Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

Metabolite Profiles of Maize Leaves in Drought,Heat, and Combined Stress Field Trials Reveal the Relationship between Metabolism and Grain Yield

The development of abiotic stress-resistant cultivars is of premium importance for the agriculture of developing countries. Further progress in maize (Zea mays) performance under stresses is expected by combining marker-assisted breeding with metabolite markers. In order to dissect metabolic responses and to identify promising metabolite marker candidates, metabolite profiles of maize leaves were analyzed and compared with grain yield in field trials. Plants were grown under well-watered conditions (control) or exposed to drought, heat, and both stresses simultaneously. Trials were conducted in 2010 and 2011 using 10 tropical hybrids selected to exhibit diverse abiotic stress tolerance. Drought stress evoked the accumulation of many amino acids, including isoleucine, valine, threonine, and 4-aminobutanoate, which has been commonly reported in both field and greenhouse experiments in many plant species. Two photorespiratory amino acids, glycine and serine, and myoinositol also accumulated under drought. The combination of drought and heat evoked relatively few specific responses, and most of the metabolic changes were predictable from the sum of the responses to individual stresses. Statistical analysis revealed significant correlation between levels of glycine and myoinositol and grain yield under drought. Levels of myoinositol in control conditions were also related to grain yield under drought. Furthermore, multiple linear regression models very well explained the variation of grain yield via the combination of several metabolites. These results indicate the importance of photorespiration and raffinose family oligosaccharide metabolism in grain yield under drought and suggest single or multiple metabolites as potential metabolic markers for the breeding of abiotic stress-tolerant maize.The increasing world population coupled to environmental deterioration is creating ever greater pressure on our capacity for sustainable food productivity. Alongside biotic stresses, abiotic stresses such as drought, heat, salinity, and nutrient deficiency greatly reduce yields in crop fields either when present alone or in combination. Breeding for more resilient crops, therefore, is one of the major approaches to cope with the increasing challenges in world agriculture. Considerable research effort has thus been invested in order to dissect plant responses to individual stresses at various levels (for review, see Urano et al., 2010; Lopes et al., 2011; Obata and Fernie, 2012), but the interaction between different stresses has been far less investigated (Cairns et al., 2012b, 2013; Suzuki et al., 2014). In general, the combination of stresses additively affects plant physiology (i.e. the symptoms of the individual stresses appear simultaneously) and synergistically diminishes the yield and productivity of plants (Keleş and Öncel, 2002; Giraud et al., 2008; Vile et al., 2012; Suzuki et al., 2014). The molecular responses, however, are not simply additive and are rarely predicted from the responses to individual stresses (Rizhsky et al., 2002, 2004; Prasch and Sonnewald, 2013; Rasmussen et al., 2013). Information from carefully controlled greenhouse experiments has begun to dissect the molecular mechanisms by which plants, in particular Arabidopsis (Arabidopsis thaliana), respond to drought and temperature stresses (Skirycz et al., 2010, 2011; Skirycz and Inzé, 2010; Bowne et al., 2012; Tardieu, 2012; Verkest et al., 2015). Our knowledge of the molecular basis of the responses of crop species in a field environment, however, is considerably less well advanced (Araus et al., 2008; Cabrera-Bosquet et al., 2012). That said, a large number of genotypes have been generated on the basis of their resistance to both biotic and abiotic stresses (for review, see Bänziger et al., 2006; Takeda and Matsuoka, 2008; Cooper et al., 2014), and the genome sequencing and molecular characterization of a range of stress-tolerant plant species have recently been reported (Wu et al., 2012; Ma et al., 2013; Bolger et al., 2014; Tohge et al., 2014). These studies are not only important as basic research for further studies in crops but also are a prerequisite in the development of molecular marker-based approaches to improve crop tolerance to stress.As a first step toward this goal, a deeper understanding of the plant responses to the stressful environment, especially those to multiple stress conditions under field conditions, is crucial for the improvement of stress-tolerant crops. This is important on two levels: (1) in the field, singular abiotic stresses are rare; and (2) yield and stress adaptation are complex traits that render breeding gains slower than would be expected under optimal conditions (Bruce et al., 2002). Recent studies have revealed that the response of plants to combinations of two or more stress conditions is unique and cannot be directly extrapolated from their responses to the different stresses when applied individually. This would be a result of complex combinations of different, and sometimes opposing, responses in signaling pathways, including those that may interact and inhibit one another (Prasch and Sonnewald, 2013; Rasmussen et al., 2013; Suzuki et al., 2014).Maize (Zea mays) is grown in over 170 million ha worldwide, of which 130 million ha are in less-developed countries (FAO, 2014). In sub-Saharan Africa, maize is a staple crop; however, yields in this region have stagnated at less than 2 tons ha⁻¹, while maize yields worldwide have continued to increase (Cairns et al., 2012a). Low yields in sub-Saharan Africa are largely associated with drought stress (DS) and low soil fertility (Bänziger and Araus, 2007). Additionally, simulation studies indicate that maize yield in Africa is likely to be significantly impaired by heat stress (HS; Lobell and Burke, 2010; Lobell et al., 2011), such as can be anticipated as a result of the changes in climate predicted for the coming decades (Müller et al., 2011). Moreover, the sensitivity of maize yield to heat is exacerbated under drought conditions (Lobell et al., 2011; Cairns et al., 2012a, 2012b, 2013). Therefore, the development of maize germplasm tolerant to drought and heat conditions is of utmost importance to both increase yields and offset predicted yield losses under projected climate change scenarios (Easterling et al., 2007), especially in sub-Saharan Africa. While direct selection for grain yield under DS has resulted in admirable gains in grain yield under stress (Bänziger et al., 2006; Cairns et al., 2013), further improvement requires the incorporation of additional selection traits (Cairns et al., 2012a, 2012b). In recent years, genetic and phenotypic markers have been searched extensively for drought tolerance of maize by high-throughput genomic and phenotyping approaches, respectively (Tuberosa and Salvi, 2006; Wen et al., 2011; Araus et al., 2012; Cairns et al., 2013; Prasanna et al., 2013; Araus and Cairns, 2014; Tsonev et al., 2014). Moreover, metabolic markers started to draw attention due to their close relationship with yield phenotypes (Fernie and Schauer, 2009; Redestig et al., 2011; Riedelsheimer et al., 2012a, 2012b; Witt et al., 2012; Degenkolbe et al., 2013). The accumulation of some metabolites has been reported to be directly related to the performance of potato (Solanum tuberosum) cultivars in beetle resistance in the field (Tai et al., 2014). Additionally, identical genomic regions were mapped as both agronomic and metabolic quantitative trait loci in field-grown maize and wheat (Triticum aestivum), indicating the utility of metabolic traits for breeding selection (Riedelsheimer et al., 2012b; Hill et al., 2015). A recent study showed that genetic gains in maize grain yield under DS were higher using a molecular marker-based approach than conventional breeding (Beyene et al., 2015).Here, we focused on the relationship between leaf metabolites and grain yield under drought, heat, and simultaneous drought and heat conditions in the field. The negative effect of DS on maize yield is especially acute during the reproductive stage between tassel emergence and early grain filling (Grant et al., 1989), when it is believed to induce premature seed desiccation and to limit grain filling. Grain is more susceptible to DS than vegetative tissues; therefore, the prediction of grain yield from the physiological parameter of leaves is a challenge (Sangoi and Salvador, 1998; Khodarahmpour and Hamidi, 2011). Nevertheless, maize yield is dependent on both the assimilate supply to the kernel (source) and the potential of the kernel to accommodate this assimilate (sink potential; Jones and Simmons, 1983). Breeding for modern temperate hybrids has focused more on the sink potential, particularly under stress conditions (Tollenaar and Lee, 2006); therefore, there should be considerable potential remaining to improve source ability. DS and HS would be anticipated largely to affect leaf metabolism and especially photosynthesis, compromising the source capacity of leaves (Chaves et al., 2009; Lawlor and Tezara, 2009; Osakabe et al., 2014). In keeping with this, drought was found to have the most dramatic effect on the metabolite composition in leaves compared with other organs in our previous greenhouse experiments (Witt et al., 2012). Since the source ability is closely related to leaf metabolism, the leaf metabolite profile should have a close relationship to grain yield particularly under conditions of stress. Given that several recent studies have indicated the importance of metabolic preadaptation to various stress tolerances in plants (Sanchez et al., 2011; Benina et al., 2013), we also postulate that basal metabolite levels under optimal growth conditions could be correlated to stress tolerance. In order to test this, metabolite profiles of the leaf blades of 10 hybrids were analyzed in field experiments conducted at the International Maize and Wheat Improvement Center (CIMMYT) subtropical experimental station in 2010 and 2011 in which the plants were exposed to singular or combined drought and heat stresses (DS+HS; Cairns et al., 2012a, 2013). The results are discussed both in the context of current models of stress tolerance and with respect to their practical implications for future breeding strategies.     

Characterization of the Possible Roles for B Class MADS Box Genes in Regulation of Perianth Formation in Orchid

To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, three paleoAPETALA3 genes, O. Gower Ramsey MADS box gene5 (OMADS5; clade 1), OMADS3 (clade 2), and OMADS9 (clade 3), and one PISTILLATA gene, OMADS8, were characterized. The OMADS8 and OMADS3 mRNAs were expressed in all four floral organs as well as in vegetative leaves. The OMADS9 mRNA was only strongly detected in petals and lips. The mRNA for OMADS5 was only strongly detected in sepals and petals and was significantly down-regulated in lip-like petals and lip-like sepals of peloric mutant flowers. This result revealed a possible negative role for OMADS5 in regulating lip formation. Yeast two-hybrid analysis indicated that OMADS5 formed homodimers and heterodimers with OMADS3 and OMADS9. OMADS8 only formed heterodimers with OMADS3, whereas OMADS3 and OMADS9 formed homodimers and heterodimers with each other. We proposed that sepal/petal/lip formation needs the presence of OMADS3/8 and/or OMADS9. The determination of the final organ identity for the sepal/petal/lip likely depended on the presence or absence of OMADS5. The presence of OMADS5 caused short sepal/petal formation. When OMADS5 was absent, cells could proliferate, resulting in the possible formation of large lips and the conversion of the sepal/petal into lips in peloric mutants. Further analysis indicated that only ectopic expression of OMADS8 but not OMADS5/9 caused the conversion of the sepal into an expanded petal-like structure in transgenic Arabidopsis (Arabidopsis thaliana) plants.The ABCDE model predicts the formation of any flower organ by the interaction of five classes of homeotic genes in plants (Yanofsky et al., 1990; Jack et al., 1992; Mandel et al., 1992; Goto and Meyerowitz, 1994; Jofuku et al., 1994; Pelaz et al., 2000, 2001; Theißen and Saedler, 2001; Pinyopich et al., 2003; Ditta et al., 2004; Jack, 2004). The A class genes control sepal formation. The A, B, and E class genes work together to regulate petal formation. The B, C, and E class genes control stamen formation. The C and E class genes work to regulate carpel formation, whereas the D class gene is involved in ovule development. MADS box genes seem to have a central role in flower development, because most ABCDE genes encode MADS box proteins (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994; Purugganan et al., 1995; Rounsley et al., 1995; Theißen and Saedler, 1995; Theißen et al., 2000; Theißen, 2001).The function of B group genes, such as APETALA3 (AP3) and PISTILLATA (PI), has been thought to have a major role in specifying petal and stamen development (Jack et al., 1992; Goto and Meyerowitz, 1994; Krizek and Meyerowitz, 1996; Kramer et al., 1998; Hernandez-Hernandez et al., 2007; Kanno et al., 2007; Whipple et al., 2007; Irish, 2009). In Arabidopsis (Arabidopsis thaliana), mutation in AP3 or PI caused identical phenotypes of second whorl petal conversion into a sepal structure and third flower whorl stamen into a carpel structure (Bowman et al., 1989; Jack et al., 1992; Goto and Meyerowitz, 1994). Similar homeotic conversions for petal and stamen were observed in the mutants of the AP3 and PI orthologs from a number of core eudicots such as Antirrhinum majus, Petunia hybrida, Gerbera hybrida, Solanum lycopersicum, and Nicotiana benthamiana (Sommer et al., 1990; Tröbner et al., 1992; Angenent et al., 1993; van der Krol et al., 1993; Yu et al., 1999; Liu et al., 2004; Vandenbussche et al., 2004; de Martino et al., 2006), from basal eudicot species such as Papaver somniferum and Aquilegia vulgaris (Drea et al., 2007; Kramer et al., 2007), as well as from monocot species such as Zea mays and Oryza sativa (Ambrose et al., 2000; Nagasawa et al., 2003; Prasad and Vijayraghavan, 2003; Yadav et al., 2007; Yao et al., 2008). This indicated that the function of the B class genes AP3 and PI is highly conserved during evolution.It has been thought that B group genes may have arisen from an ancestral gene through multiple gene duplication events (Doyle, 1994; Theißen et al., 1996, 2000; Purugganan, 1997; Kramer et al., 1998; Kramer and Irish, 1999; Lamb and Irish, 2003; Kim et al., 2004; Stellari et al., 2004; Zahn et al., 2005; Hernandez-Hernandez et al., 2007). In the gymnosperms, there was a single putative B class lineage that duplicated to generate the paleoAP3 and PI lineages in angiosperms (Kramer et al., 1998; Theißen et al., 2000; Irish, 2009). The paleoAP3 lineage is composed of AP3 orthologs identified in lower eudicots, magnolid dicots, and monocots (Kramer et al., 1998). Genes in this lineage contain the conserved paleoAP3- and PI-derived motifs in the C-terminal end of the proteins, which have been thought to be characteristics of the B class ancestral gene (Kramer et al., 1998; Tzeng and Yang, 2001; Hsu and Yang, 2002). The PI lineage is composed of PI orthologs that contain a highly conserved PI motif identified in most plant species (Kramer et al., 1998). Subsequently, there was a second duplication at the base of the core eudicots that produced the euAP3 and TM6 lineages, which have been subject to substantial sequence changes in eudicots during evolution (Kramer et al., 1998; Kramer and Irish, 1999). The paleoAP3 motif in the C-terminal end of the proteins was retained in the TM6 lineage and replaced by a conserved euAP3 motif in the euAP3 lineage of most eudicot species (Kramer et al., 1998). In addition, many lineage-specific duplications for paleoAP3 lineage have occurred in plants such as orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009), Ranunculaceae, and Ranunculales (Kramer et al., 2003; Di Stilio et al., 2005; Shan et al., 2006; Kramer, 2009).Unlike the A or C class MADS box proteins, which form homodimers that regulate flower development, the ability of B class proteins to form homodimers has only been reported in gymnosperms and in the paleoAP3 and PI lineages of some monocots. For example, LMADS1 of the lily Lilium longiflorum (Tzeng and Yang, 2001), OMADS3 of the orchid Oncidium Gower Ramsey (Hsu and Yang, 2002), and PeMADS4 of the orchid Phalaenopsis equestris (Tsai et al., 2004) in the paleoAP3 lineage, LRGLOA and LRGLOB of the lily Lilium regale (Winter et al., 2002), TGGLO of the tulip Tulipa gesneriana (Kanno et al., 2003), and PeMADS6 of the orchid P. equestris (Tsai et al., 2005) in the PI lineage, and GGM2 of the gymnosperm Gnetum gnemon (Winter et al., 1999) were able to form homodimers that regulate flower development. Proteins in the euAP3 lineage and in most paleoAP3 lineages were not able to form homodimers and had to interact with PI to form heterodimers in order to regulate petal and stamen development in various plant species (Schwarz-Sommer et al., 1992; Tröbner et al., 1992; Riechmann et al., 1996; Moon et al., 1999; Winter et al., 2002; Kanno et al., 2003; Vandenbussche et al., 2004; Yao et al., 2008). In addition to forming dimers, AP3 and PI were able to interact with other MADS box proteins, such as SEPALLATA1 (SEP1), SEP2, and SEP3, to regulate petal and stamen development (Pelaz et al., 2000; Honma and Goto, 2001; Theißen and Saedler, 2001; Castillejo et al., 2005).Orchids are among the most important plants in the flower market around the world, and research on MADS box genes has been reported for several species of orchids during the past few years (Lu et al., 1993, 2007; Yu and Goh, 2000; Hsu and Yang, 2002; Yu et al., 2002; Hsu et al., 2003; Tsai et al., 2004, 2008; Xu et al., 2006; Guo et al., 2007; Kim et al., 2007; Chang et al., 2009). Unlike the flowers in eudicots, the nearly identical shape of the sepals and petals as well as the production of a unique lip in orchid flowers make them a very special plant species for the study of flower development. Four clades (1–4) of genes in the paleoAP3 lineage have been identified in several orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009). Several works have described the possible interactions among these four clades of paleoAP3 genes and one PI gene that are involved in regulating the differentiation and formation of the sepal/petal/lip of orchids (Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009). However, the exact mechanism that involves the orchid B class genes remains unclear and needs to be clarified by more experimental investigations.O. Gower Ramsey is a popular orchid with important economic value in cut flower markets. Only a few studies have been reported on the role of MADS box genes in regulating flower formation in this plant species (Hsu and Yang, 2002; Hsu et al., 2003; Chang et al., 2009). An AP3-like MADS gene that regulates both floral formation and initiation in transgenic Arabidopsis has been reported (Hsu and Yang, 2002). In addition, four AP1/AGAMOUS-LIKE9 (AGL9)-like MADS box genes have been characterized that show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis (Hsu et al., 2003; Chang et al., 2009). Compared with other orchids, the production of a large and well-expanded lip and five small identical sepals/petals makes O. Gower Ramsey a special case for the study of the diverse functions of B class MADS box genes during evolution. Therefore, the isolation of more B class MADS box genes and further study of their roles in the regulation of perianth (sepal/petal/lip) formation during O. Gower Ramsey flower development are necessary. In addition to the clade 2 paleoAP3 gene OMADS3, which was previously characterized in our laboratory (Hsu and Yang, 2002), three more B class MADS box genes, OMADS5, OMADS8, and OMADS9, were characterized from O. Gower Ramsey in this study. Based on the different expression patterns and the protein interactions among these four orchid B class genes, we propose that the presence of OMADS3/8 and/or OMADS9 is required for sepal/petal/lip formation. Further sepal and petal formation at least requires the additional presence of OMADS5, whereas large lip formation was seen when OMADS5 expression was absent. Our results provide a new finding and information pertaining to the roles for orchid B class MADS box genes in the regulation of sepal/petal/lip formation.     

Reduced Root Cortical Cell File Number Improves Drought Tolerance in Maize

We tested the hypothesis that reduced root cortical cell file number (CCFN) would improve drought tolerance in maize (Zea mays) by reducing the metabolic costs of soil exploration. Maize genotypes with contrasting CCFN were grown under well-watered and water-stressed conditions in greenhouse mesocosms and in the field in the United States and Malawi. CCFN ranged from six to 19 among maize genotypes. In mesocosms, reduced CCFN was correlated with 57% reduction of root respiration per unit of root length. Under water stress in the mesocosms, genotypes with reduced CCFN had between 15% and 60% deeper rooting, 78% greater stomatal conductance, 36% greater leaf CO₂ assimilation, and between 52% to 139% greater shoot biomass than genotypes with many cell files. Under water stress in the field, genotypes with reduced CCFN had between 33% and 40% deeper rooting, 28% lighter stem water oxygen isotope enrichment (δ¹⁸O) signature signifying deeper water capture, between 10% and 35% greater leaf relative water content, between 35% and 70% greater shoot biomass at flowering, and between 33% and 114% greater yield than genotypes with many cell files. These results support the hypothesis that reduced CCFN improves drought tolerance by reducing the metabolic costs of soil exploration, enabling deeper soil exploration, greater water acquisition, and improved growth and yield under water stress. The large genetic variation for CCFN in maize germplasm suggests that CCFN merits attention as a breeding target to improve the drought tolerance of maize and possibly other cereal crops.Drought is a primary constraint to global crop production (Schmidhuber and Tubiello, 2007), and global climate change is likely to increase the risk of drought, especially in rain-fed agriculture (Battisti and Naylor, 2009; Burke et al., 2009; Mishra and Cherkauer, 2010; Lobell et al., 2011). Therefore, the development of crops with greater drought tolerance is an important global objective. Yield under drought is often not an efficient selection criterion in drought breeding programs, since yield is affected by many elements of the phenotype and the environment, interacting in complex and often unknown ways. Trait-based selection or ideotype breeding is generally a more efficient selection strategy, permitting the identification of useful sources of variation among lines that have poor agronomic adaptation, elucidation of genotype-by-environment interactions, and informed trait stacking (Araus et al., 2002, 2008; Manschadi et al., 2006; Lynch, 2007b, 2011; York et al., 2013).In most agroecosystems, the topsoil dries before the subsoil as drought progresses. In such environments, plants with deeper roots are able to acquire water available in deeper soil domains that may not be available to plants with shallower roots (Ludlow and Muchow, 1990; Ho et al., 2005; Hammer et al., 2009). An ideotype has been proposed to guide the breeding of crops with deeper roots and, therefore, greater water acquisition from drying soil, called Steep, Cheap, and Deep, integrating architectural, anatomical, and physiological phenes (Lynch, 2013). The term Cheap denotes phenes that reduce the metabolic cost of soil exploration, which is an important limitation to the acquisition of scarce soil resources, including water in dry soil (Fan et al., 2003; Lynch, 2007b; Zhu et al., 2010; Postma and Lynch, 2011a, 2011b; Jaramillo et al., 2013). Plant resource allocation to root growth typically increases under drought to enhance water acquisition; therefore, the metabolic cost of root growth becomes a significant component of plant fitness and adaptation under drought (Lynch, 2007b, 2013). Therefore, a plant that is able to access water in deep soil domains at reduced metabolic cost will have superior productivity, because it will have more metabolic resources available for further resource acquisition, growth, and reproduction. Evidence in support of this hypothesis comes from empirical and modeling studies for maize (Zea mays) under water and edaphic stress (Lynch, 2007a; Zhu et al., 2010; Postma and Lynch, 2011a, 2011b; Jaramillo et al., 2013).Root cortical aerenchyma (RCA) is the enlarged air space in the root cortex that forms either through cell death or cell separation (Evans, 2004). RCA is associated with a disproportionate reduction of root respiration in maize by converting living cortical tissue to air volume (Fan et al., 2003; Zhu et al., 2010). Reduction of root metabolic costs permits more internal resources to be allocated to greater root growth and, consequently, greater soil resource acquisition. RCA formation is also associated with a reduction of phosphorus content in root tissue on a volume basis, since air spaces do not contain phosphorus (Fan et al., 2003), and with improved growth in low-phosphorus soil (Lynch, 2011). RCA also reduces the nitrogen content of root tissue and is beneficial for nitrogen capture and maize growth on low-nitrogen soils (Saengwilai, 2014a). Modeling studies suggest that RCA improves crop adaptation to suboptimal nutrient availability by reducing the metabolic costs of soil exploration (Postma and Lynch, 2011a, 2011b). Under drought, Zhu et al. (2010) found that maize genotypes with more RCA had five times greater biomass and eight times greater yield than genotypes with less RCA. Living cortical area (LCA) is total transverse root cortical area minus RCA area. Jaramillo et al. (2013) found that root respiration is positively correlated with LCA, and a 3.5-fold reduction in LCA is associated with a 2.5-fold improvement in plant growth under drought. These results indicate that the metabolic demand of living cortical tissue is a primary determinant of root growth, soil exploration, and resource acquisition in soil environments with suboptimal resource availability.This study builds on earlier studies indicating that substantial reduction of root metabolic cost is associated with variation in LCA. The cortex of the maize root is composed of several concentric layers of parenchyma cells, the number of which we refer to as the cortical cell file number (CCFN). Recently, Burton et al. (2013) reported that there is 3-fold variation for CCFN in Zea spp. In that study, the variation was wider in maize landraces (six to 16 cell files) than in wild Zea spp. (seven to 13 cell files). It has been proposed that reduced CCFN would decrease the metabolic costs of root growth and maintenance, in terms of both the carbon cost of root respiration and the nutrient content of living tissue, by reducing the proportion of root volume occupied by living cortical tissue, which has greater metabolic demands than the stele (Lynch, 2013). However, the physiological utility of CCFN has not been explored.The objective of this study was to test the hypothesis that reduced CCFN would reduce root respiration, permitting greater rooting depth, thereby enhancing water acquisition and improving both plant growth and yield under water stress.     

Mechanical Failure of Fine Root Cortical Cells Initiates Plant Hydraulic Decline during Drought

Root systems perform the crucial task of absorbing water from the soil to meet the demands of a transpiring canopy. Roots are thought to operate like electrical fuses, which break when carrying an excessive load under conditions of drought stress. Yet the exact site and sequence of this dysfunction in roots remain elusive. Using in vivo x-ray computed microtomography, we found that drought-induced mechanical failure (i.e. lacunae formation) in fine root cortical cells is the initial and primary driver of reduced fine root hydraulic conductivity (Lp_r) under mild to moderate drought stress. Cortical lacunae started forming under mild drought stress (−0.6 MPa Ψ_stem), coincided with a dramatic reduction in Lp_r, and preceded root shrinkage or significant xylem embolism. Only under increased drought stress was embolism formation observed in the root xylem, and it appeared first in the fine roots (50% loss of hydraulic conductivity [P₅₀] reached at −1.8 MPa) and then in older, coarse roots (P₅₀ = −3.5 MPa). These results suggest that cortical cells in fine roots function like hydraulic fuses that decouple plants from drying soil, thus preserving the hydraulic integrity of the plant’s vascular system under early stages of drought stress. Cortical lacunae formation led to permanent structural damage of the root cortex and nonrecoverable Lp_r, pointing to a role in fine root mortality and turnover under drought stress.Root systems of woody plants consist of both coarse and nonwoody fine roots. Fine roots can constitute as little as 1% of the total root surface area (Kramer and Bullock, 1966), yet are critically important for biogeochemical cycling in terrestrial ecosystems as they constitute the primary exchange surface between plants and soil (Jackson et al., 1997). They are responsible for the vast majority of water absorption in woody root systems (Gambetta et al., 2013; Kramer and Boyer, 1995; Kramer and Bullock, 1966) and mediate backward flow of water from a plant to the soil via a process called hydraulic redistribution, which can alter regional climate (Richards and Caldwell, 1987; Lee et al., 2005). Fine roots also modify the soil through carbon exudation and stimulation of microbial activity (McCormack et al., 2015), and their production and annual turnover represent 33% of global net primary productivity (Jackson et al., 1997; McCormack et al., 2015). Elucidating details of fine root function and responses to stress can thus improve our understanding of how these plant organs can influence ecosystem carbon, nutrient, and water cycles.Fine roots are traditionally defined as all roots <2-mm diameter, but recent work has emphasized the need to delineate this diameter class into distinct functional groups. By separating fine roots into a shorter-lived absorptive pool and a longer-lived transport pool, McCormack et al. (2015) showed that fine root functionality can alter estimates of global net primary productivity by 30%. This work highlights our still-limited understanding of fine root functionality, the mechanisms underlying their lifespan and turnover, and how those traits respond to abiotic stress (Lukac, 2012; Tierney and Fahey, 2002; Guo et al., 2008). Fine root mortality during drought has been linked to increased root respiration and inhibited photosynthate transport to roots (e.g. Marshall, 1986) but could also be attributed to hydraulic dysfunction (Jackson et al., 2000). Portions of root systems are thought to operate analogously to a hydraulic fuse in an electrical circuit and designed to fail hydraulically when carrying excessive current under drought stress (Zimmermann, 1983; Jackson et al., 2000). However, the exact location of these hydraulic fuses in the root system has yet to be identified. Axial water transport in the xylem is considered a weak link, as roots of numerous species have been shown to be more susceptible to drought-induced xylem embolism compared to other organs within the same plant (i.e. trunks, stems, tap roots; Alder et al., 1996; Hacke and Sauter, 1996; McElrone et al., 2004; Pratt et al., 2015; Johnson et al., 2016). Moreover, Sperry and Ikeda (1997) found that smaller roots were the most vulnerable plant organ to xylem embolism, which would localize failure to inexpensive, distal, and easily replaceable portions of a root system. Such a design is considered effective, because it is widely assumed that the hydraulic capacity of smaller distal roots is readily repaired upon rewatering via xylem embolism removal (Domec et al., 2006; Jackson et al., 2000). While much work has demonstrated that xylem embolism reduces hydraulic capacity under severe drought stress (Brodribb et al., 2016a, 2016b; Choat et al., 2012), its contribution under mild to moderate stress is less clear (Choat et al., 2016; Cochard and Delzon, 2013; Cochard et al., 2013; McElrone et al., 2012; Wheeler et al., 2013; Choat et al., 2010; Torres-Ruiz et al., 2015). Work is still needed to resolve the location and sequence of root hydraulic dysfunction under drought and what tissues are involved in each stage of this process, especially under mild stress where fine root hydraulic conductivity (Lp_r) is known to decrease dramatically (Aroca et al., 2012).Before entering the xylem for long distance transport, water absorbed by roots must traverse a series of cell layers that include the epidermis, cortex, and endodermis (Steudle and Peterson 1998). Hydraulic resistance is much greater along this radial pathway compared to the axial transport pathway in the xylem (e.g. Frensch and Hsiao, 1993; Frensch and Steudle, 1989). The resistance differential between radial and axial pathways persists or increases in magnitude as fine Lp_r decreases under mild to moderate drought stress. While decreased fine root permeability under drought has been attributed to root shrinkage (Passioura, 1988; Nobel and Cui, 1992), changes in membrane permeability via aquaporins (Maurel et al., 2015; Aroca et al., 2012; North, 2004), development of suberized apoplastic barriers over longer periods of drought (Barrios-Masias et al., 2015; North and Nobel, 1991), or mechanical damage in cortical cells (i.e. lacunae formation; North and Nobel, 1991), the integration of these responses particularly under mild stress is still lacking. Elucidating the physiological mechanism that drives this response could help to resolve long-standing questions about fine root functionality, lifespan, and turnover.Here, we originally aimed to study whether fine roots function as the primary hydraulic fuse that disconnects a plant from drying soil. We studied the sequence of events during soil drying from saturated to severe drought conditions in coarse and fine roots of grapevines, which are considered a model species and have long been characterized as highly susceptible to drought-induced embolism. While performing these experiments, we discovered that the fine root cortex was radically changing under mild drought stress that preceded any embolism formation. We then performed hydraulic measurements and fluorescence light microscopy to investigate how fine Lp_r is affected by the formation of cortical lacunae that should significantly increase radial hydraulic resistance to flow.