Transparent Testa16Plays Multiple Roles in Plant Development and Is Involved in Lipid Synthesis and Embryo Development in Canola    

Abstract

Transparent Testa16 (TT16), a transcript regulator belonging to the B sister MADS box proteins, regulates proper endothelial differentiation and proanthocyanidin accumulation in the seed coat. Our understanding of its other physiological roles, however, is limited. In this study, the physiological and developmental roles of TT16 in an important oil crop, canola (Brassica napus), were dissected by a loss-of-function approach. RNA interference (RNAi)-mediated down-regulation of tt16 in canola caused dwarf phenotypes with a decrease in the number of inflorescences, flowers, siliques, and seeds. Fluorescence microscopy revealed that tt16 deficiency affects pollen tube guidance, resulting in reduced fertility and negatively impacting embryo and seed development. Moreover, Bntt16 RNAi plants had reduced oil content and altered fatty acid composition. Transmission electron microscopy showed that the seeds of the RNAi plants had fewer oil bodies than the nontransgenic plants. In addition, tt16 RNAi transgenic lines were more sensitive to auxin. Further analysis by microarray showed that tt16 downregulation alters the expression of genes involved in gynoecium and embryo development, lipid metabolism, auxin transport, and signal transduction. The broad regulatory function of TT16 at the transcriptional level may explain the altered phenotypes observed in the transgenic lines. Overall, the results uncovered important biological roles of TT16 in plant development, especially in fatty acid synthesis and embryo development.The plant MADS box family genes, which are named after the characterization of four members in this group, MINICHROMOSOME MAINTENANCE1, AGAMOUS, DEFICIENS and SERUM RESPONSE FACTOR, encode transcription factors that share a common DNA-binding domain (the MADS box) and play multiple roles in flower pattern formation, gametophyte cell division, and fruit wall differentiation (Ng and Yanofsky, 2001;Dinneny and Yanofsky, 2005;Colombo et al., 2008).A number of MADS domain proteins from vascular plants share a conserved structural organization, the so-called MIKC-type domain structure, where the MADS (M) domain is followed by an Intervening (I), a Keratin-like (K) and a C-terminal domain (Theissen et al., 1996). MIKC-type MADS box genes are involved in important aspects of plant reproductive development, such as flower initiation, specification of floral meristem and organ identity, and ovule and fruit development (Becker and Theissen, 2003).MADS box genes are the major members of plant floral organ identity genes, which have been divided into five classes according to the ABCDE model (Theissen, 2001;Krizek and Fletcher, 2005). The ABCDE model explains flower formation by the interaction of five classes of homeotic genes (A-E), with A controlling sepal, A+B+E controlling petal, B+C+E controlling stamen, C+E controlling carpel, and D controlling ovule development (Theissen, 2001;Krizek and Fletcher, 2005). Therefore, MADS box genes appear to have a central role in flower development. The functions of floral organ ...

Brassica napus TT16 homologs with different genomic origins and expression levels encode proteins that regulate a broad range of endothelium‐associated genes at the transcriptional level

“…To further explore the physiological functions of the BnTT16 homologs with regards to seed coat development, we analyzed the seed coat morphology of Bntt16 RNA interference (RNAi) B . napus lines, which were generated using a hairpin RNAi construct and previously confirmed to down‐regulate the expression of all four BnTT16 genes simultaneously (Deng et al ., ; Figures S4 and S5). In B. napus , the inner integument comprises the PA‐accumulating endothelium (ii1) and parenchyma (ii2), while the outer integument is composed of epidermis (oi3), parenchyma (oi2) and palisade (oi1) layers (Nesi et al ., ; Auger et al ., ).…”

“…In addition, its function in the development of other plant organs has also been reported. While the down‐regulation of this transcription factor in B. napus caused abnormal embryo development and a reduction in silique and seed numbers (Deng et al ., ), a double knock‐out mutant of the MADS‐box genes TT16 and SEEDSTICK in Arabidopsis resulted in a complete lack of endothelium and subsequent defects in the female gametophyte, reduced fertilization, impediments during seed formation and a severely reduced seed set (Mizzotti et al ., ). Since the endothelium plays a fundamental role in the interaction between the maternally derived integuments and the next generation, functioning to protect and nourish the embryo, it seems likely that the reduction or absence of an endothelium may be largely responsible for these phenotypes (Stadler et al ., ; Morley‐Smith et al ., ; Mizzotti et al ., ).…”

“…Although the four BnTT16 homologs have previously been found to play broad roles in regulating plant development (Deng et al ., ), their function in seed coat development has not been fully characterized and it remains unclear whether all of the homologs are functional in B. napus . The objective of this study, therefore, was to: (i) characterize the expression patterns and genome origins of BnTT16 homologs from B. napus , as well as their role in endothelial development, PA biosynthesis and PA polymerization; and (ii) identify their regulatory function with regard to genes involved in endothelial development and PA biosynthesis at different seed developmental stages and in distinct subseed components.…”

A lipid transfer protein, OsLTPL36, is essential for seed development and seed quality in rice

“…Most of the mineral nutrients were transferred into seed through seed coat, and were further delivered from endosperm to embryo [36]. It was reported that decreased lipid content accompanied abnormal embryo development in canola rape [37]. In Arabidopsis, the total lipid content of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 18 the kasI seeds was decreased to 33.6% of the WT which led to defective embryo development [38].…”

Genome-Wide Identification, Localization, and Expression Analysis of Proanthocyanidin-Associated Genes in Brassica

“…In Brassica species homologous cloning is used to isolate PA genes by such as DFR / TT3 (Yan et al, 2008; Akhov et al, 2009), ANS/TT18 (Yan et al, 2011), ANR/BAN (Nesi et al, 2009), TT10 (Zhang et al, 2013), TT2 (Wei et al, 2007), TT8 (Padmaja et al, 2014), TT12 (Chai et al, 2009), TT16 (Deng et al, 2012; Chen et al, 2013), TTG1 (Zhang et al, 2009; Yan et al, 2014) and TTG2 (Li et al, 2015). However, homologous cloning has drawbacks.…”