Short-Root1 Plays a Role in the Development of Vascular Tissue and Kranz Anatomy in Maize Leaves

Molecular Plant 7, 1388 1392, August 2014 LETTER TO THE EDITOR Short-Root1 Plays a Role in the Development of Vascular Tissue and Kranz Anatomy in Maize Leaves Dear Editor, Understanding how Kranz anatomy develops in C 4 plants is a critical part of the current worldwide effort to transfer C 4 photosynthesis into C 3 plants, including rice. Recently, it was proposed that the Kranz architecture that supports C 4 photosynthesis in maize leaves is an extension of the endodermal program, which is active in roots, stems, and petioles, and is ubiquitous in angiosperms (Slewinski et al., 2012; Slewinski, 2013). Support for this hypothesis was shown in maize in which mutations in the Scarecrow (SCR) gene, a primary regulator of endodermal formation, resulted in the development of ectopic bundle sheath cells (BS), merged veins without intervening mesophyll (M) cells, starch-less BS cells, and loss of minor veins (V) (Slewinski et al., 2012). Each of these phenotypes altered the normal Kranz-associated cell patterning (V-BS-M-M-BS-V) in the leaves. This finding led to the hypothesis that SCR and its upstream interacting partner SHORT-ROOT (SHR) function in the establishment of Kranz anatomy (Slewinski et al., 2012; Slewinski, 2013). However the role of SHR1 needs to be verified. Here we report a disruption in Kranz anatomy associated with a mutant allele of the maize SHR1 (ZmShr1, GRMZM2g132794) gene, supporting our hypothesis that the endodermal SCR/SHR signaling mechanism that patterns the Arabidopsis thaliana root underlies the development of Kranz anatomy in maize leaves. In A. thaliana, the Shr gene is required for the formation and specification of the endodermis, but its expression is limited to the stele (Nakajima et al., 2001; Gallagher et al., 2004). The SHR protein moves through plasmodesmata into the cells immediately adjacent to the stele where it up-regulates the transcription of SCR (Helariutta et al., 2000; Nakajima et al., 2001; Vaten et al., 2011). In that single cell layer, SCR and SHR then interact to regulate the development of that layer into endodermis both in the roots and shoots. In the shoot, the endodermis is referred to as the starch sheath, which extends from the root shoot junction to the petiole leaf blade junction (Esau, 1953). In A. thaliana, mutations in the SHR gene disrupt the development of the endodermis in the root and starch sheath of the stem (Helariutta et al., 2000; Morita et al., 2007; Gardiner et al., 2011). Other abnormalities associated with loss of SHR have thus far been detected in the root where SHR is required for the proper formation and maintenance of the cortex, quiescent center cells, xylem, and phloem (Helariutta et al., 2000; Morita et al., 2007; Gardiner et al., 2011). In order to elucidate the role of ZmSHR1 in establishing Kranz anatomy in Zea mays, a C 4 species, we characterized a mutant allele of the ZmSHR1 gene harboring a Mutator transposon insertion in the single exon of the gene (Supplemental Figures 1 and 2). Plants homozygous for the mutant zmshr1 allele display a dramatic reduction in growth (Figure 1A), as well as disruptions in overall vascular patterning and spacing (Figure 1B and 1C), leaf development, and BS and M cell patterning and development (Figure 1F 1L). In zmshr1 mutant leaves, veins frequently depart from the normal parallel track (Figure 1B) and merge with one another (Figure 1C), leading to a pattern that excludes M cells from the interveinal space (V-BS- BS-V). These alterations in vascular structure are similar to the vascular perturbations to Kranz anatomy previously found in the zmscr mutant alleles of maize (Slewinski et al., 2012). In contrast to the zmscr mutant, ectopic BS cells were infrequent in the zmshr1 mutant leaves, suggesting that ZmSCR and ZmSHR1 have different functions in BS cell division or recruitment. Additionally, xylem formation and polarity are also altered in the zmshr1 mutant (Figure 1H, 1L, 1N, and 1O), suggesting a similar role for ZmSHR1 in vascular development in maize leaves to that previously described for vascular formation in A. thaliana roots where a reciprocal signaling pathway with SHR and mirna165/66 patterns the xylem (Carlsbecker, et al., 2010; Vaten et al., 2011). Altered vascular patterning and anatomy occur in all vein classes but are most pronounced in the intermediate and minor veins within the zmshr1 mutant leaf. Similarly to the zmscr mutant (Slewinski et al., 2012), intermediate and minor veins are also reduced in number in zmshr1 mutant leaves (Figure 1C, 1E, and 1P). Many of the minor veins that do form in the mutant have incomplete BS cell layers allowing cells that are part of the vascular core, frequently the xylem elements, to come into direct contact with M cells (Figure 1I 1L). Other veins have extra layers of M cells (Figure 1F 1H). The increased number of M cells is similar to the phenotype in A. thaliana roots in which a partial reduction of SHR leads to the formation of extra layers of cortex (Koizumi et al., 2012). Secondary peripheral layers of M cells are usually larger than those in wild-type, The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/ssu036, Advance Access publication 7 April 2014 Received 23 December 2013; accepted 27 March 2014

Molecular Plant Letter to the Editor 1389 Figure 1 Whole-Plant and Leaf Vascular Phenotypes Associated with the zmshr1 Mutant. Wild-type (WT) (B, D, F, I, J, K) and zmshr1 mutant (C, D, G, H, L R) maize. (A) Photograph of 3-week-old maize plants. zmshr1 mutants plants are smaller than WT siblings, have thinner leaves, and reduced root growth.

1390 Letter to the Editor Molecular Plant are highly vacuolated, and have greatly reduced plastid numbers (Figure 1G, 1H, and 1R). These M cells take on a stem and midrib parenchyma cell-like appearance. Overall, these phenotypic data suggest that ZmSHR1 plays a role in the development of many of the essential features of Kranz anatomy including the C 4 BS as well as the establishment of C 4 M cells. A notable feature of the zmshr1 mutant is the presence of files of BS cells without associated vascular cores (Figure 1P 1U), similar to the distinctive cells (C 4 BS-like cells that occur without association with vasculature) found in the C 4 grasses Arundinella hirta (Dengler et al., 1996). These isolated BS cells accumulate starch (Figure 1P, 1Q, 1S, and 1T), have suberized cell walls (Figure 1U), and have unstacked thylakoid grana identical to plastids found in wild-type BS cells (Figure 1R). In both the zmshr1 mutant and in the C 4 grasses that have distinctive cells, these cells can occur in single files, or as small bundles with no vascular cores. In the zmshr1 mutant, these files of cells are found in place of minor veins (Figure 1P and 1T), suggesting that their formation results from a disruption of normal minor vein formation. As is the case in A. hirta (Dengler et al., 1996), the files of isolated BS cells may end blindly (Figure 1M, 1P, and 1T). These data also suggest that the designation of cells that will become the vascular core occurs after vein initiation indicating that formation of minor veins proceeds by initiating the external cells (endodermis/bs) first, followed by the initiation and subsequent differentiation of the vascular core. If the vascular core fails to initiate, only linear files of cells with an endodermal identity, namely BS cells, remain at the patterned site for normal minor veins. A model for minor vein and distinctive cell formation is detailed in Supplemental Figure 3. This is most likely due to a disruption in the SHR/SCR signaling mechanism previously described that regulates the external-to-internal signaling mechanism involved in vascular development (Vaten et al., 2011; Carlsbecker et al., 2010). In the leaf of A. thaliana, SHR is one of the first genes expressed in response to auxin (Gardiner et al., 2011). If SHR were to turn on SCR in the BS cells, both SCR and SHR proteins could then activate the expression of mirna165/6 that then signals laterally to specify the internal vascular core and xylem. This model suggests that the zmshr1 mutant (BS without associated veins) and distinctive cells, both exceptions from canonical Kranz anatomy, have a common foundation in signaling and development. This model for minor vein formation also suggests that the initial events that signal the development of a minor vein are centripetal from the outside to the inside generating BS first, then the vascular core. After the vascular core is initiated, the internal-to-external signaling begins and integrates with external-to-internal signaling (Nakajima et al., 2001; Carlsbecker et al., 2010; Gardiner et al., 2011). This gives rise to a mutually reinforcing signaling system required for normal development of both the vascular core, including the specification and differentiation of transport tissue such as xylem and phloem (Carlsbecker et al., 2010), as well as the perpetuation of the of the signaling required for endodermis (i.e. BS) and cortex (i.e. M) cell development. Proper signaling may be required to maintain complete radial patterning as the vascular core differentiates and enlarges and new endodermal (BS) cells divide, or are recruited, as the vascular core elongates and radially expands. The phyllode hypothesis of monocot evolution suggests that the monocot leaf blade is an extrapolation of the petiole and/or lower-leaf zone of the canonical dicot leaf (Arber, 1918; Kaplan, 1973). In the monocots, the structure corresponding to the dicot leaf blade is the vorlauferspitze the tip of the leaf seen early in development, but which may not persist to maturity. If the maize leaf blade is homologous to the dicot petiole, the (B E) Light micrographs of cleared leaf tissue (IKI-stained to reveal starch) showing that the regular parallel vein pattern (B, D) is disrupted in mutant leaves (C, D). (F H) Free-hand cross-sections of fresh leaf tissue illuminated with UV light. (H) Stained with berberine-aniline blue staining to show suberization of BS and lignified xylem. Mutants display altered M cell development and plastid density (G, H) when compared to WT (F). Yellow arrows show an additional mutant parenchyma-like, mesophyll cell layer. Minor veins in mutants leaves frequently have an incomplete BS/endodermal cell layer (L O) which normally fully encircles minor veins in WT leaves (I K). (I) and (J) show WT BS layers in cleared IKI-stained leaves (I) and in cross-section illuminated by UV light (J). (K) Transmission electron micrograph (TEM) of WT minor vein showing normal Kranz anatomy and plastid structures. (L O) zmshr1 mutant leaf showing incomplete Kranz anatomy and direct contact between xylem elements and M cells. (M) Cleared, IKI-stained leaves also shown in transverse cross section illuminated by UV light without staining (N) and with berberine-aniline blue staining (O) to illuminate lignin within the xylem elements and suberin in the BS cell walls. Black solid triangle shows incomplete Kranz anatomy along a minor vein. (L) TEM of a minor vein in a mutant leaf. Open triangles show absence of BS cells where M cells are in direct contact with xylem elements. Asterisk shows empty BS cell. (P U) BS cells without associated veins. IKI starch stained mutant leaves in paradermal view in (P) and (T) and in transverse cross section in (Q) and (S). (R) Transverse cross-section illuminated by UV light with berberine-aniline blue staining to show suberization of BS with no associated veins. (R) TEM of mutant leaf. Red arrows show isolated BS cells not associated with vascular cores. M, mesophyll; BS, bundle sheath; X, xylem; XP, xylem parenchyma. Scale bars in (B, C, P) = 400 μm; (D, E, I, M) = 225 μm; (F, G, T) = 60 μm; (Q, S) = 50 μm; (H, J, N, O, U) = 40 μm; (K, L, R) = 15 μm.

Molecular Plant Letter to the Editor 1391 BS is homologous to, and therefore may exhibit many of the same anatomical and physiological features as, the starch sheath (Slewinski, 2013). This may explain why all of the C 4 grasses and sedges are Kranz-type C 4 species (Slewinski, 2013). However, not all grasses utilize the C 4 photosynthetic mechanism. Some species may have selected against the energetically costly C 4 photosynthetic system in environments where it did not confer an advantage over the energetically cheaper C 3 mechanism (Slewinski, 2013). Remnants of the initial C 4 preconditioning event and the starch sheath developmental program could nonetheless persist in C 3 species (Slewinski, 2013). For example, all grasses, including those with C 3 photosynthesis, have suberized vascular BS cells along the major veins, consistently with some endodermal identity in the BS cells. Additionally, disruptions in the ZmSCR or ZmSHR genes in maize lead to a reduction in minor veins similar to vascular arrangements found in C 3 grasses (Slewinski et al., 2012). Furthermore, the starch-less BS cells in the leaves of the zmscr mutant (Slewinski et al., 2012) have a striking resemblance to the non-photosynthetic vascular BS of rice. The phyllode theory (Arber, 1918; Kaplan, 1973; Slewinski, 2013) along with AtScr and AtShr expression data from dicot leaves such as A. thaliana (Wysocka-Diller et al., 2000; Gardiner et al., 2011) suggests that SHR expression in the leaf vasculature, and subsequent protein movement into the cells that surround the veins, as well as SCR expression in the BS cells, are the most common states of gene expression in angiosperm leaves. From these data, it is reasonable to hypothesize that anatomical alterations linked to alterations or disruptions in either SCR or SHR can explain the much of the variation in vascular, BS, and M cellular arrangements and physiologies documented in C 3 and C 4 dicot and grass leaf blades. In the case of the ZmSHR1, dramatic alterations in minor vein structure in the zmshr1 mutants mirror anatomies found in non-canonical Kranz C 4 grasses such as A. hirta (Dengler et al., 1996). It is tempting to speculate that species that have distinctive cells, such as A. hirta, may be species that are derived from ancestral species that had canonical Kranz anatomy, but arose when alterations in the SHR signaling mechanisms occurred. Rice and other C 3 grasses may have also experienced a disruption in the SCR/SHR signaling mechanism. However, much more research is needed to explore this hypothesis of grass leaf blade evolution and signaling mechanisms involved in the development of C 3 grass leaves. Data from the phenotypic analysis of the zmshr1 mutant support our hypothesis that Kranz anatomy in C 4 plants is an extension of the endodermis in the roots, stems, and petioles (Slewinski et al., 2012; Slewinski, 2013). Additionally, we show that the same underlying developmental genetic pathway that gives rise to the endodermis in the roots and starch sheath in the stem and petiole, the SHR/SCR radial patterning mechanism, also plays a role in the formation of Kranz anatomy in C 4 maize leaves. FUNDING This work was supported by National Science Foundation (grant IOS-1127017 to R.T.); United States Department of Agriculture-National Institute of Food and Agriculture [post-doctoral fellowship (2011-67012-30774) to T.L.S.]. The authors declare no conflict of interest. Hypothesis, data and analysis were all generated and conducted prior to T.L.S. employment at Monsanto Company. Thomas L. Slewinski a,b,1, Alyssa A. Anderson a, Simara Price c, Jacob R. 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