The steady decline in the distance between meristems as previously observed, may simply reflect the decreasing size of the central area. Such an effect may be widespread among angiosperms, as bud rates increased following induction in chrysanthemums and Chenopodium amaranticolor, Protea spp., while increased meristem sizes following induction have been found in apple, strawberry, chrysanthemums, and Protea spp.. At about the time the first flower reaches anthesis, the inflorescence meristem then begins a long steady decline in activity. This begins well before the rosette leaves begin to senesce, and continues unperturbed well after most leaves are gone .Both of these patterns are poorly correlated with the supply of carbohydrates, which suggests that this growth pattern is actually the product of a different developmental program. One clue about the nature of the other developmental mechanism might come from an age-related response of the meristem maintenance gene WUSCHEL, which was reduced over time in the clv3-2 background. This may reflect a broader phenomenon, as the IM tissues compared in the present study were of considerably different ages. The failure to detect more than 8000 genes in the present study could thus be a result of an age-related decline in transcriptional activity, rather than the result of fruitload effects. Additional evidence of a non-carbohydrate limited developmental mechanism comes from the analysis of de-fruited plants, which displayed a peculiar pattern of prolonged activity that began at almost just before WT plants began to reach meristem arrest . This also corresponds to a time when the majority of leaves have already senesced, implying that the trigger for meristem arrest is at least partially independent of carbohydrate supply. This can also be seen in the soc1/ful double mutant, which maintains large numbers of leaves throughout its lifespan, and thus has an abundant carbohydrate supply. However, under continuous light, it was found that the soc1/ful IM began to break down after producing approximately 60 lateral organs ,raspberry grow in pots a figure that is remarkably similar to the maximum number obtained with de-fruited Col-0 plants , and with male sterile plants in the Ler background.

Thus it would appear that maximum lifespan of an IM is genetically determined, though carbohydrate supplies probably trigger arrest at a predictable point long before the maximum is reached. However, it remains unclear why that predictable point occurs at approximately 40 flowers, as leaf senescence patterns would favor an earlier time point. Interestingly, calculations based on the diameter of the Arabidopsis meristem at induction combined with the reported 10% decrease between spacing of successive flower primorida and the minimum size of meristem cells suggest that the IM is capable of producing about 20 flowers before the size of the meristem drops below a single cell diameter. Although this is only half of the actual number of buds that are produced on most plants, this estimate is a close match for the number of flowers produced after the start of the declining trend, 15 days after induction. This suggests that meristem maintenance pathways may be turned off as early as the first open flower, while subsequent flower bud production merely consumes the remaining stem cells.Once an inflorescence meristem has reached a quiescent phase, it will eventually be permanently inactivated by one of two different mechanisms. In the Ler ecotype, it was reported that certain meristems eventually produced a number of carpel-like bracts before being entirely lost . A similar pattern is observed in the soc1/ful mutant, which often produces carpel-like bracts over an extended period of time, and even rosette-like whorls of small leaves in place of the flowers. These anomalous carpel-like structures have also been reported for other Brassica spp., suggesting the existence of a single mechanism derived from a common ancestor. In this case however, even carbohydrate starvation can be ruled out, as soc1/ful double mutants retain large numbers of green leaves throughout the time of growth arrest . The mixing of different developmental programs in these terminal structures further implies that the meristem gradually loses its ability to define organ identities. Another potential route of meristem termination is the targeted senescence of the apical tissues. This is perhaps not unique to Arabidopsis, as it has also been observed in vegetative apices of a broad array of angiosperm species, including blueberries, elms, willows, mulberry, peas, kiwifruit, lilac, Brownea ariza, maple trees, Tilia chordata, Theobroma cacao and alternate bearing pistachios. The localized nature of the senescent pattern suggests that only a small group of cells actually perceive the triggering event. Combined with the observation that multiple nearby tissues senescence simultaneously, even when they are not in direct contact with each other, this also strongly suggests the involvement of the ethylene hormone.

The most likely hypothesis to explain this pattern is an ethylene “burst”, similar to the pattern of ethylene production that occurs in climacteric fruit. Provided ethylene biosynthesis was of short duration, this would be sufficient to explain the simultaneous senescence of the meristem and adjacent flower buds and the limited range of the senescent signal. Intriguingly, the ethylene biosynthesis gene ACS5 does appear to be upregulated in the apices of old inflorescence meristems, which correlates well the up-regulation of copper-related genes found in the present study, which may result in enhanced ethylene perception. The weakening of the cell wall predicted suggested by xyloglucan hydrolase TCH4, the starch degradation and even the expression of PAP2 to produce red pigments in the present study also closely parallel the process of fruit ripening in other species, which involve an increase in pectinesterases, an increase in free sugars and the synthesis of anthocyanin pigments.Leaf lengths were measured every two days until they began to senesce. This was then used to plot the average growth rates, identifying their length at the time of their maximum growth rate. Growth rates during their first six days were accurately predicted by the empirically determined formula: Leaf length = 1.05t2 , where length is in millimeters and “t” is measured in days. This was used to calculate the age of the leaf at the time of its first measureable length, then subtracted from the chronological plant age to obtain the time of leaf initiation. Leaf tip removal was timed to occur just after maximum leaf growth, on a leaf by leaf basis. Roughly 1/3 of the total leaf area was removed, using two cuts to remove a diamond-shaped section, reflecting the pattern of senescence. Stem heights, buds and fruits numbers were measured every other day until meristem arrest became visible. Meristem collection for microarray analysis used Col-0 plants grown in both long day conditions and in continuous light. In order to maximize fruit load, but to avoid collecting senescent tissues, all plants were harvested simultaneously when a single meristem anywhere in the flat was found to be arrested, under the expectation that the rest would become senescent within 48 hours as previously established. Meristem tissue collection proceeded in a stepwise manner, first by harvesting 3-5 cm sections of Arabidopsis branch tips,square plastic pots followed by micro-dissection of the SAM in batches of 10-15 meristems. These included all 1°, 2°, 3° and higher order branch meristems, as available. All flowers and fruits older than stage 2-3 were surgically removed from each section using a dissecting microscope, first removing mature flower buds and fruits, then by collecting a volume of SAM tissue of equal height and width, measuring roughly 0.2mm3 .

The same blade and cutting surface were used for all cuts. The SAM tissue was flash-frozen in liquid N2 within 30 seconds of dissection. A total of about 1500 meristems were pooled for each biological replicate, for a total of five replicates. RNA was extracted from the plant tissue by grinding the tissue under liquid nitrogen with Triazol reagent, then extracted with a Qaigen RNA easy kit. The RNA was submitted to UCR core facility http://genomics.ucr.edu for hybridization to ATH1 affymetrix microarrays. The collection of control tissues was previously described by pg 68. To summarize, the meristems were collected from bolting shoots just after the first flower reached anthesis, using Col-0 plants grown in long days . Each replicate contained 100-120 wild-type meristems collected at the end of the day, removing all flowers older than stage 6. RNA extraction and hybridization were as a described above.The ability of plants to grow from seemingly nothing at all has fascinated people for millenia, and was perhaps most famously demonstrated by Johannes Baptista van Helmont’s 1648 potted willow experiment. Although he incorrectly deduced that plants obtained their mass from water, it is known today that plants obtain much of their bulk from carbon dioxide in the atmosphere. This gas is then used by the plant to make sugars, cellulose and other molecules needed to grow and reproduce. However, it is also equally clear that plants do not simply swell up like a sponge, because their seedlings bear little resemblance to mature trees. Instead, plants actually confine much of their growth and development to very small parts of their anatomy, which can be found by tracing the stem from the ground up, so to speak. After passing the trunk, the larger branches, and the slender twigs, the actual tip of the branch is often found to be obscured by a dense cluster of small scales or leaves, collectively known as a bud. When these leaves are peeled off layer-bylayer, one will eventually find a smooth rounded dome in the center, often a mere fraction of a millimeter wide, surrounded by tiny organs in various stages of development. This is the primary unit of plant growth, and it is known as the shoot apical meristem . Appearances can be deceiving however, as the SAM actually performs many critically important activities necessary for survival.

The more obvious of these include the production of all new leaves, branches, and flowers, which replace lost or damaged organs, and are necessary to produce the next generation. The SAM is also the site of many developmentally important decisions, regulating aspects such as how fast the plant grows, how many leaves are produced, and when to flower. These decisions are in turn are based on a wide variety of information sources, such as temperature, photoperiod, disease, age, and the current nutritional state of the plant. Unlike an animal brain though, very little of this decision-making process is evident in the cellular anatomy of the SAM. When examined from longitudinal sections, its tissues are slightly smaller and denser than average, but there is otherwise little to attract attention. The most obvious feature is a subtle layered arrangement of cells near the surface, which are distinguishable by the fact that their cells always divide at right angles to the surface. In most flowering plants, two such layers are present, each of which is a single cell thick, and both are draped over an interal dome-shaped mound of irregularly shaped cells. For the sake of convenience, the layers are numbered from the outside-in starting with Layer 1 , and then proceeding through L2, L3, and so on. However, this system becomes less useful with tissue depth, because the presence of the irregularly shaped cells deep inside the SAM make it increasingly difficult to identify the individual layers. As a result, many authors simply stop counting at L3, but it is commonly accepted that “L3” refers to the entire inner volume of irregularly shaped cells, rather than to a single layer . The three-layered description has some support in terms of known gene expression patterns, and to avoid confusion the remainder of this dissertation will also stop counting at L3. The only exception occurs in chapter 4, where a longitudinal analysis of protein distributions made it necessary to describe cell layers as deep as L11 . In addition to the cell layers, there is also another discrete set of patterns in the SAM that cannot be seen by the naked eye. They are instead recognized by differences in gene expression patterns and cell division rates. The very center of the SAM for example, contains a vertical column of cells that divide at rates 2-3x slower than those in the periphery. This division rate, along with the appearance of new organs on the periphery, have long served as landmarks to identify the meristem structure, and they are conveniently known as the Central Zone and Peripheral Zone . Typically the CZ is further subdivided into upper and lower portions, such that cells in the L1 and L2 are recognized as the “upper” CZ, while those in the underlying L3 are known as the Rib Meristem .