WHIRLIES Are Multifunctional DNA-Binding Proteins With Impact on Plant Development and Stress Resistance

Abstract

WHIRLIES are plant-specific proteins binding to DNA in plastids, mitochondria, and nucleus. They have been identified as significant components of nucleoids in the organelles where they regulate the structure of the nucleoids and diverse DNA-associated processes. WHIRLIES also fulfil roles in the nucleus by interacting with telomers and various transcription factors, among them members of the WRKY family. While most plants have two WHIRLY proteins, additional WHIRLY proteins evolved by gene duplication in some dicot families. All WHIRLY proteins share a conserved WHIRLY domain responsible for ssDNA binding. Structural analyses revealed that WHIRLY proteins form tetramers and higher-order complexes upon binding to DNA. An outstanding feature is the parallel localization of WHIRLY proteins in two or three cell compartments. Because they translocate from organelles to the nucleus, WHIRLY proteins are excellent candidates for transducing signals between organelles and nucleus to allow for coordinated activities of the different genomes. Developmental cues and environmental factors control the expression of WHIRLY genes. Mutants and plants with a reduced abundance of WHIRLY proteins gave insight into their multiple functionalities. In chloroplasts, a reduction of the WHIRLY level leads to changes in replication, transcription, RNA processing, and DNA repair. Furthermore, chloroplast development, ribosome formation, and photosynthesis are impaired in monocots. In mitochondria, a low level of WHIRLIES coincides with a reduced number of cristae and a low rate of respiration. The WHIRLY proteins are involved in the plants' resistance toward abiotic and biotic stress. Plants with low levels of WHIRLIES show reduced responsiveness toward diverse environmental factors, such as light and drought. Consequently, because such plants are impaired in acclimation, they accumulate reactive oxygen species under stress conditions. In contrast, several plant species overexpressing WHIRLIES were shown to have a higher resistance toward stress and pathogen attacks. By their multiple interactions with organelle proteins and nuclear transcription factors maybe a comma can be inserted here? and their participation in organelle-nucleus communication, WHIRLY proteins are proposed to serve plant development and stress resistance by coordinating processes at different levels. It is proposed that the multifunctionality of WHIRLY proteins is linked to the plasticity of land plants that develop and function in a continuously changing environment.

Keywords: DNA-binding; WHIRLY; development; nucleoid; stress.

Figure 1

Schematic comparison of motifs in selected WHIRLY proteins. AtWHIRLY1 (Arabidopsis thaliana, NP_172893), AtWHIRLY2 (A. thaliana, NP_177282.2), AtWHIRLY3 (A. thaliana, NP_178377), HvWHIRLY1 (Hordeum vulgare, BAJ96655), HvWHIRLY2 (H. vulgare, BF627441.2), SlWHIRLY1 (Solanum lycopersicum, AFY24240.1), SlWHIRLY2 (S. lycopersicum, XP_010313085.1), StWHIRLY1 (Solanum tuberosum, NP_001275155.1), StWHIRLY2 (S. tuberosum, NP_001275393.1), NtWHIRLY1 (Nicotiana tabacum, XP_016453689.1), NtWHIRLY2 (N. tabacum, XP_016511175.1), ZmWHIRLY1 (Zea mays, NP_001123589.1), ZmWHY2 (Z. mays, NP_001152589.2), OsWHIRLY1 (Oryza sativa, BAD68418.1), OsWHIRLY2 (O. sativa, NP_001045956.1). The WHIRLY domain is colored in light blue. The N-terminal organelle targeting peptides (OTP) are illustrated in grey. Below the scheme, the positions of selected amino acid residues and motifs in different WHIRLIES are listed: the PRAPP motif in yellow, the DNA-binding motif (DBM) and a putative nuclear localization motif (pNLS) in blue, a putative copper-binding motif in purple and a putative transactivation domain (pAD) in green. The organelle targeting peptides (OTP) were predicted with TargetP-2.0 (https://services.healthtech.dtu.dk/service.php?TargetP-2.0) or UniProt, (https://www.uniprot.org/), the putative nuclear localization signal (pNLS) with NLStradamus (http://www.moseslab.csb.utoronto.ca/NLStradamus/) and the putative copper-binding motif (CBM) with Motif Scan (https://myhits.sib.swiss/cgi-bin/motif_scan). The putative autoregulatory domain (pAD) was defined by Desveaux et al. (2005).

Figure 2

Detection of WHIRLY1:GFP and WHIRLY3:RFP fusion proteins in epidermal cells of the cotyledons of stably transformed Arabidopsis plants. The constructs were overexpressed under the control of the CaMV 35S promoter. The constructs were overexpressed under the control of the CaMV 35S promoter. Leaves were imbedded in PBS:glycerol (1:1) without fixation and analyzed by a LEICA SP5 laser scanning microscope and a HCX PL Apo 63x/1.2 W objective. Sequential scans per frame for GFP [Ex 488 nm (6%), Em 510–550 nm], mRFP [Ex 543nm (14%), Em 580–610nm] and chlorophyll [Ex 633 (5%), Em 690–750 nm] were carried out. Chlorophyll fluorescence is shown in red while signals of WHIRLY1:GFP and WHIRLY3:RFP displayed both in green. A projection out of five optical layers representing 3 μm in z-direction are created by the LAS X software. The bars represent 10 μm each.

Figure 3

Morphology of nucleoids in sections from albino leaves of maize transposon mutants. The Zmwhy1-1 mutant (Prikryl et al., 2008) is compared with selected mutants lacking PEP-associated proteins (PAP; Williams-Carrier et al., 2014). All proteins are transcriptionally active chromosomes (TAC) components and hence have pTAC names. DNA was stained by SYBR GREEN as described (Krupinska et al., 2014b). Bars in low magnification images represent 20 μm, in the higher magnifications on the left, bars represent 5 μm. Frames in the transmission images on the right indicate the sections shown in the high magnification images on the left.

Figure 4

Impact of WHIRLY1 on the structure of prolamellar bodies and prothylakoids of etioplasts (A) and thylakoids of barley chloroplasts (B,C). Ultrathin sections were prepared from primary foliage leaves of RNAi-W1-7 and wild-type plants. For analysis of etioplast structure, plants were grown in darkness for 8 days. For studies of chloroplasts, plants were grown in continuous light of either 120 (B) or 320 μmol m⁻² s⁻¹ (C). Ultrastructural analyses were performed as described (Golin et al., 2020). Bars represent 500 nm.