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.

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 Arabidopsis transcription factors AtPHL1 and AtHB23 act together promoting carbohydrate transport from pedicel-silique nodes to seeds.

Carbohydrates are produced in green tissues through photosynthesis and then transported to sink tissues. Carbon partitioning is a strategic process, fine regulated, involving specific sucrose transporters in each connecting tissue. Here we report that a screening of an Arabidopsis transcription factor (TF) library using the homeodomain-leucine zipper I member AtHB23 as bait, allowed identifying the TF AtPHL1 interacting with the former. An independent Y2H assay, and in planta by BiFC, confirmed such interaction. AtHB23 and AtPHL1 coexpressed in the pedicel-silique nodes and the funiculus. Mutant plants (phl1, and amiR23) showed a marked reduction of lipid content in seeds, although lipid composition did not change compared to the wild type. While protein and carbohydrate contents were not significantly different between mutants and control mature seeds, we observed a reduced carbohydrate content in mutant plants young siliques (7 days after pollination). Moreover, using a CFDA probe, we revealed an impaired transport to the seeds, and the gene encoding the carbohydrate transporters SWEET10 and SWEET11, usually expressed in connecting tissues, was repressed in the amiR23 and phl1 mutant plants. Altogether, the results indicated that AtHB23 and AtPHL1 act together, promoting sucrose transport, and the lack of any of them provoked a reduction in seeds lipid content.

The sunflower TLDc-containing protein HaOXR2 confers tolerance to oxidative stress and waterlogging when expressed in maize plants.

The sunflower (Helianthus annuus L.) genome encodes six proteins containing a TLDc domain, typical of the eukaryotic OXidation Resistance (OXR) protein family. Expression of sunflower HaOXR2 in Arabidopsis generated plants with increased rosette diameter, higher number of leaves and increased seed production. Maize inbred lines expressing HaOXR2 also showed increased total leaf area per plant. In addition, heterologous expression of HaOXR2 induced an increase in the oxidative stress tolerance in Arabidopsis and maize. Maize transgenic plants expressing HaOXR2 experienced less oxidative damage and exhibited increased photosynthetic performance and efficiency than non-transgenic segregant plants after treatment of leaves with the reactive oxygen species generating compound Paraquat. Expression of HaOXR2 in maize also improved tolerance to waterlogging. The number of expanded leaves, aerial biomass, and stem height and cross-section area were less affected by waterlogging in HaOXR2 expressing plants, which also displayed less aerial tissue damage under these conditions. Transgenic plants also showed an increased production of roots, a typical adaptive stress response. The results show the existence of functional conservation of OXR proteins in dicot and monocot plants and indicate that HaOXR2 could be useful to improve plant performance under conditions that increase oxidative stress.

Maize expressing the sunflower transcription factor HaHB11 has improved productivity in controlled and field conditions.

HaHB11 is a sunflower transcription factor from the homeodomain-leucine zipper I family. Transgenic Arabidopsis plants expressing HaHB11 had larger rosettes and improved seed yield. In this work maize plants from hybrid HiII were transformed with 35S:HaHB11, ZmUBI:HaHB11 and ProHaHB11:HaHB11 and then backcrossed to B73 to obtain a more homozygous inbred phenotype. Transgene expression levels were stable at least during three generations. Greenhouse-grown HaHB11 transgenic lines had larger leaf area and delayed senescence than controls, together with increased total biomass (up to 25%) and seed yield (up to 28%). Field trials conducted with T2 and T4 generations indicated that enhanced leaf area (up to 18%), stem diameter (up to 28%) and total biomass (up to 40%) as well as delayed leaf senescence were maintained among transgenic individuals when upscaling from pots in the greenhouse to communal plants in the field. The T4 field-grown transgenic generation had increased light interception and radiation use efficiency as well as seed yield (43-47% for events driven by the 35S promoter). Results suggest that HaHB11 is a promising tool for crop improvement because differential traits observed in the Arabidopsis model plant were preserved in a crop like maize independently of growth conditions and backcross level.

A sunflower WRKY transcription factor stimulates the mobilization of seed-stored reserves during germination and post-germination growth.

KEY MESSAGE: The sunflower transcription factor HaWRKY10 stimulates reserves mobilization in Arabidopsis. Gene expression and enzymes activity assays indicated that lipolysis and gluconeogenesis were increased. Microarray results suggested a parallelism in sunflower. Germinating oilseeds converts stored lipids into sugars, and thereafter in metabolic energy that is used in seedling growth and establishment. During germination, the induced lipolysis linked to the glyoxylate pathway and gluconeogenesis produces sucrose, which is then transported to the embryo and driven through catabolic routes. Herein, we report that the sunflower transcription factor HaWRKY10 regulates carbon partitioning by reducing carbohydrate catabolism and increasing lipolysis and gluconeogenesis. HaWRKY10 was regulated by abscisic acid and gibberellins in the embryo leaves 48 h after seed imbibition and highly expressed during sunflower seed germination and seedling growth, concomitantly with lipid mobilization. Sunflower leaf disks overexpressing HaWRKY10 showed repressed expression of genes related to sucrose cleavage and glycolysis compared with controls. Moreover, HaWRKY10 constitutive expression in Arabidopsis seeds produced higher decrease in lipid reserves, whereas starch and sucrose were more preserved compared with wild type. Gene transcripts abundance and enzyme activities involved in stored lipid mobilization and gluconeogenesis increased more in transgenic than in wild type seeds 36 h after imbibition, whereas the negative regulator of lipid mobilization, ABI4, was repressed. Altogether, the results point out a functional parallelism between tissues and plant species, and reveal HaWRKY10 as a positive regulator of storage reserve mobilization in sunflower.

The sunflower transcription factor HaWRKY76 confers drought and flood tolerance to Arabidopsis thaliana plants without yield penalty.

KEY MESSAGE: Arabidopsis transgenic plants expressing the sunflower transcription factor HaWRKY76 exhibit increased yield and tolerance to drought and flood stresses. The genetic construct containing HaWRKY76 is proposed as a potential biotechnological tool to improve crops. Water deficit and water excess are abiotic stress factors that seriously affect crops worldwide. To increase the tolerance to such stresses without causing yield penalty constitutes a major goal for biotechnologists. In this survey, we report that HaWRKY76, a divergent sunflower WRKY transcription factor, is able to confer both dehydration and submergence tolerance to Arabidopsis transgenic plants without yield penalty. The expression pattern of HaWRKY76 was analyzed in plants grown in standard conditions and under different watering regimes indicating a regulation by water availability. The corresponding cDNA was isolated and cloned under the control of a constitutive promoter and Arabidopsis plants were transformed with this construct. These transgenic plants presented higher biomass, seed production and sucrose content than controls in standard growth conditions. Moreover, they exhibited tolerance to mild drought or flood (complete submergence/waterlogging) stresses as well as the same or increased yield, depending on the stress severity and plant developmental stage, compared with controls. Drought tolerance occurred via an ABA-independent mechanism and induction of stomatal closure. Submergence tolerance can be explained by the carbohydrate (sucrose and starch) preservation achieved through the repression of fermentation pathways. Higher cell membrane stability and chlorenchyma maintenance could be the nexus between tolerance responses in front of both stresses. Altogether, the obtained results indicated that HaWRKY76 can be a potential biotechnological tool to improve crops yield as well as drought and flood tolerances.

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.

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.