Down Regulation and Loss of Auxin Response Factor 4 Function Using CRISPR/Cas9 Alters Plant Growth, Stomatal Function and Improves Tomato Tolerance to Salinity and Osmotic Stress

Abstract

Auxin controls multiple aspects of plant growth and development. However, its role in stress responses remains poorly understood. Auxin acts on the transcriptional regulation of target genes, mainly through Auxin Response Factors (ARF). This study focuses on the involvement of SlARF4 in tomato tolerance to salinity and osmotic stress. Using a reverse genetic approach, we found that the antisense down-regulation of SlARF4 promotes root development and density, increases soluble sugars content and maintains chlorophyll content at high levels under stress conditions. Furthermore, ARF4-as displayed higher tolerance to salt and osmotic stress through reduced stomatal conductance coupled with increased leaf relative water content and Abscisic acid (ABA) content under normal and stressful conditions. This increase in ABA content was correlated with the activation of ABA biosynthesis genes and the repression of ABA catabolism genes. Cu/ZnSOD and mdhar genes were up-regulated in ARF4-as plants which can result in a better tolerance to salt and osmotic stress. A CRISPR/Cas9 induced SlARF4 mutant showed similar growth and stomatal responses as ARF4-as plants, which suggest that arf4-cr can tolerate salt and osmotic stresses. Our data support the involvement of ARF4 as a key factor in tomato tolerance to salt and osmotic stresses and confirm the use of CRISPR technology as an efficient tool for functional reverse genetics studies.

Keywords: ARF4; Auxin; CRISPR-Cas9; osmotic stress; salt; tolerance; tomato.

Figure 1

Phenotype of cv. Micro-Tom tomato plants (wild-type, WT) and isogenic ARF4 antisense transgenic line (ARF4-as), 35 days after germination. (a) WT and (b) ARF4-as plants at the same stage of development, (c) number of leaves to first inflorescence, (d) plant height to first inflorescence, (e) plant internode length and (f) stem diameter. Bars are mean values (n = 7) ± s.e.m. Asterisks indicate values that were determined by Student’s t test to be significantly different (p < 0.05) from WT.

Figure 2

Dry weight and parameters related with leaf area for Micro-Tom (WT) and isogenic ARF4 antisense transgenic line (ARF4-as), 45 days after germination. (a) leaf dry weight, (b) stem dry weight, (c) root dry weight, (d) total dry weight, (e) total leaf area and (f) specific leaf area (SLA). Values are means ± s.e.m (n = 8). Asterisks indicate values that were determined by Student’s t test to be significantly different (p < 0.05) from the Micro-tom (WT).

Figure 3

Leaf anatomy and morphology is altered in an ARF4-as transgenic line. Representative leaf cross-sections of (a) Micro-Tom (WT) and (b) ARF4-antisense line (ARF4-as). Scale bars= 20 µm. (c) Total thickness of the leaf blade, (d) thickness of palisade, (e) thickness of spongy and (f) ratio of palisade to spongy mesophyll, (g) proportion of intercellular air spaces, (h) adaxial epidermis and (i) abaxial epidermis and (j) mesophyll thickness in WT and ARF4-as plants and (k) Time series of a representative leaf illustrating blade curling in ARF4-as plants. Day 0 represents the day when the leaf was fully expanded. Values are means ± s.e.m (n = 6). Asterisks indicate values that were determined by student’s t test to be significantly different (p < 0,05) from WT while n.s indicates non significant difference between ARF4-as and WT.

Figure 4

Net CO₂ assimilation rate (A) and stomatal conductance (g_s) in Micro-Tom (WT) and isogenic ARF4 antisense transgenic line (ARF4-as). (a) CO₂ assimilation rate (A) as a function of stomatal conductance (gs), each point represents one measurement on an individual plant. Line fitted by linear regression. (b) net CO2 assimilation rate (A). (c) stomatal conductance (g_s). (d) intrinsic water efficiency (A/g_s). Values are means ± s.e.m (n = 8). Asterisks indicate values that were determined by Student’s t test to be significantly different (p < 0.05) between genotypes.

Figure 5

Photosynthetic assimilation rate and stomatal conductance in Micro-Tom (WT) and ARF4-antisense silencing line. (a) Net photosynthesis (A) curves in response to sub-stomatal (C_i) CO₂ concentration in WT and ARF4-as plants. Values are presented as means ± s.e.m. (n = 5) obtained using the fully expanded fifth leaf. Two-branch curves: the biochemically based leaf photosynthesis model [24] was fitted to the data based on C_i, values of A/C_i for five plants of WT (gray) and ARF4-as (black). (b) Response of stomatal conductance (g_s) to changes in leaf-to-air vapor pressure deficit (VPD). Vapor pressure difference was varied by changing the humidity of air, keeping leaf temperature constant. Each point represents one measurement on an individual plant.

Figure 6

SlARF4 expression in response to salt and drought stresses. Gene expression in leaves (a) and roots (b) of WT plants exposed to salt stress, in leaves (c) and roots (d) of WT plants exposed to osmotic stress. ΔΔCt refers to differences in gene expression relative to untreated plants. Values are mean ± SD of three biological replicates. GUS activity in pARF4::GUS tomato lines in salt (e) or osmotic (f) stress conditions. Bars scale (1mm). Stars (*) indicate the statistical significance (p < 0.05) according to Student’s t-test.

Figure 7

Growth parameters of tomato wildtype (WT) and ARF4-as in response to salt and osmotic stresses. (a,b) shoot fresh weight in salt and osmotic stress conditions respectively, (c,d) root fresh weight in salt and osmotic stress conditions respectively, (e,f) primary root length root in salt and osmotic stress conditions respectively, (g,h) root density in salt and osmotic stress conditions respectively. Salt and osmotic stresses were performed on three weeks tomato plants for two weeks by adding 100 mM of NaCl or 150 mM of NaCl for salt stress or 5% or 15% of Polyethylene Glycol (PEG) 20,000 for osmotic stress. Values are mean ± SD of three biological replicates. Bars with different letters indicate the statistical significance (p < 0.05) according to Student Newman-Keuls test.

Figure 8

Stomatal conductance and relative water content in WT and ARF4-as plants. (a,b) stomatal conductance in salt and osmotic stress conditions, (c) relative water content in response to dehydration. Bars with different letters indicate the statistical significance (p < 0.05) according to Student Newman-Keuls test.

Figure 9

Expression of SlNCED1, SlNCED2, SlCYP707A1, SlCYP707A2 and SlCYP707A3 genes in WT and ARF4-as leaves and roots after 2 h and 24 h of salt stress and 48 h of osmotic stress application. ΔΔCt refers to fold differences in gene expression relative to untreated plants. Values presented are mean ± SD of three biological replicates. Asterisks (*) indicate the statistical significance (p < 0.05) according to Student’s t-test.

Figure 10

CRISPR-Cas9 mediated gene editing in tomato Micro-Tom. (a) PCR genotyping of plants at the T1 generation of line-108. Deletion mutations of SlARF4 were found in plants #4, #5, #6 and #7. Among these, the T-DNA insertion (CRISPR-Cas9 transgene) was segregated out in plant #5 while still bearing the desired mutation in the SlARF4 gene. (b) Sequencing data of ARF4-PCR products from #5 and #6 plants. The PCR products from plant #5 yielded a single fragment type containing the expected 49bp DNA deletion, whereas in the case of plant #6, only 4 out of 10 PCR clones contain the desired mutation, the remaining 6 PCR clones exhibited a small deletion in both target regions. Red dashed indicated the expected cleavage sites for CRISPR-Cas9. (c) Leaf phenotype of CRISPR-Cas9 generated ARF4 mutants. (d) All four plants showed dramatic leaf curling, similar to the phenotype observed in ARF4-as plants.

Figure 11

Dry weight and parameters related with leaf area for Micro-Tom (WT) and arf4-cr, 45 days after germination. (a) leaf dry weight, (b) stem dry weight, (c) root dry weight, (d) leaf area and (e) Specific leaf area (SLA). Values are means ± s.e.m (n = 8). Asterisks indicate values that were determined by Student’s t test to be significantly different (p < 0.05) from the Micro-tom (WT).

Figure 12

Relationship between net CO₂ assimilation rate (A) and stomatal conductance (g_s) for Micro-Tom (WT) and arf4-cr. (a) CO₂ assimilation rate (A) as a function of stomatal conductance (g_s), (b) net CO₂ assimilation rate (A), (c) stomatal conductance (g_s), (d) intrinsic water efficiency (A/g_s). Values are means ± s.e.m (n = 8). Asterisks indicate values that were determined by Student’s t test to be significantly different (p < 0.05) from the wild type (WT).