Source-to-sink partitioning is altered by changes in the expression of the transcription factor AtHB5 in Arabidopsis.

Carbohydrates are transported from source to sink tissues. The efficiency of this transport determines plant growth and development. The process is finely regulated and transcription factors are crucial in its modulation. AtHB5 is a homeodomain-leucine zipper I transcription factor that is repressed during stem maturation. However, its function in this developmental event is unknown. Here, we investigated the expression pattern and role of AtHB5. AtHB5 was expressed in roots, hypocotyls, stems, petioles, pedicels, and central leaf veins. athb5 mutant plants exhibited wider and more lignified stems than controls, whereas AtHB5 overexpressors showed the opposite phenotype. Cross sections of athb5 mutant stems showed enlarged vascular bundle, xylem, phloem, and petiole areas, whereas AtHB5 overexpressors had callose deposits. Several genes involved in starch biosynthesis and degradation had altered transcript levels in athb5 mutants and AtHB5 overexpressors. Rosette and stem biomass was enhanced in athb5 mutants, positively impacting seed yield, protein, and lipid content. Moreover, these effects were more evident in debranched plants. Finally, transport to roots was significantly slowed in AtHB5 overexpressors. Altogether, the results indicated that AtHB5 is a negative modulator of carbon partitioning and sucrose transport from source to sink tissues, and its overexpression diminished plant biomass and seed yield.

Successful field performance in warm and dry environments of soybean expressing the sunflower transcription factor HB4.

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.

AtHB23 participates in the gene regulatory network controlling root branching, and reveals differences between secondary and tertiary roots.

In Arabidopsis, lateral root (LR) development is mainly controlled by several known auxin-regulated transcription factors (TFs). Here, we show that AtHB23 (a homeodomain-leucine zipper I TF) participates in this intricate network. Our study of the expression pattern of AtHB23 revealed that it is transcriptionally activated in the early stages of secondary LR primordium (LRP). We found that AtHB23 directly limits the expression of LBD16, a key factor in LR initiation, and also directly induces the auxin transporter gene LAX3. We propose that this HD-Zip I mediates the regulation of LAX3 by ARF7/19. Furthermore, AtHB23 plays distinct roles during the formation of secondary and tertiary roots, exhibiting differential expression patterns. ATHB23 is expressed throughout the tertiary root primordium, whereas it is restricted to early stages in secondary primordia, likely later repressing LBD16 in tertiary LR development and further inhibiting root emergence. Our results suggest that different genetic programs govern the formation of LRP from the main or secondary roots, thereby shaping the global dynamic architecture of the root system.

Arabidopsis and sunflower plants with increased xylem area show enhanced seed yield.

Plant architecture plasticity determines the efficiency at harvesting and plays a major role defining biomass and seed yield. We observed that several previously described transgenic genotypes exhibiting increased seed yield also show wider stems and more vascular bundles than wild-type plants. Here, the relationship between these characteristics and seed yield was investigated. Hanging weight on the main stem of Arabidopsis plants provoked significant stem widening. Such widening was accompanied by an increase in the number of vascular bundles and about 100% of yield increase. In parallel, lignin deposition diminished. Vascular bundle formation started in the upper internode and continued downstream. AUX/LAX carriers were essential for this response. The increase of vascular bundles was reverted 3 weeks after the treatment leading to an enlarged xylem area. Aux1, lax1, and lax3 mutant plants were also able to enlarge their stems after the treatment, whereas lax2 plants did not. However, none of these mutants exhibited more vascular bundles or seed yield compared with untreated plants. Weight-induced xylem area enhancement and increased seed yield were also observed in sunflower plants. Altogether these results showed a strong correlation between the number of vascular bundles and enhanced seed yield under a long-day photoperiod. Furthermore, changes in the levels of auxin carriers affected both these processes in the same manner, suggesting that there may be an underlying causality.

A role for LAX2 in regulating xylem development and lateral-vein symmetry in the leaf.

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 sunflower transcription factor HaHB11 confers tolerance to water deficit and salinity to transgenic Arabidopsis and alfalfa plants.

Homeodomain-leucine zipper (HD-Zip) transcription factors are unique to the plant kingdom; members of subfamily I are known to be involved in abiotic stress responses. HaHB11 belongs to this subfamily and it was previously shown that it is able to confer improved yield and tolerance to flooding via a quiescent strategy. Here we show that HaHB11 expression is induced by ABA, NaCl and water deficit in sunflower seedlings and leaves. Arabidopsis transgenic plants expressing HaHB11, controlled either by its own promoter or by the constitutive 35S CaMV, presented rolled leaves and longer roots than WT when grown under standard conditions. In addition, these plants showed wider stems and more vascular bundles. To deal with drought, HaHB11 transgenic plants closed their stomata faster and lost less water than controls, triggering an enhanced tolerance to such stress condition and also to salinity stress. Concomitantly, ABA-synthesis and sensing related genes were differentially regulated in HaHB11 transgenic plants. Either under long-term salinity stress or mild drought stress, HaHB11 transgenic plants did not exhibit yield penalties. Moreover, alfalfa transgenic plants were generated which also showed enhanced drought tolerance. Altogether, the results indicated that HaHB11 was able to confer drought and salinity tolerance via a complex mechanism which involves morphological, physiological and molecular changes.

The sunflower transcription factor HaHB11 improves yield, biomass and tolerance to flooding in transgenic Arabidopsis plants.

HaHB11 is a member of the sunflower homeodomain-leucine zipper I subfamily of transcription factors. The analysis of a sunflower microarray hybridized with RNA from HaHB11-transformed leaf-disks indicated the regulation of many genes encoding enzymes from glycolisis and fermentative pathways. A 1300bp promoter sequence, fused to the GUS reporter gene, was used to transform Arabidopsis plants showing an induction of expression after flooding treatments, concurrently with HaHB11 regulation by submergence in sunflower. Arabidopsis transgenic plants expressing HaHB11 under the control of the CaMV 35S promoter and its own promoter were obtained and these plants exhibited significant increases in rosette and stem biomass. All the lines produced more seeds than controls and particularly, those of high expression level doubled seeds yield. Transgenic plants also showed tolerance to flooding stress, both to submergence and waterlogging. Carbohydrates contents were higher in the transgenics compared to wild type and decreased less after submergence treatments. Finally, transcript levels of selected genes involved in glycolisis and fermentative pathways as well as the corresponding enzymatic activities were assessed both, in sunflower and transgenic Arabidopsis plants, before and after submergence. Altogether, the present work leads us to propose HaHB11 as a biotechnological tool to improve crops yield, biomass and flooding tolerance.

Tolerancia oral en artritis experimental inducida por antígeno en conejos por administración de hidrolizado de cartílago articular / Oral tolerance in antigen induced arthritis (AIA) in rabbits by administration of articular cartilage hydrolysate

La artritis reumatoide es una patología autoinmune caracterizada por inflamación poliarticular, tumefacción e inflamación que afecta a más del 1% de la población mundial. La patobiología de la artritis reumatoide involucra varias poblaciones celulares como linfocitos T, B, macrófagos y fibroblastos, así como una compleja interacción de citoquinas proinflamatorias. Las actuales terapias convencionales y biológicas no siempre funcionan o producen solo una mejora parcial. La tolerancia inmunológica es un mecanismo por el cual el sistema inmune previene la autorreactividad. El objetivo de este estudio piloto fue evaluar la eficacia de péptidos provenientes de un hidrolizado enzimático de cartílago articular extraído del tarso bovino (HCA) para el tratamiento de artritis reumatoide en un modelo de artritis reumatoide (AAE) en conejos. Los animales AAE presentaron inflamación y dolor dentro del primer mes de la inmunización primaria que fue revertida en el grupo AAE+HCA. El grupo control mostró un tejido sinovial normal sin afecciones de ningún tipo. El grupo AAE reveló un proceso inflamatorio severo con hiperplasia sinovial, infiltrado de linfocitos y proliferación vascular. El grupo tratado redujo la inflamación, proliferación linfocítica y neoangiogénesis significativamente. Los conejos artríticos incrementaron significativamente los niveles marcadores inflamatorios como óxido nítrico, interferon γ (INF-γ) y factor de necrosis tumoral α (TNF- α) respecto del control y redujeron significativamente los niveles de interleukina 4 (IL-4). El tratamiento mostró una reducción significativa de óxido nítrico, IFN-γ y TNF-α y un aumento de IL-4. Este trabajo sugiere que esta terapia podría resultar útil en el aspecto clínico y en los parámetros bioquímicos y podría inhibir específicamente la respuesta inmune. Futuros estudios con mayor número de animales y otros parámetros de laboratorio complementarios podrán brindar evidencias en este sentido (AU)

Rheumatoid arthritis is an autoimmune disease characterized by polyarticular inflammation, swelling and inflammation that affects more than 1% of the world population. The pathobiology of rheumatoid arthritis involves several cell populations as T lymphocytes, B, macrófagosy fibroblasts, and a complex proinflammatory cytokines interactions. Conventional and biologic therapies do not always work or produce only a partial improvement. Immunological tolerance is a mechanism by which the immune system prevents autoreactivity. The aim of this pilot study was to evaluate the efficacy of peptides from an from articular cartilage hydrolysate extracted of tarsus (HCA) for the treatment of rheumatoid arthritis in a model of rheumatoid arthritis (AAE) in rabbits. AAE animals showed inflammation and pain within de first month of the primary immunization that was reversed in the AAE + HCA group. The control group showed a normal unnaffected synovial tissue. The AAE group revealed an inflammatory process with synovial hyperplasia, filtering in lymphocytes and vascular proliferation. The treated group decreased significantly inflammation, lymphocyte proliferation and angiogenesis. Arthritic rabbits increased the levels inflammatory markers as nitric oxide, interferon gamma (INF-γ) and tumor necrosis factor alpha (TNF-α) compared to control and significantly reduced levels of interleukin 4 (IL-4). The treatment showed a significant reduction of nitricoxide, IFN-gamma and TNF-alpha and an increase in IL-4. This work suggests that this therapy may be useful in the clinical aspect and the biochemical and immune parameters. Future studies with larger numbers of animals and other laboratory parameters may provide additional evidence in this regard (AU)

Novel perspectives for the engineering of abiotic stress tolerance in plants.

Adverse environmental conditions pose serious limitations to agricultural production. Classical biotechnological approaches towards increasing abiotic stress tolerance focus on boosting plant endogenous defence mechanisms. However, overexpression of regulatory elements or effectors is usually accompanied by growth handicap and yield penalties due to crosstalk between developmental and stress-response networks. Herein we offer an overview on novel strategies with the potential to overcome these limitations based on the engineering of regulatory systems involved in the fine-tuning of the plant response to environmental hardships, including post-translational modifications, small RNAs, epigenetic control of gene expression and hormonal networks. The development and application of plant synthetic biology tools and approaches will add new functionalities and perspectives to genetic engineering programs for enhancing abiotic stress tolerance.

The homologous homeodomain-leucine zipper transcription factors HaHB1 and AtHB13 confer tolerance to drought and salinity stresses via the induction of proteins that stabilize membranes.

Transgenic approaches to conferring tolerance to abiotic stresses have mostly resulted in some degree of plant yield penalty under normal or mild stress conditions. Recently, we have reported that the homeodomain-leucine zipper transcription factors (TFs) HaHB1 and AtHB13 were able to confer tolerance to freezing temperatures via the induction of glucanase (GLU and PR2) and chitinase (PR4) proteins. In the present study, we show that the expression of these TFs, as well as that of their putative targets AtPR2, AtPR4 and AtGLU, is up-regulated by drought and salinity stresses. Transgenic plants overexpressing separately these five genes exhibited tolerance to severe drought and salinity stresses, displaying a cell membrane stabilization mechanism. Under normal or mild stress conditions, these plants achieved an improved yield associated with higher chlorophyll content. Moreover, overexpression of the sunflower HaHB1 gene from its own, inducible, promoter conferred a high drought-stress tolerance without yield penalty under normal or mild stress conditions. We propose these TFs as potential biotechnological tools to breed crops for tolerance to multiple stresses and for increased yield.

The homologous HD-Zip I transcription factors HaHB1 and AtHB13 confer cold tolerance via the induction of pathogenesis-related and glucanase proteins.

Plants deal with cold temperatures via different signal transduction pathways. The HD-Zip I homologous transcription factors HaHB1 from sunflower and AtHB13 from Arabidopsis were identified as playing a key role in such cold response. The expression patterns of both genes were analyzed indicating an up-regulation by low temperatures. When these genes were constitutively expressed in Arabidopsis, the transgenic plants showed similar phenotypes including cell membrane stabilization under freezing treatments and cold tolerance. An exploratory transcriptomic analysis of HaHB1 transgenic plants indicated that several transcripts encoding glucanases and chitinases were induced. Moreover, under freezing conditions some proteins accumulated in HaHB1 plants apoplasts and these extracts exerted antifreeze activity in vitro. Three genes encoding two glucanases and a chitinase were overexpressed in Arabidopsis and these plants were able to tolerate freezing temperatures. All the obtained transgenic plants exhibited cell membrane stabilization after a short freezing treatment. Finally, HaHB1 and AtHB13 were used to transiently transform sunflower and soybean leading to the up-regulation of HaHB1/AtHB13-target homologues thus indicating the conservation of cold response pathways. We propose that HaHB1 and AtHB13 are involved in plant cold tolerance via the induction of proteins able to stabilize cell membranes and inhibit ice growth.

Uncharacterized conserved motifs outside the HD-Zip domain in HD-Zip subfamily I transcription factors; a potential source of functional diversity.

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.

Patents on plant transcription factors.

Transcription factors are clue elements in the regulation of signal transduction pathways in living organisms. These proteins are able to recognize and bind specific sequences in the promoter regions of their targets and subsequently activate or repress entire metabolic or developmental processes. About 1500 TFs were informatically identified in plants, analysis mainly based in the presence of DNA-binding domains in the translated sequences. However, only a few of these 1500 were functionally characterized and clearly classified as TFs. Among these, several seem to be powerful biotechnological tools in order to improve agronomic crops via the obtaining of transgenic plants or as molecular markers. Such TFs have become the objects of patents presentations in the whole world. The assigned uses present a variety of purposes including the improvement in yield, abiotic and biotic stresses tolerances as well as a combination of them. Some examples are commented in the present overview. Most of these TFs confer to transgenic plants complex phenotypes due to a combination of different regulated pathways. In this sense, the use of inducible promoters instead of constitutive ones seems in some cases to be useful to limit the changed phenotype to the desired one, avoiding lateral effects. None of these TFs was converted up to now in a market product since time-consuming experiments and regulation permits are required to arrive to such point. Moreover, a considerable money investment must be done, not justified in all cases. However, it is likely that these molecules will become in the near future the first choice for breeders since it was demonstrated that TFs are very efficient conferring desired traits to transgenic plants. Additionally, for the public perception the over or ectopic expression of a plant gene should be more accepted than the use of molecules from other species.

The intron of the Arabidopsis thaliana COX5c gene is able to improve the drought tolerance conferred by the sunflower Hahb-4 transcription factor.

Hahb-4 is a member of Helianthus annuus (sunflower) subfamily I of HD-Zip proteins. Transgenic Arabidopsis thaliana plants constitutively expressing this gene exhibit a strong tolerance of water stress in concert with morphological defects and a delay in development. In order to obtain a drought-tolerant phenotype without morphological associated phenotype, several stress inducible promoters were isolated and transgenic plants expressing Hahb-4 controlled by them were obtained and analyzed. These plants showed unchanged morphology in normal growth conditions and enhanced drought tolerance compared with non-transformed plants, but no as high as the one exhibited by the constitutively transformed genotype. A chimerical construction between the Hahb-4 promoter and the leader intron of the Arabidopsis Cox5c gene was made either directing gus or Hahb-4 expression. GUS activity increased in transgenic plants after induction, showing the same distribution pattern as in plants transformed with a construction lacking the intron. Transgenic plants, bearing the chimerical construct, are indistinguishable from wild type plants in normal growth conditions whereas the water stress tolerance achieved was as strong as the one shown by the constitutive genotype. This enhanced stress tolerance seemed to be due to a combination of an increase in transcription and translation rates in comparison to those of plants transformed with the Hahb-4 promoter. Similar strategies could be applied in the future for the obtaining of suitable promoters responsive to other external agents.