Transcriptional coordination between leaf cell differentiation and chloroplast development established by TCP20 and the subgroup Ib bHLH transcription factors

Abstract

The establishment of the photosynthetic apparatus during chloroplast development creates a high demand for iron as a redox metal. However, iron in too high quantities becomes toxic to the plant, thus plants have evolved a complex network of iron uptake and regulation mechanisms. Here, we examined whether four of the subgroup Ib basic helix-loop-helix transcription factors (bHLH38, bHLH39, bHLH100, bHLH101), previously implicated in iron homeostasis in roots, also play a role in regulating iron metabolism in developing leaves. These transcription factor genes were strongly up-regulated during the transition from cell proliferation to expansion, and thus sink-source transition, in young developing leaves of Arabidopsis thaliana. The four subgroup Ib bHLH genes also showed reduced expression levels in developing leaves of plants treated with norflurazon, indicating their expression was tightly linked to the onset of photosynthetic activity in young leaves. In addition, we provide evidence for a mechanism whereby the transcriptional regulators SAC51 and TCP20 antagonistically regulate the expression of these four subgroup Ib bHLH genes. A loss-of-function mutant analysis also revealed that single mutants of bHLH38, bHLH39, bHLH100, and bHLH101 developed smaller rosettes than wild-type plants in soil. When grown in agar plates with reduced iron concentration, triple bhlh39 bhlh100 bhlh101 mutant plants were smaller than wild-type plants. However, measurements of the iron content in single and multiple subgroup Ib bHLH genes, as well as transcript profiling of iron response genes during early leaf development, do not support a role for bHLH38, bHLH39, bHLH100, and bHLH101 in iron homeostasis during early leaf development.

Molecular mechanisms governing Arabidopsis iron uptake

“…To exert its function, FIT can form heterodimers with one of the four subgroup Ib bHLH proteins bHLH038, bHLH039, bHLH100, or bHLH101 [27,28] (Figure 1). The genes encoding these bHLH proteins are induced by Fe deficiency; however, unlike FIT, they are also strongly expressed in leaves, where they have Fe-deficiency-and leaf-development-related functions [30][31][32]. Analyses of overexpressing lines have shown that FIT collaborates with at least bHLH038 and bHLH039 to upregulate Fe-uptake genes [28].…”

Forever Young: The Role of Ubiquitin Receptor DA1 and E3 Ligase BIG BROTHER in Controlling Leaf Growth and Development

“…3B). BHLH038 and BHLH100 have previously been shown to be induced when proliferating leaf cells stop dividing and start to expand (Andriankaja et al, 2012(Andriankaja et al, , 2014. These observations at the transcriptome level confirm the developmental shift in da1-1, bb/eod1-2, and da1-1_bb/eod1-2 compared with Col-0, showing that despite their larger size, leaves of the single mutants and especially da1-1_bb/eod1-2 were still in a younger developmental stage than those of Col-0.…”

“…Genes promoting cell division in leaves, such as AN3 and ANT (Horiguchi et al, 2005;Mizukami and Fischer, 2000) and the mitotic marker CYCB1-1, which is highly expressed in proliferating leaves (Donnelly et al, 1999), were expressed more highly in da1-1, bb/eod1-2, and da1-1_bb/eod1-2 than in Col-0. Genes that are up-regulated during this transition, such as the negative regulator of cell division, KRP1, and two iron deficiency-responsive BHLH transcription factor genes, BHLH038 and BHLH100, that are linked to the sink/source transition in leaves (Andriankaja et al, 2012(Andriankaja et al, , 2014, were less expressed in these mutants. In a complementary fashion, 24 h after artificial induction of untagged DA1 and BB expression, downstream transcriptional responses related to the transition from cell proliferation to cell expansion, such as the up-regulation of BHLH038, BHLH039, BHLH100, and BHLH101 (Andriankaja et al, 2012(Andriankaja et al, , 2014, were observed.…”

“…Genes that are up-regulated during this transition, such as the negative regulator of cell division, KRP1, and two iron deficiency-responsive BHLH transcription factor genes, BHLH038 and BHLH100, that are linked to the sink/source transition in leaves (Andriankaja et al, 2012(Andriankaja et al, , 2014, were less expressed in these mutants. In a complementary fashion, 24 h after artificial induction of untagged DA1 and BB expression, downstream transcriptional responses related to the transition from cell proliferation to cell expansion, such as the up-regulation of BHLH038, BHLH039, BHLH100, and BHLH101 (Andriankaja et al, 2012(Andriankaja et al, , 2014, were observed. Additionally, induction of BB resulted in the down-regulation of several mitotic cell cycle genes and the increase of transcript levels of negative regulators of the cell cycle and promoters of endoreduplication.…”

Mechanistic dissection of plant embryo initiation

“…While a post-embryonic function has been described for bHLH63, also known as CRYPTOCHROME-INTERACTING bHLH1 (CIB1; Liu et al, 2008) and bHLH100 (Andriankaja et al, 2014;Sivitz et al, 2012;Wang et al, 2007), no embryonic function has been reported for any of these genes. To determine whether these four bHLH genes are involved in S>E transformation, we first studied their embryonic expression domains in detail.…”