**1. Introduction**

Waterlogging can damage most crops, creating one of the most significant problems in agriculture worldwide. During the heavy rainy season in the plain area, soil can quickly become waterlogged due to poor drainage, creating a low oxygen environment in the root area underground. Low oxygen stress leads to the induction of a particular set of genes involved in carbohydrate utilization, energy metabolism, and fermentation to sustain ATP production [1]. Over the long term, low oxygen stress means morphological adaptation is required to keep the level of oxygen under control [2]. Since global climate change could increase the number of flooding events, improved crop varieties with waterlogging tolerance are essential [3,4].

The ethylene response factor (*ERF*) family is one of the largest plant-specific transcription factor families characterized by a single DNA-binding domain, APETALA2 (AP2), with expanded functions in hormonal response, development, and tolerance to biotic and abiotic stresses [5–7]. Group VII ERF (ERFVII) transcription factors is a subgroup of ERF*s* that has been recognized as a critical factor controlling the expression of numerous genes involved in an adaptive response to low oxygen stress in model plants [8–11]. A characteristic feature of all Arabidopsis *ERFVIIs* (*RAP2.2*, *RAP2.3*, *RAP2.12, HRE1*, and *HRE2*) is a conserved N-terminal motif (N-degron; [5,12]), which enables them to be degraded by oxygen and nitric oxide (NO)-dependent N-end rule pathways [13–15]. Overexpression of the Arabidopsis *ERFVIIs* enhances flooding or low oxygen stress tolerance in transgenic Arabidopsis plants [13,14,16–21]. Interestingly, overexpression of the stabilized (N-terminal mutation) *HRE1* and *HRE2* in Arabidopsis further improved low oxygen tolerance compared to Arabidopsis lines overexpressing the wildtype *HRE1* and *HRE2* [14]. However, enhanced stability of *RAP2.12* resulted in reduced biomass under aerobic conditions and did not increase tolerance to low oxygen stress in transgenic Arabidopsis plants [13,22]. Transcription of genes encoding for fermentative and starch degradation enzymes were constitutively activated in transgenic Arabidopsis overexpressing stable *RAP2.12*, which negatively affected growth and development under the aerobic condition and reduced tolerance to low oxygen stress [22]. In rice, previous studies identified *ERFVIIs*, *Snorkels*, and *Sub1A*, as a key player orchestrating the escape and quiescence response needed to survive flash-flood and prolonged submergence, respectively [23,24]. Although Sub1A contained an N-degron, it was not a substrate of the N-end rule pathway [11,14]. It has recently been shown that *Sub1A* transcriptionally activates the other *ERFVII*s, *ERF66*, and *ERF67*, resulting in transcriptional accumulation of anaerobic survival genes and improved submergence tolerance in rice [11]. Remarkably, constitutive expression of the stabilized wheat *ERFVII*, *TaERFVII.1*, enhanced tolerance to waterlogging in transgenic wheat without negative impacts on development and grain yield under aerobic conditions [25]. Thus, identification, selection, and modification of the *ERFVII* genes could be a valuable approach to improve crop waterlogging tolerance.

*Jatropha curcas* is a drought-tolerant oilseed crop for biodiesel production. It can be grown on marginal land without competing with other food crops [26]. However, waterlogging caused a significant reduction of growth and biomass yield, suggesting that Jatropha is extremely sensitive to waterlogging [27,28]. Undoubtedly, genetic improvement of waterlogging tolerant Jatropha is needed to increase Jatropha oil production. Previously, we transcriptionally profiled gene expression in Jatropha and found that waterlogging promoted anaerobic respiration, but inhibited carbohydrate synthesis, cell wall biogenesis, and plant growth [29]. Based on our previous study, *ERFVIIs* had been proposed as candidate genes for genetic engineering of waterlogging tolerant Jatropha [29].

In this study, we cloned and evaluated the tissue-specific expression and waterlogging responsive pattern of Jatropha *ERFVIIs* (*JcERFVIIs*). Next, we followed up by examining the N-end rule regulated protein stability of *JcERFVIIs* and overexpressing the *JcERFVII2* genes in Arabidopsis to evaluate the flooding and low oxygen tolerant phenotype. Finally, the molecular function of *JcERFVII2* was further investigated by transcriptome profiling of transgenic Arabidopsis lines.

#### **2. Results**

#### *2.1. Cloning and Bioinformatics Analysis of JcERFVII Genes*

Previously, we identified three *JcERFVIIs*, namely *JcERVII1* (*Jcr4S00420.40*), *JcERFVII2* (*Jcr4S00982.160*), and *JcERFVII3* (*Jcr4S01651.60*), from the Jatropha genome [29]. All three *JcERFVIIs* possess a conserved N-degron signal [NH2-MCGGAII(A/S)D] [29]. The full-length open reading frames (ORFs) of *JcERFVIIs* were cloned. Sequence analysis reveals that *JcERFVII1*, *JcERFVII2*, and *JcERFVII3* are composed of 1158, 762, and 945 nucleotides, respectively (Supplementary Materials Data S1). Deduced amino acid sequences of *JcERFVII1*, *JcERFVII2*, and *JcERFVII3* provided encoded proteins with 385, 253, and 314 amino acids with predicted molecular weights of 43, 29, and 36 kD, respectively. The amino acid sequences of the *JcERFVIIs* were aligned with amino acid sequences from all five members of the *Arabidopsis ERFVIIs*, including *RAP2.2*, *RAP2.3*, *RAP2.12*, *HRE1*, and *HRE2*, and the phylogenetic relationship was evaluated. The results revealed that *JcERFVII1* clustered with *RAP2.2* and *RAP2.12*, *JcERFVII2* clustered with *RAP2.3*, and *JcERFVII3* clustered with *HRE2* (Figure 1A). Based on the previously reported Arabidopsis ERFVII protein domain data [5], MEME assisted domain analysis also showed the similarity among each phylogenetic cluster (Figure 1B).

**Figure 1.** Phylogenetic and domain architecture analysis of Jatropha ERFVIIs. (**A**) The phylogenetic tree based on the amino acid sequence of Arabidopsis and Jatropha ERFVIIs. The numbers are bootstrap values after 1000 replicates. Scale bar represents genetic distance. (**B**) Diagram representing domain architecture of Jatropha ERFVIIs following previously published motifs [5].
