Cellular Basis and Signalling Processes in the Control of Extension Growth

How does the repression of elongation growth by light take place? It is important to note that although the photoreceptors acting in shade avoidance are the same as those acting in de-etiolation, recent work has also shown that signalling elements, and probably elementary processes, unique to each process also exist (Roig-Villanova et al. 2007). An elegant study (Gendreau et al. 1998) analysed the cellular basis of the differential growth response of the hypocotyl in darkness and light, and identified a number of key differences. The dramatically accelerated hypocotyl elongation in the dark was due to increased cellular expansion. However, the extra growth took place almost exclusively at the top of the hypocotyl, i.e. a clear gradient of extensibility was seen longitudinally in the dark. Furthermore, although no new cells were formed, cells endoreduplicated more frequently under those conditions, with 16C (16 times the haploid genome content) nuclei being detected only in the dark. A general correlation between cell size and degree of endoreduplication is well known (Melaragno et al. 1993; Sugimoto-Shirasu et al. 2002). Recently a protein, IPD1, which is actively involved in promoting endoreduplication in hypocotyls in the dark (exclusively) and whose expression is suppressed by light, has been identified (Tsumoto et al. 2006). The study of Gendreau and collaborators also observed differentiation of epidermal hypocotyl cells in the light: endoreduplication became less prominent, with nuclei never exceeding 8C, and all epidermal cells, sampled longitudinally, elongated equally. In other words, a coordinated change of elongation programme along the complete organ took place in the light.

Such an organ-wide change in programme again suggests the possibility of involvement of hormonal growth regulators. Auxins and GAs are the best known hormones in the control of elongation growth. Intercellular transport is central to auxin biology. Interestingly, auxin transport inhibitors have no effect on the ability of etiolated seedlings to elongate, while they reduce the height of hypocotyls in the light (Jensen et al. 1998; Shinkle et al. 1998). This indicates that dark growth has little sensitivity to changes in auxin concentration, either because it is auxin-independent, or because it is supersaturated for auxin levels, while upon transition to light, auxin transport becomes central to growth control. Classical auxin transporters belong to the PIN family, but members of the ATP-binding cassette (ABC) family of transmembrane proteins are also involved in auxin transport (Lin and Wang 2005). Loss of function mutations in the AtPGP1 and AtMDR1 ABC transporters results in enhanced growth inhibition in the light, or what could be described as an enhanced light-sensitivity phenotype (Lin and Wang 2005). This suggests that de-etiolation may be accompanied by a broad loss of auxin flow, presumably from the site of highest auxin concentration, around the seedling apex. Possible support for this notion is the recent observation that shoot phytochrome can control the growth of lateral roots, and that the DR5:GUS reporter, a well-established auxin biosensor, is expressed in phytochrome-deficient phyB seedlings closer to the base of the hypocotyl than it is in the wild type (Salisbury et al. 2007). Shade (FR-rich) light, i.e. loss of active phytochrome, promotes a rapid and large auxin response, as revealed in a global transcriptome analysis of whole seedlings by the dramatic elevation of HAT2 and HAT4 (Devlin et al. 2003). HAT2 and HAT4 are homeodomain-Leu zipper transcription factors which are strongly induced by auxin. The site of this elevation in seedlings is not known, but is likely to be the hypocotyl, given the later expression of these genes in shoot internodes (Carabelli et al. 1993). Shade also rapidly stimulates the expression of two atypical beta helix-loop-helix transcriptional regulators, PARI and PAR2, in a negative feedback loop in which they repress specific auxin responsive genes (Roig-Villanova et al. 2007).

Further evidence for a negative role of light in the control of auxin responses is provided by SHY2, also known as IAA3. This protein acts as a repressor of auxin responses. A mutant with constitutively active SHY2 was identified as a suppressor of the phyB mutant, causing suppression of the long etiolated hypocotyl of phyB (Tian et al. 2002). Auxin responses are mediated by the targeted proteolysis of AUX/IAA proteins, including SHY2/IAA3, and phytochrome interacts with SHY2 and the ubiquitin ligase that specifically targets it for degradation (although no light control of the degradation was observed, Tian et al. 2002).

The sensitivity of tissues to auxin is regulated in other ways. One notable, potentially important observation is that two related transcription factors, HY5 and HYH, desensitise auxin responses (Sibout et al. 2006). HY5-deficient plants appear partially etiolated in the light, and the level of HY5 protein closely correlates with the extent of light response, specifically in the hypocotyl, HY5 levels being minimal in the dark and maximal under the highest fluence rate of light tested (Osterlund et al. 2000). The role of HY5 in auxin responses provides an explanation for the surprising fact that its mutant was re-identified, and the mutated gene cloned, due to its altered root architecture (Oyama et al. 1997).

GA is a second hormone whose role in the suppression of elongation in the light has been extensively tested. Importantly, a study that examined the phenomenon in both Arabidopsis and pea observed that chemical or genetic removal of active GA was sufficient to cause a de-etiolated-like phenotype (Alabadi et al. 2004). Could GAs fully account for such a process? This is unlikely to be the case, GA action being more likely one of several growth-triggering pathways under light control. This was serendipitously observed in a study (Lopez-Juez et al. 1995) using the first plant identified to be de-

fective in the phyB protein, the cucumber long hypocotyl (lh) mutant. Grown under a moderately low fluence rate light, mutant hypocotyls grew to just over twice the length of wild-type ones, with a very small increase in DNA content (quantified from all hypocotyl cells) and with no increase (in fact with a reduction) in active GA levels. Clearly the increased elongation in the phyB-deficient mutant was not mediated by increased GA levels. However, under a higher light fluence a second growth component emerged: elongation was sustained over a longer period, epidermal cells increased in number and total hypocotyl DNA content increased in the mutant. Under those conditions active GA levels were two- to threefold higher in the mutant than in the wild type. This suggested the possibility of GA acting as a mitogen or as a cell proliferation signal, and as an "optional" component of the response to reduced phytochrome activity (Lopez-Juez et al. 1995).

Figure 2 shows the same phenomena in internodes of wild-type and phyB-deficient cucumber. At this time, active GA levels are fourfold greater in apices (where the second internode is developing). Meanwhile the preceding, first internode is already threefold longer, but actually shows smaller epidermal cells in which new transverse cell walls, the result of recent mitosis, are easy to observe (Fig. 2a). As a consequence of this greater cell pool, this internode is capable of much greater additional growth when entering the phase of cell expansion (Fig. 2b; Lopez-Juez, Kobayashi and Kamiya, unpublished observations). This phenomenon strongly resembles the role of active GA in the response to flooding of internodes of deep water rice. In such in-ternodes, when they undergo extraordinarily rapid elongation, GA promotes both cell cycle activation and cell expansion, activating the expression of genes like Replication Protein A1—for DNA synthesis—or expansins—for cell wall expansion (van der Knaap et al. 1997). Growth responsive factor 1, or GRF1, is the prototype of a novel class of plant transcription factors and was identified in rice through its role in this response (van der Knaap et al. 2000). Members of the GRF family in Arabidopsis also control growth, and distinct GRFs play roles either during cell proliferation, involving both cell growth and division (Horiguchi et al. 2005), or during cell wall expansion (Kim et al. 2003; Doerner, this volume. Indeed, at least two GRF genes appear to be lightregulated in Arabidopsis (Dillon, Bögre and Lopez-Juez, unpublished results).

GA activity could also be modulated by regulation of GA signalling. Phyto-chrome action has been shown to limit the response to externally applied GAs (Lopez-Juez et al. 1995; Reed et al. 1996). GAs target growth-repressive DELLA proteins for degradation (see Sect. 3). DELLAs have recently been shown to constrain growth in shade, and their degradation, shown to take place in response to far-red light supplementation, is necessary but not sufficient to account for the accelerated growth response (Djakovic-Petrovic et al. 2007).

Unravelling the roles of plant hormones is complicated by their mutual interdependence. This brings about a bewildering array of light effects on hormonal growth responses, including brassinosteroids and ethylene, some

Fig. 2 Elevated active gibberellin (GA) levels precede an increased cell proliferation in extremely elongated internodes of a phytochrome-deficient mutant. A Seedlings of wild type (left) or phytochrome B-deficient long hypocotyl (lh) cucumber mutant (right) are shown, after dissecting the first internode, the second leaf and the remainder of the shoot apex (including the third leaf, the second internode and the shoot meristem). The first internode of lh is already at least threefold longer that that of the wild type, yet its epidermal cells are equal or smaller in size, and in far greater numbers, this pointing to increased cell proliferation. This is preceded, in the shoot apex, by enhanced levels of active GA, in this species GA4. B The increased cell proliferation in the lh internode allows for much greater final size after the phase of cell expansion. Tracking of marks placed before this phase started indicates that the entire organ is capable of growth. C The lh mutant shows increased biomass allocation into hypocotyl and petioles, and reduced into leaf blades and roots (arrows)

Fig. 2 Elevated active gibberellin (GA) levels precede an increased cell proliferation in extremely elongated internodes of a phytochrome-deficient mutant. A Seedlings of wild type (left) or phytochrome B-deficient long hypocotyl (lh) cucumber mutant (right) are shown, after dissecting the first internode, the second leaf and the remainder of the shoot apex (including the third leaf, the second internode and the shoot meristem). The first internode of lh is already at least threefold longer that that of the wild type, yet its epidermal cells are equal or smaller in size, and in far greater numbers, this pointing to increased cell proliferation. This is preceded, in the shoot apex, by enhanced levels of active GA, in this species GA4. B The increased cell proliferation in the lh internode allows for much greater final size after the phase of cell expansion. Tracking of marks placed before this phase started indicates that the entire organ is capable of growth. C The lh mutant shows increased biomass allocation into hypocotyl and petioles, and reduced into leaf blades and roots (arrows)

of which may be indirect (Halliday and Fankhauser 2003; Nemhauser and Chory 2002). A detailed discussion of these is beyond the scope of this chapter. The hormone ethylene may also be one primary mediator of light-dependent growth responses, as shown by the fact that phytochrome defects cause ethylene overproduction in pea, and that restricting ethylene biosynthesis rescues many of the phytochrome deficiency phenotypes and mimics a full light response (Foo et al. 2006). In this case ethylene appears to act, at least in part, by suppressing GA production. Relationships between light and ethylene, and its interactions with GAs and auxin, are discussed in detail by Dugardeyn and Van Der Straeten elsewhere in this volume.

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