Comparative transcriptome analysis reveals carbohydrate and lipid metabolism blocks in Brassica napus L. male sterility induced by the chemical hybridization agent monosulfuron ester sodium

Abstract

Background: Chemical hybridization agents (CHAs) are often used to induce male sterility for the production of hybrid seeds. We previously discovered that monosulfuron ester sodium (MES), an acetolactate synthase (ALS) inhibitor of the herbicide sulfonylurea family, can induce rapeseed (Brassica napus L.) male sterility at approximately 1% concentration required for its herbicidal activity. To find some clues to the mechanism of MES inducing male sterility, the ultrastructural cytology observations, comparative transcriptome analysis, and physiological analysis on carbohydrate content were carried out in leaves and anthers at different developmental stages between the MES-treated and mock-treated rapeseed plants.

Results: Cytological analysis revealed that the plastid ultrastructure was abnormal in pollen mother cells and tapetal cells in male sterility anthers induced by MES treatment, with less material accumulation in it. However, starch granules were observed in chloroplastids of the epidermis cells in male sterility anthers. Comparative transcriptome analysis identified 1501 differentially expressed transcripts (DETs) in leaves and anthers at different developmental stages, most of these DETs being localized in plastid and mitochondrion. Transcripts involved in metabolism, especially in carbohydrate and lipid metabolism, and cellular transport were differentially expressed. Pathway visualization showed that the tightly regulated gene network for metabolism was reprogrammed to respond to MES treatment. The results of cytological observation and transcriptome analysis in the MES-treated rapeseed plants were mirrored by carbohydrate content analysis. MES treatment led to decrease in soluble sugars content in leaves and early stage buds, but increase in soluble sugars content and decrease in starch content in middle stage buds.

Conclusions: Our integrative results suggested that carbohydrate and lipid metabolism were influenced by CHA-MES treatment during rapeseed anther development, which might responsible for low concentration MES specifically inducing male sterility. A simple action model of CHA-MES inducing male sterility in B. napus was proposed. These results will help us to understand the mechanism of MES inducing male sterility at low concentration, and might provide some potential targets for developing new male sterility inducing CHAs and for genetic manipulation in rapeseed breeding.

Figure 1

Transmission Electron Microscope (TEM) micrographs of the anthers from the mock-treated (fertile) and MES-treated (sterile) plants. (A) The fertile anthers at pollen mother cell (PMC) stage; (B) Enlarged fertile meiocytes in (A); and (C) Enlarged fertile tapetum in (A) showing numerous plastids dispersed in cytoplasm (white arrow). (D) The sterile anthers at PMC stage; (E) Enlarged sterile meiocytes in (D) showing less plastids in condensed cytoplasm separated from the cell wall; (F) Enlarged sterile tapetum in (D) showing little abnormal plastids (white arrow) and more large vacuoles in cytoplasm, and with a little plasmolysis at meiocyte side (black arrow). (G) The fertile anthers at vacuolated-microspore stage; (H) The degraded tapetum in (G) showing elaioplasts and tapetsomes with abundant lipids; (I) Plastids in tapetum located in a crown showing filled with globular low electron-dense metabolites and surrounded by rich endoplasmic reticulum (ER). (J) The sterile anthers at vacuolated-microspore stage (type I); (K) The undegraded tapetum in (J) showing elaioplasts and tapetsomes with abundant lipids; (L) Plastids in tapetum located in a crown showing irregular shaped low electron-dense material. (M) The sterile anthers at vacuolated-microspore stage (type II); (N) The degraded tapetum in (M) showing scattered elaioplasts and tapetsomes with fuzzy structure; (O) The fertile anthers at mature pollen grain stage; (P) The pollen grain in (O) showing profuse globular particles; (Q) The enlarged globular particles in (P). (R) The sterile anthers at mature pollen grain stage (type II); (S) The undegraded tapetum in (R) died but cell wall still existed (black arrow); (T) The sterile anthers at mature pollen grain stage (type I). (U) The epidermis and endothecium cells in fertile plants at vacuolated-microspore stage; (V) The epidermis cells in (U) showing normal oval-shaped chloroplastids with distinct thylakoid structure and little starch granules in thylakoid; (W) The endothecium cells in (U) showing oval-shaped chloroplastids with distinct thylakoid structure. (X) The epidermis and endothecium cells in sterile plants at vacuolated-microspore stage; (Y) The epidermis cells in (X) showing abnormal chloroplastids with large starch granules in thylakoid; (Z) The endothecium cells in (X) showing fusiform-shaped chloroplastids with linear thylakoid structure. PMC, pollen mother cell; N, nucleus; T, tapetum; Msp, microspore; Ep, elaioplast; Ts, tapetosome; PG, pollen grain; TCW, tapetum cell wall; E, epidermis; En, endothecium; Ch, chloroplast. Scale bars = 10 μm (A, D, G, J, M, N, O and T), 5 μm (C, F, P, R, S, U and X), 2 μm (B, E and K), and 1 μm (H, I, L, Q, V, W, Y and Z).

Figure 2

Photographs of the leaves and developmental anthers for microarrays from the mock-treated (A, C, E, G) and MES-treated (B, D, F, H) plants. A-B, young leaves from the main inflorescences (Ls); C-D, small buds (SBs); E-F, anthers from middle buds (An-MBs); G-H, anthers form large buds (An-LBs). Scale bar in leaves was 1 cm, scale bars in SBs, An-MBs, and An-LBs were 1 mm.

Figure 3

Strategies for identification of differentially expressed transcripts (DETs) involved in microgametogenesis between the MES-treated and mock-treated plants by two sets of student’s t-test comparisons. (A) Comparisons within groups. The pair-wise comparisons of Student’s t-test between tissues (organs) were carried out within mock-treatment groups and MES-treatment groups, respectively, to detected DETs related to anther development under mock-treatment (control, fertile) and MES-treatment (male sterile) conditions. The criteria for screening DETs were p-value <0.001 and fold change ≥ 2. mock, mock-treatment; MES, MES-treatment; (B) Comparisons between the MES-treated and mock-treated groups. The pair-wise comparisons of Student’s t-test were performed between the corresponding tissues (organs) of the mock-treated group and the MES-treated group to identify DETs related to MES-treatment. The screen criteria were same as above. (C) Venn diagram showing the DETs involved in microgametogenesis between the MES-treated and mock-treated groups. Comparisons within groups produced two sets of DETs, development-related genes in the MES-treated plants and in the mock-treated plants (the left and right cycles). Comparisons between groups also produced two sets of DETs, up-regulated genes and down-regulated genes in MES-treated group (the up and down cycles). These four sets of DETs were all collected, respectively. The common sections (totally 1501 unique DETs, the red and green parts, (2) indicates 2 DETs existing in the both data sets) were considered to be anther development-related genes affected by MES-treatment.

Figure 4

Subcellular localization and functional categories of the 1087 differentially expressed unigenes between the MES-treated and the mock-treated rapeseed plants. (A) Subcellular localization, (B) Functional categories, (C) Enrichment analysis of the functional categories list in B. The scale bar indicates -log (P-value), with highly enriched categories in red color, and invalid values in gray, whereas the P-value was calculated according to a hypothesis test using cumulative hypergeometric distribution. Left panel, enrichment analysis of all the unigenes in functional categories listed in B, except for those in unclassified category; the right panels, enrichment analysis of the sub-categories from metabolism and cellular transport (blue rectangles), respectively. The prominent enriched sub-categories were circled in black ellipses.

Figure 5

Transcripts involved in stress (A) and metabolism (B) assigned by MapMan in rapeseed leaves and developmental anthers treated by MES. Positive fold change values (red) indicate up-regulation, whereas negative fold change values (blue) denote down-regulation. Color saturates at 4.5-fold change. Each square represents a differentially expressed transcript.

Figure 6

Comparison of carbohydrate content between mock-treated and MES-treated plants. Ls, young leaves from the main inflorescences; SBs, small buds with length less than 1 mm; MBs, middle buds with length of 1–3 mm in. *, **, represents significant difference at 0.05 level and at 0.01 level, respectively.

Figure 7

A putative action model for MES-treatment inducing male sterility. Some important functions and genes affected by MES treatment in leaf tissue (left rectangle) and developing anther tissues (right rectangle) are listed (see text for details). Two vertical dashed lines in the right rectangle separate three anther tissues. ‘↑’ after function categories or genes means up-regulation; ‘↓’ after function categories or genes means down-regulation. Two aspects of putative reasons for carbohydrate and lipid metabolism alteration were showed by dashed arrows ‘?’ represents unclear MES transport pathway. MES, Monosulfuron Ester Sodium; ALS, acetolactate synthase; BCAAs: Branch-Chain Amino Acids; AGP, ADP glucose pyrophosphorylase; PMG, Phosphoglucomutase; DPE1, Disproportionating enzyme; PHS2, alpha-glucan phosphorylase 2; SWEET 11, Nodulin MtN3 family protein; BXL1, beta-xylosidase; pectin lyases, pectin lyase superfamily protein; VGDH2, VANGUARD 1 homolog 2; PPME1: Pectin lyase-like superfamily protein; FLA5: FASCICLIN-like arabinogalactan protein 5 precursor; VGDH1: Plant invertase/pectin methylesterase inhibitor; VGD1: Plant invertase/pectin methylesterase inhibitor; PGA4, Polygalacturonase 4; UGE3, UDP-D-glucose/UDP-D-galactose 4-epimerase 3; HA9, H(+)-ATPase 9.