Late Embryogenesis Abundant Protein-Client Protein Interactions

Abstract

The intrinsically disordered proteins belonging to the LATE EMBRYOGENESIS ABUNDANT protein (LEAP) family have been ascribed a protective function over an array of intracellular components. We focus on how LEAPs may protect a stress-susceptible proteome. These examples include instances of LEAPs providing a shield molecule function, possibly by instigating liquid-liquid phase separations. Some LEAPs bind directly to their client proteins, exerting a holdase-type chaperonin function. Finally, instances of LEAP-client protein interactions have been documented, where the LEAP modulates (interferes with) the function of the client protein, acting as a surreptitious rheostat of cellular homeostasis. From the examples identified to date, it is apparent that client protein modulation also serves to mitigate stress. While some LEAPs can physically bind and protect client proteins, some apparently bind to assist the degradation of the client proteins with which they associate. Documented instances of LEAP-client protein binding, even in the absence of stress, brings to the fore the necessity of identifying how the LEAPs are degraded post-stress to render them innocuous, a first step in understanding how the cell regulates their abundance.

Keywords: desiccation; late embryogenesis abundant; natural protection and repair mechanism; protein interaction; seed; stress.

Figure 1

If one accepts that some LEAPs have an affinity for specific client proteins (CP), physically binding to them, the consequence of this is traditionally thought to be client protein protection. (a) In Wild Type (WT) organisms, deviations from unstressed (normal) conditions by some stressor upregulates LEAP expression, increasing LEAP protein, and the LEAP chaperonin protects the client protein that would otherwise be decimated by the stress. The client protein continues exerting at least some of its action. (b) One alternative to this chaperonin role for LEAPs is that of a surreptitious rheostat of cellular homeostasis. Upon upregulation by the stressor, LEAPs’ affinity for the client protein competes with the client protein affinity for one of its targets, sequestering sufficient client protein away from its target to consequentially mitigate its action. Biotechnological alteration of the LEAP and client protein amounts can provide insights into the nature of the LEAP–client protein relationship. (c) If the LEAP protects the client protein from a stressor, a parity of phenotypes results for mutation of either the leap or the client protein (cp). In one instance, the client protein is unprotected, decimated by the stress, and the action is attenuated. In the other, there is no client protein and the action is diminished to a basal response. (d) Overexpression of either the LEAP (LEAP OE) or the client protein (CP OE) results in either endogenous client protein amounts that are maximally protected by superabundant LEAP or a superabundance of client protein with endogenous LEAP protection, respectively. Either scenario results in a substantially greater portion of the client protein surviving the stress, relative to WT (compare Action arrow in (d) eliciting peak client protein action). (e,f) The client protein binds to some cellular entity and prevents/initiates an action leading to a phenotype. Here, we assume initiation of an action. The LEAP binds some portion of the client protein population, interfering with the initiation of the action (conflict). (e) A parity of phenotypes results from the leap mutant, allowing the entire client protein population to initiate action or the client protein overexpressor that overwhelms the LEAP binding capacity and can still initiate peak action. (f) The elimination of the client protein minimizes the action to its basal response, which, depending on the affinity of the LEAP for the client protein, is similar to LEAP overexpression, which sequesters the entire endogenous client protein population, preventing the initiation of the action. Maximal client protein amounts, initiating peak action, are designated by a solid black border. Dashed lines separate the genotypes eliciting the same phenotype.

Figure 2

A dehydrin from Arabidopsis thaliana EARLY RESPONSE TO DEHYDRATION 14 (ERD14) (At1g76180; pfam dehydrin) interacts with the C-terminal domain of the Arabidopsis Phi9 GLUTATHIONE-S-TRANSFERASE9 (At2g30860). Phi9 associates with the dehydrin in ratios of 1:5, respectively (or greater), to acquire protection from H₂O₂-related structural damage. GST binding: the glutathione-binding site (G-site).

Figure 3

A histidine-rich dehydrin from Arabidopsis thaliana HISTIDINE-RICH DEHYDRIN OF 11 KDA (HIRD11) (At1g54410) interacts with the Arabidopsis leucine-rich repeat receptor-like kinase (LRR-RLK) PHLOEM INTERCALATED WITH XYLEM-LIKE 1 (AtPXL1; AT1G08590). Plasma membrane-localized AtPXL1 phosphorylates (P) cytoplasmically localized (or plasma membrane associated?) HIRD11. At least, a sub-population of HIRD11 associates with the plasma membrane or AtPXL1 on the plasma membrane. The aP in a yellow circle represents the autocatalyzed phosphate group that is present on most LRR-RLK proteins, though this remains to be defined for this particular enzyme as present or as the phosphate group that gets transferred to the substrate.

Figure 4

A CALCIUM/CALMODULIN BINDING CYTOPLASMIC RECEPTOR-LIKE KINASE2 (CB-RLK2) and the LEAP. GLYCINE SOJA PHYSIOLOGICAL MATURE30 (GsPM30) that binds GsCBRLK2; both positively influence plant tolerance to hypersaline (NaCl) conditions when overexpressed. It is not currently known if the association leads to phosphorylation (P) of GsPM30 by CB-RLK2 (see question mark above). The aP in a yellow circle represents the autocatalyzed phosphate group that may be present on both the receptor kinases, though this remains to be defined for these particular enzymes as present or as the phosphate group that gets transferred to the substrate.

Figure 5

The Oryza sativa gene, OsZFP36, a zinc-finger transcription factor, is upregulated by the shock hormone abscisic acid (ABA) and by the reactive oxygen species H₂O₂. OsLEA5 (LOC_Os05g50710; pfam LEA 2) is upregulated by ABA, at least partially due to the action of the rice ZINC FINGER C2H2 PROTEIN OsZFP36 (LOC_Os03g32230). When OsLEA5 is present, it stabilizes OsZFP36. This results in enhanced OsZFP36 residency and increased OsLEA5 expression, a positive feedback loop (circular arrows). Another target upregulated by OsZFP36 is the peroxidase OsAPX1. This enzyme detoxifies H₂O₂, a reactive oxygen species that upregulates OsZFP36 expression. By recognizing the stabilization of the transcription factor during stress by the protein product of its LEAP target, many of the reported physiological alterations and differences in ABA sensitivity and redox poise in common between mutants in the oslea5 or in oszfp36 can be explained. ZBF motif: zinc finger binding motif.

Figure 6

A dehydrin from the cactus Opuntia streptacantha (Genbank ID: HM581971) or any of three homologs from Arabidopsis thaliana COLD-REGULATED 47 (COR47), EARLY RESPONSIVE TO DEHYDRATION 10 (ERD10), and RESPONSIVE TO ABA 18 (RAB18) (dehydrins), encoded by At1g20440; At1g20450, and At5g66400, respectively, interact with the Arabidopsis membrane intrinsic protein (MIP) aquaporin ARABIDOPSIS THALIANA PLAMAMEMBRANE INTRINSIC PROTEIN2B (AtPIP2B), encoded by At2g37170. The subcellular distribution of both the LEAPs and this client protein along the plasma membrane is consistent with biochemical evidence of these interactions. The functional significance of this interaction has not yet been elucidated. The three different dehydrins can also associate with each other in homo- or hetero-dimers. The implications of these interactions are also unknown.

Figure 7

Unlike (a) Wild Type Arabidopsis seeds, (b) mutant seed maturation protein1 (smp1) seeds lose the capacity to enter thermo-dormancy (invoked by 4 days at 42 °C while hydrated (blue pool of water)) when subsequently removed to a permissive temperature, 25 °C. The soybean LEAP GLYCINE MAX PHYSIOLOGICAL MATURE 28 (GmPM28) and its Arabidopsis orthologue, SMP1, were both able to bind several of the same client proteins. The protein most frequently bound by both orthologs was the CANCER SUSCEPTIBILITY CANDIDATE3 (CASC3), capable of marking the splice junction on mature mRNA and enhancing translation. Presumably, loss of the LEAP destabilized CASC3 during high-temperature stress and this may lead to the inability of a (presumably repressive) protein to subsequently exert thermo-dormancy on a sub-population of the seeds.

Figure 8

Immunoprecipitation of proteins physically associating with a chloroplast-stroma-localized LEAP, COR15Am (At2g42540), consistently retrieved both the small and the large subunits of RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXIDASE (RUBISCO) from cold-acclimated (b,c) but not (a) unacclimated plants. This association (b) has been positively correlated with a greater capacity to tolerate freezing, a capacity that is enhanced in both protoplasts and chloroplasts overexpressing COR15Am (Table S1). However, (c) RNAi lines reducing COR15A and COR15B simultaneously did not suffer a greater loss of RUBISCO activity during freezing after cold acclimation but rather the opposite was true. L₈: Eight large RUBISCO subunits; S₈: Eight small RUBISCO subunits; 8L and 8S are these subunits disassembled from the holoenzyme.

Figure 9

Both COR47 and ERD10 dehydrins were shown to bind ACTIN filaments. A series of experiments demonstrated that ERD10 was probably capable of binding the plus and minus ends as well as the body of the filament. Although COR47 was also shown to bind the filament, where it binds is still unknown. ERD10 binding inhibits nucleation, slowing the initiation of growth while it also slows the depolymerization of the filament. It can interfere with the capacity of latrunculin B to prevent polymerization [108].

Figure 10

The Medicago truncatula dehydrin gene, MtCAS31 (Medtr6g084640; highly similar to the Arabidopsis ERD10 (At1g20450) and ERD14 (At1g76180) pfam dehydrin) is upregulated by drought stress. When MtCAS31 protein is present, it binds one of several leghemoglobin proteins (MtLb120-1; Medtr5g080440), protecting it from dehydration induced denaturation. The continued functioning of leghemoglobin, safeguarded by MtCAS31, maintains the appropriate oxygen level in nodules conducive to both nitrogenase activity and bacterial respiration, supporting nodule persistence.

Figure 11

MtCAS31 (described in Figure 10) is upregulated by drought stress. When MtCAS31 dehydrin protein is present, it binds INDUCER OF CBF EXPRESSION (ICE1, i.e., SCREAM) as well as the Medicago basic HELIX-LOOP-HELIX -LEUCINE ZIPPER transcription factor most homologous to ICE1, Medtr7g083900. The molecular consequences of MtCAS31:ICE1 binding is that the number of ICE1 proteins available to hetero-dimerize with any of the basic HELIX-LOOP-HELIX transcription factors involved in meristemoid initiation, progression to guard mother cells, or the formation of two guard cells (in Arabidopsis: SPEECHLESS, MUTE, or FAMA), decreases. The phenotypic consequence for leaves formed when MtCAS31 is present is fewer stomata per unit leaf area, resulting in a physiology tuned to reduce evapotranspiration. X = orthologs of Arabidopsis basic HELIX-LOOP-HELIX transcription factors, SPEECHLESS, MUTE, and FAMA but not the basic HELIX-LOOP-HELIX -LEUCINE ZIPPER proteins ICE1 (i.e., SCREAM) and SCREAM2. bHLH-LZ: basic HELIX-LOOP-HELIX-LEUCINE ZIPPER transcription factor; CBFs: C-REPEAT BINDING FACTORs. Green TF: Binding with its partner basic HELIX-LOOP-HELIX transcription factor to cognate DNA motifs. Target gene(s) are transcriptionally active (green). Red TF: Bound by MtCAS31 and sequestered away from its basic HELIX-LOOP-HELIX partner and DNA motifs. Target gene(s) are transcriptionally repressed (red).

Figure 12

MtCAS31 (described in Figure 10) is: (a) upregulated upon dehydration and the protein it encodes; (b) binds the plasmamembrane intrinsic protein aquaporin MtPIP2;7 (Medtr2g094270) as well as the Medicago AUTOPHAGY-RELATED GENE8 Protein (ATG8a; Medtr4g037225) through the 2 AIM-like motifs (Autophagy Interacting Motifs) located near the MtCAS31 amino terminus. Thus, MtCAS31 acts as an adaptor protein; (c) directly linking its cargo protein MtPIP2;7 with ATG8a in phagophores, which leads to the destruction of the aquaporins (and MtCAS31) through selective autophagy. While stress is present, so too is MtCAS31, which ensures a continuous turnover of MtPIP2;7. The molecular consequences are a reduction in the number of aquaporins located in the plasma membrane of drought-stressed Medicago cells. The phenotypic consequences for the cell when MtCAS31 is present are: (d) an altered hydraulic conductivity of the cell membrane, resulting in a physiology tuned to reduce water transport across the cell membrane.

Figure 13

Alteration of phenotypes for a LEAP and one of its client proteins, depending on the physiology of the cell being examined. (a) The AtPP2-B11 F-BOX protein targets the SUCROSE NON-FERMENTING KINASEs (SnRK2.2, 2.3), positive regulators of ABA signal transduction, reducing the titer of these kinases in the cell. Both atpp2-b11 RNAi lines and LEA1-overexpressing lines have enhanced ABA sensitivity. Reducing the F-BOX titer through RNAi stabilizes the SnRK2s titer and results in increased ABA sensitivity. When the LEA1 binds AtPP2-B11, it may sequester the F-BOX protein away from SnRK2.2 and 2.3, preventing their polyubiquitination, and also increasing ABA sensitivity. (b) ATPP2-B11 F-BOX overexpression results in greater salt tolerance as does LEA1 overexpression. Reasons for the similar phenotypes under salt stress are, at this time, a matter of speculation, but a model presented in the reference supplied in the figure suggests that a transcriptional repressor is targeted by the F-BOX, and the F-BOX is stabilized during salt stress by LEA1. Green: protein stimulatory for ABA sensitivity or salt tolerance; Red: protein inhibitory for ABA sensitivity or salt tolerance; “P” = phosphorylated; tfs: transcription factors; PP2C: PROTEIN PHOSPHATASE 2C; SnRK: SUCROSE NON-FERMENTING RECEPTOR KINASES; PYR: PYRABACTIN RESISTANT PROTEIN; Rep: transcriptional repressor; UBI: UBIQUITIN.