Further analysis of XBAT32, an Arabidopsis RING E3 ligase, involved in ethylene biosynthesis

Abstract

The Arabidopsis RING E3 ligase, XBAT32, was previously characterized as a regulator of lateral root initiation. However, how XBAT32 function to modulate lateral root initiation was unknown. In our recent paper, we demonstrated that XBAT32 is involved in ethylene biosynthesis and it is through this function that XBAT32 is able to regulate lateral root production. Here we discuss a few other findings, observed in the ethylene overproducing mutant, xbat32, that reflect the effect of elevated ethylene levels on plant growth and development. Ethylene signaling also regulates plant responses to adverse environmental conditions such as high salinity. Consistent with ethylene's role as a stress hormone, xbat32 exhibited increased sensitivity to salt stress during seed germination and postgerminative growth. Thus, XBAT32 may also play a role in ethylene mediated response to abiotic stresses.

Protein ubiquitination is an integral part of numerous cellular processes and is required for proper growth and development of all eukaryotic organisms. The ubiquitination pathway covalently attaches ubiquitin, a small regulatory peptide, to selected proteins. The fate of the ubiquitinated protein differs depending upon the number of attached ubiquitin molecules as well as polyubiquitin chain topology.^1,2 Most notable is degradation of proteins modified by the attachment of a lysine 48 linked polyubiquitin chain by the 26S proteasome, a multi-catalytic protease. Ubiquitination is a multi-step reaction involving the sequential action of three enzymes, E1 (ubiquitin activating enzyme) which initiates the conjugation cascade and E2 (ubiquitin conjugating enzyme) along with the substrate binding E3 (ubiquitin ligase), transfers ubiquitin from the E2-ubiquitin intermediate to the substrate.² Almost 6% of the Arabidopsis thaliana proteome is dedicated to the ubiquitin-26S proteasome system (UPS).³ The majority of these proteins are ubiquitin ligases which, as the substrate recruiting component, govern specificity of the pathway. There are four main types of ubiquitin ligases found in plants, single subunit E3s containing either a U-box, Really Interesting New Gene (RING) or Homologous to E6-AP C-Terminus (HECT) E2-binding domain and multi-subunit cullin-RING ligases (CRL).³ The CRLs are further divided into four subtypes each containing a cullin, an E2-binding RING protein and a substrate recognition subunit. The SCF type, for example, contains cullin 1 (CUL1), RING-box1 (RBX1) protein, S phase kinase-associated proteins 1 (SKP1) adaptor protein that allows for CUL1 interaction with the substrate recognition F-box protein (FBX).³ The large number and diversity of the ubiquitin ligases illustrate the importance of this regulatory system to plant cellular biology.

The importance of the UPS to plant growth, development and survival under unfavorable conditions is due largely to its function in regulating the biosynthesis, perception and/or signaling pathways of major growth and stress hormones including abscisic acid (ABA), ethylene, jasmonic acid, gibberellin and auxin. The regulatory role of the UPS in hormone signaling is exemplified by its involvement in both the biosynthesis and signal transduction pathway of ethylene. Within the ethylene signaling pathway, the abundance of ethylene receptor ETHYLENE RESPONSE2 (ETR2), integral membrane protein ETHYLENE INSENSITIVE2 (EIN2) and transcription factors EIN3 and EIN3-like1 (EIL1) are all regulated by the UPS.^4–8 Ethylene signaling blocks the degradation of EIN2 and EIN3 by SCF type CRL E3 ligases containing EIN2-TARGETING PROTEIN1/2 (ETP1/2) and EIN3-BINDING F-BOX1/2 (EBF1/2) substrate recruiting F-box proteins, respectively.^4–6 The UPS regulates ethylene production by modulating the stability of the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) enzymes which convert S -adenosylmethionine (SAM) to the ethylene precursor ACC. The ACSs are grouped into three classes: type-1 (ACS1, ACS2 and ACS6), type-2 (ASC4, ASC5, ACS8 and ASC9) and type-3 (ACS7 and ACS11).⁹ Type-1 and -2 ACSs are short-lived in the absence of ethylene but are stable in the presence of the hormone.^9–11 E3 ligases responsible for the turnover of type-1 ACSs are unknown. The stability of the type-2 ACSs, particularly ACS5 and ACS9, is regulated mainly by ETHYLENE OVER PRODUCER 1 (ETO1) and ETO1-like (EOL1/2) broad complex/tramtrack/bric-a-brac (BTB) proteins which are the substrate recruiting subunit of a CRL E3.¹¹ UPS regulation of type-3 ACS has not previously been established.

In our recent paper we showed that RING type E3 ligase, XBAT32 (for XB3 ortholog 2 in Arabidopsis thaliana), is involved in maintaining appropriate levels of ethylene.¹² Here we discuss other effects of ethylene overproduction on plant development exhibited by xbat32 seedlings. XBAT32 is a member of the Arabidopsis RING ankyrin repeat subgroup of E3 ligases.¹³ With the exception of one family member KEEP ON GOING (KEG), which also contains a kinase domain and a series of HERC2-like repeats, the XBAT proteins contain a series of amino terminal ankyrin repeats that facilitates protein-protein interactions followed by a RING-HC domain (Fig. 1). XBAT32 was found to interact with type-2 (ACS4) and type-3 (ACS7) but not with type-1 ACSs in yeast two hybrid assays.¹² XBAT32 can catalyze the attachment of ubiquitin to both ACS4 and ACS7 in in vitro ubiquitination assays.¹² Phenotypic, interaction and biochemical analyses suggest that XBAT32 modulate the stability of ACS4 and ACS7 via ubiquitination and degradation by the 26S proteasome. Our results also indicate that similar to the type-1 and -2 ACSs, type-3 ACSs, in particular ACS7, are also under proteolytic control.

Figure 1.

A protein similarity tree depicting the relationship between Arabidopsis XBAT and XBAT-related proteins from other plant species. Neighbor joining tree was generated using an alignment of the amino acid sequences of ankyrin repeats from each protein. Bootstrap values, based on 1,000 replicates, are indicated below each node. Representative schematic for each sub-group is shown to the right. Characterized proteins are shown in bold and functions are also listed. Species: Ad, Artemisia desertorum Spreng; At, Arabidopsis thaliana; Ll, Lilium longiflorum (lily); Mt, Medicago truncatula; Mj, Mirabilis jalapa; Os, Oryza sativa; Pt, Populus trichocarpa; St, Solanum tuberosum; Vv, Vitis vinifera. The locus ID is shown for Arabidopsis thaliana and Oryza sativa and accession numbers are given for other species.

Proteins with similar domain architecture to Arabidopsis XBAT32 have been identified in a number of other plant species including rice (Oryza sativa), lily (Lilium longiflorum) and the drought tolerant plant Artemisia desertorum (Fig. 1). Of the RING ankyrin repeat proteins studied so far XBAT32 is the only member found to be involved in ethylene synthesis/signaling. Other XBAT or XBAT-related proteins are involved in disease resistance, drought tolerance, flower senescence and fertilization^14–17 (Fig. 1). Loss of XBAT32 produces a number of phenotypes that can be attributed to increase ethylene synthesis. Typically, dark-grown wild-type seedlings exposed to high levels of ethylene or ethylene overproducing mutants, such as eto1-1, demonstrate a constitutive triple response consisting of a shortened and thickened hypocotyl, exaggerated apical hook and shortened root.¹⁸ Consistent with an ethylene overproducing phenotype, etiolated xbat32 seedlings grown in air have shorter and thicker hypocotyls than wild-type seedlings. Similar to eto1-1, light grown xbat32 seedlings exhibit up to a two-fold increase in ethylene production compared to wildtype.^12,18 It has been known for sometime that ethylene suppresses primary root growth by inhibiting cell elongation.^19–21 The effects of ethylene on root development are dependent upon the modulation of auxin synthesis and transport.^20,21 Recently, it was shown that increased ethylene synthesis or signaling inhibits lateral root formation.^22,23 eto1-1 and the constitutive ethylene signaling mutant, ctr1-1, show reduced lateral root formation.²² Similarly, xbat32 seedlings exhibited a reduced lateral root phenotype.^12,24 Unlike eto1-1 and ctr1-1, the XBAT32 mutation did not affect primary root growth.¹² During root branching, elevated ethylene synthesis or signaling alter auxin transport leading to suboptimal levels of auxin for lateral root initiation.^22,23 This is consistent with the fact that complete rescue of the xbat32 lateral root defect is achieved only when auxin treatment is coupled with the inhibition of ethylene synthesis/signaling. Auxin treatment alone only partially rescues xbat32 lateral root production^12,24 (Fig. 2), suggesting that the added auxin is not fully available for initiation of lateral roots. The addition of low levels of abscisic acid (0.5 µM) during auxin treatment allowed xbat32 to produce numbers of lateral root similar to wild-type¹² (Fig. 2). This concentration of ABA (0.5 µM) did not alter the number of lateral roots produced by auxin treated wild-type roots. Consistent with ABA's role as an inhibitor of root growth and development,²⁶ higher concentrations of ABA (1 to 5 µM) reduced auxin stimulated lateral root production for both wild-type and xbat32 (Fig. 2). Since ABA is antagonistic to ethylene, treatment with low concentrations of ABA may have inhibited ethylene synthesis/signaling.²⁵ This may restore proper auxin transport and allows the exogenous auxin to promote xbat32 lateral root initiation and growth. Further analysis is required to determine if auxin transport is altered in xbat32 roots. Microarray analysis of roots from wild-type, ethylene insensitive mutant ein2-5 and auxin transport mutant aux1-7 etiolated seedlings identified several levels of interactions between ethylene and auxin.²⁷ The types of interaction found include auxin-dependent, -independent and -mediate ethylene responses or ethylene-dependent, -independent and -mediated auxin responses. From this analysis XBAT32's expression was found to be regulated by ethylene, but not by auxin. Ethylene repression of XBAT32 expression was altered in the aux1-7 mutant background indicating that the regulation by ethylene is auxin-dependent. These results are somewhat contrary to those of Nodzon et al.²⁴ where using a promoter β-glucuronidase (GUS) reporter system, auxin was found to increase the expression of XBAT32 in primary roots of light grown seedlings.²⁴ The discrepancies between the two studies could be explained by the differences in the experimental systems, for example light versus dark-grown roots. Overall, the results from the expression analyses indicate that XBAT32 may mediate tissue or cell specific cross-talk between ethylene and auxin to modulate lateral root initiation.

Figure 2.

ABA coupled with auxin treatment can fully rescue the lateral root defect of xbat32. Three day old wild-type and XBAT32 mutant seedlings were treated with increasing concentrations of abscisic acid (ABA) in the presence 2 µM naphthalenacetic acid (NAA), a synthetic auxin analog, for three days after which the number of visible lateral roots were quantified.

Several studies have illustrated the requirement for ethylene signaling in plant survival under salt stress.^28–33 Mutation in ethylene signaling components alters plant response to salt stress. For example, ctr1-1 is tolerant of salt stress and ein3-1 exhibit reduce salt tolerance during postgerminative development.³³ The ethylene over-producer, eto1-1, is salt-tolerant but only during seed germination and not during postgerminative growth.³⁴ Transgenic plants expressing a constitutively activated form of MKK9, a mitogen-activated protein kinase kinase that modulates ethylene synthesis, produce more ethylene than wild-type and exhibit enhanced sensitivity to salt stress.^35,36 Similarly, the XBAT32 mutation renders plants sensitive to salt stress during germination and early seedling growth (Fig. 3). After 48 hours of growth on medium containing 100 mM sodium chloride (NaCl) less than 60% of xbat32 seeds germinated compared to 80% of wild-type seeds (Fig. 3A). Only 20% of xbat32 seedlings on high salt medium exhibited proper cotyledon expansion compared to 80% of wild-type seedlings (Fig. 3B and C). Crosstalk between ethylene and ABA occurs during biosynthesis and signaling.²⁵ Therefore, it is likely that the altered sensitivity of XBAT32 mutants to salt stress may in part be due to ethylene's interactions with ABA. However, disruption of XBAT32 expression does not alter the plants response to ABA,¹² suggesting that XBAT32 does not modulate plant stress responses through ABA.

Figure 3.

Response of xbat32 and wild-type to salt stress. (A) Graph showing percentage (%) seed germination for wild-type (wt) and xbat32 after 48 hours of growth on medium supplemented with 100 mM sodium chloride (NaCl). (B and C) Seven-day-old wild-type and xbat32 seedlings grown on medium with or without 100 mM NaCl. Graph (C) shows percentage of seedlings with expanded cotyledons.

Overall our studies provide evidence to support the role of XBAT32, a RING E3 ligase, in maintaining appropriate levels of ethylene during plant growth and development. XBAT32 may also be involved in modulating ethylene mediated responses to salt stress.

Abbreviations

ACS

1-aminocyclopropane-1-carboxylic acid synthase

ABA

abscisic acid

RING

really interesting new gene

UPS

ubiquitin-26S proteasome

XBAT32

XB3 ortholog 2 in Arabidopsis thaliana

Addendum to: Prasad ME, Schofield A, Lyzenga W, Liu H, Stone SL. Arabidopsis RING E3 ligase XBAT32 regulates lateral root production through its role in ethylene biosynthesis. Plant Physiol. 2010;153:1587–1596. doi: 10.1104/pp.110.156976.