Promoting Factors

A multitude of Arabidopsis mutants show dwarfism because of reduced cell numbers and/or cell size (e.g., Autran et al. 2002; Nelissen et al. 2003). However, for many of them overexpression of the respective gene—where tested— does not lead to extra growth, making it difficult to assess whether these genes fulfill a regulatory role. They will therefore not be discussed further here. Similarly, while activity of the core cell cycle machinery is obviously required for growth by proliferation, overexpression of individual cell cycle stimulators does not generally lead to larger organs, suggesting that organ size is limited by other factors (Dewitte et al. 2003; Doerner et al. 1996). While not increasing final organ size, overexpression of the cell cycle stimulator CyclinD2 in tobacco was reported to accelerate plant growth, both in terms of leaf initiation by the meristem and of growth of individual leaves up to their normal sizes (Cockcroft et al. 2000).

A prominent stimulator of growth by proliferation is the APETALA2 (AP2)-domain protein AINTEGUMENTA (ANT). Loss of function ant mutants form smaller leaves and floral organs, whereas ANT overexpression increases final organ size (Elliott et al. 1996; Klucher et al. 1996; Krizek 1999; Mizukami and Fischer 2000). The larger organs are due to a prolonged period of cell proliferation, even though the response may vary somewhat amongst different organ types (Krizek 1999; Mizukami and Fischer 2000). The delayed arrest of cell division correlates with the sustained expression of a major cell cycle regulator, CycD3;1, although this is unlikely to explain the enlarged organs, as constitutive CycD3;1 overexpression does not lead to larger organs (Dewitte et al. 2003). ANT is likely to function as a transcription factor, as the protein localizes to the nucleus, can bind to DNA in a sequence-specific manner, and activates gene transcription by virtue of an N-terminal domain (Krizek and Sulli 2006). The identification of ANT target genes that underlie its growth-promoting activity remains an important task for future studies.

The ARGOS gene appears to function as an upstream activator of ANT (Hu et al. 2003). ARGOS was identified as an auxin-induced gene expressed in developing organs, and its downregulation causes reduced organ growth. By contrast, ARGOS overexpression is sufficient to increase organ size by stimulating excess cell proliferation, yet this effect is abolished in an ant mutant background. ARGOS encodes a small protein with no discernable functional domains, and how ARGOS acts biochemically is presently unknown.

The ANGUSTIFOLIA3 (AN3)/GRF-INTERACTING FACTOR1 (GIF1) gene encodes a predicted transcriptional co-activator that stimulates organ growth (Horiguchi et al. 2005; Kim and Kende 2004). AN3/GIF1 interacts with the pu tative transcription factor GROWTH REGULATING FACTOR5 (GRF5), and both genes are expressed in developing organs in the region of maximum cell division activity. Similar to ANT, loss of an3/gif1 or grf5 function reduces organ size, while overexpression of either gene leads to larger organs, with the changes in size being due to changes in cell numbers (Horiguchi et al. 2005; Kim and Kende 2004). Interestingly, the effects of an3/gif1 and grf5 mutations are not isotropic, but affect the leaf width direction more strongly than growth in length, suggesting that growth along the transverse and longitudinal axes of plant organs may be under partly independent control. GRF5 is part of a gene family in Arabidopsis (GRF1-GRF9), the members of which are expressed in actively growing organs and tissues of the plant (Horiguchi et al. 2005; Kim et al. 2003). Besides for GRF5, a growth promoting effect has also been described for GRF1, GRF2 and GRF3 (Kim et al. 2003). While overexpression of GRF1 and GRF2 is sufficient to enlarge leaf size, triple mutants of grf1 grf2 grf3 form smaller leaves. However, in contrast to the effect of GRF5 on cell numbers, the changes in organ size due to altered GRF1-GRF3 activity result from increased or reduced cell expansion, which may suggest that growth by proliferation and by expansion share common regulatory elements (see also the discussion on ARGOS-LIKE below).

Another putative transcriptional regulator with a growth-promoting function is encoded by the JAGGED (JAG) gene (Dinneny et al. 2004; Ohno et al. 2004). jag mutants form smaller floral organs, most prominently petals, because of a premature arrest of cell proliferation in the distal petal blade region. Plants with increased JAG activity develop ectopic bracts, i.e., leaves subtending flowers, and leaf lamina tissue on the leaf petiole. However, strong JAG overexpression in developing flowers leads to severe developmental defects, including lack of organ differentiation. This suggests that either the main function of JAG is to prevent premature differentiation—and the concomitant cell cycle arrest—or that JAG has additional functions in tissue patterning and specific differentiation events besides a role in growth control. The recent finding that, together with organ polarity factors, JAG is involved in controlling tissue differentiation in the developing gynoecium (Dinneny et al. 2005) can be taken as evidence for the latter (see below). A homolog to JAG, called NUBBIN (NUB), functions redundantly in stimulating tissue growth, most prominently in leaves, stamens and carpels (Dinneny et al. 2006). While nub single mutants do not have obvious phenotypes, jag nub double mutants form strongly reduced stamens and carpels because of insufficient growth. Both proteins have a C2H2-zinc finger domain and for JAG nuclear localization has been demonstrated (Dinneny et al. 2004; Ohno et al. 2004), suggesting that JAG and NUB regulate transcription.

Work in yeast and animals has demonstrated a crucial role for ribosome biogenesis by the nucleolus in the control of cell and tissue growth (Rudra and Warner 2004). Although Arabidopsis plants with a mutation in a riboso-mal protein have been reported to show a reduced rate of growth, similar to the classical Minute mutants from Drosophila, no detailed measurements of final organ sizes have been reported (Van Lijsebettens et al. 1994; Weijers et al. 2001). Very recently, a potato protein was identified that provides a putative link between ribosome biogenesis and organ size control in plants (Horvath et al. 2006). Plant EBP1 is homologous to the human nucleolar ribosome biogenesis factor EBP1 and localizes to the nucleolus (Pendle et al. 2005). While its downregulation led to smaller organs in both potato and Arabidopsis because of a premature arrest of proliferation, its overexpression could increase organ size, although extremely high levels of overexpression were required for this effect. Increased organ size was the result of accelerated proliferation up to wild-type cell numbers followed by enhanced cell expansion (Horvath et al. 2006). Thus, this protein provides initial evidence that the central role of ri-bosome biogenesis in growth control is not limited to yeast and animals, but also extends to plants. In this respect, it is interesting to note that a class I TCP (TEOSINTE BRANCHED1, CYCLOIDEA, PCF) family protein, TCP20, has been shown to bind to functionally relevant cis-regulatory elements in the promoters of important cell cycle regulators and of ribosomal proteins, suggesting a possibility for how cellular growth and cell division could be coordinated (Li et al. 2005).

In addition to the above factors that can be assumed to act cell-autonomously, intercellular signaling via the ERECTA (ER)-family of leucin-rich-repeat receptor-like kinases (LRR-RLKs) appears to play an important role in promoting cell proliferation in developing organs (Shpak et al. 2004). Triple mutants in er, erecta-like1 (erl1) and erecta-like2 (erl2) are severely dwarfed because of insufficient cell proliferation. The three genes show overlapping yet distinct expression patterns in young, growing tissues, suggestive of expression-dependent partial redundancy of the three genes. Based on expression analyses, the ER-family kinases appear to act independently of ANT. The triple mutants also exhibit defects in stomatal patterning, suggesting that activity of the ER kinase family is not only required for the proliferative cell divisions that increase overall organ cell numbers, but that it also influences the asymmetric divisions that lead to the formation of stomata (Shpak et al. 2005). The downstream signaling components and presumed extracellular ligands for the ER-family receptors are still unknown, although signal transduction has been speculated to involve a MAP-kinase cascade (Ingram and Waites 2006). It will be exciting to see whether ER ligands represent the plant-equivalent to animal mitogens and growth factors with the potential to coordinate growth at an organ-wide level. Further details on LRR-RLKs in plant growth control can be found in the chapter by Ingram in this volume.

Several plant hormones influence either cell proliferation or expansion in developing organs. Cytokinins are prominent for their cell cycle stimulating activity, and both reduced cytokinin content due to overexpression of an inactivating enzyme (Werner et al. 2001,2003) and insensitivity to the hormone (Riefler et al. 2006) cause dwarfism due to reduced cell numbers. However, to our knowledge it has not been unambiguously demonstrated that increased cytokinin levels lead to larger organs. Auxin is also known for promoting cell proliferation and growth, and the reader is referred to the relevant chapter by Offringa in this volume.

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