Basic helix-loop-helix transcription factor OsbHLH110 positively regulates abscisic acid biosynthesis and salinity tolerance in rice.

Salinity is a significant abiotic stress factor affecting plant growth, consequently reducing crop yield. Abscisic acid (ABA), a well-known phytohormone, is crucial in conferring resistance to abiotic stress, thus, understanding the mechanisms underlying ABA biosynthesis is crucial. In rice (Oryza sativa L.), OsABA2, a short-chain dehydrogenase protein, has a pivotal role in modulating ABA biosynthesis and salt tolerance by undergoing phosphorylation at Ser197 through mitogen-activated protein kinase OsMPK1. However, the interaction between OsABA2 and other proteins in regulating ABA biosynthesis remains unclear. We employed OsABA2 as a bait in yeast two-hybrid screening: a basic helix-loop-helix transcription factor interacting with OsABA2, named OsbHLH110, was identified. Our results showed that firefly luciferase complementary imaging, pull-down, and co-immunoprecipitation assays validated the interaction between OsbHLH110 and OsABA2, affirming their interaction in vivo and in vitro. Moreover, the expression of OsbHLH110 significantly increases in response to salt and ABA treatments. Additionally, OsbHLH110 can directly bind to the G-box element in the OsABA2 promoter. This binding enhances luciferase activity controlled by the OsABA2 promoter, thereby increasing the expression of the OsABA2 gene and content of the OsABA2 protein, resulting in an increase in ABA content. OsABA2 enhanced the interaction between OsbHLH110 and OsABA2 promoter. This collaborative effect enhanced the regulation of ABA biosynthesis. Subsequent genetic analysis demonstrated that OsbHLH110 improved the tolerance of rice to salt stress.

A J-Protein OsDjC46 Interacts with ZFP36 to Participate in ABA-Mediated Antioxidant Defense in Rice.

ZFP36 has been shown to be involved in ABA-induced antioxidant defense and enhance rice tolerance to drought, salt stress and oxidative stress. Using ZFP36 as bait, a yeast two-hybrid system was used to obtain the interacting protein OsDjC46, which belongs to heat shock protein and usually exists in the form of molecular chaperone, was identified. Further Co-IP (co-immunoprecipitation), BiFC (bimolecular fluorescence complement) and GST (glutathione-S-transferase) pull-down experiments verified that ZFP36 interacted with OsDjC46 in vivo and in vitro. Heat shock protein has been shown to increase plant resistance to stresses, but whether OsDjC46 was a key factor in plant response to various stresses has not been reported. Here, various stimuli, such as abscisic acid (ABA), hydrogen peroxidase (H2O2), polyethylene (PEG) and sodium chloride (NaCl) markedly induced the expression of OsDjC46 in the seedlings. Overexpression of OsDjC46 in rice can enhance the tolerance to salinity and drought; in contrast, knockout of OsDjC46 rice plants was more sensitive to salt stress and drought. Further investigation revealed that OsDjC46 could participate in regulating the expression and activities of antioxidant of SOD and CAT under drought and salt stress. Taken together, these findings reveal a novel function of OsDjC46 in adjusting ABA-induced antioxidant defense.

An Atypical Late Embryogenesis Abundant Protein OsLEA5 Plays a Positive Role in ABA-Induced Antioxidant Defense in Oryza sativa L.

OsLEA5 acts as a co-regulator of a transcriptional fact ZFP36 to enhance the expression and the activity of ascorbate peroxidase OsAPX1 to regulate seed germination in rice, but it it unknown whether OsLEA5 is also crucial in plant seedlings under stress conditions. To determine this, we generated OsLEA5 overexpression and knockdown rice plants. We found that overexpression of OsLEA5 in rice plants enhanced the tolerance to drought and salt stress; in contrast, an RNA interference (RNAi) mutant of OsLEA5 rice plants was more sensitive to drought and salinity. Further investigation found that various stimuli and ABA could induce OsLEA5 expression, and OsLEA5 acted downstream of ZFP36 to be involved in ABA-induced generation of hydrogen peroxide (H2O2), and the regulation of the expression and the activities of antioxidant defense enzymes in plants leaves, and OsLEA5 contributed to stabilize ZFP36. Additionally, OsLEA5 participates in the accumulation of ABA by up-regulating ABA biosynthesis genes and down-regulating ABA metabolism genes. Moreover, we found that two homologs of OsLEA5 (5C700, short for Os05g0526700; and 5C300, short for Os05g0584300) which were induced by ABA also interacted with ZFP36 separately; interestingly, the nuclear-located 5C700 could also act as a co-activator of ZFP36 to modulate OsAPX1, while 5C300 which was down-regulated by ABA induction acted as an ABA-induced inhibitor of ZFP36 to regulate OsAPX1. Hence, our conclusion is that OsLEA5 participates in the ABA-mediated antioxidant defense to function in drought and salt stress response in rice, and the 5C subgroup of LEAs contribute by acting as co-regulators of the transcription factor ZFP36.

The ascorbate peroxidase APX1 is a direct target of a zinc finger transcription factor ZFP36 and a late embryogenesis abundant protein OsLEA5 interacts with ZFP36 to co-regulate OsAPX1 in seed germination in rice.

Seed germination is a vital developmental process. Abscisic acid (ABA) is an essential repressor of seed germination, while ROS (reactive oxygen species) also plays a vital role in regulating seed germination. ABA could inhibit the production of ROS in seed germination, but the mechanism of ABA reduced ROS production in seed germination was hitherto unknown. Here, by ChIP (chromatin immunoprecipitation)-seq, we found that ZFP36, a rice zinc finger transcription factor, could directly bind to the promoter of OsAPX1, coding an ascorbate peroxidase (APX) which has the most affinity for H2O2 (substrate; a type of ROS), and act as a transcriptional activator of OsAPX1 promoter. Moreover, ZFP36 could interact with a late embryogenesis abundant protein OsLEA5 to co-regulate the promoter activity of OsAPX1. The seed germination is highly inhibited in ZFP36 overexpression plants under ABA treatment, while an RNA interference (RNAi) mutant of OsLEA5 rice seeds were less sensitive to ABA, and exogenous ASC (ascorbate acid) could alleviate the inhibition induced by ABA. Thus, our conclusion is that OsAPX1 is a direct target of ZFP36 and OsLEA5 could interact with ZFP36 to co-regulate ABA-inhibited seed germination by controlling the expression of OsAPX1.