Arabidopsis Lateral Root Development 3 is ... - Wiley Online Library

The Plant Journal (2011) 38, 455–467

doi: 10.1111/j.1365-313X.2011.04700.x

Arabidopsis Lateral Root Development 3 is essential for early phloem development and function, and hence for normal root system development Paul Ingram1,†, Jan Dettmer2,‡, Yrjo Helariutta2 and Jocelyn E. Malamy1,* Department of Molecular Genetics and Cell Biology, The University of Chicago, 5812 S. Ellis Street, Chicago, IL 60637, USA, and 2 Plant Biology, Viikinkaari 1 (PL 65), University of Helsinki, Helsinki 00014, Finland

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Received 4 May 2011; revised 28 June 2011; accepted 4 July 2011; published online 19 August 2011. *For correspondence (fax +1 773 702 0904; e-mail [email protected]). † Present address: GrassRoots Biotechnology, 302 E. Pettigrew St, Ste A200, Durham NC 27701, USA. ‡ Present address: Department of Plant Systems Biology, VIB, 9052 Gent, Belgium.

SUMMARY We have identified a gene, Lateral Root Development 3 (LRD3), that is important for maintaining a balance between primary and lateral root growth. The lrd3 mutant has decreased primary root growth and increased lateral root growth. We determined that the LRD3 gene encodes a LIM-domain protein of unknown function. LRD3 is expressed only in the phloem companion cells, which suggested a role in phloem function. Indeed, while phloem loading and export from the shoot appear to be normal, delivery of phloem to the primary root tip is limited severely in young seedlings. Abnormalities in phloem morphology in these seedlings indicate that LRD3 is essential for correct early phloem development. There is a subsequent spontaneous recovery of normal phloem morphology, which is correlated tightly with increased phloem delivery and growth of the primary root. The LRD3 gene is one of very few genes described to affect phloem development, and the only one that is specific to early phloem development. Continuous growth on auxin also leads to recovery of phloem development and function in lrd3, which demonstrates that auxin plays a key role in early phloem development. The root system architecture and the pattern of phloem allocation in the lrd3 root system suggested that there may be regulated mechanisms for selectively supporting certain lateral roots when the primary root is compromised. Therefore, this study provides new insights into phloem-mediated resource allocation and its effects on plant root system architecture. Keywords: root system architecture, phloem, lateral root, resource allocation, auxin, callose.

INTRODUCTION The architecture of the plant root system is highly plastic, and is determined by a combination of developmental and environmental factors (reviewed in Malamy, 2005, 2010). Root system architecture is defined by the growth of the primary root, and the number, placement and growth of secondary and higher order lateral roots. Some of the molecules that influence these characteristics must travel to the root system from a distance. For example, the plant hormone auxin transported from aerial tissues is important for regulation of lateral root emergence (Bhalerao et al., 2002; Swarup et al., 2008). Phytochrome signaling in the shoot regulates lateral root emergence, perhaps by altering auxin movement (Salisbury et al., 2007). Carbohydrates, which must be translocated from photosynthetic tissue, can be limiting for the growth of root systems (Bingham and Steª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd

venson, 1993; Freixes et al., 2002), and several studies have demonstrated that root system architecture is related closely to root carbon status. For example, shade-grown plants show a reduction in root system soluble sugar content and reduced root growth (Muller et al., 1998), while plants grown under higher light or CO2 levels, or in the presence of exogenously supplied sugars, display increased root sugar concentrations and increased primary and lateral root growth (Bingham et al., 1998; Freixes et al., 2002; Lee-Ho et al., 2007; MacGregor et al., 2008). Changes in endogenous sucrose transport have also been suggested to cause changes in root system architecture in response to nutrient stress (Hammond and White, 2008). Nevertheless, long-distance mechanisms for regulating root system architecture have not been examined closely. 455

456 Paul Ingram et al. In this work, we show that phloem plays an important role in defining root system architecture. Phloem is a major conduit for long-distance movement of sucrose and other molecules from aerial tissues to the root system. Phloem is comprised of two distinct and intimately connected cell types: the companion cells (CCs) and the sieve tube elements (SEs), which are derived from the asymmetric division of a common progenitor (Knoblauch and Van Bel, 1998; Schulz, 1998; Evert, 2006). SEs lose their nuclei and most organelles during differentiation, joining end-to-end to form enucleate pipelines that allow for the movement of phloem fluid (Sjolund, 1997; Schulz, 1998). All cellular functions that are essential for maintenance of the SEs are provided by CCs (Oparka and Turgeon, 1999; Van Bel, 2003). CCs are directly connected to SEs by special plasmodesmata called pore plasmodesmal units (PPUs; Oparka and Cruz, 2000), which provide for both regulated and unregulated movement of cargo from the CCs into the SEs (Zambryski, 2004; Aoki et al., 2005; Lough and Lucas, 2006; Lucas et al., 2009). Movement of molecules out of the phloem at sinks such as root tissues occurs either apoplastically or through symplastic connections to surrounding cells facilitated by PPUs (Oparka and Cruz, 2000; Stadler et al., 2005). Although several genes have been identified as molecular markers for different phases of phloem development (Bauby et al., 2007), our understanding of this process is extremely limited. Phloem cells are derived from the procambium, which is established during embryogenesis through a coordinated pattern of cell divisions (Berleth and Mattsson, 2000). The MYB-coiled-coil transcription factor APL is the only gene with a known role in phloem specification, as apl mutants contain cells with hybrid xylem and phloem-like morphological characteristics in the location where phloem is normally formed (Bonke et al., 2003; Truernit et al., 2008). In tomato, antisense suppression of FRUCTOKINASE2 (LeFRK2) resulted in shorter, narrower sieve elements, reduced callose deposition, and reduced phloem transport (DamaraWeissler et al., 2009), suggesting that sugar metabolism genes may play a role in vascular development. The size of the PPU aperture can be regulated during development by the production and degradation of callose (b-1,3-glucan) at the plasmodesmal opening, by the accumulation of proteins that localize to the periphery of the PPU or by the addition of branches to the plasmodesmal structure (Benitez-Alfonso et al., 2009; Lucas et al., 2009; Stonebloom et al., 2009; Zavaliev et al., 2010). However, relatively little is known about the molecular pathways that control PPU development or regulation. Here we describe the identification of LRD3 (LATERAL ROOT DEVELOPMENT 3) as an essential component of early phloem development, long-distance delivery of phloem content, and proper maintenance of Arabidopsis root system architecture.

RESULTS lrd3 mutant displays reduced primary root growth and altered root system architecture The lrd3 mutant was isolated in a previously described screen for mutants in root system architecture (Deak and Malamy, 2005). Approximately 4000 T-DNA mutagenized lines (Weigel et al., 2000; ABRC CS21995) were screened on a mild osmotic stress media used to repress lateral root formation. Mutants that showed three or more lateral roots were selected. lrd3 was selected because of reduced primary root length (PRL) in addition to increased lateral root formation. Furthermore, approximately 25% of the time (4/16 plants), one to four lateral or anchor roots (defined as roots emerging from the hypocotyl-root junction) become very long and outgrow the primary root (Figure S1). Mild osmotic stress medium was chiefly used because it suppresses lateral root formation in wild-type plants and therefore facilitates root system mutant identification. However, this medium creates a unique growth condition, with relatively high exogenous sucrose (4.5%) and nitrate (40 mM) content. For detailed characterization, lrd3 and wild-type (Col) plants were instead grown on medium with relatively low sucrose (1%) and nitrate content (10 mM), hereafter referred to as ‘standard media’. lrd3 plants displayed two consistent root system phenotypes on standard media. First, the average PRL of lrd3 was shorter than Col at all times during an 11 day experiment (Figure 1a,b). This change was accompanied by reduction in the size of the meristematic zone and a significant reduction in the average length of elongated epidermal cells in the mature region of the root (Figure S2), indicating that reduced cell division and elongation both contribute to the reduced PRL in lrd3. Second, lrd3 plants produced more lateral roots than Col by 7 days (Figure 1c). The combination of these two phenotypes resulted in greater lateral root density (number of visible lateral roots per cm primary root) in lrd3 plants compared with Col at all time points (Figure 1d), despite the fact that by 11 days the longer Col primary roots had a higher total number of lateral roots (Figure 1c). The increase in lateral root density at 11 days was due to increases in both lateral root initiation/PRL and emergence of lateral root primordia to form lateral roots (Figure S2). Closer inspection of the mutants revealed two distinct classes. In the larger class (19/24 plants, Class A), the reduction in lrd3 PRL was accompanied by an increase in the density of lateral roots along the entire length of the primary root (Figure 1a, Class A). The primary root growth rate increased over the course of the experiment in these lrd3 plants, with mean primary root growth rates of 0.26, 0.43, and 0.73 cm per day at 5–7, 7–9, and 9–11 days, respectively. Col had significantly higher mean growth rates at all time points and the rate also increased over the course of the experiment, but not as precipitously as in Class A lrd3 plants

ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 38, 455–467

LRD3 is essential for root phloem function 457

Figure 1. lrd3 plants show an altered root system architecture on standard media. (a) Col and lrd3 were grown for 11 days. Lateral or anchor roots growing beyond the primary root are indicated with a white arrowhead for Class B mutants. (b) lrd3 plants show reduced primary root length compared with Col at all time points (P < 0.01). (c) lrd3 plants have significantly more (7 days, P < 0.01) or fewer (11 days, P < 0.01) visible lateral roots than Col. (d) lrd3 plants have a higher lateral root density than Col at all time points (P < 0.01). (a) Bar = 5 mm; (b–d) n ‡ 23; Error bars = means SE.

(0.69, 0.98, and 1.02 cm per day, respectively). Hence, from 5–11 days there is a rapid recovery in the growth rate of Class A lrd3 primary roots (growth rate relative to Col is 38, 44 and 71.5% at 5–7, 7–9, and 9–11 days, respectively). In the smaller class (5/24 plants, Class B), lrd3 plants showed severely altered root system architecture, with one to four lateral or anchor roots surpassing the length of the primary root (Figure 1a, Class B). In these plants the mean PRL was significantly shorter than Col and Class A lrd3 plants at all time points and the growth rate increased very little if at all over the course of the experiment (mean primary root growth rates of 0.15, 0.13, and 0.23 cm per day at 5–7, 7–9, and 9–11 days respectively). Meristem sizes of Class B plants were significantly shorter than Class A mutants (data not shown; P < 0.001) while elongated cell lengths were not significantly different, suggesting that the slow growth was due primarily to a decrease in cell proliferation at the meristem.

In summary, lrd3 primary roots grow slowly after germination and then recover in most but not all cases. When the primary roots fail to recover, one or more lateral or anchor roots appear to replace the primary root in terms of dominant growth. lrd3 phenotype is not due to altered sucrose uptake Mutants previously identified from this screen turned out to have cuticle defects that allow greater uptake of sucrose from the media through the leaf surface; the increased aerial sucrose stimulates emergence of lateral root primordia to form lateral roots (MacGregor et al., 2008). Importantly, these mutants are distinct from lrd3 in that they invariably have increased rather than decreased PRLs (MacGregor et al., 2008). To directly test the role of sucrose uptake in the lrd3 mutant phenotype we grew mutants on a strip of Parafilm that blocked contact between the aerial tissues and the sucrose-containing media, as cuticle mutants require


458 Paul Ingram et al. such contact to display a root system architecture phenotype. The lrd3 mutant did not show a drastic change in root system architecture when aerial tissues were prevented from contacting the media (Figure S3). A more rigorous assay monitoring 14C-sucrose uptake was also performed to confirm that lrd3 is not affecting the permeability of the aerial tissues to sucrose (MacGregor et al., 2008). Indeed, the uptake of 14C-sucrose was not significantly different between Col and mutant (Figure S4). Therefore, the lrd3 phenotypes do not appear to be associated with alterations in sucrose uptake. Consistent with this, similar lrd3 phenotypes were seen when sucrose was omitted from the plate (Figure S3). lrd3 phenotype is not hypersensitive to auxin The decreased PRL and increased lateral root formation seen in the lrd3 mutant is reminiscent of the effects of auxin on the root system. Therefore, we wondered whether the mutant was hypersensitive to auxin. To test this, we grew Col and lrd3 plants on indole-acetic acid (IAA) in concentration ranging from 10)10 to 10)5 M and measured PRL. Interestingly, auxin concentrations of 10)8 and 10)7 M showed less inhibitory effect on PRL in lrd3 than in Col (Figure 2). Although the reasons for the reduced auxin sensitivity in the mutant are not immediately obvious, these findings eliminate the possibility that the lrd3 phenotype can be explained by auxin hypersensitivity. It should also be noted that exogenous auxin did not rescue the PRL defect in lrd3; the similar appearance of Col and lrd3 at 10)7 M IAA is due to the greater inhibition of root growth in Col, which causes it to resemble lrd3. lrd3 is a mutant allele of At2g39830/DAR2 We identified the gene carrying the mutation in lrd3 using thermal asymmetric interlaced (TAIL)-PCR (Liu et al., 1995). Sequencing of TAIL-PCR fragments identified a T-DNA

insertion site in the 9th intron of the uncharacterized gene At2g39830 (Figure 3a). A second mutant allele of LRD3 (salk 016122C, signal.salk.edu; Figure 3a) showed reduced PRL and altered root system architecture, similar to lrd3 (Figure 3b). F1 plants from a cross between the salk 016122C and lrd3 alleles failed to complement the primary root phenotype (Figure 3b). We also generated a construct containing 2 kb of the LRD3 promoter driving the LRD3 cDNA, and found that approximately three-quarters (14/19) of T2 plants from an individual T1 plant showed complete rescue of all root phenotypes (Figure 3c). These results provide genetic proof that mutations in At2g39830 are responsible for decreased PRL in lrd3. RT-PCR revealed that lrd3 fails to accumulate detectable full-length LRD3 transcript, suggesting a null mutation (Figure 3d). The occurrence of distinct Class A and Class B phenotypes in lrd3 suggests the possibility of a second mutation, perhaps closely linked to LRD3 and therefore co-segregating through the three back-crosses performed. To test this idea, we analyzed the phenotype of the unrelated Salk allele of LRD3. As in the original lrd3 allele, an anchor or lateral root supplanted a non-recovering primary root approximately 25% of the time in salk_016122C (12/55). Therefore, it is unlikely that the two phenotypes represent genetic segregation, and suggests instead a certain level of stochasticity in the lrd3 phenotype. The LRD3 sequence is predicted to encode a 526 amino acid protein that contains a single LIM domain about 150 amino acids from the N-terminus and a zinc binding domain near the C-terminus (Figure 3e). LIM domains contain a zincfinger motif shown to facilitate protein-protein interactions (Bach, 2000), a function with too general a description to help assign a function to LRD3. The LRD3 gene was reported to contain significant amino acid identity to the gene DA1 (At1g19270), a gene involved in the control of organ size, and was named DAR2 (DA1-RELATED 2) (Li et al., 2008). However, no phenotype was reported and no function was ascribed to LRD3/DAR2 in that study. LRD3 is expressed in the phloem

Figure 2. lrd3 plants show reduced sensitivity to auxin. At 11 days, primary root length of lrd3 was less inhibited by 10)8 and 10)7 M IAA than Col [analysis of variance (ANOVA) GxE P < 0.001]. Error bars = means SE.

To determine the potential site(s) of action of LRD3, we visualized the LRD3 transcription pattern by cloning 2 kb upstream of the LRD3 start site in front of the b-glucuronidase (GUS) gene uidA (see Experimental Procedures). The same promoter rescued the lrd3 phenotype when driving LRD3 cDNA expression in transgenic complementation experiments (Figure 3c). pLRD3:GUS plants revealed LRD3 expression in the vasculature of leaves, inflorescence stems, flowers, hypocotyls, and primary and lateral roots (Figure 4a–f). In roots, LRD3 expression appeared to be highest in the vasculature of younger tissues of primary and lateral roots (Figure 4c,f). LRD3 expression was not observed in the primary or lateral root meristems (Figure 4c,f), lateral root primordia or newly emerged lateral


LRD3 is essential for root phloem function 459 Figure 3. lrd3 is a mutant allele of At2g39830. (a) Predicted intron/exon structure of At2g39830. Exons are shown as shaded boxes. Positions of the original lrd3 mutation and the Salk allele (salk 016122C) are shown. (b) A cross between the lrd3 and Salk alleles failed to complement the mutant root phenotypes. (c) The LRD3 cDNA under control of its own promoter rescued the lrd3 primary root phenotype. Bars = means SE (d) RT-PCR of Col and lrd3 seedling mRNA. (e) Domain structure of the LRD3 protein.

roots (Figure 4d,e). The lack of expression in root meristems suggests that the LRD3 gene plays a non-autonomous role in regulating root growth. Cross-sections of pLRD3:GUS primary roots revealed that expression was tightly localized to the phloem CCs and SEs (Figure 4g, white arrows C). As SEs are enucleate, SE staining likely reflects movement of the GUS enzyme or the substrate cleavage product from the CCs. This type of movement of markers from CCs to SEs has been clearly demonstrated for green fluoresent protein (GFP; Imlau et al., 1999; Stadler et al., 2005). Hence, we conclude that LRD3 expression is specific to CCs in the root. Phloem transport assays reveal an early defect in lrd3 primary root tip unloading CCs are required for maintenance and function of their intimately connected SEs, which form a network of tubes through which sucrose and other important growth and regulatory molecules are transported (Oparka and Turgeon, 1999; Van Bel, 2003; Lough and Lucas, 2006; Turgeon and Wolf, 2009). To test if LRD3 is involved in the long-distance movement of phloem content, we performed three different assays for phloem delivery and unloading at primary root tips. Five-day-old seedlings were examined as strongest differences in primary root growth rate were seen at this time. First, as sucrose is the most abundant phloem cargo, we assayed the delivery of 14C-sucrose through the root phloem. Second, to gain insight into general phloem transport and delivery, we used the fluorescent dye carboxyflu-

orescein diacetate (CFDA) that has been extensively described for its phloem-specific transport and unloading at root tips (Oparka et al., 1994). Finally, to avoid any possible differences in application or uptake of an externally applied tracer, we used a transgenic line where GFP is expressed specifically in CCs from the promoter of the sucrose transporter SUC2. In these plants, GFP is translated in the CCs and the protein enters the adjoining SEs through plasmodesmata; GFP is then carried in the phloem stream to sink tissues such as actively-growing root tips (Imlau et al., 1999; Stadler et al., 2005). All three phloem transport assays revealed reduced phloem delivery to primary root tips in 5-day-old lrd3 plants. For 14C-sucrose, the autoradiogram revealed that sucrose was delivered to 12/12 primary root tips of Col seedlings, as evidenced by a bolus of label concentrated near the primary root tip (Figure 5a,b). In contrast, sucrose delivery to the primary root tip was only apparent in 1/12 lrd3 seedlings (Figure 5e). CFDA was successfully transported and unloaded in 8/8 Col primary root tips within 45 min of application (Figure 5c) but in 0/8 lrd3 primary root tips, even 3 h after application (Figure 5f). Finally, GFP delivery to 5-day-old pSUC2:GFP primary root tips was observed in 29/ 29 Col plants (Figure 5d), but was never observed (0/29) in lrd3;pSUC2:GFP primary root tips (Figure 5g). We used confocal microscopy to image the CCs and SEs in 5-day-old Col and lrd3 root tips in the pSUC2:GFP background. CCs and SEs both show GFP fluorescence in Col, as expected (Figure 5h). In contrast, near the root tip GFP is


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Figure 4. LRD3 is expressed in the phloem. pLRD3:GUS plants were grown for 14 days on standard media and stained for GUS activity. LRD3 expression was observed in the vasculature of leaves (a), flowers (b), and roots (c). LRD3 expression was absent from lateral root primordia (d) and young, emerged lateral roots (e), but present in more mature lateral roots (f). A cross-section through the primary root in (c) reveals GUS staining in SEs and CCs of the phloem (g, black arrows).

sequestered to the CCs in lrd3 (Figure 5i). Hence, GFP is neither moving into the SE’s from the adjacent CCs, nor is it reaching the SE’s from the more distal SE’s. This finding is consistent with the functional assays described above in which we saw abrogated phloem delivery. Reduction in delivery of sucrose to the root system might be expected to result in accumulation of carbohydrate in the leaves, as demonstrated for tdy1, tdy2, and the sucrose transporter mutant sut1 in maize (Slewinski et al., 2009) and

Figure 5. lrd3 plants show an early defect in phloem unloading at the primary root tip. Plants were grown for 5 days on standard media. (a) The black box represents the region depicted in (b–g). (b–g) 14C-sucrose (b, c), or CFDA (d, e) unloaded from the phloem at Col (b, d) but not lrd3 primary root tips (c, e). (f, g) Phloem-mobile GFP unloaded at Col but not lrd3 primary root tips. (h, i). pSUC2:GFP and lrd3;pSUC2:GFP plants were observed by confocal microscopy. Magnified regions outlined by white boxes in (h, i) are shown as insets (dashed white boxes). Plants were stained with aniline blue to visualize sieve plates in SEs (white arrows). GFP was visible in SEs and CCs in Col but absent in SEs of lrd3 root tips. (j) Aerial tissues of lrd3 show increased starch accumulation. (k) Phloem exudates of pSUC2:GFP and lrd3;pSUC2:GFP plants grown for 5 days on standard media. No significant difference was observed in GFP concentrations (n ‡ 14, P = 0.21). Bars = means SE.

suc2 in Arabidopsis (Gottwald et al., 2000). Indeed, when lrd3 and Col leaves were stained with iodine to reveal starch accumulation, the lrd3 leaves were clearly more highly stained (Figure 5j).


LRD3 is essential for root phloem function 461 lrd3 shows normal phloem loading and export from the shoot To isolate the location of the phloem transport defect, we cut 5-day-old pSUC2:GFP and lrd3;pSUC2:GFP plants at the hypocotyls and collected phloem exudates for 18 h. Loading and movement through the hypocotyl phloem was then quantified based on the amount of GFP exuded (see Experimental Procedures). No significant difference could be observed between the phloem exudates of lrd3 and Col (Figure 5k). This outcome suggests that phloem loading and export from the shoot system in lrd3 is unimpaired. Therefore, the defect that causes reduced phloem delivery to the root tip is probably specific to the root. lrd3 phloem delivery recovers in concert with an increase in primary root growth If phloem delivery to the primary root tip correlates with root growth rate, we would expect delivery to be relatively poor in lrd3 seedlings at 5 days (as shown above) but to recover by 11 days. Indeed, at 11 days the majority (19/24) of lrd3 plants (Class A) in the pSUC2:GFP background showed a dramatic increase in phloem delivery to primary root tips (Figure 6a). 14 C-sucrose labeling showed a similar recovery of phloem delivery, with 10/12 plants unloading labeled sucrose by 11 days (not shown). The lrd3 plants that failed to unload either labeled sucrose or GFP to primary root tips also failed to recover normal primary root growth rates (Class B). The correlation between recovery of phloem delivery and growth is clearly seen when fluorescence intensity of GFP at primary root tips and PRLs were measured in the same plants at 5 through 11 days. lrd3 (Class B) plants showed consistently low GFP delivery to primary root tips (5/24 showed detectable delivery by day 11; Figure 6b) and reduced primary root growth throughout the experiment (Figure 6c). lrd3 (Class A) plants displayed increasing GFP delivery over time (19/24 showed detectable delivery by day 11; Figure 6b) and increasing primary root growth (Figure 6c). Furthermore, there is a strong statistical association of fluorescence and PRL in lrd3 plants at 11 days (Figure 6d). In contrast, 11-dayold Col plants delivered GFP (Figure 6a) and 14C-sucrose (not shown) to primary root tips in all (12/12 and 24/24, respectively) of the plants tested by 11 days and root tip delivery of GFP in Col plants remained statistically unchanged at the four time points tested (Figure 6b), while root growth steadily increased (Figure 6c). (There is a lack of significant association in Col plants between primary root growth and phloem delivery due to the lack of variation in either variable.) Hence, primary root growth rate and phloem delivery are well correlated, and this correlation is best revealed in lrd3. lrd3 shows altered phloem callose deposition Callose is a polysaccharide that is deposited at phloem SE sieve plates, where it impacts the movement of phloem, and

Figure 6. Primary root growth is correlated with phloem unloading. pSUC2:GFP and lrd3;pSUC2:GFP plants were grown for 11 days on standard media. (a) Col root tips show constant GFP phloem unloading over time (top row). lrd3 root tips show dramatically decreased GFP phloem unloading that increases over time (bottom row). (b) Fluorescence intensity at the primary root tip of Col and lrd3 plants. Most lrd3 plants recover phloem unloading of GFP over time (Class A), but some do not (Class B) (n ‡ 23). (c) PRL for plants shown in (b). (d) PRL plotted against primary root tip fluorescence intensity for the plants in (b) and (c). A strong correlation was observed in lrd3 plants (R2 = 0.78).

at SE plasmodesmata, where it reduces the size exclusion limit and may inhibit unloading of phloem content (Chen and Kim, 2009; Barratt et al., 2011). Given the reduced


462 Paul Ingram et al. It must be noted that the decrease in callose is not limited to the SEs, but is seen throughout the lrd3 root tip (Figure 7). This situation may be a consequence of a smaller number of dividing cells in the root meristem, as callose is deposited along the cell plates of dividing cells (Samuels et al., 1995; Chen and Kim, 2009). Alternatively, overall diminished callose deposition throughout the root tip could be caused by reduced delivery of some phloem-mobile callose precursor such as sucrose in lrd3. Phloem delivery and lateral root growth are correlated in lrd3

Figure 7. Callose deposition at the primary root tip is decreased and shows altered patterning early in lrd3 development. At 5 days (top row), Col plants show a regular pattern of aniline blue staining at sieve plates (left panel, arrows) and PPUs (arrowheads), while lrd3 plants sieve plates show irregular spacing (right panel, arrows) and reduced staining of PPUs (arrowheads absent). At 11 days (bottom row) lrd3 staining resembles Col.

phloem delivery in lrd3 and the expression of lrd3 in the phloem, it seemed reasonable to hypothesize that lrd3 phenotypes in 5-day-old seedlings might be associated with increased callose production or deposition. This model is theoretically sound, as the mutant gat1 shows increased callose accumulation and lack of GFP delivery to pSUC2:GFP primary root tips (Benitez-Alfonso et al., 2009). To test this model, we visualized callose in the root tips of 5- and 11-day-old Col and lrd3 plants using aniline blue. At 5 days, callose staining at the sieve plates occurred at regular intervals in Col, but appeared to show increased spacing in lrd3 (Figure 7, arrows). There was also an apparent decrease in the PPUs, seen as stained puncta, in the SEs of lrd3 versus Col (Figure 7, arrowheads). Sieve elements also appeared to be narrower. In contrast, by 11 days, callose staining of sieve plates and apparent PPUs in lrd3 resembled Col (Figure 7). The normal phloem phenotype in lrd3 plants at 11 days strongly suggests that the altered morphology seen in 5-day-old lrd3 is associated with the phloem delivery defect. The above results allowed us to rule out the model that increased callose deposition interferes with phloem delivery to root tips at 5 days. Instead, the abnormal staining suggested that SEs and/or their PPUs are delayed in development or are defective in lrd3. This proposal could explain the lrd3 phenotype, as these structures are required for proper phloem delivery to root tips and for unloading of phloem content symplastically to surrounding cells.

While Col plants sustain growth of both the primary and lateral roots under standard growth conditions, the majority of the lrd3 plants show an early restriction of primary root growth and an overall increase in lateral root density (Class A), while the remainder of the lrd3 plants fail to maintain primary root growth at all and shift growth to a small number of anchor or lateral roots (Class B). This observation, together with the demonstrated reduction in phloem delivery to the primary root tip, suggests that lrd3 has an early shift in the allocation of resources to the lateral organs. To test whether the lrd3 mutant phenotype(s) is consistent with altered resource allocation, we looked for evidence of compensation between primary and lateral root growth. We measured total root system lengths by adding the lengths of all individual lateral roots per plant. When this value was plotted against PRL for each individual, we found that indeed lrd3 plants with lower PRLs showed higher total root system lengths (Figure 8a). The greatest shift from primary to lateral root growth was in the Class B lrd3 plants in which one or two lateral or anchor roots surpassed the primary root (Figure 8a, purple squares). However, the trend held in plants with a more even distribution of lateral root length (Figure 8a, red squares). In contrast, Col plants with the largest PRLs had the highest total root system lengths (Figure 8a, blue diamonds),which indicates coordinated growth of all root types in Col. This finding supports the idea that some defect in lrd3 is responsible for an increase in lateral root growth in lieu of primary root growth. Formation of lateral roots occurs in several stages. Lateral root primordia (LRP) are first initiated, these primordia emerge from the parent root through cell division and expansion, and a new lateral root apical meristem is then activated (Malamy, 2010). Although phloem delivery to the LRP was clearly observed with CFDA, 14C-sucrose and GFP, we were not able to distinguish whether there were any differences in delivery between Col and lrd3 (not shown). However, we saw clear differences in phloem delivery to lateral root tips by observation of GFP in the pSUC2:GFP background. In Col, diffuse GFP fluorescence could be seen in LRP as they emerged from the primary root (Figure 8b, top row). Soon thereafter, phloem development in young lateral


LRD3 is essential for root phloem function 463

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Some lrd3 lateral or anchor roots recover phloem unloading eventually, as observed for most lrd3 primary roots. The lrd3 plants that showed recovery of phloem delivery to the primary root tip showed the least lateral roots with phloem delivery (1/41 lateral roots on three plants). In contrast, lrd3 plants that failed to recover phloem delivery to the primary root tip had the highest number of lateral roots with clear phloem delivery (14/31 lateral roots on four plants, or approximately four roots per plant). An example of one such plant is shown (Figure 8c). It should be noted that in all cases the lrd3 lateral roots that recovered phloem GFP delivery grow substantially faster than those that did not, once again recapitulating results observed for the primary root. These results are consistent with the idea that in lrd3 there is a trade-off between phloem delivery to various organs, and that phloem delivery is tightly correlated with root growth at each organ. Taken together, these findings suggest that when phloem delivery is limited, phloem is unequally allocated to root tips within the root system. Auxin rescues the phloem defects of lrd3

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Figure 8. lrd3 plants show a shift from primary root to lateral root growth and phloem allocation. (a) PRL and total lateral root length for Col and lrd3 plants grown for 11 days on standard media (n = 12). Col plants show a correlation between increasing lateral root growth and increasing primary root growth (R2 = 0.51), while lrd3 plants show a strong correlation between increasing lateral root growth and decreasing primary root growth (R2 = 0.71). (b) GFP unloading is seen in young lateral roots in pSUC2:GFP (top) but not in lrd3; pSUC2:GFP (bottom). (c) A representative 10-day-old lrd3;pSUC2:GFP plant. The two longest lateral roots both emerged at 7 days, yet the lateral with greater GFP phloem unloading at the root tip (red) grew much faster than the other (black) and surpassed the primary root (which shows poor phloem unloading).

roots became apparent as intensely fluorescent strands that extended from the base to the tip of the growing lateral root. GFP unloading at the lateral root tip then became apparent as a bolus of GFP fluorescence. In contrast, in lrd3 lateral roots, there is a delay in the appearance of phloem, and GFP unloading at lateral root tips was not observed (Figure 8b, bottom row). Thus, there was a delay in the development of the phloem and phloem delivery in lrd3 lateral roots that recapitulates the primary root phenotype.

The early defect in phloem development in lrd3 is, to our knowledge, the first reported example of this phenotype. To further probe the mechanism underlying the defect, we tested whether it could be rescued with auxin. Auxin was chosen because of its known role in promoting vascular development (see Aloni, 2010 for review). Therefore, lrd3 and Col plants in the pSUC2:GFP background were germinated on 10)8 M IAA and GFP delivery was observed at 5 days. Indeed, GFP unloading in lrd3 plants grown on auxin closely resembled Col, with GFP extending much closer to the root tip than in the absence of auxin (Figure 9a), although the intensity of fluorescence still appeared lower than in Col (not shown). Consistent with the rescue of phloem function, the phloem morphological defects in lrd3 were also apparently rescued by auxin (Figure 9b). These results indicate that auxin either acts downstream of lrd3 or in a separate pathway to promote functional phloem development in young seedlings. DISCUSSION LRD3 regulation of phloem unloading at the root tip In this paper, we describe a detailed analysis of a new mutant, lrd3, with reduced primary root growth and reduced phloem delivery to the primary root tip. Both phenotypes are repeated for lateral roots later during the development of the lrd3 root system. Hence, the primary role of the LRD3 protein appears to be in establishing phloem delivery to young root tips. As the majority of lrd3 mutants recovers phloem delivery later in development, other gene(s) must compensate for the mutation at later times. Staining with aniline blue revealed abnormal SE and PPU morphology in 5-day-old primary root tips in lrd3. Like


464 Paul Ingram et al. carbon and the primary source of amino acids and chemical energy for non-photosynthetic tissues such as the root. Alternatively, other LRD3-mediated mechanisms may determine the activity of root meristems, which in turn may control the strength of the sink and therefore the delivery of phloem by bulk flow. However, the expression pattern of LRD3 in the phloem and not in root meristems argues in favor of the former model. It may initially seem counterintuitive to argue that root meristems of plants grown on a 1% sucrose medium are carbohydrate deficient. However, our previous work showed that sucrose in the medium has limited effects on root system development when only roots are exposed, which suggests that direct uptake of sucrose from the medium through the roots is not an efficient mechanism for delivery of sucrose to the cells at the root meristem (MacGregor et al., 2008). In contrast, uptake of sucrose through leaves dramatically affects root system growth (MacGregor et al., 2008), and this sucrose would be expected to reach the roots via the phloem. Therefore, we would expect that defects in phloem delivery to the root meristem would not be compensated by exogenous sucrose from the media. Figure 9. Auxin rescues phloem function and morphology in lrd3. Wild-type (Col) and lrd3 mutant plants in a pSUC2:GFP background were grown for 5 days on standard media with or without 10)8 M IAA. (a) Distance between visible GFP and the top of the root cap. Bars = means SE; n > 32. (b) Plants grown as in (a) stained with aniline blue. SEs are indicated by arrows. Phloem defects appear to be rescued in the presence of IAA. Scale bar = 50 lM.

phloem delivery, these abnormalities apparently recover by 11 days. Phloem delivery requires an open SE, while symplastic unloading requires PPUs. Therefore, a delay in development of SEs or PPUs could explain the lrd3 phenotype. The phloem-specific expression pattern of LRD3 is consistent with this idea. Another possibility is that there are phloem-localized mechanisms that determine the extent of phloem unloading to various sinks, and that these mechanisms operate incorrectly in the LRD3 mutant, and shift phloem allocation from primary to lateral organs (see below). Phloem delivery correlates with growth The delivery of phloem to the root tips correlated well with root growth. This correlation is seen within populations of lrd3 mutant plants, when comparing lrd3 root growth to Col, and when comparing growth of primary and lateral roots within an lrd3 root system. It is possible that the delivery of phloem determines the growth rate, and phloem is therefore limiting for cell growth and division. This idea is consistent with the decreased meristem size in lrd3 mutants, especially the Class B mutants in which primary root growth is extremely limited, and the decreased cell expansion. This proposal is reasonable, as phloem is the sole source of

Role of LRD3 in regulating root system architecture The increase in lateral root formation in lrd3 is seen as early as 7–9 days, even though at this time there is little apparent delivery of phloem to the lateral roots. This situation suggests that there is an early stimulation of lateral root formation, which is either phloem-independent or involves subtle increases in phloem delivery to LRP. Indeed, it has long been known that damage or removal of the primary root tip results in increased lateral root formation in most plants (i.e. Thimann, 1936). Hence, the increase in early lateral root formation could well be secondary to the compromised growth of the primary root. At later times, lateral roots appear to grow at the expense of the primary root in lrd3, and this is accompanied by a re-allocation of phloem delivery (Figure 8). It is important to note that there is no evidence for this kind of trade-off in Col plants. It is possible that the phloem defects in lrd3 lead to carbohydrate deficit in the root, and phloem allocation between the roots is only evident when carbohydrate is limited. Phloem allocation changes within the root system could be either the result or the cause of increased lateral root growth. Lack of phloem delivery to the lrd3 primary root may restrict its growth, which in turn may lead to stimulation of lateral root growth by an unknown ‘loss of apical dominance’ signaling mechanism. Stimulation of the lateral roots then makes them better sinks for phloem. An alternative model is that loss of active growth at the primary root allows weaker sinks such as LRP and lateral roots to compete more effectively for phloem, and these increases in phloem delivery are sufficient to increase lateral root growth. In this case, no


LRD3 is essential for root phloem function 465 further signaling mechanism need be invoked to explain the increase in lateral root growth upon loss or damage to the primary root tip. In lrd3 Class B mutants, one or more rapidly growing lateral roots extend beyond the length of the primary root and have strong phloem delivery. It is unclear how these roots are selected to supplant the primary root. It is possible that a stochastic process establishes a ‘winner’ of a competition for phloem resources, and that once strong delivery of phloem to a lateral root is established the consequent increase in growth creates greater sink strength, reinforcing phloem delivery and an altered root system architecture. In this case, the primary root may no longer have access to phloem content, even if there is recovery of functional phloem. Is phloem unloading at root tips a regulated process? It is tempting to propose that allocation of phloem delivery could be a regulatory mechanism for shaping root system architecture in wild-type plants in response to a variety of stimuli. Our results suggest that conditions that limit carbohydrate flow through the root system (i.e. low light or photosynthetic capacity) may lead to the selection of certain roots for sustained growth. Other work has demonstrated that phosphate starvation leads to drastic alterations in root system architecture including reduced primary root growth and increased lateral root growth (Lopez-Bucio et al., 2002; Sanchez-Calderon et al., 2005). These phenotypic changes are accompanied by increased translocation of sucrose in the phloem to the roots (Hammond and White, 2008). The application of exogenous sucrose to phosphate-starved plants stimulates lateral root production significantly, but not primary root growth (Jain et al., 2007; Karthikeyan et al., 2007), which may indicate differential allocation of phloem content to lateral versus primary roots. The lrd3 mutant has demonstrated that modulation of phloem delivery to root tips may have profound effects on root growth, and similar regulation of phloem delivery in wild-type plants would be an elegant mechanism to connect environmental events at the shoot and root with optimization of root system architecture. Mechanism of LRD3 and its interaction with auxin signaling The function of the LRD3 gene remains unknown, although it is clearly necessary for early phloem development and function. The narrowing of the sieve elements is reminiscent of the phenotype of antisense LeFRK2 tomato plants, and it is therefore possible that LRD3 is involved in some aspect of sucrose metabolism. LRD3 may also be essential for callose synthesis, as the lrd3 mutant shows a reduction in overall callose, and it was recently shown that callose is necessary for normal phloem transport (Barratt et al., 2011). Interestingly, our results indicate that the phloem morphological and functional defects in lrd3 are both

rescued by auxin. This finding could indicate that there is an auxin-mediated step downstream of LRD3. Indeed, as lrd3 has reduced sensitivity to auxin (Figure 3), it is possible that increased auxin is necessary for appropriate phloem development in the mutant, and that LRD3 is involved in auxin perception in the phloem. Alternatively, auxin may be involved in later stages of phloem development, a process that is apparently unimpaired in lrd3 as phloem appears normal by 11 days, even in the absence of functional LRD3. In this case, application of exogenous auxin may hasten the onset of this later developmental pathway. In keeping with this hypothesis, Bhalerao et al. (2002) demonstrated a transient ‘spike’ in endogenous shoot-derived auxin in the root at 5–7 days. It is possible that this spike plays a critical role in normal phloem development. Given the rescue of phloem development by auxin, it is surprising that PRL is not also rescued in the lrd3 mutant. It is possible that primary root growth is indeed rescued, but that it is counteracted by the ability of the rescued phloem to deliver inhibitory levels of auxin to the root tip. Alternatively, rescue may not be early or complete enough to restore primary root growth to wild-type levels. Further experiments are necessary to further elucidate the role of auxin in phloem development and the nature of its interaction with LRD3. EXPERIMENTAL PROCEDURES All materials will be made available upon request.

Plant materials and growth conditions Seeds were sterilized and grown as described (MacGregor et al., 2008). Where indicated, seedlings were transplanted with aerial tissues on Parafilm after 4 days of growth. For experiments in which no sucrose was added to the media, plates were wrapped with porous filter tape (Carolina Biological, http://www.carolina.com).

Medium composition Media simulating mild osmotic stress was composed of 100 ml L)1 macronutrients (0.3322 g L)1 CaCl2Æ6H2O, 0.1807 g L)1 MgSO4, and 0.17 g L)1 KH2PO4), 100 ml L)1 MS basal salt micronutrient solution (Sigma-Aldrich, http://www.sigmaaldrich.com), 45 g L)1 sucrose, 0.5 g L)1 MES, 20 ml L)1 1 M NH4NO3, and 20 ml L)1 1 M KNO3. Standard medium was composed of 100 ml L)1 macronutrients (0.3322 g L)1 CaCl2Æ6H2O, 0.1807 g L)1 MgSO4, and 0.17 g L)1 KH2PO4), 100 ml L)1 MS basal salt micronutrient solution (SigmaAldrich), 10 g L)1 sucrose, 0.5 g L)1 MES, 5 ml L)1 1 M NH4NO3, and 5ml L)1 1 M KNO3. For all media, the pH was adjusted to 5.7 using 1 N KOH, and 0.7% Difco agar, granulated (Fisher, http:// www.fishersci.com) was added before autoclaving. For auxin experiments, 3-indole-acetic acid (Sigma I2886) was dissolved on 0.1 N NaOH and added to autoclaved medium.

Root imaging and measurements For observation of LRP, elongated cell lengths and meristem lengths, roots were cleared as described (MacGregor et al., 2008). Elongated cells and root tips were imaged using Leica DMR (http:// www.leica-microsystems.com) or Zeiss Axioskop (http://www.zeiss. com) microscopes. For phloem delivery images and measurements, plants were imaged using a Zeiss M2 Bio microscope equipped with


466 Paul Ingram et al. an ultraviolet (UV) light source. All measurements were made using IMAGEJ (Rasband, 1997–2009).

Genetic analysis and mapping Thermal asymmetric interlaced (TAIL) PCR was performed as outlined previously (Liu et al., 1995) using primers with the following sequences: TAIL1: 5¢-ATCTAAGCCCCCATTTGGACG-3¢; TAIL2: 5¢CGTGAATGTAGACACGTCGAA-3¢; and TAIL3: 5¢-GCCTATAAATACGACGGATCG-3¢. Arbitrary degenerate primer AD2: 5¢-NGTCGA(G/C)(A/T)GANA(A/T)GAA-3¢. The presence of the T-DNA was confirmed using the TAIL primers listed above and FWD2 5¢TGTCCGGGAAATCTACATGG-3¢. The insertion site was confirmed using primers LP 5¢-CCTCAGGGTTAAGGTTCCTA-3¢ and RP 5¢GGGATGTGAGATATTACACG-3¢.

Cloning and expression analysis of LRD3 The region 1964 bp upstream of the LRD3 gene start site was amplified with LRD3ProF 5¢- CACCGCAATGGAAAATAGTCGTCTC-3¢ and LRD3ProR 5¢- GAGGATGGAAAAATGGCCCCC-3¢ and cloned into the destination vector pMDC162 (Curtis and Grossniklaus, 2003) upstream of the gene for GUS. LRD3ProF and ProR primers with NotI restriction sites added were used to clone the same promoter upstream of the LRD3 cDNA in the destination vector pMDC123 (Curtis and Grossniklaus, 2003) for complementation. Full-length LRD3 cDNA was amplified with primers with the following sequences LRD3F 5¢-CACCCATGGATTCTTCTTCCTC TTCC-3¢ and LRD3Rstop 5¢-TCACAAAGGAAAAGTTCCAGTTAAGC3¢ and cloned into pMDC123.

GUS staining Plants were stained for GUS activity and cleared as described in Malamy and Benfey (1997).

Phloem transport and collection assays A 1-ll drop composed of 50% 14C-sucrose (Sigma-Aldrich) was applied to the crimped, abaxial side of one cotyledon. After 2 h the application site was washed and the plant exposed to X-ray film for 1–3 days. A 1-ll drop of 100 lg ml)1 5(6)-Carboxyfluorescein diacetate (Sigma) in distilled water was added to the crimped, abaxial surface of one cotyledon. Plants were observed under epifluorescence microscopy (Carl Zeiss Stemi SV 11 Apo, http://www.zeiss.com). For collection of phloem exudates, hypocotyls were cut and placed into 10 mM EDTA for 18 h. GFP in exudates was quantitated in a fluorimeter (Tecan Safire 2, http://www.tecan.com).

Callose visualization with aniline blue staining Seedlings were incubated for 2 h in aniline blue solution (0.1 mg ml)1 aniline blue (Biosupplies, http://www.biosupplies. com.au) in H2O diluted 1:3 with 67 mM K3PO4 pH 9.5). For confocal microscopy the seedlings were placed in a drop of anti-fading medium (Citifluor, http://www.citifluor.co.uk). UV laser (351– 364 nm) was used to excite aniline blue and emission was recorded at 480–520 nm.

ACKNOWLEDGEMENTS We would like to thank Steven Bernacki and Lisa Pusateri for valuable assistance with the experiments described in this paper and Jonathan Fitz Gerald and Jean Greenberg for helpful discussion. This research was supported by USDA-CSREES 2007-03026 and NSF IOS-0951302 grants to JEM.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. The lrd3 mutant has altered root system architecture on mild osmotic stress media. Figure S2. The lrd3 phenotypes are caused by multiple physiological factors. Figure S3. The lrd3 root system architecture phenotype is independent of aerial tissue sucrose uptake. Figure S4. lrd3 aerial tissues show no significant difference in permeability to sucrose compared to Col. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

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