A Casparian strip domain-like gene, CASPL, negatively alters growth and cold tolerance

A cold-induced transcript encoding a Casparian strip membrane domain (CASP)-like protein (ClCASPL) was identified in watermelon (Citrullus lanatus). Fluorescence microscopy analysis showed that ClCASPL-GFP is localized in the plasma membrane. The orthologous gene in Arabidopsis thaliana (AtCASPL4C1) was also found to play an important role in cold tolerance. Expression analysis using a β-glucuronidase (GUS) reporter reveals that AtCASPL4C1 is widely expressed in a variety of organs and is cold inducible. Analysis of AtCASPL4C1 T-DNA knock-out plants showed altered growth dynamics, faster growth, increased biomass (dry weight) and earlier flowering compared to wild type (Col-0) and ClCASPL overexpressing plants. AtCASPL4C1 knock-out plants showed elevated tolerance to cold stress, while overexpressing CICASPL resulted in increased sensitivity to cold stress in Arabidopsis. Interestingly, AtCASPL4C1 knock-out plants did not display significant alterations in the Casparian strip formation in roots. Thus, the combination of these results suggests a role for CICASPL and AtCASPL4C1 beyond Casparian strip formation in roots, possibly indicating a more fundamental role in vascular tissue.

proposed to form an extensive, transmembrane polymeric platform and were speculated to guide the assembly and activity of lignin biosynthetic enzymes 9 . Recently, evolutionary analysis of CASP family genes indicated that CASPL genes belong to the MARVEL (MAL and related proteins for vesicle trafficking and membrane link) protein family, which has only been experimentally described in metazoans, to date 12 .
Previously, we identified a gene encoding a cold-induced integral membrane protein in watermelon 13 . The orthologue to this gene was identified to be At3g55390 in Arabidopsis thaliana, where it is annotated as the CASP-LIKE PROTEIN 4C1. At3g55390 belongs to the CASP protein family containing the five CASP genes (CASP1/2/3/4/5), which are known to mediate Casparian strip formation in plants 9 . Thereafter, these are referred to as ClCASPL (from watermelon) and AtCASPL4C1 (CASP-like) or AtCASPL4C1 from Arabidopsis, respectively. In this study, we investigated the role of ClCASPL and AtCASPL4C1 in growth and cold tolerance in watermelon and Arabidopsis, respectively.

Materials and Methods
Plant Materials and treatment. An IVSM-9 inbred line of watermelon (Citrullus lanatus) and wild type (WT) of Arabidopsis (Arabidopsis thaliana) (Col-0) were used for amplification of ClCASPL and AtCASPL4C1 (At3G55390) genes. WT Arabidopsis thaliana (Col-0) was used for transformation to construct OX-ClCASPL of Arabidopsis. The SALK_034800C line was used for screening of homozygous Atcaspl4c1 knock-out mutant plants. 3-week-old plants in Jiffy seedling culture substrate or 2-week-old seedlings cultured in 1/2 MS medium of WT, Atcaspl4c1 and OX-ClCASPL Arabidopsis were used for analysis of cold stress and phenotypes evaluations. Tobacco (Nicotianaben thamiana) was used for transient expression of the ClCASPL gene. 5  Phylogenetic Tree Construction. The ClustalW program was used for alignment of ClCASPL with the AtCASPL4C1 protein sequence, which was obtained from the TAIR database. After alignment by ClustalW, a Neighbour-Joining tree was constructed by using MEGA 6.0, with 1000 as the number of bootstrap replications. The 39 members of CASP family genes were included from TAIR database. The TMHMM program was used for transmembrane region identification for ClCASPL and AtCASPL4C1.
Transient Expression of ClCASPL Gene in Tobacco. The amplification of ClCASPL coding sequence without a termination codon was linked to pMDC83 binary expression vector to generate the ClCASPL-pMDC83 by Gateway cloning 14 . The ClCASPL-GFP plasmid was introduced into Agrobacterium tumefaciens strain (GV1301). Leaves from tobacco (Nicotianaben thamiana) were used for transformation and for checking transient expression according to the procedures 15 . Various organelle-targeted markers fused with RFP were used as controls to determine sub-cellular localization 16 . These tobacco leaves were observed using a confocal laser scanning microscope (ZEISS). The primers used in this study for GFP fusion are listed in Supplementary Table 1.

Expression of CASPL.
In silico transcript abundance analysis for genes encoding proteins of the CASP family was carried out as outlined in Narsai et al. 17 . The promoter sequences were searched by A Database of Plant Promoter Sequences (http://linux1.softberry.com/berry.phtml?topic= plant-prom&group= data&subgroup= plantprom). The promoter region of At3g55390 was amplified and inserted into a region upstream of the GUS gene of within the pMDC162 binary expression vector using the gateway system 14 . The resulting construct was transformed in the Agrobacterium tumefaciens strain GV3101. Transformation of Arabidopsis was conducted according to the floral-dip method 18 . Tissues from transgenic plants were collected in microcentrifuge tubes. Subsequently, the samples were stained as previously published procedures 19 .
Cold-induced expression of CASPL was analyzed using qPCR and CASPL-promoter analysis using ClCASPL promoter-GUS activity after cold stress. GUS activity was measured as previously published procedures 20 . The primers used for promoter amplification are listed in Supplementary Table 1.
AtCASPL4C1 and OX-ClCASPL Construction. cDNA amplification of the ClCASPL gene was inserted to pMDC32 binary expression vector to generate the ClCASPL-pMDC32 by gateway approaches 14 . The ClCASPL-pMDC32 plasmid was introduced into Agrobacterium tumefaciens strain (GV1301). The floral dipping method was used to generate OX-ClCASPL of Arabidopsis 18 . For screening of transgenic plants, seeds harvested from transformed Arabidopsis plants were surface-sterilized and plated onto MS medium containing 50 mg/L hygromycin B. The plated seeds were vernalized at 4 °C for 2-4 days in the dark to synchronize germination and then transferred to a growth chamber at 22 °C (16/8 h photoperiod). 10-day-old in vitro-grown anti-hygromycin B seedlings were then transferred to soil mix. PCR and RT-PCR amplifications of the ClCASPL gene from candidate transgenic Arabidopsis plants were employed to confirm the successful transformation. WT and transgenic Arabidopsis T 3 lines were used in this study. The SALK_034800C line was used for the screening of homozygous AtCASPL4C1 knockout mutants of Arabidopsis using the three primers sets designed from the online service (http://signal. salk.edu/tdnaprimers.2.html). Primers used in this study for cDNA amplification of ClCASPL gene and screening of AtCASPL4C1 knock-out mutant of Arabidopsis are listed in Supplementary Table 1.
Casparian Strip Analysis. Propidium iodide (PI) staining was used to check Casparian strip formation in root as previously published procedures 9 . Roots from 5-day-old seedlings grown in MS medium were incubated in the dark for 10 min in 15 μ M (10 μ g/ml) PI (Invitrogen) and then were rinsed twice in water. The stained roots were observed using a fluorescence microscope (OLYMPUS BX51, Japan). Excitation and emission wavelengths are 488 nm and 500-550 nm.
Chlorophyll Fluorescence Analysis. 4-week-old seedlings of WT, AtCASPL4C1 and OX-ClCASPL of plants were cold treated at 10 °C and Fv/Fm measurements were taken. Individual plants were dark-adapted for 20 min prior to measurement. Chlorophyll fluorescence was measured in dark-adapted plants at 0, 6, 12, 24, 48 and 72 h under 10 °C cold stresses using a chlorophyll fluorescence system (M-Series Imaging-PAM, Germany).

Subcellular Localization of ClCASPL Protein.
ClCASPL is predicted to be targeted to the plasma membrane, according to the TargetP and SignalP subcellular localization prediction programs 22,23 . The subcellular localization of ClCASPL was investigated by the generation of a ClCASPL-green fluorescent protein (GFP) fusion and transient expression in tobacco (Nicotianaben thamiana) leaves. Expression of the constructed ClCASPL-GFP gene revealed a fluorescence signal exclusively in the plasma membrane, co-localizing with the plasma membrane marker 16 that was tagged with red fluorescent protein (RFP) (Fig. 2).
Expression Analysis of CASPL. Transcriptomic analysis of public microarrays from the AtGenExpress developmental dataset 5 and over seed germination 17 indicated that AtCASPL4C1 (At3G55390) is expressed in various tissues, but unlike some genes in this family it could not be defined as being predominantly expressed in root tissues (Fig. 3, black box). In contrast, a strong root expression profile is observed for several genes in this family, including CASP1/2/3/4/5 and other CASP-like genes (Fig. 3, yellow box). To determine spatial expression more precisely AtCASPL4C1 expression patterns were determined using an AtCASPL4C1-promoter-GUS (for β -glucuronidase) reporter. Histochemical analysis expression in planta, with 12 independent lines all showed a very similar expression pattern, varied only slightly in the intensity of GUS staining. GUS-staining revealed that AtCASPL4C1 expression is visible in roots and leaves (Fig. 4A). We can observe AtCASPL4C1 expression in the vascular cylinder of roots, but no expression was detected in root tip (Fig. 4A,B). AtCASPL4C1 expression was also detected in emerged lateral root (Fig. 4C). The filament, stigma and sepal of flowers also displayed intense staining (Fig. 4D), and signal could also be readily detected in siliques, but not in seeds (Fig. 4E). These results indicate that AtCASPL4C1 is expressed in a wide range of variety of tissues and is not restricted to the vascular tissue of roots alone.

CASPL Negatively Regulates Growth Dynamics. To investigate the function of CASPL in plants,
AtCASPL4C1 knockout mutants and transgenic plants overexpressing ClCASPL in Arabidopsis were generated ( Supplementary Figure 1 and 2). Growth and development parameters were measured to quantify any changes in phenotype due to the absence or over-expression of ClCASPL. The germination rate among wild type (Col-0), AtCASPL4C1 knockout and OX-ClCASPL plants did not significantly differ (Fig. 5A,C). The loss of AtCASPL4C1, however, did result in a small but significant increase in primary root length (Fig. 5D). In agreement with this, overexpressing ClCASPL in Arabidopsis significantly decreased primary root length (Fig. 5D). AtCASPL4C1 plants displayed significantly faster growth in several parameters, bigger plants (Fig. 5B,E), more biomass (Fig. 5H) and earlier rosette leaves development and flowering (Fig. 5F,G) compared to wild type (Col-0) and OX-ClCASPL plants. Thus, the loss of AtCASPL4C1 resulted in a positive growth effect, whereas over-expression gave the opposite effect. Thus, while only a single T-DNA insertion mutant is available for this gene in Arabidopsis, the complementary results of the knock-out and over-expressing plants strongly indicate that the phenotypes observed are due to the absence or increased abundance of CASPL.  Figure 3A). CASP1/2/3/4/5 transcript abundance was examined in root of wild type, AtCASPL4C1 and OX-ClCASPL. The transcript abundance of CASP1 was significantly altered, with increased transcript abundance in AtCASPL4C1 knock-outs and reduced abundance observed in OX-ClCASPL. The transcript abundance of CASP2, CASP3, CASP4 and CASP5 were significantly increased transcript abundance in AtCASPL4C1 knock-outs (Supplementary Figure 3B Figure 4).
The transcript abundance of ClCASPL and AtCASPL4C1 was examined under cold stress in watermelon and Arabidopsis. In watermelon, ClCASPL was induced and peaked in expression 12 h after cold treatment, while in Arabidopsis, AtCASPL4C1 was induced and peaked in expression 48 h after  Transcript abundance is shown over germination and development. Expression levels (log 2 ) are shown for the 28 genes (represented on microarrays) that encoding CASP or CASP-like genes. Hierarchical clustering shows strong co-expression of specific CASP like genes with known CASP genes (strongly co-expressed genes indicated by an asterisk*). The transcript abundance for At3g55390 is shown in a black box, and genes that display a root enriched expression profile are shown in a yellow box. White boxes indicate no expression was detected.
cold treatment (Fig. 6A). Moreover, the AtCASPL4C1 promoter-GUS activities after cold stress showed that expression of AtCASPL4C1 was induced by cold stress in Arabidopsis (Fig. 6B). To study the role of CASPL in cold tolerance, different parameters were used to evaluate cold tolerance in wild type, AtCASPL4C1 and OX-ClCASPL plants. Growth was monitored for 5-day-old plants exposed to cold at 10 °C for 7 days in medium. This examination showed that AtCASPL4C1 grows better under cold stress, based on the significantly longer primary root length (Fig. 7A-C). Furthermore, 3-week-old AtCASPL4C1 plants showed enhanced growth treated with cold stress, at 10 °C for 10 days (Fig. 7D,E), keeping relatively higher chlorophyll fluorescence parameters compared to WT (Fig. 7F), as well as, more rosette leaves and greater biomass (Fig. 7G,H). These results displayed greater cold tolerance than wild type and OX-ClCASPL.

Discussion
The Casparian strip in the root endodermis is a barrier that restricts the free diffusion of molecules between the inner cell layers of the root. Recent studies revealed that this endodermal barrier is not only a regulator of water and nutrient uptake, but also probably acts as signal center for hormone-mediated control of growth 24,25 . It may be considered as a signal communication hub in response to the external environment, facilitating the activation of hormone signaling pathways and the propagation of calcium waves 24,25 . It also plays a central role in mediating cross talk between different cell layers during the  26 . The molecular mechanisms involved in the deposition of the Casparian strip in endodermal cells are beginning to be uncovered 27,28 . The Casparian strip domain protein (CASP) anchors the plasma membrane to the cell wall and recruits the enzymes necessary for lignin biosynthesis 27,28 .
Evolutionary analysis indicated plant CASP family is associated with the MARVEL protein family described only in metazoans with similarity in specific transmembrane domains, in the overall tetraspan protein structure and putative Cys bridge in the second extracellular loop 12 . CASPs in this family are the earliest known proteins responsible for the CSD (Casparian strip membrane domain) formation 9 . The localizing at the CSD when expressed in the endodermis of CASPLs in this family prompts us to predict the role of yet-undiscovered CASPLs in mediating membrane subdomain formation and other plasma membrane domains in other cell types 12 . Here, we identified the membrane-localizing AtCASPL4C1 and ClCASPL genes that encode proteins of the CASP family. Overall, the absence of AtCASPL4C1 results in significantly increased growth under normal conditions. Based on analysis of CASP1/2/3/4/5 transcript abundance and lignin staining in roots, it is suggested that mutation in AtCASPL4C1 (At3G55390) did not dramatically affect the Casparian strip formation in roots. It has previously been shown in Atcasp1 or Atcasp3 single mutants, that Casparian strip formation was unaltered compared to WT, however, casp double mutants (casp1/casp3) displayed disorganized Casparian strip 9 . Thus, due to redundancy it is not possible to conclude if there is a direct role in Casparian strip formation for AtCASPL4C1. However, loss of function of AtCASPL4C1 was observed to result in earlier vegetative and reproductive development and increased biomass (Fig. 5).
Plants respond to environmental changes by altering tissue differentiation and development, referred to as plant phenotypic plasticity. Here, we showed CASPL gene also regulates enhanced growth under cold stress conditions. While AtCASPL4C1 belongs to this family based on predicted protein homology, the expression pattern based on in silico analyses and GUS reporter gene expression patterns suggest that the role of AtCASPL4C1 may extend well beyond the Casparian strip in roots (Figs 3 and 4). The widespread expression pattern in vascular tissues also suggests that it plays a more fundamental role in plant growth and development, which is supported by the altered phenotype when it is inactivated or over-expressed.
In conclusion, we identified a CASP-like ClCASPL gene in Citrullus lanatus and its ortholog AtCASPL4C1 in Arabidopsis are associated with growth and cold tolerance. These findings offered us clues to further explore the distinguished role of the Casparian strip protein family genes in growth, development and environmental communications.