Constant distance between leaf initiation sites permits non-destructive analysis of apical meristem activity during cactus shoot growth

Abstract

Background and Scope

Trunks of saguaro cacti (Carnegiea gigantea) grow for many years, and during this time the shoot apical meristem (SAM) of each trunk not only grows in diameter, it also initiates new orthostichies (ribs). Several questions were examined. Is a saguaro SAM’s diameter correlated with the number of orthostichies/ribs it is producing? Is SAM diameter tightly controlled, or does it vary among individuals of the same age? When saguaro trunks are ~3 m tall, their SAMs stop adding new orthostichies/ribs: do SAMs stop growing only after reaching a critical diameter, or do the SAMs vary in diameter when each stops growing?

Methods

Ribs were counted at various heights (corresponding to various ages) on saguaro plants in habitat. Shoot apical meristem diameter was measured by light microscopy in sectioned material. Shoot apical meristems of Echinocactus grusonii were also studied.

Key Results

Shoot apical meristem diameter is strongly correlated with the number of ribs being maintained: the circumferential distance between newly initiated leaf primordia remains constant (145 ± 10.6 µm in C. gigantea; 193 ± 10.7 µm in E. grusonii) even as an SAM grows in diameter. An SAM’s diameter and circumference can be estimated by counting the number of ribs it is maintaining. The diameter of each SAM of C. gigantea increases for many years but it eventually stabilizes; the final, stable diameter of each C. gigantea SAM varies from shoot to shoot.

Conclusions

Shoot apical meristem diameter in both species can be estimated non-destructively by simply counting the number of orthostichies/ribs the SAM is producing (or produced in the past). The growth rate of C. gigantea SAMs varies from plant to plant and can change with age. All C. gigantea SAMs stop increasing in diameter at some point, but that diameter varies from plant to plant.

INTRODUCTION

The research here explores the hypothesis that shoot apical meristems (SAMs) of saguaro cacti (Carnegiea gigantea) and golden barrel cacti (Echinocactus grusonii) grow for many years, and as the basal circumference of each SAM increases, it periodically has enough room available to initiate a new rib (a rib is a phyllotactic orthostichy). This research was prompted by the observation that SAMs of both species repeatedly initiate new ribs for many years (during the production of the first 3 m of trunk longitudinal growth in C. gigantea; Fig. 1A–D; Mauseth, 2021). Furthermore, mature shoots of many species of cacti have giant SAMs (diameters >900 µm are common and a diameter of 2565 µm was measured in E. grusonii; Mauseth, 2004), but young epicotyls of cactus seedlings have much smaller SAMs (Mauseth, 1978a, b, 1979). Thus, in many cactus species, each SAM itself grows larger even as it adds cells to the shoot axis as part of shoot longitudinal growth. The prolonged period during which shoots of C. gigantea and E. grusonii initiate new ribs may correspond to a prolonged period of growth of the SAM itself.

Fig. 1.

(A) Trunk of Carnegiea gigantea, showing numerous longitudinal ribs (orthostichies). Each spine cluster is an axillary bud (the subtending foliage leaf of each is much too small to be visible here). When this trunk’s SAM was located at the mid-level of this region (arrow), it initiated a new orthostichy, after which the SAM continued to add new leaf primordia to the distal ends of each orthostichy. (B) The SAM of this C. gigantea trunk initiated two new orthostichies (arrows) almost simultaneously and close to each other laterally. (C) Apex of a young C. gigantea trunk in which the SAM is producing only 16 orthostichies. (D) Apex of an older C. gigantea trunk in which the SAM is producing 27 orthostichies. (E) Scanning electron micrograph of an E. grusonii SAM maintaining 25 orthostichies. The apical meristems of succulent stems are located in an apical depression, so the young ribs and leaves initially extend upwards above the SAM. Orthostichies extend distally to the base of the SAM. A new orthostichy could be accommodated at the base of this SAM either if the SAM grew to have a larger circumference or if future leaf primordia were to be narrower such that new primordia could fit more closely together than did older primordia. Arrows indicate three leaf primordia. Many trichomes were removed during preparation, but a few long, slender ones remain; young trichomes occur as clusters located in the axils of young leaves. Scale bar = 300 µm. (F) The initiation sites of five new orthostichies are indicated by arrows. After initiating the first new orthostichy (lowest arrow), the SAM added 28 new leaf primordia to all orthostichies, then initiated the next new rib (next higher arrow; no new initiation sites are out of view on the back side of the trunk), then it added ten new leaf primordia to all orthostichies before initiating the third new orthostichy followed soon after by a fourth then a fifth new orthostichy. These can be represented in a map, similar to that in Fig. 2.

The phyllotactic pattern of leaf primordia on a shoot is a record of SAM activity. Each leaf primordium of a ribbed shoot is part of three phyllotactic series: two parastichies (one clockwise, one counterclockwise) and a longitudinal orthostichy; the orthostichies are the ribs (Mauseth, 2016). Orthostichies are continuous distally to the base of the SAM, which is the location in which new leaf primordia are added to each orthostichy (Fig. 1E). The initiation of a new orthostichy must indicate that a significant event has occurred at the SAM’s base (new orthostichies are never initiated at any point below the SAM; new ribs are never intercalated among regions of developed, mature ribs in proximal parts of a shoot).

The initiation of a new orthostichy involves the initiation of the first leaf primordium of that new orthostichy. The formation of a leaf primordium requires a site that has a particular circumferential width at the SAM’s base, and consequently a new orthostichy can be initiated and intercalated among the distal ends of the already-existing orthostichies only after an SAM’s circumference has enough space to accommodate the width of an additional leaf primordium (Fig. 1E).

Two of the hypotheses explored here concern the spacing of leaf primordia at the SAM’s base (the base of the peripheral zone, the region in which leaf primordia are initiated). The first postulates that the width required for each leaf primordium remains constant throughout the lifetime of an SAM; if this is correct, then the relationship between an SAM’s basal circumference and rib number will be constant over time. The second, alternative hypothesis is that the width required by each leaf primordium becomes narrower as a shoot ages such that a greater number of leaf primordia—and orthostichy apices—can fit onto the base of an older SAM even if that SAM has not increased its circumference. If so, then the initiation of new ribs on shoots neither requires nor indicates growth of the SAM.

An earlier study of the initiation of new orthostichies by C. gigantea SAMs (Mauseth, 2021) mapped the location of each leaf/axillary bud (often called areoles in cacti) on each rib such that the location of the sites of initiation of each new orthostichy could be identified precisely. That study found that new orthostichies are not initiated at uniform intervals along a shoot’s length. Instead, new orthostichies are initiated most often one at a time at irregular intervals. Occasionally, an SAM also initiates two new orthostichies simultaneously (shown in Fig. 1B here). The initiation of each new orthostichy is followed by a rather prolonged but temporary stable growth period during which the SAM does not initiate any more new orthostichies even though it is adding new leaf primordia to the distal ends of all existing orthostichies (Fig. 1F here illustrates the irregularity of new rib initiations, and Fig. 2 here shows this type of irregularity in map form). In Fig. 2 here, new orthostichies # 3 and # 7 (initiation sites labelled 56a and 56b) were initiated simultaneously at a height of 56 areoles above soil level (areole spacing along the crest of each rib tends to be 1.77 cm from centre to centre). At some point, the SAM’s temporary stable period ends, and the SAM initiates one or two additional new orthostichies followed by another temporary stable period. Eventually, when a C. gigantea trunk’s SAM has produced ~3 m of shoot length, the SAM becomes permanently stable, after which it initiates no new orthostichies whatsoever (in Fig. 2 here, the region above 150 areoles above soil level), even though the shoot continues to grow in length for many more years and many more metres, and the SAM continues to add new leaf primordia to each of the many existing orthostichies (Fig. 3A here).

Fig. 2.

Map of orthostichy initiation sites in a trunk of C. gigantea. Orthostichies were numbered at a high level above the soil (at 175 areoles above soil level: labels on Y-axis; height here is measured as the number of areoles above soil level, counted along a reference rib). This trunk had 28 ribs; note that ribs # 1 and # 2 appear near the left side of the map and are repeated on the right side of the map, just as ribs # 25–28 also appear on both sides (this is done just to make patterns more visible). The SAM of this plant initiated 15 new orthostichies (orthostichy # 4 was initiated at 30 areoles above soil level followed almost immediately by orthostichy # 26 at a height of 31 areoles above soil level; orthostichy # 26 is one of the ribs shown on both sides of the map), then orthostichies # 3 and # 7 were initiated simultaneously at 56 areoles above soil level (because these two initiation events were simultaneous, they are labelled ‘a’ and ‘b’). Maps like this are explained more fully in Mauseth (2021).

Fig. 3.

(A) This plant of C. gigantea is typical of those examined in this study. The three counting heights (163 cm, 245 cm and 4 m above soil level) are indicated. Rib number was typically constant slightly above the location of the branches and maximum rib number could be counted at a height of 4 m, where branches did not interfere with viewing of ribs on the trunk. (B) Echinocactus grusonii with 41 orthostichies; plants of E. grusonii are globose and almost never grow to a height exceeding 1 m. (C) The emergence of branches from axillary buds in C. gigantea distorts and obscures the trunk’s ribs in that region, making it difficult to count rib numbers accurately at that height.

A study of SAM growth (Mauseth, 2020) in cactus seedlings found that in some species the full complement of ribs (i.e. the number typical of a mature shoot) is present on epicotyls shortly after seed germination, whereas in other species seedling epicotyls have few ribs but then initiate several new ribs quickly. Seedlings of C. gigantea have only 11 or 12 ribs, many fewer than is recorded as being typical of older shoots (up to 30 ribs according to Hunt et al., 2006); presumably C. gigantea seedling SAMs need to grow much larger before they can accommodate 30 ribs. All the seedlings of C. gigantea in that study were young, <8.5 cm tall (Mauseth, 2020), so studies of the growth of older SAMs were needed and are presented here. Carnegiea gigantea is of special interest because large populations of thousands of plants occur in both natural habitats and in cultivation in Arizona and California, and the phyllotaxy of these plants is easy to observe: they could be a valuable source of information about the activity of thousands of SAMs over a period of many years in diverse natural and horticultural conditions.

Plants of another cactus species, E. grusonii (golden barrel cactus; Fig. 3B), also initiate new ribs for many years and are abundant in cultivation. This species too might be a particularly useful research subject. The correlation between rib number and SAM circumference was studied here in this species as well as in C. gigantea.

Background on cactus ribs and axillary buds (areoles)

In most columnar cacti, an embryo’s epicotyl SAM persists for a plant’s lifetime, producing a central trunk (Mauseth, 2006, 2020). The foliage leaves are tiny in columnar cacti (Mauseth, 2007), but the position of each is marked by its persistent axillary bud, the areole (spine cluster; Fig. 1A, B, F; Mauseth, 2007, 2017a, b). Cacti are stem-photosynthetic, and their cortex and epidermis are perennial, being retained for years; consequently the axillary buds/areoles are also retained, providing a permanent record of the location of every foliage leaf ever produced, even at the base of very old shoots (Mauseth, 2006, 2020, 2021).

Phenomena studied here

(1) As SAMs of C. gigantea and E. grusonii increase in diameter with age, is SAM circumference correlated with the number of ribs the SAM is maintaining such that the circumference of a shoot’s SAM can be reliably estimated by counting the number of ribs it is maintaining (or that it maintained at some time in the past)? (‘Maintaining’ means that the SAM is adding leaf primordia to all existing orthostichies.) This was studied by measuring SAM diameter by light microscopy in median longitudinal sections of 15 SAMs of C. gigantea and 13 SAMs of E. grusonii; the number of ribs present at the apex of each was recorded and compared with SAM diameter.

(2) Is the rate at which a saguaro SAM grows in circumference correlated with the shoot’s growth in length? That is, in the time it takes for trunks to grow to a particular height (1 m, for example), do all SAMs grow to the same circumference (have the same rib number)? This was studied only in C. gigantea, by counting rib numbers at specific heights in many plants growing in habitat.

(3) When a saguaro’s trunk has grown to a length of ~3 m, its SAM enters its permanently stable phase and stops initiating new orthostichies even though it continues to perform its other functions normally, adding new leaf primordia to all existing ribs and generating cells that will be involved in the shoot’s continued, normal longitudinal growth (Fig. 3A). Do C. gigantea SAMs enter their permanent stable phase only after they have grown to a particular, specific size and are maintaining a specific number of ribs? If so, the plant may have some mechanism that detects when an SAM has achieved a critical circumference and then inhibits further growth of the SAM itself. Alternatively, the number of ribs an SAM is maintaining when it stabilizes its own circumference might be variable. If so, then whatever mechanism causes an SAM to stop its own growth is not related to the SAM’s circumference or the number of ribs it is maintaining. It may be some other factor in the plant’s biology, such as shoot length or age, perhaps. This was studied by examining plants growing in habitat and counting rib numbers at a height above the point at which new rib initiation had ceased.

MATERIALS AND METHODS

Plants of Carnegiea gigantea were studied in three localities in Arizona, USA: (1) in or near Tucson Mountain Park, (2) Saguaro National Park, and (3) south of Why, near Organ Pipe Cactus National Park. The abundance of C. gigantea plants made it possible to study the sites of new rib initiations on many plants (71 plants were examined).

Only cultivated plants of Echinocactus grusonii were available so it was not possible to carry out field studies. It was possible, however, to ascertain that in this species also new ribs are initiated even in very old plants. Thirteen plants were purchased so that their SAM diameter could be measured by light microscopy and compared with rib number.

Examination of the diameter of SAMs

Six shoot tips from a single large, branched plant of C. gigantea were obtained from the Arizona-Sonora Desert Museum, and eight plants of various sizes, each ~1 m tall, were purchased from a commercial nursery (B and B Cactus Farm, Tucson, AZ, USA). The apex of a large, old shoot (Fig. 1D) was provided by D. Swann of Saguaro National Park. Thus 15 SAMs of C. gigantea were studied by light microscopy to measure SAM diameter and compare that with the number of ribs being maintained by each. The plants had orthostichy numbers ranging from 11 to 23.

Shoot apical meristems were also obtained from 13 cultivated plants of E. grusonii that had been obtained from commercial nurseries. These plants had orthostichy numbers ranging from 13 to 33 (Fig. 3B).

The SAMs of both species were isolated by dissection, fixed in Navashin’s solution, dehydrated through a tertiary-butanol series, then embedded in Paraplast Plus. Sections were stained with Safranin and Fast Green (Mauseth et al., 1984). Each SAM’s basal circumference was measured as the distance from the axil of the distalmost leaf primordium on one side of the SAM to the axil of the distalmost leaf primordium on the other side.

One SAM of E. grusonii was examined by scanning electron microscopy. It was fixed in Navashin’s solution, critical-point dried, then coated with gold/palladium.

Data were analysed with the statistical functions of Microsoft Excel^©. Sample size for C. gigantea was 86 shoots (15 for light microscopy, 71 for counting changes in rib numbers); for E. grusonii it was 13 shoots, all for light microscopy.

Rib numbers at various heights in C. gigantea trunks were counted to determine variability in the rate of initiation of new orthostichies

In 71 tall, old plants of C. gigantea growing in habitat, the total number of ribs present at 163 cm above soil level on the trunk was counted. This height was chosen because it was eye level for me, so rib numbers could be counted easily and accurately (Fig. 3A, lowest arrow). This height was also chosen because no SAM at this height had yet reached its full complement of ribs: all had initiated new ribs above this level, and all continued to add new leaf primordia to the distal ends of all ribs.

In a subset of 30 of these 71 plants of C. gigantea, the number of ribs was also counted at the base of the region in which branches had grown out. The lowest actual height at which branches grew out varied slightly from plant to plant but was ~245 cm above soil level (82 cm above eye level of 163 cm). This height was chosen for counting ribs for three reasons: first, rib number could be counted reliably at this height; second, at this height each trunk had grown for several years after having reached the 163 cm height; and finally, this level was ~0.5 m below the height at which new rib initiation typically ceased (Mauseth, 2021). The great expansion of branch bases distorted the ribs on the trunk, making it difficult or impossible to count the number of ribs exactly at the height at which the last new rib was initiated (i.e. the height at which SAM circumference and phyllotaxy became permanently stable). Counting rib numbers immediately above the branches could not be done reliably because the branches obscured the view of the trunk in that region (Fig. 3C).

From among the 30 plants of C. gigantea for which rib number was counted both at the 163 and 245 cm height, a subset of 12 plants was chosen and their maximum number of ribs was counted at a height of 4 m above soil level, well above the branches and above the point at which no new ribs had been initiated for at least 1 m (Fig. 3A, highest arrow). These 12 plants represent the ones whose ribs could be counted accurately at such a high level, without the use of a ladder or a drone. Rib counts at 4 m above soil level represent the maximum number of ribs produced by each SAM.

The terms ‘orthostichy’ and ‘rib’ are used as synonyms here.

RESULTS

Shoot apical meristem growth in diameter; constancy of spacing between leaf primordia

In both C. gigantea and E. grusonii, SAMs of older plants had both more ribs and a greater basal circumference as compared with the SAMs of younger plants. In both species, a strong correlation existed between an SAM’s basal circumference and the number of ribs it was maintaining (Tables 1 and 2; Fig. 4A, B). When each SAM’s basal circumference was divided by the number of ribs that the SAM was maintaining, the mean value was constant in both species: 145 ± 10.6 µm for C. gigantea and 193 ± 10.7 for E. grusonii (Tables 1 and 2; Fig. 5A, B; this is the width of the field in which one new leaf primordium would form at the distal end of each orthostichy). An SAM of C. gigantea generated an extra 145 µm at its base before it initiated the first leaf primordium for a new orthostichy (this is in addition to the space generated for each new leaf primordium of existing orthostichies). The low standard deviation in both species indicated a considerable constancy of the spacing between leaf primordia. The fields within which leaf primordia of E. grusonii arose were wider than those of C. gigantea; their SAMs generated an extra 193 µm of circumference before they initiated a new orthostichy. In both species, the spacing between leaf primordia at the SAM’s base neither increased nor decreased as the SAMs grew in circumference; the distal ends of orthostichies were not closer together in older SAMs (Fig. 5A, B).

Table 1.

Characters of 15 Carnegiea gigantea SAMs with several ages and rib numbers¹.

Plant number2	Number of ribs	SAM diameter (µm)	SAM circumference (µm)	Circumference (µm)/rib number
1834	11	524	1646	1503
1820	12	570	1791	149
1821	12	547	1719	143
1822	12	513	1612	134
1854	12	616	1935	161
1833	13	570	1791	138
1835	13	524	1646	127
1853	13	673	2115	163
1602	15	718	2256	150
1789a4	19	878	2759	145
1789d	19	889	2793	147
1789e	19	866	2721	143
1789c	22	912	2866	130
1789f	22	946	2972	135
1789b	23	1140	3582	156

Statistics for rightmost column: mean, 145 ± 10.6 µm; range, 127–163 µm.

¹Samples are sorted by number of ribs.

²These plant numbers are the fixation numbers in my microscopy records; each fixed specimen has a unique number.

³150 here is the circumference of 1646 µm divided by the number of ribs (11): 1646 µm/11 = 149.6 µm of circumferential space between the distalmost leaf primordium of one rib and that of an adjacent rib.

⁴The six samples labelled ‘1789’ are from a single large, old, branched plant.

Table 2.

Characters of 13 Echinocactus grusonii SAMs with several ages and rib numbers¹.

Plant number2	Number of ribs	SAM diameter (µm)	SAM circumference (µm)	Circumference (µm)/rib number
1857a	13	798	2507	1933
1858	13	866	2722	209
1859	15	889	2793	186
1860	15	878	2758	184
1836	17	1094	3437	202
1851	18	1186	3725	207
1847	19	1129	3546	186
1850	19	1174	3689	194
1837	20	1197	3760	188
1849	21	1254	3940	188
1846	21	1151	3617	172
1852	22	1368	4298	195
1176	33	2166	6805	206

Statistics for rightmost column: mean, 193 ± 10.7 µm; range, 172–209 µm.

¹Samples are sorted by number of ribs.

²These plant numbers are the fixation numbers in my microscopy records; each fixed specimen has a unique number.

³193 here is the circumference of 2507 µm divided by the number of ribs (13): 2507 µm/13 = 192.8 µm of circumferential space between the distalmost leaf primordium of one rib and that of an adjacent rib.

Fig. 4.

(A) Carnegiea gigantea, rib number vs SAM circumference in micrometres. n = 15. (B) Echinocactus grusonii, rib number vs SAM circumference in micrometres. n = 13.

Fig. 5.

(A) Carnegiea gigantea, rib number vs width of space between leaf primordia in micrometres (i.e. SAM circumference in micrometres divided by rib number). n = 15. (B) Echinocactus grusonii, rib number vs width of space between leaf primordia in micrometres (i.e. SAM circumference in micrometres divided by rib number). n = 13.

Rib number varied among C. gigantea trunks that had grown to the same height

In the 71 plants of C. gigantea old enough to have trunks at least 4 m tall, the mean and standard deviation of the numbers of ribs each trunk had at the low height of 163 cm above ground level (Fig. 3A) was 20.6 ± 2.7 (Table 3, bottom row).

Table 3.

Rib numbers at three heights in Carnegiea gigantea trunks.

Height of measurement	Sample size (number of plants)	Range of rib counts	Mean ± standard deviation	Mode
Stable rib number, 4m	12	24–35	27.8 ± 3.3	26
Branch level, 245 cm	30	21–31	25.3 ± 2.6	27
Eye level, 163 cm	71	16–27	20.6 ± 2.7	20

In a subset of 30 of these 71 plants, the number of ribs was also counted at a height of 245 cm above soil level, immediately below the region in which branches had grown out. This second counting level was 82 cm above the lower level of 163 cm and represented an unknown number of years of continued growth for each SAM. The mean number of ribs at 245 cm above soil level was greater than at 163 cm: 25.3 ± 2.6 (Table 3, middle row), and the range was about the same width, just with higher numbers. The mode at 245 cm height was 27 ribs.

Rib number and SAM circumference did not converge at one value in trunks of very old plants of C. gigantea

In a subset of 12 of the 30 plants whose ribs were counted at 245 cm above soil level, rib number was also counted at a height of ~4 m above soil level (Fig. 3A, upper arrow), a height that had been produced after the rib number had stabilized in each trunk. In these 12 trunks, rib number was still variable from plant to plant at this very tall height: the SAMs of the 12 plants had not converged to have a single rib number or even a narrow range of rib numbers (Table 3, top row). The maximum number of ribs being maintained by any of these 12 SAMs during this stable period was 35, but the SAMs of the other 11 plants had stabilized at smaller rib numbers, some maintaining as few as 24 ribs. The greatest number of SAMs were maintaining 26 ribs during this permanent stable phase (Table 3, top row, Mode).

DISCUSSION

Shoot apical meristems grow in diameter as they age, but the mean width of space between leaf primordia remains constant

The sample of 15 C. gigantea shoot apices studied by light microscopy clearly indicates that, as saguaro shoots age, their increased numbers of ribs are correlated with increased SAM diameter and basal circumference. An SAM’s basal circumference—the base of the peripheral zone, the region in which leaf primordia are initiated—increases from 1595 µm in seedlings with 11 ribs [calculated as (11 ribs) (145 µm per rib)] to 3335 µm when old enough to produce 23 ribs [(23 ribs) (145 µm per rib)].

It is especially important that the amount of space needed for each new leaf primordium does not change with a shoot’s age, being ~145 µm in all samples of C. gigantea (Table 1 and Fig. 5A). Because of this, the number of orthostichies that an SAM is maintaining can be used to calculate that SAM’s basal circumference and diameter: an SAM’s size can be measured non-destructively. This, combined with the retention and easy visibility of axillary buds/areoles even on old regions of a shoot, makes it possible to calculate the size an SAM had at each point in its past and to correlate that size with the environmental or cultural conditions that occurred. Schnablová et al. (2020) found that SAM shape varies phylogenetically but is size-independent, so it may be that in C. gigantea SAM shape remains constant as it undergoes its extensive growth, but only basal diameter and circumference were studied here, and they are the only two dimensions that, at this time, can be estimated from rib number.

In E. grusonii also, spacing between leaf primordia is constant throughout the lifetime of a shoot, and rib number can be used to calculate an SAM’s basal diameter (Table 2 and Fig. 5B). Consequently, E. grusonii can also be used to study non-destructively the effects of various factors on SAM activity: the SAM of the plant in Fig. 3B would have had a circumference of (41 ribs) (193 µm per rib) = 7913 µm with a diameter of (7913 µm)/π = 2519 µm.

The amount of space occupied by each new leaf primordium in E. grusonii is larger than that occupied by each new leaf primordium in C. gigantea. An earlier survey of many species of cacti (Mauseth, 2004) found that the amount of space occupied by each new leaf primordium varies from species to species. In that publication, Table 1 lists 27 species with ribbed shoots in which leaf primordia were wide (range 108–432 µm) and Table 2 in that paper lists 21 species with ribbed shoots in which leaf primordia were much narrower (range 44–98 µm). Thus the values reported here for C. gigantea (145 µm) and E. grusonii (193 µm) fit within the range of values previously reported.

In the carpellate inflorescences of maize (Zea mays), in which primordia are also arranged in orthostichies, Bommert et al. (2013) also found that the number of kernel orthostichies, which varied from 10 to 20, was positively correlated with SAM diameter, which varied from 170 to 340 µm. The circumferential spacing of primordia at the base of the Z. mays inflorescence SAM was ~53 µm, which is much smaller than in either of the two cacti studied here but within the range of narrower leaf primordia of the cacti studied earlier (Mauseth, 2004).

Shoot apical meristem basal circumference varies among C. gigantea trunks that have grown to the same height

By using the constant correlation between rib number and SAM circumference of Fig. 5A and the rib counts of Table 3, growth dynamics of C. gigantea SAMs could be calculated.

The rate of growth of an SAM itself in trunks of C. gigantea varies from plant to plant and from one time (height) to another within a single trunk. The rate at which a shoot grows in length is not closely correlated with the SAM’s own rate of growth in basal circumference. In the time that 71 plants grew to a height of 163 cm above soil level, their 71 SAMs had themselves grown to such different sizes that they were producing very different numbers of ribs (16–27, corresponding to circumferences of 2320–3915 µm; Table 3). Six of these plants had the lowest measured value of 16 ribs (Fig. 6A, leftmost column) and three plants had 27 ribs (Fig. 6A, rightmost column). Thus, many SAMs had grown rapidly in basal circumference, whereas other SAMs had enlarged themselves very slowly. Most SAMs (mode, 20 ribs; Table 3) had grown to have a diameter of 923 µm and a circumference of 2900 µm. The fraction of cells that an SAM retains as part of its own growth versus the fraction of cells that flow out and become part of the shoot varies greatly from plant to plant.

Fig. 6.

(A) Number of ribs at 163 cm above soil level in trunks of 71 plants. The number above each bar indicates the number of plants in that size class (e.g. six plants had 16 ribs at 163 cm above soil level; at the other extreme, three had 27 ribs at the same height). (B) Number of new ribs initiated between eye level (163 cm) and just below branch level (245 cm) in trunks of 30 C. gigantea plants. Each bar represents a single shoot; the number above each bar (and the length of each bar) indicates the number of new ribs initiated by that particular shoot’s SAM in the interval between the heights of 163 and 245 cm above soil level.

In 30 of these 71 plants, the number of ribs was also counted at a height of 245 cm above soil level to determine how much more each SAM had grown in circumference after the 163 cm growth period. The counts are paired samples: the exact number of new ribs initiated by each SAM after height 163 cm until height 245 cm is known for each SAM. These values are presented in Fig. 6B, in which trunks that had few ribs while young (i.e. when the SAM was located only 163 cm above ground level) are on the left side; those that had greater numbers of ribs while young are on the right side. Figure 6B has 30 bars: each bar represents one individual trunk, and whereas each bar’s position near the left or right side indicates that SAM’s growth in circumference up to the height of 163 cm, each bar’s height indicates its growth in circumference after the 163 cm height until the 245 cm height. The mean number of new ribs initiated by each SAM in this region of intermediate age was extremely variable. For example, three SAMs initiated only one new rib each during this period (all these are located in the right half of Fig. 6B); the basal circumference of each of these three SAMs must have increased by only 145 µm during the time each produced a trunk 82 cm longer. In contrast, a single SAM initiated ten more new ribs (the tall centre bar in Fig. 6B), so its basal circumference increased by 1450 µm in this same growth interval. Furthermore, this SAM was already so large that it was maintaining 20 ribs by the time it had produced a trunk 163 cm tall, so it reached a total of 30 ribs by the time its trunk had grown to be 245 cm tall; the circumference of this SAM grew from 2900 to 4350 µm. On the other hand, one SAM that had 26 ribs at 163 cm (next to the rightmost side of Fig. 6B) initiated another five new ribs before reaching the height of 245 cm, so it was ultimately maintaining 31 ribs at the higher measuring level (SAM circumference increased from 3330 µm to 4495 µm).

The mean number of new ribs initiated per SAM during the growth interval from 163 to 245 cm was 4.5 ± 2.7 (mean increase of SAM circumference, 652.5 ± 391.5 µm) with a range from 1 to 10 ribs (range of increase in circumference, 145–1450 µm). But Fig. 6B shows an important trend: those plants that had the fewest ribs at height 163 cm (left side of Fig. 6B) tended to initiate a greater number of new ribs above the 163 cm height than did the plants with many ribs at 163 cm (Fig. 6B, right side; dotted line is the trend line). The mean number of new ribs initiated by the nine SAMs that had grown the most slowly up to the 163 cm level (the 9 leftmost columns) was 6.44 ± 2.60 (SAM circumference increase of 933 ± 377 µm), whereas the mean number of new ribs initiated after height 163 cm by the 11 SAMs that had grown the fastest up to the 163 cm level (the 11 rightmost columns in Fig. 6B) was only 3.0 ± 1.61 (SAM circumference increase of only 435.0 ± 233.4 µm). The SAMs that had enlarged themselves most slowly before reaching 163 cm height tended to enlarge themselves more rapidly above this level as compared with those SAMs that had already grown rapidly before 163 cm height, which then slowed down. However, the accelerated growth of the previously slowly growing SAMs and the reduced growth rate of the previously fast-growing SAMs were not enough to cause all SAMs to converge to the point of having the same numbers of ribs at the 245 cm level (Table 3, middle row). The range was still broad (low of 21 ribs to high of 31 ribs; SAM basal circumference, 3045–4495 µm), but the sizes of SAMs within the population were no longer diverging as much as they had been while younger.

There is no reason to assume that the height of 163 cm represents a special inflexion point in the growth rate of an SAM’s diameter and circumference. This height was chosen simply because the number of ribs at this level could be counted accurately in a large number of plants. Perhaps if rib numbers had been counted at several other heights (such as 40, 80 and 120 cm above soil level), it might have been found that SAM growth rate changes several times.

Shoot apical meristems of C. gigantea eventually stop growing but not at a specific SAM circumference

It was rare to see any new ribs initiated after shoots had elongated to a length of 3 m (Mauseth, 2021 and Fig. 2 here); SAM circumference stabilized permanently at about this shoot length. The number of ribs above this point typically was constant for the rest of the shoot’s elongation. Rib numbers at the very tall height of 4 m still varied from plant to plant in the population; SAMs had not converged to having a single rib number or even a narrow range of rib numbers by the time the SAM’s own circumference had stopped increasing. The greatest number of ribs being maintained by any SAM during this permanent, stable period was 35 (calculated SAM diameter and circumference of 1615 and 5075 µm; no sample of a 35-rib SAM was available for dissection and measurement). The SAMs of other C. gigantea trunks had stabilized at much smaller rib numbers, some producing as few as only 24 ribs at the 4 m height (SAM diameter 1107 µm and circumference 3480 µm), even though the trunks were very old and very tall. The greatest number of SAMs had stabilized at 26 ribs (SAM diameter and circumference 1200 and 3770 µm) during this phase of permanent stability.

Theoretically, an SAM producing 24 ribs at 4 m height might have been producing a greater number of ribs while younger if the SAM could terminate one or several ribs (perhaps by the SAM becoming smaller with age). But rib termination was found to be extremely rare in C. gigantea plants growing in habitat (Mauseth, 2021). No rib terminations were encountered in the current study.

The size of an SAM is controlled by the interactions of several genes, with CLAVATA3 (CLV3), WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM) being especially important (Fletcher, 2018; Kim et al., 2022). In most species that have been studied, the interactions of these genes appear to remain constant during normal vegetative shoot growth, and they change only when a vegetative SAM converts itself into a floral or inflorescence apical meristem with a different phyllotaxy. CLV3 encodes a small peptide that moves within the apical meristem, and WUS is required for CLV3 expression; the two are part of a negative feedback loop. Zhang et al. (2020) described a mutant allele, big-shoot meristem, of the gene BIG in Arabidopsis thaliana that causes meristems to increase in size; BIG functions upstream of WUS and STM.

It seems reasonable that similar gene interactions occur within the SAMs of cacti. If so, then these gene interactions must have some unusual aspects in C. gigantea: they must allow C. gigantea SAMs to grow for many years and to reach sizes much greater than anything that occurs in model plants. Even within the relatively small SAMs of young trunks of C. gigantea (with only 11 ribs), the peptide encoded by the cactus equivalent of CLV3 presumably must move though a much greater volume of meristem cells, and it also must exert its effect on a greater number of WUS-expressing cells than in an A. thaliana SAM. Furthermore, after having permitted the SAM’s growth for many years, the gene interactions must then stop that growth and permanently stabilize the cactus SAM’s circumference. Whatever factor causes these genes to stop the growth of an SAM of C. gigantea and then stabilize its circumference, the end result is that stable SAM circumferences of individual trunks vary greatly within a population, even though each SAM functions normally and exhibits no aberrations. Studies of the genes controlling SAM size in C. gigantea would be extremely valuable.

Hypotheses for future research

An important consideration now is the manner in which extra circumference accumulates until an SAM has grown large enough to initiate a new orthostichy. The research here may help explain several phenomena evident in the maps of orthostichy initiation sites in C. gigantea (Fig. 2 here and Fig. 2A–D in Mauseth, 2021). It had been assumed (by me) that new ribs would be initiated individually (one at a time) at regular longitudinal intervals; i.e. that SAM circumference would increase at a constant rate correlated with shoot growth in length. That assumption was too simple. Although C. gigantea SAMs most often initiate one new orthostichy at a time, presumably after the SAM’s circumference has grown by 145 µm, an SAM can also occasionally initiate two new orthostichies simultaneously [in Fig. 2 here, ribs # 3 and # 7 (labelled 56a and 56b) were initiated simultaneously at 56 areoles above soil level, and ribs # 1 and # 5 were initiated simultaneously at 76 areoles above soil level]. In all observed cases of pairs of simultaneous initiations, the two new orthostichies were always separated laterally by at least one pre-existing rib (Fig. 1B here); the two new orthostichies were never side by side. Furthermore, about half of all orthostichies that are initiated individually are initiated shortly after a previous new orthostichy (‘shortly’ here is defined as the second initiation occurring at a height of three areoles or less above the first): the SAM’s temporary stable phase is exceptionally brief. For example, in Fig. 2 the new rib that was initiated at height 126 areoles above soil level was initiated not long after the one initiated at height 124, but the two were not initiated simultaneously (the orthostichy initiated at height 124 brought the SAM to 22 ribs, and the orthostichy initiated at height 126 brought the total to 23 ribs; between these two initiations the SAM added two leaf primordia to all 22 ribs, so the second orthostichy initiation occurred 44 plastochrons after the first). The data are as follows: of the 235 orthostichy initiation sites mapped earlier (Mauseth, 2021), 201 (85.5 %) were initiated individually, 34 (14.5 %) were initiated as simultaneous pairs, and of the 201 initiated individually, 140 (70 %) were initiated shortly after a previous new orthostichy (less than three areoles above the previous new orthostichy).

Two further unexpected phenomena also exist. First, when new orthostichies are initiated individually, they are located neither at random around the SAM’s circumference nor in radially symmetrical patterns. Instead each new orthostichy is most often initiated quite close laterally to the most recently initiated new orthostichy (Fig. 4 in Mauseth, 2021). The second phenomenon is that each SAM has one or several regions of its circumference in which new orthostichies are initiated frequently (in Fig. 2 here, the region between rib # 6 and rib # 14 contains seven initiation sites) and other regions in which no new orthostichies are initiated at all (in Fig. 2 here, the region between rib # 14 and rib # 21; a discussion of these initiation-free sectors is presented in Mauseth, 2021).

These various observations appear at first glance to be evidence that C. gigantea SAMs might grow slowly for extended periods followed by occasional bursts of rapid growth during which new orthostichies are initiated either as simultaneous pairs or sets that are initiated shortly after each other. Having circumferential regions in which new orthostichies are either plentiful or absent might indicate that an SAM undergoes asymmetrical radial growth (in which some circumferential sectors grow greatly and initiate many new orthostichies, whereas other sectors grow little or not at all and so do not initiate any new orthostichies). But an alternative hypothesis could accommodate these phenomena within the context of an SAM growing radially symmetrically at a uniform rate.

The important aspect may be the manner in which ‘extra’ space accumulates at an SAM’s basal circumference as it grows (‘extra’ space is explained immediately below). This in turn is related to the morphogenetic pattern that governs the locations of incipient leaf primordia. Consider the time interval in which a C. gigantea SAM grows from a circumference of e.g. 1450 µm (enough space for 10 orthostichies) to 1595 µm (enough space for 11 orthostichies): how is the surplus circumference distributed during this growth phase (during which the SAM has more than enough space for 10 orthostichies but not enough for 11 and during which new leaf primordia are being added to the 10 existing orthostichies)? It could be that all new ‘extra’ circumference accumulates at just one site in the morphogenetic pattern that governs incipient leaf primordia (perhaps e.g. between the site for rib # 1 and the site for rib # 2 while the sites for the incipient primordia of all remaining ribs continue to remain close to each other). This would require that in one particular region of an SAM’s basal circumference the sites of incipient leaf primordia (for ribs # 1 and # 2 in this example) would become located farther apart than normal. In this hypothesis, as soon as that one region of circumference accumulates 145 µm of ‘extra’ space, the first leaf primordium of one new orthostichy (the 11th) could form, and the phyllotactic pattern for the sites of the incipient leaf primordia of all 11 orthostichies would temporarily revert to one in which every site is as close to its neighbours as possible. Afterwards, continued SAM growth would cause another single new site to gradually enlarge. A mechanism such as this should not result in the simultaneous initiation of two new orthostichies nor of sets of new orthostichy initiations that are close to each other longitudinally (Table 4, entry 1).

Table 4.

Hypotheses concerning the morphogenetic patterns that govern the location of sites of incipient leaf primordia in C. gigantea, using 145 µm as the width of space needed for each new orthostichy.

1. All extra space accumulates at one site. No rearrangement of the morphogenetic pattern is necessary. One new orthostichy can be initiated as soon as the site becomes 145 µm wide.

2. Extra space accumulates at multiple sites around the SAM’s circumference. A rearrangement of the morphogenetic pattern of incipient leaf primordia would be necessary to generate one or several sites, at least one of which is 145 µm wide.

a. Rearrangement occurs as soon as enough space is available for one new orthostichy (145 µm). New orthostichies would not arise in simultaneous pairs.

b. Rearrangement occurs at some time other than when 145 µm of extra space has accumulated.

i. Rearrangement occurs when enough extra space has accumulated for two new orthostichies (290 µm). Orthostichies would arise as simultaneous pairs.

ii. Rearrangement occurs at any time after 145 µm of extra space has accumulated. If rearrangement creates one site 145 µm wide, a new orthostichy will form and a subsequent new orthostichy can form soon after, once the second site has also grown to be 145 µm wide. Orthostichies would arise soon after each other but not simultaneously.

iii. Rearrangement occurs early. If rearrangement produces two sites of unequal width, then the two will probably not grow to be 145 µm at the same time, and two new orthostichies will be initiated, one soon after the other but not simultaneously.

Alternatively, it might be that as an SAM grows from a circumference of 1450 µm to 1595 µm, the sites of the incipient leaf primordia for all orthostichies remain more or less equally spaced and as far apart as possible around the base of the SAM: no single site would become especially large. In this scenario, at the time the SAM has grown to have a circumference of 1595 µm, the ‘extra’ 145 µm of circumference would be divided into ten bits of extra space each ~14.5 µm wide between every neighbouring pair of the ten existing orthostichies. The initiation of a new 11th orthostichy would require that the pattern of sites of the 10 incipient leaf primordia rearrange itself to a pattern of 11 sites of incipient leaf primordia in which all sites of incipient leaf primordia are as close together as possible, thus creating one site sufficiently large for the initiation of the new 11th orthostichy (Table 4, entries 2 and 2a).

A variation of this second hypothesis is that, rather than rearranging immediately after 145 µm of extra space is available, the pattern of incipient leaf primordia persists temporarily as one in which all sites continue to remain as far apart as possible. If so, no new orthostichy could be initiated as soon as the SAM has grown sufficiently to accommodate an 11th orthostichy: no single site has accumulated the necessary 145 µm of width. Perhaps the SAM continues to grow until its circumference is e.g. a further 145 µm larger and the sites of incipient leaf primordia of all ten orthostichies are far apart. It might be that as sites become more greatly separated, it becomes increasingly difficult to maintain that pattern of wide separation. Once the SAM’s circumference is large enough to accommodate two new orthostichies, perhaps then the pattern rearranges into one with a more close-packed configuration, thus consolidating all the ‘extra’ new space either into one single site that is 290 µm wide (enough for two new orthostichies side by side) or into two sites, each 145 µm wide (both of which being wide enough for one new orthostichy, and the two new sites would be separated from each other by one or several existing orthostichies; Table 4, entry 2bi). This accumulation of 290 µm ‘extra’ circumference before the incipient leaf primordium pattern rearranges could explain the occurrence of pairs of orthostichies initiated simultaneously. As mentioned above, 14.5 % of all orthostichy initiations occur as simultaneous pairs (Mauseth, 2021). No instance of two new orthostichies arising simultaneously side by side has ever been observed (all have at least one pre-existing orthostichy between them), so apparently SAMs in C. gigantea can accumulate an ‘extra’ 290 µm of circumference, but never at one site.

A further possibility exists. It could be that at any point after an SAM ‘overgrows’ the extra 145 µm needed for a single new orthostichy, the pattern of incipient leaf primordia can rearrange itself and generate one site that is 145 µm wide and one that is narrower than that: one new orthostichy could be generated immediately after such a rearrangement and a second new orthostichy could be generated soon after, but not simultaneously (Table 4, entry 2bii). Rearrangement might even occur before an overgrowth of 145 µm occurs, resulting in two sites that are both too narrow to allow the initiation of a new orthostichy immediately; after further growth, the two sites might each initiate one new orthostichy, either simultaneously or one soon after the other, depending on whether the two sites were the same or different sizes after rearrangement. As mentioned above, in the 18 trunks of C. gigantea mapped in the earlier study (Mauseth, 2021), of the 201 new orthostichies that were initiated individually, 140 were initiated shortly after but not simultaneously with a previous new orthostichy.

These several hypotheses are focused on the morphogenetic patterns that control the locations of incipient leaf primordia. In each hypothesis, an SAM itself could grow larger uniformly and symmetrically, without sudden bursts of growth being necessary to explain the occasional simultaneous initiation of orthostichy pairs or the instances in which one orthostichy is initiated soon after a previous new orthostichy. Also, radially asymmetrical growth of the SAM is not needed to explain why many orthostichies are initiated in certain longitudinal sectors whereas none is initiated in other sectors. Examination of SAMs with scanning electron microscopy will probably be required to test these hypotheses and determine how the extra space accumulates as an SAM’s circumference grows.

In conclusion, SAMs of both C. gigantea and E. grusonii grow in diameter and basal circumference for many years and during this time they periodically initiate new orthostichies such that the SAM’s basal circumference divided by rib number remains constant. Consequently, the basal circumference and diameter of the SAMs of both species can be determined non-destructively merely by counting the number of ribs it is maintaining. Because cactus shoots retain their cortex, ribs and axillary buds indefinitely, the SAM’s circumference at any point in the past can also be determined by counting the number of ribs present on older, more basal regions of the shoot. This makes it possible to carry out studies of SAM growth dynamics over extended periods of time and involving variations of ecological factors such as cultivation conditions and weather patterns. In populations of C. gigantea in habitat, the SAMs of each plant grow in circumference at different rates such that shoots of a particular length have different rib numbers and SAM circumferences. The rate of growth of SAMs changes with time, with the SAMs of some plants growingly slowly at first then growing faster, whereas in other plants SAM growth slows after an initial period of rapid growth. The same relationships between SAM circumference and rib number are true in E. grusonii and are possibly true for other ribbed cacti.