A METHOD FOR DETERMINING PLASTOCHRON INDICES DURING HETEROBLASTIC SHOOT GROWTH

A plastochron is defined as the time interval between two successive recurring events during the growth of plant shoots, such as leaf initiation. The plastochron index (PI) formulated by Erickson and Michelini (1957, American Journal of Botany 44: 297–305) provides a method for determining 1) morphological equivalence in a developmentally variable sample of shoots and 2) rates of development in microscopic tissues and organs, by expressing shoot age as a function of plastochron number. The PI assumes that homologous organs at successive nodes grow exponentially, at equal rates, and the plastochron remains constant. These three conditions are not met in many shoots that exhibit heteroblasty in their plastochron and the growth rate of organs at successive nodes. An alternative computational method for the PI is presented that uses two measurements taken at different times from the same organ during its exponential growth phase. The method does not assume that the PI is a linear function of time. Results of an analysis of cyme internode growth in two races of Arenaria uniflora (Caryophyllaceae) demonstrate that the method proposed is in good agreement with Erickson and Michelini's (1957) method when shoot growth is not markedly heteroblastic. The current method is also used to determine the nonlinear relation between PI and time in a race of A. uniflora that has heteroblastic cyme growth. The results generalize the PI for use in studies of heteroblasty, and for shoots where the relative plastochron rate cannot be directly determined.     

Development of the shoot apex ofChenopodium rubrum L. after photoperiodic induction in the cotyledon stage

Development of the shoot apex up to floral differentiation was investigated in the short-day plantChenopodium rubrum. The changes occurring in the apex from energence until full opening of the cotyledons (Figs 1–4), development during photoperiodic induction (Figs. 5–8), as well as the resulting floral differentiation (Figs. 9–10) are described. It was aimed at excluding the influence of plastochron changes on the interpretation of ontogeny of the shoot apex. For that reason two planes of longitudinal sections and two plastochron stages were compared. In young plants zonation does not become fully evident prior to floral differentiation. The anatomical structure of the shoot apex does not change substantially during the first two inductive cycles which proved to be obligatory under the given experimental conditions. The changes occurring during two further inductive cycles correspond to the total activation of the meristems as manifested by the growth and branching of the apex preceeding floral differentiation proper.     

CHANGES IN EPILOBIUM PHYLLOTAXY INDUCED BY N-1-NAPHTHYLPHTHALAMIC ACID AND α-4-CHLOROPHENOXYISOBUTYRIC ACID

Preliminary studies establishing relationships between leaf plastochron index and Epilobium hirsutum L. shoot growth provide a method for rigorous selection of plants utilized in experiments designed to test the working hypothesis that endogenous auxin gradient interactions are factors of phyllotactic control in this species. Application of N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, to one of the youngest bijugate primordia on the shoot meristem results in increased growth of the treated primordium. Fasciation between the treated primordium and one of the next primordia to be initiated alters relative vertical spacing of primordia. Angular shifts between subsequent primordia result in spiral transformation of Epilobium bijugate phyllotaxy. Application of α-4-chlorophenoxyisobutyric acid (CPIB), an auxin antagonist, to one of the youngest bijugate primordia on the shoot meristem results in decreased growth of the treated primordium that alters both radial and vertical spacing of primordia. This is followed by angular shifts between subsequent primordia resulting in spiral transformation of the bijugate phyllotaxy. Changes in the growth parameters of NPA- and CPIB-treated shoots are similar. Relative plastochron rates of radial and vertical shoot growth of induced spiral shoots are about half those of lanolin paste control shoots, as are the plastochrons and relative plastochron rates of leaf elongation. Treated shoot meristems have eccentricities of 0.5 as compared to bijugate control meristem eccentricities of 0.7. No significant difference is apparent between basal transverse areas of treated and control shoot meristems. The relative chronological rates of growth of treated shoots are not significantly different from those rates of control shoots. Spiral transformation results from changes in relative positions of leaf primordia insertion on the shoot meristem, not from changes in growth of treated shoots. These changes are accompanied by an increased rate of leaf initiation on a more circular shoot meristem. Existing theoretical models of phyllotaxy are discussed in relation to these chemically induced changes of Epilobium leaf arrangement.     

PLASTOCHRON2 regulates leaf initiation and maturation in rice

In higher plants, leaves initiate in constant spatial and temporal patterns. Although the pattern of leaf initiation is a key element of plant shoot architecture, little is known about how the time interval between initiation events, termed plastochron, is regulated. Here, we present a detailed analysis of plastochron2 (pla2), a rice (Oryza sativa) mutant that exhibits shortened plastochron and precocious maturation of leaves during the vegetative phase and ectopic shoot formation during the reproductive phase. The corresponding PLA2 gene is revealed to be an orthologue of terminal ear1, a maize (Zea mays) gene that encodes a MEI2-like RNA binding protein. PLA2 is expressed predominantly in young leaf primordia. We show that PLA2 normally acts to retard the rate of leaf maturation but does so independently of PLA1, which encodes a member of the P450 family. Based on these analyses, we propose a model in which plastochron is determined by signals from immature leaves that act non-cell-autonomously in the shoot apical meristem to inhibit the initiation of new leaves.     

RELATIONSHIPS BETWEEN SHOOT GROWTH AND CHANGING PHYLLOTAXY OF RANUNCULUS

Growth of Ranunculus shoots through ontogeny is quantified by techniques utilizing scanning electron microscopy and studies on living plant material. The order of the contact parastichy phyllotaxy in the apical system is related to the relative plastochron rates of growth of the shoot. There is a change in the (2, 3) contact parastichy pattern of vegetative phyllotaxy to a transitional (3, 5) contact pattern which is maintained through sepal production. Formation of a 5(1, 1) whorl of petal primordia establishes a (5, 8) contact pattern with the sepal primordia. Subsequent initiation of stamen primordia, in spiral sequence, results in (5, 8, 13) triple contacts between petal and stamen primordia. The stamen primordia and carpel primordia arrangement is characterized by a (8, 13) contact parastichy pattern of phyllotaxy. Through ontogeny the volume of the shoot apex progressively increases but the shape of the apex, described by a second degree polynomial, remains constant. The plastochron and the relative plastochron rates of radial and vertical displacement of primordia progressively decrease during transition but there is no alteration of the chronological rate of apical expansion. The change in contact parastichy phyllotaxy through ontogeny is interpreted as a change in the relative positions of primordia insertion on the apex resulting from an increase in apical volume and an increased rate of primordia initiation.     

Growth of the Shoot Apex of Agropyron repens (L.) Beauv. During Successive Plastochrons

Two kinds of size change occur in the apical dome of Agropyronrepens during development of the shoot. A cyclic increase anddecrease in size results from the production of a new stem segmentand associated leaf primordium during each plastochron. A progressiveincrease and then decrease in size, which occur over a periodof several plastochrons, is attributable to discrepancies betweenthe size increment during each plastochron and the size of thestem segment formed at the end of the plastochron. The volumedoubling time of the dome remains constant at approximatelyone plastochron. Fluctuations in mean cell generation time correlatewith changes in mean cell volume and do not contribute to thesize changes of the dome. Agropyron repens (L.), Beauv, couch grass, shoot apex, cell growth, cell divisions     

Growth changes in the shoot apex of Sinapis alba during transition to flowering

The aim of the work was to report morphological changes whichoccur in the shoot apex during the morphogenetic switch to floweringin the model long day (LD) plant, Sinapis alba. During the floraltransition induced by 1 LD the growth rate of all componentsof the shoot apex is modified profoundly. The earliest changes,detected at 24 h after start of LD, include a decrease in plastochronduration and an increase of growth of leaf primordia. One daylater, the meristem dome starts to increase in volume, apicalinternodes have an increased height and there is a precociousoutgrowth of axillary meristems. All these changes precede initiationof flower primordia, which starts at about 60 h after the startof LD. Later changes include meristem doming, a decrease inthe plastochron ratio and a shift to a more complex phyllotaxis.All the changes, except the decreased plastochron ratio, arecharacteristics of an apex with an increased tempo of growth.The stimulation of longitudinal growth (height of apical intemodes)is more marked and occurs earlier than the reduction of radialgrowth (plastochron ratio). Key words: Axillary meristem, internode growth, leaf growth, plastochron ratio, plastochron duration     

Transgenic tobacco over-expressing a homeobox gene shows a developmental interaction between leaf morphogenesis and phyllotaxy.

The tobacco gene, NTH1, encodes a polypeptide of 326 amino acids and is a member of the class1 KN1-type family of homeobox genes. Expression of NTH1 has mainly been observed in vegetative and reproductive shoot apices, not observed in roots or expanded leaves. Over-expression of NTH1 in transgenic plants caused abnormal leaf morphology, consisting of wrinkling and curvature. Interestingly, the direction of leaf curvature tended to be conserved among almost all of the leaves in any given transformant. In transgenic plants exhibiting clockwise or anticlockwise phyllotaxy, leaves curved to the right or left, respectively, when looking from the shoot apex toward the base. Micro-surgical experiments demonstrated that the presence of the shoot apex is necessary for the development of leaf curvature, indicating that the order of formation of leaves on the stem (the generative spiral) affects leaf development. We found a correlation between the severity of leaf curvature and the value of the plastochron ratio, a parameter of phyllotaxy. Transformants with more severe phenotypes had larger plastochron ratios. From these findings, we discuss the possibility that an increase in the plastochron ratio, caused by over-expression of NTH1 in the shoot apex, may be involved in leaf curvature.     

CHANGE IN EPILOBIUM PHYLLOTAXY DURING REPRODUCTIVE TRANSITION

The ontogeny of Epilobium hirsutum grown under natural summer photoperiod in a glasshouse was divided into vegetative, early transitional, transitional, and floral stages. Bijugate phyllotaxy, common to both the vegetative and early transitional stages, is transformed into spiral phyllotaxy during the transitional stage by an initial change in the divergence angle of a single primordium inserted at a unique level on the shoot. Leaf primordia subsequently are inserted in a spiral arrangement in the indeterminate floral shoot apex. The early transitional shoot apical meristem is about 1.5 times the volume of the vegetative meristem but expands at about two-thirds the relative plastochron rate of volume increment of the vegetative meristem. There are progressive decreases in the plastochron and relative plastochron rates of radial and vertical shoot growth through ontogeny. Relative chronological rates of shoot growth, however, are not altered during ontogeny. Spiral transformation results from changes in the relative points of insertion of leaf primordia on the shoot meristem. These changes are accompanied by an increased rate of primordia initiation on a more circular shoot meristem. The change in phyllotaxy during ontogeny is similar to that which was artificially induced by chemical modification of auxin concentration gradients in the shoot apex, with the additional feature that there is an initial increase in the volume of the shoot meristem prior to the natural spiral transformation. Size of the shoot apical meristem, however, appears to have little influence on Epilobium phyllotaxy; but the geometric shape of the meristem is well correlated with bijugate to spiral transformations. This suggests that geometric parameters of the shoot meristem should be considered in theoretical models of phyllotaxy.     

SLOW MOTION Is Required for Within-Plant Auxin Homeostasis and Normal Timing of Lateral Organ Initiation at the Shoot Meristem in Arabidopsis

The regular arrangement of leaves and flowers around a plant''s stem is a fascinating expression of biological pattern formation. Based on current models, the spacing of lateral shoot organs is determined by transient local auxin maxima generated by polar auxin transport, with existing primordia draining auxin from their vicinity to restrict organ formation close by. It is unclear whether this mechanism encodes not only spatial information but also temporal information about the plastochron (i.e., the interval between the formation of successive primordia). Here, we identify the Arabidopsis thaliana F-box protein SLOW MOTION (SLOMO) as being required for a normal plastochron. SLOMO interacts genetically with components of polar auxin transport, and mutant shoot apices contain less free auxin. However, this reduced auxin level at the shoot apex is not due to increased polar auxin transport down the stem, suggesting that it results from reduced synthesis. Independently reducing the free auxin level in plants causes a similar lengthening of the plastochron as seen in slomo mutants, suggesting that the reduced auxin level in slomo mutant shoot apices delays the establishment of the next auxin maximum. SLOMO acts independently of other plastochron regulators, such as ALTERED MERISTEM PROGRAM1 or KLUH/CYP78A5. We propose that SLOMO contributes to auxin homeostasis in the shoot meristem, thus ensuring a normal rate of the formation of auxin maxima and organ initiation.     

Dual effects of miR156-targeted SPL genes and CYP78A5/KLUH on plastochron length and organ size in Arabidopsis thaliana

Leaves of flowering plants are produced from the shoot apical meristem at regular intervals, with the time that elapses between the formation of two successive leaf primordia defining the plastochron. We have identified two genetic axes affecting plastochron length in Arabidopsis thaliana. One involves microRNA156 (miR156), which targets a series of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. In situ hybridization studies and misexpression experiments demonstrate that miR156 is a quantitative, rather than spatial, modulator of SPL expression in leaf primordia and that SPL activity nonautonomously inhibits initiation of new leaves at the shoot apical meristem. The second axis is exemplified by a redundantly acting pair of cytochrome P450 genes, CYP78A5/KLUH and CYP78A7, which are likely orthologs of PLASTOCHRON1 of rice (Oryza sativa). Inactivation of CYP78A5, which is expressed at the periphery of the shoot apical meristem, accelerates the leaf initiation rate, whereas cyp78a5 cyp78a7 double mutants often die as embryos with supernumerary cotyledon primordia. The effects of both miR156-targeted SPL genes and CYP78A5 on organ size are correlated with changes in plastochron length, suggesting a potential compensatory mechanism that links the rate at which leaves are produced to final leaf size.     

The determination of pea leaves,leaflets, and tendrils

Pea leaf determination was examined by culturing excised leaf, leaflet, and tendril primordia of different ages on a nutrient medium. Pinna primordia were designated as 1) determined, if they grew normally in culture; 2) undetermined, if they grew into differentiated structures that were morphologically and anatomically different from either leaflet or tendril; or 3) partially determined, if the two pinnae of an opposite pair developed unequally in isolation, or for leaflet pinnae only, if laminae were initiated but did not develop completely. The compound pea leaf as a whole is determined over four plastochrons of development. Proximal pinnae are determined during the second leaf plastochron, approximately 0.8 plastochron after their initiation. The second most proximal pair of pinnae is determined during the third plastochron, and the terminal portion of the rachis is determined last, during the fourth plastochron. Determination of leaflet dorsiventrality is gradual, requiring a critical minimum period with the leaf in physiological contact with the shoot system. The rachis primordium, when isolated from the shoot, does not affect determination of its pinnae as leaflets or tendrils. Afila and tendril-less homeotic mutations do not alter the timing of pinna determination.     

A new method to measure leaf age: Leaf measuring-interval index

We propose a new method, the leaf measuring-interval index (LMI), to estimate leaf age in morphological and physiological studies of leaves. When the plastochron, the interval between the initiation of successive leaves, is constant, the well-known leaf plastochron index (LPI) provides a robust measure of leaf age. When the duration of the plastochron is not uniform, however, we show that the LPI can (in simulations) and does (with actual data) turn variation in duration of the plastochron into variance about the regression estimates of leaf growth curves. The method we present in this paper, the LMI, is plastochron independent. This new method is particularly suited, therefore, for studies of plants growing in natural environments rather than in controlled growth facilities where the assumptions of the LPI method can be met.     

DNA, RNA, and Protein in the Pea Shoot Apex in Relation to Leaf Initiation

The average amounts of DNA, RNA, and protein per cell measuredhistochemically in each region of the shoot apical meristemremained unchanged during the course of a plastochron and duringthe early development of the leaf primordium. The average contentof DNA, RNA, and protein per cell was the same in all regionsof the shoot apical meristem.     

COMPARISON OF EARLY LATERAL VEIN FORMATION IN LINUM USITATISSIMUM LEAVES WITH THEORETICAL NETWORK MODELS

The plastochron age of the Linum leaf that first exhibited lateral leaf vein divergences, the divergent leaf, increased through shoot ontogeny, but the size of the divergent leaf remained constant. There were progressive decreases in the plastochron and relative plastochron rate of leaf elongation, but no significant change in relative chronological rate of leaf elongation, through ontogeny. Thus, divergent leaves of similar sizes occupied different relative positions in the array of leaves on stems of different plastochron ages. These observations are partially consistent with theoretical network model predictions on early leaf vein development. The empirical data of this study suggest additional features of leaf development that should be incorporated into future simulation models for leaf vein development.     

A dynamical model for characterising seasonality effects on eelgrass plastochron intervals

The plastochron interval is widely used to calculate age and rates of productivity in many plants, including seagrasses. However, plant responses to changing environmental conditions, including seasonal patterns, can introduce substantial errors in methods for calculating age and rates of growth. We propose a generalised method for characterising seasonal variability in eelgrass plastochron values based on a model that consists of a linear combination of a trend, a seasonality component and a stochastic noise component. The model was validated using data obtained biweekly during 1998–2003 in a Zostera marina meadow in a coastal lagoon in northwestern Baja California. Plastochron intervals exhibited marked interannual and seasonal variability as well as in the timing of plastochron interval (PI) minima and maxima. Correlation analyses indicated that sea surface temperature is a fundamental forcing factor for the plastochron interval, whose local variability is influenced by the onset of ‘El Niño’ and ‘La Niña’ events. The proposed model provided reliable interpretations that elicited the existence of seasonal processes which are usually masked by multimodal changes in the plastochron interval. Using successive averages of seasonal PI to describe annual cycles resulted in reliable leaf-growth assessments as well as in better determinations of shoot age than those calculated using a single annual mean.     

Control of Plant Architecture: The Role of Phyllotaxy and Plastochron

Plants display a wide variety of three dimensional forms, or architectures, that are critical for their survival in competitive environments or, in the case of crops, for their productivity. Architecture is generated after embryogenesis through the activities of shoot apical meristems and root apical meristems. Leaves are the principal lateral organ that determines the plant shoot morphology, and they normally develop in very regular patterns in time and space. The spatial pattern of leaf arrangement is called phyllotaxy, and the temporal pattern is determined by the plastochron, which is the time between successive leaf initiation events. Both programs involve many gene activities as well as the hormones auxin and cytokinin. Apparently, the mechanisms controlling phyllotaxy and plastochron share some regulatory components. In this review, the molecular mechanisms for both patterning programs will be discussed.     

SPECIAL PAPER: THE PLASTOCHRON INDEX: A REVIEW AFTER TWO DECADES OF USE

The plastochron index provides a morphological time scale which has proved more reliable than chronological age in studies relating morphological and physiological development of a whole plant or plant organ. Since its inception in 1957, the index has been utilized in a variety of investigations from leaf ontogeny in cottonwood trees to rhizoid cluster initiation in algae. The plastochron index has been extensively used in studies involving source and sink relationships, leaf anatomy, cell differentiation, and primary vascularization. It has been used in investigations of hormonal regulation of plant growth and in studies of the effects of various environmental factors on developmental processes in crops. This paper reviews some of the literature from 1957 to present concerning the development and use of the plastochron index.     

RELATION OF PLASTOCHRON TO ANATOMY AND GROWTH IN THE SHOOT APEX OF CHRYSANTHEMUM

The relation between plastochron stage, apical anatomy, and thymidine-C¹⁴ incorporation was studied in the shoot apex of Chrysanthemum morifolium ‘Albatross.’ Apices were sorted into early, middle, and late plastochron stage under a dissecting microscope, fixed, and sectioned longitudinally so that median sections included known sectors of the apical flank. Study of these sections revealed no discernible difference between apices in early, middle, or late plastochron with respect to regularity of cell pattern, presence of a cambium-like zone, appearance of the second tunica layer or staining pattern with pyronin or with toluidine blue. Likewise, apices that had been treated with thymidine-C¹⁴ for 2-4 hr showed no differences between the three stages in number or distribution of labeled cells.