Research Note: Observations on a terrestrial charophyte in a temperate environment

This study describes the unusual growth forms of Chara (Charales) in a natural terrestrial environment in a temperate region (southern Siberia). Chara vulgaris had a moss‐like habit and grew on wet soil in small compact turfs probably germinated from oospores. The upper branchlets were curved in a hook‐liked manner and convergent above the apex of the thallus. The lowest internode and branchlets of fertile plants were ecorticate, whereas all other parts were normally corticated; the completely ecorticate plants were sterile. The terrestrial growth of charophytes had an irregular appearance but was successfully maintained in culture, providing a potential model of the first steps of land colonization by the ancestors of embryophytes.     

Intercellular Transport and Photosynthetic Differentiation in Chara corallina

Intercellular transport of ¹⁴C-labelled photoassimilates, bothin isolated upper shoots and in isolated internode-branchletcomplexes of Chara corallina, was measured. The isolated uppershoots were composed of a primary apex, two mature internodes,and three branchlet whorls. A 10 min loading of the isolatedupper shoot with H¹⁴CO₃^– resulted in a greater accumulationof ¹⁴C in the apical complex and branchlets than in the internodes,while a subsequent 50 min chase with unlabelled solution inthe light resulted in a greater accumulation of ¹⁴C in internodesthan in other parts of the shoot. In the isolated internode-branchlet complex, when the apex wasnot detached, the amount of ¹⁴C transported from branchletsto internodes was about fives times that transported from internodesto branchlets. Removal of the apex resulted in a decrease intransport from branchlets to internodes and an increase in transportin the opposite direction. In an attempt to explain the mechanism of the polar transportof photosynthetically fixed carbon between branchlets and internodes,photosynthetic activities of both types of cells were investigated.Detached branchlets have higher photosynthetic ¹⁴C-fixationactivities than those of internodes. Chlorophyll contents, measuredin terms of surface area, in internodes and branchlets werealmost identical. The ribulose-l,5-bisphosphate carboxylase(RuBPCase) activity of branchlets was 1.6 times that of internodes,and the rate of ferricyanide-dependent evolution of oxygen inbranchlets was 1.4 times that in internodes. Key words: Chara, internode, branchlet, polar transport, photosynthesis     

Intercellular Transport and Subcellular Distribution of Photoassimilates in Chara corallina

The mechanism of polar transport of ¹⁴C-photoassimilates betweenChara corallina branchlets and internodes was investigated usingan intracellular perfusion procedure. When the apex remained,more ¹⁴C-photoassimilates were transported from branchlet tointernode than in the opposite direction. When the apex wasdetached, this polarity disappeared. To study the polar transportmechanism, we examined the subcellular distribution of ¹⁴C-photoassimilatesin the three main intracellular compartments, the cortical chloroplastlayer, streaming sol endoplasm and vacuole. When the internode or branchlet of an internode-branchiet complexwas exposed to ¹⁴C-bicarbonate for 10 min, 70 to 80% of thetotal fixed carbon was found in the sol endoplasm. In the subsequent3 h chase, the ¹⁴C-photoassimilates in the sol endoplasm ofthe source cell decreased greatly to 20 to 40% of the totallabelled substances. When the apex remained, the branchlet showed higher photosyntheticactivity than the internode, which resulted in a concentrationgradient of 13·5 mol m^–3 in the sol endoplasm frombranchlet to internode after photosynthesis for 10 mm. In the3 h chase period, this gradient decreased to about 1·0mol m^–3. In the other three cases, involving transportfrom branchlet to internode when the apex was detached, or transportfrom internode with or without apex to branchlet, the intercellulargradient of ¹⁴C after 10 mm photosynthesis was about 70 molm^–3 in the sol endoplasm. Thus, when the apex remains,the greater gradient of photoassimilates between the sol endoplasmof branchlet and internode seems to be one of the reasons forpolar intercellular transport. Key words: Chara corallina, polar transport, ¹⁴C photoassimilates     

The Relationships between Fertility and Contents of Gibberellic Acid, Sugars and Dry Mass in Apical Parts of Chara Vulgaris Thalli

The contents of endogenous gibberellic acid (GA₃), sugars, and dry mass in apical parts of fertile and sterile thalli of Chara vulgaris were estimated. The GA₃ concentration in the first node of fertile thallus, determined by capillary electrophoresis, was about 70.0 mg kg^–1 of fresh mass (f.m.). Pisum sativum-bioassay showed GA₃ concentration of 80.0 mg kg^–1 (f.m.) which was about 3 times higher than in the first node of sterile thallus. The higher amount of GA₃, glucose, and the lower starch content and dry mass in fertile plants than in sterile ones suggest the interdependence between fertility and contents of studied components.     

Source—sink characteristics of carbon transport in Chara hispida

Abstract Rates of uptake of ¹⁴C-labelled inorganic carbon were measured for whole Chara hispida plants, detached parts of the shoot and isolated (split-chamber technique) apices, lateral branchlets and rhizoid—node complexes. The rates of inorganic carbon uptake by the rhizoid—node complex expressed per gram fresh weight whole plant were three to four orders of magnitude less than the uptake for the whole plant. Up to 70% of the carbon taken up by the rhizoid—node complex was translocated to the shoot. After 12 h exposure to ¹⁴C-labelled inorganic carbon the concentration of ¹⁴C was greater in apices than in uppermost or central internodal cells and in all lateral branchlets, regardless of whether label was supplied to the whole plant or isolated rhizoid—node complexes. Measurement of inorganic carbon uptake by detached internodal cells and detached and isolated apices and lateral branchlets showed that lateral branchlets had the greatest rates of inorganic carbon uptake. During 12 h exposure to ¹⁴C, isolated lateral branchlets translocated to the attached shoot 55% of the labelled carbon taken up; for isolated apices this value was only 13%. It is concluded that it is highly unlikely that the rhizoid of Chara hispida could acquire a significant fraction of the whole plant requirement for inorganic carbon and that apices are sink regions for photosynthate while lateral branchlets are source regions.     

Kinetin-promoted transport of assimilates in stems of Phaseolus vulgaris L.

Kinetin, applied as a dispersion in aqueous lanolin to the stumps of decapitated stems of P. vulgaris plants with their roots removed, was found to promote the transport of ¹⁴C- and ³²P-labelled assimilates to the site of hormone application. Measurement of photosynthetic rate of, and assimilate export rate from the source leaves, indicated that kinetin was not acting to promote assimilate transport by stimulating these processes. Moreover, it was found that the time between kinetin application and detection of an enhanced transport flux was independent of the distance over which kinetin would need to move to be present throughout the length of the transport pathway. These observations, together with the finding that lateral applications of kinetin to the stems resulted in an enhanced localized accumulation of assimilates, provided evidence that kinetin acted locally at its point of application to stimulate assimilate transfer.Abbreviations GA₃ gibberellic acid - IAA indol-3yl-acetic acid     

Zum Assimilattransport in wachsende Früchte von Phaseolus vulgaris

Zusammenfassung Nach ¹⁴CO₂-Assimilation im Blatt wurde der Eintransport markierter Assimilate in wachsende Früchte von Phaseolus vulgaris histoautoradiographisch untersucht. Die ¹⁴C-markierten Substanzen werden danach im Phloem der Fruchtstiele transportiert, in keinem Falle konnte eine Assimilatleitung in anderen Geweben des Fruchtstiels bemerkt werden. Hierzu im Widerspruch stehende Befunde von Bachofen und Wanner werden diskutiert.

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On the translocation of assimilates into ripening fruits of Phaseolus vulgaris

Summary Leaves of fruiting plants of Phaseolus vulgaris were exposed to ¹⁴CO₂ in the light. The movement of ¹⁴C-labelled assimilates from these leaves into the growing fruits was studied by histoautoradiography (dry technique). The fact that ¹⁴C-activity could be detected within the fruit stalks exclusively in the areas of the phloem may favor the hypothesis that also in this case assimilates are transported only via the phloem. The xylem was always free of ¹⁴C. The contradictory results of Bachofen and Wanner are discussed.

     

Effects of shoot bending of apple trees on accumulation and translocation of14C-labelled assimilates

The translocation of¹⁴C-labelled assimilates from a single leaf in bent and intact apple shoots was studied in varying stages of shoot development. In actively growing shoots¹⁴C-labelled assimilates translocated from the treated leaf and accumulated mainly in the shoot apex. In moderately growing apple shoots radioactive assimilates were translocated from the treated leaf in both directions towards and down the shoot. In apple shoots showing only slight growth activity the¹⁴C-labelled assimilates were transported from the treated leaf mainly to the base of the shoot, stem and roots. Bending shoots changed the pattern of distribution of radioactive assimilates. Bending actively and moderately growing shoots resulted in higher concentration of 14-carbon in the shoot apex than in controls. In slowly growing and non-growing apple shoots bending caused a higher accumulation of radioactive assimilates in the bent section than in an equal section of control shoots.     

Distribution of labelled assimilates within a young apple tree after supplying14CO2to a leaf or shoot

Labelled carbon dioxide was supplied for 22 hrs to a leaf of the leader or to the lateral shoot in two-year-old apple seedlings. The distribution of radioactive assimilates within the plant following this treatment was investigated by using radioautography. The transport of labelled assimilates from the young leaf of the leader was very meagre and affected only parts of the stem and the leaves situated in the close vicinity of the treated leaf. The¹⁴C-labelled assimilates from the mature leaf of the leader were transported in a considerable amount to the apex and to the other leaves of the leader. They were also found in an appreciable amount in the stem and the roots, as well as in some lateral shoots. After supplying¹⁴CO₂ to the lateral shoot remarkable transport of labelled assimilates was observed. Radioactivity was detected in the tip and in the youngest leaves of the leader, as well as in the roots. Their path in the stem was studied by dissecting the plant and examining the cross section from each internode. This method revealed that the assimilates from the treated leaf or shoot were transported downward only on one side of the stem in a helical pattern. The lateral shoots situated on the radioactive side of the stem were also labelled, whereas those situated on the opposite (non-radioactive) side were not labelled.     

13C labelling reveals different contributions of photoassimilates from infructescences for fruiting in two temperate forest tree species

The pathways of currently fixed carbon in fruit bearing branchlets were investigated in two temperate forest tree species (CARPINUS BETULUS and FAGUS SYLVATICA), which differ in texture of their vegetative infructescence tissues (leaf-like in CARPINUS vs. woody in FAGUS). During late spring, (13)C pulse-labelling was conducted on girdled, defoliated, girdled plus defoliated and untreated fruiting branchlets of mature trees IN SITU, to assess changes in C relations in response to the introduced C source-sink imbalances. At harvest in early August, 75 - 100 % of the recovered (13)C label was bound to infructescences (either fruits or vegetative infructescence tissue), revealing them as the prime C sinks for current photoassimilates. Leaves on girdled branchlets were not stronger labelled than on ungirdled ones in both species, indicating no upregulation of the leaves' photosynthetic capacity in response to the prevention of phloemic transport, which was also supported by measurements of light saturated photosynthesis. In contrast, (13)C labels tended to be higher after complete defoliation in the vegetative infructescence tissues of CARPINUS, suggesting enhanced net photosynthesis of green infructescence parts as compensation for the loss of regular leaves. The total labelling-derived (13)C content of whole infructescences was very similar between foliated and defoliated CARPINUS branchlets. Cupulae of FAGUS, on the other hand, remained almost unlabelled on defoliated branchlets, indicating the photosynthetic inactivity of this woody infructescence tissue. Consequently, CARPINUS still produced relatively high fruit masses on girdled plus defoliated branchlets, while in FAGUS fruit development ceased almost completely at this most severe treatment. Our results highlight that green vegetative infructescence tissue assimilates substantial amounts of C and can partly substitute regular leaves as C sources for successful fruit development.     

Correlative inhibition of lateral bud growth in Phaseolus vulgaris L. timing of bud growth following decapitation

Summary On intact, 3-week-old plants of Phaseolus the larger bud in the axils of the primary leaves shows slow, continuous elongation growth. Release from correlative inhibition can be detected within 30 min following decapitation. When 0.1% indoleacetic acid in lanolin is applied to the decapitated stem stump, the lateral bud shows slow growth during the first 7 h, then stops completely for a further 15 h but after 2 days a further gradual increase in length is observed.The movement of ¹⁴C-labelled assimilates from the subtending primary leaf into the lateral bud increases following removal of the shoot apex. When indole acetic acid is applied to decapitated plants the ability of the buds to import ¹⁴C increases for 5–7 h and then declines to a negligible amount. Little or no radioactivity from tritiated indoleacetic acid is transported into the lateral buds of decapitated plants during the first 48 h following removal of the apex and it appears that rapid metabolism of the compound occurs in the stem tissues.     

PERTICA,A NEW GENUS OF DEVONIAN PLANTS FROM NORTHERN MAINE

A new genus of Devonian age fossil plants is described from the Trout Valley Formation of northern Maine. Abundant compression material permits a rather complete understanding of its morphology. Pertica quadrifaria Kasper and Andrews, gen. et sp. nov., was an erect plant, perhaps a meter tall, with a pseudomonopodial main axis and dichotomous side branches. The side branches were arranged in a clockwise spiral (from base to apex) and were tetrastichous. They dichotomized numerous times, with the intervals between dichotomies decreasing distally. The ultimate branchlets bore numerous sporangia in dense clusters. Other side branches were completely sterile. Pertica quadrifaria is classified in the Subdivision Trimerophytina of Banks. Its evolutionary significance rests in the fact that it is a link in the chain of increasingly complex early vascular land plants.     

Seasonal differences in the pattern of assimilate movement in branches of Coffea arabica L

At Ruiru, Kenya, ¹⁴CO₂ was fed to single leaves at different distances from the apex on branches of mature fruiting and non-fruiting trees of Coffea arabica on six occasions from the Short Rains (November) 1966 to the Long Rains (April-May) 1967. The location of ¹⁴C-labelled assimilates in the treated branches was determined 26 h later by autoradiography. The direction of movement of labelled assimilates indicated large seasonal differences in the relative sink strengths of the shoot tip, fruit and trunk-root systems. On vegetative trees the sink strength of trunk-roots was much smaller, as compared with the shoots, at the beginning of the Long Rains than at the end of the previous Short Rains or in the intervening dry season. Assimilate use by growing fruits did not alter the pattern of distribution of assimilates to the other sinks at the end of the Short Rains, but it did restrict assimilate movement to both shoot tips and trunk-roots at the_ beginning of the Long Rains. In the dry season, virtually all assimilates were utilized by growing fruits when these were present. Vegetative secondary shoots provided assimilates to growing fruits and trunk-roots at the end of the Short Rains and in the dry season. Some practical implications are noted.     

Quantitative Analysis of Intercellularly-Transported Photoassimilates in Chara corallina

Using a thin-layer chromatographic technique, we have identifiedthe photoassimilates that are transported intercellularly frombranchlets to internodes in Chara corallina. An internode-branchletcomplex having a primary apex was used in these experiments.After feeding 1 mol m³ NaH¹⁴CO₃ to a branchlet for 10 min, the¹⁴C-labelled photoassimilates (¹⁴C-photoassimilates) found inthe sol endoplasm of the branchlet were composed of sucrose,amino acids, malate, and sugarphosphates. The composition ofthe ¹⁴C-photoassimilates transported from the source branchletto the sink internode in 10 min was the same as that in thesol endoplasm of the source branchlet. From the proportion ofeach ¹⁴C-photoassimilate in both the source branchlet and thesink internode, it was deduced that the main photoassimilatesinvolved in the intercellular transport were sucrose and aminoacids. We found previously that polar transport of photoassimilatesoccurs from a branchlet to an internode with an apex. Determinationof the amount of sucrose, amino acids, glucose-6-phosphate,and malate in both branchlet and internode with or without anapex revealed that there were gradients in the concentrationsof sucrose, serine, and glutamic acid between the sol endoplasmof the two cells. The levels were higher in the branchlet andlower in the internode and the gradients decreased when theapex was detached. Therefore, it is concluded that sucrose andthese amino acids are the compounds involved in the polar intercellulartransport. Key words: Chara corallina, intercellular transport, photoassimilates, ¹⁴C     

Influence of galls of Phanacis taraxaci on carbon partitioning within common dandelion, Taraxacum officinale

We examined how leaf galls, induced by the cynipid wasp Phanacis taraxaci, influence the partitioning of photoassimilates within the host, the common dandelion, Taraxacum officinale. Galled and ungalled plants were exposed to ¹⁴CO₂ and the labelled photoassimilates accumulating within galls and other parts of the host were measured. During the growth phase of the gall they were physiological sinks for photoassimilates, accumulating 9% to 70% of total carbon produced by the host, depending upon the number of galls per plant. High levels of ¹⁴C assimilation in the leaves of galled plants compared to controls, suggest that galls actively redirect carbon resources from unattacked leaves of their host plant. This represents a significant drain on the carbon resources of the host, which increases with the number and size of galls per plant. Active assimilation of ¹⁴C by the gall is greatest in the growth phase and is several orders of magnitude lower in the maturation phase. This finding is consistent with physiological and anatomical changes that occur during the two phases of gall development and represents a key developmental strategy by cynipids to ensure adequate food resources before larval growth begins.     

Studies on Genetic Male-Sterile Soybeans : IV. Effect of Male Sterility and Source of Nitrogen Nutrition on Accumulation, Partitioning, and Transport of Nitrogen

Soybean (Glycine max [L.] Merr.) germplasm, isogenic except for loci controlling male sterility (ms₁) and nodulation (rj₁), was used to investigate the effects of reproductive tissue development and source of nitrogen nutrition on accumulation, transport, and partitioning of nitrogen in a greenhouse experiment. Nodulated plants were supplied nitrogen-free nutrient solution, and nonnodulated plants were supplied nutrient solution containing 20 millimolar KNO₃. Plants were sampled from flowering until maturity (77 to 147 days after transplanting).

Accumulation rates of nitrogen in whole plants during reproductive growth were not significantly different among the four plant types. Nitrogen accumulation in the sterile, nonnodulated plants, however, ceased 2 weeks earlier than in fertile, nonnodulated or fertile and sterile, nodulated plants. This early cessation in nitrogen accumulation resulted in sterile, nonnodulated plants accumulating significantly less whole plant nitrogen by 133 days after transplanting (DAT) than fertile, nonnodulated plants. Thus, changing the site of nitrogen assimilation from nodules (N₂-fixing plants) to roots and leaves (NO₃-fed plants) resulted in similar whole-plant nitrogen accumulation rates in fertile and sterile plants, despite the absence of seed in the latter.

Leaflet and stem plus petiole tissues of both types of sterile plants had significantly higher nitrogen concentrations after 119 DAT than both types of fertile plants. Significantly higher concentrations and exudation rates of nonureide, reduced-nitrogen in xylem sap of sterile than of fertile plants after 105 DAT were observed. These latter results indicated possible cycling of nonureide, reduced-nitrogen from the downward phloem translocation stream to the upward xylem translocation stream in roots of sterile plants. Collectively, these results suggest a lack of sinks for nitrogen utilization in the shoots of sterile plants. Hence, comparison of nitrogen accumulation rates for sterile and fertile plants does not provide a definitive test of the hypothesis that reproductive tissue development limits photosynthate availability for support of N₂ fixation and nitrate assimilation in determinate soybeans.

Nitrogen assimilation during reproductive growth met a larger proportion of the reproductive-tissue nitrogen requirement of nitrate-dependent plants (73%) than of N₂-fixing plants (63%). Hence, vegetative-tissue nitrogen mobilization to reproductive tissue was a more prominent process in N₂-fixing than in nitrate-dependent plants. N₂-fixing plants partitioned nitrogen to reproductive tissue more efficiently than nitrate-dependent plants as the reproductive tissues of the former and latter contained 65 and 55%, respectively, of the whole-plant nitrogen at the time that nitrogen accumulation in reproductive parts had ceased (133 DAT).

     

Vacuoles of Mesophyll Cells as a Transient Reservoir for Assimilates

We studied the efflux of radioactive photosynthetic products from the central vacuole into the cytosol of protoplasts isolated from the mesophyll tissue of the sugar beet (Beta vulgaris L.) after their darkening and subsequent cessation of photosynthesis. Among the products accumulated in the vacuole were the ¹⁴C-labelled sugars malate and alanine, small amounts of citric, glutamic, and aspartic acids, and some other amino acids. During the initial 20–30 min of darkness, there was no substantial utilization of photoassimilates accumulated in the vacuole during the preceding light period. An efflux of assimilates occurred later, after 30–40 min of darkness. A decrease in the vacuolar ¹⁴C-sucrose occurred not only due to its exit into the cytosol but also because of its conversion into ¹⁴C-monosaccharides by the vacuolar invertase. In fact, this decrease in the sucrose content correlated well with the accumulation of monosaccharides. Immediately after photosynthesis ceased, the chloroplastic ¹⁴C-starch was utilized for the maintenance of cytoplasmic metabolism. After 30-min darkness, the content of starch in the chloroplasts decreased by several times. We believe that the vacuoles of sugar-beet mesophyll cells are transient reservoirs for assimilates and the products of their conversion (glucose and fructose), which can rapidly leave the vacuole to maintain homeostasis in the cytosol under varying environmental conditions.     

The role of the shoot apex in controlling rhizogenesis in vitro

This paper reports that rhizogenesis in woody plant species in vitro was mediated through the basipetal transport of auxin from the shoot apex. This can directly induce roots in easy-to-root species such as Betula pendula, but was dependent upon an interaction with exogenous auxin in more difficult-to-root species such as Daphne cneorum, and to a lesser extent in Quercus robur. Shoot apex removal reduced rhizogenesis in Quercus, and inhibited it in Daphne, even in the presence of exogenous auxin, whereas rooting in Betula was unaffected. That basipetally transported auxin modulates rhizogenesis was demonstrated by the inhibition of root induction in Betula shoots by the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA), and by the substitution of indole-3-acetic acid (IAA) for a bud in Betula internodal sections.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - TIBA 2,3,5-triiodobenzoic acid - MS Murashige and Skoog medium - WPM woody plant medium     

Distribution of photoassimilates in the pea plant: chronology of events in non-fertilized ovaries and effects of gibberellic acid

The short-lived isotope¹¹C (t_1/2=20.4 min) has been used to study assimilate distribution in intact pea plants (Pisum sativum L.). Radiolabel was measured at the leaf fed with¹¹CO₂ (feed-leaf), at the ovary of the flower subtended by this leaf, and in shoot apex and roots of individual plants. Considerable¹¹C-radiolabel was detected in the young ovaries during the first days after anthesis. Thereafter, when the ovaries stopped growing the uptake of¹¹C rapidly decreased. At this developmental stage only apex and roots were competing for the photoassimilates. Fertilization, however, restored the strong sink activity of the ovaries. The same effect could be achieved by applying gibberellic acid to non-fertilized ovaries. About 2 h after treatment the residual¹¹C-radiolabel entering the ovary started to increase and, at about the same time, the ovary resumed growth. Feed-leaf photosynthesis, as well as export of¹¹C-radiolabel out of the leaf, was not changed by the treatment. The¹¹C experiments show the dynamic behaviour of the sinks during developmental stages from the day of anthesis until 5 d later and demonstrate that phytohormones may play an important role in regulating carbon distribution.     

Toward a mechanistic understanding of competition in vascular-feeding herbivores: an empirical test of the sink competition hypothesis

Recent evidence suggests that competitive interactions among herbivores are mostly indirect and mediated by plant responses to herbivory. Most studies, however, emphasize chewing insects and secondary chemistry, thus ignoring the diverse group of vascular-parasites that may be more likely to compete through induced changes in phytonutrients. Using an aboveground phloem-feeding aphid (Myzus persicae) and a belowground gall-forming nematode (Meloidogyne incognita) on tobacco plants, we assessed the importance of competition via induced host–plant sinks. In a series of experimental trials, nematode root herbivory caused 55 and 72% declines in the growth and fecundity of aphids, respectively. Aphids, on the other hand, did not impact nematode performance. Therefore, we predicted that nematodes out-compete M. persicae by attenuating the magnitude of aphid-induced sinks. Through a combination of invertase enzyme measurements and stable isotope (¹³C and ¹⁵N) enrichment, we found evidence that both herbivores act as mobilizing sinks. Aphids attracted photoassimilates to feeding aggregations on leaves and nematode galls accumulated resources in the roots. Levels of invertase enzymes, for example, were more than fourfold higher in nematode galls than in surrounding root tissue. Yet we found no evidence supporting a sink competition model for aphid–nematode interactions. The strength of aphid-induced leaf sinks was entirely unaffected by nematode presence, and vice versa. Thus, induced host–plant sinks appear to be a common strategy employed by vascular parasites to manipulate the physiology of their host, but multi-sink competition may be limited to herbivores that co-occur on the same tissue type and/or plants under growth-limited abiotic conditions.