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A high leaf-starch mutant in alfalfa has altered invertase activity.

PARTITIONING of photosynthetic assimilates among tissues is a critical process in plant development and one of the primary factors in economic yield. Based on their ability to produce or consume assimilates, plant tissues are classified as either source or sink tissues. Leaves exhibit a sink to source transition during their growth; meristems and emerging leaves are considered sinks; young, expanding leaves are heterotrophic, whereas mature leaves are source tissues (Turgeon, 1989).

The end products of carbon fixation in photosynthetic tissues of alfalfa (Medicago sativa L.) are starch and sucrose. Starch in leaves serves as a temporary storage of recently fixed carbon, whereas sucrose is the principal transport carbohydrate. Thus, sucrose is present in the cytosol during biosynthesis, in the vacuole during transient storage, and in the apoplast and phloem tissue during long distance transport. The transition of a leaf from metabolic sink to source is accompanied by changes in the activities of the carbohydrate metabolizing enzymes: sucrose hydrolyzing enzymes decrease, whereas enzymes involved in sucrose biosynthesis increase (Huber, 1989).

Many physiological studies have inhibited phloem transport or altered source-sink balance in whole plants to gain insight into the sink-source transition in plants. However, inherent complications such as changes in movement of nitrogen, minerals, and hormones have complicated the interpretation of these experiments. Recently, manipulation of source-sink relationships was achieved by molecular techniques with transgenic tobacco (Nicotiana tabacum L.) and Arabidopsis thaliana (L.) Heynh expressing a yeast invertase gene, such (von Schaewen et al., 1990; Sonnewald et al., 1991).

Medicago satira is a perennial forage legume and, therefore, its source-sink relationships are quite dynamic. Sink tissues include the meristematic regions and [N.sub.2]-fixing nodules, and the principal storage sink is the taproot. Carbohydrates stored in the taproot serve as the major source of carbon assimilates during regrowth of the shoot following harvest or winter dormancy, and are replenished as the shoot matures. Therefore, carbohydrate levels in roots have been implicated in the yield and winter hardiness of this crop (Heichel et al., 1988). An alfalfa mutant has been isolated that appeared to have dramatic changes in these source-sink relations (McKersie et al., 1992). The leaves emerged normally, but after full expansion, regions of chlorophyll depletion appeared on the adaxial leaf surface. Ultrastructural observations indicated that many palisade cells had accumulated starch and subsequently exhibited changes that resembled senescence, leading to cellular disintegration and the formation of necrotic regions. In contrast, the mesophyll and other tissues remained normal.

From the previous analysis of the [F.sub.1] crosses between the original hls plant, Ex139, and the normal phenotype, C2-4, a two independent dominant gene model was proposed to explain the inheritance of this phenotype in autotetraploid alfalfa (McKersie et al., 1992). Based on this model, the genetic constitution of the original mutant was postulated to be Aaaa Bbbb and the normal parent a double recessive (aaaa bbbb) with respect to the two genes involved. However, there were uncertainties in the data that were not fully explained by this model. Although [F.sub.1] progeny had both leaf phenotypes, mutant and normal, the observed ratios of the mutant phenotype were higher when the mutant was used as a female parent, indicating preferential maternal inheritance. Therefore, there was a possibility that the genes encoding the hls phenotype were present in the chloroplast genome. In this study, we report the further characterization of this mutant phenotype, which has been designated as high leaf-starch (his), by examining the genetic and biochemical basis for the starch accumulation in the palisade layer.


The original mutant genotype from the cv. Excalibur, designated as Ex139, was compared with and cross pollinated with a normal phenotype, C2-4, a selection from the alfalfa breeding program at the University of Guelph that was developed for its ability to form somatic embryos in culture. A second random selection from cv. Excalibur was also used in some comparisons as an additional control. These plants were propagated by cuttings to maintain the original genotype and grown in 1-L pots containing "Turface", an inert silica clay (Plant Products Co. Ltd., Bramelea, ON). Plants were watered daily and supplemented twice weekly with 20-20-20 N-P-K nutrient solution containing micronutrients (Plant Products Co. Ltd.). Plants were grown in a controlled environment at 20:18 [degrees] C (light:dark), a relative humidity of 60%, and irradiance of 250 [micro] mol [m.sup.-2] [s.sup.-1], and day length of 16 h. Three developmental stages of the leaves were sampled: L0, the very young emerging leaves; L1, the partially expanded leaves, i.e., when the leaves were 30 to 50% unfolded; and L2, the fully expanded leaves.


All flowers were emasculated by the suction method (Tysdal and Garl, 1940) to prevent self-pollination. Reciprocal crosses were made between genotypes Ex139 and C2-4. The [F.sub.1] progenies were screened and two plants were selected from each reciprocal cross, one plant showing the mutant phenotype and one plant having normal leaf appearance. Each of the four [F.sub.1] plants were subsequently backcrossed to Ex139 and C2-4 producing 16 types of [BC.sub.1] progeny, four from each of the [F.sub.1] plants. In addition, four F: generations were obtained by selfing the [F.sub.1] progenies (Table 1). Seeds of each family were scarified in liquid [N.sub.2], germinated on moistened filter paper, and transplanted to the growth conditions described above. The L2 leaves of each plant were classified into one of the two leaf phenotype classes. Chi-square tests using `Yates Correction' (Sokal and Rohlf, 1981) were used to test the goodness of fit of the observed phenotypic class frequencies to the expected phenotypic ratios in each of the crosses.

Table 1. Chi-square goodness of fit values for the B[C.sub.1] and [F.sub.1] alfalfa (Medicago sativa L.) progenies from the crosses involving [F.sub.1] plants derived from the cross between Ex139 and C2-4 segregating for the his character.
Cross                                           Observed number

B[C.sub.1] (hls [F.sub.1] x hls parent)         hls      Normal


(Ex139 x C2-4) x Ex 139                         28         30
Ex139 x (Ex139 x C2-4)                          21         12
(C2-4 x Ex139) x Ex139                          43         43
Ex139 x (C2-4 x Ex139)                          45         29
Pooled -hls [F.sub.1] as female                 71         73
Pooled -hls [F.sub.1] as male                   66         41

B[C.sub.1] (hls [F.sub.1] x normal parent)

(Ex139 X C2-4) x C2-4                            9         32
C2-4 x (Ex139 x C2-4)                            5         32
(C2-4 x Ex139) x C2-4                            3         30
C2-4 x (C2-4 x Ex139)                            2         35
Pooled -hls [F.sub.1] as female                 12         62
Pooled -hls [F.sub.1] as male                    7         78

B[C.sub.1] (hls [F.sub.1] x normal parent)

(Ex139 x C2-4) x Ex139                          10         47
Ex139 x (Ex139 x C2-4)                          13         27
(C2-4 x Ex139) x Ex139                           7         46
Ex139 x (C2-4 x Ex139)                           2         32
Pooled -hls parent as female                    15         59
Pooled -hls parent as male                      17         93

B[C.sub.1] (normal [F.sub.1] x
normal parent)

(Ex139 x C2-4) x C2-4                            0         36
C2-4 x (Ex139 x C2-4)                            0         33
(C2.4 x Ex139) x C2-4                            0         59
C2-4 x (C2-4 x Ex139)                            0         33
Pooled                                           0        161

[F.sub.2] (hls [F.sub.1] x his [F.sub.1])                 3:1

(Ex139 x C2-4) x (Ex139 x C2-4)                 22         31
(C2-4 x Ex139) x (C2-4 x Ex139)                 30         72
Pooled                                          52        103

[F.sub.2] (normal [F.sub.1] x [F.sub.1])

(Ex139 x C2-4) x (Ex139 x C2-4)                  0         94
(C2-4 x Ex139) x (C2-4 x Ex139)                  0         41
Pooled                                           0        135

Cross                                             Ratio tested

B[C.sub.1] (hls [F.sub.1] x hls parent)         3:1          9:7


(Ex139 x C2-4) x Ex 139                       20.69(**)    1.19 NS
Ex139 x (Ex139 x C2-4)                         1.71 NS     0.46 NS
(C2-4 x Ex139) x Ex139                        27.35(**)    1.13 NS
Ex139 x (C2-4 x Ex139)                         7.21(**)    0.45 NS
Pooled -hls [F.sub.1] as female               49.34(**)    2.55 NS
Pooled -hls [F.sub.1] as male                  9.42(**)    1.07 NS

B[C.sub.1] (hls [F.sub.1] x normal parent)     1:1         1:3

(Ex139 X C2-4) x C2-4                         11.80(**)    0.07 NS
C2-4 x (Ex139 x C2-4)                         28.52(**)    4.69(*)
(C2-4 x Ex139) x C2-4                         20.48(**)    3.65 NS
C2-4 x (C2-4 x Ex139)                         27.68(**)    6.57(*)
Pooled -hls [F.sub.1] as female               32.45(**)    2.59 NS
Pooled -hls [F.sub.1] as male                 57.65(**)   11.86(**)

B[C.sub.1] (hls [F.sub.1] x normal parent)     1:01        1:3

(Ex139 x C2-4) x Ex139                        22.74(**)    1.32 NS
Ex139 x (Ex139 x C2-4)                         4.23(*)     0.83 NS
(C2-4 x Ex139) x Ex139                        27.25(**)    3.33 NS
Ex139 x (C2-4 x Ex139)                        24.74(**)    5.65(*)
Pooled -hls parent as female                  24.99(**)    0.65 NS
Pooled -hls parent as male                    51.14(**)    4.85(*)

B[C.sub.1] (normal [F.sub.1] x
normal parent)

(Ex139 x C2-4) x C2-4                         0:infinity
C2-4 x (Ex139 x C2-4)                             -
(C2.4 x Ex139) x C2-4                             -
C2-4 x (C2-4 x Ex139)                             -
Pooled                                            -

[F.sub.2] (hls [F.sub.1] x his [F.sub.1])     9:7         33:15

(Ex139 x C2-4) x (Ex139 x C2-4)               29.94(**)    4.10(*)
(C2-4 x Ex139) x (C2-4 x Ex139)              110.64(**)   28.77(**)
Pooled                                       139.84(**)   31.54(**)

[F.sub.2] (normal [F.sub.1] x [F.sub.1])      0:infinity

(Ex139 x C2-4) x (Ex139 x C2-4)                     -
(C2-4 x Ex139) x (C2-4 x Ex139)                     -
Pooled                                              -

Cross                                             Ratio tested

B[C.sub.1] (hls [F.sub.1] x hls parent)       33:15        105:33


(Ex139 x C2-4) x Ex 139                      10.38(**)    16.61(**)
Ex139 x (Ex139 x C2-4)                        0.20 NS      1.01 NS
(C2-4 x Ex139) x Ex139                       13.21(**)    21.72(**)
Ex139 x (C2-4 x Ex139)                        1.82 NS      4.90(*)
Pooled -hls [F.sub.1] as female              24.44(**)    39.46(**)
Pooled -hls [F.sub.1] as male                 2.17 NS      6.28(*)

B[C.sub.1] (hls [F.sub.1] x normal parent)    3:5          5:7

(Ex139 X C2-4) x C2-4                         3.59 NS      5.77(*)
C2-4 x (Ex139 x C2-4)                        13.89(**)    18.02(**)
(C2-4 x Ex139) x C2-4                        10.18(**)    13.10(**)
C2-4 x (C2-4 x Ex139)                        14.92(**)    18.55(**)
Pooled -hls [F.sub.1] as female              13.41(**)    18.69(**)
Pooled -hls [F.sub.1] as male                29.82(**)    37.72(**)

B[C.sub.1] (hls [F.sub.1] x normal parent)    3:5          5:7

(Ex139 x C2-4) x Ex139                        8.85(**)    12.67(**)
Ex139 x (Ex139 x C2-4)                        0.24 NS      1.03 NS
(C2-4 x Ex139) x Ex139                       12.33(**)    16.51(**)
Ex139 x (C2-4 x Ex139)                       13.18(**)    16.47(**)
Pooled -hls parent as female                  8.65(**)    13.07(**)
Pooled -hls parent as male                   21.88(**)    30.03(**)

B[C.sub.1] (normal [F.sub.1] x
normal parent)

(Ex139 x C2-4) x C2-4
C2-4 x (Ex139 x C2-4)
(C2.4 x Ex139) x C2-4
C2-4 x (C2-4 x Ex139)

[F.sub.2] (hls [F.sub.1] x his [F.sub.1])    105:39

(Ex139 x C2-4) x (Ex139 x C2-4)              17.06(**)    24.91(**)
(C2-4 x Ex139) x (C2-4 x Ex139)              71.64(**)    95.57(**)
Pooled                                       87.77(**)   119.66(**)

[F.sub.2] (normal [F.sub.1] x [F.sub.1])

(Ex139 x C2-4) x (Ex139 x C2-4)
(C2-4 x Ex139) x (C2-4 x Ex139)

(*), (**) means significant different at P = 0.05 and P = 0.01, respectively.

NS, means not significantly different P = 0.05.

Determination of Leaf and Root Starch

Leaf starch content was determined from leaves at the L1 and L2 stages of development, whereas the starch content of taproots was determined with flowering plants. The roots and leaflets were cut into small pieces, dried at 70 [degrees] C for 48 h and ground to pass a 1-mm screen and stored in a sealed container at -20 [degrees] C until starch was extracted. Starch was extracted and determined according to the enzymatic procedure of Rose et al. (1991). The leaf experiment was arranged as a split-plot (L1 and L2 were subplots; genotypes were the main plots) in a completely randomized design with four replications. The root experiment was arranged as a randomized design with three replications.

Characterization of Starch and Sucrose Metabolizing Enzymes

Amylase was extracted following the methods of Steup and Latzko (1979) and native PAGE-activity staining and total amylase activity were conducted according to Lin et al. (1988). Starch phosphorylases were extracted following the methods used for amylase and activity measured as described by Steup and Latzko (1979). Native-PAGE gels were incubated at 30 [degrees] C in one of the following mixtures: (i) 1 g [kg.sup.-1] soluble starch in 0.1 M citrate buffer (pH 6.0) containing 20 mmol glucose 1-phosphate (GIP), (ii) 4 g [kg.sup.-1] starch in 0.1 M citrate buffer containing 50 mmol inorganic orthophosphate (Pi). After incubation, the gels were stained with a solution containing 10 mmol [I.sub.2] and 14 mmol KI and stored in a mixture of 300 g [kg.sup.-1] methanol and 50 g [kg.sup.-1] acetic acid.

Total leaf extracts for hexokinase, phosphoglucomutase, hexose phosphate isomerase, and phosphofructokinase were collected by homogenizing the leaf tissues in 40 mM Tris-HCl buffer (pH 7.0). Similarly, total aldolase and triosephosphate isomerase were extracted by homogenizing the leaf tissues with 100 mM imidazole-HCl buffer (pH 7.0). Uridine diphosphate glucose pyrophosphorylase was extracted in a buffer containing 50 mM Tris-HCL (pH 7.5), 1 mM EDTA, and 10 mM 2-mercaptoethanol and analyzed according to Gordon and Ordin (1974). Sucrose phosphate phosphatase extraction was conducted as described by Whitaker (1984).

Hexokinase and phosphoglucomutase were assayed as described by Stitt et al. (1978); phosphoglucose isomerase, triosephosphate isomerase, aldolase, and uridine diphosphate glucose pyrophosphorylase assayed according to Bergmeyer et al. (1974); phosphofructokinase assayed according to Kelly and Latzko (1977); fructose-1,6-bisphosphatase assayed according to Zimmermann et al. (1976), and sucrose phosphate phosphatase assayed by the discontinuous method of Whitaker (1984).

Leaf invertases were extracted at the L0, L1, and L2 developmental stages, quantified by enzyme specific activity as described by Krishnan et al. (1985), and characterized by native PAGE-activity staining as described by yon Schaewen et al. (1990). Images of the PAGE gels were captured with a CCD video camera and Northern Exposure Imaging System (Empix Imaging Inc., Mississauga, ON).

Sucrose synthase and sucrose phosphate synthase were assayed in extracts from leaves (0.3-0.6 g FW) that were frozen and ground in liquid [N.sub.2], homogenized in extraction buffer, centrifuged, frozen in liquid [N.sub.2] and stored until use. Samples were then assayed either as a crude extract, partially purified by affinity chromatography on an aminohexylagarose column, partially purified by size exclusion chromatography (Bio-Rad Biogel P6 column; Bio-Rad Lab., Richmond, CA), or "deactivated" by boiling for 5 min as a control to distinguish between apparent enzyme activity versus non-enzymatic reactions. Reactions for sucrose synthase and sucrose phosphate synthase were conducted according to Stitt et al. (1988). Products of the reactions were measured by one or more of the following methods: anthrone (Huber et al., 1996, Guy et al., 1992) or resorcinol (Rufty and Huber, 1983) spectrophotometric detection of sugar products, uridine diphosphate (UDP) spectrophotometric detection via pyruvate kinase and lactate dehyrogenase (Morell and Copeland, 1985), or direct measurement of sugars by gas chromatography. Highly purified sucrose synthase (Sigma Chemical S6379; Sigma Chemical Co., St. Louis) was added to some samples as an internal control.


Genetic Basis of the Mutant Phenotype

To test the two-gene nuclear model against the chloroplast-gene model, backcrosses of the parents (Ex139 and C2-4) with their F1 progeny (visually classified as either his or normal) were conducted as shown in Table 1. Assuming the two gene model, a cross between the original hls genotype, Ex139, and a [F.sub.1] plant with the hls phenotype would give progeny in the ratio of 9:7 (hls:normal). Similarly, self-pollination of an [F.sub.1] his phenotype would also give progeny in the ratio of 9:7 (hls:normal). If chloroplast genes controlled the expression of the phenotype, this frequency would not be expected in the progeny. In addition, chimeric plants would be expected because of the random sorting of the plastids carrying the mutation. Evidence for chloroplast inheritance was not observed. All progenies from crosses between [F.sub.1] his plants and Ex139 segregated according to the expected 9:7 ratio (Table 1) supporting the nuclear gene model that the original Ex139 genotype was a diallelic simplex at both mutated loci (Aaaa Bbbb). However, fewer progeny from self-pollination of the [F.sub.1] plants exhibited hls than expected from the 9:7 ratio (Table 1), perhaps as a consequence of inbreeding depression that is common in alfalfa.

Crosses of the [F.sub.1] progeny exhibiting the hls phenotype with a plant exhibiting a normal phenotype, C2-4, segregated in the 1:3 (hls:normal) ratio as predicted by the two-gene nuclear model, but only when hls was the female parent (Table 1). When hls was the male parent, transmission of his was less than predicted from this genetic model, confirming the original observation (McKersie et al., 1992).

[F.sub.1] plants not exhibiting the hls phenotype were backcrossed to the normal-leaf phenotype, C2-4, and self-pollinated. In the 296 progeny examined, none exhibited the hls phenotype (Table 1). This further supports the dominant two-gene model for his indicating that his was expressed only when both loci have at least one dominant allele.

Alteration in Starch Dynamics Between Leaves and Taproots

The starch content of the leaves in Ex139 was not significantly different from that in C2-4 when the leaves were 30 to 50% expanded (L1 stage; Table 2). However, fully expanded leaves (L2) of Ex139 had six times more starch than C2-4. The starch content in taproots of Ex139 was less that of C2-4 at flowering.

Table 2. Starch content of partially expanded and fully expanded leaves and taproots of two alfalfa (Medicago sativa L.) genotypes with normal leaf type (C2-4) and hls leaf type (Ex139).
                              Partially     Fully
                              expanded      expanded
Genotype        Phenotype      leaf         leaf        Taproot

                                       g/kg dry matter

C2-4            normal          3.7           4.3         243
Ex139           hls             5.7          25.3          89
Significance of t-test           NS          (**)         (**)

(*), (**) means significantly different at P = 0.05 and P = 0.01, respectively.

NS, means not significantly different P = 0.05.

Crosses between Ex139 and C2-4 produced two types of [F.sub.1] progeny: those with symptoms of hls, such as necrotic sectors on fully expanded leaves, and those with visually normal leaves. To determine if the increased starch accumulation co-segregated with the visual appearance of the hls trait in the [F.sub.1] generation, normal and mutant-leaf phenotypes were visually selected and starch quantified in the fully expanded leaves. The [F.sub.1] progeny with the visual hls phenotype had 14.4 g [kg.sup.-1] starch in their L2 leaves, which was three fold greater than that found in those [F.sub.1] progeny exhibiting the normal-leaf type (Table 3). Thus, the elevated starch level in the leaves was coincident with the visual appearance of the hls phenotype.

Table 3. Leaf starch content in fully expanded leaves and invertase activity of partially expanded leaves from normal and his mutant [F.sub.1] progeny from the cross of Ex139 x C2-4.
                         Number of
Phenotype                progeny      Starch          Invertase

                            no.     g [kg.sup.-1]     protein

Normal                      10          4.8             62.1
hls                          5         14.4             79.3
Significance of t-test                 (**)             (**)

(*), (**) means significantly different at P = 0.05 and P = 0.01, respectively.

Enzymes of the Pathways of Starch Degradation and Carbohydrate Metabolism

Electron micrographs showed the accumulation of starch in the palisade layer of the hls phenotype as the leaves matured (McKersie et al., 1992). These observations suggested that the accumulation of starch in the source leaves was due to a restriction at some point along the path between photosynthesis in the source leaf and starch accumulation in the root. No morphological differences were observed previously in the phloem (McKersie et al., 1992) suggesting that long distance transport was not disrupted. Therefore, our hypothesis was that the restriction in carbohydrate translocation from the source leaf was due to a change in the activity of one or more carbohydrate metabolizing enzyme(s). Transitory starch in the source leaf is degraded by starch degrading enzymes including amylase and starch phosphorylase. No difference was detected between the mutant and normal leaves for activity of either of these enzymes (Table 4). Native PAGE followed by activity staining for these two enzymes also did not show any distinction between the two phenotypes (data not shown).

Table 4. Activities of starch-degrading, glycolytic and sucrose-synthesizing enzymes in total leaf extracts from the hls genotype Ex139 and the normal genotype C2-4 of alfalfa.
                                      Enzyme activity

Ensyme                            C2-4 (normal)    Ex139 (hls)

                                  units [g.zup.-1] protein

Amylase                               34.7           35.3
Starch phosphorylase                  15.7           15.3
Hexokinase                            0.95           1.01
Phosphoglucomutase                    0.84           0.80
Phosphohexose isomerase               1.88           1.87
Phosphofruetokinase                   0.84           0.83
Aldolase                              6.9            6.5
Triosephosphate isomerase            125            126
Fructose-1,6-bisphosphatase            2.8            2.7
UDP glucose pyrophosphorylase          0.5            0.5
Sucrose-P phosphalase                 18             17
Invertase                             61.4           87.3

Ensyme                               of t-test

Amylase                                  NS
Starch phosphorylase                     NS
Hexokinase                               NS
Phosphoglucomutase                       NS
Phosphohexose isomerase                  NS
Phosphofruetokinase                      NS
Aldolase                                 NS
Triosephosphate isomerase                NS
Fructose-1,6-bisphosphatase              NS
UDP glucose pyrophosphorylase            NS
Sucrose-P phosphalase                    NS
Invertase                               (**)

(*), (**) means significantly different at P = 0.05 and P = 0.01, respectively.

NS, means not significantly different P = 0.05.

The products from hydrolysis and/or phosphorolysis of starch are subsequently metabolized through the glycolytic and oxidative pentose phosphate pathways to give products that are either metabolized by the chloroplasts or exported to the cytoplasm for sucrose biosynthesis (Stitt, 1990). The activities of the enzymes of the glycolytic pathway in Ex139 were similar to those in plants with normal leaf morphology in both total leaf extracts (Table 4) and the chloroplastic fraction (data not shown). Similarly, the enzyme activities of the sucrose biosynthetic pathway were not significantly different between Ex139 and the normal-leaf phenotype.

None of the assays detected sucrose synthase or sucrose phosphate synthase activity in crude or purified leaf extracts from alfalfa. If exogenous UDP was added to the reaction mixture, the UDP was detected, indicating that the leaf extract was not inhibiting the actual detection of the enzyme product. Between 0.1 and 5 units of commercial sucrose synthase could be detected in a standard assay. If the same amount of this sucrose synthase was added to the alfalfa leaf extracts, no sucrose synthase activity was detected by measuring either UDP spectrophotometrically or sugars by gas chromatography. Therefore, the alfalfa leaf extract contained some factor that inhibited the sucrose synthase and sucrose phosphate synthase enzymes, and therefore their activities were not measured in the hls mutants.

Altered Acid Invertase Activity in Mutant Leaves

Sucrose synthesized in the cytosol is either exported from the leaf or transiently stored in the vacuole where it can be hydrolyzed by the enzyme invertase. At the L1 (heterotrophic) stage, leaves of Ex139 had 40% more invertase activity than did the leaves of C2-4 (Table 4). At the L1 stage of leaf development, there were no chlorotic symptoms on the his leaves of Ex139. In the [F.sub.1] progeny of Ex139 x C2-4, his phenotypes had an average invertase activity of 79.3 units [g.sup.-1] protein compared with 62.1 units [g.sup.-1] protein for progeny with the normal phenotype (Table 3). Thus, both elevated starch level and increased invertase activity co-segregated with visual leaf necrosis among these [F.sub.1] progeny.

The native PAGE separation of the total leaf extract followed by activity staining for invertase revealed two isozymes with different patterns of expression during development (Fig. 1). The fast moving invertase isozyme (F) was active in leaves from the C2-4 normal-leaf phenotype at the L0 stage but was not present at the L1 stage. In contrast, this isozyme in the Ex139 hls mutant was active at both the L0 and L1 stages. Furthermore, in the [F.sub.1] progeny showing normal-leaf phenotype from the cross, Ex139 x C2-4, the fast moving invertase isozyme (F) was present at the L0 stage, but it was not present at the L1 stage (data not shown). As was the case with Ex139, all [F.sub.1] progeny with the hls phenotype showed the fast moving band at the L1 stage (data not shown). The slow moving band (S) was not differentially expressed during development nor was it differentially expressed among the [F.sub.1] genotypes.



The inheritance data presented in this study confirm that the two-locus dominant gene model best explains the segregation of the his phenotype, with the original mutant genotype of Ex139 being Aaaa Bbbb (McKersie et al., 1992). In some crosses, notably those to the C2-4 normal phenotype parent, there was reduced transmission of the hls trait through the male parent. The trait does not appear to be encoded in the chloroplast genome. Because chloroplasts originate from the pre-existing chloroplasts by replication and partition randomly during mitosis, one would anticipate cell lineage variegation and chimeric plants if the mutation was in the chloroplast genome. However, this was not observed in any plant expressing the hls trait. Similarly, the direction of the initial cross might have influenced inheritance of the hls trait in subsequent generations (i.e., progeny of Ex139 x C2-4 would differ from those of C2-4 x Ex139), but this was not observed either. Therefore, the most probable cause of the difference between maternal and paternal inheritance of the hls phenotype in some crosses is that the mutation caused a competitive disadvantage in either the male gamete or zygote. Inbreeding depression and self-incompatibility are further complicating factors that may have contributed to the lower than expected transmission of hls to the progeny in some crosses.

Removal of leaf starch from the chloroplast and carbohydrate transport to the taproot involves several metabolic pathways, including starch degradation, glycolysis, and sucrose synthesis. We postulated that hls mutants had a blockage somewhere along this complex sequence of pathways. The starch degrading enzymes, amylase and starch phosphorylase, have been associated with transitory starch breakdown, but these had identical activities in his and normal-leaf plants, and identical native PAGE banding patterns. Similarly, analysis of enzymes associated with glycolysis or sucrose biosynthesis did not give any indication of their involvement in hls. The only measured enzyme activity that was changed in the hls mutants was invertase (Table 4). Surprisingly, the enzyme activity was increased by the mutation.

In experiments with transgenic tobacco plants, expression of the yeast invertase gene, suc2, caused dramatic phenotypic changes including increased soluble sugars and starch in the leaves, and necrosis of older leaves (von Schaewen et al., 1990). The similarity of the transgenic phenotype to the his trait in alfalfa further supports the concept that the hls mutation involves changes in the regulation of invertase activity. Finally, the co-segregation of the altered expression pattern of the F isozyme of invertase with visual necrosis and starch accumulation confirms that changes in the regulation of invertase activity are associated with the expression of the hls phenotype.

Only the F invertase isozyme appeared to be deregulated in his, so that its activity remained high as the leaf shifted from being a sink to a source leaf. The two genes that appeared to be linked to hls from the inheritance study are probably not the genes for invertase per se, but instead encode proteins that act in the regulation of the invertase enzyme or gene, either directly or indirectly. Transacting factors or invertase inhibitors would be among the candidates (Elliot et al., 1993; Sampietro, 1995).

The altered invertase activity could restrict carbohydrate export from the source leaf in either of two ways. Since the differences in invertase activities were detected in hls when the leaf changed from an importing sink to an exporting source, we propose that in hls the F invertase, which is active in the young sink leaf, remains active in the source leaf, causing the palisade cells to retain the characteristic of a sink tissue even though the leaf is mature. If the F invertase were localized in the vacuole, hexose sugars would accumulate there and subsequently be phosphorylated by hexokinases in the cytosol. This futile cycling of hexose sugars would result in the sequestration of cytosolic [P.sup.i] limiting its counterexchange with triose phosphates within the chloroplasts. The increased ratio of triose phosphates to [P.sub.i] in the chloroplasts would activate the ADP-glucose pyrophosphorylase enzyme for starch synthesis resulting in the accumulation of starch in the chloroplasts. On the other hand, if F invertase were localized in the apoplasm, hydrolysis of apoplastic sucrose would prevent phloem loading. These models can be tested experimentally because they predict changes in the quantities of specific carbohydrates in the hls leaves and the subcellular localization of acid invertase activity.

Although the restriction in carbohydrate export from the source leaf may be due to altered invertase activity alone, there remain several other possible explanations for the his mutation that can not be discounted by the present experimental data. Sucrose synthesis may be blocked at some site that was not measured. Our inability to detect sucrose synthase and sucrose phosphate synthase activity in these leaves does not eliminate this possibility. Even though there were no anatomical differences observed previously in the minor vein phloem of the hls mutant (McKersie et al., 1992), short or long distance transport of sucrose through the phloem may still be blocked. Also, the utilization of carbohydrates in the root may be altered changing the demand for carbohydrate export from the leaf. These alternative hypotheses imply that the correlation of invertase activity with the mutant phenotype occurs because invertase activity is a consequence, not a cause of starch accumulation. Because two independent genes are associated with the mutant phenotype, it is quite probable that more than one enzyme or protein must be modified to create the mutant phenotype.

Although its biochemical basis is not precisely defined, the his mutant exhibited a form of premature senescence in a specific leaf tissue, associated with changes in carbohydrate metabolism. Senescence is genetically "programmed" (Nooden, 1988). The disappearance of chlorophyll and plastid ribosomes, the occurrence of large osmiophilic globuli, dilation of thylakoids, and loss of the chloroplast envelope are common ultrastructural changes observed during leaf senescence. The fully expanded leaves of hls plants showed many of these symptoms that are characteristic of senescence, but only in the palisade cell layer (McKersie et al., 1992). However, starch accumulation as observed in hls does not occur during natural senescence. The biochemical or molecular link between starch accumulation and senescence in hls is not clear at this time, but carbohydrate-modulated repression has been reported for several photosynthetic genes, including chlorophyll a/b binding proteins (Jang and Sheen, 1994). It is interesting to note that in the transgenic tobacco plants ex pressing the invertase suc2 gene (Polle, 1996), there was a correlation between carbohydrate accumulation and oxidative stress, which has also been implicated in mediating senescence in leaves (Leshem et al., 1986).


The authors thank Ms. Lori Wright for technical assistance with the enzyme assays.


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Shankar B. Das, Agriculture and Agri-Food Canada, Saskatoon Res. Centre, 107 Science Place, Saskatoon, SK S7N OX2; Stephen R. Bowley and Bryan D. McKersie, Dep. of Crop Science, Univ. of Guelph, Guelph, ON, N1G 2W1, Canada. Financial support was provided by the Natural Sciences and Engineering Research Council of Canada and the Ontario Ministry of Agriculture, Food and Rural Affairs. Received 8 July, 1996. Bryan D. McKersie, Corresponding author (mckersie@crop.
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Author:Das, Shankar B.; Bowley, Stephen R.; McKersie, Bryan D.
Publication:Crop Science
Date:May 1, 1998
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