The WHY family is a group of plant-specific transcription factors, that can bind to single-stranded DNA molecules and play a variety of functions in plant nuclei and organelles, participating in the regulation of plant leaf senescence (Ruan et al., 2022). p24 (24kD), a single stranded DNA (ssDNA) binding subunit of plant defense transcription factor PBF2 (PR-10a Binding Factor 2, also called as StWhy1) is the first member of this family to be studied in Solanum tuberosum. In response to elicitation, the pathogenesis related gene PR-10a is positively regulated by specific binding of p24 subunit to GTCAAAAA/T PB (PBF-2 binding) core element in the PR-10a promoter region. However, in absence of elicitation the inactive form of PBF2 is stored in the fresh tuber nuclei (Desveaux et al., 2000, Desveaux et al., 2004).

The name WHIRLY is derived from a crystallographic study of the p24 subunit of PBF2, where the tetramer of p24 protomer gives the whirligig appearance to the PBF2 quaternary protein structure (Desveaux et al., 2002). Arabidopsis, potato, soybean, wheat, rice and maize consist of 3, 2, 3, 2, 2 and 2 WHIRLY protein members respectively. Based on multiple sequence alignment and p24 mutational studies, KGKAAL, YDW sequence and Lys188 forms a most conserved WHIRLY domain and plays a crucial role in optimal binding of the WHIRLY proteins to the ssDNA. The N-termini of WHIRLY family members contains proline rich or serine rich (polyglutamate for StWhy1) putative transactivation domain, contributing to their diverse regulatory role  and a predicted organelle transit peptide targeting them to mitochondria or chloroplast or both. However, the variable C-termini contains a putative autoregulatory domain (Desveaux et al., 2005, Triezenberg.,1995., Krause et al., 2005).

In Arabidopsis, the expression of AtWhy1 (an ortholog of StWhy1) is induced in response to salicylic acid mediated plant defense response and its induction is independent of transcription factor associated NPR1 (Nonexpressor of pathogenesis-related genes 1) protein, required for systemic acquired resistance (SAR).  This finding implies a role for  AtWhy1 in plant defense and SAR (Desveaux et al., 2004). AtWhy1 also appears to play a role in maintaining telomere length homeostasis (Yoo et al., 2007).

In Maize, there appears to be just 2 WHIRLY family members. ZmWhy1 was identified by mass spectrometry analysis of CRS1 (Chloroplast RNA splicing 1) coimmunoprecipitated proteins. ZmWhy1 binds to both DNA and RNA and localizes to the thylakoid membrane and chloroplast stroma. ZmWhy1 mutant exhibited normal chloroplast DNA and RNA levels but specific rRNA deficiencies  like ribosome less plastids, low plastid ribosome content and abnormal 23S rRNA metabolism, thus implying a ZmWhy1 role in biogenesis of large ribosomal subunit rather than plastid DNA replication or transcription (Prikryl et al., 2008).

In tomato, a genome-wide study identified two whirly genes namely SlWhy1 and SlWhy2 showing characteristic whirly domain. Based on qRT-PCR results, expression of both SlWhy1 and SlWhy2 was upregulated in response to salt and drought stress specifically in roots and leaves (Akbudak and Filiz., 2019).

Based on immunogold labeling, Barley WHY1 protein localizes to the leaf base nuclei. Transcript and metabolite abundance profiling along the developmental leaf gradient for wild type WHY1 and barley lines without WHY1 (W1-1 and W1-7) highlighted the role of WHY1 in regulating nuclei encoded transcription factors involved in chloroplast development namely GATA, GLK-like and ARF, since these protein levels were reduced in WHY1 lacking W1-1 and W1-7 barley lines (Karpinska et al., 2022). Also, Barley HvWHY1 plays a role in drought stress induced senescence signaling pathways by acting on H3K9ac euchromatin mark on the promoter and coding region of  senescence associated barley gene HvS40. WHY1 acts as an upstream regulator of WRKY and NAC families of senescence and stress responsive signaling pathways respectively, since RNAi mediated knockdown of HvWHY1 resulted in impaired WHY1 accumulation and affected expression pattern of these transcription factor families (Janack et al., 2016).

In Arabidopsis, binding of WHIRLY1 to the leaf senescence associated WRKY53 promoter region causes epigenetic changes like repression of  H3K4me3 enrichment before senescence initiation and enhanced H3K9ac enrichment and RNAPII recruitment at WHIRLY1 binding site at senescence initiation. Thus WHIRLY1 regulates the expression of WRKY53 leaf senescence associated genes through chromatin sites modifications (Huang et al., 2018)

Last updated June 2023 by John Gray and Ankita Abnave

References:

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Krause K, Kilbienski I, Mulisch M, Rödiger A, Schäfer A, Krupinska K. DNA-binding proteins of the Whirly family in Arabidopsis thaliana are targeted to the organelles. FEBS Lett. 2005 Jul 4;579(17):3707-12. doi: 10.1016/j.febslet.2005.05.059. PMID: 15967440.

Karpinska B, Razak N, James EK, Morris JA, Verrall SR, Hedley PE, Hancock RD, Foyer CH. WHIRLY1 functions in the nucleus to regulate barley leaf development and associated metabolite profiles. Biochem J. 2022 Mar 18;479(5):641-659. doi: 10.1042/BCJ20210810. PMID: 35212355; PMCID: PMC9022988.

Janack B, Sosoi P, Krupinska K, Humbeck K. Knockdown of WHIRLY1 Affects Drought Stress-Induced Leaf Senescence and Histone Modifications of the Senescence-Associated Gene HvS40. Plants (Basel). 2016 Sep 6;5(3):37. doi: 10.3390/plants5030037. PMID: 27608048; PMCID: PMC5039745.

Huang D, Lan W, Li D, Deng B, Lin W, Ren Y, Miao Y. WHIRLY1 Occupancy Affects Histone Lysine Modification and WRKY53Transcription in Arabidopsis Developmental Manner. Front Plant Sci. 2018 Oct 19;9:1503. doi: 10.3389/fpls.2018.01503. PMID: 30405658; PMCID: PMC6202938.

Ruan Q, Wang Y, Xu H, Wang B, Zhu X, Wei B, Wei X. Genome-wide identification, phylogenetic, and expression analysis under abiotic stress conditions of Whirly (WHY) gene family in Medicago sativa L. Sci Rep. 2022 Nov 4;12(1):18676. doi: 10.1038/s41598-022-22658-3. PMID: 36333411; PMCID: PMC9636397.