Plant Physiol. (1995) 108: 125-1 28
Similarities in Gene Expression during the Postharvest-lnduced Senescence of Spears and Natural Foliar Senescence of Asparagus’ Graeme A. King*, Kevin M. Davies, Richard J. Stewart2, and Wilhelmina M. Borst
New Zealand lnstitute for Crop & Food Research Limited, Levin Research Center, Private Bag 4005, Levin, New Zealand corresponding transcripts substantially increased in abundance by 12 h (King and Davies, 1992): pTIP9, function unknown; pTIPl1, a P-galactosidase homolog (King and Davies, 1995); pTIP12, an AS homolog (Davies and King, 1993).We are interested in changes in the abundance of the transcripts encoded by these cDNA clones during normal plant development, because this may give insight into the factors regulating gene expression after harvest. When left unharvested, asparagus spears develop into mature, photosynthetic foliar structures comprising many needle-like branches (cladophylls; Robb, 1984) arising in whorls from nodes of the stem and branches (the fern; Blasberg, 1932). During autumn in temperate climates, the fern senesces, returning nitrogen, phosphorus, and potassium to the underground crown to support new spear growth during the following spring (Robb, 1984).Nitrogen is often transported in plants as Asn (Sieciechowicz et al., 1988), and so AS transcripts may also accumulate during natural fern senescence in asparagus. Here we report changes in Chl, protein, sugars, and gene expression during the development and natural senescence of the asparagus fern. Transcripts corresponding to pTIP9, pTIP11, and pTIP12 accumulated during natural fern senescence, suggesting links between the harvest-induced senescence of spears and natural foliar senescence.
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Changes in gene expression and tissue composition were investigated during foliar development and natural senescence of asparagus (Asparagus officinalis L.). Three phases in development and senescence of the foliage were characterized: early fern growth, mature fern, and senescence, when a marked loss of chlorophyll, sucrose, and protein occurred and major changes in translatable mRNAs were detected. Transcripts for three asparagus spear harvest-induced cDNA clones, pTIP9, pTIP11, and pTIP12 (G.A. King and K.M. Davies [1992] Plant Physiol 100: 1661-1669), accumulated during natural foliar senescence, suggesting that the underlying regulatory mechanisms may be similar in both developmental situations. We have used our knowledge of asparagus spear physiology, the probable proteins encoded by the cDNA clones, and our fern development data to propose that sugar depletion regulates the accumulation of at least pTlP12 transcripts in senescing asparagus tissue.
Senescence is a poorly understood sequential process. It may occur naturally, as in senescing leaves and ripening fruit (Biggs et al., 1986; Kamachi et al., 1991), or be artificially induced by a variety of stresses including dark treatment or excision of plant organs (Huber, 1987; Azumi and Watanabe, 1991). Senescence is generally accepted to be a programmed process involving changes in gene expression, although marked differences may occur during natural and artificially induced senescence of particular organs (Becker and Apel, 1993). We are investigating the early physiological, biochemical, and genetic changes following harvest of asparagus (Asparagus oficinalis L.) spears to identify factors regulating postharvest senescence (an artificially induced senescence situation). We previously constructed cDNA libraries from mRNA that was extracted from tips of spears at harvest and from spears held in the dark at 20°C for 12 h. Differential hybridization screening of these libraries isolated severa1 cDNA clones for transcripts that had altered abundance after harvest, including three cDNA clones, whose
MATERIALS A N D M E T H O D S Plant Material
Asparagus (Asparagus officinnlis L. cv Limbras 10) fern growing in a commercial field at the Levin Research Center was sampled at 3-week intervals from the time of cladophyll opening, which was mid-January (4 weeks after the end of commercial spear harvesting; FST l), until midMarch (FST 4). As the cladophylls neared senescence, samples were taken every 2 weeks until cladophylls were yellow (FST 5-FST 7). At each sampling time, approximately 2 g of cladophyll and branch tissues were excised from ferns from each of three separate plants, frozen in liquid nitrogen, and held at -80°C until required.
Supported by a grant in aid from the Scientific Research Committee of the New Zealand Asparagus Council. This is Levin Research Center paper No. 94/002. Present address: Department of Plant Science, Waite Campus, University of Adelaide, Australia. * Corresponding author; e-mail
[email protected]; fax 646368-3578.
RNA Extraction and Analysis
The methods for RNA extraction, in vitro translation, and dot-blot hybridization were as described by King and Abbreviations: AS, Asn synthetase; FST, fern sampling time. 125
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Davies (1992). pTIP9, pTIPl1, and pTIPl2 were us;ed as probes for the RNA hybridization studies. These CDNA clones encode transcripts that markedly accumulate in the tips of harvested asparagus spears (King and Davies, 1992). pTIPll and pTIP12 are of identical nucleotide sequence to the same regions of the corresponding full-length cDNA clones pTIP31 (King and Davies, 1995) and pTIP27 (Davies and King, 1993), respectively. In vitro translation and dot-blot hybridization ewperiments were carried out using three separate sets of RNA extracted from ferns in three sequential growing seasons. The dot blots shown are a typical example of the consistent trends found in each of the 3 years.
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a Chl, Total Protein, and Soluble Sugar Determination
Chl was determined on 0.5-g samples of fresh asparagus fern using the N,N-dimethylformamide method (h4oran and Porath 1980; Inskeep and Bloom, 1985). Total pxotein was estimated on duplicate 20-mg samples of powclered, freeze-dried fern using the method of Lowry et al. (1951), with BSA as the standard as described by King et al. (1990). Soluble sugars were determined on duplicate 10-mg samples of powdered freeze-dried fern as described by Hurst et al. (1993). A11 compositional analyses were replicated three times, with the plant material comprising each replicate being obtained from separate plants.
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RESULTS Chl, Protein, and Sugar Content
The Chl content of asparagus fern increased slightly between FST 1 and FST 2 and then remained at a constant level to FST 5 (Fig. 1A). Chl levels began to decline at FST 6 when cladophylls were visibly yellowing. Chl levels declined further at FST 7, at which time cladophylls and large areas of the branch tissue were yellow. Total protein levels were highest in fern at FST -1 and remained at a constant, although slightly lower, leve1 between FST 2 and FST 5 (Fig. 1B). Total protein levels declined at FST 6 and FST 7, mirroring changes in Chl content as the fern senesced. Suc was the most abundant sugar present in asparagus fern at the time of cladophyll opening (FST 1) and remained at a high level (100-140 mg g-' dry weight) until FST 5 (Fig. 1C). SUClevels declined markedly as the fern senesced. Glc and Fru levels were low (7-27 mg g-' dry weight) in comparison with SUClevels during FST 1 to 4 but increased as the fern neared and underwent senescence (Fig. 1C). In Vitro Translation
of RNA
The changes in translatable mRNA populations were investigated as an initial step in characterizing the gonetic changes accompanying fern development and senescence. The majority of the translation products detected were common at a11 FSTs; however, a number of specific changes were detected (Fig. 2). Major changes in translaitable mRNA abundance were detected during early fern devel-
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Fern sampling time 1
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Figure 1. Chl (A), total protein (B), and soluble sugar (C) content during the development and senescence of asparagus ferri. Each data point i s the mean of three samples obtained from separaie representative ferns. Bars are SE and contained within the symbols when not shown.
opment between FST 1 and FST 2. Severa1 lconsistent changes in translatable mRNA abundance were also detected as the fern senesced. Three translation products (60, 36, and 23 kD) increased in abundance at FST 6 and FST 7, and six translation products (100-69, 43, and 20 kD) decreased in abundance at the same fern sampling times. These changes were coincident with the loss of Chl, protein, and SUCfrom the fern tissue (Fig. l), indic3ting that niarked changes in physiology and gene expression accompany senescence of the fern. Abundance of Transcripts Corresponding to pTlP0, pTlP11, and pTIP12
To investigate changes in the abundance of t:.-anscripts induced by spear harvest during fern developinent and
mRNA Activity in Asparagus Fern
97.469-
1
2 3 4 5 6 7
Fern sampling time Figure 2. Changes in mRNA in vitro translation products during the development and senescence of asparagus fern. Total RNA was prepared from fern at each sampling time and translated in vitro using a rabbit reticulocyte lysate in the presence of L-[J5S]Met. Translation products (100,000 cpm/lane) were separated by SDSPAGE on 12 to 17% gels and detected by fluorography. Bars on the left indicate the positions of l4C-labeled mol wt marker proteins (in thousands). Closed triangles on the right identify translation products from mRNAs that increased in abundance during senescence (FST 5-FST 7). Open triangles mark products from mRNAs whose abundance decreased during senescence.
senescence, RNA was extracted at seven times as the fern matured and was analyzed by dot-blot hybridization. Transcripts for pTIP9 and pTIP12 were detected at high levels only at FST 6 or FST 7 (Fig. 3). Transcripts for pTIPll also accumulated during senescence but in addition were abundant at FST 1 and FST 2. This suggests that transcripts for pTIPH encode a protein that has a role in early fern development as well as fern senescence.
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son (1989) similarly identified cDNA clones for mRNAs involved in both the ripening of fruit and senescence of leaves. In addition, lusem et al. (1993) described a tomato transcript that was induced by both leaf-water deficit and during fruit ripening. Collectively, these results suggest that the regulation of senescence in different organs may share at least some similar underlying mechanisms. We have recently established that pTIP12 encodes an AS homolog (Davies and King, 1993) that is encoded by a single-copy gene in asparagus (R.L. Moyle and K.J.F. Farnden, unpublished data). The transient accumulation of transcripts for AS at FST 6 was coincident with the loss of Chl, protein, and Sue (Fig. 1) from fern tissue. Asn is commonly synthesized as a nitrogen transport compound (Joy, 1988), particularly when plants are placed in stressful situations and carbon skeletons are limiting (Asn formation requires less carbon for each nitrogen unit than Gin; Sieciechowicz et al., 1988; Brouquisse et al., 1992). We previously found that AS transcripts accumulate rapidly during the postharvest-induced senescence of asparagus spears coincident with Sue loss (Davies and King, 1993; Irving and Hurst, 1993). Furthermore, Asn accumulation in sugarstarved sycamore cell cultures is reversed by the addition of Sue back to the starved cell cultures (Genix et al., 1990). These results suggest that Asn formation is closely related to the carbon status of plant tissue. AS may be active during fern senescence to provide a carbon-efficient means of translocating nitrogen released from proteolysis in the
pTIP9 pTIP11
DISCUSSION
Three phases in the development and senescence of the asparagus fern were observed. Phase one (FST 1 and FST 2) involved the completion of branch and cladophyll opening from the spear tissue and was accompanied by an increase in Chl content and Sue levels, a decrease in total protein levels, and marked changes in the translatable mRNA population of the fern (Figs. 1 and 2). This was followed by a period of mature fern development, when there was comparatively little change in any of these parameters (FST 3-FST 5). Phase three was senescence and involved marked losses of Chl and protein and changes in the soluble sugar composition and translatable mRNA population of the fern (FST 6 and FST 7). We wanted to determine whether transcripts corresponding to three spear harvest-induced cDNA clones also accumulated during the development and senescence of asparagus fern, because this might give insight into the factors regulating gene expression in harvested spears and other senescence situations. Transcripts corresponding to pTIP9, pTIPll, and pTIP12 all accumulated during fern senescence (Fig. 3), suggesting roles for the proteins encoded by these transcripts at this stage. Davies and Grier-
pTIP12 pTIP19 1234567
Fern sampling time Figure 3. Changes in the abundance of spear harvest-induced mRNAs encoded by pTIP9, pTIP11, and pTIP1 2 during the development and senescence of asparagus fern. Total RNA was prepared from fern at each sampling time. RNA (10 /j.g) was blotted onto Hybond-N + membranes (Amersham) following the manufacturer's recommendations. Transcript abundance was detected after hybridization to [32P|dCTP-radiolabeled cDNA probes, washing of the membranes at high stringency (0.1 x SSC, 65°C), and visualization by autoradiography (King and Davies, 1992). RNA loadings were equalized by hybridization with a cDNA encoding the asparagus 25/26S rRNA (pTIP6; K.M. Davies, unpublished data). pTIP19 (encoding elongation factor 1-a; C.A. King, unpublished data) was used to demonstrate that the changes detected are not due to changes in percentage mRNA levels as the fern senesced.
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fern to the crown for subsequent metabolic use (Robb, 1984). Our most recent data have established that pTIPll encodes a P-galactosidase homolog (King and Davies, 1995), which is a member of a small multigene family in asparagus (G.A. King, unpublished data). The loss of Gal from cell walls and/or induction of P-galactosidase enzyme activity during ripening and senescence of fruit and flowers has been reported (Wiemken-Gehrig et al., 1974; Seymour et al., 1990; Redgwell et al., 1992; Ross et al., 1993). Although mobilization of Gal from the cell wall during senescence may be anticipated to aid recycling of carbon for metabolic use, the role of P-galactosidase during early fern development is unclear. Gal can inhibit cell expansion and increase carbon import into phloem in barley roots (Farrar et al., 1994) and, therefore, may have important physiological roles during normal plant development. Investig ations of P-galactosidase enzyme activity and consideration of the possibility of differential expression of P-galactosidase isoforms would be necessary to elucidate further the role of this enzyme during the development and senescence of asparagus tissue. Knowledge of the probable proteins encoded by pTIPll and pTIP12 strongly suggests that the accumulation of transcripts for enzymes involved in specific carbon and nitrogen remobilization processes are a consistent feature of both the postharvest-induced senescence of spears and natural senescence of the asparagus fern. The regulatory mechanisms may be similar in both developmental situations, and at least some of the changes may be linked to sugar depletion. The regulation of AS by tissue sugar levels might also account for the accumulation of AS transcripts during the dark cycle of plants (Tsai and Coruzzi, 1991). We are currently testing the hypothesis that sugar :$tatus regulates the accumulation of AS, P-galactosidase, and pTIP9 transcripts in asparagus tissue. ACKNOWLEDCMENTS
We thank Erin ODonoghue, Chris Downs, Tony Conner, and David Swain for helpful criticism of the manuscript. Received October 3, 1994; accepted January 30, 1995. Copyright Clearance Center: 0032-0889/95/108/0125/04. LITERATURE ClTED
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