MsTFL1A delays flowering and regulates shoot architecture and root development in Medicago sativa.

KEY MESSAGE: MsTFL1A is an important gene involved in flowering repression in alfalfa (Medicago sativa) which conditions not only above-ground plant shoot architecture but also root development and growth. Delayed flowering is an important trait for forage species, as it allows harvesting of high-quality forage for a longer time before nutritional values decline due to plant architecture changes related to flowering onset. Despite the relevance of delayed flowering, this trait has not yet been thoroughly exploited in alfalfa. This is mainly due to its complex genetics, sensitivity to inbreeding and to the fact that delayed flowering would be only advantageous if it allowed increased forage quality without compromising seed production. To develop new delayed-flowering varieties, we have characterized the three TERMINAL FLOWERING 1 (TFL1) family of genes in alfalfa: MsTFL1A, MsTFL1B and MsTFL1C. Constitutive expression of MsTFL1A in Arabidopsis caused late flowering and changes in inflorescence architecture, indicating that MsTFL1A is the ortholog of Arabidopsis TFL1. Overexpression of MsTFL1A in alfalfa consistently led to delayed flowering in both controlled and natural field conditions, coupled to an increase in leaf/stem ratio, a common indicator of forage quality. Additionally, overexpression of MsTFL1A reduced root development, reinforcing the role of MsTFL1A not only as a flowering repressor but also as a regulator of root development.We conclude that the precise manipulation of MsTFL1A gene expression may represent a powerful tool to improve alfalfa forage quality.

Improvement of alfalfa forage quality and management through the down-regulation of MsFTa1.

Alfalfa (Medicago sativa L.) is one of the most important forage crops worldwide. As a perennial, alfalfa is cut several times each year. Farmers face a dilemma: if cut earlier, forage nutritive value is much higher but regrowth is affected and the longevity of the stand is severely compromised. On the other hand, if alfalfa is cut later at full flower, stands persist longer and more biomass may be harvested, but the nutritive value diminishes. Alfalfa is a strict long-day plant. We reasoned that by manipulating the response to photoperiod, we could delay flowering to improve forage quality and widen each harvesting window, facilitating management. With this aim, we functionally characterized the FLOWERING LOCUS T family of genes, represented by five members: MsFTa1, MsFTa2, MsFTb1, MsFTb2 and MsFTc. The expression of MsFTa1 correlated with photoperiodic flowering and its down-regulation led to severe delayed flowering. Altogether, with late flowering, low expression of MsFTa1 led to changes in plant architecture resulting in increased leaf to stem biomass ratios and forage digestibility. By manipulating photoperiodic flowering, we were able to improve the quality of alfalfa forage and management, which may allow farmers to cut alfalfa of high nutritive value without compromising stand persistence.

Nicotiana attenuata NaHD20 plays a role in leaf ABA accumulation during water stress, benzylacetone emission from flowers, and the timing of bolting and flower transitions.

Homeodomain-leucine zipper type I (HD-Zip I) proteins are plant-specific transcription factors associated with the regulation of growth and development in response to changes in the environment. Nicotiana attenuata NaHD20 was identified as an HD-Zip I-coding gene whose expression was induced by multiple stress-associated stimuli including drought and wounding. To study the role of NaHD20 in the integration of stress responses with changes in growth and development, its expression was silenced by virus-induced gene silencing (VIGS), and control and silenced plants were metabolically and developmentally characterized. Phytohormone profiling showed that NaHD20 plays a positive role in abscisic acid (ABA) accumulation in leaves during water stress and in the expression of some dehydration-responsive genes including ABA biosynthetic genes. Moreover, consistent with the high levels of NaHD20 expression in corollas, the emission of benzylacetone from flowers was reduced in NaHD20-silenced plants. Additionally, bolting time and the opening of the inflorescence buds was decelerated in these plants in a specific developmental stage without affecting the total number of flowers produced. Water stress potentiated these effects; however, after plants recovered from this condition, the opening of the inflorescence buds was accelerated in NaHD20-silenced plants. In summary, NaHD20 plays multiple roles in N. attenuata and among these are the coordination of responses to dehydration and its integration with changes in flower transitions.

HAHB10, a sunflower HD-Zip II transcription factor, participates in the induction of flowering and in the control of phytohormone-mediated responses to biotic stress.

The transcription factor HAHB10 belongs to the sunflower (Helianthus annuus) HD-Zip II subfamily and it has been previously associated with the induction of flowering. In this study it is shown that HAHB10 is expressed in sunflower leaves throughout the vegetative stage and in stamens during the reproductive stage. In short-day inductive conditions the expression of this gene is induced in shoot apexes together with the expression of the flowering genes HAFT and HAAP1. Transgenic Arabidopsis plants expressing HAHB10 cDNA under regulation either by its own promoter or by cauliflower mosaic virus (CaMV) 35S exhibited an early flowering phenotype. This phenotype was completely reverted in a non-inductive light regime, indicating a photoperiod-dependent action for this transcription factor. Gene expression profiling of Arabidopsis plants constitutively expressing HAHB10 indicated that specific flowering transition genes such as FT, FUL, and SEP3 were induced several fold, whereas genes related to biotic stress responses, such as PR1, PR2, ICS1, AOC1, EDS5, and PDF1-2a, were repressed. The expression of HAHB10 and of the flowering genes HASEP3 and HAFT was up-regulated by both salicylic acid (SA) treatment and infection with a virulent strain of Pseudomonas syringae. Basal SA and jasmonic acid (JA) levels in Arabidopsis plants ectopically expressing HAHB10 were similar to those of control plants; however, SA levels differentially increased in the transgenic plants after wounding and infection with P. syringae while JA levels differentially decreased. Taken together, the results indicated that HAHB10 participates in two different processes in plants: the transition from the vegetative to the flowering stage via the induction of specific flowering transition genes and the accumulation of phytohormones upon biotic stresses.

The sunflower HD-Zip transcription factor HAHB4 is up-regulated in darkness, reducing the transcription of photosynthesis-related genes.

HAHB4 belongs to the sunflower subfamily I of HD-Zip proteins and is involved in drought-tolerance response and ethylene-mediated senescence. Cross-talk between these two processes through this transcription factor was recently described. In this study it is shown that the expression of HAHB4 is induced in darkness and quickly disappears when plants are exposed to light. This regulation of HAHB4 was confirmed at the transcriptional level through the use of transgenic Arabidopsis plants bearing constructs in which different segments of the HAHB4 promoter were fused with the reporter gene GUS. Together with electrophoretic mobility shift assays performed with sunflower nuclear proteins, these experiments allowed a cis-acting element involved in this response to be located. Transient overexpression of the HAHB4 cDNA in sunflower leaf discs and HAHB4 knockdown by iRNA were performed, demonstrating the participation of this transcription factor in the transcriptional down-regulation of a large group of photosynthesis-related genes. In accordance with the reduction in the transcripts encoding chlorophyll a/b-binding proteins, the content of these pigments is diminished in Arabidopsis HAHB4-expressing transgenic plants. Thus, it appears that HAHB4 may participate with other factors in the intricate regulation mechanism of the photosynthetic machinery in darkness.

Two ABREs, two redundant root-specific and one W-box cis-acting elements are functional in the sunflower HAHB4 promoter.

HAHB4 is a sunflower gene encoding a homeodomain-leucine zipper (HD-Zip) transcription factor. It was previously demonstrated that this gene is regulated at the transcriptional level by several abiotic factors and hormones. A previous analysis in the PLACE database revealed the presence of four putative ABREs. In this work these four elements and also one W-box and two root-specific expression elements were characterized as functional. Site-directed mutagenesis on the promoter, stable transformation of Arabidopis plants as well as transient transformation of sunflower leaves, were performed. The analysis of the transformants was carried out by histochemistry and real time RT-PCR. The results indicate that just one ABRE out of the four is responsible for ABA, NaCl and drought regulation. However, NaCl induction occurs also by an additional ABA-independent way involving another two overlapped ABREs. On the other hand, it was determined that the W-box located 5' upstream is responsive to ethylene and only two root-specific expression elements, among the several detected, are functional but redundant. Conservation of molecular mechanisms between sunflower and Arabidopsis is strongly supported by this experimental work.

HAHB4, a sunflower HD-Zip protein, integrates signals from the jasmonic acid and ethylene pathways during wounding and biotic stress responses.

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.

The true story of the HD-Zip family.

The HD-Zip family of transcription factors is unique to the plant kingdom. These proteins exhibit the singular combination of a homeodomain with a leucine zipper acting as a dimerization motif. They can be classified into four subfamilies, according to a set of distinctive features that include DNA-binding specificities, gene structures, additional common motifs and physiological functions. Some HD-Zip proteins participate in organ and vascular development or meristem maintenance. Others mediate the action of hormones or are involved in responses to environmental conditions. Here, we review recent data for this family of transcription factors from a wide variety of plant species to unravel their crucial role in plant development.

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.

Cross-talk between ethylene and drought signalling pathways is mediated by the sunflower Hahb-4 transcription factor.

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.

Hahb-10, a sunflower homeobox-leucine zipper gene, is regulated by light quality and quantity, and promotes early flowering when expressed in Arabidopsis.

Homeodomain-leucine zipper proteins constitute a family of transcription factors found only in plants. Expression patterns of the sunflower homeobox-leucine zipper gene Hahb-10 (Helianthus annuus homeobox-10), that belongs to the HD-Zip II subfamily, were analysed. Northern blots showed that Hahb-10 is expressed primarily in mature leaves, although expression is clearly detectable in younger leaves and also in stems. Considerably higher expression levels were detected in etiolated seedlings compared with light-grown seedlings. Induction of Hahb-10 expression was observed when seedlings were subjected to treatment with gibberellins. Transgenic Arabidopsis thaliana plants that express Hahb-10 under the 35S cauliflower mosaic virus promoter show special phenotypic characteristics such as darker cotyledons and planar leaves. A reduction in the life cycle of about 25% allowing earlier seed collection was also observed, and this phenomenon is clearly related to a shortened flowering time. When the number of plants per pot increased, the difference in developmental rate between transgenic and non-transformed individuals became larger. After gibberellin treatment, the relative difference in life cycle duration was considerably reduced. Several light-regulated genes have been tested as possible target genes of Hahb-10. One of them, PsbS, shows a different response to illumination conditions in transgenic plants compared with the response in wild-type plants while the other genes behave similarly in both genotypes. We propose that Hahb-10 functions in a signalling cascade(s) that control(s) plant responses to light quality and quantity, and may also be involved in gibberellin transduction pathways.

Identification of three MADS-box genes expressed in sunflower capitulum.

Three cDNA clones, HaPI, HaAG and HaAP3, were isolated from sunflower inflorescences at the R2 stage of development. The cDNAs share high sequence similarity with the PISTILLATA, AGAMOUS, and APETALA3 genes from Arabidopsis, respectively, which contain a MADS-box and are involved in floral organ development. Expression of the corresponding genes was analysed by northern blots and in situ hybridization. They are expressed preferentially in the R3 and R4 stages of capitulum development. HaAG accumulates in fertile flowers, mainly in stamens, while HaPI and HaAP3 are preferentially expressed in ray (sterile) flowers and more weakly in petals and stamens of fertile flowers.