WHIRLY protein functions in plants

Abstract

Environmental stresses pose a significant threat to food security. Understanding the function of proteins that regulate plant responses to biotic and abiotic stresses is therefore pivotal in developing strategies for crop improvement. The WHIRLY (WHY) family of DNA-binding proteins are important in this regard because they fulfil a portfolio of important functions in organelles and nuclei. The WHY1 and WHY2 proteins function as transcription factors in the nucleus regulating phytohormone synthesis and associated growth and stress responses, as well as fulfilling crucial roles in DNA and RNA metabolism in plastids and mitochondria. WHY1, WHY2 (and WHY3 proteins in Arabidopsis) maintain organelle genome stability and serve as auxiliary factors for homologous recombination and double-strand break repair. Our understanding of WHY protein functions has greatly increased in recent years, as has our knowledge of the flexibility of their localization and overlap of functions but there is no review of the topic in the literature. Our aim in this review was therefore to provide a comprehensive overview of the topic, discussing WHY protein functions in nuclei and organelles and highlighting roles in plant development and stress responses. In particular, we consider areas of uncertainty such as the flexible localization of WHY proteins in terms of retrograde signalling connecting mitochondria, plastids, and the nucleus. Moreover, we identify WHY proteins as important targets in plant breeding programmes designed to increase stress tolerance and the sustainability of crop yield in a changing climate.

Keywords: DNA damage; chloroplasts; homologous recombination; mitochondria; nucleus; pathogenesis‐related genes.

FIGURE 1

Sequence alignments of WHIRLY genes in a range of species. The Profile ALIgNmEnt (PRALINE) multiple sequence alignment toolkit with homology‐extended alignment illustrates the conserved KGKAAL DNA binding domain in WHIRLY proteins (Bawono & Heringa, ; Simossis & Heringa, 2005). PRALINE allows quick visual comparisons of the best characterized species used in WHIRLY research, or which published sequence data are available. Species are as follows: Arabidopsis thaliana (At), Brassica rapa (Br), Glycine max (Gm), Hordeum vulgare (Hv), Manihot esculenta (Me), Populus trichocarpa (Pt), Oryza sativa (Os), Sorghum bicolor (Sb), Solanum lycospericum (Sl), Solanum tuberosum (St), Triticum aestivum (Ta), Zea mays (Zm), Chlamydomonas reinhardtii (Cre). The scoring scheme works from 0 for the least conserved alignment position, up to 10 for the most conserved alignment position. The colour‐coded assignments are scored as conservation of alignment position from unconserved (blue) to highly conserved (red)

FIGURE 2

A summary of WHY1 and WHY3 expression obtained by analysis of published transcriptome data. Co‐expression of WHY1 (AT1G14410) and WHY3 (AT2G02740). (a) Identification of genes with similar expression patterns to WHY1 at https://www.michalopoulos.net/act/. Gene ontology analysis of the 119 genes with most similar expression pattern identified significant enrichment in genes encoding plastid localized proteins (adjusted p value 5.9 × 10⁻⁵³). (b–d) Comparative expression of WHY1and WHY3 identified no significant differential expression between tissues (fold difference < ±2) (b) leaf mesophyll and guard cells (c) stem and epidermis of shoot tissue (d) rosette leaves. Tissue types and normalization of expression values is described by Winter et al. (2007)

FIGURE 3

WHY1‐mediated regulation of nuclear gene expression. (a) WHY1 binds to the ERE‐like element in the promoter of the HSP21.5A gene of Solanum lycospericum (tomato) in response to heat stress to activate the heat shock response (Zhuang, Gao, et al., 2020); (b) WHY proteins bind to an upstream region in the NCED1 gene promoter and to the CIPK23 promoter of Manihot esculenta (cassava) in response to drought stress to activate abscisic acid biosynthesis (Yan, Ning, et al., 2020); (c) WHY1 binds to the ERE‐like element in the RbcS1 promoter in response to chilling stress. WHY1 is also required for the activation of the synthesis of the D1 protein of photosystem II (Zhuang, Wang, et al., 2020). WHY1 binding to the ERE‐like element of the AMY3‐L promoter to activate α‐amylase, which is required for starch degradation, and to the ERE element of the ISA2 promoter to inhibit isoamylase‐mediated starch‐synthesis (Zhuang, Gao, et al., 2020). WHIRLY proteins are upregulated upon infection of pathogens and interact in biotic defence pathways. d) WHY1 is upregulated upon fungal infection by Ustiligo maydis in Zea mays (Kretschmer et al., 2017) and WHY1 was found to have a weak interaction with the Irregular Vascular Patterning (IVP) in Cucumis sativus, which functions in Botrytis cinerea resistance. Furthermore WHY2 was upregulated by B. cinerea infection in Solanum lycospericum (Akbudak & Feliz, 2019). Both WHY1 and WHY2 were upregulated in Fragaria × ananassa cv. Primoris upon Colletotrichum acutatum infection (Higuera et al., 2019). WHY proteins have also featured in oomycete infection where e) in addition to WHY1 upregulation upon infection by Hyaloperonospora parasitica (Desveaux et al., 2004), WHY1 was first discovered to act as a trans‐acting factor on Pathogenesis Related (PR) gene expression Phytophthora infestans where it binds to an elicitor‐response element (ERE) in the promoter of the PR‐10a gene which acts on the salicylic acid‐dependent disease resistance pathway (Despres et al., ; Desveaux et al., 2000). During f) bacterial infection, WHY2 has been found to be upregulated upon infection by Ralstonia solanacearum (previously Pseudomonas solanacearum) in S. lycospericum, which induced the transcriptional expression of PR1 and PR2 defence‐related genes (Zhao et al., 2018). More recently, WHY proteins have also been found to work in allelopathy defence against g) biochemical produced by competitive plants in allelopathy. Upon release of allelochemicals by neighbouring plants WHY1 and WHY2 were bound with Histone H4 (all of which were upregulated) to the promoter of PAL2;3, potentially to an ERE element, in Oryza sativa to negatively regulate allelopathy (Fang et al., 2020)

FIGURE 4

Model of WHIRLY involvement in DNA repair pathways. WHIRLY is involved in two pathways; (a1) by blocking microhomology‐mediated end joining (MMEJ) by DNA polymerases in the presence of AtWHY2 where single‐stranded regions are longer than 12 nucleotides (García‐Medel et al., 2019) and (b1) the accumulation of RNA: DNA hybrids (Pérez Di Giorgio et al., 2019). The transcribed DNA strand (black) is associated with the RNA (green) and RNA polymerase (RNAP). WHY proteins protect cp‐ and mtDNA by preventing the error‐prone MMBIR pathway in both chloroplasts and mitochondria (Cappadocia et al., 2010). This leads to the formation of (a2) error‐free HR, or (b2) R loops. In the absence of WHY proteins the double strand break could be repaired by MMEJ/ microhomology‐mediated break‐induced replication (MMBIR) by the recruitment of PolII (a1) alone or (b3) in conjunction with RNA, which are both error‐prone pathways

FIGURE 5

The phenotypes of why mutants of (a) Arabidopsis and (b) maize (Zea mays). (a) Rosette phenotypes of the three Atwhy mutants compared to the wild type at 3 and 6 weeks post‐germination grown under a 12 h photoperiod. No phenotypic differences were observed in any of the Atwhy mutants relative to the wild type during early (3 weeks) or late development (6 weeks). Scale bars are 10 mm. Unpublished data from this laboratory group. (b) Phenotypes of 9‐day old Zmwhy1 mutant seedlings. Left to right: homozygous Zmwhy1‐1 albino‐ivory, heteroalleic complementation cross of pale yellow Zmwhy1‐1 and Zmwhy1‐2, homozygous Zmwhy1‐2 pale green phenotype and green wild type

FIGURE 6

Schematic model of the intracellular localization of the WHIRLY proteins. The WHY proteins are targeted to organelles and are found in the nucleus (blue) at different stages of plant development. During growth WHY1 and WHY3 are targeted to the chloroplasts (green and yellow) while WHY2 is targeted to the mitochondria (maroon and orange). WHY3 and WHY1 have redundant functions (green double arrow). WHY2 has also been found in the chloroplasts (blue arrow). This model infers that WHY1 and WHY2 can be relocated to the nucleus in response to environmental stress (red dashed arrow)