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Most plant growth and development processes are regulated in one way or another by auxin. The best-studied mechanism by which auxin exerts its regulatory effects is through the nuclear auxin pathway (NAP). In this pathway, Auxin Response Factors (ARFs) are the transcription factors that ultimately determine which genes become auxin regulated by binding to specific DNA sequences. ARFs have primarily been studied in Arabidopsis thaliana, but recent studies in other species have revealed family-wide DNA binding specificities for different ARFs and the minimal functional system of the NAP system, consisting of a duo of competing ARFs of the A and B classes. In this review, we provide an overview of key aspects of ARF DNA binding such as auxin response elements (TGTCNN) and tandem repeat motifs, and consider how structural biology and in vitro studies help us understand ARF DNA preferences. We also highlight some recent aspects related to the regulation of ARF levels inside a cell, which may alter the DNA binding profile of ARFs in different tissues. We finally emphasize the need to study minimal NAP systems to understand fundamental aspects of ARF function, the need to characterize algal ARFs to understand how ARFs evolved, how cutting-edge techniques can increase our understanding of ARFs, and which remaining questions can only be answered by structural biology.

In the obligate short-day potato Solanum tuberosum group Andigena (Solanum andigena), short days, or actually long nights, induce tuberization. Applying a night break in the middle of this long night represses tuberization. However, it is not yet understood how this repression takes place. We suggest a coincidence model, similar to the model explaining photoperiodic flowering in Arabidopsis. We hypothesize that potato CONSTANS (StCOL1), expressed in the night of a short day, is stabilized by the light of the night break. This allows for StCOL1 to repress tuberization through induction of StSP5G, which represses the tuberization signal StSP6A. We grew S. andigena plants in short days, with night breaks applied at different time points during the dark period, either coinciding with StCOL1 expression or not. StCOL1 protein presence, StCOL1 expression and expression of downstream targets StSP5G and StSP6A were measured during a 24-h time course. Our results show that a night break applied during peak StCOL1 expression is unable to delay tuberization, while coincidence with low or no StCOL1 expression leads to severely repressed tuberization. These results imply that coincidence between StCOL1 expression and light does not explain why a night break represses tuberization in short days. Furthermore, stable StCOL1 did not always induce StSP5G, and upregulated StSP5G did not always lead to fully repressed StSP6A. Our findings suggest there is a yet unknown level of control between StCOL1, StSP5G and StSP6A expression, which determines whether a plant tuberizes.

Late embryogenesis abundant (LEA) proteins are essential to the ability of resurrection plants and orthodox seeds to protect the subcellular milieu against irreversible damage associated with desiccation. In this work, we investigated the structure and function of six LEA proteins expressed during desiccation in the monocot resurrection species Xerophyta schlechteri (XsLEAs). In silico analyses suggested that XsLEAs are hydrophilic proteins with variable intrinsically disordered protein (IDP) properties. Circular dichroism (CD) analysis indicated that these proteins are mostly unstructured in water but acquire secondary structure in hydrophobic solution, suggesting that structural dynamics may play a role in their function in the subcellular environment. The protective property of XsLEAs was demonstrated by their ability to preserve the activity of the enzyme lactate dehydrogenase (LDH) against desiccation, heat and oxidative stress, as well as growth of Escherichia coli upon exposure to osmotic and salt stress. Subcellular localization analysis indicated that XsLEA recombinant proteins are differentially distributed in the cytoplasm, membranes and nucleus of Nicotiana benthamiana leaves. Interestingly, a LEA_1 family protein (XsLEA1-8), showing the highest disorder-to-order propensity and protective ability in vitro and in vivo, was also able to enhance salt and drought stress tolerance in Arabidopsis thaliana. Together, our results suggest that the structural plasticity of XsLEAs is essential for their protective activity to avoid damage of various subcellular components caused by water deficit stress. XsLEA1-8 constitutes a potential model protein for engineering structural stability in vitro and improvement of water-deficit stress tolerance in plants.