Kimberly L. Gallagher

Callose Biosynthesis Regulates Symplastic Trafficking during Root Development

Plant cells are connected through plasmodesmata (PD), membrane-lined channels that allow symplastic movement of molecules between cells. However, little is known about the role of PD-mediated signaling during plant morphogenesis. Here, we describe an Arabidopsis gene, CALS3/GSL12. Gain-of-function mutations in CALS3 result in increased accumulation of callose (β-1,3-glucan) at the PD, a decrease in PD aperture, defects in root development, and reduced intercellular trafficking. Enhancement of CALS3 expression during phloem development suppressed loss-of-function mutations in the phloem abundant callose synthase, CALS7 indicating that CALS3 is a bona fide callose synthase. CALS3 alleles allowed us to spatially and temporally control the PD aperture between plant tissues. Using this tool, we are able to show that movement of the transcription factor SHORT-ROOT and microRNA165 between the stele and the endodermis is PD dependent. Taken together, we conclude that regulated callose biosynthesis at PD is essential for cell signaling.

Mechanisms Regulating SHORT-ROOT Intercellular Movement

Signaling centers within developing organs regulate morphogenesis in both plants and animals. The putative transcription factor SHORT-ROOT (SHR) is an organizing signal regulating the division of specific stem cells in the Arabidopsis root. Comparison of gene transcription with protein localization indicates that SHR moves in a highly specific manner from the cells of the stele in which it is synthesized outward. Here, we provide evidence that SHR intercellular trafficking is both regulated and targeted. First, we show that subcellular localization of SHR in the stele is intrinsic to the SHR protein. Next, we show that SHR must be present in the cytoplasm to move, providing evidence that SHR movement is regulated. Finally, we describe an informative new shr allele, in which the protein is present in the cytoplasm yet does not move. Thus, in contrast to proteins that move by a process resembling diffusion, a cytoplasmic pool of SHR is not sufficient for movement.

Both the conserved GRAS domain and nuclear localization are required for SHORT‐ROOT movement

Transcription factor movement is well established in plants. Since the initial report of KNOTTED movement, more than a dozen transcription factors have been shown to move in plants. However, the developmental significance of movement is not known. Using the SHORT-ROOT (SHR) transcription factor as a tool for studying cell-to-cell trafficking, we show that movement of SHR from its site of synthesis is necessary for normal development of the Arabidopsis root. We identify multiple regions of SHR that are required for intra- and intercellular movement of SHR, including a region that is necessary for movement but not activity. We made the surprising discovery that the capacity for intercellular movement may be conserved among other GRAS family proteins. Finally, we provide evidence that movement requires both cytoplasmic and nuclear localization, strongly suggesting a mechanistic link between nuclear transport and cell-to-cell movement.

An Essential Protein that Interacts with Endosomes and Promotes Movement of the SHORT-ROOT Transcription Factor

Plant cells can communicate through the direct transport of transcription factors [1-7]. One of the best-studied examples of this phenomenon is SHORT-ROOT (SHR), which moves from the stele cells into the endodermis and root tip of Arabidopsis, where it specifies endodermal cell identity and stem cell function, respectively [8-10]. In the endodermis, SHR upregulates the transcription factors SCARECROW (SCR) [2] and JACKDAW (JKD), which in turn inhibit movement of SHR from the endodermis [11]. Although much is known about the regulatory pathways that mediate expression and activity of SHR [1, 8-14], little is known about the factors that promote its movement or the movement of other transcription factors. We have identified a novel protein, SHORT-ROOT INTERACTING EMBRYONIC LETHAL (SIEL), that interacts with SHR, CAPRICE (CPC), TARGET OF MONOPTEROUS 7 (TMO7), and AGAMOUS-LIKE 21 (AGL21). Null alleles of SIEL are embryonic lethal. Hypomorphic alleles produce defects in root patterning and reduce SHR movement. Surprisingly, both SHR and SCR regulate expression of SIEL, so that siel/scr and siel/shr double mutants have extremely disorganized roots. SIEL localizes to the nucleus and cytoplasm of root cells where it is associated with endosomes. We propose that SIEL is an endosome-associated protein that promotes intercellular movement.

Roles for polarity and nuclear determinants in specifying daughter cell fates after an asymmetric cell division in the maize leaf

Asymmetric cell divisions occur repeatedly during plant development, but the mechanisms by which daughter cells are directed to adopt different fates are not well understood [1,2]. Previous studies have demonstrated roles for positional information in specification of daughter cell fates following asymmetric divisions in the embryo [3] and root [4]. Unequally inherited cytoplasmic determinants have also been proposed to specify daughter cell fates after some asymmetric cell divisions in plants [1,2,5], but direct evidence is lacking. Here we investigate the requirements for specification of stomatal subsidiary cell fate in the maize leaf by analyzing four mutants disrupting the asymmetric divisions of subsidiary mother cells (SMCs). We show that subsidiary cell fate does not depend on proper localization of the new cell wall during the SMC division, and is not specified by positional information acting on daughter cells after completion of the division. Instead, our data suggest that specification of subsidiary cell fate depends on polarization of SMCs and on inheritance of the appropriate daughter nucleus. We thus provide evidence of a role for unequal inheritance of an intracellular determinant in specification of cell fate after an asymmetric plant cell division.

The SHORT-ROOT protein acts as a mobile, dose-dependent signal in patterning the ground tissue

A key question in developmental biology is how cellular patterns are created and maintained. During the formation of the Arabidopsis root, the endodermis, middle cortex (MC), and cortex are produced by periclinal cell divisions that occur at different positions and at different times in root development. The endodermis and cortex arise continuously from the periclinal divisions of cells that surround the quiescent center (QC) at the tip of the root. The MC arises between days 7 and 14 from periclinal divisions of the endodermis. The divisions that produce the middle cortex begin in the basal region of the root meristem away from the QC and then spread apically and circumferentially around the root. Although the transcription factor SHORT-ROOT (SHR) is required for both of these divisions, the mechanism that determines where and when SHR acts to promote cell division along the longitudinal axis of the root is unknown; SHR is present along the entire length of the root tip, but only promotes periclinal divisions at specific sites. Here we show that the abundance of the SHR protein changes dynamically as the root develops, and that the pattern of cell division within the endodermis is sensitive to the dose of this protein: high levels of SHR prevent the formation of the MC, whereas intermediate levels of SHR promote MC formation. These results provide a mechanism for the longitudinal patterning of the endodermis, and represent the first example in plants of a mobile transcription factor whose function (activator or repressor) depends upon concentration.

Mutations in two non-canonical Arabidopsis SWI2/SNF2 chromatin remodeling ATPases cause embryogenesis and stem cell maintenance defects

SWI2/SNF2 chromatin remodeling ATPases play important roles in plant and metazoan development. Whereas metazoans generally encode one or two SWI2/SNF2 ATPase genes, Arabidopsis encodes four such chromatin regulators: the well-studied BRAHMA and SPLAYED ATPases, as well as two closely related non-canonical SWI2/SNF2 ATPases, CHR12 and CHR23. No developmental role has as yet been described for CHR12 and CHR23. Here, we show that although strong single chr12 or chr23 mutants are morphologically indistinguishable from the wild type, chr12 chr23 double mutants cause embryonic lethality. The double mutant embryos fail to initiate root and shoot meristems, and display few and aberrant cell divisions. Weak double mutant embryos give rise to viable seedlings with dramatic defects in the maintenance of both the shoot and the root stem cell populations. Paradoxically, the stem cell defects are correlated with increased expression of the stem cell markers WUSCHEL and WOX5. During subsequent development, the meristem defects are partially overcome to allow for the formation of very small, bushy adult plants. Based on the observed morphological defects, we named the two chromatin remodelers MINUSCULE 1 and 2. Possible links between minu1 minu2 defects and defects in hormone signaling and replication-coupled chromatin assembly are discussed.

Intact microtubules are required for the intercellular movement of the SHORT‐ROOT transcription factor

In both plants and animals, cell-to-cell signaling controls key aspects of development. In plants, cells communicate through direct transfer of transcription factors between cells. It is thought that most, if not all, mobile transcription factors move via plasmodesmata, membrane-lined channels that connect nearly all cells in the plant. However, the mechanisms by which these proteins access the plasmodesmata are not known. Using four independent assays, we examined the movement of the SHORT-ROOT (SHR) transcription factor under conditions that affect microtubule stability, organization or dynamics. We found that intact microtubules are required for cell-to-cell trafficking of SHR. Either chemical or genetic disruption of microtubules results in a significant reduction in SHR transport. Interestingly, inhibition of microtubules also results in mis-localization of the SHR-INTERACTING EMBRYONIC LETHAL (SIEL) protein, which has been shown to bind directly to SHR and is required for SHR movement. These results show that microtubules facilitate cell-to-cell transport of an endogenous plant protein.

Transcription factors on the move

Mobile transcription factors play essential roles in plant development including the control of cell identity and tissue patterning, as well as organ initiation and the induction of major developmental switches. Within the past few years, the molecules and cellular structures that regulate the movement of these signals have emerged. Here we cover some of the major findings of the past two years as they relate to the intercellular movement of multiple different families of transcription factors.

Identification of SHRUBBY, a SHORT-ROOT and SCARECROW interacting protein that controls root growth and radial patterning

The timing and extent of cell division is particularly important for the growth and development of multicellular organisms. Roots of the model organism Arabidopsis thaliana have been widely studied as a paradigm for organ development in plants. In the Arabidopsis root, the plant-specific GRAS family transcription factors SHORT-ROOT (SHR) and SCARECROW (SCR) are key regulators of root growth and of the asymmetric cell divisions that separate the ground tissue into two separate layers: the endodermis and cortex. To elucidate the role of SHR in root development, we identified 17 SHR-interacting proteins. Among those isolated was At5g24740, which we named SHRUBBY (SHBY). SHBY is a vacuolar sorting protein with similarity to the gene responsible for Cohen syndrome in humans. Hypomorphic alleles of shby caused poor root growth, decreased meristematic activity and defects in radial patterning that are characterized by an increase in the number of cell divisions in the ground tissue that lead to extra cells in the cortex and endodermis, as well as additional cell layers. Analysis of genetic and molecular markers indicates that SHBY acts in a pathway that partially overlaps with SHR, SCR, PLETHORA1 and PLETHORA2 (PLT1 and PLT2). The shby-1 root phenotype was partially phenocopied by treatment of wild-type roots with the proteosome inhibitor MG132 or the gibberellic acid (GA) synthesis inhibitor paclobutrazol (PAC). Our results indicate that SHBY controls root growth downstream of GA in part through the regulation of SHR and SCR.