Integration of embryo-endosperm interaction into a holistic and dynamic picture of seed development using a rice mutant with notched-belly grains

The interaction between the embryo and endosperm affects seed development, an essential process in yield formation in crops such as rice. Signals that communicate between embryo and endosperm are largely unknown. Here we use the notched-belly (NB) mutant with impaired communication between embryo and endosperm to evaluate 1) the impact of embryo on developmental staging of the endosperm; 2) signaling pathways emanating from the embryo that regulate endosperm development. Hierachical clustering of mRNA datasets from embryo and endosperm samples collected through development in NB and wild type showed a delaying effect of the embryo on the developmental transition of the endosperm by extending the middle stage. K-means clustering further identified coexpression modules of gene sets specific for embryo and endosperm development. Combined gene expression and biochemical analysis showed that T6P-SnRK1, gibberellin and auxin signalling from the embryo regulate endosperm developmental transition. The data enable us to propose a new seed developmental staging system for rice and the most detailed signature of rice grain formation to date, that will direct genetic strategies for rice yield improvement.


INTRODUCTION
2 1 8 analysis, and uncovered four distinguishing groups within embryo and endosperm, with each 2 1 9 group corresponding to a specific developmental stage ( Fig. 2E-H).

1
The first cluster is formed at 5 DAF, representing the stage around differentiation (Em-S1).

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During this stage, coleoptile and SAM emerge and first leaf primordium becomes visible on as characterized by the formation of second and third leaf primordia, and organ enlargement 2 2 6 (particularly the scutellum). The third cluster at 20DAF corresponds to the maturation phase 2 2 7 (Em-S3) that shows no obviously morphological change. The fourth cluster from 25 DAF to 2 2 8 60 DAF represents the period of seed dormancy (Em-S4).

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By contrast, the duration of developmental stages in endosperm varied with genotypes.   Using transcript datasets of WT, tissue-specific genes were clustered into coexpression 2 5 3 modules by the k-means clustering algorithm (Howe et al., 2010). Gene ontology (GO) 2 5 4 annotation was performed to assign genes to functional categories for each module 2 5 5 (Ashburner et al., 2000). Twenty-two coexpression modules were generated for both embryo 2 5 6 and endosperm (Fig. 3A, B). Among these modules, 11 and 9 modules were expressed 2 5 7 broadly at more than one stage in embryo and endosperm, respectively, indicating some 2 5 8 common cellular processes across several stages. Moreover, genes from the 11 modules of 2 5 9 embryo and the 13 modules of endosperm were more prevalent at one out of the four 2 6 0 developmental stages, indicating specific function of these modules at the corresponding 2 6 1 stage.

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Cellular processes in the developing embryo 2 6 3 Cellular processes characterizing each developmental stage were identified by GO terms that 2 6 4 were overrepresented at particular coexpression modules ( Fig. 3A; Supplementary Fig. S5A).

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The early stage of Em-S1, best represented by modules DP1-DP5, was typified by the  were related to protein complex, Golgi apparatus, protein kinase activity, cellular response to 2 7 8 stimulus, RNA modification, ribosome biogenesis and endonuclease activity.

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Cellular processes in the developing endosperm The functional characterization of endosperm was found different from that of embryo as processing and signal transducer activity were overrepresented in DP14-DP22 and were 2 9 2 broadly expressed throughout development. was higher in the embryo and endosperm at 5-10DAF, and then decreased gradually    T6P level in embryo and endosperm showed a gradual decrease in the development course.

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The SnRK1 activity in endosperm maintained a low level at 5-20 DAF, and then increased mainly expressed in endosperm. In addition, SnRK1A was broadly expressed across the time 3 2 7 points in embryo. By contrast, SnRK1B and OSK35 were more prevalent in endosperm at 5-20 3 2 8 DAF.

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Amino acids and proteins showed an opposite correlation with storage proteins content, suggesting that their encoding  GAs showed a gradual decrease over seed developmental course. Compared with endosperm, respectively.

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CKs in endosperm was lower than those in embryo at 5-30 DAF and peaked at 5 DAF. matter, which had a higher rate of accumulation than the minerals (Wang et al., 2018).

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Compared with endosperm, contents of all minerals were high in the embryo.

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Influence of embryo on the transition of developmental stages of endosperm

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A novel method to qualify the effect of embryo on endosperm development to it. To quantitively evaluate the embryo effect, we developed a novel comparison system by 3 7 3 comparing the upper and bottom endosperms of NB, using WT as a reference (Fig. 6).

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Briefly, this system has three key components: (i) Position effect. The comparison between

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To exemplify the working principle of this comparison method, we selected three Using the new comparison method, our results showed that T6P-SnRK1 signaling pathway 3 9 2 was active in the endosperm, consistent with that of the wheat (Martínez-Barajas et al., 2011).

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As shown in Fig. 7, at early stage of 5 to 10 DAF, embryo had a negative effect on sucrose, 3 9 4 glucose and fructose levels in endosperm during 5-10 DAF, probably due to its nutrient 3 9 5 consumption. The decrease of sucrose content in endosperm was accompanied by the decline 3 9 6 in T6P levels, which in return inhibited SnRK1 activity (Fig. 7A). SnRK1 promoted catabolic 3 9 7 activity in the endosperm, as evidenced by the declining accumulation of starch and storage in endosperm (Fig. 7B).

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In addition, this comparison method revealed that the influence of embryo on IAA was 4 1 4 similar with T6P but converse with SnRK1 activity across the developmental stages,  Seed development is an orchestrated progression through a series of stages, which can be

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Notably, two recent studies in Arabidopsis suggest an independent relationship between the 4 3 7 two tissues. By analyzing mutants with defective endosperm cellularization, O'Neill et al.

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(2019) found that this endosperm process is not required for the onset of embryo maturation.  well as the enhanced gene activity of amylase, lipase, and protease in endosperm (Fig. 7).

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Conversely, at middle stage between 20 and 25 DAF, the T6P-SnRK1 signaling showed an 4 8 0 opposite trend relative to that between 5 and 10 DAF, indicating it may be involved in the 4 8 1 transition of developmental stages in endosperm (Fig. 7). Taken together, the anticorrelation formation, and thus subdivide it into two stages: embryo enlargement and endosperm filling.

0 0
Accordingly, we paint a holistic and dynamic picture of rice seed development (Fig. 8), and 5 0 1 provide a brief description of each stage and its agronomical relevance as follows.  filling into two separate months, our previous report showed that only 10 % of the grain yield with results from molecular, physiological, and anatomical investigations, we framed a 5 5 7 holistic and dynamic landscape of rice seed development, and explained its agronomical significance, in particular for the intensive systems of rice-wheat or rice-oilseed rape double 5 5 9 cropping. In contrast to the quantum leap in fundamental science of genetic control of seed The following supplementary data are available at JXB online.

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Table S1. Details of primers used in this study.         β -AMY, β -Amylase. Red and navy rectangles indicate up-and down-regulation, respectively.