Transcriptomic analysis of salt stress responsive genes in Rhazya stricta

Abstract

Rhazya stricta is an evergreen shrub that is widely distributed across Western and South Asia, and like many other members of the Apocynaceae produces monoterpene indole alkaloids that have anti-cancer properties. This species is adapted to very harsh desert conditions making it an excellent system for studying tolerance to high temperatures and salinity. RNA-Seq analysis was performed on R. stricta exposed to severe salt stress (500 mM NaCl) across four time intervals (0, 2, 12 and 24 h) to examine mechanisms of salt tolerance. A large number of transcripts including genes encoding tetrapyrroles and pentatricopeptide repeat (PPR) proteins were regulated only after 12 h of stress of seedlings grown in controlled greenhouse conditions. Mechanisms of salt tolerance in R. stricta may involve the upregulation of genes encoding chaperone protein Dnaj6, UDP-glucosyl transferase 85a2, protein transparent testa 12 and respiratory burst oxidase homolog protein b. Many of the highly-expressed genes act on protecting protein folding during salt stress and the production of flavonoids, key secondary metabolites in stress tolerance. Other regulated genes encode enzymes in the porphyrin and chlorophyll metabolic pathway with important roles during plant growth, photosynthesis, hormone signaling and abiotic responses. Heme biosynthesis in R. stricta leaves might add to the level of salt stress tolerance by maintaining appropriate levels of photosynthesis and normal plant growth as well as by the participation in reactive oxygen species (ROS) production under stress. We speculate that the high expression levels of PPR genes may be dependent on expression levels of their targeted editing genes. Although the results of PPR gene family indicated regulation of a large number of transcripts under salt stress, PPR actions were independent of the salt stress because their RNA editing patterns were unchanged.

Fig 1. Hierarchical cluster analysis of gene expression based on log ratio RPKM data for leaf transcriptome of R. stricta under water and salt (500 mM NaCl) treatments for 0, 2, 12 and 24 h.

Fig 2. Gene ontology analysis describing the three main categories “biological process”, “molecular function” and “cellular component” for the 10 functional groups with the largest number of upregulated or downregulated transcripts under salt stress.

Fig 3. Profiles of fold change values for six transcripts (alaD, pbgD, cpo, ppx, chlD and fch) encoding enzymes in the porphyrin and chlorophyll metabolism pathway in leaves of R. stricta under water and salt stress treatments for 0, 2 12 and 24 h. 0h1,2 (control), 2h-w1,2 (water treated for 2 h), 12h-w1,2,3 (water treated for 12 h), 24h-w1 (water treated for 24 h), 2h-s1,2,3 (salt stressed for 2 h), 12h-s1,2,3 (salt stressed for 12 h), 24h-s1,2,3 (salt stressed for 24 h).

Information about the different genes is shown in S1 and S5 Tables.

Fig 4. Profiles of fold change values for 25 selected R. stricta PPR leaf transcripts under water and salt stress treatments for 0, 2, 12 and 24 h.

0h1,2 (control), 2h-w1,2 (water treated for 2 h), 12h-w1,2,3 (water treated for 12 h), 24h-w1 (water treated for 24 h), 2h-s1,2,3 (salt stressed for 2 h), 12h-s1,2,3 (salt stressed for 12 h), 24h-s1,2,3 (salt stressed for 24 h). Information about the different genes is shown in S1, S2 and S5 Tables.

Fig 5. Profiles of fold change values for four selected R. stricta leaf transcripts, three of which are PPR-edited under water and salt stress treatments for 0, 2, 12 and 24 h.

Three transcripts are chloroplast (ndhF, petB and clpP), while one is mitochondrial (nad4). 0h1,2 (control), 2h-w1,2 (water treated for 2 h), 12h-w1,2,3 (water treated for 12 h), 24h-w1 (water treated for 24 h), 2h-s1,2,3 (salt stressed for 2 h), 12h-s1,2,3 (salt stressed for 12 h), 24h-s1,2,3 (salt stressed for 24 h). Information about the different genes is shown in S1 and S5 Tables.