In analogy to the previous section, overexpression of many genes has been reported to cause dwarfism because of reduced cell numbers (e.g., ICK1/KRP2, De Veylder et al. 2001; LOB1, Shuai et al. 2002), yet in the absence of a complementary loss-of-function phenotype with increased proliferation, the role of these factors in endogenous proliferation control remains unclear. The following discussion will therefore be limited to those factors that have been shown to be required for limiting proliferation in developing organs.
Several putative transcriptional regulators of the class II TCP family are required for proper arrest of cell proliferation during organ development. Loss of function of the Antirrhinum CINCINNATA (CIN) protein leads to larger organs with a wrinkled shape because of excess proliferation at the leaf margins (Nath et al. 2003). The CIN expression pattern is intriguing in that expression sweeps across the lamina of the developing leaf in a basipetal manner, preceding the cell cycle arrest front that terminates proliferation in a basipetal direction. Therefore, CIN may sensitize leaf margin cells to an unknown cell cycle arrest signal. Downregulation of Arabidopsis CIN homologs by overexpression of the JAW microRNA (miRNA) leads to a very similar leaf phenotype, suggesting that class II TCP action in limiting proliferation is conserved in plants (Palatnik et al. 2003). However, the picture is somewhat more complicated, as in petals CIN has the opposite effect, i.e., it promotes organ growth by proliferation, suggesting organ identity factors may determine the nature of its influence on growth (Crawford et al. 2004).
Recently, the model of how proliferation arrest is achieved in developing leaves has been refined by the identification of the redundant homologs PEAPOD1 (PPD1) and PEAPOD2 (PPD2) (White 2006). ppd1 ppd2 double mutants form enlarged leaves with a bell shape because of excess growth in the center of the leaf lamina. This excess growth appears to be driven by prolonged proliferation of so-called dispersed meristematic cells, such as stomatal precursors and procambial cells. By contrast, PPD overexpression reduces leaf size by causing premature cell cycle arrest of dispersed meristem-atic cells. PPD expression appears to follow the general cell cycle arrest front, i.e., expression is found in the region of organs where most cells have stopped proliferating, but dispersed meristematic cells continue to divide. These observations lead to the proposal that the first, TCP-dependent cell cycle arrest front is followed by a second, PPD-mediated arrest front that specifically affects the dispersed meristematic cells.
The BIG BROTHER (BB) gene also limits organ growth by restricting the period of cell proliferation, affecting mainly floral organs and the stem (Disch et al. 2006). Final organ size is tightly correlated with BB mRNA expression levels, with a reduction by 50% or a three-fold increase in mRNA amount leading to significant organ enlargement or reduction, respectively. BB is expressed in all actively growing tissues. The BB protein has a RING-finger domain, exhibits E3 ubiquitin-ligase activity in vitro, and mutations that abolish this activity also abrogate function in planta. Together, these findings suggest that proteasome-mediated degradation of crucial growth stimulators during the phase of cell proliferation determines when proliferation ceases. As BB acts independently of ANT, JAG and phytohormones, the identification of its substrates promises to uncover important additional growth-promoting factors.
Similar to BB, loss-of-function mutants for AUXIN RESPONSE FACTOR2 (ARF2) form thicker stems and larger organs, including leaves, integuments and, as a consequence, seeds (Ellis et al. 2005; Okushima et al. 2005; Schruff et al. 2006). The organ enlargement correlates with prolonged expression of ANT and its target gene CycD3;1 (Schruff et al. 2006), suggesting that the excess growth is due to an extended period of cell proliferation. No overexpression phenotype has been reported for ARF2. In vitro ARF2 protein has been shown to bind to auxin response elements (AuxREs) and it was able to repress transcription from synthetic AuxRE-containing promoters (Li et al. 2004; Tiwari et al. 2003). However, arf2 mutants do not exhibit global expression changes in auxin-regulated genes (Okushima et al. 2005), calling into question whether its effects on growth are due to misregulated auxin signaling.
Studies of natural variation in tomato fruit size have identified the fruit weight2.2 fw2.2) gene as another important repressor of cell proliferation (Frary et al. 2000), as will be discussed in more detail below.
As the preceding discussion has shown, there is a growing inventory of factors that positively or negatively influence cell proliferation and thus contribute to organ size regulation. However, if and how individual factors interact with each other and with the basic cell cycle and growth machinery, how many independent pathways impinge on the control of proliferation, and how the identified factors act molecularly all remain largely unknown.
One generalization that can be drawn from the studies of growth by proliferation is that the factors isolated to date—with the exception of EBP 1 and CyclinD2—all seem to influence the duration of cell proliferation, rather than the rate of cell cycling. This could suggest that cells in organ primordia normally cycle at the maximum rate that is possible under the given nutritional and environmental circumstances, and that it may be easier for example to extend the period of proliferation than to speed up cell cycling. Accelerated growth by proliferation that leads to larger organs has only been observed in plants overexpressing the putative ribosome biogenesis factor EBP1, which appears intuitively plausible, as increased ribosome production should allow for a higher rate of macromolecular synthesis by protein translation and thus faster and more overall cell growth. The first glimpses at the molecular evolution of organ size, i.e., the role of fw2.2 in tomato fruits, seem to support the idea that extending the time of proliferation may be a more accessible path than accelerating growth, as changes in fruit size due to allelic differences at the fw2.2 locus are the result of altered timing of cell cycle arrest (Cong et al. 2002). Unraveling further cases of evolutionary modification of organ size should indicate whether it is indeed the timing of growth by proliferation that has been the most frequent target of evolutionary change.
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