gravitropism involving CYCLINB and auxin transporters.

Gravitropism is a finely regulated tropistic response based on the plant perception of directional cues. Such perception allows them to direct shoot growth upwards, above ground, and root growth downwards, into the soil, anchoring the plant to acquire water and nutrients. Gravity sensing occurs in specialized cells and depends on auxin distribution, regulated by influx/efflux carriers. Here we report that AtHB40, encoding a transcription factor of the homeodomain-leucine zipper I family, was expressed in the columella and the root tip. Athb40 mutants exhibited longer primary roots. Enhanced primary root elongation was in agreement with a higher number of cells in the transition zone and the induction of CYCLINB transcript levels. Moreover, athb40 mutants and AtHB40 overexpressors displayed enhanced and delayed gravitropistic responses, respectively. These phenotypes were associated with altered auxin distribution and deregulated expression of the auxin transporters LAX2, LAX3, and PIN2. Accordingly, lax2 and lax3 mutants also showed an altered gravitropistic response, and LAX3 was identified as a direct target of AtHB40. Furthermore, AtHB40 is induced by AtHB53 when the latter is upregulated by auxin. Altogether, these results indicate that AtHB40 modulates cell division and auxin distribution in the root tip thus altering primary root length and gravitropism.

Lateral root development differs between main and secondary roots and depends on the ecotype.

Root architecture depends on the development of the main root and also on the number and density of lateral roots. Most molecular knowledge about the development of lateral roots was acquired studying primary roots, and it was implied that high order roots follow the same pattern. Recently, we informed that AtHB23 is differentially regulated in primary and secondary roots. Here we show that LBD16, a target of AtHB23, also is differentially regulated; it is expressed in the tip of secondary and tertiary roots but not in primary ones. Moreover, the key hormone auxin exhibits a different distribution pattern in secondary and tertiary roots, according to the reporter DR5. Finally, we show that in Col 0 and Ler ecotypes development of secondary and tertiary roots exhibits significant variations. Altogether, we can conclude that different genetic programs govern secondary and tertiary roots development and such processes are dependent on the Arabidopsis genotype.

An Interdisciplinary Approach to Study the Performance of Second-generation Genetically Modified Crops in Field Trials: A Case Study With Soybean and Wheat Carrying the Sunflower HaHB4 Transcription Factor.

Research, production, and use of genetically modified (GM) crops have split the world between supporters and opponents. Up to now, this technology has been limited to the control of weeds and pests, whereas the second generation of GM crops is expected to assist farmers in abiotic stress tolerance or improved nutritional features. Aiming to analyze this subject holistically, in this presentation we address an advanced technology for drought-tolerant GM crops, upscaling from molecular details obtained in the laboratory to an extensive network of field trials as well as the impact of the introduction of this innovation into the market. Sunflower has divergent transcription factors, which could be key actors in the drought response orchestrating several signal transduction pathways, generating an improved performance to deal with water deficit. One of such factors, HaHB4, belongs to the homeodomain-leucine zipper family and was first introduced in Arabidopsis. Transformed plants had improved tolerance to water deficits, through the inhibition of ethylene sensitivity and not by stomata closure. Wheat and soybean plants expressing the HaHB4 gene were obtained and cropped across a wide range of growing conditions exhibiting enhanced adaptation to drought-prone environments, the most important constraint affecting crop yield worldwide. The performance of wheat and soybean, however, differed slightly across mentioned environments; whereas the improved behavior of GM wheat respect to controls was less dependent on the temperature regime (cool or warm), differences between GM and wild-type soybeans were remarkably larger in warmer compared to cooler conditions. In both species, these GM crops are good candidates to become market products in the near future. In anticipation of consumers' and other stakeholders' interest, spectral analyses of field crops have been conducted to differentiate these GM crops from wild type and commercial cultivars. In this paper, the potential impact of the release of such market products is discussed, considering the perspectives of different stakeholders.

WITHDRAWN: Plant science with relevance to biotechnology.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/<10.1016/S0168-1656(14)00098-4>. The duplicate article has therefore been withdrawn.

Hahb-4, a sunflower homeobox-leucine zipper gene, is a developmental regulator and confers drought tolerance to Arabidopsis thaliana plants.

Homeodomain-leucine zipper proteins constitute a family of transcription factors found only in plants. Hahb-4 is a member of Helianthus annuus (sunflower) subfamily I. It is regulated at the transcriptional level by water availability and abscisic acid. In order to establish if this gene plays a functional role in drought responses, transgenic Arabidopsis thaliana plants that overexpress Hahb-4 under the control of the 35S Cauliflower Mosaic Virus promoter were obtained. Transformed plants show a specific phenotype: they develop shorter stems and internodes, rounder leaves and more compact inflorescences than their non-transformed counterparts. Shorter stems and internodes are due to a lower rate in cell elongation rather than to a stop in cell division. Transgenic plants were more tolerant to water stress conditions, showing improved development, a healthier appearance and higher survival rates than wild-type plants. Indeed, either under normal or drought conditions, they produce approximately the same seed weight per plant as wild-type plants under normal growth conditions. Plants transformed with a construct that bears the Hahb-4 promoter fused to gusA show reporter gene expression in defined cell-types and developmental stages and are induced by drought and abscisic acid. Since Hahb-4 is a transcription factor, we propose that it may participate in the regulation of the expression of genes involved in developmental responses of plants to desiccation.