Overexpressing Jatropha curcas CBF2 in Nicotiana benthamiana improved plant tolerance to drought stress

Highlights:

• JcCBF2 positively modulated the physiological response to drought.

• JcCBF2 overexpression decreased leaf area and increased leaf thickness.

• JcCBF2 increased the accumulation of CTK, IAA, ABA and JA significantly.

• Hormone related genes were enriched by RNA sequencing analysis.

• JcCBF2 increased the transcription level of MYB transcription factors.

Abstract

Jatropha curcas is an important bioenergy oil plant, and often planted on barren land to save the area of arable land. It is significant to improve the adaptability of J. curcas to various abiotic stresses. In the present study, we transferred a J. curcas gene, encoding a CBF2 transcription factor, into Nicotiana benthamiana. Under drought treatment, the JcCBF2 transgenic lines showed improved survival rate, leaf water retention and active oxygen scavenging capacity, but reduced photosynthesis and transpiration rate, suggesting that JcCBF2 played an important role in improving plant drought tolerance. Overexpressing JcCBF2 decreased leaf area and increased leaf thickness. To explore the possible mechanisms for the change of leaf anatomical structure, the leaves of wild-type and overexpression lines under drought stress were RNA sequenced. Genes involved in the plant hormones signal transduction were found to be enriched. Cytokinin and indole-3-acetic acid were the major plant hormones whose abundance increased. Quantitative RT-PCR analysis showed expression of NbMYB21, NbMYB86 and NbMYB44 and both abscisic acid (ABA) and jasmonic acid (JA) related genes in the overexpression lines were increased under drought stress. These results indicated that JcCBF2 was able to positively regulate plant drought response by changing the leaf anatomical structure and possibly through JA and ABA signalling pathways. Our work may help us to understand the drought tolerant mechanism.

Introduction

Jatropha curcas, with origin in Central and South America, has received much attention worldwide as a resource of green fuel and renewable energy. The seeds of J. curcas contain 37% oil and are used to produce biodiesel (Fairless, 2007), but also used for soap production, drug extraction and feed fermentation (Kumar and Sharma, 2008). To preserve the land for food crop production, J. curcas is usually introduced to wastelands or degraded lands (Achten et al., 2010b). However, poor soil conditions reduce seedling growing rate and seed oil content of J. curcas (Achten et al., 2010a), and improving stress-tolerance of this species will help to expand planting areas and increase the yield in poor lands.

J. curcas is exposed to a variety of abiotic stresses during its life cycle. In addition to chilling, drought is an important environmental stress factor, which restricts growth and productivity of J. curcas in poor soils. Long-term severe drought stress promotes the accumulation of reactive oxygen species (ROS) and decreases the rate of photosynthesis (Reddy et al., 2004). Plants can sense drought quickly, and respond to it by regulating various physiological mechanisms, such as relative water content, ROS scavenging, photosynthesis, transpiration, and hormone metabolism. Plant hormone signals, such as abscisic acid (ABA) and jasmonic acid (JA), play indispensable roles in plant response to drought tolerance (Kaur et al., 2016, Liu and Avramova, 2016). In addition, overexpressing downstream transcription factors (NAC, MYB, C2H2, DREB, WRKY, bHLH) can also improve plant drought resistance (Wani et al., 2018).

C-repeat-binding factor/dehydration-responsive element-binding factor (CBF/DREB) is widely considered a key regulator of plant abiotic stress response (Yang et al., 2017). CBF belongs to a subfamily of the AP2/ERF transcription factor family. The CBF protein usually contains an AP2 DNA-binding domain and “CBF signature motifs” (Gamboa et al., 2007). Elevated CBF expression levels improve significantly stress tolerance in plant. Thus, JcDREB expression is induced by drought, salt, and cold stress, and its overexpression in Arabidopsis enhances stress tolerance (Tang et al., 2011). Overexpression of ScCBF1 in Arabidopsis improves its tolerance to freezing and drought (Li et al., 2018). The analysis of DREB/CBF gene expression patterns in grape leaves and roots showed that CBF4 is more responsive to drought stress in both leaf and root tissues when compared with the expressions of CBF2 and CBF3, and it is upregulated in response to drought stress (Zandkarimi et al., 2015). Although CBF is known to be associated with stress resistance, it also affects plant development. For example, compared with wild type (wt), root growth rate and the number of rosette leaves were reduced in Arabidopsis cbf1cbf2cbf3 mutant (Zhao et al., 2016). Additionally, overexpression of CBF often results in dwarfism, delayed flowering, and formation of small and thick leaves formation (Sharabi-schwager et al., 2010, Jia et al., 2016). CBF also participates in hormone metabolism and signal transduction. ABA activates CBF expression by interacting with C-repeat cis-acting elements of the CBF promoter (Heather et al., 2004). Jasmonate ZIM-domain (JAZ) interacts with inducer of CBF expression 1 (ICE1) and ICE2 to suppress ICE (a CBF upstream control element) expression. Therefore, increased methyl jasmonate content will remove the inhibitory effect of JAZ on ICE-CBF and improve CBF gene expression (Yanru et al., 2013).

CBF2, belonging to the CBF/DREB gene family, plays an important role in improving plant tolerance to abiotic stress (including cold and drought). AmCBF2 (a gene from mangrove tree Avicennia marina) was induced by drought and ABA, whereas, AmCBF1 and AmCBF3 exhibited insignificant changes under drought stress (Peng et al., 2013). Besides, constitutive expression of MrCBF2 enhanced phytohormone ABA-independent drought tolerance in transgenic Arabidopsis (Wu et al., 2017). CBF2 expression also delays leaf senescence and extends the life span in overexpressing Arabidopsis under methyl jasmonate treatment (Sharabi-Schwager et al., 2010).

A gene with pleiotropic effect against abiotic stress is potentially a good candidate gene for genetic engineering. In a previous study, we demonstrated that JcCBF2 gene improves freezing tolerance of Arabidopsis during the early stage of stress (Wang et al., 2014). However, the effect of this gene on drought stress response has not been elucidated. In the present study, CBF2 was cloned from J. curcas and its putative role on drought stresses was conducted in transgenic tobacco. Assessment of drought response was conducted by measuring physiological responses and morphological parameters. RNA sequencing was used to explore the mechanisms that might be involved in regulating the leaf development. To understand the molecular basis of the enhanced drought tolerance, we analyzed the expression of hormone-related genes.