# MeNADP-ME3 Confers Salt and Drought Tolerance in Arabidopsis and Drives Functional Diversification of the NADP-ME Family in Cassava

## TL;DR

This study explores how a specific NADP-ME gene in cassava helps the plant tolerate drought and salt stress, offering insights into improving crop resilience.

## Contribution

The study identifies MeNADP-ME3 as a novel gene conferring stress tolerance and driving functional diversification in cassava.

## Key findings

- Arabidopsis overexpressing MeNADP-ME3 showed 30–120% higher antioxidant enzyme activities under stress.
- MeNADP-ME3 is localized in chloroplasts and responds to abiotic stresses like drought and salt.
- Cassava's NADP-ME family shows distinct expression patterns and evolutionary diversification.

## Abstract

As a typical C3-C4 intermediate plant, cassava (Manihot esculenta Crantz) exhibits high photosynthetic efficiency and low photorespiration. NADP-malic enzyme (NADP-ME) is a key enzyme in the C4 photosynthetic pathway that provides elevated CO2 concentrations for Rubisco. However, research on NADP-ME in C3-C4 intermediate species remains limited. In this study, we identified four NADP-ME genes in the cassava genome, with segmental duplication serving as the primary driving force for gene evolution. Cis-acting element analysis indicated potential roles of MeNADP-ME genes in environmental adaptation, stress responses, and growth regulation. Expression profiling using bulk RNA sequencing and single-cell RNA sequencing revealed distinct expression patterns in different tissues and cell subsets. Comparative analysis with Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) NADP-ME families demonstrated that MeNADP-ME3 exhibits bundle sheath cell-specific expression analogous to ZmchlC4NADP-ME in maize. Notably, photosynthetic genes and plasmodesmata (PD)-related genes exhibited high co-expression within mesophyll subcluster 13 and bundle sheath cells, providing molecular evidence for a limited C4 photosynthetic pathway in cassava. Protein–protein interaction predictions implicated MeNADP-ME3 in photosynthetic carbon metabolism and photorespiration regulation. Furthermore, qRT-PCR revealed significant responsiveness of MeNADP-ME3 to various abiotic stresses, and confocal imaging confirmed its chloroplast localization. Functional validation demonstrated that Arabidopsis overexpressing MeNADP-ME3 exhibited 30–120% enhanced antioxidant enzyme activities (SOD, POD, CAT) and 20–32% reduced oxidative damage markers (MDA, H2O2) under drought and salt stresses. These findings reveal the evolutionary trajectory of NADP-ME genes in C3-C4 intermediate species and provide genetic resources for developing stress-tolerant cassava cultivars.