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Soybean yield is limited primarily by abiotic constraints. No transgenic soybean with improved abiotic stress tolerance is commercially available. We transformed soybean plants with genetic constructs able to express the sunflower transcription factor HaHB4, which confers drought tolerance to Arabidopsis and wheat. One line (b10H) carrying the sunflower promoter was chosen among three independent lines because it exhibited the best performance in seed yield, and was evaluated in the greenhouse and in 27 field trials in different environments in Argentina. In greenhouse experiments, transgenic plants showed increased seed yield under stress conditions together with greater epicotyl diameter, larger xylem area, and increased water use efficiency compared with controls. They also exhibited enhanced seed yield in warm and dry field conditions. This response was accompanied by an increase in seed number that was not compensated by a decrease in individual seed weight. Transcriptome analysis of plants from a field trial with maximum difference in seed yield between genotypes indicated the induction of genes encoding redox and heat shock proteins in b10H. Collectively, our results indicate that soybeans transformed with HaHB4 are expected to have a reduced seed yield penalty when cultivated in warm and dry conditions, which constitute the best target environments for this technology., HaHB4 transgenic soybean plants significantly outyielded controls in warm and dry environments, showing increased seed number, xylem area, and water use efficiency, and induction of genes encoding redox and heat shock proteins.

Second-generation transgenic crops have the potential to transform agriculture, but progress has been limited, and particularly so in wheat where no transgenic cultivar has yet been approved. Taking on the challenge, González et al. (2019) report that transgenic wheat lines carrying a mutated version of the sunflower transcription factor (HaHB4), belonging to the homeodomain-leucine zipper family (HD-Zip I), had increased yield and water use efficiency across a range of environments, with particular benefits under stress. It is an important step forward in an area where progress is urgently needed, though it is too early to claim that transgenic wheat will form the backbone of a second Green Revolution. To meet the growing demand for food, together with the challenges imposed by climate change, substantial improvements in yields of major crops are needed. This includes wheat, where globally the multi-year tendency for growth in yield is decreasing (Passioura, 2012) or even stagnating (Driever et al., 2017). Current and expected future relative rates of progress in yield potential and drought adaptation in wheat are a matter of real concern, and insufficient to meet the projected demand for cereals by 2050 (Hall and Richards, 2013). There are three major challenges: increasing yield potential, protecting yield potential from different types of stress, and increasing resource use efficiency to ensure sustainability (Hawkesford et al., 2013). info:eu-repo/semantics/publishedVersion

Transgenic wheat expressing the sunflower transcription factor HaHB4 outyields the wild type by 6% when tested in the field during 9 years across 37 contrasting environments., 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

Moreno Piovano, G. S. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Instituto de Agrobiotecnología del Litoral. Santa Fe, Argentina. Moreno, Javier E. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Instituto de Agrobiotecnología del Litoral. Santa Fe, Argentina. Cabello, Julieta V. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Instituto de Agrobiotecnología del Litoral. Santa Fe, Argentina. Arce, Agustin L. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Instituto de Agrobiotecnología del Litoral. Santa Fe, Argentina. Otegui, María Elena. Universidad de Buenos Aires. Facultad de Agronomía. Buenos Aires, Argentina. Chan, Raquel L. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Instituto de Agrobiotecnología del Litoral. Santa Fe, Argentina. 577–590 Background and Aims The symmetry of venation patterning in leaves is highly conserved within a plant species. Auxins are involved in this process and also in xylem vasculature development. Studying transgenic Arabidopsis plants ectopically expressing the sunflower transcription factor HaHB4, it was observed that there was a significant lateral-vein asymmetry in leaves and in xylem formation compared to wild type plants. To unravel the molecular mechanisms behind this phenotype, genes differentially expressed in these plants and related to auxin influx were investigated. Methods Candidate genes responsible for the observed phenotypes were selected using a co-expression analysis. Single and multiple mutants in auxin influx carriers were characterized by morphological, physiological and molecular techniques. The analysis was further complemented by restoring the wild type (WT) phenotype by mutant complementation studies and using transgenic soybean plants ectopically expressing HaHB4. Key Results LAX2, down-regulated in HaHB4 transgenic plants, was bioinformatically chosen as a candidate gene. The quadruple mutant aux1 lax1 lax2 lax3 and the single mutants, except lax1, presented an enhanced asymmetry in venation patterning. Additionally, the xylem vasculature of the lax2 mutant and the HaHB4-expressing plants differed from the WT vasculature, including increased xylem length and number of xylem cell rows. Complementation of the lax2 mutant with the LAX2 gene restored both lateral-vein symmetry and xylem/stem area ratio in the stem, showing that auxin homeostasis is required to achieve normal vascular development. Interestingly, soybean plants ectopically expressing HaHB4 also showed an increased asymmetry in the venation patterning, accompanied by the repression of several GmLAX genes. Conclusions Auxin influx carriers have a significant role in leaf venation pattering in leaves and, in particular, LAX2 is required for normal xylem development, probablt controlling auxin homeostasis.

The Helianthus annuus (sunflower) HAHB4 transcription factor belongs to the HD-Zip family and its transcript levels are strongly induced when sunflower plants are attacked by herbivores, mechanically damaged or treated with methyl-jasmonic acid (MeJA) or ethylene (ET). Promoter fusion analysis, in Arabidopsis and in sunflower, demonstrated that induction of HAHB4 expression by these treatments is regulated at the transcriptional level. In transiently transformed sunflower plants HAHB4 expression upregulates the transcript levels of several genes involved in JA biosynthesis and defense-related processes such as the production of green leaf volatiles and trypsin protease inhibitors (TPI). In HAHB4 sunflower overexpressing tissue, increased activities of lipoxygenase, hydroperoxide lyase and TPI are detected whereas in HAHB4-silenced tissue these activities are reduced. Transgenic Arabidopsis thaliana and Zea mays plants ecotopically expressing HAHB4 also exhibit higher transcript levels of defense-related genes and when Spodoptera littoralis or Spodoptera frugiperda larvae are placed on each species, respectively, larvae consumed less and gain less mass compared with larvae feeding on control plants. Arabidopsis plants ectopically expressing HAHB4 had higher amounts of JA, JA-isoleucine and ET compared with control plants both before and after wounding, but reduced levels of salicylic acid (SA) after wounding and bacterial infection. We conclude that HAHB4 coordinates the production of phytohormones during biotic stress responses and mechanical damage, specifically by positively regulating JA and ET production and negatively regulating ET sensitivity and SA accumulation. Fil: Manavella, Pablo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Dezar, Carlos Alberto Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Bonaventure, Gustavo. Instituto Max Planck Institut für Chemische Okologie; Alemania Fil: Baldwin Ian Thomas. Instituto Max Planck Institut für Chemische Okologie; Alemania Fil: Chan, Raquel Lia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina

In the present work, we have analysed the promoter region of the sunflower nuclear gene Hahb4, encoding an homeodomain-leucine zipper protein involved in water stress responses. This region is represented in two different but highly conserved alleles of 1015 and 1221 bp, respectively, in the sunflower hybrid studied. To gain insight into the structure and function of these promoter forms, we have obtained plants stably transformed with different fragments fused to the b-glucuronidase (gus) reporter gene. Histochemical staining indicated that both protein involved in water stress responses. This region is represented in two different but highly conserved alleles of 1015 and 1221 bp, respectively, in the sunflower hybrid studied. To gain insight into the structure and function of these promoter forms, we have obtained plants stably transformed with different fragments fused to the b-glucuronidase (gus) reporter gene. Histochemical staining indicated that both Hahb4, encoding an homeodomain-leucine zipper protein involved in water stress responses. This region is represented in two different but highly conserved alleles of 1015 and 1221 bp, respectively, in the sunflower hybrid studied. To gain insight into the structure and function of these promoter forms, we have obtained plants stably transformed with different fragments fused to the b-glucuronidase (gus) reporter gene. Histochemical staining indicated that bothb-glucuronidase (gus) reporter gene. Histochemical staining indicated that both Hahb4 promoter forms direct expression in roots, cotyledons and leaves during the entire plant life cycle. No expression was observed in reproductive organs. The analysis of progressive upstream deletions of the promoters suggested that a minimal 417/421 bp fragment is required for basal expression. The presence of positive regulatory elements between nucleotides -601/608 and -818/-1024 from the transcription initiation site (depending on the promoter) and a sequence required for specific expression in the root central cylinder between transcription initiation site (depending on the promoter) and a sequence required for specific expression in the root central cylinder between reproductive organs. The analysis of progressive upstream deletions of the promoters suggested that a minimal 417/421 bp fragment is required for basal expression. The presence of positive regulatory elements between nucleotides -601/608 and -818/-1024 from the transcription initiation site (depending on the promoter) and a sequence required for specific expression in the root central cylinder between transcription initiation site (depending on the promoter) and a sequence required for specific expression in the root central cylinder between promoter forms direct expression in roots, cotyledons and leaves during the entire plant life cycle. No expression was observed in reproductive organs. The analysis of progressive upstream deletions of the promoters suggested that a minimal 417/421 bp fragment is required for basal expression. The presence of positive regulatory elements between nucleotides -601/608 and -818/-1024 from the transcription initiation site (depending on the promoter) and a sequence required for specific expression in the root central cylinder between transcription initiation site (depending on the promoter) and a sequence required for specific expression in the root central cylinder between 601/608 and -818/-1024 from the transcription initiation site (depending on the promoter) and a sequence required for specific expression in the root central cylinder between -818/-1024 and -1015/-1221 has been detected. Water stress, ABA or NaCl treatments induced Hahb4 promoter-dependent-818/-1024 and -1015/-1221 has been detected. Water stress, ABA or NaCl treatments induced Hahb4 promoter-dependent b-glucuronidase expression as observed by Northern blot hybridization experiments. Putative regulatory elements involved in the regulation of other genes were detected in the promoter fragment required for expression. These elements, together with experimental evidence, were analysed with the aim of elucidating the molecular mechanisms that participate in the expression of this gene. of other genes were detected in the promoter fragment required for expression. These elements, together with experimental evidence, were analysed with the aim of elucidating the molecular mechanisms that participate in the expression of this gene. -glucuronidase expression as observed by Northern blot hybridization experiments. Putative regulatory elements involved in the regulation of other genes were detected in the promoter fragment required for expression. These elements, together with experimental evidence, were analysed with the aim of elucidating the molecular mechanisms that participate in the expression of this gene. Fil: Dezar, Carlos Alberto Alejandro. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Fedrigo, Griselda Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina Fil: Chan, Raquel Lia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentina

Fil: Manavella, Pablo Andrés. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. HAHB4 es un factor de transcripción de girasol que pertenece a la familia HD-Zip. En trabajos previos realizados en nuestro laboratorio, se había determinado que la expresión del gen que codifica HAHB4 es regulada a nivel transcripcional por factores externos como la disponibilidad de agua, el ácido abscísico y la salinidad del suelo. .Los estudios presentados en este trabajo de Tesis tuvieron como objetivo dilucidar los mecanismos de acción de este factor de transcripción así como las vías de señalización en las que participa. Ensayos fisiológicos y moleculares por diversas técnicas, permitió establecer que la tolerancia a estrés hídrico es principalmente una consecuencia del retraso en la entrada en el estadio de senescencia de las plantas que sobreexpresan este gen, causada por una inhibición en la biosíntesis y en la percepción de la hormona etileno. Se determinó asimismo que la expresión de HAHB4 se activa en plantas de girasol por la acción de del ácido jasmónico (además de por el etileno) y que esta activación genera una respuesta frente al ataque de insectos o la producción de heridas mecánicas. En conclusión, HAHB4 es un factor de transcripción que interviene y conecta las vías de señalización de las hormonas etileno y ácido jasmónico, así como la de biogénesis de componentes de la fotosíntesis, funcionando en plantas como un disparador no sólo de tolerancia a condiciones de estrés hídrico sino también al ataque de insectos, convirtiéndose de esta forma en una poderosa herramienta biotecnológica HAHB4 is a transcription factor that belongs to the sunflower HD-Zip family (subfamily I). In previous works performed in our lab, it was demonstrated that the expression of the gene encoding HAHB4 is regulated at the transcriptional level by environmental factors such as salinity and drought and the hormone ABA. Our aim during this Thesis’ work was to elucidate the action mechanisms of this transcription factor in order to understand the drought tolerance conferred by it as well as the transduction signal pathways in which it is involved. Physiological and molecular studies, carried out with a diversity of techniques, led us to establish that the drought tolerance conferred by HAHB4 to transgenic plants is mainly a consequence of a delay that suffer the plants in their entry to the senescence stage. This delay is caused by the inhibition in the biosynthesis and perception of the ethylene hormone as shown by molecular and physiological studies. By other hand, it was experimentally demonstrated that HAHB4 expression is induced in sunflower plants by the action of jasmonic acid and that this induction produces a massive defense response against injuries caused by insects or mechanical factors. In conclusion, HAHB4 is a transcription factor that mediates the crosstalk between several signal transduction pathways including those where ethylene, jasmonic acid and photosynthesis are involved. It functions in plants as a switch, not only turned on in drought conditions, but also when insects attack, becoming in this way a valuable biotechnological tool. Agencia Nacional de Promoción Científica y Tecnológica Consejo Nacional de Investigaciones Científicas y Técnicas

Hahb-4 is a member of the Helianthusannuus (sunflower) subfamily I of HD-Zip proteins that is transcriptionally regulated by water availability and abscisic acid. Transgenic Arabidopsis thaliana plants overexpressing this transcription factor (TF) exhibit a characteristic phenotype that includes a strong tolerance to water stress. Here we show that this TF is a new component of ethylene signalling pathways, and that it induces a marked delay in senescence. Plants overexpressing Hahb-4 are less sensitive to external ethylene, enter the senescence pathway later and do not show the typical triple response. Furthermore, transgenic plants expressing this gene under the control of its own inducible promoter showed an inverse correlation between ethylene sensitivity and Hahb-4 levels. Potential targets of Hahb-4 were identified by comparing the transcriptome of Hahb-4-transformed and wild-type plants using microarrays and quantitative RT-PCR. Expression of this TF has a major repressive effect on genes related to ethylene synthesis, such as ACO and SAM, and on genes related to ethylene signalling, such as ERF2 and ERF5. Expression studies in sunflower indicate that Hahb-4 transcript levels are elevated in mature/senescent leaves. Expression of Hahb-4 is induced by ethylene, concomitantly with several genes homologous to the targets identified in the transcriptome analysis (HA-ACOa and HA-ACOb). Transient transformation of sunflower leaves demonstrated the action of Hahb-4 in the regulation of ethylene-related genes. We propose that Hahb-4 is involved in a novel conserved mechanism related to ethylene-mediated senescence that functions to improve desiccation tolerance. Fil: Manavella, Pablo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Arce, Agustín Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Dezar, Carlos Alberto Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Agrobiotecnología de Rosario; Argentina Fil: Bitton, Frédérique. Unite de Recherche en Genomique Vegetale; Francia Fil: Renou, Jean-Pierre. Unite de Recherche en Genomique Vegetale; Francia Fil: Crespi, Martin. Institut des Sciences Vegetales; Francia Fil: Chan, Raquel Lia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina