1. Introduction and History

Water deficits caused by osmotic stress, drought, and salt result in the accumulation of reactive oxygen species (ROS), which impairs the function of biochemical processes, damages organelles, and ultimately results in cell death in stressed plants ^[1]. We have previously shown that seedlings of a non-secretor mangrove, K. candel, possess an efficient oxygen scavenging system against ROS under NaCl stress ^[2][3][4]. K. candel increased transcription of CSD gene encoding a Cu/Zn superoxide dismutase to reduce ROS in the chloroplast in a long-term and high saline environment ^[2]. Moreover, salt treatment increased transcription of a f-type thioredoxin (Trx) in K. candel ^[3]. Being as small and ubiquitous proteins (12-14 kD) with the conserved redox-active site (WCXPC), Trxs serve as a crucial important redox regulators in higher plants ^[5][6]. These proteins are able to catalyze the reduction of disulfide bonds in many target proteins to regulate their structure and function ^[5][6]. We have shown that KcTrxf-transgenic plants could scavenge the salt-elicited ROS in leaf cells through the up-regulation of catalase and ascorbate peroxidase and increased the activities of MDAR (monodehydroascorbate reductase) and GR (glutathione reductase) in chloroplast AsA-GSH cycle, leading to an increase in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) and non-protein thiols (NPTs) in the leaves ^[3]. Antioxidative systems also play an important role in the defense against negative consequences of drought stress ^[7]. However, the regulatory roles of K. candel Trx family genes in osmotic and drought tolerance are not yet fully understood.

2. Development

We attempted to explore the role of KcTrxf in plant adapting to water limited environments. Increasing concentration of mannitol resulted in elevated expression of thioredoxin f (KcTrxf) in Kandelia candel. This suggests that KcTrxf may contribute to osmotic tolerance. By means of amino acid sequence and phylogenetic analysis, the mangrove Trx was classified as an f-type thioredoxin. Subcellular localization showed that KcTrxf localized to chloroplast. Trx activity was analyzed using purified recombinant KcTrxf protein. Enzymatic activity characterization revealed that KcTrxf possessed the disulfide reductase function. KcTrxf overexpression contributed to osmotic and drought tolerance in tobacco in terms of fresh weight, root length, malondialdehyde content, and H₂O₂ production. KcTrxf was shown to reduce stomatal aperture by enhancing K⁺ efflux in guard cells, which increased water retaining capacity in leaves under drought. Noteworthy, the abscisic acid (ABA) sensitivity was increased in KcTrxf-transgenic tobacco, which benefits the droughted plants to reduce water loss by promoting stomatal closure. KcTrxf-transgenic plants limited the drought-induced H₂O₂ in leaves, which could reduce lipid peroxidation and retain the membrane integrity. Noteworthy, KcTrxf-transgenic plants increased glucose and fructose under drought stress, presumably resulting from the KcTrxf-promoted starch degradation under water stress. We conclude that KcTrxf contributed to drought tolerance by increasing water status, enhancing osmotic adjustment, and maintaining reactive oxygen species homeostasis in transgene plants.

Our findings demonstrated the role of K. candel Trxf in the osmotic and drought tolerance. In particular, the following findings add new to our knowledge of mechanisms contributing to drought tolerance in higher plants, in brief,

1. KcTrxf contains a redox-active dithiol in the active site and serves as a redox regulator ^[5][6]. Therefore, KcTrxf might directly participate in the control of ROS under drought conditions.
2. KcTrxf contributes to regulating stomatal aperture by mediating the K⁺ flow through plasma membrane in guard cell. This enabled the water-stressed plants to maintain water status, thus limiting the ROS production under drought stress ^[1].
3. KcTrxf could modify the osmolytes for osmotic adjustment to deal with drought stress ^[8][9]. KcTrxf increased glucose and fructose by enhancing starch degradation. Similarly, Trx f1-regulated b-amylase triggers diurnal starch degradation in Arabidopsis guard cells, and in mesophyll cells under osmotic stress ^[10]