OsNucleolin1-L Expression in Arabidopsis Enhances Photosynthesis via Transcriptome Modification under Salt Stress Conditions

Introduction

Environmental stress such as drought and saline soils can reduce various activities essential for respiration (Criddle et al. 1989) and photosynthesis (Yeo and Flower 1982), leading to reduction of crop yield. Exposure of plants to these stresses induces expression changes in genes with widely different roles (Yamaguchi-Shinozaki and Shinozaki 2006), presumably in order to cope with the deleterious effects of stress. The plant hormone ABA mediates many plant responses to physical stress, especially the response to water status such as drought and salinity. ABA regulates stomatal closure, plant development, such as seed maturation, germination, seedling growth and also adaptive responses to osmotic stress (Zhang et al. 2006).

RNA-binding proteins play diverse roles in plant growth and development (Lu and Fedoroff 2000). Several reports described the involvement of RNA-binding proteins in the ABA response and also in plant response to extreme environments such as those involving regulation of HYL1, ABH1 and CBP20 (Lu and Fedoroff 2000, Hugouvieux et al. 2001, Papp et al. 2004). Nucleolin (NUC) is an RNA-binding protein predominant in the nucleolus particularly around fibrillar centers (FCs) and the dense fibrillar component (DFC) (Tong et al. 1997, Pontvianne et al. 2007, Stepinski 2012). NUC is composed of four domains: the nuclear localization signal (NLS); the N-terminal acidic region; two RNA-recognition motifs (RRMs); and a glycine–arginine rich (GAR) region. Arabidopsis Nucleolin1, AtNUC-L1, the predominant nucleolin in Arabidopsis, is reported to be involved in sugar-inducible expression of ribosomal proteins. The disruption of the AtNUC-L1 gene resulted in phenotypes similar to those of ribosomal protein gene mutants (Kojima et al. 2007).

Two NUC genes are found in the rice genome, OsNUC1 and OsNUC2. Only OsNUC1 responds to salt stress conditions. Rice lines with higher salt tolerance display a higher level of OsNUC1 gene expression. In a previous study, we discovered by Northern blot analysis that OsNUC1 had two mRNA forms: OsNUC1-S and OsNUC1-L, 1.9 and 2.4 kb respectively. Overexpression of the OsNUC1-S gene in rice and its expression in Arabidopsis conferred salt tolerance in both species. Enhancement of the AtSOS1 and AtP5CS1 transcripts in Arabidopsis expressing OsNUC1-S suggests that the RRM and GAR regions of OsNUC1 function in salt resistance (Sripinyowanich et al. 2013).

Here, we describe the effect of expressing the OsNUC1-L isoform in Arabidopsis thaliana. We used transcriptome analysis to determine the impact of OsNUC1-L on global mRNAs, connecting the molecular changes to the salt tolerance phenotype of the transgenic lines. The overexpression of the OsNUC1-L isoform in rice was used to demonstrate that the phenotype in Arabidopsis is not due to artificial gain of function in the heterologous system.

Results

OsNUC1-L improved salt tolerance in Arabidopsis

Three independent transgenic lines (T14, T22 and T24) were characterized for the expression of OsNUC1-L by quantitative real-time PCR (RT-PCR). OsNUC1-L was expressed at different levels in each transgenic line. T22 showed the strongest expression among the three lines (Fig. 1A). The transgenic lines expressing OsNUC1-L displayed early flowering starting 16 d after stratification, when grown on Murashige and Skoog (MS) medium (Fig. 1B). However, when they were grown on soil, the flowering date and number of rosette leaves at flowering of the transgenic lines and wild-type control were similar (Supplementary Fig. S1). The phenotype of transgenic OsNUC1 expression differed from that reported for a AtNUC1 loss-of-function mutation caused by T-DNA insertion (par1), which displayed abnormal development, retarded growth, reduced fertility and defects in leaf venation (Petricka and Nelson 2007).

Fig. 1

The relative level of OsNUC1-L transcripts in transgenic Arabidopsis with the 35SCaMV:OsNUC1-L construct (T14, T22 and T24) (A). The phenotype of 16-day-old transgenic Arabidopsis expressing OsNUC1-L and the wild type, when grown on MS medium or MS medium supplemented with 150 mM NaCl (B). The relative growth rate of wild-type (WT) and transgenic Arabidopsis on MS medium supplemented with 150 mM NaCl (C) was determined using a completely randomized design (CRD) with 10 replicates, each of which consisted of at least 10 seedlings. Means with a different letter above the bar are significantly different (P < 0.05, Duncan’s Multiple Range Test).

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All three transgenic lines expressing OsNUC1-L displayed increased salt tolerance, maintaining Chl content after growth on MS medium supplemented with 150 mM NaCl for 3 weeks (Fig. 1B). Interestingly, expression of OsNUC1-L did not prevent dry weight loss in Arabidopsis under salt stress conditions. The expression of OsNUC1-L only slightly enhanced relative growth rate during salt stress (Fig. 1C). This is in contrast to transgenic Arabidopsis expressing OsNUC1-S, which showed enhanced plant growth under stress conditions (Sripinyowanich et al. 2013).

OsNUC1-L altered the response to ABA

To explore a potential connection between OsNUC1-L and ABA, we tested seed germination of the wild type and three transgenic OsNUC1-L lines on MS medium with or without ABA supplementation. There was no effect on germination of the transgenic lines in the absence of ABA. Germination of wild-type Arabidopsis is repressed by ABA (Bewley 1997). In the presence of 0.6 µM ABA, while wild-type Arabidopsis displayed 40% reduction in germination, the transgenic lines displayed virtually complete suppression (Fig. 2).

Fig. 2

Germination response of transgenic OsNUC1 (T14, T22 and T24, dashed lines) and wild-type Arabidopsis (solid line) on MS medium (A, B) and MS medium supplemented with various concentrations of ABA (C, D).

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In order to understand the enhanced response to ABA, the expression of genes encoding ABA synthesis and ABA-responsive genes was investigated via transcriptome analysis of the T22 line. We found that OsNUC1-L expression reduced the expression of the NCED3 gene, encoding 9-cis-epoxycarotenoid dioxygenase, which catalyzes the rate-limiting step in ABA biosynthesis, conversion of 9-cis-violaxanthin to xanthoxin (Qin and Zeevaart 2002) (Supplementary Fig. S2). At the same time, expression of OsNUC1-L induced the expression of AAO3 and NCED6 (Table 1). Moreover, expression of the gene encoding CYP707A4, an enzyme mediating ABA degradation, was 5-fold higher than in the wild type. The expression of some ABA-responsive genes was also changed in the transgenic lines. We found a significant reduction of ABA-responsive gene expression, such as genes encoding the late embryogenesis abundant protein RD29A, and dehydrin RAB18-like protein (Table 2).

OsNUC1-L globally changed the gene expression

Expression of OsNUC1-L in Arabidopsis (line T22) caused significant changes in 1,967 genes under normal conditions and 999 genes under salt stress conditions (Supplementary Table S1). When these significant genes were subjected to Gene Ontology (GO) term enrichment analysis in TAIR using cellular compartment, biological function and molecular function categorization, the results shown in Fig. 3A–C were obtained. Enrichment categories of significant genes were similar in normal and salt stress conditions, except that in the latter condition genes involved in ribosome and structural molecular function (red column in Fig. 3A, B) displayed higher enrichment ratios than in normal conditions, supporting a role for OsNUC1 in RNA processing during salt stress.

Fig. 3

Gene enrichment of the significant gene expression difference between the transgenic Arabidopsis with expression of the OsNUC1-L gene and the wild type under normal and salt stress conditions. The enrichment analysis was performed for compartmentalization (A), molecular function (B) and biological function (C) categories. The red bar indicates the higher gene count under salt stress conditions in the transgenic line found in those categories.

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To study the global effect of OsNUC1-L gene expression on the transcriptome of Arabidopsis, we used the cytoscape plugin ClueGo (Bindea et al. 2009) to generate and visualize gene set enrichment under normal and salt stress conditions. The significantly enriched terms with more up- and down-regulated genes were visualized in red and blue color, respectively. The node size represents the degree of significance and the color density represents the number of genes in each node.

When using biological processes classification, stress-responsive genes were preponderant in both control and salt stress conditions (Fig. 4A, B). The stress-associated genes were divided into functional proteins and regulatory proteins. Functional proteins play adaptive roles during stress, such as stabilization of macromolecules, and included, for example, late embryogenesis abundant proteins, enzymes that regulate osmolyte biosynthesis and transporter proteins. Under normal conditions (Fig. 4A), OsNUC1-L transgenics induced a group of genes responding to stress, transporter genes, membrane docking genes and genes that regulate stomatal movement. The set of genes encoding regulatory proteins and exhibiting altered responses in the transgenics included genes encoding MAPK and LRR-RLK signal transduction pathways and transcription factors such as MYB, MYC and WRKY. Likewise, transgenics displayed increased expression of genes encoding proteins involved in cellular alkene metabolic process, polysaccharide localization, negative regulation of leaf senescence and regulation of cell communication. In contrast, under normal conditions, the transgenic line displayed a reduction in the expression of genes encoding proteins involved in growth and development, especially in root morphogenesis, photosynthesis and the endomembrane system (Fig. 4A).

Fig. 4

The gene set enrichment analysis of significantly expressed genes in transgenic OsNUC1-L under normal (A) and salt stress (B) conditions. Nodes in the network show the biological processes significantly enriched, with up- and down-regulated genes shown in red and blue, respectively. The node size represents the degree of significance and the color density the number of genes in each node. The enrichment network was generated by ClueGo (Bindea et al. 2009) with Benjamini and Hochberg correction (corrected P < 0.05).

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Under salt stress conditions, the transcriptome analysis still showed the up-regulation in stress-responsive genes and the cellular alkene metabolic process. Moreover, some genes assigned to the auxin metabolic process were also enhanced (Fig. 4B). Although overall genes responding to stress were up-regulated, genes responding to arsenic-containing substances and oxygen level were decreased in the transgenic lines. The down-regulated genes were found in negative regulation of signal transduction, seed maturation, circadian rhythm, positive regulation of post-embryonic development and carbohydrate transport categories (Fig. 4B).

We classified genes according to the subcellular location of their products. In lines expressing OSNUC1-L, induced genes included those with extracellular organelle annotation, such as exocyst subunit proteins, as well as plasma membrane components. Therefore, genes encoding intrinsic components of membrane and transmembrane proteins were over-represented among the genes induced under normal growth conditions. In contrast, genes encoding proteins in external encapsulated structures, vacuole and endoplasmic reticulum, and photosystem were over-represented among the genes suppressed under normal conditions (Fig. 5A). Under salt stress conditions, OSNUC1-L expression suppressed genes functioning in peroxisomes and the cell surface, while it induced genes encoding products working in the ribosome, light-harvesting complex and nucleolus (Fig. 5B). Interestingly, genes involved in the photosynthetic process responded differently to salt and normal conditions. Under normal conditions, PSI subunit protein, D1 and PSII reaction center proteins were down-regulated, while Chl a/b-binding protein (CAB) and PSII light-harvesting complex (LHCB) genes were up-regulated during salt stress conditions (Table 3).

Table 3

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Light-harvesting system protein and photosynthetic membrane components that displayed significant changes in transgenic OsNUC1-L Arabidopsis during normal and salt stress conditions

Experiment .	Location .	Locus name .	Gene name .	Description .	Fold change .
Control	PSII	ATCG00020	PSBA	PSII reaction center protein a	0.428
		ATCG00270	PSBD	PSII reaction center protein d	0.464
		ATCG00280	CP43	CP43 subunit of the PSII reaction center	0.437
		ATCG00340	PSAB	D1 subunit of PSI reaction center	0.566
		ATCG00350	PSAA	psaA protein comprising the reaction center for PSI	0.518
		ATCG00630	PSAJ	Subunit J of PSI	0.532
		ATCG00680	PSBB	PSII reaction center protein b	0.449
		ATCG00710	PSBH	PSII reaction center protein h	0.457
		ATCG01060	PSAC	PsaC subunit of PSI	0.394
	Cyt b6f complex	AT5G60920	COB	Glycosylphosphatidylinositol-anchored protein	0.652
		ATCG00540	PETA	Photosynthetic electron transfer a	0.640
		ATCG00720	PETB	Photosynthetic electron transfer b	0.401
		ATCG00730	PETD	Photosynthetic electron transfer d	0.490
		ATMG00160	COX2	Cyt c oxidase subunit 2	0.534
		ATMG01360	COX1	Cyt c oxidase subunit 1	0.475
Salt stress	Light-harvesting complex	AT1G29910	CAB3	Chl a/b-binding protein 3	1.403
		AT1G29920	CAB2	Chl a/b-binding protein 2	1.914
		AT2G05070	LHCB2.2	PSII light-harvesting complex gene 2.2	1.682
		AT2G05100	LHCB2.1	PSII light-harvesting complex gene 2.1	1.665
		AT3G27690	LHCB2.3	PSII light-harvesting complex gene 2.3	1.881
		AT4G31390	ACDO1	ABC1-like kinase related to Chl degradation and oxidative stress 1	0.683
		AT5G54270	LHCB3	Light-harvesting Chl b-binding protein 3	1.390
		ATCG00020	PSBA	PSII reaction center protein A	0.709

Experiment .	Location .	Locus name .	Gene name .	Description .	Fold change .
Control	PSII	ATCG00020	PSBA	PSII reaction center protein a	0.428
		ATCG00270	PSBD	PSII reaction center protein d	0.464
		ATCG00280	CP43	CP43 subunit of the PSII reaction center	0.437
		ATCG00340	PSAB	D1 subunit of PSI reaction center	0.566
		ATCG00350	PSAA	psaA protein comprising the reaction center for PSI	0.518
		ATCG00630	PSAJ	Subunit J of PSI	0.532
		ATCG00680	PSBB	PSII reaction center protein b	0.449
		ATCG00710	PSBH	PSII reaction center protein h	0.457
		ATCG01060	PSAC	PsaC subunit of PSI	0.394
	Cyt b6f complex	AT5G60920	COB	Glycosylphosphatidylinositol-anchored protein	0.652
		ATCG00540	PETA	Photosynthetic electron transfer a	0.640
		ATCG00720	PETB	Photosynthetic electron transfer b	0.401
		ATCG00730	PETD	Photosynthetic electron transfer d	0.490
		ATMG00160	COX2	Cyt c oxidase subunit 2	0.534
		ATMG01360	COX1	Cyt c oxidase subunit 1	0.475
Salt stress	Light-harvesting complex	AT1G29910	CAB3	Chl a/b-binding protein 3	1.403
		AT1G29920	CAB2	Chl a/b-binding protein 2	1.914
		AT2G05070	LHCB2.2	PSII light-harvesting complex gene 2.2	1.682
		AT2G05100	LHCB2.1	PSII light-harvesting complex gene 2.1	1.665
		AT3G27690	LHCB2.3	PSII light-harvesting complex gene 2.3	1.881
		AT4G31390	ACDO1	ABC1-like kinase related to Chl degradation and oxidative stress 1	0.683
		AT5G54270	LHCB3	Light-harvesting Chl b-binding protein 3	1.390
		ATCG00020	PSBA	PSII reaction center protein A	0.709

Table 3

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Light-harvesting system protein and photosynthetic membrane components that displayed significant changes in transgenic OsNUC1-L Arabidopsis during normal and salt stress conditions

Experiment .	Location .	Locus name .	Gene name .	Description .	Fold change .
Control	PSII	ATCG00020	PSBA	PSII reaction center protein a	0.428
		ATCG00270	PSBD	PSII reaction center protein d	0.464
		ATCG00280	CP43	CP43 subunit of the PSII reaction center	0.437
		ATCG00340	PSAB	D1 subunit of PSI reaction center	0.566
		ATCG00350	PSAA	psaA protein comprising the reaction center for PSI	0.518
		ATCG00630	PSAJ	Subunit J of PSI	0.532
		ATCG00680	PSBB	PSII reaction center protein b	0.449
		ATCG00710	PSBH	PSII reaction center protein h	0.457
		ATCG01060	PSAC	PsaC subunit of PSI	0.394
	Cyt b6f complex	AT5G60920	COB	Glycosylphosphatidylinositol-anchored protein	0.652
		ATCG00540	PETA	Photosynthetic electron transfer a	0.640
		ATCG00720	PETB	Photosynthetic electron transfer b	0.401
		ATCG00730	PETD	Photosynthetic electron transfer d	0.490
		ATMG00160	COX2	Cyt c oxidase subunit 2	0.534
		ATMG01360	COX1	Cyt c oxidase subunit 1	0.475
Salt stress	Light-harvesting complex	AT1G29910	CAB3	Chl a/b-binding protein 3	1.403
		AT1G29920	CAB2	Chl a/b-binding protein 2	1.914
		AT2G05070	LHCB2.2	PSII light-harvesting complex gene 2.2	1.682
		AT2G05100	LHCB2.1	PSII light-harvesting complex gene 2.1	1.665
		AT3G27690	LHCB2.3	PSII light-harvesting complex gene 2.3	1.881
		AT4G31390	ACDO1	ABC1-like kinase related to Chl degradation and oxidative stress 1	0.683
		AT5G54270	LHCB3	Light-harvesting Chl b-binding protein 3	1.390
		ATCG00020	PSBA	PSII reaction center protein A	0.709

Experiment .	Location .	Locus name .	Gene name .	Description .	Fold change .
Control	PSII	ATCG00020	PSBA	PSII reaction center protein a	0.428
		ATCG00270	PSBD	PSII reaction center protein d	0.464
		ATCG00280	CP43	CP43 subunit of the PSII reaction center	0.437
		ATCG00340	PSAB	D1 subunit of PSI reaction center	0.566
		ATCG00350	PSAA	psaA protein comprising the reaction center for PSI	0.518
		ATCG00630	PSAJ	Subunit J of PSI	0.532
		ATCG00680	PSBB	PSII reaction center protein b	0.449
		ATCG00710	PSBH	PSII reaction center protein h	0.457
		ATCG01060	PSAC	PsaC subunit of PSI	0.394
	Cyt b6f complex	AT5G60920	COB	Glycosylphosphatidylinositol-anchored protein	0.652
		ATCG00540	PETA	Photosynthetic electron transfer a	0.640
		ATCG00720	PETB	Photosynthetic electron transfer b	0.401
		ATCG00730	PETD	Photosynthetic electron transfer d	0.490
		ATMG00160	COX2	Cyt c oxidase subunit 2	0.534
		ATMG01360	COX1	Cyt c oxidase subunit 1	0.475
Salt stress	Light-harvesting complex	AT1G29910	CAB3	Chl a/b-binding protein 3	1.403
		AT1G29920	CAB2	Chl a/b-binding protein 2	1.914
		AT2G05070	LHCB2.2	PSII light-harvesting complex gene 2.2	1.682
		AT2G05100	LHCB2.1	PSII light-harvesting complex gene 2.1	1.665
		AT3G27690	LHCB2.3	PSII light-harvesting complex gene 2.3	1.881
		AT4G31390	ACDO1	ABC1-like kinase related to Chl degradation and oxidative stress 1	0.683
		AT5G54270	LHCB3	Light-harvesting Chl b-binding protein 3	1.390
		ATCG00020	PSBA	PSII reaction center protein A	0.709

Fig. 5

The gene set enrichment analysis of significantly regulated genes in transgenic OsNUC1-L under normal (A) and salt stress (B) conditions. Nodes in the network show the specific cellular comparment, where the significantly enriched terms with more up- and down-regulated genes are shown in red and blue, respectively. The node size represents the degree of significance and the color gradient represents the proportion of genes in each node. The enrichment network was generated by ClueGo (Bindea et al. 2009) with Benjamini and Hochberg correction (corrected P < 0.05).

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OsNUC1-S has strong effects on gene expression during salt stress

As OsNUC1-S has been shown to induce salt tolerance in both Arabidopsis and rice (Sripinyowanich et al. 2013), transcriptome analysis was performed to reveal the effects of OsNUC1-S on gene expression of Arabidopsis under normal and salt stress conditions. In normal conditions, only 85 genes were differentially expressed, but under salt stress conditions, 4,596 genes were found to be differently expressed when compared between the wild type and OsNUC1-S-expressing lines (Fig. 6; Supplementary Table S2). This is in contrast to what was found in Arabidopsis with OsNUC1-L expression, where more genes were regulated in the transgenic lines grown under normal conditions (Fig. 3). When enrichment of the genes differentially expressed under salt stress conditions was analyzed for association with biological process with the cytoscape plugin ClueGo (Bindea et al. 2009), several groups of genes were found, such as response to oxygen-containing compounds, chromosome organization, secondary metabolic process, single-organism catabolic process, photosynthesis, cellular macromolecule and carbohydrate metabolic process. Genes in stress responses were also found, including oxidative stress and osmotic stress (Fig. 7).

Fig. 6

Gene enrichment of the significant gene expression difference between the transgenic Arabidopsis with expression of the OsNUC1-S gene and the wild type under normal and salt stress conditions. The enrichment analysis was performed for compartmentalization (A), molecular function (B) and biological function (C) categories.

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Fig. 7

The gene set enrichment analysis of significantly expressed genes in transgenic OsNUC1-S under salt stress conditions. The enrichment network was generated by ClueGo (Bindea et al. 2009) with Benjamini and Hochberg correction (corrected P < 0.01). The node size represents the number of gene in each node, and the darker colors represent higher siginificant levels.

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When the differentially expressed genes under salt stress conditions were analyzed for cellular compartment enrichment, the major localization was the nucleus and chloroplast (Fig. 8A). This was in agreement with molecular function enrichment, which was found to be nucleotide binding, nucleic acid binding, Chl binding and tetrapyrrole binding. Interestingly, glutathione transferase activity was also found (Fig. 8B).

Fig. 8

The gene set enrichment analysis of significantly regulated genes in transgenic OsNUC1-S under salt stress conditions, showing cellular compartment (A) and molecular function (B). Nodes in the network show the specific cellular comparment. The node sizes represent the degree of significance.The enrichment network was generated by ClueGo (Bindea et al. 2009) with Benjamini and Hochberg correction (P < 0.01).

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OsNUC1-L improved photosynthetic activity during salt stress by regulating the expression of genes in the thylakoid membrane

Expression analysis (see above) pointed to photosynthesis as a likely mechanism affecting the altered stress response of OSNUC1-L transgenic plants. In order to assess their photosynthetic activity, we measured net photosynthetic rate (P_n), stomatal conductance (g_s), transpiration rate (E), intracellular CO₂ concentration (C_i) and water use efficiency (WUE). Furthermore, we compared the expression activity of genes encoding proteins involved in photosynthetic processes detected by transcriptome analysis. After irrigation with 150 mM NaCl for 24 h, the P_n of the wild type was slightly decreased, while the P_n of all NUC transgenic plants was significantly increased (Fig. 9A). However, no significant changes in g_s (Fig. 9B) and E (Fig. 9C) were detected among lines. These observations suggest that the increase in P_n was not due to the changes in stomatal activity, but it was caused rather by modification of other activities in the photosynthetic process. A remarkable difference between the wild type and the transgenic lines was the change in the C_i. In OsNUC1-L transgenic lines, C_i was significantly decreased, but it increased in the wild type (Fig. 9D). The transcriptomic study revealed significant enhancement of genes encoding the light-harvesting system proteins and photosynthetic membrane components during salt stress (Fig. 5B). CAB2 and CAB3 (AT1G29910 and AT1G29920) and a group of genes encoding LHCB (AT2G05070, AT2G05100, AT3G27690and ATCG00020) were distinctly up-regulated (Table 3). In addition, we found that some genes encoding thylakoid membrane components, especially PSII proteins, displayed increased expression levels. The latter included genes encoding CAB and LHCB (Table 3). These data support the role of OsNUC1-L expression in enhancing photosynthesis during salt stress, at least by up-regulation of the genes encoding proteins involved in the light reaction, especially those of PSII. However, we cannot exclude other changes in the transcriptome that can also contribute to the enhancement of photosynthesis under salt stress conditions. Interestingly, WUE, the ratio of P_n to E, was significantly enhanced in all transgenics compared with the wild type (Fig. 9E). These observations support a role for the OsNUC1-L gene in water balance adaptation during salt stress. Noticeably, the enhancement of the photosynthetic response under salt stress was not correlated with the level of OsNUC1-L expression in the transgenic lines. T22 showed the highest OsNUC1-L gene expression, but showed the lowest response in enhancement of photosynthesis (Fig. 9). However, the response of T22 was in the same direction as other transgenic lines with OsNUC1-L expression, while the wild type displayed the opposite response. These findings suggest a role for OsNUC1-L in maintenance of the stability of photosynthesis under salt stress conditions.

Fig. 9

Changes in photosynthesis parameters: (A) net photosynthesis, (B) stomatal conductivity, (C) transpiration rate, (D) intracellular CO₂ concentration and (E) water use efficiency, measured before and after 1 d of salt treatment (150 mM NaCl). Means were compared using one-way analysis of variance. All parameters ae shown as the mean ± SE. * indicates significant difference from the wild type (WT) (P < 0.05, Duncan’s Multiple Range Test). Nine plants were used for the analysis with a completely randomized design (CRD).

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Transcriptome analysis of Arabidopsis with OsNUC1-S expression also suggested changes in the chloroplast gene function (Fig. 8A, B) and photosynthetic process (Fig. 7). Unfortunately, we could not evaluate the photosynthetic responses in Arabidopsis with OsNUC1-S expression due to irregular inheritance of the transgene in the line selected for advancement.

Overexpression of OsNUC1-L enhances photosynthesis under salt stress conditions in rice

In order to test the role of OsNUC1-L in salt resistance in rice, we generated three transgenic lines, FL1, FL2 and FL3, with OsNUC1-L expressed using the ubiquitin promoter. In the wild type, the native OsNUC1 gene was up-regulated 2 d after salt stress, consistent with the report by Sripinyowanich et al. (2013). Higher expression of OsNUC1 was detected in all transgenic lines (Fig. 10). Importanly, detection of OsNUC1 gene expression was performed using primers that detect both endogenous and overexpressed copies. Expression levels in the transgenic plants varied, perhaps due to the position effects of the transgene (Filipecki and Malepszy 2006, Raveendar et al. 2007). Therefore, the result shown here is the overall result of OsNUC1 gene expression at each stage. Under salt stress conditions, overexpression of OsNUC1-L did not affect seedling shoot and root growth (Supplementary Fig. S3). This was similar to the expression of OsNUC1-L in Arabidopsis.

Fig. 10

OsNUC1 relative gene expression in wild-type (WT) (A) and OsNUC1-L-overexpressing transgenic lines, FL1 (B), FL2 (C) and FL3 (D) under normal and salt stress conditions. The experiment was performed with three biological replicates. Relative gene expression was calculated in comparison with the expression of the wild type on Day 0.

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To study the effects on photosynthesis, salt stress was applied to rice plants at the booting stage, and rate of photosynthesis and stomatal conductance were determined in the flag leaf and in the second leaf. In normal conditions, overexpression of OsNUC1-L did not affect either the rate of photosynthetic or stomatal conductance in any leaf types (Fig. 11). However, after 9 d of salt stress, the rate of photosynthesis in flag leaves was reduced to 30% of that in the wild type, while in transgenic rice the rate of photosynthesis was not significantly decreased (FL2 and FL3) or was decreased to about 50% (FL1) (Fig. 11A). The rate of photosynthesis was better retained in the second leaf of the transgenic lines (Fig. 8B). This resulted in a significantly higher rate of photosynthesis in all transgenic lines in both flag leaves and second leaves, when compared with wild-type rates (Fig. 11A, B). Significantly higher stomatal conductance in the transgenic lines was also found (Fig. 11C, D).

Fig. 11

Photosynthesis rate (A, B) and stomatal conductance (C and D) of flag leaves (A and C) and second leaves (B and D) of wild-type and transgenic rice, FL1, FL2 and FL3 at the booting stage grown in normal conditions or treated with salt stress for 9 d. The experiment was designed in random complete block design with four replications. Three individuals of each homozygous T₃ transgenic line and the wild type were used for each replication. Means with a different letter above the bar are significantly different (P < 0.05, Duncan’s Multiple Range Test).

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The salt stress effects on photosynthetic pigments and Chl fluorescence in transgenic rice with OsNUC1-L overexpression supported the function of OsNUC1-L in the maintenance of the photosynthetic apparatus. In normal conditions, OsNUC1-L overexpression did not affect photosynthetic pigment content. However, after 9 d of salt stress, a decline in Chl a, Chl b and carotenoids could be detected in the flag leaves of wild-type plants. All OsNUC1-L overexpression transgenic lines maintained Chl a and carotenoid contents, Chl b less markedly, leading to significantly higher levels in flag leaves of all transgenic lines in comparison with the wild type (Fig. 12A, C, E). In the second leaves, salt stress resulted in reduction of loss of photosynthetic pigments. No significant difference in Chl a and Chl b content was detected, but carotenoid content was significant higher in all transgenic lines (Fig. 12B, D, F).

Fig. 12

Photosynthetic pigment content, Chl a (A and B), Chl b (C and D) and carotenoid (E and F), of flag leaves (A, C, E) and second leaves (B, D, F) of wild-type and transgenic rice with OsNUC1-L overexpression, when rice plants were treated with salt stress at 8–10 dS m^–1 for 9 d at the booting stage. The experiment was designed in a random complete block design with four replications. Three individuals of each homozygous T₃ transgenic line and the wild type were used for each replication. Means with a different letter above the bar are significantly different (P < 0.05, Duncan’s Multiple Range Test).

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The measurement of Chl fluorescence (F_v/F_m) has been used in many studies to determine the effect of salt stress on plants (Sayed 2003, Yan et al. 2013, Mancarella et al. 2016, Moles et al. 2016, Sayyad-Amin et al. 2016, Zhao et al. 2016, Zhu et al. (2016). F_v/F_m in the flag leaves of all lines was normal (Fig 13A). Under salt stress conditions, however, a reduction of F_v/F_m was detected in the second leaves of the wild type, while the transgenic lines with OsNUC1-L overexpression maintained PSII efficiency (Fig. 13B).

Fig. 13

PSII efficiency determined by Chl fluorescence (F_v/F_m) after 30 min of dark adaptation in flag leaves (A) and second leaves (B) of wild-type (WT) and transgenic rice lines, FL1, FL2 and FL3 grown in normal conditions or treated with salt stress for 9 d. The experiment was designed in a random complete block design with four replications. Three individuals of each homozygous T₃ transgenic line and the wild type were used for each replication. Means with a different letter above the bar are significantly different (P < 0.05, Duncan’s Multiple Range Test).

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To explore the correlation between the changes in the photosynthesis response and gene expression in the OsNUC1-L overexpression rice lines, we performed quantitative RT-PCR of the light reaction-associated genes that are homologous to the Arabidopsis genes displaying significant differences upon OsNUC1-L overexpression. Four rice genes, LOC_Os01g52240.1, encoding a CAB, LOCOS01g57942.1 encoding Cyt b₆, LOC_Os03g39610.1 encoding a CAB and LOC_Os08g35420.1 encoding a photosynthetic reaction center protein, which respectively are homologous to AT1G29920 (CAB2), ATCG00720 (PETB), AT2G05070 (LHCB2.2) and ATCG00020 (PSBA), were examined in the flag leaf and second leaf of transgenic rice in comparison with the wild type.

In normal conditions, the expression of all four genes in the flag leaf and second leaf of the transgenic line FL3 was higher than in the wild type. After 9 d of salt stress, higher expression for all tested genes was found only in the flag leaf (Fig. 14). In the second leaf, only LOC_Os01g52240.1 showed significantly higher expression in the wild type (Fig. 14A). As shown previously, the enhancement of the rate of photosynthesis was found in both flag leaves and second leaves (Fig. 11). In conclusion, higher expression in the overexpressing OsNUC1-L transgenic lines of the photosynthesis genes supports the involvement of the photosynthetic response in salt tolerance.

Fig. 14

Relative gene expression of four rice genes, LOC_Os01g52240.1(A), encoding a CAB, LOCOS01g57942.1 (B) encoding Cyt b₆, LOC_Os03g39610.1 (C) encoding a CAB, and LOC_Os08g35420.1 (D) encoding a photosynthetic reaction center protein in transgenic rice (FL3) at the flowering stage under normal conditions or 9 d after salt stress. The experiment was performed using the T₃ transgenic line (FL3), with three biological replicates of three technical replicates used for the quantiative PCR. The non-transgenic wild type (WT) was used as controls. Means with a different letter above the bar are significantly different (P < 0.05, Duncan’s Multiple Range Test).

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Discussion

A considerable amount of information is available on the biochemistry and function of NUC. By ultrastructural cell analysis, NUC is mostly found in the nucleolus, particularly around FCs and the DFC (Tong et al. 1997, Pontvianne et al. 2007, Stepinski 2012), where it is involved in rRNA transcription and pre-ribosomal formation (Medina et al. 2010). Yeast and Arabidopsis mutants lacking NUC function display overproduction of unprocessed rRNA (Lee et al. 1992. Petricka and Nelson 2007. Pontvianne et al. 2007). At the same time, NUC also affects the 40S/60S ribosomal subunit ratio. Yeast defective in NUC gene function displayed a dramatic decrease in the 40S/60S ribosomal subunit ratio (Kondo and Inouye 1992). Thus, NUC is associated with rRNA transcription and maturation, as well as ribosome biogenesis. Consistent with this association, the level of NUC expression is directly related to cell activity and protein translation (Stepinski 2012). In alfalfa, NUC expression was absent in stationary phase cells, but high in dividing cells (Bogre et al. 1996).

The two NUC splice isoforms appear to have at least some separate functions. Transgenic expression of the large isoform of rice nucleolin in A. thaliana, OsNUC1-L, did not prevent dry weight loss under salt stress, in contrast to the effect found when overexpressing OsNUC1-S (Sripinyowanich et al. 2013). The subcellular localization of green fluorescent protein (GFP)–OsNUC1 fusion protein showed that both OsNUC1-L and OsNUC1-S could be imported into the nucleus. The difference in the phenotype of the OsNUC1-L and OsNUC1-S expression lines may result from differential binding of RNA targets (Ginisty et al. 1999). However, we cannot rule out the possibility that OsNUC1-L and OsNUC1-S can regulate transcription itself. Surprisingly, OsNUC1-L was more stable in transgenic Arabidopsis, while OsNUC1-S inheritance appeared compromised, limiting a complete examination of the physiological response to Arabidopsis OsNUC1-S transgenics.

The expression of the OsNUC1-L gene in A. thaliana resulted in both up- and down-regulation of multiple mRNAs. The mechanism through which NUC regulates the mRNA level of target genes is still unclear. NUC is a multifunctional protein involved in multiple regulatory pathways. The well-documented RNA-binding activity of NUC might affect RNA turnover and thus abundance. Binding of NUC to DNA has also been documented (Olson et al. 1983, Bouche et al. 1984, Sapp et al. 1986) as well as its ability to interact with histones and induce chromatin decondensation (Erard et al. 1988, Kharrat et al. 1991, Gaume et al. 2011, Goldstein et al. 2013). A chromatin-modifying role for NUC has been well documented in rDNA transcription (reviewed by Durut and Sáez-Vásquez 2015). Therefore, OsNUC1 may regulate the transcription of target genes by modifying local chromatin properties. Moreover, transgenic expression of OsNUC1-L during salt stress up-regulated a large group of genes involved in ribosome biogenesis (Fig. 5), suggesting that OsNUC1-L stabilized growth under saline conditions by stimulating the translation machinery, a role consistent with known NUC properties (Ginisty et al. 1999).

An interesting effect of OsNUC1-L expression in transgenic Arabidopsis was hypersensitivity to the hormone ABA. Under an ABA concentration that delays germination of wild-type seed, the OsNUC1-L transgenic lines were completely inhibited. This phenomenon is similar to that displayed by mutants of genes encoding proteins with RNA binding activities (Kuhn and Schroeder 2003). Increased responsivenes to ABA, such as delayed seed germination, root growth inhibition and rapid stomatal closure upon ABA application, link this class of proteins to ABA signal transduction. One such gene, ABA-HYPERSENSITIVE 1 (ABH1), encodes the large subunit of the nuclear cap-binding protein complex that was showed to participate in several steps of RNA processing, nuclear export and mRNA decay (Makarov et al. 2002). The abh1 knock-out mutant, identified in a screen for altered ABA responses, displays delayed seed germination, rapid stomatal closure and a reduced wilting phenotype during drought stress (Hugouvieux et al. 2001) as well as altered post-transcriptional RNA processing (Kuhn and Schroeder 2003). Another example of an RNA-binding protein affecting the ABA response and plant stress is provided by the glycine-rich RNA-binding protein, ATGRP7-1, whose mutation in A. thaliana results in increased expressoin of ABA-induced genes and in other phenotypes related to those displayed by NUC mutants. Overexpression of ATGRP7-1 increases cold tolerance (Cao et al. 2006, Kim et al. 2008, Kim et al. 2010). Complementation of the atgrp7 mutant with OsGRP1 and OSGRP4 restored mRNA export from the nucleus to the cytosol, supporting broad conservation of NUC and a relationship between RNA metabolism, hormone response and stress adaptation.

Chloroplasts and ribosomes are most directly affected by salt stress. Salt stress and high light intensity induce photoinhibition (Takahashi and Murata 2008), probably resulting from the balance between the rate of photodamage to and rate of repair of PSII (Allakhverdiev et al. 2005). Hypersaline conditions suppress multiple pathways: CO₂ fixation by inhibiting the activity of ribulose-1,5-bisphosphate carboxylase oxygenase (Solomon et al. 1994), protein translation by reducing ribosome stability (Brady et al. 1984) and the repair mechanism of PSII by inhibiting D1 protein synthesis (Allakhverdiev et al.2005). The NUC gene is strongly induced after exposure to light (Tong et al. 1997, Reichler et al. 2001) consistent with a role for NUC in the high light response. Gene enrichment analysis of salt-treated Arabidopsis OsNUC1-L transgenics revealed that genes with increased expression encode proteins important in chloroplast and ribosome function (Fig. 5B). OsNUC1-L up-regulation of these genes may contribute to the stability of these organelles.

During salt stress conditions, the net photosynthesis rate in OsNUC1-L transgenic lines was significantly increased compared with wild-type Arabidopsis (Fig. 9A). Moreover, transcriptomic analysis revealed that during salt stress the OsNUC1-L transgenic lines increased expression of the genes encoding PSII proteins, such as the PSII light-harvesting complex genes (LHCB2.1, LHCB2.2 and LHCB2.3) genes, significantly enhancing light-harvesting system protein and photosynthetic membrane components (Table 3). Genes encoding CAB2 and CAB3 (AT1G29910 and AT1G29920) and proteins of the PSII light-harvesting complex were also up-regulated, as were genes encoding photosynthetic membrane components (Table 3). The CABs (also LHCB for light-harvesting CAB) are the main component in photosystems and the most abundant proteins in plant green tissues. The T-DNA knockouts of some LHCB genes increased stomatal opening and sensitivity to drought stress (Xu et al. 2012). Plausibly, increased expression of the LHCB genes could increase tolerance to stress. Consistent with this, salt tolerance in Indian mustard was associated with higher expression of proteins involved in stabilization of photosystems (Yousuf et al. 2015, Yousuf et al. 2016), and higher salt tolerance of ‘Pokkali’ rice was associated with a higher level of proteins involved in the light reaction of photosynthesis (Saleethong et al. 2016). Higher expression of OsNUC1 is associated with the higher salt tolerance of this rice (Sripinyowanich et al. 2013). To validate the role of OsNUC1-L, we produced and characterized transgenic rice lines overxpressing OsNUC1-L. Similar to transgenic Arabidopsis expressing OsNUC1-L, we detected a higher photosynthetic rate under salt stress in this transgenic rice at the flowering stage, although not in seedlings. Based on detailed gene expression analysis of the significantly regulated photosynthesis genes in the RNA-seq data, all tested genes were expressed at a higher level in the flag leaves of the transgenic lines in salt stress conditions. This supports the role of OsNUC1-L in enhancement of photosynthesis via the protection of light reaction components under salt stress. Decreased expression of the tested genes in the second leaf suggests that the salt stress effects may be strong in that tissue. Rice flag leaf is the major photosynthetic organ responsible for grain filling. More than half of the carbohydrates in rice seeds are from flag leaf photosynthesis (Li et al. 1998). Therefore, the differential protection from salt stress may occur between the flag and second leaf. These results indicate that the protection of photosynthesis under salt stress found in OsNUC1-L-expressing Arabidopsis is not a novel and spurious NUC function acquired in the Arabidopsis heterologous system, but rather it derives from a conserved role for OsNUC1-L in photosynthesis stabilization under salt stress conditions.

In conclusion, we explored NUC function in the context of environmental stress by expressing OsNUC1-L in the background of wild-type A. thaliana. Plants expressing the rice NUC displayed altered regulation of the genes encoding key enzymes in ABA biosynthesis and degradation such as NCED3, AAO3, ABA3 and CYP707A4. Consistent with a role for NUC in ABA signaling, they also displayed changes in the expression level of some ABA-responsive genes. These changes had phenotypic consequences as the rice NUC expressors displayed hypersensitivity to ABA during germination. A question remaining to be addressed is whether this response is due to the changes in ABA accumulation or ABA sensitivity. Our transcriptome analysis of rice NUC transgenic plants indicated that OsNUC1-L plays a role in RNA metabolism and specifically regulates the expression level of genes involved in stress responses, photosynthesis, hormone response and transport. The use of cellular compartment criteria connected OsNUC1-L expression to up-regulation of genes in photosynthesis, especially in the light-harvesting complex. Our results showed that during stress conditions OsNUC1-L increased the transcript level of LCHBs proteins, leading to maintenance of photosynthetic capacity, increasing WUE and growth. The higher capacity for photosynthesis maintenance under salt stress conditions was found in both Arabidopsis and rice with OsNUC1-L overexpression. We conclude that OsNUC1 is involved in photosystem stabilization during salt stress conditions.

Materials and Methods

Generation of transgenic OsNUC1-L- and OsNUC1-S-expressing Arabidopsis

Full-length OsNUC1-L and OsNUC1-S cDNA was PCR amplified, cloned into the pCR^®- Blunt II-TOPO^® vector (Invitrogen) and sequenced. Then it was subcloned into the plant gene transformation vector, pJim19 plasmid, to generate the expression construct under the Cauliflower mosaic virus 35S promoter (35SCaMV promoter). The recombinant plasmid was used to transfer the genes into wild-type Arabidopsis (Arabidopsis thaliana ecotype Columbia-0) by floral dipping (Clough and Bent 2008). The kanamycin resistance gene was used to select the successfully transformed plants. The T₂ homozygous transgenics were used for further experiments.

Generation of OsNUC1-L-overexpressing transgenic rice

The OsNUC1-L cDNA was cloned into the pWM101 vector to generate the overexpression construct of OsNUC1-L under the ubiquitin promoter. Transformation was performed according to Sripinyowanich et al. (2013). Briefly, rice calli were regenerated from scutellum on MS basal medium containing 2.5 mg l^–1 2,4-D and 3% (w/v) sucrose. Then, Agrobacterium transformation was done to transfer the required construct to rice calli. The transformed calli were regenerated to seedlings and selected on MS basal medium containing 50 mg l^–1 hygromycin B. Three T₃ homozygous transgenic rice lines, FL1, FL2 and FL3, were used for characterization.

Germination test

After surface sterilization, seeds were placed on half-strength MS medium (Murashige and Skoog 1962), stratified at 4°C for 2 d and then transferred to growth chambers at 25°C, with a light intensity of 100 µmol m^–1 s^–1 and 16 h days/8 h nights. To determine ABA sensitivity during germination, seeds of OsNUC1-L transgenic and wild-type Arabidopsis were germinated on half-strength MS medium with or without 0.2, 0.4 and 0.6 µM ABA (Sigma).

OsNUC1-L expression quantification

The gene expression of OsNUC1 in 14-day-old rice seedlings grown under normal or salt stress conditions for 6 d was detected with the method according to Sripinyowanich et al (2013). The OsEf-1α gene (GenBank accession No. GQ848074.1) was used as internal control. The relative expression was calculated with the above formula.

Transcriptome study using RNA-seq

Three biological replicates of the wild-type and transgenic Arabidopsis were used for transcriptome analysis. Two-week-old plants grown on MS medium were transferred to MS medium containing 100 mM NaCl for salt stress conditions and to freshly perpared MS medium for control conditions. After 5 h of treatment, total RNA was isolated with Plant RNA Reagent (Invitrogen). The mRNA was uniquely isolated with Dynabead mRNA purification (Invitrogen) and converted to cDNA with SuperScript III (Invitrogen). The cDNA was fragmented by dsDNA fragmentase (NEB). Fragment sizes of 300 bp were selected and purified using the AMPure protocol. The DNA fragments were enriched by PCR for 10 cycles using adaptors with unique DNA barcodes for each cDNA library. All six libraries were generated using the Genome Analyzer (Illumina), loading them together in the same sequencing lane of the flow cell.

The transcriptome analysis compared transcripts from the wild-type and transgenic plants under salt stress and control conditions. Briefly, all short-sequence reads produced from Genome Analyzer were processed to remove the DNA barcode and grouped according to the DNA barcode in the right category using the pipeline created by Missirian et al. (2012). The sequence reads were uniquely aligned and mapped to the Arabidopsis genome (TAIR10) using Bowtie aligner (Langmead et al. 2009) and TopHat (Trapnell et al. 2009). The DESeq program (Anders and Huber 2010) was used to identify differentially expressed genes. Significantly regulated genes were considered differentially expressed.

Determination of gas exchange parameters and Chl fluorescence

To examine the effect of salt stress on photosynthetic activity in OsNUC1-L-overexpressing transgenic lines, the seeds of the wild type and three 35SCaMV:OsNUC1-L transgenic lines were grown on soil mix for 4 weeks at 25°C, 180 µmol photon m^–1 s^–1 and 16 h days/8 h nights. The net photosynthetic rate, stomatal conductance and transpiration rate were determined using a Gas Analysis System (Li-Cor, LI-6400) with a 6400-15 extended reach 1 cm diameter chamber. The gas exchange measurement was performed at 25°C under an external light source of 650 µmol photon m^–1 s^–1, the reference carbon dioxide concentration of 350 µmol m^–1 s^–1 and relative humidity of 40–44%. For the salt treatment, wild-type and transgenic Arabidopsis were exposed to 25 ml of 100 mM NaCl per pot by irrigation. The WUE was calculated by the formula is given as: WUE = P_n/E, where P_n = net rate of photosynthesis (µmol CO₂ m^–1 s^–1) and E = transpiration rate (mmol m^–2 s^–1)

The rate of photosynthesis and stomatal conductance were determined in rice at the booting stage. At this stage, rice leaves have been enlarged which is better for the measurement of photosyntheic behavior than seedling leaves. Rice plants were grown on soil in 30 cm diameter pots until flowering in the greenhouse under natural light conditions. For salt stress treatment, when rice plants reached the booting stage, 150 mM NaCl solution was poured to cover the soil and the soil electric conductivity was determined to reach 8–10 dS m^–1 during salt stress treatment. The photosynthetic rate and stomatal conductance were determined a Gas Analysis System (Li-Cor, LI-6400) after 9 d of treatment. The gas exchange measurement was performed at 30–33°C under an external light source of 1,200 µmol photon m^–1 s^–1, the reference carbon dioxide concentration of 380 µmol m^–1 s^–1 and relative humidity of 56–67%.

F_v/F_m was determined in flag leaves and second leaves of normal and salt-stressed plants using Pocket PEA (Hansatech Instruments, Ltd.) after 30 min of dark adaptation.

Determination of the content of photosynthetic pigments

Photosynthetic pigment concentration was determined by the methods of Shabala et al. (1998) and Lichtenthaler (1987). The pigments were extracted with 80% acetone, and Chl a, Chl b and carotenoids were measured using a UV–visible spectrophotometer (Agilent Technology) at absorbance wavelengths of 662, 644 and 470 nm. The pigment contents were calculated with the following equations: [Chl a] = 9:784D₆₆₂ – 0:99D₆₄₄; [Chl b] = 21:42D₆₆₄ – 4:65D₆₆₂; and [carotenoid] = (1,000D₄₇₀ – 1:90[Chl a] – 63:14[Chl b])/214.

Supplementary data

Supplementary data are available at PCP online.

Funding

This research was supported by the Ratchadaphiseksomphot Endowment Fund (2015–16) and Chulalongkorn University [RES560530061-FW, CU-58-012-FW, and, CUAASC; Ratchadaphiseksomphot Fund for Postdoctoral Fellowship; Chulalongkorn University [to T.U. and S.S]; and the Science Achievement Scholarship of Thailand [to C.B.].

Abbreviations

Abbreviations
 
• AAO3

  aldehyde oxidase 3

 
• ABH1

  ABA hypersensitive 1

 
• AtEF-1α

  Eukaryotic elongation factor1-α subunit

 
• CAB

  Chl a/b-binding protein

 
• 35SCaMV promoter

  Cauliflower mosaic virus 35S promoter

 
• CBP20

  nuclear cap-binding protein subunit 2

 
• C_i

  intracellular CO₂ concentration

 
• CYP707A4

  abscisic acid 8′-hydroxylase 4

 
• DFC

  dense fibrillar component

 
• E

  transpiration rate

 
• FC

  fibrillar center

 
• GAR

  glycine–arginine rich

 
• GO

  Gene Ontology

 
• g_s

  stomatal conductance

 
• HYL1

  Hyponastic leaves1 mutant

 
• LHCB

  PSII light-harvesting complex

 
• MS

  Murashige and Skoog

 
• NCED

  9-cis-epoxycarotenoid dioxygenase

 
• NLS

  nuclear localization signal

 
• NUC

  nucleolin

 
• OsNUC1

  Rice nucleolin 1

 
• P_n

  net photosynthetic rate

 
• PSBA

  PSII reaction center protein

 
• RRM

  RNA-recognition motif

 
• RT-PCR

  real-time PCR

 
• WUE

  water use efficiency

Acknowledgments

We would like to thanks Dr. Isabelle M. Henry and Ms. Kathie Ngo, Genome Center, University of California Davis for their valuable help in bioinformatic analysis.

Disclosures

The authors have no conflicts of interest to declare.

References