From Tip to Base: Parallel Proteomic and Phosphoproteomic Analyses of Successive Stages of Maize Leaf Development

Maize (Zea mays) leaves mature from tip to base. Thus, the entire sequence of development can be found along the length of a young, unexpanded maize leaf at the same point in time. Proliferative, symmetric cell divisions occur at the leaf base (zone 1). Distal to zone 1, symmetric divisions give way to fate-specifying asymmetric divisions as cells differentiate and begin to expand (zone 2). Further distal to zone 1, the cells no longer divide, but they continue to expand and mature (zone 3). While maize leaf development has been studied extensively by anatomical and mutant analyses as well as fate mapping (Freeling, 1992; Nelissen et al., 2012), understanding exactly what occurs during the transition from proliferative cell division to differentiation to cell expansion requires performing an exhaustive molecular analysis of each leaf zone. Analysis of transcriptomic changes during these developmental transitions would be informative. However, since transcript and protein abundance don’t always agree, fully understanding maize leaf developmental transitions requires direct analysis of all proteins (the proteotype) present in each leaf zone. A crucial component of the proteotype is posttranslational modifications affecting protein function, such as phosphorylation. Detecting changes in the phosphorylation status of a protein requires simultaneous quantification of unphosphorylated and phosphorylated forms of the protein, an arduous task barring comprehensive coverage of the proteome, which, until recently, has not been possible.

Facette et al. (pages 2798–2812) used a label-free proteomics method (Nakagami et al., 2012) to perform parallel proteomic and phosphoproteomic analyses of zone 1, 2, and 3 and mature maize leaf tissue. They identified 8005 proteins, as well as 13,925 phosphorylation sites from 3557 phosphoproteins, in the maize leaf. Using the spectral counts from the mass spectrometry data, they estimated the relative abundance of proteins and phosphoproteins, a subset of which was validated by protein gel blot analysis, and they compared the proteins in the three zones of growing leaves to one another and to those of mature maize leaves (see figure). Correlations among zone 1, 2, and 3 proteins and phosphoproteins were much higher than those between mature leaves and the younger zones. Although most proteins were present in all leaf regions, many were found in young leaf zones but not in mature leaves. The authors then performed hierarchical clustering analysis of the relative abundances of the proteins and phosphoproteins. Comparing these clusters, they discovered that few proteins but many phosphoproteins are enriched in zone 2, which suggests that some proteins are preferentially phosphorylated. Therefore, posttranslational regulation may play a key role in developmental transitions. The authors provide specific examples of proteins that change in either abundance or phosphorylation status over leaf development by analyzing specific proteins with cell wall– and hormone-related functions. For example, comparison of unmodified and phosphorylated forms of the auxin transporter PIN-FORMED1 (PIN1) suggests that there is a tissue-specific difference in phosphorylation, which correlates with changes in PIN1 polarization in epidermal cells during development.

Venn diagram of proteins present in the unmodified proteomes of the four leaf tissue segments. (Reprinted from Facette et al. [2013], Figure 2C.)

Many other important proteins are uniquely enriched or differentially phosphorylated in distinct maize leaf developmental zones. Identifying these proteins and elucidating their functions requires further studies that take advantage of this important resource.