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Heat shock transcription factors (HSF) are divided into classes A, B and C. Class A transcription factors are generally recognized as transcriptional activators, while functional characterization of class B and C heat shock transcription factors have not been fully developed in most plant species. We isolated and characterized a novel HSF transcription factor gene, TrHSFB2a (a class B HSF) gene, from the drought stress-sensitive forage crop species, white clover (Trifolium repens). TrHSFB2a was highly homologous to MtHSFB2b, CarHSFB2a, AtHSFB2b and AtHSFB2a. The expression of TrHSFB2a was strongly induced by drought (PEG6000 15% w/v), high temperature (35 °C) and salt stresses (200 mM L−1 NaCl) in white clover, while subcellular localization analysis showed that it is a nuclear protein. Overexpression of the white clover gene TrHSFB2a in Arabidopsis significantly reduced fresh and dry weight, relative water contents (RWC), maximum photosynthesis efficiency (Fv/Fm) and performance index on the absorption basis (PIABS), while it promoted leaf senescence, relative electrical conductivity (REC) and the contents of malondialdehyde (MDA) compared to a wild type under drought, heat and salt stress conditions of Arabidopsis plants. The silencing of its native homolog (AtHSFB2a) by RNA interference in Arabidopsis thaliana showed opposite trends by significantly increasing fresh and dry weights, RWC, maximum photosynthesis efficiency (Fv/Fm) and performance index on the absorption basis (PIABS) and reducing REC and MDA contents under drought, heat and salt stress conditions compared to wild type Arabidopsis plants. These phenotypic and physiological indicators suggested that the TrHSFB2a of white clover functions as a negative regulator of heat, salt and drought tolerance. The bioinformatics analysis showed that TrHSFB2a contained the core B3 repression domain (BRD) that has been reported as a repressor activator domain in other plant species that might repress the activation of the heat shock-inducible genes required in the stress tolerance process in plants. The present study explores one of the potential causes of drought and heat sensitivity in white clover that can be overcome to some extent by silencing the TrHSFB2a gene in white clover.

White clover (Trifolium repens) is one of the most widely cultivated livestock forage legumes co-cultivated worldwide with pasture grass in a mixed-sward setting, however, its persistence and aesthetic quality are severely affected by abiotic stresses. In this study, regeneration of white clover plants was conducted through a callus system for 4-5 months with a regeneration frequency of 36-41%. Inoculating 4-day-old cotyledons into MS media fortified with 0.4 mg·L-1 6-BA and 2 mg·L-1 2,4-D significantly increased the callus formation rate. Roots and cotyledons were better induced, followed by hypocotyls, leaves, and petioles. The development of differentiated structures performed effectively on MS supplemented with 1 mg·L-1 6-BA and 0.1 mg·L-1 NAA. Further, we determined factors affecting the Agrobacterium tumefaciens-mediated transient transformation for root-derived callus and 4-day-old cotyledons. The parameters that facilitated transient transformation were: Agrobacterium suspension density of 0.5 (OD600), 20 mg·L-1 AS, and 4-days co-cultivation duration. Subsequently, we developed two transformation protocols: transformation after callus formation in root segments (Protocol A) and transformation before callus initiation in 4-day-old cotyledons (Protocol B). The transformation frequencies varied from 1.92% to 3.17% in Protocol A and from 2.76% to 3.47% in Protocol B. We offer the possibility to regenerate multiple transgenic white clover from a single genetic background. In addition to assistance in identification of functional genes associated with yield, resistance and aesthetic quality, our research will also contribute to successful genetic manipulation and genome editing in white clover.

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