Arabidopsis NAC45/86 direct sieve element morphogenesis culminating in enucleation

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Science  22 Aug 2014:
Vol. 345, Issue 6199, pp. 933-937
DOI: 10.1126/science.1253736

Removing the nucleus in sieve elements

Although a cell's nucleus performs critical command and control functions, some cell types, such as enucleated red blood cells, seem to do without. Sieve element cells in plants similarly carry out their function of transporting nutrients and signals from one end of the plant to the other without the guidance of a nucleus. Furuta et al. watched how the nucleus self-destructs during the development of sieve element cells (see the Perspective by Geldner). The process is regulated under the control of transcription factors, even as the entire nuclear edifice crumbles into nothingness.

Science, this issue p. 933; see also p. 875


Photoassimilates such as sugars are transported through phloem sieve element cells in plants. Adapted for effective transport, sieve elements develop as enucleated living cells. We used electron microscope imaging and three-dimensional reconstruction to follow sieve element morphogenesis in Arabidopsis. We show that sieve element differentiation involves enucleation, in which the nuclear contents are released and degraded in the cytoplasm at the same time as other organelles are rearranged and the cytosol is degraded. These cellular reorganizations are orchestrated by the genetically redundant NAC domain–containing transcription factors, NAC45 and NAC86 (NAC45/86). Among the NAC45/86 targets, we identified a family of genes required for enucleation that encode proteins with nuclease domains. Thus, sieve elements differentiate through a specialized autolysis mechanism.

Long-distant transport sustains life in multicellular organisms. In plants, phloem sieve element cells form a transport network specialized for long-distance allocation of photoassimilates and signaling molecules (1). Unlike in the animal circulatory system, contents are transported through cells rather than between cells. Differentiation of sieve elements elaborates specialized structures (such as sieve plates with pores) and eliminates others (vacuoles, Golgi stacks, the nucleus, and some other organelles) (2). At the same time, the cytosol becomes degraded (36). Nuclear elimination in animal erythrocytes and lens fiber cells, and in some unicellular eukaryotes, occurs by extrusion from the cell body (7), programmed cell death–like process (8), or nucleophagy (9). Here, we analyze the mechanisms by which the nucleus is removed from developing sieve element cells in the plant Arabidopsis.

The two files of protophloem sieve elements in the Arabidopsis root display a longitudinal time series through development, from apparently undifferentiated cells to mature enucleated cells (1012) (Fig. 1, E and F). We reconstructed a three-dimensional (3D) representation from a series of ultrathin (40-nm) sections (n = 3) generated through serial block-face scanning electron microscopy (SEM) (13) (Fig. 1A, fig. S1, and movie S1) in parallel with live cell imaging (see supplementary materials and methods). We identified three stages of development: (i) stage one, in which the nuclear structure is modified, cell wall thickening occurs, and typical sieve element structures are formed; (ii) stage two, in which the nucleus and other organelles become distorted; and (iii) stage three, during which enucleation is completed and cytosol density decreases (Fig. 1, A and C, and figs. S2 to S4). During stage one, the nucleus deforms from a smooth sphere to crimpled, irregular, and nucleolus fragments (Fig. 1, A and C, and fig. S3), as previously noted (6, 14, 15). At the end of stage two, the nucleus shrinks, together with the signal of NUCLEAR PORE PROTEIN 1 marker (NUP1-YFP, where YFP is yellow fluorescent protein) (table S1, fig. S5, and movie S2). The spread of the NUP1 signal suggests disorganization of the nuclear envelope (fig. S5). Histone 2B (H2B)–YFP, typically confined to the nucleus, also spreads throughout the cytoplasm and becomes diluted in ~10 min (Fig. 1G and movie S3). During stage three, remnants of the disorganized nuclear envelope persist (Fig. 1C and fig. S3). Consistent with this, NUP1-YFP was retained after enucleation (fig. S5). While the nucleus is disintegrating, many organelles, including mitochondria and vacuoles, form two perinuclear clusters at opposite sides of the nucleus (Fig. 1A and fig. S4), and the mitochondria gradually change their shape (figs. S2 and S4). The dilution of the H2B-YFP signal coincides with the completion of cytosol degradation, as shown by the abrupt disappearance of electron-dense matter (Fig. 1A), and the disappearance of a cytoplasm-localized protein At3g14780-YFP (fig. S5); this suggests a type of enucleation wherein the nuclear contents are released and subsequently degraded in the cytosol, leaving behind a disorganized nuclear envelope. This differs from previously described enucleation processes in various organisms (79). Sieve elements lack the large central vacuole characteristic of programmed cell death (fig. S5); the lytic vacuoles are rather small and persist through cytoplasmic reorganization (figs. S2 and S5) (16). There is also no evidence for autophagy, based on the ATG8a marker, a homolog of which surrounds the degrading nucleus in Tetrahymena (17) (fig. S5).

Fig. 1 Dynamic enucleation and other subcellular rearrangements during sieve element differentiation.

(A and B) Overview of sieve element differentiation in the wild type (A) and nac45-2 nac86 (B). Asterisks and color coded lines indicate various stages (stages one to three). (C and D) 3D models of nuclei in the wild type (C) and nac45-2 nac86 (D) during various stages of sieve element differentiation. (E) Pattern of Arabidopsis primary root vasculature, establishing the two phloem poles with protophloem sieve elements located between two companion cells and two phloem pole pericycles. Metaphloem sieve elements develop later. (F) Modified pseudo-Schiff–propidium iodide (mPS-PI) staining (19) shows sieve element cell files by their thickened cell walls. (G) pCALS7::H2B-YFP demonstrates sieve element enucleation with diffusion (yellow arrows) and dilution (blue arrows) in the wild type, but nuclear retention in nac45-2 nac86 (yellow arrows). White arrows indicate the nuclei before nuclear degradation. Panels surrounded by dashed red boxes show magnified views of the panels at left. Scale bars represent 10 μm in (A) and (B), 2 μm in (C) and (D), 25 μm in (F), 50 μm in (G, main panels), and 10 μm in (G, dashed boxes).

To unravel the molecular mechanisms regulating cellular remodeling of sieve elements, we analyzed gene expression downstream of a key transcription factor [ALTERED PHLOEM DEVELOPMENT (APL)] (11) that controls phloem development in sorted phloem cells and identified a pair of NAC-type transcription factors [NAC045 (NAC45) and NAC086 (NAC86)] (Fig. 2I, fig. S6, and table S2). We observed specific expression of these two genes in a few sieve element cells before enucleation, as well as in phloem-pole pericycle cells (Fig. 2, A to F). This expression pattern overlaps with that of APL (11) (fig. S6), and expression was reduced in apl (Fig. 2, G and H, and fig. S6), indicating dependence of these genes on APL. NAC45 expression was up-regulated in DEX-treated pAPL::APL-GR/apl plants in the presence of the protein synthesis inhibitor cycloheximide, suggesting that NAC45 may be a direct transcriptional target of APL (fig. S6).

Fig. 2 Molecular characterization of NAC45/86.

(A to F) The expression of pNAC45::GFP-GUS and pNAC86::GFP-GUS is first detected in the developing protophloem sieve elements (white arrows) and at later stages in the phloem pole pericycles (yellow arrows) in the root. Pink arrowheads indicate the xylem axis. (G and H) Expression of pNAC45::GFP-GUS in the wild type (G) and the apl mutant (H). (I) Schematic of gene organization of NAC45 and NAC86. Transferred DNA insertion sites are marked with black arrowheads. Scale bars represent 25 μm in (A) and (D); 20 μm in (B), (C), (E), and (F); 50 μm in (G) and (H); and 100 base pairs in (I).

Next, we characterized loss-of-function mutants of NAC45 and NAC86 (NAC45/86). The single mutants appeared normal (fig. S7), but the nac45-1 nac86 double mutant exhibited delayed growth (Fig. 3A), and the nac45-2 nac86 double mutant displayed determinate growth and seedling lethality, like apl (Fig. 3A). The nac45-2 nac86 phenotype was rescued by expressing YFP-fused NAC86, specifically in the sieve element (fig. S7), suggesting that NAC86 functions cell-autonomously. Histological analysis revealed no mature protophloem sieve elements in either of the double mutants, whereas differentiation of other tissues appeared normal (Fig. 3B and fig. S7). Defective sieve element formation in nac45-2 nac86 impairs phloem transport, as demonstrated by the loss of phloem-mediated transport of free green fluorescent protein (GFP) from the companion cells into the root tip region of pAtSUC2::GFP (18) (Fig. 3C). pAtSUC2::GFP is expressed in the companion cells of nac45-2nac86, indicating normal companion cell development. Imaging of sieve element differentiation in nac45-2 nac86 by serial block-face SEM revealed that the cells in the sieve element position exhibit normal cell wall architecture, including a sieve plate with developed sieve pores (Fig. 1B and fig S2). Some organelle morphogenesis, such as gradual mitochondrial shape changes (figs. S2 and S4) and sieve element plastid development (fig. S2), also seem to occur properly; however, the perinuclear organelle clusters were not formed in nac45-2 nac86 (fig. S4). Neither enucleation nor a decrease of cytosol density were completed in nac45-2 nac86 (Fig. 1, B and D). Consistent with this, both H2B-YFP and the cytoplasmic At3g14780-YFP protein were retained in fully elongated sieve element cells in nac45 nac86 mutants (Fig. 1G and figs. S3 and S5). By contrast, NAC45/86 are not required for formation of lytic vacuoles or the expression and localization of ATG8a in sieve elements (fig. S2 and S5).

Fig. 3 NAC45 is necessary and sufficient for sieve element nuclear and cytosol degradation.

(A) WT, nac45-1 nac86, and nac45-2 nac86 seedlings, showing root-growth phenotypes. (B) Toluidine blue–stained root cross sections of the wild type and nac45-2 nac86. Protophloem sieve elements are indicated by white arrows and are magnified in inset boxes. (C) AtSUC2::GFP in the wild type and nac45-2 nac86 shows defective transport of free GFP in the mutants. (D) Transmission electron microscopy imaging of the pG1090-XVE::NAC45 line with or without induction. White arrows highlight protophloem sieve cells; red arrows mark ectopic white cells at the phloem pole. (E) Plants harboring pG1090-XVE::NAC45, pAt5g48060::H2B-YFP, and At5g48060::RFP-PIP2 with or without induction. Red cells with dotted or diffused H2B-YFP signals are occasionally seen in ectopic positions after induction. Red cells without YFP signals are shown in normal sieve element position with and without induction. The magenta signal in the left panel indicates PI staining. Scale bars represent 1 cm in (A), 20 μm in (B, main panels), 5 μm (top) and 2.5 μm (bottom) in (B, insets), 100 μm in (C), 5 μm in (D), and 10 μm in (E).

We overexpressed NAC45 in transgenic plants under an estradiol-inducible promoter (pG1090-XVE::NAC45). After 4 days of induction, the cells neighboring sieve elements in pG1090-XVE::NAC45 took on characteristics of differentiated sieve elements (Fig. 3D and fig. S7), indicating that ectopically expressed NAC45 is sufficient to promote cytosol degradation at the phloem pole. To examine the function of NAC45 in enucleation, two additional nuclear- and plasma-membrane markers (H2B-YFP and RFP-PIP2, respectively, where RFP, is red fluorescent protein) were introduced into pG1090-XVE::NAC45 under the NAC45-inducible At5g48060 promoter. After NAC45 induction, we observed that RFP-positive cells in ectopic positions occasionally exhibited several small dots of diffused YFP signals in their cytoplasm, rather than the large round signal typically observed in nuclear-localized H2B-YFP, indicating ongoing nuclear degradation (Fig. 3E and fig S7). This suggests that NAC45 is sufficient to promote enucleation in non–sieve element cells at the phloem pole. We conclude that NAC45/86 regulate sieve element differentiation, culminating in enucleation and cytosol degradation.

To understand the downstream targets of NAC45/86, we analyzed RNA expression of wild-type (WT), nac45-2 nac86, and NAC45-overexpression roots (fig. S8). We validated six genes as NAC45/86 downstream targets in plants and studied their subcellular localization (fig. S8). In addition, we identified a family of DEDDh exonuclease-like domain-containing proteins and designated them NAC45/86-DEPENDENT EXONUCLEASE-DOMAIN PROTEIN 1 to 4 (NEN1 to NEN4) (Fig. 4C and figs. S10 and S11). The expression of NEN1 and NEN2 begins in stage one and increases toward enucleation, whereas NEN4 is expressed from late stage one or stage two (fig. S9). The expression level of these genes was reduced in nac45-2nac86, indicating their dependence on NAC45/86 (fig. S9). We also confirmed that NEN2 expression is ectopically induced by NAC45 overexpression (fig. S9). The NEN4-YFP fusion protein accumulates in the nuclei of one or a few sieve element cells before enucleation, whereas both NEN1-YFP and NEN2-YFP change subcellular localization from the cytoplasm to the nucleus during enucleation (Fig. 4A). This shift of NEN2 localization does not occur in nac45 nac86 (fig. S9), suggesting that NEN2 is regulated by NAC45/86 both transcriptionally and posttranslationally.

Fig. 4 NEN4 is a downstream effector of NAC45/86-dependent enucleation.

(A) Subcellular localization of NEN1-YFP and NEN2-YFP are shifted from the cytoplasm (white arrows) to the nucleus (yellow arrows; blue and magenta signal indicate staining with 4′,6-diamidino-2-phenylindole); the signal then disappears (blue arrows). Nuclear-localized NEN4-YFP is transiently expressed before sieve element enucleation. (B) The pCALS7::H2B-YFP signal is degraded in the Ws wild type (blue arrow) but is retained in nen4-2 (yellow arrows). White arrows indicate the nuclei before nuclear degradation. (C) Protein domains of the NEN family. (D) Ws WT, nen4-1, and nen4-2 seedlings. (E) Overview of sieve element differentiation of nen4-2 by serial block-face SEM. Perinuclear clustering (white arrows) and cytosol degradation occur normally. A yellow arrow highlights the retained nucleus. (F and G) Serial block-face scanning electron microscope image of Ws wild type (F) and nen4-2 (G). The nen4-2 protophloem sieve elements show agglomerated nuclear contents associated with the nuclear envelope (red arrows). Black arrows indicate nuclear envelope disorganization. Scale bars represent 10 μm in (A), 50 μm in (B), 1 cm in (D), 10 μm in (E), and 2 μm in (F) and (G).

Next, we examined the role of NEN1 to NEN4 via gene silencing and mutant analysis (fig. S10). The nen4-1 and nen4-2 mutants, which exhibited ~28 and ~21% reduction of root length compared with the Wassilewskija (Ws) wild type (Fig. 4D and fig. S10), retained amorphous H2B-YFP signals (Fig. 4B and fig. S10). Serial block-face SEM revealed that sieve element differentiation events, including cytoplasmic degradation, nuclear envelope disorganization, and organelle clustering, occurred normally in nen4-1 (n = 4 sieve element files) and nen4-2 (n = 2) (Fig. 4E and figs. S1 and S10). However, agglomerated structures abutting on the inner side of the nuclear envelope were observed after stage three (Fig. 4G and fig. S10), which was not observed in either Ws (n = 2) (Fig. 4F) or the Columbia (Col) wild type (n = 3) (Fig. 1A and fig. S3). The amorphous H2B-YFP remnants were located in the peripheral region of the nucleus in nen4-1 and nen4-2 (Fig. 4G and fig. S10). Furthermore, the defective enucleation in nen4-1 was transgenically complemented (fig. S10). Taken together, this indicates that NEN4 is required for completing the NAC45/86-regulated enucleation process.

We have outlined the process by which sieve element cells in Arabidopsis differentiate and divest themselves of a nucleus. NAC45/86 orchestrate the enucleation, perinuclear organelle clustering, shift in NEN1 and NEN2 localization, and cytosol degradation by regulating the expression of a set of target genes, including NEN1, NEN2, and NEN4. The interdependence of these various NAC45/86-mediated subcellular processes remains to be investigated. We also observed a set of NAC45/86 independent processes, such as the formation of cell wall architecture and callose accumulation, the latter of which appears to be dependent on the upstream transcription factor APL. Our results add to the diversity of autolytic processes by which eukaryotic cells restructure their contents.

Supplementary Materials

Materials and Methods

Supplementary Text

Figs. S1 to S11

Tables S1 to S6

References (2033)

Movies S1 to S3

References and Notes

  1. Acknowledgments: We thank K. Kainulainen, M. Herpola, M. Lindman, A. Salminen, and I. Sevilem for technical assistance; R. Siligato, A. Mähönen, the University of Ghent, the Arabidopsis Biological Resource Center, and the Nottingham Arabidopsis Stock Centre for published materials; A. Mähönen, E.-L. Eskelinen, A. Suomalainen-Wartiovaara, and P. Runeberg-Roos for discussions; and P. Benfey, K. Nakajima, and S. El-Showk for their critical comments. The serial block-face electron microscopy was supported by Biocenter Finland. S.M. was financially supported by the Japanese Society for the Promotion of Science. Work in the lab of T.B. was partly financed by grants of the Research Foundation-Flanders (project FWO – GO20011N) and the Interuniversity Attraction Poles Programme from the Belgian Federal Science Policy Office. Work in the E.J. laboratory is funded by the Academy of Finland, Biocenter Finland, and the University of Helsinki. Financial support for the lab of Y.H. was provided by the Academy of Finland Centre of Excellence programme, the University of Helsinki, the European Research Council Advanced Investigator Grant Symdev, Tekes (the Finnish Funding Agency for Technology and Innovation), and the Gatsby Foundation. The supplementary materials contain additional data.

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