Genome-wide identification and expression analysis of ammonium transporter 1 (AMT1) gene family in cassava (Manihot esculenta Crantz) and functional analysis of MeAMT1;1 in transgenic Arabidopsis.

Nitrogen (N), a fundamental macronutrient for plant growth and development, is absorbed from the soil primarily in the form of ammonium (NH4 +) and uptaken through a plant's ammonium transporters (AMTs). While AMT proteins have been documented within diverse plant taxa, there has been no systematic analysis of their activity in cassava (Manihot esculenta Crantz), which is highly resistant to nitrogen deficiency. Here, we perform a comprehensive genome-wide analysis to identify and characterize the functional dynamics of cassava ammonium transporters 1 (MeAMT1). We identified a total of six AMT1 genes in the cassava genome (MeAMT1;1 to MeAMT1;6), the phylogenetic analysis of which fell into three distinct subgroups based on the conserved motifs and gene structures. Collinearity analysis showed that segmental duplication events played a key role in expansion of the MeAMT1 gene family. Synteny analysis indicated that two MeAMT1 genes were orthologous to Arabidopsis and rice. MeAMT1 promoters were additionally found to include various cis-acting elements related to light responsiveness, hormones, stress, and development processes. According to the RNA-seq data, the majority of MeAMT1 genes displayed specific patterns in the tested tissues. qRT-PCR revealed that all the tested MeAMT1 genes were up-regulated by low ammonium exposure. Furthermore, Arabidopis transformed with MeAMT1;1 gene grew well than wild-type plants in response to ammonium deficiency, suggesting that MeAMT1s play important role in response to low ammonium. Overall, our work lays the groundwork for new understanding of the AMT1 gene family in cassava and provides a basis for breeding efficient nitrogen use in other plants. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03070-6.

The protein kinase complex CBL10-CIPK8-SOS1 functions in Arabidopsis to regulate salt tolerance.

Salt tolerance in plants is mediated by Na+ extrusion from the cytosol by the plasma membrane Na+/H+ antiporter SOS1. This is activated in Arabidopsis root by the protein kinase complex SOS2-SOS3 and in Arabidopsis shoot by the protein kinase complex CBL10-SOS2, with SOS2 as a key node in the two pathways. The sos1 mutant is more sensitive than the sos2 mutant, suggesting that other partners may positively regulate SOS1 activity. Arabidopsis has 26 CIPK family proteins of which CIPK8 is the closest homolog to SOS2. It is hypothesized that CIPK8 can activate Na+ extrusion by SOS1 similarly to SOS2. The plasma membrane Na+/H+ exchange activity of transgenic yeast co-expressing CBL10, CIPK8, and SOS1 was higher than that of untransformed and SOS1 transgenic yeast, resulting in a lower Na+ accumulation and a better growth phenotype under salinity. However, CIPK8 could not interact with SOS3, and the co-expression of SOS3, CIPK8, and SOS1 in yeast did not confer a significant salt tolerance phenotype relative to SOS1 transgenic yeast. Interestingly, cipk8 displayed a slower Na+ efflux, a higher Na+ level, and a more sensitive phenotype than wild-type Arabidopsis, but grew better than sos2 under salinity stress. As expected, sos2cipk8 exhibited a more severe salt damage phenotype relative to cipk8 or sos2. Overexpression of CIPK8 in both cipk8 and sos2cipk8 attenuated the salt sensitivity phenotype. These results suggest that CIPK8-mediated activation of SOS1 is CBL10-dependent and SOS3-independent, indicating that CIPK8 and SOS2 activity in shoots is sufficient for regulating Arabidopsis salt tolerance.

Co-expression of SpSOS1 and SpAHA1 in transgenic Arabidopsis plants improves salinity tolerance.

BACKGROUND: Na+ extrusion from cells is important for plant growth in high saline environments. SOS1 (salt overly sensitive 1), an Na+/H+ antiporter located in the plasma membrane (PM), functions in toxic Na+ extrusion from cells using energy from an electrochemical proton gradient produced by a PM-localized H+-ATPase (AHA). Therefore, SOS1 and AHA are involved in plant adaption to salt stress. RESULTS: In this study, the genes encoding SOS1 and AHA from the halophyte Sesuvium portulacastrum (SpSOS1 and SpAHA1, respectively) were introduced together or singly into Arabidopsis plants. The results indicated that either SpSOS1 or SpAHA1 conferred salt tolerance to transgenic plants and, as expected, Arabidopsis plants expressing both SpSOS1 and SpAHA1 grew better under salt stress than plants expressing only SpSOS1 or SpAHA1. In response to NaCl treatment, Na+ and H+ in the roots of plants transformed with SpSOS1 or SpAHA1 effluxed faster than wild-type (WT) plant roots. Furthermore, roots co-expressing SpSOS1 and SpAHA1 had higher Na+ and H+ efflux rates than single SpSOS1/SpAHA1-expressing transgenic plants, resulting in the former amassing less Na+ than the latter. As seen from comparative analyses of plants exposed to salinity stress, the malondialdehyde (MDA) content was lowest in the co-transgenic SpSOS1 and SpAHA1 plants, but the K+ level was the highest. CONCLUSION: These results suggest SpSOS1 and SpAHA1 coordinate to alleviate salt toxicity by increasing the efficiency of Na+ extrusion to maintain K+ homeostasis and protect the PM from oxidative damage induced by salt stress.

Over-expression of a plasma membrane H+-ATPase SpAHA1 conferred salt tolerance to transgenic Arabidopsis.

The SpAHA1 gene, encoding a plasma membrane (PM) H+-ATPase (AHA) in Sesuvium portulacastrum, was transformed into Arabidopsis plants, and its expression increased salinity tolerance of transgenic Arabidopsis plants: seed germination ratio, root growth, and biomass of transgenic plants were greater compared to wild-type plants under NaCl treatment condition. Upon salinity stress, both Na+ and H+ effluxes in the roots of SpAHA1 expressing plants were faster than those of untransformed plants. Transformed plants with SpAHA1 had lower Na+ and higher K+ contents relative to wild-type plants when treated with NaCl, resulting in greater K+/Na+ ratio in transgenic plants than in wild-type plants under salt stress. Extent of oxidative stress increased in both transgenic and wild-type plants exposed to salinity stress, but overexpression of SpAHA1 could alleviate the accumulation of hydrogen peroxide (H2O2) induced by NaCl treatment in transgenic plants relative to wild-type plants; the content of malondialdehyde (MDA) was lower in transgenic plants than that in wild-type plants under salinity stress. These results suggest that the higher H+-pumping activity generated by SpAHA1 improved the growth of transgenic plants via regulating ion and reactive oxygen species (ROS) homeostasis in plant cells under salinity stress.

Expression Patterns and Identified Protein-Protein Interactions Suggest That Cassava CBL-CIPK Signal Networks Function in Responses to Abiotic Stresses.

Cassava is an energy crop that is tolerant of multiple abiotic stresses. It has been reported that the interaction between Calcineurin B-like (CBL) protein and CBL-interacting protein kinase (CIPK) is implicated in plant development and responses to various stresses. However, little is known about their functions in cassava. Herein, 8 CBL (MeCBL) and 26 CIPK (MeCIPK) genes were isolated from cassava by genome searching and cloning of cDNA sequences of Arabidopsis CBLs and CIPKs. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression levels of MeCBL and MeCIPK genes were different in different tissues throughout the life cycle. The expression patterns of 7 CBL and 26 CIPK genes in response to NaCl, PEG, heat and cold stresses were analyzed by quantitative real-time PCR (qRT-PCR), and it was found that the expression of each was induced by multiple stimuli. Furthermore, we found that many pairs of CBLs and CIPKs could interact with each other via investigating the interactions between 8 CBL and 25 CIPK proteins using a yeast two-hybrid system. Yeast cells co-transformed with cassava MeCIPK24, MeCBL10, and Na+/H+ antiporter MeSOS1 genes exhibited higher salt tolerance compared to those with one or two genes. These results suggest that the cassava CBL-CIPK signal network might play key roles in response to abiotic stresses.