We examined the substrate preference of Cuphea paucipetala acyl-ACP thioesterases, CpFatB4 and CpFatB5, and gene expression changes associated with the modification of lipid composition in the seed, using Brassica napus transgenic plants overexpressing CpFatB4 or CpFatB5 under the control of a seed-specific promoter. CpFatB4 seeds contained a higher level of total saturated fatty acid (FA) content, with 4.3 times increase in 16:0 palmitic acid, whereas CpFatB5 seeds showed approximately 3% accumulation of 10:0 and 12:0 medium-chain FAs, and a small increase in other saturated FAs, resulting in higher levels of total saturated FAs. RNA-Seq analysis using entire developing pods at 8, 25, and 45 days after flowering (DAF) showed up-regulation of genes for β-ketoacyl-acyl carrier protein synthase I/II, stearoyl-ACP desaturase, oleate desaturase, and linoleate desaturase, which could increase unsaturated FAs and possibly compensate for the increase in 16:0 palmitic acid at 45 DAF in CpFatB4 transgenic plants. In CpFatB5 transgenic plants, many putative chloroplast- or mitochondria-encoded genes were identified as differentially expressed. Our results report comprehensive gene expression changes induced by alterations of seed FA composition and reveal potential targets for further genetic modifications.

Cuphea viscosissima plants accumulate mediumchain fatty acids (MCFAs), i.e., those containing 8~14 carbons, in their seeds, in addition to the longer carbon chain fatty acids (≥16 carbons) found in a variety of plant species. Previous studies have reported the existence of three C. viscosissima MCFA-producing acyl-acyl carrier protein (ACP) thioesterases with different substrate specificities. In this study, CvFatB4, a novel cDNA clone encoding an acyl-ACP thioesterase (EC 3.1.2.14), was isolated from developing C. viscosissima seeds. Sequence alignment of the deduced amino acid sequence revealed that four catalytic residues for thioesterase activity are conserved and a putative N-terminal chloroplast transit peptide is present. Overexpression of CvFatB4 cDNA, which was under the control of the cauliflower mosaic virus 35S promoter, in Arabidopsis thaliana led to an increase in 16:0 fatty acid (palmitate) levels in the seed oil at the expense of 18:1 and other non-MCFAs.

Toll/interleukin -1 receptor (TIR) domains, which have NAD+ cleavage activity, are used as signaling modules in NOD-like receptors for defense responses. It has been shown that TIR domains not only form homo- or heterodimers with TIR domain-containing proteins but also interact with various proteins. A previous study showed that overexpression of Arabidopsis thaliana (Arabidopsis) AtTX14, encoding an N-terminal TIR domain and a C-terminal domain with unknown function, resulted in dwarfism and constitutive defense signaling or autoimmunity. Transgenic Arabidopsis overexpressing AtTX14 displays enhanced defense responses and associated dwarf phenotypes at 28 °C compared with those at 22 °C, which differs from other mutant or transgenic Arabidopsis with constitutive defense responses. We found that AtTX14 is alternatively spliced to encode three different proteins, and the TIR domain itself can induce autoimmunity and elevated defense responses to the bacterial pathogen Pseudomonas syringae pv. tomato. In addition, we revealed that the transcription of AtTX14 is regulated by a positive feedback mechanism. With transient overexpression of three AtTX14 protein forms in tobacco leaves, providing a heterologous system free from the positive feedback of AtTX14 in Arabidopsis, we demonstrated that expression of a splicing variant encoding the TIR domain-only protein is sufficient to activate defense signaling. A deeper understanding of interaction networks involving AtTX14 will broaden our knowledge on how plant defense signaling is regulated in response to pathogen infection and ambient temperature changes.

Brassinosteroids (BRs) are essential plant steroid hormones required for cell elongation, plant growth, development and abiotic and biotic stress tolerance. BRs are recognized by BRI1 receptor kinase that is localized in the plasma membrane, and the BRI1 protein will eventually autophosphorylate in the intracellular domain and transphosphorylate BAK1, which is a co-receptor in Arabidopsis thaliana. However, little is known of the role OsBRI1 receptor kinase plays in Oryza sativa, monocotyledonous plants, compared to that in Arabidopsis thaliana, dicotyledonous plants. As such, we have studied OsBRI1 receptor kinase in vitro and in vivo with recombinant protein and transgenic plants, whose phenotypes were also investigated. A OsBRI1 cytoplasmic domain (CD) recombinant protein was induced in BL21 (DE3) E.coli cells with IPTG, and purified to obtain OsBRI1 recombinant protein. Based on Western blot analysis with phospho-specific pTyr and pThr antibodies, OsBRI1 recombinant protein and OsBRI1-Flag protein were phosphorylated on Threonine residue(s), however, not on Tyrosine residue(s), both in vitro and in vivo. This is particularly intriguing as AtBRI1 protein was phosphorylated on both Ser/Thr and Tyr residues. Also, the OsBRI1 full-length gene was expressed in, and rescued, bri1-5 mutants, such as is seen in normal wild-type plants where AtBRI1-Flag rescues bri1-5 mutant plants. Root growth in seedlings decreased in Ws2, AtBRI1, and 3 independent OsBRI1 transgenic seedlings and had an almost complete lack of response to brassinolide in the bri1-5 mutant. In conclusion, OsBRI1, an orthologous gene of AtBRI1, can mediate normal BR signaling for plant growth and development in Arabidopsis thaliana.

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