A double-negative feedback loop between miR319c and JAW-TCPs establishes growth pattern in incipient leaf primordia in Arabidopsis thaliana.

The microRNA miR319 and its target JAW-TCP transcription factors regulate the proliferation-to-differentiation transition of leaf pavement cells in diverse plant species. In young Arabidopsis leaf primordia, JAW-TCPs are detected towards the distal region whereas the major mRNA319-encoding gene MIR319C, is expressed at the base. Little is known about how this complementary expression pattern of MIR319C and JAW-TCPs is generated. Here, we show that MIR319C is initially expressed uniformly throughout the incipient primordia and is later abruptly down-regulated at the distal region, with concomitant distal appearance of JAW-TCPs, when leaves grow to ~100 µm long. Loss of JAW-TCPs causes distal extension of the MIR319C expression domain, whereas ectopic TCP activity restricts MIR319C more proximally. JAW-TCPs are recruited to and are capable of depositing histone H3K27me3 repressive marks on the MIR319C chromatin. JAW-TCPs fail to repress MIR319C in transgenic seedlings where the TCP-binding cis-elements on MIR319C are mutated, causing miR319 gain-of-function-like phenotype in the embryonic leaves. Based on these results, we propose a model for growth patterning in leaf primordia wherein MIR319C and JAW-TCPs repress each other and divide the uniformly growing primordia into distal differentiation zone and proximal proliferation domain.

The TCP4 Transcription Factor Directly Activates TRICHOMELESS1 and 2 and Suppresses Trichome Initiation.

Trichomes are the first line of defense on the outer surface of plants against biotic and abiotic stresses. Because trichomes on leaf surfaces originate from the common epidermal progenitor cells that also give rise to pavement cells and stomata, their density and distribution are under strict genetic control. Regulators of trichome initiation have been identified and incorporated into a biochemical pathway wherein an initiator complex promotes trichome fate in an epidermal progenitor cell, while an inhibitor complex suppresses it in the neighboring cells. However, it is unclear how these regulator proteins, especially the negative regulators, are induced by upstream transcription factors and integrated with leaf morphogenesis. Here, we show that the Arabidopsis (Arabidopsis thaliana) class II TCP proteins activate TRICHOMELESS1 (TCL1) and TCL2, the two established negative regulators of trichome initiation, and reduce trichome density on leaves. Loss-of-function of these TCP proteins increased trichome density whereas TCP4 gain-of-function reduced trichome number. TCP4 binds to the upstream regulatory elements of both TCL1 and TCL 2 and directly promotes their transcription. Further, the TCP-induced trichome suppression is independent of the SQUAMOSA PROMOTER BINDING PROTEIN LIKE family of transcription factors, proteins that also reduce trichome density at later stages of plant development. Our work demonstrates that the class II TCP proteins couple leaf morphogenesis with epidermal cell fate determination.

Generation of Inducible Transgenic Lines of Arabidopsis Transcription Factors Regulated by MicroRNAs.

Transcription factors play key regulatory roles in all the life processes across kingdoms. In plants, the genome of a typical model species such as Arabidopsis thaliana encodes over 1500 transcription factors that regulate the expression dynamics of all the genes in time and space. Therefore, studying their function by analyzing the loss and gain-of-function lines is of prime importance in basic plant biology and its agricultural application. However, the current approach of knocking out genes often causes embryonic lethal phenotype, while inactivating one or two members of a redundant gene family yields little phenotypic changes, thereby making the functional analysis a technically challenging task. In such cases, inducible knock-down or overexpression of transcription factors appears to be a more effective approach. Restricting the transcription factors in the cytoplasm by fusing them with animal glucocorticoid/estrogen receptors (GR/ER) and then re-localizing them to the nucleus by external application of animal hormone analogues has been a useful method of gene function analysis in the model plants. In this chapter, we describe the recent advancements in the GR and ER expression systems and their use in analyzing the function of transcription factors in Arabidopsis.