Shoot morphology and leaf area during reproduction in spinach
A dioecious species, the flowering regions of staminate and pistillate spinach plants appear quite different from one another (), but prior to the appearance of the inflorescence the young leaves enclose the apical bud and it is impossible visually to distinguish staminate from pistillate plants.
Fig. 1. Photographs of male (staminate, right) and female (pistillate left) spinach plants. The lower photograph shows spinach plants in the second week of the flowering period. The upper photograph shows the upper part of the inflorescence of plants in the fourth (more ...)
Under long-day inductive conditions, inflorescences develop in the axils of the upper leaves, and the internodes elongate. The staminate plants develop small racemes of flowers consisting of little more than clusters of anthers surrounded by green calyxes in the axil of a leaf. Larger axillary inflorescences produce two tiers of flower clusters, separated by a slender stalk, with the clusters not visibly subtended by a leaf. In these male-gamete-producing inflorescences, the shape of the leaves changes gradually from the base of the inflorescence, where the leaves resemble the sagittate leaves below, to the apex, becoming extremely small and ovoid (see Supplementary Fig. S1
online). The pistillate plants produce tiny green sessile flowers, with only the stigmata visible, and the leaves remain sagittate in shape. The upper leaves decline in size compared with the lower ones (see Supplementary Fig. S1
online), but less so than in staminate plants, remaining considerably larger (). The first flowering nodes are usually located 7–10 nodes above the cotyledons. At the onset of flowering in pistillate plants stigmata become visible as the stem elongates to separate young leaves spatially from those still surrounding the apical bud. Staminate plants, in contrast, produce a convex inflorescence bud no longer enclosed by the young leaves.
In the first week following the onset of flowering, both staminate and pistillate plants produced approximately 10 flowering nodes in 10 cm of inflorescence (; see Supplementary Fig. S2
and Table S1
online). Stigmata protruded from the axils of leaves in pistillate plants, but the anthers remained enclosed in the calyxes of staminate plants. By the end of the second week after the start of flowering some anthesis and pollen shedding had begun in staminate plants, the inflorescences of both types having attained about 25 cm in length, with up to 30 flowering nodes. Minute fruits began to appear in the lower leaf axils of the inflorescences of the pistillate plants. In the third week of flowering, plants of both sexes had 50 or more flowering nodes. The staminate plants shed copious amounts of pollen and pistillate plants had several large pointed fruits in the axils of lower leaves, although, near the apex, fruits remained small or indistinguishable from the flowers. At this stage, in many plants of both sexes, age-related senescence of leaves below the inflorescence may have begun. By week 4, the staminate plants showed yellowing nearly up to the level of the inflorescence, and the apical bud had converted into flower buds. The pistillate plants either continued production of flowering nodes or ceased node initiation in week 4, with development of the fruits occurring in either case. The staminate plants exhibited signs of the late, degradative stages of senescence in week 5, with overall yellowing, and sampling of these plants ceased before this age. Pistillate plants continued green, with the fruits enlarging, until the seventh week after the onset of flowering, although the lowest leaves often yellowed and withered with age. The overall senescence stage of the pistillate plants in week 8 corresponded to a similar stage of staminate plants in week 5.
Throughout the reproductive period, the leaf area in the region below the flowering area remained relatively constant (). The large lower leaves in pollen-producing plants at weeks 4 and 5 resulted from the fact that the sturdier plants were more likely to last to that age. The reduction in leaf area in the older pistillate plants was due to the withering of the oldest leaves.
Area of leaves of staminate (black bars) and pistillate plants (grey bars). (A) Area of leaves below the inflorescence; (B) area of leaves within the inflorescence. After week 5, the staminate plants have senesced. Bars represent ±SE.
Leaves inside the inflorescence were larger in the pistillate plants than in the staminate plants (). The two types were similar in the first and second weeks of floral development, but the photosynthetic area in the females increased more rapidly than the males. The highest leaf area average for the staminate plants was 277 cm2 during the fourth week of reproductive development; the pistillate plants averaged 52% larger at that time (421 cm2), and at their maximum in week 6 were 2.2 times larger (615 cm2).
Short-term photosynthate partitioning
Leaves of plants of similar size and age were treated with 14CO2 for 1 h at 1 week intervals. At the earliest flowering stage, 22% of the photosynthate from the leaf subtending the staminate inflorescence (L–1) was partitioned into the flowers (). By comparison, in the pistillate plants, 8.6% went to the flowers, indicating that staminate plants allocated 2.5 times as much of the current photosynthate export to flowering structures than did pistillate plants of the same age (). The similar relative specific activity (RSA), which takes into account sink mass, of staminate and pistillate flowers demonstrates that they have a similar sink strength, about twice the average sink strength of all tissues (). By contrast, the leaves within the inflorescences of both types of plant (most of which were still expanding) drew about 11% of the export at this early stage (), although their RSA was below average ().
Fig. 3. Allocation of radiolabelled photosynthate to different structures from the leaf just below the inflorescence (L-1) to each importing structure at the onset of flowering, within a few days of flowers becoming visible on the plants. (A) As a percentage (more ...)
At 1–3 weeks following the onset of flowering, when staminate flowers were developing and undergoing anthesis, the differences between the partitioning patterns of staminate and pistillate plants had become increasingly large (). Reproductive organs in pollen-producing plants received 4–6.5 times more photosynthate than in pistillate individuals [40% versus 9% (week 1) to 6% (week 3), respectively; ]. The mass of both types of flowers also rose rapidly (see Supplementary Table S1
online). The RSA for the pistillate reproductive structures continued to be similar to that of the staminate flowers through the first week (), but fell to less than half of the comparable value in the second week and dropped even lower in later weeks.
Fig. 4. Allocation of radiolabelled photosynthate from the leaf just below the inflorescence (L–1), over time, from the start of flowering: (A, B) to the flowers or fruits; (C, D) to the leaves and stem below the inflorescence; (E, F) to the apical bud (more ...)
The bulk of the current carbon fixed by the leaf just below the inflorescence was distributed to the tissue below the inflorescence region, being 63% of the exported radioactivity in the earliest stage of flowering in pistillate plants () and more than 50% for pollen-producing plants. However, the proportions become more disparate at later stages. At the time when partitioning to the shoot below the inflorescence declined rapidly for staminate plants (values range from 17–31% after the initial stage) (), distribution to the comparable region in seed-bearing individuals stayed between 1.3 and 3 times higher, only falling to the 30% level well after the staminate plants had senesced. When the allocation to the lower region of the shoot was considered on a mass-corrected basis, the contrast remained: for staminate plants RSAs were between 0.4 and 1 during the period of flowering (), whereas the RSAs in pistillate plants were larger at every stage, reaching a value of over 5 in the fifth week.
The percentage exported to the apical bud, which included both leaf and flower primordia, ranged from a fraction of 1% to 3.5% (), but was, after the first week, higher in the staminate plants than the pistillate plants. The young leaves of the pistillate plants were large enough, relative to the flowers, to enclose the bud, while those of the staminate plants were not. Because of the small size of this structure, even the very small amount allocated resulted in a high RSA, but the values in staminate and pistillate plants were comparable at most stages. However, by week 7, after the staminate plants have senesced, the RSA for the apical buds of the pistillate plants exceeded 12 (). At this stage, the bud included several large fruits.
By the fourth week of flowering, the staminate plants had begun to show the yellowing that is characteristic of whole plant senescence, and the export to the reproductive structures reached 68% (). The pistillate plants were still robust, although individual older leaves had begun to senesce, and export from the leaf just below the inflorescence to the developing fruits and young flowers remained near 10% (). Vegetative structures continued to receive the larger proportion of the exported photosynthate in the pistillate plants, including 51% to the lower vegetative tissue (). However, in weeks 5, 6, and 7, the allocation to the fruits increased (), while that to the lower portion of the plant declined. In week 7, the pistillate plants showed signs of overall senescence, and by week 8, most of the pistillate plants were dead.
Long-term photosynthate partitioning
Three weeks after the start of flowering, a full day of partitioning after 14CO2 labelling of L–1 for 1 h produced a similar photosynthate allocation pattern as did a 3 h partitioning period in both staminate and pistillate plants. The staminate flowers retained 47% of the radioactive carbon, while pistillate reproductive structures had 9% (). However, whereas the amount in the pistillate flowers remained constant over the next 3 d, the proportion in the pollen-producing flowers had declined appreciably by day 2 and thereafter remained stable over the next 2 d. Whereas less than 9% of exported carbon went to the leaves within the staminate inflorescence, inflorescence leaves in the pistillate plants received 20–33% (), with no consistent changes with the time. The amount of carbon located in inflorescence stem tissue was greater in the pistillate plants (22%) than the staminate plants for the first day (14%), but the difference disappeared in subsequent days (). Even at the end of the four days, levels of total recovered radioactivity were similar to values for the 3 h partitioning period, suggesting that any respired carbon was immediately re-fixed.
Fig. 5. Allocation of radioactivity to various parts of staminate and pistillate plants at 3 weeks after the start of flowering and at various times following a 1 h exposure of L–1 to 14CO2 followed by long chase times. (A) The flowers and fruits; (B) (more ...)
The RSA of the staminate flowers still showed a substantial drop over the long chase period (). The RSAs of the pistillate flowers and fruits remained similar during partitioning over 8 d, with values of less than 1, indicating a below-average sink activity drawing from the leaf immediately below the inflorescence.
Fig. 6. Mass corrected (RSA) measure of radiolabelled photosynthate allocated to the reproductive structures of staminate and pistillate plants at 3 weeks after the start of flowering and at various times following a 1 h exposure of L–1 to 14CO2 followed (more ...)
Staminate flowers as sinks
To determine the stage of maximum photosynthate import into the staminate flowers, the flowers of staminate plants in the second week of floral development were each separated into four categories following labelling of the leaf just below the inflorescence for 1 h and a chase period of 3 h. The four categories were: bud, being those flowers and flower parts still held within the axils; extruded, which had extruded anthers; anthesis, where anthers had begun to open; and old, where the anthers were withered and had largely shed their pollen. The flowers in the bud stage received the largest amount of radioactivity, 35%, compared with less than 5% at other stages (), but they also had the largest mass. The buds were the only stage containing developing pollen, as determined by microscopic examination; in all the other stages, all of the pollen was mature. RSA differences between the staminate floral stages were not significant (). The RSA for the empty anthers was just below 1, indicating less than average but not a cessation of import, despite the completion of development. However, as the flowers of the older stages were few in number, their total drain was relatively small.
Fig. 7. Allocation of radiolabelled photosynthate from L–1 to different parts of staminate plants with the reproductive organs separated into flowers of different ages, 2 weeks after the start of flowering. (A) As a percentage of total export; (B) RSA. (more ...)
Inflorescence leaves as sources of photosynthate for the flowers and fruits
Because of the differing contributions of individual leaves to the reproductive structures, an examination of partitioning of labelled photosynthate from all the tissues within the inflorescence region was examined. Using longer plastic bags, the entire inflorescence region of young plants was exposed to 14CO2 for 1 h, followed by a 4 h chase period. The greater allocation to reproductive structures in the young staminate plants compared with the predominant allocation to vegetative structures in the young pistillate plants, which live longer, was also evident for photosynthate from within the inflorescence (). This difference showed clearly at week 1, when the staminate flowers received 2.5 times more photosynthate than pistillate flowers, but disappeared by week 2. This contrasts with the situation when a leaf below the inflorescence was labelled, when the difference persisted throughout the life of the staminate plants.
Fig. 8. Allocation to organs of staminate and pistillate plants when the entire inflorescence was exposed to 14CO2 for 1 h with a 4 h chase period. (A) 1 week after the start of flowering. (B) Plants two weeks after the start of flowering. Bars represent ±SE. (more ...)
Photosynthesis and respiration in the light and in the dark
Photosynthesis rates in the leaves of the staminate plants were constant during the first 3 weeks of flowering (). The photosynthetic rates of the leaves declined substantially in week 4 and even further, essentially to zero, in week 5. The pistillate plants, on the other hand, showed a slight reduction in activity from week 1 to week 5, but thereafter displayed noticeable loss of function in weeks 6, 7, and 8 ().
Fig. 9. Photosynthetic rates of leaves of staminate and pistillate spinach plants measured at various times following the start of flowering. Measurements occurred at mid-morning with 175 μE m−2 of light. (A) Leaves of staminate plants; (B) Leaves (more ...)
As the different regions of the inflorescence were at different stages of development, respiration was measured in consecutive 3.5 cm segments, numbering 1 at the apex down to the mature regions of the inflorescence, following the removal of the leaves from the inflorescence. Plants from the second week of flowering were measured at four consecutive segments, with the number of measurements increasing with the larger plants. The staminate plants, stripped of their leaves, had 3–3.5-fold greater rates of respiration at week 2 than did the pistillate plants near the inflorescence apices (). This difference still existed, but with less magnitude, further down the axis in young plants. At this time, some anthers had begun to shed pollen, although very few in the first two segments. In older plants, at week 4, flowers existed in a mixture of pre-and post-anthesis stages: the majority of the staminate flowers had undergone anthesis while the region nearest the tip showed the fewest open anthers. At this time the respiration rates for the pistillate plants had risen to levels comparable with that of the staminate plants (). The upper two segments of the pistillate inflorescence had very small flower clusters until week 4, at which time some of those in segment 2 had full-sized fruits.
Fig. 10. Rates of respiration per segment (A, B) of 3.5 cm segments of the inflorescence, without leaves, and rates of respiration, per unit fresh weight, of the reproductive structures (C, D) from 3.5 cm segments within the inflorescence, both numbered downwards (more ...)
As the pistillate flowers were initially extremely small, the degree to which the initial disparity in activity results from size was determined based on a calculation of the respiratory rate per gram of flower or fruit. To determine the respiration of the reproductive structures, carbon dioxide evolution of the stem regions was measured with both leaves and reproductive structures removed. This value was subtracted from the rate of respiration of the stem with flowers and fruits, and the resulting value divided by the fresh weight of the flowers removed from the segment, producing an estimate of the respiratory activity of the reproductive tissue on a tissue fresh weight basis. In the apical region, all of the tissues were very active in respiration, with the youngest plants showing the highest level of activity on a per gram basis (). Below this region, the respiration of the staminate flowers declined, but still tended to be more than in the pistillate flowers. The older fruits showed a reduced respiration on a per gram basis compared with the young pistillate flowers ().
The concentration of total non-structural carbohydrates (glucose+fructose+sucrose+starch) in the senescing staminate flowers at week 5 was as high as the highest level in pistillate fruits, which occurred close to fruit maturity at week 7 (). These non-structural carbohydrates showed a continuous increase in leaf L3 of the staminate plants until their senescence (), while in pistillate plants a very low level was maintained until the fifth week, when the amount rose, peaking 1 week later.
Fig. 11. Changes over time in the total non-structural carbohydrate content of staminate and pistillate plants. (A) Reproductive structures 15–18.5 cm below the tip of the inflorescence; (B) leaf L3 (third leaf, counting from the base, within the inflorescence). (more ...)