(1) LHT1/MAC7 contributes to proper alternative splicing under long-term heat stress and mediates variation in the heat tolerance of Arabidopsis
Natural genetic variation has facilitated the identification of genes underlying complex traits such as stress tolerances. We here evaluated the long-term (L-) heat tolerance (37°C for 5 days) of 174 Arabidopsis thaliana accessions and short-term (S-) heat tolerance (42°C, 50 min) of 88 accessions and found extensive variation, respectively. Interestingly, L-heat–tolerant accessions are not necessarily S-heat tolerant, suggesting that the tolerance mechanisms are different. To elucidate the mechanisms underlying the variation, we performed a chromosomal mapping using the F2 progeny of a cross between Ms-0 (a hypersensitive accession) and Col-0 (a tolerant accession) and found a single locus responsible for the difference in L-heat tolerance between them, which we named Long-term Heat Tolerance 1 (LHT1). LHT1 is identical to MAC7, which encodes a putative RNA helicase involved in mRNA splicing as a component of the MOS4 complex. We found one amino acid deletion in LHT1 of Ms-0 that causes a loss of function. Arabidopsis mutants of other core components of the MOS4 complex—mos4-2, cdc5-1, mac3a mac3b, and prl1 prl2—also showed hypersensitivity to L-heat stress, suggesting that the MOS4 complex plays an important role in L-heat stress responses. L-heat stress induced mRNA processing–related genes and compromised alternative splicing. Loss of LHT1 function caused genome-wide detrimental splicing events, which are thought to produce nonfunctional mRNAs that include retained introns under L-heat stress. These findings suggest that maintaining proper alternative splicing under L-heat stress is important in the heat tolerance of A. thaliana.
(2) MOS4-associated complex contributes to proper splicing and suppression of ER stress under long-term heat stress in Arabidopsis
Plants are often exposed not only to short-term (S-) but also to long-term (L-)heat stress over several consecutive days. A few Arabidopsis mutants defective in L-heat tolerance have been identified, but the molecular mechanisms are less understood for this tolerance than for S-heat stress tolerance. To elucidate the mechanisms of the former, we used a forward genetic screen for sensitive to long-term heat (sloh) mutants and isolated sloh3 and sloh63. The mutants were hypersensitive to L- but not to S-heat stress, and sloh63 was also hypersensitive to salt stress. We identified the causal genes, SLOH3 and SLOH63, both of which encoded splicing-related components of the MOS4-associated complex (MAC). This complex is widely conserved in eukaryotes and has been suggested to interact with spliceosomes. Both genes were induced by L-heat stress in a time-dependent manner, and some abnormal splicing events were observed in both mutants under L-heat stress. In addition, endoplasmic reticulum (ER) stress and subsequent unfolded protein response occurred in both mutants under L-heat stress and were especially prominent in sloh63, suggesting that enhanced ER stress is due to the salt hypersensitivity of sloh63. Splicing inhibitor pladienolide B led to concentration-dependent disturbance of splicing, decreased L-heat tolerance, and enhanced ER stress. These findings suggest that maintenance of precise mRNA splicing under L-heat stress by the MAC is important for L-heat tolerance and suppressing ER stress in Arabidopsis.