Several studies have shown that suboptimal temperatures constrain vegetative growth in temperate grasses by inhibiting the extension of leaves rather than their initiation. Thus, over-wintering Lolium spp. develop a 'stored growth' potential that becomes expressed as a surge of canopy production when spring temperatures cross a threshold, usually about 5°C or an equivalent period of thermal time (Peacock 1975,1976; Pollock and Eagles 1988, Fournier et al. 2005). L. temulentum has been a frequent subject for analysing the temperature relations of leaf extension. At 20°C under an 8 h (non-inductive) daylength in controlled-environment conditions, leaf 5 of L. temulentum line Ba3081 emerges 28 days after seed germination and reaches full expansion about 11 days later (Thomas 1983b; Thomas and Potter 1985). If plants are transferred from 20 to 5°C 21 days after germination, emergence of the fifth leaf is delayed by around 20 days and this leaf takes about 45 days to achieve a fully expanded size about 58% of that of 20°C-grown leaves. If plants are transferred from 20 to 2°C at 21 days, the fifth leaf fails to emerge at all. The response of L. temulentum leaf extension on exposure to 2°C is qualitative and almost immediate: high-resolution kinetic measurements with growth transducer instruments showed that growth fell precipitously to about 4% of the rate at 20°C and became confined to the dark phase of the photoperiod (Thomas and Stoddart 1984; Stoddart et al. 1986).
Chilling temperatures constrain leaf extension by throttling back demand for, rather than supply of, photosynthate. Pollock et al. (1983) showed that the Q10 for photosynthesis in L. temulentum between 20°C, 5°C and 2°C was in the range 2.1-4.6, whereas the corresponding range for relative growth rates was 1.7-44.6, and plants held at 5°C or 2°C accumulated high levels of soluble sugars, including fructans. Prolonged growth at 5°C reduced leaf cell number, chloroplast number and chlorophyll content but did not reduce photosynthetic capacity expressed on a unit area, fresh weight or chlorophyll basis (Pollock et al. 1984). Unlike exposure to supra-optimal temperatures, which induces the synthesis of heat shock proteins, there was no evidence of major qualitative changes in gene expression when L. temulentum was exposed to growth-limiting chilling temperatures (Ougham 1987).
Leaf extension is acutely sensitive to tissue water status, and the physiological characteristics of the limitation imposed on L. temulentum leaf extension by chilling temperatures are consistent with a direct response at the level of cell turgor and the constraints of cell wall rigidity. Thomas et al. (1989) measured turgor pressure parameters in the expanding zone of L. temulentum leaves over the temperature range 20°C-2°C and concluded that the site of thermal perception is the cell wall, the rheological properties of which are particularly sensitive to chilling temperatures. Bacon et al. (1997) came to a similar conclusion in their studies of the inhibition of leaf extension in L. temulentum by drought. These authors observed abrupt increases in the activity of cell wall-associated peroxidases in the extension zone and suggested that these enzymes have an important role in tissue rheology by making stiffening cross-links between cell wall polymers. Other plasticity-modifying factors associated with cell walls in growing leaf tissues of grasses include extensins, glycosyl transferases and gibberellins (Farell et al. 2006). Growth responses to salinity stress are also mediated in part by turgor. Baldwin and Dombrowski (2006) isolated a large number of salinity-responsive genes from leaves and crowns of L. temulentum exposed for 20 h to 500 mM NaCl administered to the roots. Of the 528 unique sequences identified, 167 were ortho-logues of previously identified plant stress response genes. Interestingly, genes with functions in cell wall cross-linking were absent from the collection. Subsequently, Dombrowski et al. (2008) focused on a gene from the library encoding smGTP, a small guanosine triphosphate-binding protein. On the basis of comparative specificity of its expression pattern with respect to salinity and other abiotic challenges, they concluded that smGTP is part of a dehydration stress signalling pathway.
It is normal for forage and amenity grasses to experience defoliation, through either grazing, or cutting for conservation, or harvest for seed production or mowing to maintain low sward height. Physiological changes in the cut herbage depend on post-harvest conditions. Herbivory by ruminants almost instantaneously changes the environment of the grazed tissue from mild temperatures in daylight and an aerobic atmosphere to darkness, anoxia and about 40°C. The immediate response of the still-viable ingesta is to trigger a characteristic cell death-like programme with far-reaching consequences for animal nutrition and its impact on the environment (Kingston-Smith and Theodoou 2000). Baldwin et al. (2007) made subtractive gene libraries from cut L. temulentum straw held under post-harvest conditions simulating those used in the field for seed production. They identified almost 600 unique cutting-specific sequences (Fig. 8). As might be expected, there was a fair amount of similarity with the profile of genes associated with leaf
■ Energy transfer ■a General matabolism
■ Maintenance genes
■ Photosynthesis sp.
1:1 Piastid/mitochondriai genome a Protein degradation, folding and transport
■ Ribosomal Signal transduction
■ Stress related n Transcription factor
Transport and membrane bound
Fig. 8 Proportions of genes in different functional categories expressed post-harvest in seed-bearing shoots of Lolium temulentum. Data of Baldwin et al. (2007), doi:10.1016/j. plantsci.2007.04.001
senescence. For example, a number of genes encoding enzymes of protein degradation were identified, including several cysteine-, serine- and metallo-endopeptidases, Clp proteases and components of the ubiquitin/proteasome system. Responses of L. temulentum to defoliation and subsequent regrowth were studied by Ourry et al. (1996). The pool sizes and mobilization patterns of C and N compounds respond to defoliation through their control by modified source/sink relationships. Nitrogen uptake and mobilization by defoliated plants were determined by 15N labelling. The difference in regrowth yield was accounted for by the initial availability of N reserves (Louahlia et al. (1999) reported similar observations for contrasting cultivars of L. perenne in a follow-up study). Changes in gene expression were visualised by comparing in vitro translation profiles of RNA extracted from meristem, sheath and lamina tissues after defoliation with those from intact L. temulentum plants. Modulations in expression pattern were observed to be dependent on the tissue and elapsed time since cutting. Some changes were detectable within 1h of defoliation, but the largest shifts in abundance of translatable transcripts occurred between 12 and 72 h after shoot removal. Amongst the activities that increase in leaves during regrowth are enzymes of amino acid and sucrose/ fructan metabolism, whereas in the stubble, which acts as a remobilizing source to support the production of new foliage, peptidases and carbohydrate-recycling enzymes become elevated.
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