Biomass Accumulation and Cell Wall Structure of Rice Plants Overexpressing a Dirigent-Jacalin of Sugarcane (ShDJ) Under Varying Conditions of Water Availability

Abstract

A sugarcane gene encoding a dirigent-jacalin, ShDJ, was induced under drought stress. To elucidate its biological function, we integrated a ShDJ-overexpression construction into the rice Nipponbare genome via Agrobacterium-mediated transformation. Two transgenic lines with a single copy gene in T₀ were selected and evaluated in both the T₁ and T₄ generations. Transgenic lines had drastically improved survival rate under water deficit conditions, at rates close to 100%, while WT did not survive. Besides, transgenic lines had improved biomass production and higher tillering under water deficit conditions compared with WT plants. Reduced pectin and hemicellulose contents were observed in transgenic lines compared with wild-type plants under both well-watered and water deficit conditions, whereas cellulose content was unchanged in line #17 and reduced in line #29 under conditions of low water availability. Changes in lignin content under water deficit were only observed in line #17. However, improvements in saccharification were found in both transgenic lines along with changes in the expression of OsNTS1/2 and OsMYB58/63 secondary cell wall biosynthesis genes. ShDJ-overexpression up-regulated the expression of the OsbZIP23, OsGRAS23, OsP5CS, and OsLea3 genes in rice stems under well-watered conditions. Taken together, our data suggest that ShDJ has the potential for improving drought tolerance, plant biomass accumulation, and saccharification efficiency.

Keywords: RT-qPCR; monocot plants; overexpression; transgenic plants; water deficit.

FIGURE 1

Quantitative polymerase chain reaction (PCR) analyses of the ShDJ gene in leaves of IACSP94-2094 and IACSP97-7065 sugarcane genotypes subjected to water deficit. (A) Screening for the ShDJ gene in global gene expression analyses using microarray and RNA-seq. (B) Field experiment, with evaluations 42, 89, and 117 days after last rainfall. (C) Greenhouse experiment, with evaluations after 15 and 21 days of water deficit and after 9 days of soil rehydration (recovery). The expression profile was evaluated by determined the difference in Ct between ShDJ and UBQ1, according to 2^−ΔCt (Livak and Schmittgen, 2001). In (B,C), data represent the mean values [n = 3 ± standard error (SE)].

FIGURE 2

Phylogenetic relationship and multiple sequence alignment of the ShDJ protein with other Dirigent-jacalin (DJs). Phylogenetic analyses of the ShDJ protein and DJs protein sequences from sorghum (Sb), rice (Os), maize (Zm), Arabidopsis thaliana (At), Setaria italica (Si), Panicum virgatum (Pavir), and Hordeum vulgare (Hv) were generated using the neighbor-joining (NJ) method in MEGA6. Bootstrap values greater than 50% (1,000 replicates) are shown for nodes in the tree. Black symbols indicate the sugarcane DJs.

FIGURE 3

Molecular characterization of ShDJ lines. (A) Schematic representation of the T-DNA region of the binary vector pUBIL::ShDJ. RB, right border; LB, left border; pUBIL, maize ubiquitin promoter; Tnos, nopaline synthase terminator; p35S, 35S promoter; hptII, hygromycin phosphotransferase gene (selectable marker); T35S, 35S terminator. (B) Analyses of ShDJ expression in rice plants of T₀ and T₄ generations. Abundance of the ShDJ transcripts in lines #17 and #29 was evaluated by real time quantitative polymerase chain reaction (RT-qPCR) analyses (n = 3 ± standard error) using gene-specific primers for ShDJ. Analyses used the 2^−ΔCt method (Livak and Schmittgen, 2001), in which ΔCt represents the relative quantification of a target gene and a reference gene (elF-1α).

FIGURE 4

Tillering (A,C) and biomass (B,D) of ShDJ transgenic lines #17 and #29 in T₁ (A,B) and T₄ (C,D) progenies. Mean values (n = 4) ± SE. Different letters indicate significant differences between plants and water conditions (t-test, P < 0.05).

FIGURE 5

Overexpression of ShDJ increased the survival of T₄ transgenic rice seedlings under drought stress. (A) Seedlings of WT and lines #17 and #29 2 days after re-watering. (B) Survival rate of WT and transgenic rice seedlings after drought. Survival rates were calculated as the ratio of surviving plants to the total number of plants after re-watering.

FIGURE 6

Effects of ShDJ overexpression on the cell wall composition and saccharification efficiency of transgenic rice lines #17 (A,C,E,G,I) and #29 (B,D,F,H,J) under varying levels of water availability. (A,B) Pectin; (C,D) hemicellulose; (E,F) cellulose; (G,H) total lignin; (I,J) saccharification. Evaluations considered the entire plant shoot of T₄ progeny. Each histogram is the mean value (n = 4) ± SE. Different letters indicate significant differences between plants and water conditions (t-test, P < 0.05).

FIGURE 7

ShDJ modified the expression of cell wall and drought-related genes in leaves and stems of transgenic rice lines (A) #17 and (B) #29. Expression profiles of OsMYB58/63, OsNST1/2, OsP5CS, OsLea3, OsbZIP23, and OsGRAS23 genes observed by RT-qPCR analyses in T₄ plants were maintained under irrigated conditions. Expression in transgenic lines is relative to that in WT plants using the 2^−ΔΔCt method (Livak and Schmittgen, 2001). Data are mean values (n = 3) ± SE, and asterisks on the top of each bar indicate significant differential expression between lines and WT as determined using REST© software (5% significance).