Carlos A. Dezar

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 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.

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.

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.

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.

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 Helianthusannuus (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.

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.

Rewiring of auxin signaling under persistent shade

Significance In agricultural crops, plants are cultivated in close proximity, exposed to mutual shading. Photosensory receptors perceive this light environment and initiate growth responses that adjust the position of plant organs within the canopy to maximize light capture. During the early hours under shade, these responses are mediated by elevated levels of the hormone auxin. Here we show that under prolonged shade, the PHYTOCHROME INTERACTING FACTOR 4 light-signaling transcription factor, selected auxin receptors, their upstream regulatory miRNA, and downstream transcriptional regulators change their abundance to enhance growth responses, while auxin levels return to those observed before shade. Thus, in the presence of persistent shade, plants elicit a system-level rearrangement of auxin signaling, which sustains growth without increased auxin. Light cues from neighboring vegetation rapidly initiate plant shade-avoidance responses. Despite our detailed knowledge of the early steps of this response, the molecular events under prolonged shade are largely unclear. Here we show that persistent neighbor cues reinforce growth responses in addition to promoting auxin-responsive gene expression in Arabidopsis and soybean. However, while the elevation of auxin levels is well established as an early event, in Arabidopsis, the response to prolonged shade occurs when auxin levels have declined to the prestimulation values. Remarkably, the sustained low activity of phytochrome B under prolonged shade led to (i) decreased levels of PHYTOCHROME INTERACTING FACTOR 4 (PIF4) in the cotyledons (the organs that supply auxin) along with increased levels in the vascular tissues of the stem, (ii) elevated expression of the PIF4 targets INDOLE-3-ACETIC ACID 19 (IAA19) and IAA29, which in turn reduced the expression of the growth-repressive IAA17 regulator, (iii) reduced abundance of AUXIN RESPONSE FACTOR 6, (iv) reduced expression of MIR393 and increased abundance of its targets, the auxin receptors, and (v) elevated auxin signaling as indicated by molecular markers. Mathematical and genetic analyses support the physiological role of this system-level rearrangement. We propose that prolonged shade rewires the connectivity between light and auxin signaling to sustain shade avoidance without enhanced auxin levels.