Control of cell proliferation, endoreduplication, cell PNAS PLUS size, and cell death by the retinoblastoma-related pathway in maize endosperm Paolo A. Sabellia,1, Yan Liua,2, Ricardo A. Dantea,3, Lucina E. Lizarragaa,4, Hong N. Nguyena, Sara W. Browna, John P. Klinglera,5, Jingjuan Yua,b, Evan LaBranta, Tracy M. Laytona, Max Feldmana,6, and Brian A. Larkinsa,1 aSchool of Plant Sciences, University of Arizona, Tucson, AZ 85721; and bState Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China Contributed by Brian A. Larkins, March 18, 2013 (sent for review February 18, 2013) The endosperm of cereal grains is one of the most valuable products death during endosperm development could potentially increase of modern agriculture. Cereal endosperm development comprises grain yield and perhaps improve its quality, yet a detailed un- different phases characterized by mitotic cell proliferation, endor- derstanding of the factors underlying control and integration of eduplication, the accumulation of storage compounds, and pro- these processes is lacking. grammed cell death. Although manipulation of these processes Cylin-Dependent Kinase (CDK) and Retinoblastoma-Related could maximize grain yield, how they are regulated and integrated (RBR) genes play fundamental roles in cell cycle regulation in all is poorly understood. We show that the Retinoblastoma-related higher eukaryotes (7, 8). They control the onset of DNA replica- (RBR) pathway controls key aspects of endosperm development in tion but also regulate other phases of the cell cycle as well as cell maize. Down-regulation of RBR1 by RNAi resulted in up-regulation cycle-independent processes (9, 10). RBR genes generally restrict of RBR3-type genes, as well as the MINICHROMOSOME MAINTE- cell cycle progression by preventing S-phase through inhibition of NANCE 2–7 gene family and PROLIFERATING CELL NUCLEAR ANTI- gene expression programs that depend on adenovirus E2 pro- GEN, which encode essential DNA replication factors. Both the moter binding factor (E2F) transcription factors. In turn, RBRs mitotic and endoreduplication cell cycles were stimulated. Develop- can be inhibited through phosphorylation by CDK activity, which ing transgenic endosperm contained 42–58% more cells and ∼70% stimulates downstream gene expression and cell cycle progression. PLANT BIOLOGY more DNA than wild type, whereas there was a reduction in cell Although these mechanisms are well known, it is not clear whether and nuclear sizes. In addition, cell death was enhanced. The DNA RBR genes play a role in cell cycle regulation during cereal en- content of mature endosperm increased 43% upon RBR1 down- dosperm development or whether they function in a pathway fi regulation, whereas storage protein content and kernel weight that depends on speci c CDKs. The cereal RBR gene family is were essentially not affected. Down-regulation of both RBR1 and more complex than that in other plants. According to phylo- CYCLIN DEPENDENT KINASE A CDKA 1 CDKA;1 genetic, gene expression, and functional studies, at least two ( ); indicated that is RBR1 RBR3 epistatic to RBR1 and controls endoreduplication through an RBR1- distinct types of RBR genes, and , have been identi- dependent pathway. However, the repressive activity of RBR1 on downstream targets was independent from CDKA;1, suggesting Significance diversification of RBR1 activities. Furthermore, RBR1 negatively regulated CDK activity, suggesting the presence of a feedback Cereal endosperm is a key source of dietary calories and raw loop. These results indicate that the RBR1 pathway plays a major materials for countless manufactured goods. Understanding role in regulation of different processes during maize endo- how the cell cycle is regulated during endosperm development sperm development and suggest the presence of tissue/organ- could lead to increased crop yield. We show that a maize level regulation of endosperm/seed homeostasis. Retinoblastoma-related gene, RBR1, plays a central role in regulating gene expression, endoreduplication, and the num- seed development | endocycle ber, size, and death of endosperm cells. RBR1 is genetically coupled to Cyclin Dependent Kinase A;1 in controlling endor- he seed endosperm is a triploid tissue resulting from the fusion eduplication but not gene expression. Seeds down-regulated Tof one haploid sperm nucleus with the diploid central cell nu- for RBR1 develop normally, which suggests higher-order con- cleus within the female gametophyte. Development of the endo- trol mechanisms regulating endosperm development that are sperm in flowering plants is characterized by acytokinetic mitoses superimposed on cell cycle regulation. of the primary endosperm nucleus, resulting in a syncytium, cel- lularization of syncytial nuclear domains, and cell proliferation Author contributions: P.A.S., Y.L., R.A.D., and B.A.L. designed research; P.A.S., Y.L., R.A.D., through mitotic activity that is coupled to cell division (1, 2). Ad- L.E.L., H.N.N., S.W.B., J.P.K., J.Y., E.L., T.M.L., and M.F. performed research; P.A.S., Y.L., R.A.D., ditionally, in the Poaceae (grass) family, the endosperm undergoes H.N.N., J.P.K., and J.Y. analyzed data; and P.A.S., R.A.D., and B.A.L. wrote the paper. a rapid growth phase that coincides with accumulation of storage The authors declare no conflict of interest. compounds, such as starch and storage proteins, during a special- Freely available online through the PNAS open access option. ized type of cell cycle known as endoreduplication. Endor- 1To whom correspondence may be addressed. E-mail: [email protected] or blarkins2@ eduplication is characterized by one or more rounds of DNA unl.edu. synthesis in the absence of mitosis, resulting in polyploid cells (3– 2Present address: Herman B. Wells Center for Pediatric Research, Indiana University School 5). Endoreduplication is highly correlated with cell size in many of Medicine, Indianapolis, IN 46202. plant and animal tissues, but its role in endosperm development 3Present address: Embrapa Agricultural Informatics, Campinas, SP 13083-886, Brazil. has not been established. Upon completion of endoreduplication 4Present address: Pharmacology and Toxicology Department, College of Pharmacy, Uni- and storage metabolite synthesis, cereal endosperm cells undergo versity of Arizona, Tucson, AZ 85721. programmed cell death (PCD), resulting in extensive DNA deg- 5Present address: Plant Stress Genomics Research Center, King Abdullah University of radation (5, 6). In maize (Zea mays L.), endosperm cells transition Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia. from a mitotic to an endoreduplication cell cycle at approximately 6Present address: Donald Danforth Plant Science Center, St. Louis, MO 63132. 8 d after pollination (DAP), and PCD becomes obvious at ap- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. proximately 16 DAP. Manipulation of cell cycle regulation and cell 1073/pnas.1304903110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1304903110 PNAS Early Edition | 1of10 Downloaded by guest on October 1, 2021 fied in grasses. Additionally, the maize genome possesses almost Results identical paralogs to RBR1 and RBR3, which are termed RBR2 Generation of RBR1DS1 Transgenic Endosperm and RBR-Specific RBR4 – and , respectively (9, 11 13). Studies of cell cycle regulation Antibodies. A transgenic maize line that down-regulated RBR1 in tissue culture showed that, in contrast to the cell cycle- in developing endosperm, termed RBR1DS1, was obtained by RBR1 RBR3 repressive activity of , plays a positive role in E2F- embryo transformation with a DNA construct producing a dou- dependent gene expression, DNA replication, and the regeneration ble-stranded hairpin RNA under control of the maize 27-kDa of transformed plant cells (13), which is uncharacteristic for γ-zein promoter (Fig. 1A). This well-characterized promoter is a member of a family of known cell cycle inhibitors. This situation highly active throughout the endosperm from approximately 8 is clearly more complex than in most dicots, such as Arabidopsis DAP (Fig. S1A), which typically coincides with the onset of the thaliana, which typically possess only a single RBR gene with endoreduplication cell cycle (21, 22). Because of the high degree a clear cell cycle-inhibitory function. Both the potential in- of nucleotide sequence identity (∼93%) between the region of activation by phosphorylation of the maize RBR1 gene product RBR1 targeted by RNAi and the corresponding domain of the (14) and an increase in its expression (11, 15) during endosperm closely related RBR2 gene, this construct could have conceivably development have been reported. However, whether RBRs play simultaneously down-regulated both RBR1 and RBR2. However, any role in regulating the cell cycle, endoreduplication, or other collateral down-regulation of the more distantly related RBR3- aspects of cereal endosperm development is unknown. type genes (∼63% identity with RBR1), RBR3 and RBR4, would How cereal RBR proteins are regulated by CDKs is also un- be unlikely. clear. Although there is compelling evidence that A- and D-type Selected domains of RBR1 and RBR3 proteins were expressed cyclins form complexes with CDKs that target RBR proteins for as GST fusions in Escherichia coli (Fig. 1 A and B) and the corre- inhibitory phosphorylation (7), the identity of the kinase moiety is sponding antisera generated. Two affinity-purified antibodies