Journal of Experimental Botany, Vol. 62, No. 9, pp. 3197–3212, 2011 doi:10.1093/jxb/err021 Advance Access publication 16 February, 2011 RESEARCH PAPER Development of structural and biochemical characteristics of C4 photosynthesis in two types of Kranz anatomy in genus Suaeda (family Chenopodiaceae) Nuria K. Koteyeva1, Elena V. Voznesenskaya1, James O. Berry2, Simon D. X. Chuong3,†, Vincent R. Franceschi3 and Gerald E. Edwards3,* 1 Laboratory of Anatomy and Morphology, V. L. Komarov Botanical Institute of Russian Academy of Sciences, Professor Popov Street 2, 197376, St Petersburg, Russia 2 Department of Biological Sciences, State University of New York, Buffalo, NY 14260, USA Downloaded from 3 School of Biological Sciences, Washington State University, Pullman, WA 9164-4236, USA y Present address: Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada * To whom correspondence should be addressed. E-mail: [email protected] Received 15 November 2010; Revised 14 January 2011; Accepted 19 January 2011 http://jxb.oxfordjournals.org/ Abstract Genus Suaeda (family Chenopodiaceae, subfamily Suaedoideae) has two structural types of Kranz anatomy consisting of a single compound Kranz unit enclosing vascular tissue. One, represented by Suaeda taxifolia, has mesophyll (M) and bundle sheath (BS) cells distributed around the leaf periphery. The second, represented by Suaeda eltonica, has M and BS surrounding vascular bundles in the central plane. In both, structural and at OUP site access on April 22, 2013 biochemical development of C4 occurs basipetally, as observed by analysis of the maturation gradient on longitudinal leaf sections. This progression in development was also observed in mid-sections of young, intermediate, and mature leaves in both species, with three clear stages: (i) monomorphic chloroplasts in the two cell types in younger tissue with immunolocalization and in situ hybridization showing ribulose bisphosphate carboxylase oxygen- ase (Rubisco) preferentially localized in BS chloroplasts, and increasing in parallel with the establishment of Kranz anatomy; (ii) vacuolization and selective organelle positioning in BS cells, with occurrence of phosphoenolpyruvate carboxylase (PEPC) and immunolocalization showing that it is preferentially in M cells; (iii) establishment of chloroplast dimorphism and mitochondrial differentiation in mature tissue and full expression of C4 biochemistry including pyruvate, Pi dikinase (PPDK) and NAD-malic enzyme (NAD-ME). Accumulation of rbcL mRNA preceded its peptide expression, occurring prior to organelle positioning and differentiation. During development there was sequential expression and increase in levels of Rubisco and PEPC followed by NAD-ME and PPDK, and an increase in the 13C/12C isotope composition of leaves to values characteristic of C4 photosynthesis. The findings indicate that these two forms of NAD-ME type C4 photosynthesis evolved in parallel within the subfamily with similar ontogenetic programmes. Key words: C3 plants, C4 plants, Chenopodiaceae, chloroplast ultrastructure, immunolocalization, in situ mRNA hybridization, NAD-ME type, photosynthetic enzymes, Suaeda. Introduction C4 photosynthesis evolved as a means of increasing the would otherwise be limiting for photosynthesis. Limitations supply of CO2 to ribulose bisphosphate carboxylase oxy- can occur by reduced stomatal conductance (e.g. in re- genase (Rubisco) and the C3 cycle under conditions that sponse to a water deficit), and by warm temperatures where Abbreviations: BS, bundle sheath.d13C values, a measure of the carbon isotope composition; LSU, large subunit; M, mesophyll; NAD-ME, NAD-malic enzyme; NADP- ME, NADP-malic enzyme; PEPC, phosphoenolpyruvate carboxylase; PEP-CK, phosphoenolpyruvate carboxykinase; PPDK, pyruvate, Pi dikinase; PPFD, photosynthetic photon flux density; RT, room temperature; Rubisco, ribulose bisphosphate carboxylase oxygenase; SEM, scanning electron microscope/microscopy; TEM, transmission electron microscope/microscopy. ª The Author [2011]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: [email protected] 3198 | Koteyeva et al. photorespiration and the Rubisco oxygenase/carboxylase a compound Kranz unit with two layers of chlorenchyma activity ratio is increased. characteristic of species with C4 photosynthesis, BS and M C4 plants evolved independently multiple times, with cells, occurring at the leaf periphery surrounding water convergence in a number of specialized features. These storage tissue in which the vascular bundles are embedded. shared features include fixation of atmospheric CO2 into C4 Schoberia type leaves have a compound Kranz unit acids within mesophyll (M) cells through phosphoenolpyr- characterized by two concentric layers of chlorenchyma uvate carboxylase (PEPC), and decarboxylation of C4 acids cells, BS and M, encircling the vascular bundles, and by and donation of released CO2 to Rubisco and the C3 cycle a layer of enlarged water storage hypodermal cells just within bundle sheath (BS) cells. Common to this process is beneath the epidermis. The chloroplasts in BS cells are the selective expression of the carboxylation phase of C4 located in the centripetal position in the Salsina type, and in enzymes in the M cells, with Rubisco and other C3 cycle the centrifugal position in the Schoberia type (Freitag and enzymes accumulating in the chloroplasts, and glycine Stichler, 2000; Schu¨tze et al., 2003; Voznesenskaya et al., decarboxylase in the mitochondria, of the BS. This pathway 2007). Transmission electron microscopy (TEM) shows that is also correlated with distinctive cellular structural features chlorenchyma cells in species representing the two different that provide diffusive resistance to leakage of CO2, so that C4 anatomical types have features characteristic of NAD- it can be effectively donated from the C4 cycle to Rubisco ME type C4 chenopods: the M cells contain chloroplasts (Edwards and Walker, 1983; Hatch, 1987; Sage, 2004). with reduced grana, while BS cells have chloroplasts with The independent evolution of C4 photosynthesis from C3 well-developed grana and large, specialized mitochondria ancestors across 19 different plant lineages has led to (Fisher et al., 1997; Voznesenskaya et al., 2007). Biochem- Downloaded from considerable diversity in biochemistry, with three types of ical and physiological studies show that the availability and C4 cycle operating through the C4 decarboxylases, NADP- selective immunolocalization of key photosynthetic malic enzyme (NADP-ME), NAD-malic enzyme (NAD- enzymes, CO2 compensation points, photosynthetic re- ME), and phosphoenolpyruvate carboxykinase (PEP-CK), sponse curves to varying CO2 and light were similar for as well as diversity in structural features. There are at least Salsina and Schoberia anatomical types, and these were http://jxb.oxfordjournals.org/ 25 forms of Kranz anatomy, and two forms of single-cell C4 typical for C4 plants (Voznesenskaya et al., 2007; Smith without Kranz (Edwards and Voznesenskaya, 2011). The et al., 2009). forms of Kranz anatomy can be classified into two major The establishment of complex C4 systems requires highly groups: one in which two layers of Kranz chlorenchyma coordinated expression of many genes during development, are formed around each vein (multiple Kranz units per under the control of regulatory factors that must function leaf); the other in which layers of Kranz chlorenchyma concurrently with differentiation of Kranz anatomy surround all the veins, forming a single compound Kranz (Hibberd and Covshoff, 2010; Berry et al., 2011). Studies at OUP site access on April 22, 2013 unit, according to the classification of Peter and Katinas on development of leaf structure relative to enzyme (2003). Current information, though limited, indicates that expression have been performed on a limited number of C4 there is also diversity in the ways different enzymes were species belonging to different structural and biochemical recruited during evolution to function in the C4 cycles, and subtypes. Except for Salsola richteri, which has a structural in the mechanism leading to control of selective expres- form of C4 called Salsoloid type with a compound Kranz sion of C4 in M versus BS cells, e.g. transcriptional, post- unit and NADP-ME biochemistry (Voznesenskaya et al., transcriptional, translation control (Hibberd and Covshoff, 2003b), most studies on C4 development have been on C4 2010; Berry et al., 2011; Gowik and Westhoff, 2011). species that have multiple simple Kranz units with chloren- The family Chenopodiaceae includes many species, with chyma surrounding individual veins. Among the eudicots great diversity in forms of C4 and C3 photosynthesis, studied this includes Atriplex rosea (Dengler et al., 1995) including different anatomical, physiological, and biochem- and Amaranthus hypochondriacus (Ramsperger et al., 1996), ical features. Subfamily Suaedoideae is especially interesting which have atriplicoid type leaf anatomy and the NAD-ME as among ;85 species there are four structural forms of C4 biochemical subtype. In monocots, this includes develop- (two different forms having a compound Kranz unit, and mental studies on Arundinella hirta (Wakayama et al., 2003) two single-cell forms without Kranz anatomy, all NAD-ME and Zea mays (Langdale et al., 1988a; Brutnell et al., 1999; biochemical subtype), as well as C3 species (Kadereit et al., Covshoff et