Ian Sussex, Department of Molecular, Cellular, and Developmental Biology, Yale University

May, 2006

Evolving concepts of the organization and function of plant meristems

Caspar Wolff first observed the shoot apical meristem of flowering plants in 1759 and recognized that it was the center of organ and cell formation in the shoot. Almost two and a half centuries since Wolff's discovery, the understanding of how meristems are organized and function has increased enormously. Much of this progress resulted from the development of new technologies that allowed previously unanswered questions to be answered, but which often raised new questions that remained unanswered until even newer technologies became available. Thus, progress in understanding meristems has been punctuated by periods of rapid progress followed by periods of relative stasis.

Studies on meristems can be divided into three periods that relate to the available technology. The earliest was observational analysis that had its major impact from the mid 19^th century to the mid 20^th; this was followed by a period of experimental manipulation of meristems that spanned the 1940s to the 1970s; most recently genetic and molecular analysis that began actively in the 1980s has become the major method of analysis. The following discussion focuses on studies on the shoot apical meristem. Comparable work on the apical meristem of the root is described in Chapter 17.

The Era of Direct Observation

The first studies of meristem structure were made on living tissue sections cut with a sharpened knife or straight razor and observed microscopically. The meristems appeared to consist of homogeneous populations of undifferentiated, isodiametric cells. However, the development of improved methods of tissue preparation and staining, associated with the invention of the microtome in the mid 19^th century, it was possible to cut and observe serial sections of apices. These revealed distinct regions within meristems. In angiosperms the shoot apical meristem was described as consisting of one to several superficial layers that covered a central core. These two regions were named tunica and corpus, respectively. In the 1930s more detailed examination revealed cytological and histological differences between cells in three different regions of the tunica and corpus. The areas were termed zones and consisted of the central zone, a surrounding peripheral zone, and an underlying file zone. Apical meristems were then described as having a tunica-corpus organization subdivided into central, peripheral, and file cytohistological zones. The meristems of a large number of angiosperm species were classified according to variations in these parameters. Eventually observational analysis of meristems diminished as an active field of research because the descriptions seemed to be complete and the research on killed and fixed tissues was unable to analyze dynamic relations within different parts of the meristem, or between the meristem and the derivative tissues and organs.

The Experimental Era

In the 1940s and 50s three new experimental technologies were introduced for studying apical meristems. These resulted in a renewed interest in meristem organization and function, and provided answers to questions remaining from the direct observation era. These technologies were microsurgery, radioisotope labeling, and chimeric analysis.

Microsurgical studies were performed by making punctures or ablations that destroyed subsets of meristem cells, or incisions that separated parts of the meristem from each other. In other studies meristems or their parts were surgically excised from the plant and grown in aseptic culture.

When punctures or ablations were made into the extreme summit of the meristem, destroying cells in the central zone, the results were quite dramatic. Growth from the punctured meristem ceased and new meristems that initiated leaf primordia were formed on the flanks of the original apex. This was interpreted as demonstrating control over meristem function by the centrally located cells. This interpretation was supported when marginal parts of the meristem were separated from the center by an incision and formed independent functional meristems, each initiating new leaf primordia. This suggested that the distal region of the meristem was able to suppress precocious outgrowth of its flanking regions.

When entire apical meristems or subsets of meristem cells were excised from the plant and grown in sterile culture they produced shoots. These shoots had normal morphology and indicated that the information to organize and construct a shoot with its various organs and cell types existed within the apical meristem.

Radioisotope labeling, in which ³H-thymidine was used as an indicator of DNA synthesis, resolved questions of mitotic activity within the meristem. Observational studies implied that the distal-most cells in the meristem were the ultimate source of all cells by virtue of their mitotic activity and were named "initial cells." However in the first experiments on ³H-thymidine labeled apices, there was a complete absence of DNA synthesis in these cells. As a result, some scientists believed that these apparently mitotically quiescent cells did not contribute cells to the vegetative shoot. Resolution of the difference between the observational and labeling studies came from two different experiments. First, when the time period of ³H-thymidine labeling was extended beyond twenty four hours, some cells at the meristem summit did incorporate the label, indicating that these cells had a long cell cycle time, and therefore a low frequency of division. Secondly, chimeric analysis, in which plants were subject to the action of mutagens to induce phenotypically marked cells, typically albino or other pigment loss, also demonstrated that the initial cells for the entire shoot were located at the meristem summit.

The ability to mark cells within meristems by genetic means had another important use. It enabled researchers to relate each of the meristem layers to the differentiated cells to which they gave rise. Thus it was shown that the surface layer of the tunica generated the epidermal layer of the shoot, the underlying layer of the tunica generated most of the cortex and some of the vascular tissue, and the corpus generated the central core of the shoot. These three meristem layers were designated L1, L2 and L3 respectively, a terminology that is still in common use.

These experimental technologies greatly expanded the understanding of how meristems are organized and function, and in particular how one part of the meristem influences function within another part. But studies using these methods failed to advance beyond this point because the technologies that would have allowed investigation of the nature of these influences did not exist.

The Molecular Genetic Era

Since the 1970s there has been an explosion of new information on plant meristems. This resulted from the conjunction of several factors. First, there was a focus on a few reference species such as Arabidopsis and maize in which large numbers of mutants could easily be generated or already existed. Second, the technologies for gene isolation, characterization, and modification were already developed in animal and microbial systems, and these were easily transferred to plant systems. Protein localization and in situ hybridization were also significant advancements during this era. New technologies for plant transformation by Agrobacterium or the gene gun were developed as well. Together these technologies allowed a depth of analysis that was previously not possible.

There are three questions that were identified but not entirely resolved by observational and experimental studies: do the central, peripheral, and file meristem zones each correspond to specific patterns of gene expression?; how is the meristem maintained as a population of pluripotant cells that undergo continued self-renewal while their derivative cells differentiate?; and, how do the different regions of the meristem interact with each other to maintain the meristem as a cohesive unit?

Genes that are expressed exclusively or predominantly in a meristem zone-specific manner have been identified in several species including Arabidopsis, maize, tobacco, tomato, and rice. Many of these belong to classes of considered to be regulatory, controlling the expression of down-stream genes. Thus current research is beginning to explore the gene networks that underlie meristem function and cell fate decisions.

In tobacco three genes belonging to the KNOTTED1 family of homeobox genes have expression patterns that localize to specific zones of the meristem. One is restricted to the corpus; another is expressed predominantly in the peripheral zone, and the third in the file meristem. Whether these genes specify function of the meristem zones in which they are expressed is still uncertain because the level of resolution of in situ hybridization, and particularly of the boundaries, is less precise than that of histological observation. So it is still not clear if the molecular expression fields and the histological zones correspond exactly. For this reason the areas of expression are usually referred to as domains rather than zones.

In Arabidopsis numerous genes that are expressed in the meristem but not in differentiating cells, or in specific meristem zones have been identified. The principal research approaches have been to identify and characterize mutants that have altered meristem morphology, clone the relevant genes, and determine their expression patterns by in situ hybridization. SHOOTMERISTEMLESS (STM) is expressed throughout the meristem but not in differentiating cells. CLAVATA3 (CLV3) is expressed in a small number of the distal cells of the layers L1, L2, and L3. These cells are the initial cells for the shoot which have been renamed "stem cells" because of their functional similarity to stem cells in animals. WUSCHEL (WUS) is expressed in a subset of cells within the corpus just below the CLV3 expressing cells. MGOUN is expressed at the outer margin of the peripheral zone. We will discuss interactions between these and other genes later, but it is evident that different histologically distinct zones within the meristem are characterized by distinct patterns of gene expression.

The second question asks how the meristem is maintained as a population of pluripotent self-renewing cells. This is a longstanding question in the biology of the shoot apical meristem and recent studies that combined mutant and molecular analysis shed light on this question. The maintenance of stem cell function in a regulated way depends on a feedback loop involving the CLV1, –2 and –3 genes and WUS. In clavata mutants the meristem becomes greatly enlarged due to excessive accumulation of stem cells. This suggested that in a wildtype meristem the size of the stem cell population is regulated to a small number. In wuschel mutants stem cell function ceases and all meristem cells differentiate. Thus the wuschel gene product was proposed to be required for stem cell identity. It appears that the CLV gene products together form a signal transduction pathway that limits the expression region of WUS. WUS expression is required for stem cell function and is also able to restrict the lateral spread of CLV expressing cells. Thus the interaction between CLV and WUS maintains stem cell function and the maintenance of the meristem as a source of cells for the shoot.

The third question of how genes within the meristem interact with each other to maintain the meristem as a cohesive unit is only now beginning to be addressed. So far it appears that a combination of negative and positive interactions between genes in different zones within the meristem and between meristem genes and those expressed in differentiating regions of the shoot may help to explain this question. Figure 1 diagrams some of the gene interactions in the shoot apical meristem of Arabidopsis. It is expected that future research will reveal how meristem specific gene networks intersect with cell division regulating genes and with the genes that control growth processes and thus produce a more coherent picture of meristem organization and function than we now have.

Figure 1   Cellular and molecular characteristics of the shoot apical meristem of Arabidopsis. The boundary of the L1 cell layer is shown within the meristem and into the adjacent primordium. For clarity the boundaries of the layers L2 and L3 are shown within the central zone only. The three cytohistological zones, CZ, PZ, and FZ are indicated. The tunica is composed of the cell layers L1 and L2 and the corpus is composed of underlying L3 cells. A positive signal from WUS maintains the CLV3 expression domain and stem cell identity in L1, L2, and L3 layers at the summit of the meristem. A signal from CLV3 limits the expression domain of WUS to a small region within the corpus. Arabidopsis homologs of the rice gene SHO are proposed to regulate the rate of cell transition from the CZ to the PZ. MGO1 and MGO2 promote the transition of cells from the PZ into the primordium. The lower part of the figure shows the proposed role of cytokinin in stimulating mitotic activity in the SAM by increasing the levels of D-cyclins. Cytokinin is also proposed to increase expression of KNAT1 and KNAT2, which results in increased cytokinin accumulation. AS1 and AS2 are expressed in founder cells of the primordium but negative interactions between them and STM, KNAT1, and KNAT2 maintain the SAM/primordium boundary. (Adapted from Fletcher, 2002.)

The Next Era

What are likely to be the next technological breakthroughs that will result in a new era of rapid progress in our understanding of plant meristems? Of course we do not know this or we would have applied them already. However, it is clear that the new areas of genomics and proteomics are likely to yield major insights, and in the future what is being called "cellomics" will also be important. Because these technologies allow simultaneous display of hundreds or thousands of gene products their use should permit elucidation of complex gene networks. One new technology that is being used in plant biology is laser capture microscopy (LCM). This technology allows a scientist to capture and remove single cells from specific tissues or regions in tissue sections and process them for molecular analysis. Thus, it will be possible to determine precisely which genes and proteins are expressed in each cell type. The incorporation of these and other new technologies into plant meristem research is likely to maintain this as an active and fascinating field of discovery.

References

Fletcher, J. C. (2002) Coordination of cell proliferation and cell fate decisions in the angiosperm shoot apical meristem. Bioessays 24: 27–37.

Howell, S. H. (1998) Molecular Genetics of Plant Development. Cambridge Univ. Press, UK.

Lyndon, R. F. (1998) The Shoot Apical Meristem. Cambridge Univ. Press, UK.

Steeves, T. A., and Sussex, I. M. (1978) Patterns in Plant Development. Cambridge Univ. Press, NY.

Weigel, D., and Jürgens, G. (2002) Stem cells that make stems. Nature 415: 751–754.