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a Hermitage Research Station, Dep. of Primary Industries, Warwick Queensland 4370, Australia
b QDPI/CSIRO Agricultural Production Systems Research Unit, Toowoomba Queensland 4350, Australia
borrela{at}dpi.qld.gov.au
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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and rate
of leaf senescence under terminal water deficit.
Abbreviations: DAE, days after emergence • GLAM, green leaf area at maturity (cm2/m2) • LAI, leaf area index • LNC, leaf nitrogen concentration (mg N g-1) • ND, No water deficit • NUE, Nitrogen use efficiency (kg kg-1) • PFD, Post-flowering water deficit • SLN, Specific leaf nitrogen (g N m-2) • SLW, Specific leaf weight (g m-2) • TD, Terminal water deficit
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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Longevity of a leaf is intimately related to its nitrogen status (Thomas and Rogers, 1990). During senescence, amino acids cease to be formed, existing protein is degraded and not replaced, and the resultant amino acids are translocated out of the leaf (Thomas and Rogers, 1990). A considerable proportion of leaf protein is bound in pigment-protein complexes of the photosynthetic apparatus, resulting in the characteristic yellowing of the leaf as chlorophyll is released from this association and subsequently broken down. It is likely that the coordination and triggering of senescence in the whole plant is regulated by an increased demand for nitrogen elsewhere which is communicated to source leaves (Thomas and Rogers, 1990).
Foliar development can be analyzed in terms of its supply and demand relations with the rest of the plant (Thomas and Smart, 1993). A clear sequence is followed, commencing with the import of assimilate from older to younger leaves. The youngest leaf then attains photosynthetic competence, and the new organ becomes a net contributor to the carbon budget of the whole plant. Finally, assimilation declines as the leaf yellows. It appears that attainment of photosynthetic capability and the subsequent reduction in assimilation are linked to, and may even be caused by, the rate of export of nitrogen. Thomas and Smart (1993) divided leaf development into segments, each delimited by a condition of net nitrogen balance. The career of a normal leaf is summarized by progression through the following stages: juvenile, carbon export, nitrogen remobilization (first phase of senescence), and breakdown of integrity (terminal phase of senescence).
Stay-green can also be viewed as a consequence of the balance between N demand by the grain and N supply during grain filling. Nitrogen uptake is related to the demand for N within the plant and the availability of soluble carbohydrates in the roots (Tolley-Henry and Raper, 1991). During the grain filling period, there are two sources of N for grain growth: concurrently absorbed N from the soil and remobilized N from vegetative tissues (Ta and Weiland, 1992). Rajcan and Tollenaar (1999b) reported that the proportion of N derived from the soil during grain filling in maize (Zea mays L.) was 60% for Pioneer 3902 (stay-green hybrid) and 40% for Pride 5 (senescent hybrid). Under conditions of abiotic stress such as drought or N deficiency, remobilization of N from vegetative tissues becomes particularly important for grain growth (Ta and Weiland, 1992). Delayed remobilization of N from leaves maintains photosynthetic capacity for longer, possibly resulting in higher grain yield. Under post-anthesis drought, grain yield in sorghum was positively correlated
with green leaf area at maturity in northern Australia (Borrell et al., 2000b) and with green leaf area at mid grain filling
in southern India (Borrell et al., 1999). In a study of recurrent selection for drought tolerance in six tropical maize populations, however, Bolanos and Edmeades (1996) reported a lack of association between green leaf longevity and grain yield. Their results may indicate that increased demand for N by the larger ear resulting from selection are met by mobilization of N from the leaves, resulting in senescence (Muchow, 1994). Banziger et al. (1999) raised an important question about the N supply-demand framework for stay-green in maize. Does the larger N accumulation at maturity in drought-tolerant selections result in delayed leaf senescence (Wolfe et al., 1988a), or is it a result of delayed leaf senescence (Ta and Weiland, 1992)? This question will be addressed in the current paper.
During post-anthesis drought, hybrids containing the stay-green trait maintain more photosynthetically active leaves compared with hybrids not containing this trait (Rosenow et al., 1983; McBee, 1984; Borrell et al., 2000a,b). Since photosynthesis is closely linked with the nitrogen content of leaves (Sinclair and Horie, 1989; Thomas and Smart, 1993; Muchow and Sinclair, 1994), it is important to determine the contribution of nitrogen to yield improvement in stay-green hybrids. The positive relationship between specific leaf nitrogen (leaf N content per unit leaf area) and specific leaf weight (leaf dry mass per unit leaf area) observed in a number of crops suggests that thicker leaves contain more N (Nageswara Rao and Wright, 1994; Wright and Hammer, 1994). For example, Gardner et al. (1994) found that thicker sorghum leaves resulted in more mesophyll per unit leaf area and could have contributed towards increased photosynthesis rate per unit leaf area. Furthermore, a hyperbolic relationship between SLN and leaf carbon dioxide assimilation has been reported for sorghum and maize (Muchow and Sinclair, 1994).
The primary aim of the research outlined in this paper is to determine the effects of water regime on nitrogen uptake and partitioning in nine hybrids varying in rate of leaf senescence. Second, there is a need to clarify the role of nitrogen in extending leaf greenness. Do the leaves in stay-green hybrids take up more nitrogen simply because they grow for longer, or do they stay green for longer because their leaves contain more nitrogen? In particular, the role of specific leaf nitrogen and its components (leaf nitrogen concentration and specific leaf weight) in retaining leaf greenness will be examined.
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Materials and methods |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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The water regime treatments were No Deficit (ND), Post-Flowering Deficit (PFD) and Terminal (pre- and post-flowering) Deficit (TD). The two contrasting water-limited environments (PFD and TD) were based on the classifications of Ludlow and Muchow (1990) for crop production in the semi-arid tropics. Intermittent stress reflects a variable environment, with stress occurring at any time and with varying intensities throughout crop growth. PFD represents one pattern of intermittent stress. Terminal stress typifies crop growth on a progressively depleted soil moisture profile. All water regime treatments were covered with black plastic prior to sowing to exclude rainfall and prevent evaporation losses, although an additional 80 mm of water entered the soil profile through the plastic in series of rainfall events near anthesis. Post anthesis biomass production was significantly less (P < 0.01) under TD compared with ND (756 vs. 1089 g m-2) (Borrell et al., 2000b).
Nine hybrids were examined from crosses of three females varying in stay-green (AQL39, senescent; AQL41, intermediate; A35, stay-green) and three males similarly varying (R69264, senescent; RQL36, intermediate; RQL12, stay-green). A35 is the male-sterile version of B35. The B35 and KS19 sources of stay-green are derived from sorghum lines native to Ethiopia and Nigeria, respectively. B35 (SC 35-6) is a derivative from the sorghum conversion program BC1 (Stephens et al., 1967) of `IS 12555', a durra landrace from Ethiopia. RQL12 is a BC4 derivative of KS19, which in turn was derived from the cross between Combine Kafir-60 and Short Kaura, the latter being from Nigeria. AQL41, a female line with an intermediate level of the B35 source of stay-green, was derived from the cross between QL33 and B35. RQL36 (TAM2566*2//KS19*4/Krish13///Tx2767), a male line with an intermediate level of the KS19 source of stay-green, was derived from the cross between QL27 and Tx2767, the former being a KS19 derivative. Hybrid parents were initially characterized for stay-green by visually rating these lines on green leaf area at maturity in a range of multi-environment trials over a number of years within the Australian sorghum breeding program. This 3-by-3 matrix enabled the B35 source of stay-green to be examined in crosses with three males (R69264, RQL36 and RQL12) and the KS19 source of stay-green to be examined in crosses with three females (AQL39, AQL41, and A35).
Soil type was a cracking and self-mulching grey clay with abundant calcium carbonate concretions (Elphinstone depositional, McKeown, 1978; Ug 5.16, Northcote, 1974). At the surface (0–0.1 m) pH, EC, and Cl were 7.9, 0.125 mS/cm, and 15 mg/kg, respectively, and increased to 9.1, 0.366 mS/cm, and 74 mg/kg, respectively, at depth (0.8–0.9 m). Organic carbon was 1.3% at the surface (0–0.1 m), decreasing to 0.6% at depth (0.8–0.9 m). Nitrate-N and ammonium-N were 10.4 and 4 mg/kg, respectively, at the surface (0–0.1 m), and decreased to 7.4 and 2 mg/kg, respectively, at depth (0.8–0.9 m). The experiment site was fertilized on, 19 Oct. 1994 with 300 kg N ha-1 as urea. Two days later a mixture containing P, 40; K, 30; Zn, 10 kg ha-1 was applied. The site was rotary-hoed immediately after fertilizer application to break down larger clods for ridge construction and seedbed preparation. The experiment was hand-sown on 15 Dec. 1994, emerged on 18 Dec. 1994, and seedlings were thinned to 10 plants per meter row on 3 Jan. 1995.
Sampling and Analyses
Chemical Analyses of Plant Tissue for N Concentration
A single row of length 1 m was cut from the center three rows of each plot at 30, 46, 59 (A+3d), 87, and 114 d after emergence (DAE). Green leaf area was determined for each plot at all harvest times with an electronic planimeter (Delta-T DIAS image analysis system, Cambridge England). For partly senesced leaves, the senesced portion was cut away from the leaf prior to measurement so that only green leaf area was determined. Harvests at 30, 59, and 114 DAE corresponded to the phenological stages of panicle initiation, anthesis, and physiological maturity. Each sample was dried in a forced draft oven at 80 °C for 48 h before weighing. All samples at 59 and 114 DAE, and TD samples also at 87 DAE, were divided into mainstem and tiller components, then further partitioned into green leaf, senesced leaf, stem (including leaf sheaths), and panicle. Dry matter samples harvested at 59, 87, and 114 DAE were analyzed for concentration of N and, together with dry matter yields, were used to calculate content of N in the various organs. Nitrogen in the senesced tissue was included in the total aboveground N content for all harvests subsequent to the commencement of leaf senescence. To determine N concentration in the various tissues (e.g., leaf N concentration, LNC), a subsample (about 0.25 g) of finely ground (1 mm) tissue was digested in sulphuric acid–sodium sulphate mixture using a selenium catalyst in a semi-micro Kjeldahl apparatus. The digestate was diluted prior to automated colorimetric determination of N using the indophenol reaction with salicyclate and a nitroprusside catalyst after modification of the Bertholot reaction.
Nitrogen Use Efficiency
Nitrogen use efficiency (NUE) is defined as aboveground dry mass divided by aboveground nitrogen content (kg kg-1).
Specific Leaf Nitrogen and Specific Leaf Weight
Specific leaf nitrogen (SLN) is positively correlated with photosynthetic capacity in maize (Sinclair and Horie, 1989) and sorghum (Muchow and Sinclair, 1994), and was used in this study as such an indicator. SLN is defined as leaf N content per unit leaf area (g N m-2). Specific leaf weight (SLW) is defined as leaf mass per unit area, and is usually directly proportional to leaf thickness.
Statistical Analyses
Unless otherwise stated, statistical differences are referred to as significant when they differ at the
level, i.e, P < 0.05 denotes significance. Data were analyzed by standard analyses of variance and pairwise comparisons of means were performed by the protected LSD procedure at
(Carmer and Swanson, 1973). Correlations among variables were computed.
Total N content was the sum of the N contents of various plant components, i.e., green leaf, dead leaf, stem, rachis, and grain. At anthesis, stem N% was missing in one of 27 plots, green leaf N% was missing in three of 27 plots, and deaf leaf N% was missing in one of 27 plots, resulting in four of 27 missing values for total N content (in one case both stem N% and green leaf N% were missing from the same plot). At maturity, stem N% was missing in five of 27 plots, and grain N% was missing in one of 27 plots, resulting in six of 27 missing values for total N content. To calculate total N content, these missing values were replaced with a missing value estimate obtained from individual analyses of variances of these variables. The output from these analyses was used for Tables 6 and 7 .
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Results and discussion |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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,
). Under these conditions, stay-green hybrids (A35/RQL36, AQL39/RQL12, AQL41/RQL12, and A35/RQL12; filled symbols) took up more (P < 0.01) nitrogen from the soil compared with the senescent hybrids (AQL39/R69264, AQL41/R69264, A35/R69264, AQL39/RQL36, and AQL41/RQL36; open symbols). Since green leaf area at maturity explained over 70% of the variation in grain growth rate under TD (Borrell et al. 2000b), the relation between N content and green leaf area index (LAI) under TD was examined at anthesis, mid grain fill and maturity (Fig. 2)
. At anthesis, N content was positively correlated
with green LAI, although there was no trend for higher N content in stay-green types (filled symbols) at this time (Fig. 2a). By mid grain filling, there was a trend for stay-green hybrids to exceed senescent hybrids (open symbols) in N content (Fig. 2b), although the correlation between N content and green LAI was weaker
than at anthesis. At maturity, N content was correlated
with green LAI, and was significantly higher in the stay-green hybrids compared with the senescent hybrids (260 vs. 210 kg N ha-1), and this was associated with higher green LAI in the stay-green than senescent hybrids (1.85 vs. 0.87) (Fig. 2c).
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Partitioning of Nitrogen among Organs
Expression of stay-green is a consequence of the balance between N demand by the grain and N supply during grain filling. To better understand this balance in the B35 and KS19 sources of stay-green, the partitioning of nitrogen among the leaf, stem and panicle was examined in the TD treatment. Only the results for the B35 source of stay-green will be discussed since trends were similar for the KS19 source. N concentration in organs will be discussed first, followed by N content in organs.
Three females varying in rate of leaf senescence (AQL39, senescent; AQL41, intermediate; A35, stay-green) were examined in crosses with a common male (RQL36, intermediate) to determine the patterns of nitrogen partitioning in hybrids varying in the B35 source of stay-green. The N concentration in the green leaf declined from about 3% at anthesis to 1.5% at maturity (Fig 3a) . Green leaf N concentration at mid-grain fill was higher in A35/RQL36 (stay-green) compared with AQL41/RQL36 (intermediate) and AQL39/RQL36 (senescent), and a similar trend existed at anthesis. At maturity, green leaf N concentration was higher in the stay-green hybrid compared with the intermediate hybrid. No differences in dead leaf N concentration among genotypes were observed at any of the harvest times (Fig. 3b).
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The N concentration in the panicle was consistently higher in the stay-green and intermediate hybrids compared with the senescent hybrid throughout the grain filling period, although these differences were only significant at mid grain fill (Fig. 3d).
During the first half of the grain filling period, green leaf N content increased in A35/RQL36 (Fig. 4a) . Over the same period, N content declined in AQL41/RQL36 (intermediate) and AQL39/RQL36 (senescent), so that green leaf N content at mid grain fill was higher in the stay-green hybrid (75 kg ha-1) than in the intermediate (51 kg ha-1) or senescent (44 kg ha-1) hybrids. Green leaf N content declined markedly in all hybrids during the second half of the grain filling period.
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Nitrogen content in the stem was always higher in the stay-green than in the senescent hybrid (Fig. 4c). There was no difference in stem N content between the stay-green and intermediate hybrids at anthesis or mid grain fill. N content in the stem was less, however, in the intermediate hybrid at maturity.
During the first half of the grain filling period, N accumulated in the panicle at a greater rate in the stay-green and intermediate hybrids than in the senescent hybrid (Fig. 4d). Nitrogen continued to accumulate in the panicle of the stay-green hybrid at a high rate during the second half of grain filling, but accumulation slowed in the intermediate and senescent hybrids, resulting in less grain N content at maturity in the senescent hybrid (132 kg ha-1) compared with the stay-green hybrid (201 kg ha-1).
For both the B35 and KS19 sources of stay-green, N concentration in the green leaves was higher in the stay-green than senescent hybrids at mid grain fill and maturity, and a similar trend was observed at anthesis. Indeed, N content in the green leaf increased during the first half of the grain filling period in the stay-green hybrids, yet declined in the intermediate and senescent hybrids, indicating a period of active photosynthesis in the stay-green types according to the leaf development model proposed by Thomas and Smart (1993). It is not surprising then that differences in leaf N concentration and content were observed at maturity among hybrids varying in rate of leaf senescence, since increased growth in stay-green hybrids would be expected to be associated with increased N uptake during the period of trait expression in the latter half of the grain filling period. Before the trait was apparent visually, however, differences in leaf N status were observed at anthesis between stay-green and senescent hybrids. While this may at first appear surprising, it can be explained by viewing stay-green as a consequence of the balance between N supply and demand. This will be discussed later in the section Balancing Nitrogen Supply and Demand.
Specific Leaf Nitrogen
Water regime and genotype did not interact for specific leaf nitrogen (SLN) at anthesis or maturity. SLN was not affected by water regime at anthesis or maturity. Significant genotypic variation in SLN, however, was observed at anthesis and maturity across all water regimes (Table 2)
, and at mid-grain filling under TD (SLN was not measured in ND or PFD at mid-grain filling). SLN was higher (P < 0.01) in stay-green (A35 and RQL12) hybrids compared with intermediate (AQL41 and RQL36) and senescent (AQL39 and R69264) hybrids at all sampling times.
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with green LAI at maturity under terminal water deficit (Fig. 5)
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. This suggests that, under drought, the retention of green leaf area during the latter half of the grain filling period is dependent on SLN and, if SLN falls below a critical level, the leaves will senesce. Thomas and Smart (1993) proposed a model of foliar development in which the career of a normal leaf terminates with nitrogen remobilization and subsequent breakdown of integrity. They suggested that the attainment of photosynthetic capability and the subsequent reduction in assimilation may be driven by the rate of export of nitrogen. The concept of a threshold SLN level below which senescence will occur supports this proposition. Maintaining adequate levels of leaf N is important because of the relationship between SLN and leaf CO2 assimilation rate observed in a number of crops, including sorghum (Muchow and Sinclair, 1994). A substantial fraction of the leaf N is associated with the photosynthetic apparatus (Sinclair and Horie, 1989). For example in C4 leaves, ribulose 1,5-biphosphate carboxylase accounts for 10 to 20% of the soluble protein (Schmitt and Edwards, 1981), and it is estimated that phosphoenolpyruvate carboxylase accounts for another 10% (Brown, 1978). Therefore, SLN reflects the leaf potential for CO2 assimilation rate per unit leaf area.
Components of Specific Leaf Nitrogen
Why do leaves of stay-green hybrids have a higher SLN than their intermediate and senescent counterparts? In an attempt to answer this question, SLN was examined in terms of its components: leaf nitrogen concentration (LNC) and specific leaf weight (SLW). Although SLN, LNC, and SLW are not independent variables, it is instructive nonetheless to examine the relationships among these variables.
No interaction was observed between genotype and water regime for SLW at anthesis or maturity. At anthesis, SLW was lower in ND (49.8 g/m2) and TD (50.5 g/m2) compared with PFD (51.7 g/m2), and at maturity was lower in ND (66.6 g/m2) compared with PFD (74.4 g/m2) and TD (76.9 g/m2). Genotypic variation in SLW was observed at anthesis and maturity (Table 3) , although these differences were only associated with the B35 source of stay-green.
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with LNC (Fig. 6a)
, and stay-green hybrids exceeded senescent hybrids in both LNC (3.11 vs. 2.83 mg) and SLN (1.54 vs. 1.43 g N m-2). SLN was also correlated
with SLW (Fig. 6b), although the response varied between the B35 and KS19 sources of stay-green. For the B35 source, SLN was correlated (P < 0.001) with LNC in two of three backgrounds and was correlated (P < 0.05) with SLW in all three backgrounds (Table 4)
, indicating that leaves with this type of stay-green were thicker and contained more N compared with their senescent counterparts. For the KS19 source of stay-green, SLN was correlated (P < 0.01) with LNC in all three backgrounds and was correlated (P < 0.05) with SLW in two of three backgrounds (Table 5)
. Although leaves of hybrids with this type of stay-green contained more N than those of related senescent hybrids, as evidenced by higher LNC, SLW was equivalent in both stay-green and senescent hybrids. This suggests that the leaves of hybrids with the KS19 source of stay-green were not thicker than their senescent counterparts, but rather, they contained more N per unit thickness. This is evidenced by the high proportion of points representing the KS19 source of stay-green above the SLW-SLN regression line in Fig. 6b.
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Leaf Thickness
The ability to adjust leaf thickness to the water status of the environment may be an adaptive mechanism for plants to increase productivity under drought. Alagarswamy et al. (1988) reported a similar response for a landrace cultivar of pearl Millet [Pennisetum glaucum (L.) R. Br.] which reacted opportunistically by increasing leaf thickness when supplied with additional N in greenhouse studies. But how does a leaf increase its thickness? During early crop growth, there is generally a trade-off between individual leaf size and leaf thickness. For example, Sinclair and Horie (1989) reported that N was used competitively for the construction of either large leaf area or high leaf N content. In the current study, however, A35 hybrids produced both larger (Borrell et al. 2000a) and thicker leaves compared with their senescent counterparts, suggesting that these hybrids assimilated carbon more efficiently. At anthesis, SLW was equivalent between AQL39 (senescent) and A35 (stay-green) hybrids in crosses with R69264 and RQL36, yet area per leaf was higher in the A35 hybrids (Borrell et al. 2000a). Interestingly, SLN was also higher in the A35 hybrids at anthesis. By maturity, however, SLW, in addition to leaf size, was higher in the A35 hybrids, suggesting that more carbon and N had been partitioned to these leaves during grain filling compared with senescent hybrids. Borrell et al. (2000b) provide data to support this case. They found A35 hybrids increased in green leaf dry mass between anthesis and mid-grain filling, leading to increased leaf thickness, since leaf expansion had already ceased. An alternative explanation is that less assimilates were remobilized out of the leaf during this period in the stay-green compared with the senescent hybrid, thereby maintaining relatively thicker leaves in the stay-green hybrid. This was not the case for AQL39 (senescent) or AQL41 (intermediate) hybrids. The story was somewhat different in crosses with RQL12. Area per leaf was equivalent in the AQL39 and A35 hybrids, yet SLW and SLN were higher in the stay-green than senescent hybrid at both anthesis and maturity. In this case, additional carbon and nitrogen appear to have contributed largely to increased leaf thickness, with little effect on leaf area. Thicker leaves in peanut (Arachis hypogaea L.) crops have been found to be associated with lower minimum temperatures (Bell et al., 1992; Nageswara Rao and Wright, 1994), which may induce deposition of starch and other non-structural carbohydrates in chloroplasts (Araus et al., 1989). The mechanism for increased leaf thickness in A35 hybrids is not clear, although it may simply be the result of increased leaf dry matter production after leaf expansion has ceased.
Associations with Grain Yield
The impact of SLN at anthesis on grain yield at maturity was examined in six crosses under the TD treatment. SLN was correlated with grain yield in five of six crosses, and was correlated to a lesser extent (P < 0.1) in the remaining cross (Tables 4 and 5). The effects of the components of SLN (LNC and SLW) on grain yield were also examined. For the B35 source, yield was correlated with LNC in two of three crosses, and was correlated with SLW in one of three crosses (Table 4). For the KS19 source, yield was correlated with LNC in all three crosses, but was not correlated with SLW in any of the crosses examined (Table 5). Hence, LNC was correlated with yield in five of six crosses, yet SLW was correlated with yield in only one of six crosses. This suggests that, overall, leaf nitrogen status was more critical than leaf thickness for yield determination under post-anthesis drought, since higher LNC was associated with increased retention of green leaf area for both sources of stay-green, yet SLW was associated with stay-green only for the B35 source. The strong association between LNC at anthesis and grain yield under drought suggests that measuring LNC at flowering could be used to screen for drought-resistance in sorghum breeding programs. Chapman and Barreto (1997) have shown that a SPAD chlorophyll meter can be used to estimate LNC in maize, and preliminary studies in sorghum have found good correlations with SLN. In addition, the chlorophyll meter could be used to rate stay-green in breeding lines during the latter half of the grain filling period, instead of the visual approach that is currently used (Borrell et al., 1996).
Marker Assisted Selection of Stay-Green
Structural analysis of genes can be linked to the underlying physiological mechanisms through genome analysis (McCouch and Xiao, 1998). Now that we have the ability to isolate and clone genes, and to map quantitative trait loci (QTL), geneticists and physiologists can glimpse the prize of making the vital connection between the gene and the character (Jones et al., 1997). Marker assisted selection of stay-green should greatly enhance the efficiency of selection for the trait, since expression only occurs in those environments in which post-anthesis drought is sufficiently severe. In a recombinant inbred line population developed from a cross between BQL39 (senescent) and BQL41 (stay-green), three of the four QTL associated with improved stay-green were derived from the BQL41 parent (Tao et al., 2000). BQL41 (B35/BQL33) contains stay-green genes derived from the B35 source. This source of stay-green is currently used in commercial sorghum hybrids in Australia. If stay-green is a consequence of the balance between N supply and demand, then one hypothesis is that genomic regions associated with leaf N status (e.g., SLN, LNC, leaf chlorophyll) will map to one or more of the stay-green QTLs that have already been identified. Leaf chlorophyll at anthesis is currently (1999–2000 season in Australia) being estimated with a SPAD meter in a set of recombinant inbred lines varying in the B35 source of stay-green, with the aim of testing the above hypothesis. If this hypothesis is valid, and molecular markers for leaf greenness are subsequently identified, the efficiency of selecting for stay-green should be further improved. The concept of linking markers to causal processes (e.g., SLN in the stay-green phenomenon) rather than to the phenotype (e.g., retention of green leaf area during grain filling), should enable traits to be more closely linked to the causal genes compared with the phenotyping approach alone.
Balancing Nitrogen Supply and Demand
During the pre-anthesis period, N uptake was correlated with crop growth rate for hybrids grown under ND
, PFD
, and TD
. Although there was no genotypic variation in N content or biomass at anthesis, SLN varied significantly among hybrids, indicating that partitioning of N between leaf and non-leaf components during the pre-anthesis period varied among hybrids, with proportionally more N being allocated to the leaf in stay-green compared with senescent hybrids. Why was more N partitioned to leaf compared with non-leaf components in stay-green than in senescent hybrids before anthesis? One possible explanation is that leaf structure was sufficiently different in stay-green and senescent hybrids to cause differences in sink strength of leaves for N. For example, the correlation between SLN and SLW indicates that leaves of hybrids with the B35 source of stay-green were thicker than their senescent counterparts, and this factor alone could have increased the demand of these leaves for N, ultimately leading to higher leaf N status at anthesis.
During the grain filling period, N uptake was also correlated with crop growth rate for hybrids grown under ND
, PFD
, and TD
. In all water regimes, N uptake during grain filling was correlated with grain number per square meter, but not with grain size. Perhaps sink demand, indicated by grain number per square meter, determined N uptake during grain filling, i.e., those hybrids which set more grains per square meter at anthesis took up more N after anthesis to meet the increased demand for grain growth. For example under TD, an additional 0.57 mg N was taken up for each additional grain that was filled
.
For grain crops such as sorghum, onset and rate of leaf senescence can be viewed as the balance between N demand by the grain and N supply during grain filling. During this period, N is supplied from the soil and from the stem and leaves via translocation (Pan et al., 1986; Ta and Weiland, 1992; Rajcan and Tollenaar, 1999a). If stay-green is a consequence of the balance between N supply and demand, then variation in these components might be observed among hybrids prior to the expression of stay-green. This situation is confirmed in this study by the fact that stay-green and senescent hybrids already differed in SLN at anthesis (supply component) and grain number per square meter (demand component).
On the supply side, N uptake from the soil during grain filling under TD averaged 116 and 82 kg ha-1 for the stay-green (A35/RQL36, AQL39/RQL12, AQL41/RQL12, and A35/RQL12) and senescent (AQL39/R69264, AQL41/R69264, A35/R69264, AQL39/RQL36, and AQL41/RQL36) hybrids, respectively (Table 6). In addition, green leaf N content decreased by 41 and 52 kg N ha-1 during the grain filling period in stay-green and senescent hybrids. This provides an estimate of the amount of N available for grain filling via translocation from the leaf, assuming that all N found its way to the grain (Table 6). On the basis of this assumption, it is also estimated that a further 26 and 28 kg N ha-1 were available via translocation from the stems of stay-green and senescent hybrids (Table 6). Grain N content, the sink for N supply from these various sources, was 10% higher
in the stay-green than senescent hybrids (183 vs. 166 kg N ha-1). Of the N taken up by the grain, 49% was extracted from the soil in senescent hybrids compared with 64% in stay-green hybrids (Table 6). Similarly, in a study of two maize hybrids varying in rate of leaf senescence, Rajcan and Tollenaar (1999b) reported that the proportion of N in the grain derived from post-silking N uptake was 40% for Pride 5 (senescent) and 60% for Pioneer 3902 (stay-green). Weiland and Ta (1992) also observed that increased N supply from the soil during grain filling in maize can lower the amount of N derived from vegetative tissue. On the demand side, grain numbers were 16% higher in the four stay-green hybrids (mean of 33 300 per m-2) than in the five senescent hybrids (mean of 28 700 per m-2). Hence, N content per grain was similar in stay-green (0.55 mg N/grain) and senescent (0.58 mg N/grain) hybrids (Table 6).
If, as we have suggested, the demand for grain N is largely determined by grain number rather than grain size, how then is this demand met? There are three supply-sources of N during grain filling. Understanding how the plant determines which source of N to draw upon, and in what priority, is critical to the stay-green story. Our hypothesis is that, first, N is mobilized from the leaf and stem as part of the ageing process, i.e., age-related senescence. Second, available N is taken up from the soil. Third, if these two sources of N are still unable to meet grain N demand, then additional N can be mobilized from the leaf and, to a lesser extent, the stem, resulting in accelerated leaf senescence. Studies in maize have found that the relative contribution of mobilization and uptake to N supply during grain filling varies among lines (Beauchamp et al., 1976; Below et al., 1981; Pan et al., 1984; Ta and Weiland, 1992).
Data from the current study can be used to support this hypothesis. N supply from age-related (normal) leaf senescence can be estimated for each hybrid by the difference in green leaf N content between anthesis and maturity when both water and nitrogen are non-limiting (ND treatment) (Table 7, column 1). Similarly, the potential amounts of N translocated from the stem to fill the grain during normal leaf senescence can also be estimated for the ND treatment by the difference in stem N content between anthesis and maturity (Table 7, column 2). This component of N supply (baseline for age-related leaf senescence) can then be added to N uptake from the soil during grain filling under TD, providing an estimate of N supply from these two sources (Table 7, column 4). Matching the N supply from these two sources with grain N content under TD reveals that the shortfall in N supply for grain filling in the senescent and stay-green hybrids averaged 41 and 32 kg N ha-1 (Table 7, column 6). This shortfall in N supply was met primarily by the translocation of additional N from the leaves of senescent (34 kg N ha-1) and stay-green (28 kg N ha-1) hybrids, resulting in more accelerated leaf senescence in the senescent than stay-green hybrids (Borrell et al., 2000a). The remaining shortfall was met by translocation of N from the stems. Hence a greater proportion of N was translocated from the leaves compared with the stem. In a study of two maize hybrids differing in leaf canopy senescence, Ta and Weiland (1992) used labeled 15N to measure the rate of N remobilization under field conditions in various maize tissues, including roots. They found that leaves and stem each provided about 45% of the N remobilized during grain filling, while roots contributed about 10%.
Viewing leaf senescence in terms of the balance between N supply and N demand raises another question. Can the genotypic differences in delayed onset and reduced rate of leaf senescence observed by Borrell et al. (2000a) be explained in terms of differences in SLN at anthesis, or variation in N uptake during grain filling, or something else? Onset of leaf senescence was correlated with SLN at anthesis (r = 0.749**, n = 27), but not with N uptake during grain filling. SLN can further be analyzed in terms of its components: leaf nitrogen concentration (LNC) and specific leaf weight (SLW). LNC at anthesis was correlated (r = 0.751**, n = 27) with onset of leaf senescence, but SLW was not. Relative rate of leaf senescence was correlated with SLN at anthesis (r = -0.714*, n = 27), and rate of senescence was correlated with N uptake during the grain filling period (r = -0.720*, n = 27). LNC at anthesis was correlated (r = -0.783**, n = 27) with rate of senescence, but SLW was not. These correlations suggest that leaf N status at anthesis, in particular LNC, was an important determinant of both the onset and rate of leaf senescence during grain filling. It appears that SLN in stay-green hybrids remained above the threshold senescence level for longer than in senescent hybrids for at least three reasons. First, the leaf N benchmark at anthesis was higher in stay-green than senescent hybrids; second, N uptake during grain filling was higher in stay-green than senescent hybrids; and third, the remobilization of N from leaves of stay-green hybrids during grain filling was less compared with that of senescent hybrids.
We are still left with the question as to why stay-green genotypes take up more N during grain filling compared with senescent hybrids. According to Banziger et al. (1999), larger N accumulation at maturity in drought-tolerant maize selections can be interpreted either as (i) causing delayed leaf senescence (Wolfe et al., 1988a), leading to a larger biomass and grain yield; (ii) resulting from delayed senescence, as N assimilation by roots and root growth may have been prolonged (Ta and Weiland, 1992); or (iii) resulting from a more extensive root system and hence larger aboveground biomass (O'Toole and Bland, 1987). Our hypothesis is an integration of the above three interpretations: increased N uptake by stay-green hybrids is a result of greater biomass accumulation during grain filling in response to increased sink demand (higher grain numbers) which, in turn, is the result of increased radiation use efficiency due to higher SLN. Delayed leaf senescence resulting from higher SLN should, in turn, allow more carbon and nitrogen to be allocated to the roots of stay-green hybrids during grain filling, thereby maintaining a greater capacity to extract N from the soil compared with senescent hybrids. Enhanced N uptake during the grain filling period might reflect the ability of the plant to supply the root system with assimilates (Pan et al., 1986; Osaki, 1995), assisting in both root growth and N uptake.
Benefits of Stay-Green under Low Nitrogen Conditions
The current study was conducted under a high N environment (application of 300 kg N ha-1). Would the stay-green trait still enhance grain yield if N supply was significantly less, e.g., 50-100 kg N ha-1 applied? For example, under N-limited conditions, if the developing grain and photosynthetic apparatus are competing for the same pool of N in the leaves, would the stay-green trait still be beneficial? Or would grain be shriveled and low in protein because N is retained in the leaves rather than being remobilized? Borrell et al. (1999) examined 160 recombinant inbred sorghum lines varying in rate of leaf senescence under severe post-anthesis drought at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) in southern India. The experimental site was fertilized prior to sowing with only 40 kg N ha-1. They found that grain size was negatively correlated (r = -0.632***, n = 110) with relative rate of leaf senescence, such that reducing rate of leaf senescence from 3 to 1% loss of leaf area per day resulted in doubling grain size from about 15 to 30 mg. In addition, grain yield was positively correlated (r = 0.643***, n = 110) with green leaf area at 25 d after anthesis, such that increasing GLA from 2000 to 10000 cm2 m-2 resulted in doubling yield from 2.5 to 5 t ha-1. This study at ICRISAT provides evidence that the stay-green trait enhances yield under severe post-anthesis drought and low N conditions. Further work is required to assess the value of the stay-green trait under N-limiting conditions.
Implications for Grain-Growers
Increased N uptake during the grain filling period by stay-green compared with senescent hybrids has implications for nitrogen management of grain sorghum under water-limited conditions. Enhanced predictive ability of seasonal climatic patterns in northern Australia (Stone et al., 1996) now provides sorghum growers with probabilities of rainfall occurrence for their cropping areas prior to sowing. Armed with this knowledge, growers are now able to modify their fertilizer application according to expected yield potential for a given season (Hammer et al., 1996). Genotypic variation in yield and N uptake under drought should also be considered by growers when determining their fertilizer strategy. For example, it is important that the yield potential of stay-green hybrids under drought not be limited by applying insufficient nitrogen.
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ACKNOWLEDGMENTS |
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Received for publication June 22, 1999.
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TOPABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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