Jorge I. Giacomelli

Role of recently evolved miRNA regulation of sunflower HaWRKY6 in response to temperature damage

MicroRNAs (miRNAs) are small 21-nucleotide RNAs that post-transcriptionally regulate gene expression. MiR396 controls leaf development by targeting GRF and bHLH transcription factors in Arabidopsis. WRKY transcription factors, unique to plants, have been identified as mediating varied stress responses. The sunflower (Helianthus annuus) HaWRKY6 is a particularly divergent WRKY gene exhibiting a putative target site for the miR396. A possible post-transcriptional regulation of HaWRKY6 by miR396 was investigated. Here, we used expression analyses, performed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and northern blots together with computational approaches to establish the regulatory interaction between HaWRKY6 and the identified sunflower miR396. Arabidopsis plants expressing a mi396-resistant version of HaWRKY6 confirmed the miRNA-dependency of the HaWRKY6 silencing. Sunflower plants exposed to high temperatures or salicylic acid presented opposite expression of HaWRKY6 and miR396. Experiments using the wildtype and miRNA-resistant versions of HaWRKY6 showed altered stress responses. Our results showed a role of the recently evolved miR396 regulation of HaWRKY6 during early responses to high temperature. Our study reveals how a miRNA that normally regulates development has been recruited for high-temperature protection in sunflower, a plant particularly well adapted to this type of stress.

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 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.

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.