Overexpression of RING Domain E3 Ligase ZmXerico1 Confers Drought Tolerance through Regulation of ABA Homeostasis

Abstract

Drought stress is one of the main environmental problems encountered by crop growers. Reduction in arable land area and reduced water availability make it paramount to identify and develop strategies to allow crops to be more resilient in water-limiting environments. The plant hormone abscisic acid (ABA) plays an important role in the plants' response to drought stress through its control of stomatal aperture and water transpiration, and transgenic modulation of ABA levels therefore represents an attractive avenue to improve the drought tolerance of crops. Several steps in the ABA-signaling pathway are controlled by ubiquitination involving really interesting new genes (RING) domain-containing proteins. We characterized the maize (Zea mays) RING protein family and identified two novel RING-H2 genes called ZmXerico1 and ZmXerico2 Expression of ZmXerico genes is induced by drought stress, and we show that overexpression of ZmXerico1 and ZmXerico2 in Arabidopsis and maize confers ABA hypersensitivity and improved water use efficiency, which can lead to enhanced maize yield performance in a controlled drought-stress environment. Overexpression of ZmXerico1 and ZmXerico2 in maize results in increased ABA levels and decreased levels of ABA degradation products diphaseic acid and phaseic acid. We show that ZmXerico1 is localized in the endoplasmic reticulum, where ABA 8'-hydroxylases have been shown to be localized, and that it functions as an E3 ubiquitin ligase. We demonstrate that ZmXerico1 plays a role in the control of ABA homeostasis through regulation of ABA 8'-hydroxylase protein stability, representing a novel control point in the regulation of the ABA pathway.

Figure 1.

Classification and representation of consensus domains of maize RING-domain-containing proteins. Graphical representation of multiple sequence alignments of maize RING protein domains for each RING protein type using HMMlogo. The height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino acid at that position. Interleaved zinc coordination sites are indicated. The number of proteins for each category of RING domain and percentage of the total number of RING proteins are indicated.

Figure 2.

Transmembrane domain of ZmXerico1, ZmXerico2, and related proteins and subcellular localization of ZmXerico1 in the ER. A, Alignment of ZmXerico protein sequences and related protein sequences from Arabidopsis (AtXerico, RHA2a, and RHA2b) and rice (OsRHP1). Putative transmembrane domains identified using TMHMM2.0 are indicated by blue boxes. RING-H2 domains are identified by a red box and position of C and H by asterisks. Non-similar, conserved, similar, identical and weakly similar amino acids are represented by black letters on white background, blue letters on blue background, black letters on green background, red letter on yellow background, or green letters on white background respectively. Arrows indicate aa mutated in ZmXerico1 mutant proteins used in Figure 12. B, The first 40aa of ZmXerico1, including its predicted transmembrane domain, were fused to GFP (ZmXerico1 (40aa):GFP) and the protein fusion was transiently co-expressed in maize protoplasts with an ER-targeted RFP fusion marker (CHIT:RFP:HDEL). Merged confocal microscopy pictures show co-localization of GFP and RFP signals. Bar is 5 microns.

Figure 3.

Response of ZmXerico, ZmXerico1, and ZmXerico2 gene expression to drought stress and recovery in leaf and root of B73 seedlings. Expression of ZmXerico genes was measured using qRT-PCR-specific assays in maize B73 seedlings grown in well-watered conditions (0), seedlings after 24 h (24), and 48h (48) of water withdrawal and seedlings 72 h after rewatering from a 48 h stress (72). Error bars represent se from the mean (sem; n = 3).

Figure 4.

Diurnal expression pattern of ZmXerico, ZmXerico1, and ZmXerico2 in maize. Expression of ZmXerico genes (A, ZmXerico; B, ZmXerico1; and C, ZmXerico2) was measured in B73 seedlings grown in growth chambers over a 3-d period in 12-h light and 12-h dark (shaded areas) conditions. Leaf and root samples were collected every 2 h from plants grown in well-watered (blue lines) or drought-stressed (red lines) conditions. Drought stress was applied at zt = 0 by stopping watering until the end of day 3. NextGen Illumina reads were mapped to proprietary B73 gene models corresponding to the different ZmXerico genes and expression quantified as reads per kilobase of transcript per ten million mapped reads (RPKtM). Error bars indicate sem (n = 4).

Figure 5.

Delayed drought-induced senescence of Ubi::ZmXerico1 maize transgenics compared to control plants. Representative pictures of Ubi::ZmXerico1 transgenic and control plants grown under managed drought stress conditions in 2009 and 2011. Transgenic plants displayed reduced leaf rolling and visibly healthier lower canopy leading to statistically significant staygreen phenotypes compared to controls.

Figure 6.

Drought stress recovery phenotypes of Ubi::ZmXerico1 and control maize plants. A, Comparison of a representative transgenic Ubi::ZmXerico1 overexpressing event (Event 5) and control null plants 4 h after recovery from water stress showing faster recovery of transgenics. B, Comparison of the recovery of a transgenic non-expressing Ubi::ZmXerico1 event (Event 15) and control null plants 4 h after water stress.

Figure 7.

Drought tolerance and reduced water loss of drought-stressed 35S::ZmXerico transgenics compared to control Arabidopsis plants. A, Phenotype of Arabidopsis plants overexpressing ZmXerico1 and ZmXerico2 (TG) compared to controls (C) after 14 d of water withdrawal showing enhanced drought tolerance of transgenic plants. B, Cumulated water loss of Arabidopsis plants overexpressing ZmXerico1 compared to control plants over a 9-d period after withholding water. Error bars represent sem. Statistically significant difference (Student's t test; P < 0.001) is indicated by asterisks (n = 36).

Figure 8.

Physiological analysis and water use efficiency of maize Ubi::ZmXerico1 and control plants grown in well-watered conditions. A, Photosynthetic rate; B, stomatal conductance; and C, WUE of Ubi:ZmXerico1 events and control null plants grown in well-watered conditions in the greenhouse (n = 26). Measurements were obtained from leaves of V8 plants with a LICOR portable photosynthesis system; photosynthetic rate and stomatal conductance were used to calculate WUE. Error bars represent sem. Asterisks indicate significant statistical differences (Student's t test; P < 0.05).

Figure 9.

Water usage and grain yield of maize Ubi::ZmXerico1 events and control plants grown in the greenhouse. A, Plant pots were watered to capacity and pot weight measured. After withholding water for 3 d, pot weights were measured again and water use was measured as the difference in pot weight at the two time points. B, After rewatering for several days, the same cycle was repeated and water use calculated similarly (n = 9 to 12 for each event; n = 35 for controls). C, Yield of Ubi:ZmXerico1 events compared to control plants grown in well-watered conditions or subjected to 5 drought stress recovery cycles in the greenhouse (n = 6 for drought-stresed plants and n = 15 for well-watered plants). Error bars represent sem. Asterisks indicate significant statistical differences (Student's t test; P < 0.05).

Figure 10.

Measurement of Arabidopsis 35S::ZmXerico and maize Ubi::ZmXerico1 overexpresser sensitivity to ABA treatments. A, Percentage germination of Arabidopsis 35S::ZmXerico transgenics (black symbols) and control (white symbols) seeds after 3 d on plates containing different ABA concentrations. B, Percentage germination of Arabidopsis 35S::ZmXerico transgenics (black symbols) and control (white symbols) seeds on plates containing 0.6 µm ABA over a 5-d period. A and B are representative graphs of three independent experiments. C, Root elongation rate of Ubi::ZmXerico1 transgenic (black bars) and control plants (white bars) grown in germ paper imbibed with a solution containing 0 or 50 µm ABA. Error bars represent sem. Statistical significance of P < 0.01 (Student's t test) is indicated by asterisk (n = 15).

Figure 11.

ABA, ABA-GE, DPA, and PA levels in leaves of maize Ubi::ZmXerico1 and Ubi::ZmXerico2 transgenics and control plants. A, ABA metabolites measured from three independent events of Ubi::ZmXerico1 (textured bars; n = 3) and control plants (white bars; n = 3) and from (B) three independent events of Ubi::ZmXerico2 (textured bars; n = 3) and control plants (white bars; n = 6). Error bars represent sem. Construct average is presented (black bars), and statistical significance (Student's t test; P < 0.05) is indicated by asterisks. nd = not detected.

Figure 12.

ZmXerico1 E3 ubiquitin ligase activity. A, Recombinant MBP-ZmXerico1 fusion protein catalyzes self-ubiquitination in presence of E1, E2, and Ubiquitin. B, Mutations in amino acids critical for E2 and RING-H2 interaction inactivate self-ubiquitination of MBP-ZmXerico1. C, Mutation in amino acids critical for the stability of the RING-H2 domain inactivate self-ubiquitination of MBP-ZmXerico1.

Figure 13.

ZmXerico1 destabilizes ZmABA8ox1a and ZmABA8ox3a proteins. A, The steady-state level of ZmABA8ox1a, ZmABA8ox2, and ZmABA8ox3a proteins in maize protoplasts coexpressed with either ZmXerico1-FLAG-HA or ZmXerico1 (105 aa)-GFP. B, The steady-state level of ZmABA8ox1a and ZmABA8ox3a protein in maize protoplast with increasing ZmXerico1 expression. GFP was used as a control.

Figure 14.

ZmXerico1 interacts with ZmABA8ox1 and ZmABA8ox3. A, Coimmunoprecipitation of ZmXerico1 and ZmABA8ox1a. B, Coimmunoprecipitation of ZmXerico1 and ZmABA8ox3a. Protein extracts from maize protoplasts expressing ZmXerico1-FLAG-HA and ZmABA8ox-HA were immunoprecipitated with an anti-FLAG antibody, and the immunoprecipitated proteins were analyzed by immunoblotting using an anti-HA antibody.